Incompatible and sterile insect techniques combined eliminate mosquitoes.

The radiation-based sterile insect technique (SIT) has successfully suppressed field populations of several insect pest species, but its effect on mosquito vector control has been limited. The related incompatible insect technique (IIT)-which uses sterilization caused by the maternally inherited endosymbiotic bacteria Wolbachia-is a promising alternative, but can be undermined by accidental release of females infected with the same Wolbachia strain as the released males. Here we show that combining incompatible and sterile insect techniques (IIT-SIT) enables near elimination of field populations of the world's most invasive mosquito species, Aedes albopictus. Millions of factory-reared adult males with an artificial triple-Wolbachia infection were released, with prior pupal irradiation of the released mosquitoes to prevent unintentionally released triply infected females from successfully reproducing in the field. This successful field trial demonstrates the feasibility of area-wide application of combined IIT-SIT for mosquito vector control.

Long noncoding RNAs C2dat1 enhances vascular smooth muscle cell proliferation and migration by targeting MiR-34a-5p.

Deregulated proliferation of vascular smooth muscle cells (VSMCs) is one common phenomenon of atherosclerosis progression. Long noncoding RNAs (lncRNAs) are one group of noncoding RNAs that play essential roles in many cell biological processes, including cell development, growth, and migration. However, the role of a novel calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D)-associated lncRNA, CAMK2D-associated transcript 1 (C2dat1), in VSMCs is still uncovered. In this study, we showed that the expression level of C2dat1 was higher in coronary artery disease (CAD) tissues than in normal arterial tissues and the C2dat1 expression level was upregulated in the proliferating VSMC after being treated with PDGF-bb or TNF-α. In addition, we indicated that overexpression of C2dat1 promoted VSMC growth and enhanced proliferating cell nuclear antigen (PCNA) expression in VSMC. Moreover, ectopic expression of C2dat1 increased VSMC migration. Furthermore, we showed that elevated expression of C2dat1 suppressed microRNA-34a (miR-34a) expression and promoted sirtuin 1 (SIRT1) expression, which was a direct target gene of miR-34a. We demonstrated that the expression level of miR-34a was lower in CAD tissues than in normal arterial tissues and the expression of miR-34a was negatively correlated with C2dat1 expression. Restored expression of C2dat1 increased VSMC proliferation and migration through promoting SIRT1 expression. These data suggested that lncRNA C2dat1 might be a potential therapeutic target to promote VSMC growth and migration in CAD.

Long non-coding RNA BRE-AS1 represses non-small cell lung cancer cell growth and survival via up-regulating NR4A3.

Recently, several long non-coding RNAs (lncRNAs) have been revealed to play crucial roles in tumorigenesis and progression of many cancers. Nevertheless, more than 50,000 lncRNAs were identified in human cells and the roles of majority of these lncRNAs in non-small cell lung cancer (NSCLC) are unknown. In this study, using public NSCLC microarray data we identified a novel lncRNA BRE antisense RNA 1 (BRE-AS1). BRE-AS1 is significantly down-regulated in NSCLC tissues and cell lines. Gain-of-function and loss-of-function assays showed that BRE-AS1 reduces NSCLC cell viability, represses NSCLC cell proliferation, and induces NSCLC cell apoptosis in vitro, and represses NSCLC tumor growth in vivo. Mechanistic investigation revealed that BRE-AS1 physically binds STAT3, reduces the binding of STAT3 to the promoter of NR4A3, relieves the repression of NR4A3 caused by STAT3, and up-regulates NR4A3 expression. Consistently, NR4A3 is significantly down-regulated in NSCLC tissues and the expression of NR4A3 is positively correlated with the expression of BRE-AS1 in NSCLC tissues. In addition, depletion of NR4A3 attenuates the tumor suppressive roles of BRE-AS1 in NSCLC. Collectively, our data demonstrate that BRE-AS1 represses NSCLC cell growth and survival via up-regulating NR4A3 and suggest that enhancing BRE-AS1 may be potential therapeutic strategy for NSCLC.

Loss of MiR-424-3p, not miR-424-5p, confers chemoresistance through targeting YAP1 in non-small cell lung cancer.

