Comparison of the RE and B1 gene for detection of Toxoplasma gondii infection in children with cancer

Early, accurate and effective diagnosis of toxoplasmosis can make an important contribution to the prevention and control of disease, especially in people who are at risk. In this study, two commonly used genomic repeats of Toxoplasma gondii, RE (GenBank accession number AF146527) and B1, were compared to each other in nested-PCR assay. Five hundred and thirty-five blood samples from children with leukemia were tested for the presence of T. gondii antibodies using enzyme immunoassays. One hundred and ten DNA samples of these patients (50 IgM +, IgG +, 10 IgM −, IgG +, and 50 IgM −, IgG −) were analyzed by nested-PCR. The specificity of two nested PCR assays was determined using the DNA samples of other parasites and human chromosomal DNA. As a result, 82% (41/50) and 68% (34/50) of the IgM +, IgG + samples were positive on duplicate RE and B1-nested PCR analyses, respectively. None of the 10 IgM −, IgG + seropositive samples was detected positive after testing RE and B1-nested PCR assays in duplicate. One (2%) of the 50 seronegative samples was positive by duplicate RE-nested PCR but none of them were positive by duplicate B1-nested PCR. The detection limit of the RE-nested PCR assay was 640 fg of T. gondii DNA whereas this rate for B1-nested PCR was 5.12 pg of the DNA template. No cross-reactivity with the DNA of other parasites and human chromosomal DNA was found. The results indicate that an RE-based nested PCR assay is more sensitive than B1 genomic target, of those tested, for detection of T. gondii. It is noteworthy that in comparison with B1-nested PCR, RE-nested PCR could detect the T. gondii DNA in seronegative samples too.     

Development and evaluation of a reverse dot blot assay for the simultaneous detection of common toxic microalgae along the Chinese coast

Toxic microalgae currently pose a great threat to human health, ecosystem, fishery, tourism, and aquaculture along the Chinese coast. The detection of toxic microalgae by routinely monitoring natural waters is necessary to provide timely mitigation. Therefore, an effective, simultaneous detection protocol should be established for the simple, rapid, and accurate identification of causative algae. This study developed and evaluated a reverse dot blot assay (RDBA) combined with a low-density membrane-based DNA array for the rapid and simultaneous detection of toxic microalgae that are commonly distributed along the Chinese coast. The large subunit rDNA D1–D2 regions of the target species were first sequenced to design taxonomic probes. Probe specificity was validated by performing a cross-reactivity test with dot blot hybridization. The tailed probes were immobilized onto a nylon membrane to prepare a low-density DNA array for RDBA. The established detection procedure involved DNA extraction, biotin (Bio)-labeling of objective sequences by multiple polymerase chain reaction (M-PCR), RDBA, coloration, and judgment of hybridization by the naked eye. Bio-labeled primer-based labeling proved to be an economical and effective method to prepare Bio-labeled PCR products for RDBA. The detection limits of RDBA using the M-PCR-labeling products from DNA templates prepared by different methods were also compared, and a kit-based DNA extraction method displayed the lowest detection limit of 0.5 cells. Simulation results showed that RDBA can recover all target species and was not affected by background DNA. RDBA was proven effective, specific, and sensitive for the simultaneous detection of toxic microalgae in the field samples. Therefore, this method may be used in the field monitoring of natural samples.     

Partial cloning and production of polyclonal antiserum against recombinant capsid protein of Hepatopancreatic Parvovirus (HPV) and its application for diagnostics in penaeid shrimp

Hepatopancreatic Parvovirus (HPV) causes infection in the early stages of shrimp leading to retarded growth, ultimaltely resulting in monetary loss to the shrimp farmers. To over come this situation screening of post-larvae (PL) by immunology-based diagnostics is required. Hence, the specific gene of capsid protein for HPV was cloned into pRSET B expression vector and rHCP overexpressed with 6-histidine tagged fusion protein in Escherichia coli BL21(DE3). Immunology-based methods like Western blot, dot blot and ELISA techniques were employed to detect HPV in infected samples using the antiserum raised in rabbits against recombinant HCP of HPV. The dot blot assay using anti-rHCP was found to be capable of detecting HPV in HPV infected post-larvae as early as at 24 h post infection. The antiserum could detect the HPV in the infected samples at 1 ng of total protein. HPV infection estimated by ELISA using anti-HCP and pure r-HCP as a standard was found to increase gradually during the course of infection from 24 h post infection. The sensitivity of antibody-based diagnostics employed in the present study was compared with that of PCR diagnostic method to screen the post-larvae for the detection of HPV.     

