Molecular identification of severe fever with thrombocytopenia syndrome viruses from tick and bitten patient in Southeast China.

BACKGROUND: Severe fever and thrombocytopenia bunyavirus (SFTSV) infection causes severe fever and thrombocytopenia syndrome with high mortality. It is extremely rare that a transmitting tick can be directly captured in bite wounds, and that SFTSV can be isolated from both the captured tick and patient's serum to establish a solid pathogen diagnosis. CASE PRESENTATION: We report a case infected with severe fever and thrombocytopenia bunyavirus. The 69-year-old male patient presented with fever and tenderness on two lymph nodes in the right groin. A visible tick bite mark appeared on right upper quadrant of the patient's abdomen, and a live tick was captured in the bite wound upon physical examination. The virus was detected in both the blood of the patient and in the tick that stayed in the bite wound for 7 days. The phylogenetic analysis indicated that the SFTSV isolated from the tick and the patient's serum sample belonged to type B, in which the L/S segment of these two isolates shared 100% homology, while the M segment had 99.9% homology. The bitten patient was given various supportive care, but eventually died of multiple organ failure. CONCLUSION: The present case provides strong evidence of SFTSV transmission from H. longicornis to humans, and suggests that direct cross-species transmission can occur without additional intermediate hosts.

Development and evaluation of a rapid detection assay for severe fever with thrombocytopenia syndrome virus based on reverse-transcription recombinase polymerase amplification.

Rapid detection of severe fever with thrombocytopenia syndrome virus (SFTSV) is crucial for its control and surveillance. In this study, a rapid isothermal real-time reverse-transcription recombinase polymerase amplification (RT-RPA) assay was developed for the detection of SFTSV. The detection limit at 95% probability was 241 copies per reaction. A test of 120 serum samples of suspected severe fever with thrombocytopenia syndrome (SFTS) patients revealed that the sensitivity and specificity of the RT-RPA assay was approximately 96.00% (95%CI: 80.46%-99.79%) and 98.95% (95% CI: 94.28%-99.95%), respectively; the kappa value was 0.9495 (P＜0.001). The Bland-Altman analysis showed that 87.50% of the different data points were located within the 95% limits of agreement, indicating a good correlation between the results from RT-RPA assays and those of RT-qPCR assays. In conclusion, the rapid and efficient RT-RPA assay can be a promising candidate for point-of-care detection method of SFTSV.

Characterizing nitric oxide emissions from two typical alpine ecosystems.

A portion of alpine meadows has been and will continue to be cultivated due to the concurrent increasing demands for animal- and crop-oriented foods and global warming. However, it remains unclear how these long-term changes in land use will affect nitric oxide (NO) emission. At a field site with a calcareous soil on the Qinghai-Tibetan Plateau, the authors measured the year-round NO fluxes and related variables in a typically winter-grazed natural alpine meadow (NAM) and its adjacent forage oat field (FOF). The results showed that long-term plow tillage, fertilization and growing forage oats significantly yielded ca. 2.7 times more (p < 0.01) NO emissions from the FOF than the NAM (conservatively 208 vs. 56 g N/(ha·year) on average). The spring freeze-thaw period and non-growing season accounted for 17%-35% of the annual emissions, respectively. The Q10 of surface soil temperature (Ts) was 8.9 in the NAM (vs. 3.8 in the FOF), indicating increases of 24%-93% in NO emissions per 1-3 °C increase. However, the warming-induced increases could be smaller than those due to land use change and management practices. The Ts and concentrations of ammonium, nitrate and water-extractable organic carbon jointly explained 69% of the variance in daily NO fluxes from both fields during the annual period (p < 0.001). This result indicates that temporally and/or spatially distributed NO fluxes from landscapes with calcareous soils across native alpine meadows and/or fields cultivated with forage oats can be predicted by simultaneous observations of these four soil variables.

Apoptosis of Acanthamoeba castellanii Trophozoites Induced by Oleic Acid.