MiR-424 has been discovered to be involved in the chemoresistance of lung cancer. However, the underlying mechanism by which miR-424 played role in chemoresistance has been unknown. Here, in our study, to investigate the role of miR-424 in non-small cell lung cancer (NSCLC), we have detected the expression of miR-424-3p and -5p in NSCLC tissues and paired normal control. Moreover, to explore the role of miR-424-3p in NSCLC cells, miR-424-3p and -5p were both re-expressed and knocked down using transient transfection with their respective mimics and inhibitors. Cell viability, migration, and invasion were evaluated using MTT, wound-healing and Transwell assays, respectively. It was found that down-regulation of miR-424-3p was pronouncedly associated with NSCLC progression and overall prognosis; and that both miR-424-3p and -5p were markedly capable of preventing the proliferation, migration, and invasion in NSCLC cells. Additionally, it is miR-424-3p but not miR-424-5p that enhances the chemo-sensitivity of NSCLC cells through targeting YAP1. Mechanistically, YAP1 was identified as down-stream target of miR-424-3p. Together, it was for the first time in our study found that it is loss of miR-424-3p not miR-424-5p that enables chemoresistance through targeting YAP1 in NSCLC, supporting that miR-424-3p could be used as therapeutic target in the curing of NSCLC with chemoresistance. © 2016 Wiley Periodicals, Inc.

Reference Intervals of and Relationships among Essential Trace Elements in Whole Blood of Children Aged 0-14 years.

BACKGROUND: Essential trace elements are vital to human health. In this study, our aim was to establish reference intervals of and to evaluate relationships among Ca, Fe, Cu, Mg, and Zn for children. METHODS: We collected blood samples of 3210 children aged 0-14 years from Lu'an, China, and concentrations of the above elements were determined by atomizer absorption spectrophotometer. A nonparametric method was used to establish the reference intervals. RESULTS: Gender-related differences in concentrations were not statistically significant for the elements, except for Fe. There were strong positive and negative correlations between age and Fe (R = 0.305, P < 0.001), Zn (R = 0.573, P < 0.001); and age and Ca (R = -0.372, P < 0.001), Cu (R = -0.127, P < 0.001), respectively. Correlations between Ca-Mg (r = 0.222~0.384, P < 0.001), Fe-Mg (r = 0.495~0.614, P < 0.001), and Fe-Zn (r = 0.239~0.471, P < 0.001) were the strongest compared with others. In multivariable linear regression, after adjusted for confounding factors, the associations between Zn-Fe and Mg-Fe were the strongest with per concentration quintile increase of Fe caused Zn and Mg increasing by 4.19% (ß = 0.041; 95% CI: 0.037, 0.045; P < 0.001) and 3.87% (ß = 0.038; 95% CI: 0.036, 0.040; P < 0.001), respectively. CONCLUSIONS: Gender- and age-based reference intervals of Ca, Fe, Cu, Mg, and Zn for children were established, and correlations between them were quite complex. More works are needed to illuminate these relationships and their impacts on children's health.

Seasonal Variation of Blood Calcium Levels in Children Aged 1-10.

BACKGROUND: In this study, the associations of seasons with blood calcium levels in children aged 1-10 have not been evaluated. METHODS: In 2012-2014, whole blood samples were collected from 2,562 children and calcium concentrations were determined by flame atomic absorption spectrometry. The associations of seasons with calcium levels were analyzed by multivariable regression. RESULTS: The mean value of calcium concentrations was 1.61 ± 0.13 mmol/l and the overall deficiency was 29.3%. Overall, compared to those in winter, children in spring and summer had significant lower calcium concentrations that decreased by 1.2% (ß = -0.012; 95% CI: -0.021, -0.002) and 1.4% (ß = -0.014; 95% CI: -0.023, -0.005), respectively; and corresponding higher calcium deficiencies than those in spring, summer, and autumn with odds ratios (OR) were 1.93 (95% CI: 1.39, 2.66), 1.65 (95% CI: 1.21, 2.24), and 1.57 (95% CI: 1.14, 2.15), respectively. Moreover, this seasonality was more significant in girls in whom calcium concentration in summer decreased by 1.9% (ß = -0.019; 95% CI: -0.036, -0.003) and OR for calcium deficiencies in summer was 2.46 (1.38-4.41), compared to the girls in winter. CONCLUSIONS: The seasons have significant association with blood calcium levels, especially in girls. However, the impact of this seasonality on children's health is still unknown.

