Genetic diversity and natural selection on the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium falciparum on Bioko Island, Equatorial Guinea and global comparative analysis.

BACKGROUND: Thrombospondin-related adhesive protein (TRAP) is a transmembrane protein that plays a crucial role during the invasion of Plasmodium falciparum into liver cells. As a potential malaria vaccine candidate, the genetic diversity and natural selection of PfTRAP was assessed and the global PfTRAP polymorphism pattern was described. METHODS: 153 blood spot samples from Bioko malaria patients were collected during 2016-2018 and the target TRAP gene was amplified. Together with the sequences from database, nucleotide diversity and natural selection analysis, and the structural prediction were preformed using bioinformatical tools. RESULTS: A total of 119 Bioko PfTRAP sequences were amplified successfully. On Bioko Island, PfTRAP shows its high degree of genetic diversity and heterogeneity, with π value for 0.01046 and Hd for 0.99. The value of dN-dS (6.2231, p < 0.05) hinted at natural selection of PfTRAP on Bioko Island. Globally, the African PfTRAPs showed more diverse than the Asian ones, and significant genetic differentiation was discovered by the fixation index between African and Asian countries (Fst > 0.15, p < 0.05). 667 Asian isolates clustered in 136 haplotypes and 739 African isolates clustered in 528 haplotypes by network analysis. The mutations I116T, L221I, Y128F, G228V and P299S were predicted as probably damaging by PolyPhen online service, while mutations L49V, R285G, R285S, P299S and K421N would lead to a significant increase of free energy difference (ΔΔG > 1) indicated a destabilization of protein structure. CONCLUSIONS: Evidences in the present investigation supported that PfTRAP gene from Bioko Island and other malaria endemic countries is highly polymorphic (especially at T cell epitopes), which provided the genetic information background for developing an PfTRAP-based universal effective vaccine. Moreover, some mutations have been shown to be detrimental to the protein structure or function and deserve further study and continuous monitoring.

Genetic polymorphism of Plasmodium falciparum circumsporozoite protein on Bioko Island, Equatorial Guinea and global comparative analysis.

BACKGROUND: Plasmodium falciparum circumsporozoite protein (PfCSP) is a potential malaria vaccine candidate, but various polymorphisms of the pfcsp gene among global P. falciparum population become the major barrier to the effectiveness of vaccines. This study aimed to investigate the genetic polymorphisms and natural selection of pfcsp in Bioko and the comparison among global P. falciparum population. METHODS: From January 2011 to December 2018, 148 blood samples were collected from P. falciparum infected Bioko patients and 96 monoclonal sequences of them were successfully acquired and analysed with 2200 global pfcsp sequences mined from MalariaGEN Pf3k Database and NCBI. RESULTS: In Bioko, the N-terminus of pfcsp showed limited genetic variations and the numbers of repetitive sequences (NANP/NVDP) were mainly found as 40 (35%) and 41 (34%) in central region. Most polymorphic characters were found in Th2R/Th3R region, where natural selection (p > 0.05) and recombination occurred. The overall pattern of Bioko pfcsp gene had no obvious deviation from African mainland pfcsp (Fst = 0.00878, p < 0.05). The comparative analysis of Bioko and global pfcsp displayed the various mutation patterns and obvious geographic differentiation among populations from four continents (p < 0.05). The global pfcsp C-terminal sequences were clustered into 138 different haplotypes (H_1 to H_138). Only 3.35% of sequences matched 3D7 strain haplotype (H_1). CONCLUSIONS: The genetic polymorphism phenomena of pfcsp were found universal in Bioko and global isolates and the majority mutations located at T cell epitopes. Global genetic polymorphism and geographical characteristics were recommended to be considered for future improvement of malaria vaccine design.

Genetic diversity and allele frequencies of Plasmodium falciparum msp1 and msp2 in parasite isolates from Bioko Island, Equatorial Guinea.

