The AalNix3&4 isoform is required and sufficient to convert Aedes albopictus females into males.

Aedes albopictus is one of the most invasive insect species in the world and an effective vector for many important arboviruses. We reported previously that Ae. albopictus Nix (AalNix) is the male-determining factor of this species. However, whether AalNix alone is sufficient to initiate male development is unknown. Transgenic lines that express each of the three AalNix isoforms from the native promoter were obtained using piggyBac transformation. We verified the stable expression of AalNix isoforms in the transgenic lines and confirm that one isoform, AalNix3&4, is sufficient to convert females into fertile males (pseudo-males) that are indistinguishable from wild-type males. We also established a stable sex-converted female mosquito strain, AalNix3&4-♂4-pseudo-male. The pseudo-male mosquitoes can fly and mate normally with wild-type female, although their mating competitiveness is lower than wild-type. This work further clarifies the role of AalNix in the sex determination pathway and will facilitate the development of Ae. albopictus control strategies that rely on male-only releases such as SIT and sex-ratio distortion.

The dynamics of deltamethrin resistance evolution in Aedes albopictus has an impact on fitness and dengue virus type-2 vectorial capacity.

BACKGROUND: Worldwide invasion and expansion of Aedes albopictus, an important vector of dengue, chikungunya, and Zika viruses, has become a serious concern in global public health. Chemical insecticides are the primary means currently available to control the mosquito populations. However, long-term and large-scale use of insecticides has selected for resistance in the mosquito that is accompanied by a genetic load that impacts fitness. RESULTS: A number of laboratory strains representing different resistance mechanisms were isolated and identified from laboratory-derived, deltamethrin-resistant Ae. albopictus recovered in previous work. Resistance levels and fitness costs of the strains were evaluated and compared to characterize the evolution of the resistance genotypes and phenotypes. The heterozygous F1534S mutation (1534F/S) in the voltage gated sodium channel (vgsc) gene product (VGSC), first detected in early stages of resistance evolution, not only confers high-level resistance, but also produces no significant fitness costs, leading to the rapid spread of resistance in the population. This is followed by the increase in frequency of homozygous F1534S (1534S/S) mosquitoes that have significant fitness disadvantages, prompting the emergence of an unlinked I1532T mutation with fewer side effects and a mating advantage better adapted to the selection and reproductive pressures imposed in the experiments. Metabolic resistance with no significant fitness cost and mediating a high-tolerance resistance phenotype may play a dominant role in the subsequent evolution of resistance. The different resistant strains had similar vector competence for dengue virus type-2 (DENV-2). Furthermore, a comparative analysis of vectorial capacity revealed that increased survival due to deltamethrin resistance balanced the negative fitness cost effects and contributed to the risk of dengue virus (DENV) transmission by resistant populations. The progressive evolution of resistance results in mosquitoes with both target-site insensitivity and metabolic resistance with lower fitness costs, which further leads to resistant populations with both high resistance levels and vectorial capacity. CONCLUSIONS: This study reveals a possible mechanism for the evolution of deltamethrin resistance in Aedes albopictus. These findings will help guide practical strategies for insecticide use, resistance management and the prevention and control of mosquito-borne disease.

Genomic Shifts, Phenotypic Clines, and Fitness Costs Associated With Cold Tolerance in the Asian Tiger Mosquito.

Climatic variation is a key driver of genetic differentiation and phenotypic traits evolution, and local adaptation to temperature is expected in widespread species. We investigated phenotypic and genomic changes in the native range of the Asian tiger mosquito, Aedes albopictus. We first refine the phylogeographic structure based on genome-wide regions (1,901 double-digest restriction-site associated DNA single nucleotide polymophisms [ddRAD SNPs]) from 41 populations. We then explore the patterns of cold adaptation using phenotypic traits measured in common garden (wing size and cold tolerance) and genotype-temperature associations at targeted candidate regions (51,706 exon-capture SNPs) from nine populations. We confirm the existence of three evolutionary lineages including clades A (Malaysia, Thailand, Cambodia, and Laos), B (China and Okinawa), and C (South Korea and Japan). We identified temperature-associated differentiation in 15 out of 221 candidate regions but none in ddRAD regions, supporting the role of directional selection in detected genes. These include genes involved in lipid metabolism and a circadian clock gene. Most outlier SNPs are differently fixed between clades A and C, whereas clade B has an intermediate pattern. Females are larger at higher latitude yet produce no more eggs, which might favor the storage of energetic reserves in colder climate. Nondiapausing eggs from temperate populations survive better to cold exposure than those from tropical populations, suggesting they are protected from freezing damages but this cold tolerance has a fitness cost in terms of egg viability. Altogether, our results provide strong evidence for the thermal adaptation of A. albopictus across its wide temperature range.