Biased virus transmission following sequential coinfection of Aedes aegypti with dengue and Zika viruses.

BACKGROUND: Mosquito-borne arboviruses are expanding their territory and elevating their infection prevalence due to the rapid climate change, urbanization, and increased international travel and global trade. Various significant arboviruses, including the dengue virus, Zika virus, Chikungunya virus, and yellow fever virus, are all reliant on the same primary vector, Aedes aegypti. Consequently, the occurrence of arbovirus coinfection in mosquitoes is anticipated. Arbovirus coinfection in mosquitoes has two patterns: simultaneous and sequential. Numerous studies have demonstrated that simultaneous coinfection of arboviruses in mosquitoes is unlikely to exert mutual developmental influence on these viruses. However, the viruses' interplay within a mosquito after the sequential coinfection seems intricated and not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We conducted experiments aimed at examining the phenomenon of arbovirus sequential coinfection in both mosquito cell line (C6/36) and A. aegypti, specifically focusing on dengue virus (DENV, serotype 2) and Zika virus (ZIKV). We firstly observed that DENV and ZIKV can sequentially infect mosquito C6/36 cell line, but the replication level of the subsequently infected ZIKV was significantly suppressed. Similarly, A. aegypti mosquitoes can be sequentially coinfected by these two arboviruses, regardless of the order of virus exposure. However, the replication, dissemination, and the transmission potential of the secondary virus were significantly inhibited. We preliminarily explored the underlying mechanisms, revealing that arbovirus-infected mosquitoes exhibited activated innate immunity, disrupted lipid metabolism, and enhanced RNAi pathway, leading to reduced susceptibility to the secondary arbovirus infections. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that, in contrast to simultaneous arbovirus coinfection in mosquitoes that can promote the transmission and co-circulation of these viruses, sequential coinfection appears to have limited influence on arbovirus transmission dynamics. However, it is important to note that more experimental investigations are needed to refine and expand upon this conclusion.

Laccase2 is required for sclerotization and pigmentation of Aedes albopictus eggshell.

Laccase (EC 1.10.3.2) is a member of multicopper oxidases that have been found in higher plants, fungus, bacterium, and insects. Two types of laccase genes have been detected in many species of insects: laccase1 and laccase2. It has been identified that laccase2 enzyme may play a key role in sclerotization and pigmentation of insect cuticle. But few attentions were given to the biological role of laccase2 in the synthesizing of similar structures, such as oothecae, eggshell, or silk cocoons. We cloned laccase2 gene from Aedes albopictus, one main mosquito vector of dengue virus in China. An upregulation of laccase2 gene was observed after a blood meal in female adult mosquitoes, suggesting that laccase2 gene may have an involvement in the development of ovary. RNA interference experiment was performed by using adult female mosquitoes. Female mosquitoes were injected with 20 ng of double-strain RNA into the thorax. Pigmentation of mosquito eggshell was blocked that these eggs never became dark. And the incomplete sclerotization of eggshell weakened the stability and flexibility of the eggs. These eggs without enough protection were deformed and died in water. These results demonstrate that laccase2 plays a critical role in the development of eggs of A. albopictus. Laccase2 may provide a novel target for mosquito control and management.

Molecular characterization and immunolocalization of a protein disulfide isomerase from Angiostrongylus cantonensis.

Protein disulfide isomerases (PDIs), belonging to the thioredoxin superfamily, are oxidoreductases that catalyze the formation, reduction, and isomerization of disulfide bonds among cysteine residues of proteins. In this study, we report the cloning and characterization of a cDNA encoding a protein disulfide isomerase (AcPDI) from a cDNA library of fourth-stage larvae of Angiostrongylus cantonensis. The deduced amino acid sequence contains two thioredoxin domains and exhibits high identity to the homologues from other species. Quantitative real-time PCR (qRT-PCR) was performed at the third-stage larvae, fourth-stage larvae, and adult stage of A. cantonensis, and the results revealed that the AcPDI mRNA, while expressed at all three stages, is expressed at a significantly higher level in female adult worms. Results of immunohistochemical studies indicated that the AcPDI expression was specifically localized in the tegument and uterus wall of female adult worms. Biochemical analysis showed that recombinant AcPDI was biologically active in vitro and exhibited the typical biochemical functions of PDIs: oxidase/isomerase and reductase activities. Collectively, these results implied that AcPDI may be a female-enriched protein and associated with the reproductive development of A. cantonensis. In addition, considering its biochemical properties, AcPDI may be involved in the formation of the cuticle of A. cantonensis.