BACKGROUND: Malaria is still a serious public health problem on Bioko Island (Equatorial Guinea), although the number of annual cases has been greatly reduced since 2004 through the Bioko Island Malaria Control Project (BIMCP). A better understanding of malaria parasite population diversity and transmission dynamics is critical for assessing the effectiveness of malaria control measures. The objective of this study is to investigate the genetic diversity of Plasmodium falciparum populations and multiplicity of infection (MOI) on Bioko Island 7 years after BIMCP. METHODS: A total of 181 patients with uncomplicated P. falciparum malaria diagnosed with microscopy were collected from Bioko Island from January 2011 to December 2014. Parasite DNA was extracted using chelex-100 and species were identified using a real-time PCR followed by high-resolution melting. Plasmodium falciparum msp1 and msp2 allelic families were determined using nested PCR. RESULTS: Three msp1 alleles (K1, MAD20, and RO33) and two msp2 alleles (FC27 and 3D7) were analysed in all samples. In msp1, the MAD20 allelic family was predominant with 96.69% (175/178) followed respectively by the K1 allelic family with 96.07% (171/178) and R033 allelic family with 70.78% (126/178). In msp2, the FC27 allelic family was the most frequently detected with 97.69% (169/173) compared to 3D7 with 72.25% (125/173). Twenty-six different alleles were observed in msp1 with 9 alleles for K1, 9 alleles for MAD20 and 8 alleles for R033. In msp2, 25 individual alleles were detected with 5 alleles for FC27 and 20 alleles for 3D7. The overall MOI was 5.51 with respectively 3.5 and 2.01 for msp1 and msp2. A significant increase in overall MOI was correlated with the age group of the patients (P = 0.026) or parasite densities (P = 0.04). CONCLUSIONS: The present data showed high genetic diversity and MOI values among the P. falciparum population in the study, reflecting both the high endemic level and malaria transmission on Bioko Island. These data provide valuable information for surveillance of P. falciparum infection and for assessing the appropriateness of the current malarial control strategies in the endemic area.

Design and development of a rapid meat detection system based on RPA-CRISPR/Cas12a-LFD.

In recent years, meat adulteration safety incidents have occurred frequently, triggering widespread attention and discussion. Although there are a variety of meat quality identification methods, conventional assays require high standards for personnel and experimental conditions and are not suitable for on-site testing. Therefore, there is an urgent need for a rapid, sensitive, high specificity and high sensitivity on-site meat detection method. This study is the first to apply RPA combined with CRISPR/Cas12a technology to the field of multiple meat identification. The system developed by parameter optimization can achieve specific detection of chicken, duck, beef, pork and lamb with a minimum target sequence copy number as low as 1 × 100 copies/µL for 60 min at a constant temperature. LFD test results can be directly observed with the naked eye, with the characteristics of fast, portable and simple operation, which is extremely in line with current needs. In conclusion, the meat identification RPA-CRISPR/Cas12a-LFD system established in this study has shown promising applications in the field of meat detection, with a profound impact on meat quality, and provides a model for other food safety control programs.

Comparison of physicochemical properties of different tissues from China climbing perch Anabas testudineus and crucian carp Carassius auratus.

This study aimed to investigate nutrition in climbing perch Anabas testudineus which is an important nutritious economic freshwater fish in Asia and compare with Carassius auratus (crucian carp). Three kinds of tissues, including muscle, livers, and eggs, were isolated, respectively. Physicochemical properties including moisture, ash, protein, amino acids, fat, vitamins, and calcium contents in those tissues were determined. The results showed climbing perch muscle and liver contained less moisture, but more protein, amino acids, and vitamins than crucian carp muscle and liver. While moisture, ash, protein, and total amino acids contents of climbing perch egg were lower than those of crucian carp egg. Climbing perch egg had more fat, vitamins, and calcium than crucian carp egg. The amino acid profile was also performed, and 16 amino acids were identified and quantified in muscle, liver, and egg. Among tissues, the highest and lowest concentration of total amino acid content was found in crucian carp eggs and livers, respectively. Glutamic acid (Glu) and histidine (His) were the most and least amino acids in climbing perch and crucian carp tissues, respectively. Sixteen amino acids in climbing perch egg were less than those in crucian carp egg while it is an opposite case in muscle and liver, which amino acids of climbing perch tissues were more than those of crucian carp muscle and liver. Vitamin A of climbing perch was more than crucian carp in all three tissues, but vitamin E content in climbing perch liver was lower than that of crucian carp liver. Calcium content of muscle had no difference between two species. The abovementioned comparison of physicochemical properties of different tissues from China climbing perch and crucian carp will provide a necessary supplementary of freshwater muscle nutrition research, also was helpful for application of climbing perch.