Hyperbranched rolling circle amplification as a novel method for rapid and sensitive detection of Amphidinium carterae

High quality of coastal water is critical to marine ecosystems, marine fisheries, public health, and aquatic environment. Specially, bio-toxin derived from toxic microalgae is currently threatening many coastal countries. Therefore, development of rapid and sensitive methods for the detection of toxin-producing microalgae is necessary for warning of water quality. In this paper, we established a novel method for rapid and sensitive detection of Amphidinium carterae by hyperbranched rolling circle amplification (HRCA). The partial large subunit rDNA (LSU D1–D2) of A. carterae was sequenced to design species-specific padlock probe (PLP). The PLP-coupled with two amplification primers were employed for HRCA. The optimized HRCA conditions were as follows: padlock concentration, 20 pM; ligation temperature, 65 °C; ligation time, 15 min; amplification temperature, 61 °C; and amplification time, 15 min. The developed HRCA was confirmed to be specific for A. carterae by tests with other algae. The sensitivity of HRCA was 100-fold higher than regular PCR, exhibiting a detection limit of 1 fg/μL representing 283 copies for the recombinant plasmid containing the target LSU D1–D2, and 1 cell for target species. Finally, a simplified protocol was applied to the simulated field and environmental materials, and exhibited a good performance. The whole detection could be completed within 1.5 h, displaying a repeated detection limit of 1 cell. The positive HRCA results could be visualized through coloration reaction by adding the fluorescent dye SYBR Green I to the amplification products. The HRCA provides a useful tool to quickly screen large sample sets for A. carterae, as well as other toxic species.     

Can human papillomavirus DNA testing of self-collected vaginal samples compare with physician-collected cervical samples and cytology for cervical cancer screening in developing countries?

Background: To determine human papillomavirus (HPV) types by polymerase chain reaction (PCR)-reverse line blot assay and examine the concordance between HPV by Hybrid Capture 2 (HC2) and PCR on self-collected vaginal and physician-collected cervical samples and cytology. Methods: This was a cross-sectional study of 546 sexually active women aged ≥30 years with persistent vaginal discharge, intermenstrual or postcoital bleeding or an unhealthy cervix. Participants self-collected vaginal samples (HPV-S) and physicians collected cervical samples for conventional Pap smear and HPV DNA (HPV-P) testing and performed colposcopy, with directed biopsy, if indicated. HPV testing and genotyping was done by HC2 and PCR reverse line blot assay. Concordance between HC2 and PCR results of self- and physician-collected samples was determined using a Kappa statistic (κ) and Chi-square test. Results: Complete data were available for 512 sets with 98% of women providing a satisfactory self-sample. PCR detected oncogenic HPV in 12.3% of self- and 13.0% of physician-collected samples. Overall, there was 93.8% agreement between physician-collected and self-samples (κ = 76.31%, 95% confidence interval [CI]: 64.97–82.29%, p = 0.04)—complete concordance in 473 cases (57 positive, 416 negative), partial concordance in seven pairs and discordance in 32 pairs. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of self-sampling for detection of cervical intraepithelial neoplasia (CIN)2+ disease were 82.5%, 93.6%, 52.4% and 98.4%, respectively; for physician-sampling they were 87.5%, 93.2%, 52.2% and 98.9%, respectively; and for cytology they were 77.5%, 87.3%, 34.1% and 97.9%, respectively. Concordance between HC2 and PCR was 90.9% for self-samples (κ = 63.7%, 95% CI: 55.2–72.2%) and 95.3% for physician-collected samples (κ = 80.4%, 95% CI: 71.8–89.0%). Conclusions: Self-HPV sampling compares favourably with physician-sampling and cytology. A rapid, affordable, HPV self-test kit can be used as the primary method of cervical cancer screening in low-resource situations.     

Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification

Trichinella spiralis is a tissue-dwelling nematode parasite. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the sensitive and rapid detection of T. spiralis larvae in muscle samples. Sixteen sets of primers were designed to recognise distinct sequences of a conserved gene, a 1.6 kb repetitive element of the Trichinella genome. One set of primers was selected as the most appropriate for rapid detection. The specificity and sensitivity of the primers in LAMP reactions for T. spiralis larvae and muscle samples of mice infected with T. spiralis were determined. Another 10 heterologous parasites were selected for specificity assays. The results showed that target DNA was amplified and visualised by monitoring turbidity and adding calcein detection methods within 70 min at an isothermal temperature of 63 °C. The sensitivity of LAMP with the detection limit of 362 fg/μl was >10 times higher than that for PCR. The designed primers had a good specificity. No cross-reactivity was found with the DNA of any other parasites. The assay was able to detect T. spiralis in all mouse muscle samples infected with 10 T. spiralis larvae on day 20 p.i. We believe this is the first report regarding the application of the LAMP assay for detection of T. spiralis larvae in muscle samples from experimentally infected mice. This method demonstrates a potentially valuable means for the direct detection of T. spiralis larvae in meat inspection.     

Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples

Clinical microbiology laboratories rely on quantitative PCR for its speed, sensitivity, specificity and ease-of-use. However, quantitative PCR quantitation requires the use of a standard curve or normalisation to reference genes. Droplet digital PCR provides absolute quantitation without the need for calibration curves. A comparison between droplet digital PCR and quantitative PCR-based analyses was conducted for the enteric parasite Cryptosporidium, which is an important cause of gastritis in both humans and animals. Two loci were analysed (18S rRNA and actin) using a range of Cryptosporidium DNA templates, including recombinant plasmids, purified haemocytometer-counted oocysts, commercial flow cytometry-counted oocysts and faecal DNA samples from sheep, cattle and humans. Each method was evaluated for linearity, precision, limit of detection and cost. Across the same range of detection, both methods showed a high degree of linearity and positive correlation for standards (R² ⩾ 0.999) and faecal samples (R² ⩾ 0.9750). The precision of droplet digital PCR, as measured by mean Relative Standard Deviation (RSD;%), was consistently better compared with quantitative PCR, particularly for the 18S rRNA locus, but was poorer as DNA concentration decreased. The quantitative detection of quantitative PCR was unaffected by DNA concentration, but droplet digital PCR quantitative PCR was less affected by the presence of inhibitors, compared with quantitative PCR. For most templates analysed including Cryptosporidium-positive faecal DNA, the template copy numbers, as determined by droplet digital PCR, were consistently lower than by quantitative PCR. However, the quantitations obtained by quantitative PCR are dependent on the accuracy of the standard curve and when the quantitative PCR data were corrected for pipetting and DNA losses (as determined by droplet digital PCR), then the sensitivity of both methods was comparable. A cost analysis based on 96 samples revealed that the overall cost (consumables and labour) of droplet digital PCR was two times higher than quantitative PCR. Using droplet digital PCR to precisely quantify standard dilutions used for high-throughput and cost-effective amplifications by quantitative PCR would be one way to combine the advantages of the two technologies.     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 μg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 μg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 μg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 × 10² to 1 × 10⁵ genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 × 10⁵ CFU/ml at 4, 20, and 37 °C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 °C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 °C to a minority of the mixed population compared to membrane damage.     

Opisthorchis viverrini and Haplorchis taichui: Development of a multiplex PCR assay for their detection and differentiation using specific primers derived from HAT-RAPD

Specific primers for the detection of Opisthorchis viverrini and Haplorchis taichui were investigated by using the HAT-RAPD PCR method. Fourteen arbitrary primers (Operon Technologies) were performed for the generation of polymorphic DNA profiles. The results showed that a 319 bp fragment generated from the OPA-04 primer was expected to be O. viverrini-specific while a 256 bp fragment generated from the OPP-11 primer was considered to be H. taichui-specific. Based on each sequence data, two pairs of specific primers were designed and sequences of each primer were as follows; H. taichui; Hapt_F5′-GGCCAACGCAATCGTCATCC-3′and Hapt_R1 5′-CTCTCGACCTCCTCTAGAAT-3′ which yielded a 170 bp PCR product. For O. viverrini, OpV-1F: 5′-AATCGGGCTGCATATTGACCGAT-3′ and OpV-1R: 5′-CGGTGTTGCTTATTTTGCAGACAA-3′ which generated a 319 bp PCR product. These specific primers were tested for efficacy and specific detection for all parasites DNA samples. The results showed that 170 and 319 bp specific PCR products were generated as equivalent to positive result in H. taichui and O. viverrini, respectively by having no cross-reaction with any parasites tested. PCR conditions are recommended at 68 °C annealing temperature and with 0.5 mM magnesium chloride (Mg Cl₂). Additionally, specific primers developed in this study were effective to determine the presence of both parasites in fish and snail intermediate hosts, which the DNA of O. viverrini was artificially spiked since it is rarely found in northern Thailand.The H. taichui and O. viverrini-specific primers successfully developed in this study can be use for epidemiological monitoring, preventing management and control programs.     