Comparative study on the mutation spectrum of tissue DNA and blood ctDNA in patients with non-small cell lung cancer.

Background: To study the mutations detected in tissue DNA and blood circulating tumor DNA (ctDNA) of patients with advanced lung cancer and analyze the correlations between gene mutations, clinical features, and treatment. Methods: Targeted next-generation sequencing (NGS) technology based on probe hybrid capture and Illumina high-throughput sequencing was used to detect the DNA of tumor tissue samples (n=24) and blood samples (n=15) of ctDNA from 28 lung cancer patients with a clear pathological diagnosis. For mutations, the detection range included 4 types of mutations (point mutations, small indels, copy number variations) in all exon regions and partial intron regions of the 556 genes panel. Results: The most frequently detected mutant genes in 24 lung cancer tissue samples were TP53 (58.3%, 14/24), EGFR (33.3%, 8/24), LRP1B (25.0%, 6/24), KRAS (20.8%, 5/24), and ARID1A (16.7%, 4/24). The common mutant genes detected in 15 ctDNA samples were TP53 (60%, 5/15), EGFR (33.3%, 5/15), LRP1B (20.0%, 3/15), ERBB4 (3/15), and ARID1A (13.3%, 2/15). Among the 28 patients, 11 patients underwent both tissue DNA and ctDNA detection. The average co-mutation frequency of paired tissue DNA with ctDNA was 38.9% (0-83.3%), and the median value was 37.5%. Conclusions: Tissue DNA and ctDNA samples could detect genetic mutations and be consistent. Therefore, ctDNA detection as a method for disease diagnosis and evaluation of tumor molecular status may be helpful for the clinical diagnosis and treatment of lung cancer patients.

Case report: Acute Talaromyces marneffei mediastinitis in an HIV-negative patient.

Talaromyces marneffei (T. marneffei) is one of the most important opportunistic human pathogens endemic in Southeast Asia. Talaromycosis, which was once regarded as an opportunistic infectious disease in patients with acquired immunodeficiency syndrome, is being increasingly reported in HIV-negative populations. Since T. marneffei infection can be localized or disseminated, patients may present with a variety of symptoms. However, mediastinal infection attributed to T. marneffei is extremely rare. We report the case of a 32-year-old female who manifested a large mediastinal mass and was eventually diagnosed as acute T. marneffei mediastinitis. The patient was HIV-negative and had no direct contact with intermediate hosts. We successfully managed to treat the patient with inhaled amphotericin B deoxycholate and observed lesion absorption in subsequent CT examinations. To our knowledge, this is the first published case of T. marneffei mediastinitis and first use of inhaled antifungal monotherapy on patients with T. marneffei infection.

Wolbachia wAlbB remains stable in Aedes aegypti over 15 years but exhibits genetic background-dependent variation in virus blocking.

The ability of the maternally transmitted endosymbiotic bacterium Wolbachia to induce cytoplasmic incompatibility (CI) and virus blocking makes it a promising weapon for combatting mosquito-borne diseases through either suppression or replacement of wild-type populations. Recent field trials show that both approaches significantly reduce the incidence of dengue fever in humans. However, new questions emerge about how Wolbachia-mosquito associations will co-evolve over time and whether Wolbachia-mediated virus blocking will be affected by the genetic diversity of mosquitoes and arboviruses in the real world. Here, we have compared the Wolbachia density and CI expression of two wAlbB-infected Aedes aegypti lines transinfected 15 years apart. We have also assessed wAlbB-mediated virus blocking against dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) viruses and examined whether host genetic backgrounds modulate viral blocking effects by comparing ZIKV infection in mosquitoes with a Mexican genetic background to those with a Singaporean background. Our results show that over 15 years, wAlbB maintained the capacity to form a stable association with Ae. aegypti in terms of both density and CI expression. There were variations in wAlbB-induced virus blocking against CHIKV, DENV, and ZIKV, and higher inhibitory effects on ZIKV in mosquitoes on the Singaporean genetic background than on the Mexican background. These results provide important information concerning the robustness and long-term stability of Wolbachia as a biocontrol agent for arbovirus disease control.