Virome and nrEVEome diversity of Aedes albopictus mosquitoes from La Reunion Island and China.

BACKGROUND: Aedes albopictus is a public health threat for its worldwide spread and ability to transmit arboviruses. Understanding mechanisms of mosquito immunity can provide new tools to control arbovirus spread. The genomes of Aedes mosquitoes contain hundreds of nonretroviral endogenous viral elements (nrEVEs), which are enriched in piRNA clusters and produce piRNAs, with the potential to target cognate viruses. Recently, one nrEVE was shown to limit cognate viral infection through nrEVE-derived piRNAs. These findings suggest that nrEVEs constitute an archive of past viral infection and that the landscape of viral integrations may be variable across populations depending on their viral exposure. METHODS: We used bioinformatics and molecular approaches to identify known and novel (i.e. absent in the reference genome) viral integrations in the genome of wild collected Aedes albopictus mosquitoes and characterize their virome. RESULTS: We showed that the landscape of viral integrations is dynamic with seven novel viral integrations being characterized, but does not correlate with the virome, which includes both viral species known and unknown to infect mosquitoes. However, the small RNA coverage profile of nrEVEs and the viral genomic contigs we identified confirmed an interaction among these elements and the piRNA and siRNA pathways in mosquitoes. CONCLUSIONS: Mosquitoes nrEVEs have been recently described as a new form of heritable, sequence-specific mechanism of antiviral immunity. Our results contribute to understanding the dynamic distribution of nrEVEs in the genomes of wild Ae. albopictus and their interaction with mosquito viruses.

Benzobis(imidazole) derivatives as STAT3 signal inhibitors with antitumor activity.

Polycyclic aromatic systems have been considered good biological probes, but some may also be good scaffolds for drug development. In this study, a series of benzobis(imidazole) derivatives were identified as STAT3 signal inhibitors, among which compound 24 showed significant inhibition of IL-6 induced JAK/STAT3 signalling pathway activation. Moreover, 24 inhibited cancer cell growth and migration, and induced cell apoptosis as well as cycle arrest in human hepatocellular carcinoma cells (HepG2) and oesophageal carcinoma cells (EC109). Compound 24 also displayed obvious antitumor activity in a mouse HepG2 cell xenograft tumor model without affecting the body weight. These results confirmed that 24 was a potential STAT3 signal inhibitor with certain antitumor activity.

Druggability of lipid metabolism modulation against renal fibrosis.

Renal fibrosis contributes to progressive damage to renal structure and function. It is a common pathological process as chronic kidney disease develops into kidney failure, irrespective of diverse etiologies, and eventually leads to death. However, there are no effective drugs for renal fibrosis treatment at present. Lipid aggregation in the kidney and consequent lipotoxicity always accompany chronic kidney disease and fibrosis. Numerous studies have revealed that restoring the defective fatty acid oxidation in the kidney cells can mitigate renal fibrosis. Thus, it is an important strategy to reverse the dysfunctional lipid metabolism in the kidney, by targeting critical regulators of lipid metabolism. In this review, we highlight the potential "druggability" of lipid metabolism to ameliorate renal fibrosis and provide current pre-clinical evidence, exemplified by some representative druggable targets and several other metabolic regulators with anti-renal fibrosis roles. Then, we introduce the preliminary progress of noncoding RNAs as promising anti-renal fibrosis drug targets from the perspective of lipid metabolism. Finally, we discuss the prospects and deficiencies of drug targeting lipid reprogramming in the kidney.

Design, synthesis and biological evaluation of dipeptides as novel non-covalent 20S proteasome inhibitors.

Based on the interaction modes of the natural 20S proteasome inhibitors TMC-95A, we have previously discovered a dipeptide 1. To explore the SAR around compound 1, we designed and synthesized a series of dipeptides (8-38) with a fragment-based strategy. Among them, nine compounds showed significant inhibitory activities against the chymotrypsin-like activity of human 20S proteasome with IC50 values at the submicromolar level, which were comparable or even superior to the parent compound 1. Meanwhile, they displayed no significant inhibition against trypsin-like and caspase-like activities of 20S proteasome. The results suggested the feasibility to design dipeptides as novel and potent 20S proteasome inhibitors.[Formula: see text].

[1,2,4]Triazolo[1,5-a]pyrimidine derivative (Mol-5) is a new NS5-RdRp inhibitor of DENV2 proliferation and DENV2-induced inflammation.