Acanthamoeba spp. can be parasitic in certain situations and are responsible for serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis, and cutaneous acanthamoebiasis. We analyzed the fatty acid composition of Acanthamoeba castellanii trophozoites and tested the inhibitory activity of the main fatty acids, oleic acid and arachidonic acid, in vitro. Oleic acid markedly inhibited the growth of A. castellanii, with trophozoite viability of 57.4% at a concentration of 200 µM. Caspase-3 staining and annexin V assays showed that apoptotic death occurred in A. castellanii trophozoites. Quantitative PCR and dot blot analysis showed increased levels of metacaspase and interleukin-1ß converting enzyme, which is also an indication of apoptosis. In contrast, arachidonic acid showed negligible inhibition of growth of A. castellanii trophozoites. Stimulated expression of Atg3, Atg8 and LC3A/B genes and monodansylcadaverine labeling suggested that oleic acid induces apoptosis by triggering autophagy of trophozoites.

Evaluation of a PCR/ESI-MS platform to identify respiratory viruses from nasopharyngeal aspirates.

Acute respiratory tract infection is a major cause of morbidity and mortality worldwide, particularly in infants and young children. High-throughput, accurate, broad-range tools for etiologic diagnosis are critical for effective epidemic control. In this study, the diagnostic capacities of an Ibis platform based on the PCR/ESI-MS assay were evaluated using clinical samples. Nasopharyngeal aspirates (NPAs) were collected from 120 children (<5 years old) who were hospitalized with lower respiratory tract infections between November 2010 and October 2011. The respiratory virus detection assay was performed using the PCR/ESI-MS assay and the DFA. The discordant PCR/ESI-MS and DFA results were resolved with RT-PCR plus sequencing. The overall agreement for PCR/ESI-MS and DFA was 98.3% (118/120). Compared with the results from DFA, the sensitivity and specificity of the PCR/ESI-MS assay were 100% and 97.5%, respectively. The PCR/ESI-MS assay also detected more multiple virus infections and revealed more detailed subtype information than DFA. Among the 12 original specimens with discordant results between PCR/ESI-MS and DFA, 11 had confirmed PCR/ESI-MS results. Thus, the PCR/ESI-MS assay is a high-throughput, sensitive, specific and promising method to detect and subtype conventional viruses in respiratory tract infections and allows rapid identification of mixed pathogens.

Prevalence and genetic diversity of Entamoeba species infecting macaques in southwest China.

Many colonies of macaques (Macaca fascicularis and Macaca mulatta) are maintained in China, especially in Guangxi and Guizhou. A total of 803 fresh stool samples infected with Entamoeba were obtained from three big colonies of macaques located in southwest China. The samples were examined for the presence of five Entamoeba species using PCR. Entamoeba nuttalli, Entamoeba dispar, Entamoeba coli, and Entamoeba chattoni infections were detected, but Entamoeba histolytica infection was not. This study is the first to report on the prevalence of E. nuttalli in wild macaques from China. Eighteen E. nuttalli isolates and five E. dispar isolates were obtained by culturing the samples in Tanabe-Chiba medium. The serine-rich protein (SRP), ribosomal RNA (rRNA), hexokinase (HXK), glucose-6-phosphate isomerase (GPI), and phosphoglucomutase (PGM) genes of E. nuttalli isolates were compared with other reported isolates. The results showed clear differences among the Chinese E. nuttalli isolates and other isolates based on the SRP gene sequences. However, HXK, GPI, and PGM genes of these strains were similar to those of other isolates. The rRNA genes of E. coli and E. chattoni were also amplified and analyzed from these samples. The results suggested that host species might be a more important factor than geographic location in amebic genetic diversity.

Unique short tandem repeat nucleotide sequences in Entamoeba histolytica isolates from China.