Mechanism of enrofloxacin-induced multidrug resistance in the pathogenic Vibrio harveyi from diseased abalones.

Vibrio harveyi infection had caused severe economic losses in aquaculture. A pathogenic V. harveyi strain had been successfully induced to be a multiple-resistant strain by enrofloxacin (EFX), then the mechanism of multidrug resistance was analyzed. It suggested that the minimum inhibitory concentration of EFX increased by 32-folds. Results of the Kirby-Bauer test showed that the inhibitory zone diameter was 25.3 mm for the sensitive strain (labeled as HL-S) and 8.5 mm for the resistant strain (labeled as HL-R). After 20 serial passages, even when the stress of EFX was removed, the resistance persisted. After induction of EFX, HL-R resisted to other fluoroquinolones, it even resisted to furazolidone and streptomycin, although it was sensitive to these antibiotics initially. Its sensitivity to rifampicin and doxycycline also decreased obviously. Results showed that 3522 differentially expressed genes were identified. Expression of the multidrugs efflux resistance-nodulation-cell division was significantly upregulated (164.61-folds) in HL-R. Other key genes connected with drug efflux were also upregulated significantly (p<0.05). Notably, recA encoded for recombination protein was upregulated significantly, lexA was downregulated significantly in HL-R. Research results showed that the efflux system and the save our souls system have played crucial roles during the development of multidrug resistance of V. harveyi.

Molecular Surveillance of Artemisinin-Based Combination Therapies Resistance in Plasmodium falciparum Parasites from Bioko Island, Equatorial Guinea.

Artemisinin-based combination therapies (ACTs) resistance has emerged and could be diffusing in Africa. As an offshore island on the African continent, the island of Bioko in Equatorial Guinea is considered severely affected and resistant to drug-resistant Plasmodium falciparum malaria. However, the spatial and temporal distribution remain unclear. Molecular monitoring targeting the Pfcrt, Pfk13, Pfpm2, and Pfmdr1 genes was conducted to provide insight into the impact of current antimalarial drug resistance on the island. Furthermore, polymorphic characteristics, haplotype network, and the effect of natural selection of the Pfk13 gene were evaluated. A total of 152 Plasmodium falciparum samples (collected from 2017 to 2019) were analyzed for copy number variation of the Pfpm2 gene and Pfk13, Pfcrt, and Pfmdr1 mutations. Statistical analysis of Pfk13 sequences was performed following different evolutionary models using 96 Bioko sequences and 1322 global sequences. The results showed that the prevalence of Pfk13, Pfcrt, and Pfmdr1 mutations was 73.68%, 78.29%, and 75.66%, respectively. Large proportions of isolates with multiple copies of Pfpm2 were observed (67.86%). In Bioko parasites, the genetic diversity of Pfk13 was low, and purifying selection was suggested by Tajima's D test (-1.644, P > 0.05) and the dN/dS test (-0.0004438, P > 0.05). The extended haplotype homozygosity analysis revealed that Pfk13_K189T, although most frequent in Africa, has not yet conferred a selective advantage for parasitic survival. The results suggested that the implementation of continuous drug monitoring on Bioko Island is an essential measure. IMPORTANCE Malaria, one of the tropical parasitic diseases with a high transmission rate in Bioko Island, Equatorial Guinea, especially caused by P. falciparum is highly prevalent in this region and is commonly treated locally with ACTs. The declining antimalarial susceptibility of artemisinin-based drugs suggested that resistance to artemisinin and its derivatives is developing in P. falciparum. Copy number variants in Pfpm2 and genetic polymorphisms in Pfk13, Pfcrt, and Pfmdr1 can be used as risk assessment indicators to track the development and spread of drug resistance. This study reported for the first time the molecular surveillance of Pfpm2, Pfcrt, Pfk13, and Pfmdr1 genes in Bioko Island from 2017 to 2019 to assess the possible risk of local drug-resistant P. falciparum.

A visual method to detect meat adulteration by recombinase polymerase amplification combined with lateral flow dipstick.

