Functional analysis of Ornithine decarboxylase in manipulating the wing dimorphism in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

The brown planthopper (BPH, Nilaparvata lugens), a major insect pest of rice, can make a shift in wing dimorphism to adapt to complex external environments. Our previous study showed that NlODC (Ornithine decarboxylase in N. lugens) was involved in wing dimorphism of the brown planthopper. Here, further experiments were conducted to reveal possible molecular mechanism of NlODC in manipulating the wing dimorphism. We found that the long-winged rate (LWR) of BPH was significantly reduced after RNAi of NlODC or injection of DFMO (D, L-α-Difluoromethylornithine), and LWR of males and females significantly decreased by 21.7% and 34.6%, respectively. Meanwhile, we also examined the contents of three polyamines under DFMO treatment and found that the contents of putrescine and spermidine were significantly lower compared to the control. After 3rd instar nymphs were injected with putrescine and spermidine, LWR was increased significantly in both cases, and putrescine was a little bit more effective, with 5.6% increase in males and 11.4% in females. Three days after injection of dsNlODC, injection of putrescine and spermidine rescued LWR to the normal levels. In the regulation of wing differentiation in BPH, NlODC mutually antagonistic to NlAkt may act through other signaling pathways rather than the classical insulin signaling pathway. This study illuminated a physiological function of an ODC gene involved in wing differentiation in insects, which could be a potential target for pest control.

Comparison of the signaling pathways of wing dimorphism regulated by biotic and abiotic stress in the brown planthopper.

Wing polymorphism is an evolutionary trait that is widely present in various insects and provides a model system for studying the evolutionary significance of insect dispersal. The brown planthopper (BPH, Nilaparvata lugens) can alter its wing morphs under biotic and abiotic stress. However, whether differential signaling pathways are induced by the 2 types of stress remain largely unknown. Here, we screened a number of candidate genes through weighted gene co-expression network analysis (WGCNA) and found that ornithine decarboxylase (NlODC), a key enzyme in the synthesis of polyamines, was associated with wing differentiation in BPH and mainly responded to abiotic stress stimuli. We analyzed the Kyoto Encyclopedia of Genes and Genomes enrichment pathways of differentially expressed genes under the 2 stresses by transcriptomic comparison, and found that biotic stress mainly influenced insulin-related signaling pathways while abiotic stress mainly influenced hormone-related pathways. Moreover, we found that insulin receptor 1 (NlInR1) may regulate wing differentiation of BPH by responding to both biotic and abiotic stress, but NlInR2 only responded to biotic stress. Similarly, the juvenile hormone epoxide hydrolase associated with juvenile hormone degradation and NlODC may regulate wing differentiation mainly through abiotic stress. A model based on the genes and stresses to modulate the wing dimorphism of BPH was proposed. These findings present a comprehensive molecular mechanism for wing polymorphism in BPH induced by biotic and abiotic stress.

[Application of personalized digital analog assisted acetabular prosthesis precise implantation in Crowe typeâ and â ¡ hip dysplasia].

OBJECTIVE: To explore the effect of personalized digital analog assisted acetabular prosthesis precise implantation in hip dysplasia. METHODS: From February 2017 to July 2019, 11 patients(12 hips) with hip dysplasia underwent total hip arthroplasty, including 4 males(5 hips) and 7 females(7 hips), aged from 27 to 61 years old, with an average of (46.64±12.93) years old;Crowe classification:8 hips in typeⅠ and 4 hips in typeⅡ. The preoperative thin-layer CT scan was imported into Mimics 10.01 software. The appropriate size and placement angle of acetabular prosthesis were selected through preoperative simulation, and the acetabular bone defect was understood to determine whether structural bone grafting was needed during the operation. The length of both lower limbs, the anteversion angle of acetabular prosthesis, the abduction angle, the height of acetabular rotation center and the horizontal distance of hip joint center before and after the operation were measured, and the postoperative dislocation, bone graft healing and acetabular cup loosening were observed. The hip Harris score was used to evaluate the joint function. RESULTS: All patients were followed up for 18 to 30 months with an average of (23.45±3.70) months. There was no prosthesis dislocation, loosening and bone graft healing after operation. One case had numbness in the innervation area due to the traction of sciatic nerve during operation, and was treated with neurotrophic drugs and recovered one month after operation. The length difference of both lower limbs decreased from (31.73±5.98) mm before operation to (4.73±1.90) mm 3 months after operation (t=15.268, P<0.01). The anteversion angle of acetabular cup and acetabulum was (17.45±3.62)°and abduction angle was (40.10 ± 2.30)° after operation. In all cases, the abduction angle and anteversion angle were within the safe range of Lewinek. The height of hip rotation center was (20.64±2.58) mm and the horizontal inward displacement of hip was (33.46±3.61) mm. Harris score increased from (45.36±2.34) before operation to (91.27±2.37) 3 months after operation (P<0.05). CONCLUSION: Through preoperative personalized digital analog reconstruction of acetabulum in patients with hip dysplasia, we can better understand the acetabular defect, help to evaluate the size and placement angle of acetabular prosthesis and whether structural bone grafting is needed, and obtain satisfactory clinical curative effect.