A few PCR-based DNA typing methods using repetitive elements contained within both protein-coding genes and noncoding DNAs have been reported for Entamoeba histolytica over the years. The serine-rich E. histolytica protein and tRNA-linked short tandem repeats (STRs) are most commonly used to investigate the relationship between parasite genotype and E. histolytica infection outcome. Many E. histolytica infections in China have been reported; however, little genome information has been provided. In the current paper, five Chinese E. histolytica samples were reported: three amoebic liver abscess cases, one combined case and one asymptomatic case. Our study is the first to report on the DNA typing information of E. histolytica in China. We included two city, one imported, and two country cases. Sequence analysis of serine-rich protein genes confirmed the presence of seven sequence types in five isolates. The STRs amplified from the samples revealed five STR variations in the A-L, four in the N-K2, and R-R loci, three in D-A, S(TGA)-D and S-Q loci. Two country patients were found to have a different outcome of infection with the same genotypes of E. histolytica, whereas in a city case, one E. histolytica strain had led to different outcome of the infection in one patient. Analyses of the results suggest that more genome information of E. histolytica strains from China through accurate methods is needed to interpret how the parasite genome plays a role in determining the outcome of E. histolytica infections.

Cloning, expression, and characterization of salivary apyrase from Aedes albopictus.

Apyrases (ATP diphosphohydrolase) hydrolyze the phosphodiester bonds of nucleoside tri- and diphosphates to orthophosphate and mononucleodides. They can inhibit platelet activation by depletion of adenosine diphosphate. In the current study, the Escherichia coli expression vector pET-19b equipped with an N-terminal histidine tag was applied to express the apyrase of Aedes albopictus. The gene-coding mature apyrase protein was amplified by RT-PCR and cloned into pET-19b. Soluble apyrase protein with high purity was successfully obtained by utilization of the suitable renaturation approach and Ni-NTA purification column. Four monoclonal antibodies to apyrase from A. albopictus were produced in male BALB/c mice immunized with the renatured apyrase. Using immunofluorescence assay and immunoblotting analysis, recombinant apyrase showed fine consistency with native apyrase. From kinetic analysis, it had a K (m) of 11.6 µM and V (max) of 1.02 nM/S/µg protein for adenosine triphosphate. Adenosine diphosphate-induced platelet aggregation was inhibited by approximately 6% when 0.4 µM recombinant apyrase was added and by about 9.5% when the concentration of recombinant apyrase was 0.8 µM. The effect on platelet aggregation was dose dependent. In conclusion, the apyrase of A. albopictus was cloned and expressed highly in the E. coli expression system. Recombinant apyrase protein showed biological activity, and anti-apyrase monoclonal antibody was also prepared.

[Serological survey of Toxoplasma gondii infection among public health practitioners in Xuhui District of Shanghai].

448 public health practitioners in the district were selected randomly from Dec. 2010 to Mar. 2011. Blood specimens were collected and tested for anti-T. gondii IgG by ELISA. The result showed that the positive rate was 10.3% (46/448). No significant difference was found between males and females, so as different cities of origin (P > 0.05). The positive rate was higher in > or = 30 age group (14.9%, 29/195) than that in < 30 age group (6.7%, 17/253)(P < 0.05), and the highest sero-prevalence was recorded in 30-39 age group (15.8%, 16/101). The positive rate was higher in subjects engaged in the food production and processing enterprises (12.6%, 36/286) than those in other industries (6.2%, 10/162) (P < 0.05).

Toxoplasma gondii Rhoptry Protein 7 (ROP7) Interacts with NLRP3 and Promotes Inflammasome Hyperactivation in THP-1-Derived Macrophages.

Toxoplasma gondii is a common opportunistic protozoan pathogen that can parasitize the karyocytes of humans and virtually all other warm-blooded animals. In the host's innate immune response to T. gondii infection, inflammasomes can mediate the maturation of pro-IL-1ß and pro-IL-18, which further enhances the immune response. However, how intercellular parasites specifically provoke inflammasome activation remains unclear. In this study, we found that the T. gondii secretory protein, rhoptry protein 7 (ROP7), could interact with the NACHT domain of NLRP3 through liquid chromatography-mass spectrometry analysis and co-immunoprecipitation assays. When expressing ROP7 in differentiated THP-1 cells, there was significant up-regulation in NF-κB and continuous release of IL-1ß. This process is pyroptosis-independent and leads to inflammasome hyperactivation through the IL-1ß/NF-κB/NLRP3 feedback loop. The loss of ROP7 in tachyzoites did not affect parasite proliferation in host cells but did attenuate parasite-induced inflammatory activity. In conclusion, these findings unveil that a T. gondii-derived protein is able to promote inflammasome activation, and further study of ROP7 will deepen our understanding of host innate immunity to parasites.