Determining the animal source in meat and meat products is crucial to prevent meat adulteration and fraud. Conventional methods require considerable operator skills, expensive instruments and are unable to provide fast mobile on-site detection systems to detect contamination of meat products. We developed a visual method based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) to identify beef (Bos taurus), sheep (Ovis aries), pork (Sus scrofa), duck (Anas platyrhynchos) and chicken (Gallus gallus). The reaction was completed within 20 min. The results were determined by the naked eye. The detection limits of the RPA-LFD assays for duck, beef, sheep, chicken and pork were 101/µL, 102/µL, 102/µL, 101/µL and 101/µL, respectively. Furthermore, the RPA-LFD assays could differentiate species in boiled, microwaved, pressure-cooked or fried samples. These RPA-LFD assays represent a rapid, mobile detection system for determining meat product contamination.

The Mechanisms of Yu Ping Feng San in Tracking the Cisplatin-Resistance by Regulating ATP-Binding Cassette Transporter and Glutathione S-Transferase in Lung Cancer Cells.

Cisplatin is one of the first line anti-cancer drugs prescribed for treatment of solid tumors; however, the chemotherapeutic drug resistance is still a major obstacle of cisplatin in treating cancers. Yu Ping Feng San (YPFS), a well-known ancient Chinese herbal combination formula consisting of Astragali Radix, Atractylodis Macrocephalae Rhizoma and Saposhnikoviae Radix, is prescribed as a herbal decoction to treat immune disorders in clinic. To understand the fast-onset action of YPFS as an anti-cancer drug to fight against the drug resistance of cisplatin, we provided detailed analyses of intracellular cisplatin accumulation, cell viability, and expressions and activities of ATP-binding cassette transporters and glutathione S-transferases (GSTs) in YPFS-treated lung cancer cell lines. In cultured A549 or its cisplatin-resistance A549/DDP cells, application of YPFS increased accumulation of intracellular cisplatin, resulting in lower cell viability. In parallel, the activities and expressions of ATP-binding cassette transporters and GSTs were down-regulated in the presence of YPFS. The expression of p65 subunit of NF-κB complex was reduced by treating the cultures with YPFS, leading to a high ratio of Bax/Bcl-2, i.e. increasing the rate of cell death. Prim-O-glucosylcimifugin, one of the abundant ingredients in YPFS, modulated the activity of GSTs, and then elevated cisplatin accumulation, resulting in increased cell apoptosis. The present result supports the notion of YPFS in reversing drug resistance of cisplatin in lung cancer cells by elevating of intracellular cisplatin, and the underlying mechanism may be down regulating the activities and expressions of ATP-binding cassette transporters and GSTs.

Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus.

Background: The emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed. Methods: Targeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively. Results: The limit of detection was 1 copies/µl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples. Conclusion: This work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.

Epidemiology, evolutionary origin, and malaria-induced positive selection effects of G6PD-deficient alleles in Chinese populations.

BACKGROUND: Although glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited disorder in the Chinese population, there is scarce evidence regarding the epidemiology, evolutionary origin, and malaria-induced positive selection effects of G6PD-deficient alleles in various Chinese ethnic populations. METHODS: We performed a large population-based screening (n = 15,690) to examine the impact of selection on human nucleotide diversity and to infer the evolutionary history of the most common deficiency alleles in Chinese populations. RESULTS: The frequencies of G6PD deficiency ranged from 0% to 11.6% in 12 Chinese ethnic populations. A frequency map based on geographic information showed that G6PD deficiency was highly correlated with historical malaria prevalence in China and was affected by altitude and latitude. The five most frequently occurring G6PD gene variants were NM_001042351.3:c.1376G>T, NM_001042351.3:c.1388G>A, NM_001042351.3:c.95A>G, NM_001042351.3:c.1311T>C, and NM_001042351.3:c.1024C>T, which were distributed with ethnic features. A pathogenic but rarely reported variant site (NM_001042351.3:c.448G>A) was identified in this study. Bioinformatic analysis revealed a strong and recent positive selection targeting the NM_001042351.3:c.1376G>T allele that originated in the past 3125 to 3750 years and another selection targeting the NM_001042351.3:c.1388G>A allele that originated in the past 5000 to 6000 years. Additionally, both alleles originated from a single ancestor. CONCLUSION: These results indicate that malaria has had a major impact on the Chinese genome since the introduction of rice agriculture.