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 × 10⁰ cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.     

Human Papillomavirus (HPV) 16 E6 seropositivity is elevated in subjects with oral HPV16 infection

IntroductionHuman Papillomavirus (HPV) 16 E6 serum antibodies are common in people with HPV-related oropharyngeal cancers (HPV-OPC), but not the general population. We explored HPV16 seroprevalence in people with and without oral HPV16 infection, the cause of HPV-OPC.MethodsOral rinse samples were collected semiannually and tested for 36 types of HPV DNA by PCR. HPV16 E6 serum antibodies were tested at the visit of first oral HPV detection in participants with prevalent (n = 54), or incident (n = 39) oral HPV16 DNA; or at baseline in matched participants with no oral HPV16 DNA (n = 155) using multiplex serology assay. Predictors of seropositivity were examined using logistic regression.ResultsHPV16 E6 seropositivity (7.5% vs 0.7%; p = 0.005) but not seropositivity to the other HPV16 antigens, was significantly more common in those with than without oral HPV16 infection. There were only 8 HPV16 E6 seropositive participants, but oral HPV16 DNA remained a strong predictor of E6 seropositivity after adjustment for other risk factors (aOR = 14.6 95%CI, 1.7–122.5). Seroprevalence was similar in those with prevalent (7.4%; 4/54), and incident (7.7%; 3/39) oral HPV16 infection (p = 1.00). E6 seroprevalence was associated with reduced oral HPV16 clearance, but was not statistically significant (HR = 0.65 95% CI, 0.16–2.70).Seropositive participants were primarily male (87.5%), HIV-positive (75.0%; median CD4 cell-count of 840) and had oral HPV16 DNA (87.5%). History of an HPV-related cancer (0/8) or HPV-related anogenital dysplasia (1/8) was rare, and 4 participants had recent screening showing no anogenital dysplasia.DiscussionHPV16 E6 seropositivity was higher among people with than without oral HPV16 infection, despite no known anogenital disease in these participants.     

DNA integrity of fresh and frozen canine epididymal spermatozoa

The aims of this study were to evaluate: (1) the effect of cryopreservation on DNA fragmentation of canine epididymal spermatozoa, and (2) the potential protective effect of melatonin on post-thaw sperm quality (motility, morphology, acrosomal and DNA integrity). Epididymal spermatozoa were collected after orchiectomy of ten dogs. Sperm samples were frozen in the presence or absence of melatonin (1 mM). DNA fragmentation index (percentage of spermatozoa with fragmented DNA) was similar in fresh samples (3.3 ± 3.6) and samples frozen with (4.2 ± 3.8) or without (3.6 ± 3.7) melatonin. Sperm motility was significantly (p < 0.0001) higher in fresh compared to frozen samples. The presence of melatonin in the freezing extender did not affect the sperm motility. Proportions of spermatozoa with normal morphology were similar in fresh and frozen samples, irrespective of the presence of melatonin in the extender. Acrosome integrity was significantly decreased (p < 0.01) by cryopreservation, and melatonin did not exert any beneficial effects. In conclusion, DNA fragmentation of canine epididymal spermatozoa was not affected by the freezing procedure, and the presence of melatonin did not preserve motility and acrosome integrity which were adversely affected by cryopreservation. The evaluation of DNA status of thawed gametes is particularly relevant for epididymal spermatozoa since these spermatozoa are usually stored and used in assisted reproductive techniques.     