Dengue fever is an acute infectious disease caused by dengue virus (DENV) and transmitted by Aedes mosquitoes. There is no effective vaccine or antiviral drug available to date to prevent or treat dengue disease. Recently, RNA-dependent RNA polymerase (RdRp), a class of polymerases involved in the synthesis of complementary RNA strands using single-stranded RNA, has been proposed as a promising drug target. Hence, we screened new molecules against DENV RdRp using our previously constructed virtual screening method. Mol-5, [1,2,4]triazolo[1,5-a]pyrimidine derivative, was screened out from an antiviral compound library (~8000 molecules). Using biophysical methods, we confirmed the direct interactions between mol-5 and purified DENV RdRp protein. In luciferase assay, mol-5 inhibited NS5-RdRp activity with an IC50 value of 1.28 ± 0.2 µM. In the cell-based cytopathic effect (CPE) assay, mol-5 inhibited DENV2 infectivity with an EC50 value of 4.5 ± 0.08 µM. Mol-5 also potently inhibited DENV2 RNA replication as observed in immunofluorescence assay and qRT-PCR. Both the viral structural (E) and non-structural (NS1) proteins of DENV2 were dose-dependently decreased by treatment with mol-5 (2.5-10 µM). Mol-5 treatment suppressed DENV2-induced inflammation in host cells, but had no direct effect on host defense (JAK/STAT-signaling pathway). These results demonstrate that mol-5 could be a novel RdRp inhibitor amenable for further research and development.

Metabolomic analysis of raw Pinelliae Rhizoma and its alum-processed products via UPLC-MS and their cytotoxicity.

Alum-processing is a traditional method to attenuate the toxicity of Pinelliae Rhizoma (tubers of Pinellia ternate, PT). The present study aimed at investigating the chemical and cytotoxic changes during alum processing. Metabolomic profiles of raw and alum-processed PT were studied based on ultra-performance liquid chromatography coupled with Orbitrap mass spectrometry. More than 80 chemicals in positive MS mode and 40 chemicals in negative MS mode, such as organic acids, amino acids, glucosides and nucleosides, were identified after multivariate statistical analysis, including principal component analysis and orthogonal partial least-square discriminant analysis. Almost all of the identified chemical markers were significantly decreased ~10- to 100-fold after alum processing. Meanwhile, the correlations between the chemical markers were assimilated to a positive coefficient from disorderly distribution during the processing. Raw PT extracts could inhibit the proliferation of human carcinoma cells (HCT-116, HepG2, and A549) at the rate of 40.5, 24.8 and 31.6% more strongly than processed PT. It was concluded that the alum processing of PT could decrease the number of actively water-soluble principles at the same time as decreasing toxicity. Given the water-insoluble property of toxic calcium oxalate raphides in PT, we suggest that a more scientific processing method should be sought.

Bacterial microbiota assemblage in Aedes albopictus mosquitoes and its impacts on larval development.

Interactions between bacterial microbiota and mosquitoes play an important role in mosquitoes' capacity to transmit pathogens. However, microbiota assemblages within mosquitoes and the impact of microbiota in environments on mosquito development and survival remain unclear. This study examined microbiota assemblages and the effects of aquatic environment microbiota on the larval development of the Aedes albopictus mosquito, an important dengue virus vector. Life table studies have found that reducing bacterial load in natural aquatic habitats through water filtering and treatment with antibiotics significantly reduced the larva-to-adult emergence rate. This finding was consistent in two types of larval habitats examined-discarded tires and flowerpots, suggesting that bacteria play a crucial role in larval development. Pyrosequencing of the bacterial 16S rRNA gene was used to determine the diversity of bacterial communities in larval habitats and the resulting numbers of mosquitoes under both laboratory and field conditions. The microbiota profiling identified common shared bacteria among samples from different years; further studies are needed to determine whether these bacteria represent a core microbiota. The highest microbiota diversity was found in aquatic habitats, followed by mosquito larvae, and the lowest in adult mosquitoes. Mosquito larvae ingested their bacterial microbiota and nutrients from aquatic habitats of high microbiota diversity. Taken together, the results support the observation that Ae. albopictus larvae are able to utilize diverse bacteria from aquatic habitats and that live bacteria from aquatic habitats play an important role in larval mosquito development and survival. These findings provide new insights into bacteria's role in mosquito larval ecology.

Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution.