Generation of a neutralizing human monoclonal antibody Fab fragment to surface antigen 1 of Toxoplasma gondii tachyzoites.

A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant surface antigen 1 (SAG1) of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy-chain gene revealed that the closest germ line V segments were VH3-23. The germ line D segment was D1-7, and the closest germ line J segment was JH4. In the light-chain genes, the closest germ line V segment was Vκ1-17 with the Jκ1 or Jκ4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09 × 10(-9) M for Tox203 and 2.01 × 10(-8) M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.

High prevalence of Entamoeba infections in captive long-tailed macaques in China.

Long-tailed macaques (Macaca fascicularis) are bred in China for export and for use in experiments. Entamoeba infections in captive long-tailed macaques were surveyed in one of the biggest colonies located in Guangxi Province, China. One stool sample was obtained from each of the 152 different cages representing >3,000 macaques in the colony. The samples were examined by PCR for five Entamoeba species. The number of detected Entamoeba coli infections comprised 94% of the samples, 93% for Entamoeba chattoni, and 83% for Entamoeba dispar. In contrast, Entamoeba histolytica and Entamoeba nuttalli were not detected. Six isolates of E. dispar were obtained by culture in Tanabe-Chiba medium. Analysis of serine-rich protein genes in these isolates showed two genotypes, one of which is identical to that of the E. dispar SAW760 strain in humans. This suggests transmission of E. dispar between humans and nonhuman primates. These results demonstrate that Entamoeba infections are common, but virulent Entamoeba species are absent in this colony. This work also confirms the need for monitoring with PCR-based identification of Entamoeba species for captive macaques in breeding colonies to ensure animal health and protection of humans from zoonotic hazards.

High Co-Infection Rate of Trichomonas vaginalis and Candidatus Mycoplasma Girerdii in Gansu Province, China.

Trichomonas vaginalis (Tv) is a flagellated protist parasite that infects the human urogenital tract. The symbiotic relationship between Tv and Mycoplasma hominis has been reported. Recent studies have identified a new Mycoplasma strain, Candidatus Mycoplasma girerdii (Ca. M. girerdii), present in the vaginal secretions of women and have shown that this strain may be related to trichomoniasis. Here, we evaluated the presence of Tv, M. hominis and Ca. M. girerdii in 312 clinical samples from adult women diagnosed with vaginitis in Zhangye, Gansu province, China. Among these samples, 94, 153, and 48 were Tv, M. hominis and Ca. M. girerdii positive, respectively. Moreover, Tv was highly frequent in 17-30-year-old women in this region. Forty samples (83.3%) positive for Ca. M. girerdii were also positive for Tv. Six Tv isolates were successfully cultured, including five isolates that showed symbiotic relationships with Mycoplasma. This is the first report to evaluate the genetic characteristics of Ca. M. girerdii in China and may therefore provide insights into the effects of Ca. M. girerdii on the reproductive health of women.

A QoT prediction technique based on machine learning and NLSE for QoS and new lightpaths in optical communication networks.

In this paper, we proposed a quality of transmission (QoT) prediction technique for the quality of service (QoS) link setup based on machine learning classifiers, with synthetic data generated using the transmission equations instead of the Gaussian noise (GN) model. The proposed technique uses some link and signal characteristics as input features. The bit error rate (BER) of the signals was compared with the forward error correction threshold BER, and the comparison results were employed as labels. The transmission equations approach is a better alternative to the GN model (or other similar margin-based models) in the absence of real data (i.e., at the deployment stage of a network) or the case that real data are scarce (i.e., for enriching the dataset/reducing probing lightpaths); furthermore, the three classifiers trained using the data of the transmission equations are more reliable and practical than those trained using the data of the GN model. Meanwhile, we noted that the priority of the three classifiers should be support vector machine (SVM) >K nearest neighbor (KNN) > logistic regression (LR) as shown in the results obtained by the transmission equations, instead of SVM > LR > KNN as in the results of the GN model.