SMC4 enhances the chemoresistance of hepatoma cells by promoting autophagy.

Background: Drug resistance is a major contributing factor to chemotherapy failure in hepatocellular carcinoma (HCC) patients. However, the exact mechanism underlying the chemoresistance of HCC remains unknown. Methods: HepG2 cells were incubated with different concentrations of 5-fluorouracil (5-FU), and the Cell Counting Kit-8 assay was used to test the cell survival rate. The expression level of structural maintenance of chromosome 4 (SMC4) in drug-resistant cells was analyzed by real-time quantitative polymerase chain reaction (PCR) and western blotting. To assess autophagy, immunofluorescence was applied to detect the light chain 3 beta (LC3B) level in HepG2/5-FU cells. To further study the upstream regulation of miR (microRNA)-219/SMC4, a gene chip assay was performed. A luciferase reporter assay was used to determine whether long non-coding RNA-XIST (lncRNA-XIST) functions as a competitive endogenous RNA (ceRNA) for miR-219. Cellular proliferation was evaluated using MTT [3-(4,5)-dimethylthiahiazo (-z-y1)-2,5-di-phenytetrazoliumromide] and colony formation assays, wound healing and invasion assays were performed to study the invasion and migration ability of the cells, and flow cytometry assays were carried out to evaluate cell apoptosis. Results: In the present study, we established a drug-resistant hepatoma cell line named HepG2/5-FU. We confirmed that SMC4 may play an important role in hepatoma cell autophagy and could promote autophagy to increase the drug resistance of hepatoma cells. We also demonstrated that lncRNA-XIST may competitively bind to miR-219 by acting as a miRNA sponge, thereby preventing miR-219 from effectively reducing the expression of SMC4 and further affecting the autophagy and drug resistance of hepatoma cells via the adenosine 5'-monophosphate (AMP)-activated protein kinase/mechanistic target of rapamycin (AMPK/mTOR) pathway. Conclusions: Our study suggests that SMC4 may be a potential marker of a poor HCC response to chemotherapy and a novel therapeutic target for HCC chemotherapy.

CRISPR/Cas9-mediated knockout of the NlCSAD gene results in darker cuticle pigmentation and a reduction in female fecundity in Nilaparvata lugens (Hemiptera: Delphacidae).

In insects, cuticular pigmentation genes have been exploited as potential visible markers for constructing genetic manipulation systems. Here, we cloned cysteine sulfinic acid decarboxylase (CSAD), an orthologue of melanin metabolism pathway genes, and performed RNAi experiments in the brown planthopper Nilaparvata lugens (Hemiptera: Delphacidae). The results showed that a decrease in the level of transcription of NlCSAD increased melanin deposition in the body compared to the control group, resulting in darker cuticle pigmentation. Female adults treated with dsNlCSAD and mated with wild-type males laid significantly fewer eggs than the dsGFP-treated group, and lower hatchability of the eggs was also observed. In addition, two melanic mutant N. lugens strains (NlCSAD-/+ and NlCSAD-/-) constructed by the CRISPR/Cas9 genome editing system showed darker cuticular melanisation and a reduced oviposition and hatching rate, but the homozygotes had a darker body colour, fewer eggs and lower hatchability than heterozygotes or individuals after RNAi. Thus, we have provided the first evidence that NlCSAD is required for normal body pigmentation in adults and has a role in the fecundity of females and hatchability of eggs in N. lugens via a combination of RNAi and knockout of target genes based on the CRISPR/Cas9 genome editing system. Our results suggest that NlCSAD is a candidate visual reference gene for genetic manipulation of this important crop pest.