The Asian tiger mosquito, Aedes albopictus, is a highly successful invasive species that transmits a number of human viral diseases, including dengue and Chikungunya fevers. This species has a large genome with significant population-based size variation. The complete genome sequence was determined for the Foshan strain, an established laboratory colony derived from wild mosquitoes from southeastern China, a region within the historical range of the origin of the species. The genome comprises 1,967 Mb, the largest mosquito genome sequenced to date, and its size results principally from an abundance of repetitive DNA classes. In addition, expansions of the numbers of members in gene families involved in insecticide-resistance mechanisms, diapause, sex determination, immunity, and olfaction also contribute to the larger size. Portions of integrated flavivirus-like genomes support a shared evolutionary history of association of these viruses with their vector. The large genome repertory may contribute to the adaptability and success of Ae. albopictus as an invasive species.

Re-engineering and synthesis of cytotoxic 2,3:7,8-di(alkylenedioxy)-extended analogs of quaternary sanguinarine chloride.

A method was developed to synthesize 2,3:7,8-di(alkylenedioxy)-extended analogs of quaternary sanguinarine chloride. 1-Bromo-2-bromomethyl-3,4-alkylenedioxy benzenes and 6,7-alkylenedioxynaphthalen-1-amines were synthesized first. Reactions to construct the target compounds with these two series of synthons involved alterations on a published method for synthesizing 2,3,7,8-tetraoxygenated derivatives of benzo[c]phenanthridinium, substituting benzyl bromides for benzoic aldehydes, prolonging the radical annulation time, and conducting N-methylation with formic acid and NaBH4. All the target compounds showed the same or better in vitro growth inhibitory activities against cancer cell lines compared with the positive compound. The structure activity relationship relevant to cytotoxicity and lipophilicity of the target compounds was produced.

Two new saponins from the leaves of Panax notoginseng.

Two new saponins, notoginsenosides Ng1 (1) and Ng2 (2), together with seven known compounds (3-9), were isolated from the leaves of Panax notoginseng. Their structures were elucidated by UV, IR, HRESIMS, and NMR experiments. Compounds 6 and 7 showed moderate cytotoxic activities against HCT-116, with IC50 values of 4.98 and 0.64 µmol/L, respectively.

Competence of Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus Mosquitoes as Zika Virus Vectors, China.

In China, the prevention and control of Zika virus disease has been a public health threat since the first imported case was reported in February 2016. To determine the vector competence of potential vector mosquito species, we experimentally infected Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus mosquitoes and determined infection rates, dissemination rates, and transmission rates. We found the highest vector competence for the imported Zika virus in Ae. aegypti mosquitoes, some susceptibility of Ae. albopictus mosquitoes, but no transmission ability for Cx. quinquefasciatus mosquitoes. Considering that, in China, Ae. albopictus mosquitoes are widely distributed but Ae. aegypti mosquito distribution is limited, Ae. albopictus mosquitoes are a potential primary vector for Zika virus and should be targeted in vector control strategies.

Azacyclo-indoles and Phenolics from the Flowers of Juglans regia.

Seven new azacyclo-indoles and phenolics and four known alkaloids were isolated from the flowers of Juglans regia. Spectroscopic and chromatographic data revealed that the structures of the new compounds are 5,6,11,12-tetrahydropyrrolo[1',2':1,2]azepino[4,5-b]indole-3-carbaldehyde (1), (±)-5,6,7,11c-tetrahydro-1H-indolizino[7,8-b]indol-3(2H)-one (2), (±)-9-hydroxy-5-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepine-2-carboxamide (3), 5-(ethoxymethyl)-1-(4-hydroxyphenethyl)-1H-pyrrole-2-carbaldehyde (4), (±)-5,8-dihydroxy-4-(1H-indol-3-yl)-3,4-dihydronaphthalen-1(2H)-one (5), (±)-4-(6-amino-9H-purin-9-yl)-5,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one (6), and (±)-4-(6-amino-9H-purin-9-yl)-5-hydroxy-3,4-dihydronaphthalen-1(2H)-one (7). The five pairs of enantiomers were resolved, and the absolute configurations of the enantiomers were assigned via electronic circular dichroism data. Compound 1 exhibited significant in vitro growth inhibition against the HCT-116, HepG2, BGC-823, NCI-H1650, and A2780 cancer cell lines, with IC50 values of 2.87, 1.87, 2.28, 2.86, and 0.96 µM, respectively, and low cytotoxicity toward normal IEC-6 cells, with a 79.6% survival rate at a 10 µM concentration.

Comparative performance of transcriptome assembly methods for non-model organisms.