Trends in Molecular Markers Associated with Resistance to Sulfadoxine-Pyrimethamine (SP) Among Plasmodium falciparum Isolates on Bioko Island, Equatorial Guinea: 2011-2017.

PURPOSE: Antimalarial drug resistance is one of the major challenges in global efforts to control and eliminate malaria. In 2006, sulfadoxine-pyrimethamine (SP) replaced with artemisinin-based combination therapy (ACT) on Bioko Island, Equatorial Guinea, in response to increasing SP resistance, which is associated with mutations in the dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes. PATIENTS AND METHODS: To evaluate the trend of molecular markers associated with SP resistance on Bioko Island from 2011 to 2017, 179 samples collected during active case detection were analysed by PCR and DNA sequencing. RESULTS: Pfdhfr and Pfdhps gene sequences were obtained for 90.5% (162/179) and 77.1% (138/179) of the samples, respectively. For Pfdhfr, 97.5% (158/162), 95.7% (155/162) and 98.1% (159/162) of the samples contained N51I, C59R and S108N mutant alleles, respectively. And Pfdhps S436A, A437G, K540E, A581G, and A613S mutations were observed in 25.4% (35/138), 88.4% (122/138), 5.1% (7/138), 1.4% (2/138), and 7.2% (10/138) of the samples, respectively. Two classes of previously described Pfdhfr-Pfdhps haplotypes associated with SP resistance and their frequencies were identified: partial (IRNI-SGKAA, 59.4%) and full (IRNI-SGEAA, 5.5%) resistance. Although no significant difference was observed in different time periods (p>0.05), our study confirmed a slowly increasing trend of the frequencies of these SP-resistance markers in Bioko parasites over the 7 years investigated. CONCLUSION: The findings reveal the general existence of SP-resistance markers on Bioko Island even after the replacement of SP as a first-line treatment for uncomplicated malaria. Continuous molecular monitoring and additional control efforts in the region are urgently needed.

Evidence of positively selected G6PD A- allele reduces risk of Plasmodium falciparum infection in African population on Bioko Island.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) is an essential enzyme that protects red blood cells from oxidative damage. Although G6PD-deficient alleles appear to confer a protective effect of malaria, the link with clinical protection against Plasmodium infection is conflicting. METHODS: A case-control study was conducted on Bioko Island, Equatorial Guinea and further genotyping analysis used to detect natural selection of the G6PD A- allele. RESULTS: Our results showed G6PD A- allele could significantly reduce the risk of Plasmodium falciparum infection in male individuals (adjusted odds ratio [AOR], 0.43; 95% confidence interval [CI], 0.20-0.93; p < .05) and homozygous female individuals (AOR, 0.11; 95% CI, 0.01-0.84; p < .05). Additionally, the parasite densities were significantly different in the individuals with different G6PD A- alleles and individual levels of G6PD enzyme activity. The pattern of linkage disequilibrium and results of the long-range haplotype test revealed a strong selective signature in the region encompassing the G6PD A- allele over the past 6,250 years. The network of inferred haplotypes suggested a single origin of the G6PD A- allele in Africans. CONCLUSION: Our findings demonstrate that glucose-6-phosphate dehydrogenase (G6PD) A- allele could reduce the risk of P. falciparum infection in the African population and indicate that malaria has a recent positive selection on G6PD A- allele.

Rapid identification of apolipoprotein E genotypes by high-resolution melting analysis in Chinese Han and African Fang populations.

Apolipoprotein E (APOE) gene polymorphism can affect APOE gene transcription, serum lipid levels and repair of tissue damage, which could place individuals at serious risk of cardiovascular disease or certain infectious diseases. Recently, high-resolution melting (HRM) analysis was reported to be a simple, inexpensive, accurate and sensitive method for the genotyping or/and scanning of rare mutations. For this reason, an HRM analysis was used in the present study for APOE genotyping in the Southern Chinese Han and African Fang populations. A total of 100 healthy Southern Chinese Han and 175 healthy African Fang individuals attended the study. Polymerase chain reaction-DNA sequencing was used as a reference method for the genotyping of these samples. The six APOE genotypes could all be rapidly and efficiently identified by HRM analysis, and 100% concordance was found between the HRM analysis and the reference method. The allele frequencies of APOE in the Southern Chinese Han population were 7.0, 87.5 and 5.5% for ɛ2, ɛ3 and ɛ4, respectively. In the African Fang population, the allele frequencies of APOE were 24.3, 65.7 and 10.0% for ɛ2, ɛ3 and ɛ4, respectively. A statistically significant difference was found between the allele frequencies between the populations (P<0.05). In conclusion, the present study revealed the molecular characterization of APOE gene polymorphism in the Han population from the Chaozhou region of Southern China and the Fang population from Equatorial Guinea. The findings of the study indicated that HRM analysis could be used as an accurate and sensitive method for the rapid screening and identification of APOE genotypes in prospective clinical and population genetic analyses.