Occurrence of toxic Prymnesium parvum blooms with high protease activity is related to fish mortality in Hungarian ponds

The unicellular alga Prymnesium parvum has been responsible for toxic incidents with severe ecological impacts in many parts of the world, and causes massive fish kills worldwide. Recently the haptophyte microalgae have caused water-bloom (4.3 × 10⁴ cells ml⁻¹) in 6 fish ponds with high conductivity in Hungary, and caused fish mortality with typical symptoms. Toxicity of P. parvum from water samples was quantified by the assay of the influence of its cell-free filtrates on haemolysis (346 ± 42.2) and in fish and daphnia toxicity tests. High amount of proteases in P. parvum containing waterbloom samples were detected with the help of activity gel electrophoresis. The proteases of investigated P. parvum samples (125–18 kDa) showed high gelatinolytic activity and some of them showed sensitivity to EDTA (inhibitors of metalloproteases) and to PMSF (inhibitors of serine proteases).     

Comparison of PCR-based diagnoses for visceral leishmaniasis in Bangladesh

The diagnosis of visceral leishmaniasis (VL) is performed using multiple methods encompassing parasitological, serological and nucleic acid-based diagnostic tools, each method with its own unique advantages and disadvantages. Conventional parasitological methods are risky for the patient and require skilled personnel to collect specimens from spleen or bone marrow, and hence they are not generally available in impoverished areas. Polymerase chain reaction (PCR) has been validated as an excellent alternative to microscopy in terms of sensitivity and specificity. Here, we evaluate four different PCR assays targeting ITS1, ITS2, mini-exon and small subunit-rRNA (SSUrRNA) using DNA extracted from peripheral blood buffy coat in order to avoid more invasive processes. A total of 61 VL patients and 75 non-VL infected control individuals were enrolled. The VL patients were confirmed to be positive for Leishmania amastigotes in splenic smears by microscopy. Sensitivities of the PCR targeting ITS1, ITS2, SSUrRNA and mini-exon were 96.7%, 91.8%, 88.5% and 34.4%, respectively, while the specificity was 98.7% for all methods. Nested PCR for ITS1 resulted in 100% sensitivity. The efficacy of each PCR was evaluated with various Leishmania amastigote parasite loads in each spleen smear, graded from 1 + to 5 +. The PCR targeting ITS1 showed 100% sensitivity for the detection of Leishmania donovani in all samples from grades ≥ 3, ≥ 4, and ≥ 5, respectively. The restriction fragment length polymorphism observed in ITS1 amplicons digested by HaeIII classified the parasite into L. donovani complex. The ITS1 PCR was found to be equal to conventional, but very invasive and risky parasitological diagnoses and superior to other PCR based methods in sensitivity and examination of genetic heterogeneity. We recommend the PCR targeting ITS1 using peripheral blood buffy coat DNA as an alternate, less invasive diagnostic choice for the confirmation of L. donovani infection.     

Residual DNA Quantification in Clinical Batches of BBG2Na,a Recombinant Subunit Vaccine Against Human Respiratory Syncytial Virus

BBG2Na, a well-defined recombinant protein produced in Escherichia coli, is a promising human respiratory syncytial virus subunit vaccine candidate. This study describes the quantification of residual DNA in large scale batches used in phase I to III clinical trials. Two different analytical methods were developed and applied on five different final bulks of Drug Substance and their associated in process control samples, namely a chemiluminescent hybridisation assay and the total DNA Threshold™ System assay. These two complementary methods demonstrated the clearance of residual DNA during the downstream purification process. The amount of residual DNA found in the final bulks was below 20 pg of DNA per 300 μg BBG2Na, the highest tested clinical dose of antigen. This is very low level of residual DNA for a recombinant subunit vaccine produced in a bacteria and contribute to make for BBG2Na a well-characterised biopharmaceutical. This study also provides data concerning the validation of the hybridisation dot blot assay and the total DNA Threshold^™assay.     