BACKGROUND: The technological revolution in next-generation sequencing has brought unprecedented opportunities to study any organism of interest at the genomic or transcriptomic level. Transcriptome assembly is a crucial first step for studying the molecular basis of phenotypes of interest using RNA-Sequencing (RNA-Seq). However, the optimal strategy for assembling vast amounts of short RNA-Seq reads remains unresolved, especially for organisms without a sequenced genome. This study compared four transcriptome assembly methods, including a widely used de novo assembler (Trinity), two transcriptome re-assembly strategies utilizing proteomic and genomic resources from closely related species (reference-based re-assembly and TransPS) and a genome-guided assembler (Cufflinks). RESULTS: These four assembly strategies were compared using a comprehensive transcriptomic database of Aedes albopictus, for which a genome sequence has recently been completed. The quality of the various assemblies was assessed by the number of contigs generated, contig length distribution, percent paired-end read mapping, and gene model representation via BLASTX. Our results reveal that de novo assembly generates a similar number of gene models relative to genome-guided assembly with a fragmented reference, but produces the highest level of redundancy and requires the most computational power. Using a closely related reference genome to guide transcriptome assembly can generate biased contig sequences. Increasing the number of reads used in the transcriptome assembly tends to increase the redundancy within the assembly and decrease both median contig length and percent identity between contigs and reference protein sequences. CONCLUSIONS: This study provides general guidance for transcriptome assembly of RNA-Seq data from organisms with or without a sequenced genome. The optimal transcriptome assembly strategy will depend upon the subsequent downstream analyses. However, our results emphasize the efficacy of de novo assembly, which can be as effective as genome-guided assembly when the reference genome assembly is fragmented. If a genome assembly and sufficient computational resources are available, it can be beneficial to combine de novo and genome-guided assemblies. Caution should be taken when using a closely related reference genome to guide transcriptome assembly. The quantity of read pairs used in the transcriptome assembly does not necessarily correlate with the quality of the assembly.

Cytotoxic Lignans and Sesquiterpenoids from the Rhizomes of Acorus tatarinowii.

Five new compounds, including a rare phenyldihydronaphthalene lignanamide (1), an unusual hybrid-norlignan derivative (2), a rare cycloheptenone oxide derivative (3), one new acorane-type sesquiterpenoid (4), and one new guaiane-type sesquiterpenoid (5), together with seven known compounds (6-12), have been isolated from the rhizomes of Acorus tatarinowii. The structures of compounds 1-5 were determined by means of extensive spectroscopic methods. To the best of our knowledge, this is first report of a phenyldihydronaphthalene lignanamide and hybrid-norlignan and cycloheptenone oxide derivatives from the genus Acorus. In addition, compound 5 represents the first guaiane-type sesquiterpenoid with an epoxy group located between C-6 and C-9 from natural sources. Compounds 1-12 were evaluated for their in vitro cytotoxicity against five tumor cell lines. Among them, 2, 3, 5, and 10 exhibited moderate cytotoxicity with IC50 values of 2.11-9.23 µM.

[Olaparib potentiates the antitumor effect of Taxol on 4T1 breast cancer].

Poly (ADP-ribose) polymerase 1/2 (PARP1/2) can catalyze the poly (ADP ribose) (PAR) substrate protein modification and play an important role in the regulation of DNA damage repair, cell death and transcriptional activity. The PARP inhibitor olaparib (AZD2281) can be used as a sensitizer of radiotherapy and chemotherapy in the cancer treatment. Through establishment of biological fluorescent labeled 4T1 ectopic breast tumor model, we found that olaparib exhibited a poor effect on 4T1 breast cancer alone. However, in the combination with Taxol, olaparib significantly increased the anti-tumor effect of Taxol, and reduced the PAR levels of the tumor tissues. Importantly, olaparib did not amplify the toxicity of chemotherapy drugs. This study suggests that olaparib is a representative of the PARP inhibitor that can enhance Taxol's antitumor effect in the 4T1 ectopic breast tumor model, which sets the foundation for future study of the mechanism of olaparib action.

[Establishment of a glioma orthotopic xenograft model based on imaging technology].

Orthotopic xenograft model of human brain cancer cells is a good preclinical model for evaluation of antitumor compounds. In the present study, an orthotopic xenograft model of U87MG-m Cherry-luc was established in Balb/c nude mice and the tumor growth was monitored via imaging technology including magnetic resonance imaging (MRI), in vivo imaging (IVI) and micro-CT. Furthermore, the model was evaluated with a positive drug temozolomide. Our data suggest that integrated imaging technology including MRI, IVI and micro-CT in orthotopic xenograft model can be used to facilitate monitoring of cancer progression and evaluate antitumor activity of drugs against glioma.