DNA-methylation-induced silencing of DIO3OS drives non-small cell lung cancer progression via activating hnRNPK-MYC-CDC25A axis.

DNA methylation is a class of epigenetic modification manner, which is responsible for the inactivation of various tumor suppressors. Recently, long non-coding RNAs (lncRNAs) were revealed to be implicated in a variety of malignancies, including non-small cell lung cancer (NSCLC). However, the contributions of lncRNAs to DNA-methylation-induced oncogenic effects in NSCLC remain largely unknown. In this study, we identified a DNA-methylation-repressed lncRNA DIO3 opposite strand upstream RNA (DIO3OS) in NSCLC. DIO3OS is downregulated in NSCLC, and its low expression is related to poor prognosis. Ectopic expression of DIO3OS repressed NSCLC cell growth and motility and promoted NSCLC cell apoptosis in vitro. DIO3OS also repressed NSCLC tumorigenesis and metastasis in vivo. DIO3OS knockdown exhibited opposite biological effects. DIO3OS competitively bound heterogeneous nuclear ribonucleoprotein K (hnRNPK), repressed the binding of hnRNPK to MYC DNA and MYC mRNA, reduced the promoting roles of hnRNPK on MYC transcription and translation, led to the repression of MYC transcription and translation, and therefore remarkably decreased the expression of MYC and CDC25A, a downstream target of MYC. Additionally, depletion of hnRNPK blocked the tumor-suppressive roles of DIO3OS in NSCLC. In conclusion, these findings identified DIO3OS as an important protective factor against NSCLC via modulating hnRNPK-MYC-CDC25A axis.

Toward implementation of combined incompatible and sterile insect techniques for mosquito control: Optimized chilling conditions for handling Aedes albopictus male adults prior to release.

Combined incompatible and sterile insect technique (IIT-SIT) has been considered to be an effective and safe approach to control mosquito populations. Immobilization of male adults by chilling is a crucial process required for the packing, transportation and release of the mosquitoes during the implementation of IIT-SIT for mosquito control. In this study, effects of chilling on the Aedes albopictus males with triple Wolbachia infections (HC line), a powerful weapon to fight against the wild type Ae. albopictus population via IIT-SIT, were evaluated under both laboratory and field conditions. Irradiated HC (IHC) males were exposed to 1, 5 and 10°C for 1, 2, 3, 6 and 24 h. The survival rate of the post-chilled IHC males was then monitored. Longevity of post-chilled IHC males was compared to non-chilled males under laboratory and semi-field conditions. Mating competitiveness of IHC/HC males after exposure to 5 or 10°C for 0, 3 and 24 h was then evaluated. Effects of compaction and transportation under chilled conditions on the survival rate of IHC males were also monitored. The optimal chilling conditions for handling IHC males were temperatures between 5 and 10°C for a duration of less than 3 h with no negative impacts on survival rate, longevity and mating competitiveness when compared to non-chilled males. However, the overall quality of post-chilled IHC/HC males decreased when exposed to low temperatures for 24 h. Reduced survival was observed when IHC males were stored at 5°C under a compaction height of 8 cm. Transportation with chilling temperatures fluctuating from 8 to 12°C has no negative impact on the survival of IHC males. This study identified the optimal chilling temperature and duration for the handling and transportation of Ae. albopictus IHC male adults without any detrimental effect on their survival, longevity and mating competitiveness. Further studies are required to develop drone release systems specific for chilled mosquitoes to improve release efficiency, as well as to compare the population suppression efficiency between release of post-chilled and non-chilled males in the field.

Water-induced strong protection against acute exposure to low subzero temperature of adult Aedes albopictus.