Characterizations of cholinesterases in golden apple snail (Pomacea canaliculata).

Cholinesterases (ChEs) have been identified in vertebrates and invertebrates. Inhibition of ChE activity in invertebrates, such as bivalve molluscs, has been used to evaluate the exposure of organophosphates, carbamate pesticides, and heavy metals in the marine system. The golden apple snail (Pomacea canaliculata) is considered as one of the worst invasive alien species harmful to rice and other crops. The ChE(s) in this animal, which has been found recently, but poorly characterized thus far, could serve as biomarker(s) for environmental surveillance as well as a potential target for the pest control. In this study, the tissue distribution, substrate preference, sensitivity to ChE inhibitors, and molecular species of ChEs in P. canaliculata were investigated. It was found that the activities of both AChE and BChE were present in all test tissues. The intestine had the most abundant ChE activities. Both enzymes had fair activities in the head, kidney, and gills. The BChE activity was more sensitive to tetra-isopropylpyrophosphoramide (iso-OMPA) than the AChE. Only one BChE molecular species, 5.8S, was found in the intestine and head, whereas two AChE species, 5.8S and 11.6S, were found there. We propose that intestine ChEs of this snail may be potential biomarkers for manipulating pollutions.

High resolution melting analysis: a rapid screening and typing tool for common ß-thalassemia mutation in Chinese population.

ß-thalassemia is a common inherited disorder worldwide including southern China, and at least 45 distinct ß-thalassemia mutations have been identified in China. High-resolution melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. However, there was no systemic study on the diagnostic capability of HRM to identify ß-thalassemia. Here, we used an improved HRM method to screen and type 12 common ß-thalassemia mutations in Chinese, and the rapidity and reliability of this method was investigated. The whole PCR and HRM procedure could be completed in 40 min. The heterozygous mutations and 4 kinds of homozygous mutations could be readily differentiated from the melting curve except c.-78A>G heterozygote and c.-79A>G heterozygote. The diagnostic reliability of this HRM assay was evaluated on 756 pre-typed genomic DNA samples and 50 cases of blood spots on filter paper, which were collected from seven high prevalent provinces in southern China. If c.-78A>G heterozygote and c.-79A>G heterozygote were classified into the same group (c.-78&79 A>G heterozygote), the HRM method was in complete concordance with the reference method (reverse dot blot/DNA-sequencing). In a conclusion, the HRM method appears to be an accurate and sensitive method for the rapid screening and identification of ß-thalassemia mutations. In the future, we suggest this technology to be used in neonatal blood spot screening program. It could enlarge the coverage of ß-thalassemia screening program in China. At the same time, its value should be confirmed in prospectively clinical and epidemiological studies.

Characterization of acetylcholinesterase in Hong Kong oyster (Crassostrea hongkongensis) from South China Sea.

Acetylcholinesterase (AChE) activity has been used to evaluate the exposure of mollusk bivalves to organophosphates, carbamate pesticides, and heavy metals. Crassostrea hongkongensis is a Hong Kong endemic oyster, and has a high commercial value along the coastal area of South China. The use of this species as a bio-indicator of the marine environment, and the use of AChE activity measurements in tissues of C. hongkongensis require prior characterization of AChE in this species. Here, we report that gill tissue contains the highest AChE activity in C. hongkongensis, and that the molecular form of AChE is most likely to be a dimeric form. In addition, the effect of the pesticide acephate on AChE activity in the gill of C. hongkongensis was analyzed, and the mean inhibition concentration (IC50) value was determined. This study suggests that AChE activity in the gill tissue of C. hongkongensis might be used as a biomarker in monitoring organophosphate contamination in the marine fauna of South China.