RNA interference of a trehalose-6-phosphate synthase gene reveals its roles in the biosynthesis of chitin and lipids in Heortia vitessoides (Lepidoptera: Crambidae).

Trehalose-6-phosphate synthase (TPS), an enzyme that hydrolyzes two glucose molecules to yield trehalose, plays a pivotal role in various physiological processes. In this study, we cloned the trehalose-6-phosphate synthase gene (HvTPS) and investigated its expression patterns in various tissues and developmental stages in Heortia vitessoides Moore (Lepidoptera: Crambidae). HvTPS was highly expressed in the fat body and after pupation or before molting. We knocked down TPS in H. vitessoides by RNA interference and found that 3.0 µg of dsHvTPS resulted in optimal interference at 24 h and 36 h post-injection and caused a sharp decline in the survival rate during the 5th instar larval-pupal stage and obviously abnormal or lethal phenotypes. Additionally, compared to the controls, TPS activity and trehalose contents were significantly lower and the glucose content was significantly higher 24 h or 36 h after injection with 3.0 µg of dsHvTPS. Furthermore, the silencing of HvTPS suppressed the expression of six key genes in the chitin biosynthesis pathway and one key gene related to lipid catabolism. The expression levels of two genes associated with lipid biosynthesis were upregulated. These results strongly suggest that HvTPS is essential for the normal growth and development of H. vitessoides and provide a reference for further studies of the utility of key genes involved in chitin and lipid biosynthesis for controlling insect development.

Chitin deacetylase 1 and 2 are indispensable for larval-pupal and pupal-adult molts in Heortia vitessoides (Lepidoptera: Crambidae).

Heortia vitessoides Moore is a notorious defoliator of Aquilaria sinensis (Lour.) Gilg trees. Chitin deacetylases (CDAs) catalyze the N-deacetylation of chitin, which is a crucial process for chitin modification. Here, we identified and characterized HvCDA1 and HvCDA2 from H. vitessoides. HvCDA1 and HvCDA2 possess typical domain structures of CDAs and belong to the Group I CDAs. HvCDA1 and HvCDA2 were highly expressed before and after the larval-larval molt. In addition, both exhibited relatively high mRNA expression levels during the larval-pupal molt, the pupal stage, and the pupal-adult molt. HvCDA1 and HvCDA2 transcript expression levels were highest in the body wall and relatively high in the larval head. Significant increases in the HvCDA1 and HvCDA2 transcript expression levels were observed in the larvae upon exposure to 20-hydroxyecdysone. RNA interference-mediated HvCDA1 and HvCDA2 silencing significantly inhibited HvCDA1 and HvCDA2 expression, with abnormal or nonviable phenotypes being observed. Post injection survival rates of the larvae injected with dsHvCDA1 and dsHvCDA2 were 66.7% and 46.7% (larval-pupal) during development and 23.0% and 6.7% (pupal-adult), respectively. These rates were significantly lower than those of the control group insects. Our results suggest that HvCDA1 and HvCDA2 play important roles in the larval-pupal and pupal-adult transitions and represent potential targets for the management of H. vitessoides.

Candidate olfactory genes identified in Heortia vitessoides (Lepidoptera: Crambidae) by antennal transcriptome analysis.