Electrochemical oxidation behavior of guanine and adenine on graphene–Nafion composite film modified glassy carbon electrode and the simultaneous determination

The electrochemical behavior of guanine and adenine on the graphene and Nafion composite film modified glassy carbon electrode was investigated by differential pulse voltammetry (DPV). The results indicated that the modified electrode exhibited an excellent electrocatalytic activity towards the oxidation of guanine and adenine, testified by the increased oxidation peak current and decreased oxidation potential. The experimental conditions were optimized. The separation of the two oxidation peaks was 0.364 V in 0.1 M pH 4.4 acetate buffer solution (ABS). Based on this, a novel electrochemical method was proposed to simultaneously determine guanine and adenine with the detection limit of 0.58 (guanine) and 0.75 (adenine) μM (S/N = 3). The proposed method was applied to determine guanine and adenine in milk powder, urine and herring sperm DNA samples with satisfactory results. The value of (G + C)/(A + T) in herring sperm DNA was calculated to be 0.8065. The fabricated electrode showed excellent reproducibility, stability and anti-interference.     

Properties of cold-active uracil-DNA glycosylase from Photobacterium aplysiae GMD509, and its PCR application for carryover contamination control

Owing to its selective uracil-excision property, uracil-DNA glycosylase (UDG) has been widely utilized in diagnostic PCR applications as an effective decontamination method. Since mesophilic UDGs in PCR has been shown to degrade not just contaminant DNA but also target amplicon, there has been an increase in demand for cold-active UDGs. We characterized UDG from Photobacterium aplysiae GMD509 (Pap GMD509 UDG) expressed in Escherichia coli BL21 (DE3). The optimal temperature range of the enzyme was 25–30 °C, which is considerably lower than any other reported UDG, and the half-life of the enzyme at 40 °C and 50 °C was approximately 77 s and 33 s, respectively. These results clearly demonstrate the fragility of this enzyme upon heating. In addition, we compared the carryover contamination control property of Pap GMD509 UDG with other commercialized UDGs. The results indicate that Pap GMD509 UDG is capable of degrading contaminant DNA without a preincubation step before the main PCR reaction. These attributes imply that the Pap GMD509 UDG is a highly adequate enzyme to prevent carryover contamination during PCR.     

Comparative assessment of single and joint effects of diuron and Irgarol 1051 on Arctic and temperate microalgae using chlorophyll a fluorescence imaging

Ship groundings and ice-breakers can cause pollution of the polar environment with antifouling biocides such as diuron and Irgarol 1051. The present study used pulse amplitude modulated fluorometry to compare single and joint toxicities of diuron and Irgarol 1051 on two freshwater taxa of microalgae (Chlorella and Chlamydomonas) originating from Arctic and temperate regions. 30 min acute toxicity tests using chlorophyll a (Chl a) fluorescence revealed that Arctic strains of microalgae were more sensitive to herbicides than their temperate counterparts. Diuron and Irgarol 1051 had equal toxicities in the Arctic species, while Irgarol 1051 was more toxic (EC₅₀ = 5.55–14.70 μg L⁻¹) than diuron (EC₅₀ = 12.90–>40 μg L⁻¹) in the temperate species. Toxicity assessment of various mixtures of diuron and Irgarol 1051 revealed antagonistic, additive, and synergistic effects. Our data suggest that herbicides can adversely affect photosynthesis in Arctic microalgae at relatively low levels, and their impact can increase under complex mixture conditions.     

Tissue factor pathway inhibitor-2 induced hepatocellular carcinoma cell differentiation

To investigate the effect of over-expression of tissue factor pathway inhibitor-2 (TFPI-2) on the differentiation of hepatocellular carcinoma (HCC) cells (Hep3B and HepG2). The TFPI-2 recombinant adenovirus (pAd-TFPI-2) was constructed using the pAdeasy-1 vector system. Transfected by pAd-TFPI-2, the cell proliferation of HCC cells was evaluated by CCK-8 assay, flow cytometry was used to detect cell apoptosis and CD133 expression. Real-time PCR and Western blot were used to detect the expression levels of markers of hepatocellular cancer stem cells (CSC) and hepatocytes. The over-expression of TFPI-2 significantly suppressed cell proliferation, induced apoptosis, and dramatically decreased the percentage of CD133 cells, which was considered as CSC in HCC. Real-time PCR and Western blot showed that the expression of markers of CSC in Hep3B cells and HepG2 cells infected with pAd-TFPI-2 was markedly lower than those of the control group (P < 0.05), while the expression of markers of hepatocytes was significantly increased (P < 0.05). Hence, TFPI-2 could induce the differentiation of hepatocellular carcinoma cells into hepatocytes, and is expected to serve as a novel way for the treatment of HCC.