As an important vector of dengue and Zika, Aedes albopictus has been the fastest spreading invasive mosquitoes in the world over the last 3-4 decades. Cold tolerance is important for survival and expansion of insects. Ae. albopictus adults are generally considered to be cold-intolerant that cannot survive at subzero temperature. However, we found that Ae. albopictus could survive for several hours' exposure to -9 to -19 oC so long as it was exposed with water. Median lethal time (LT50) of Ae. albopictus exposed to -15 and -19 oC with water increased by more than 100 times compared to those exposed to the same subzero temperature without water. This phenomenon also existed in adult Aedes aegypti and Culex quinquefasciatus. Ae. albopictus female adults which exposed to low subzero temperature at -9 oC with water had similar longevity and reproductive capacity to those of females without cold exposure. Cold exposure after a blood meal also have no detrimental impact on survival capacity of female adult Ae. albopictus compared with those cold exposed without a blood meal. Moreover, our results showed that rapid cold hardening (RCH) was induced in Ae. albopictus during exposing to low subzero temperature with water. Both the RCH and the relative high subzero temperature of water immediate after cold exposure might provide this strong protection against low subzero temperature. The molecular basis of water-induced protection for Ae. albopictus might refer to the increased glycerol during cold exposure, as well as the increased glucose and hsp70 during recovery from cold exposure. Our results suggested that the water-induced strong protection against acute decrease of air temperature for adult mosquitoes might be important for the survival and rapid expansion of Ae. albopictus.

[Pathological change in the brain of mice infected with Angiostrongylus cantonensis].

OBJECTIVE: To observe the pathological change in the brain of Angiostrongylus cantonensis-infected mouse. METHODS: Forty-eight mice were orally infected each with 40 third stage larvae of A. cantonensis, 3 mice were sacrificed at 7, 10, 13, 16, 19, 22, 25, 28 days postinfection respectively for worm recovery, and another 3 mice were for observing the histopathological change in tissue sections of the brain. RESULTS: Ten days postinfection, worms were found in the brain of the infected mice with a mean worm number of (7.0+/-1.7) per mouse. The highest number of worms was found at 16 days postinfection, with a mean of (23.7+/-4.9) per mouse. Notable symptoms of nervous system were seen on 15 days postinfection. Most mice died around 22 days postinfection. Histological examination revealed mechanical damages. Cavities and inflammation were observed in the brain parenchyma. Worms were seen in the subarachnoid space. Meningitis-like signs started at 13 days and aggravated then. CONCLUSIONS: Infection of A. cantonensis causes pathological change in mouse brain and the process is aggravating with postinfection time.

[Relationship between ERCC1 expression and cisplatin intervention in human lung adenocarcinoma cell lines].

BACKGROUND: The excision repair cross-complementing gene 1 (ERCC1), which is important in the repair of cisplatin-DNA adducts, is reported to be related to cisplatin resistance in tumor cells. The aim of this study is to investigate the influence of low-dose cisplatin on expression of ERCC1 gene and to confirm the correlation between ERCC1 and cisplatin resistance in human lung adenocarcinoma cell lines. METHODS: A549 and A549/DDP cell lines were treated with 10 µmol/L cisplatin for 12, 24, 48 and 72 h, or treated with 5, 10, 20, 40 µmol/L cisplatin for 24 h respectively. Then the expression of ERCC1 mRNA and protein was measured by RT-PCR and immunocytohistology SABC assay respectively. The resistance of A549/DDP cells was measured by MTT assay. RESULTS: After treating with 10 µmol/L cisplatin for 12 h, up-regulation of ERCC1 mRNA and protein was observed in A549 cells, then reached the peak levels in 72 h group. After treating with 5 µmol/L cisplatin for 24 h, up-regulation of ERCC1 mRNA and protein was observed in A549 cells, and when treated with 20 µmol/L cisplatin for 24 h, the ERCC1 mRNA and protein reached the peak levels. Comparing with the parental cells, ERCC1 expression increased obviously in A549/DDP cells, which were established by continuous low-dose cisplatin treatment. CONCLUSIONS: Up-regulation of ERCC1 expression can be induced by low-dose cisplatin in human lung adenocarcinoma cell line A549, and ECRR1 may play roles in cisplatin resistance.

Establishment of a medium-scale mosquito facility: optimization of the larval mass-rearing unit for Aedes albopictus (Diptera: Culicidae).