Heortia vitessoides Moore is the most severe defoliating pest of Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae) forests. Olfaction in insects is essential for host identification, mating, and oviposition, in which olfactory proteins, including odorant-binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs), are responsible for chemical signaling. Here, we determined the transcriptomes of male and female adult antennae of H. vitessoides. We assembled 52,383 unigenes and annotated their putative gene functions based on the gene ontology (GO), eukaryotic ortholog groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Overall, 61 olfactory-related transcripts, including nine OBPs, 10 CSPs, 28 ORs, 12 IRs, and two SNMPs, were identified. Expression patterns of OBPs and CSPs in the female antennae, male antennae, and legs were performed using reverse transcription quantitative PCR (RT-qPCR). The results revealed that HvitOBP1, HvitOBP6, and HvitGOBP1 were enriched in the female antennae, while HvitOBP2, HvitOBP3, HvitOBP5, HvitGOBP2, and HvitPBP1 were enriched in the male antennae. HvitOBP4 was expressed at nearly the same level in the antennae of both males and females. Four CSPs (HvitCSP3, HvitCSP5, HvitCSP7, and HvitCSP10) and two CSPs (HvitCSP1 and HvitCSP4) were expressed at higher levels in the female and male antennae, respectively. HvitCSP6 was expressed at higher levels both in the female antennae and legs. Three CSP genes (HvitCSP2, HvitCSP8, and HvitCSP9) were expressed at higher levels in the legs. These results provide a basis for further studies on the molecular olfactory mechanisms of H. vitessoides.

Identification and characterization of the catalase gene involved in resistance to thermal stress in Heortia vitessoides using RNA interference.

To elucidate the role of catalase (CAT) in Heortia vitessoides Moore, which is one of the most destructive defoliating pests in Aquilaria sinensis (Loureiro) Sprenger forests, a CAT gene (HvCAT) was identified in the transcriptome of adult H. vitessoides. Sequence analyses indicated that HvCAT encodes a protein containing 507 amino acids, including a proximal active site sequence (FXRERIPERVVHAKGXGA), heme-ligand sequence (RLFSYNDTX), heme-binding residues (H73, S112, N146, F151, F159, R352, and Y356), and NADPH-binding residues (P149, H192, Y196, G199, R201, N211, H233, K235, I300, W301, P302, H303, Q442, and L445). A phylogenetic analysis indicated that CAT from lepidopteran species could be assigned to one well-supported cluster. Regarding its stage- and tissue-specific expression profiles, HvCAT was expressed at high levels in fifth-instar larvae, fat body of larvae, and abdomen of adults. Furthermore, when fifth-instar larvae were exposed to thermal stress at 35, 37, and 39 °C, hydrogen peroxide and malondialdehyde content significantly increased. HvCAT mRNA was upregulated when the larvae were exposed to temperatures of 31, 33, 35, 37, and 39 °C. The enzymatic activity of HvCAT was significantly elevated following thermal stress (35 and 37 °C). After the knockdown of HvCAT by double-stranded RNA interference, the expression of thioredoxin peroxidase (Tpx) increased, whereas that of copper zinc superoxide dismutase (Cu/ZnSOD) decreased. Additionally, knocking down HvCAT transcripts in fifth-instar larvae resulted in accelerated death following thermal stress at 35 °C. In summary, the results suggest that HvCAT plays a major role in the thermotolerance of H. vitessoides.

TNM stages and prognostic features of colorectal mucinous adenocarcinomas: a meta analysis.

AIM: The significance of the mucinous adenocarcinoma in TNM staging and prognosis for colorectal tumor patients is still controversial. The aim was to provide a meta-analysis for TNM staging and prognostic features of colorectal tumors. METHODS: 30 individual case-control studies were finally included into this meta-analysis, involving a total of 444,489 cancer cases and 45,050 mucinous adenocarcinomas, of relations with TNM staging and prognostic features. RESULTS: Compared to non-mucinous adenocarcinoma patients, the TNM IV stage accounted for a larger percentage of mucinous adenocarcinomas (OR=1.48, 95%CI 1.28-1.71, POR<0.001) and the prognosis was significantly poor (HR=1.06, 95%CI 1.04-1.08, P<0.001). After heterogeneity testing, the results was similar to the holistic approach outcome (HR=1.48, 95%CI 1.35-1.62, P<0.001). CONCLUSION: Compared to patients with non-mucinous adenocarcinomas, mucinous adenocarcinoma patients with later TNM staging make up a big percentage, and mucinous adenocarcinoma is an independent risk factor for poor prognosis.