An Oxygen-Concentration-Controllable Multiorgan Microfluidic Platform for Studying Hypoxia-Induced Lung Cancer-Liver Metastasis and Screening Drugs.

Various cancer metastasis models based on organ-on-a-chip platforms have been established to study molecular mechanisms and screen drugs. However, current platforms can neither reveal hypoxia-induced cancer metastasis mechanisms nor allow drug screening under a hypoxia environment on a multiorgan level. We have developed a three-dimensional-culture multiorgan microfluidic (3D-CMOM) platform in which the dissolved oxygen concentration can be precisely controlled. An organ-level lung cancer and liver linkage model was established under normoxic/hypoxic conditions. A transcriptomics analysis of the hypoxia-induced lung cancer cells (A549 cells) on the platform indicated that the hypoxia-inducible factor 1α (HIF-1α) pathway could elevate epithelial-mesenchymal transition (EMT) transcription factors (Snail 1 and Snail 2), which could promote cancer metastasis. Then, protein detection demonstrated that HIF-1α and EMT transcription factor expression levels were positively correlated with the secretion of cancer metastasis damage factors alpha-fetoprotein (AFP), alkaline phosphatase (ALP), and gamma-glutamyl transpeptidase (γ-GT) from liver cells. Furthermore, the cancer treatment effects of HIF-1α inhibitors (tirapazamine, SYP-5, and IDF-11774) were evaluated using the platform. The treatment effect of SYP-5 was enhanced under the hypoxic conditions with fewer side effects, similar to the findings of TPZ. We can envision its wide application in future investigations of cancer metastasis and screening of drugs under hypoxic conditions with the potential to replace animal experiments.

A flux-adaptable pump-free microfluidics-based self-contained platform for multiplex cancer biomarker detection.

Microfluidics drives technological advancement in point-of-care (POC) bioanalytical diagnostics towards portability, fast response and low cost. In most microfluidic bioanalytical applications, flowing antigen/antibody reacts with immobilized antibody/antigen at a constant flux; it is difficult to reach a compromise to simultaneously realize sufficient time for the antigen-antibody interaction and short time for the entire assay. Here, we present a pump-free microfluidic chip, in which flow is self-initialized by capillary pumping and continued by imbibition of a filter paper. Microfluidic units in teardrop shape ensure that flow passes through the reaction areas at a reduced flux to facilitate the association between antigen and antibody and speeds up after the reaction areas. By spotting different antibodies into the reaction area, four types of biomarkers can be measured simultaneously in one microfluidic chip. Moreover, a small-sized instrument was developed for chemiluminescence detection and signal analysis. The system was validated by testing four biomarkers of colorectal cancer using plasma samples from patients. The assay took about 20 minutes. The limit of detection is 0.89 ng mL-1, 1.72 ng mL-1, 3.62 U mL-1 and 1.05 U mL-1 for the assays of carcinoembryonic antigen, alpha-fetoprotein, carbohydrate antigen 125 and carbohydrate antigen 19-9, respectively. This flux-adaptable and self-contained microfluidic platform is expected to be useful in various POC disease-monitoring applications.

Generation of flow and droplets with an ultra-long-range linear concentration gradient.

In the chemical and biological fields, the creation of concentration gradient microenvironments is an important approach for many applications, such as crystal growth and drug screening. Although many concentration gradient generators have been demonstrated, current generators can hardly produce ultra-long linear concentration gradients. In this paper, we propose a concentration-gradient flow/droplet generator which consists of a microfluidic flow switch, a cavity array for stage-by-stage concentration dilution, and an optional T-junction for droplet formation. The generator can realize an ultra-long continuously-varying concentration gradient along the flow direction. Generation of a 38 mm concentration gradient was demonstrated. The length can be further extended by enlarging the capacity of the cavities and increasing the number of the stages. The concentration gradient showed high linearity in the range of 10% to 90%. Moreover, cyclic generation of a concentration gradient flow and droplets with different concentrations was realized by the generator. In a demonstration of drug screening, the generator was employed to produce paclitaxel in different concentrations. A negative correlation between the 4T1 cell viability and the paclitaxel concentration was observed after the treatment. We envision that the concentration gradient generator will be a promising candidate for various drug screening applications.