BACKGROUND: Standardized larval rearing units for mosquito production are essential for the establishment of a mass-rearing facility. Two larval rearing units, developed respectively by the Guangzhou Wolbaki Biotech Co. Ltd. (Wolbaki) and Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture (FAO/IAEA-IPCL), are tested to assess their potential uses to mass-rear the larval stages of Aedes albopictus in support of the establishment of a medium-scale mosquito facility for the application of mosquito genetic control strategies. METHODS: The triple Wolbachia-infected Ae. albopictus strain (HC strain) was used in this study. The effects of larval densities of two larval rearing trays (corresponding to 2.4, 3.0 and 3.6 larvae/cm2) and tray size/position (top, middle and bottom layers) on the pupae production and larval survival were assessed when trays were stacked within the larval rearing units. The male pupae production, female pupae contamination after sex separation, and male mating competitiveness were also studied by using both larval rearing units in their entirety. RESULTS: The optimal larval rearing density for Wolbaki-tray (Wol-tray) was 6,600 larvae (equal to 3.0 larvae/cm2) and 18,000 larvae (3.6 larvae/cm2) for the FAO/IAEA-IPCL tray (IAEA-tray). No significant difference in pupae production was observed when trays were stacked within top, middle or bottom layers for both units. At thirty-four hours after the first pupation, the average male pupae production was (0.89 × 105) for the Wol-unit and (3.16 × 105) for the IAEA-unit. No significant difference was observed in female pupae contamination between these two units. The HC males showed equal male mating competitiveness to wild type males for mating with wild type females in large cages, regardless of whether they were reared in the Wol-unit or IAEA-unit. CONCLUSIONS: The current study has indicated that both the Wol-unit and IAEA-unit are suitable for larvae mass-rearing for Ae. albopictus. However, the IAEA-unit, with higher male production and less space required compared to the Wol-unit, is recommended to be used in support of the establishment of a medium-sized mosquito facility.

[Effect of down-regulation of survivin gene on apoptosis and cisplatin resistance in cisplatin resistant human lung adenocarcinoma A549/CDDP cells].

OBJECTIVE: To investigate the effects of survivin antisense oligodeoxynucleoties (ASODN) transfection mediated by cytofectin on apoptosis and cisplatin resistance in cisplatin resistant human lung adenocarcinoma A549/CDDP cells in vitro. METHODS: A549/CDDP cells were cultured routinely in RPMI-1640 medium. Survivin ASODN mediated by cytofectin was transfected into the A549/CDDP cells. Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry SABC assays were performed to determine the regulation of survivin expression by ASODN. The influence of ASODN transfection on apoptosis was determined by fluoroscence microscopy and Hoechst staining, agarose gel electrophoresis, flow cytometry and caspase-3 colorimetric assay. MTT assay was performed to detect the cell viability, half-maximum inhibitory concentration (IC50) and cisplatin resistance index (RI) were thereby calculated. RESULTS: Transfected by survivin ASODN for 48 h, down-regulation of survivin expression was measured, of which mRNA and protein expression was significantly down-regulated to 41.56% and 0.864 +/- 0.045, respectively (P < 0.05). Transfection with survivin ASODN caused typical apoptotic changes, including characteristic chromatin condensation, nuclear shrinkage, nuclear cleavage and the cells grew more regularly, and some cells were floating. Typical DNA ladder pattern was observed by agarose gel electrophoresis. Furthermore, apoptotic index and caspase-3 activity was enhanced to 34.03% and 1.1298 +/- 0.2502, respectively (P < 0.05). It was significantly different as compared with the control group. While combination with ASODN and 10 micromol/L cisplatin caused far more distinctive apoptotic alterations, of which AI and caspase-3 activity reached to 65.85% and 1.6805 +/- 0.2758, respectively (P < 0.05), and even compared with the single ASODN group, the difference was still significant (P < 0.05). Transfected with survivin ASODN only or with combination of cisplatin for 48 h, the inhibitory rate of cell growth was enhanced to 59.3% and 83.7% (P < 0.05), respectively, while inversely, the cell viability reduced to a lowest value. The half-maximum inhibitory concentration of cisplatin was reduced from 225.03 +/- 10.59 micromol/L to 158.84 +/- 4.26 micromol/L, and the resistant index was conversely reduced from 11.9 to 8.39. Non-sense oligodeoxynucleotides (NSODN) and liposome had no effect on the cells growth (P > 0.05). CONCLUSION: Transfection with survivin ASODN can to a great extent reverse the cisplatin resistance in human cisplatin resistant lung adenocarcinoma cells A549/CDDP in vitro, and can thereby significantly inhibit the growth of the cells. The mechanism of reversal of resistance to cisplatin by this transfection can be associated with specific down-regulation of survivin expression, which decreases the threshold of apoptosis, induces more pronounced apoptosis,and reverses the resistance to apoptosis induced by cisplatin in A549/CDDP cells in vitro.