High-Throughput Cell Trapping in the Dentate Spiral Microfluidic Channel.

Cell trapping is a very useful technique in a variety of cell-based assays and cellular research fields. It requires a high-throughput, high-efficiency operation to isolate cells of interest and immobilize the captured cells at specific positions. In this study, a dentate spiral microfluidic structure is proposed for cell trapping. The structure consists of a main spiral channel connecting an inlet and an out and a large number of dentate traps on the side of the channel. The density of the traps is high. When a cell comes across an empty trap, the cell suddenly makes a turn and enters the trap. Once the trap captures enough cells, the trap becomes closed and the following cells pass by the trap. The microfluidic structure is optimized based on the investigation of the influence over the flow. In the demonstration, 4T1 mouse breast cancer cells injected into the chip can be efficiently captured and isolated in the different traps. The cell trapping operates at a very high flow rate (40 µL/s) and a high trapping efficiency (>90%) can be achieved. The proposed high-throughput cell-trapping technique can be adopted in the many applications, including rapid microfluidic cell-based assays and isolation of rare circulating tumor cells from a large volume of blood sample.

EGFR inhibitors regulate Ca2+ concentration and apoptosis after PM2.5 exposure based on a lung-mimic microfluidic system.

Air pollution has side effects on human health. Epidemiology studies indicate a positive association between ambient fine particle (PM2.5, or particles less than 2.5 µm in diameter) concentration and lung cancer. However, how fine particles affect lung cancer at the molecular level and related therapeutic methods to address these diseases are unclear. Here, the multi-omics analysis (DNA methylation and transcriptomic) was used to detect human bronchial epithelial cells (HBE), that were exposed to PM2.5 using a quantified, small, portable, and organ-level air-liquid interface microfluidic system that mimics lung functions. The results indicate that 36,838 differentially methylated genes were detected. Of these 33,796 genes were hypomethylated (beta < 0), and 2862 genes were hypermethylated (beta > 0). RNA-Seq analysis demonstrated that 19,489 genes were upregulated (log2FC > 0), and 16,659 were downregulated. Furthermore, the calcium and apoptosis pathways were activated according to multi-omics analysis. The change in EGFR gene expression after PM2.5 exposure was the result of alterations of the cellular DNA methylome in the promoter. Inhibition or down-regulation of EGFR could result in the regulation of the downstream intracellular Ca2+ concentration and apoptosis via the EGFR/PLCγ and EGFR/STAT/Bcl-XL pathways after PM2.5 exposure. EGFR inhibitors decrease the Ca2+ concentration of cells, thereby strengthening the effects of fine particles on apoptosis. In short, the Ca2+ concentration and the apoptosis of cells can be regulated via EGFR related pathway after PM2.5 exposure. The EGFR may be a potentially promising therapeutic target for the treatment of air pollution-induced lung cancer through regulation of the intracellular Ca2+ concentration and apoptosis.

Comparison of serine-rich protein genes of Entamoeba histolytica isolates obtained from institutions for the mentally retarded in Kanagawa and Shizuoka Prefectures, Japan.

In Japan, amebiasis has been observed in homosexual men, in institutionalized persons, and in overseas travelers. We have previously reported an outbreak of amebiasis that occurred from 1986 to 1994 in institutions for the mentally retarded in Kanagawa and Shizuoka Prefectures in Eastern Japan. Entamoeba histolytica but not Entamoeba dispar was identified in Entamoeba cultures obtained from cyst passers in four institutions located in different municipalities in this region. In the present study, serine-rich protein genes of eight isolates from the four institutions were sequenced, and their polymorphism was analyzed. The results showed that all the sequences from the E. histolytica isolates were identical. This retrospective study led us to conclude that the outbreak of amebiasis in different municipalities was derived from a single source of E. histolytica.