RNF125­mediated ubiquitination of MCM6 regulates the proliferation of human liver hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated mortality worldwide. Minichromosome maintenance proteins (MCMs), particularly MCM2-7, are upregulated in various cancers, including HCC. The aim of the present study was to investigate the role of MCM2-7 in human liver HCC (LIHC) and the regulation of the protein homeostasis of MCM6 by a specific E3 ligase. Bioinformatics analyses demonstrated that MCM2-7 were highly expressed in LIHC compared with corresponding normal tissues at the mRNA and protein levels, and patients with LIHC and high mRNA expression levels of MCM2, MCM3, MCM6 and MCM7 had poor overall survival rates. Cell Counting Kit-8 and colony formation assays revealed that the knockdown of MCM2, MCM3, MCM6 or MCM7 in Huh7 and Hep3B HCC cells inhibited cell proliferation and colony formation. In addition, pull-down, co-immunoprecipitation and ubiquitination assays demonstrated that RNF125 interacts with MCM6 and mediates its ubiquitination. Furthermore, co-transfection experiments indicated that RNF125 promoted the proliferation of HCC cells mainly through MCM6. In summary, the present study suggests that the RNF125-MCM6 axis plays an important role in the regulation of HCC cell proliferation and is a promising therapeutic target for the treatment of LIHC.

Camrelizumab and apatinib combined with chemotherapy in perioperative effective therapy for advanced gastric carcinoma with peritoneal metastasis: a case report.

Background: Peritoneal metastases (PMs) are the most frequent metastatic pattern with a very poor prognosis in stage IV gastric cancer (GC). An effective therapeutic option has yet to be established. Combination therapy of anti-angiogenesis therapy, immunotherapy and chemotherapy was first used in advanced GC for perioperative treatment in this case. Case Description: A 39-year-old man was diagnosed with stage IV GC with PM (CY1, P1) and the patient had an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 1 and adequate organ function. After disease progression on the first-line chemotherapy of paclitaxel plus S-1, the patient received a transformational therapy of camrelizumab (immune checkpoint inhibitors) plus apatinib (anti-angiogenic agent) combined with chemotherapy leading to macroscopic disappearance of the peritoneal lesions, negative peritoneal cytology, and the absence of other distant metastases, rendering him suitable to accept a radical gastrectomy with D2 lymph node dissection. The postoperative histopathology showed a tumor regression rate of more than 90%. Later, the patient was admitted for adjuvant therapy of camrelizumab plus apatinib combined with chemotherapy and relapsed at around 8 months after surgery. Treatment was well tolerated with no significant adverse effects. Conclusions: Camrelizumab plus apatinib combined with chemotherapy as second-line treatment demonstrated feasible anti-tumor activity and manageable safety in this advanced GC patient with PM and provided an opportunity for radical resection, improving the objective response rate (ORR) and prolonging patient survival. The incorporation may overcome resistance to treatment with therapy alone and produce synergistic effects, indicating a promising treatment option in the clinic to GC with PM.

Genome-wide in vitro and in vivo RNAi screens reveal Fer3 to be an important regulator of kkv transcription in Drosophila.

Krotzkopf verkehrt (kkv) is a key enzyme that catalyzes the synthesis of chitin, an important component of the Drosophila epidermis, trachea, and other tissues. Here, we report the use of comprehensive RNA interference (RNAi) analyses to search for kkv transcriptional regulators. A cell-based RNAi screen identified 537 candidate kkv regulators on a genome-wide scale. Subsequent use of transgenic Drosophila lines expressing RNAi constructs enabled in vivo validation, and we identified six genes as potential kkv transcriptional regulators. Weakening of the kkvDsRed signal, an in vivo reporter indicating kkv promoter activity, was observed when the expression of Akirin, NFAT, 48 related 3 (Fer3), or Autophagy-related 101(Atg101) was knocked down in Drosophila at the 3rd-instar larval stage; whereas we observed disoriented taenidial folds on larval tracheae when Lines (lin) or Autophagy-related 3 (Atg3) was knocked down in the tracheae. Fer3, in particular, has been shown to be an important factor in the activation of kkv transcription via specific binding with the kkv promoter. The genes involved in the chitin synthesis pathway were widely affected by the downregulation of Fer3. Furthermore, Atg101, Atg3, Akirin, Lin, NFAT, Pnr, and Abd-A showed that the potential complex mechanism of kkv transcription is regulated by an interaction network with bithorax complex components. Our study revealed the hitherto unappreciated diversity of modulators impinging on kkv transcription and opens new avenues in the study of kkv regulation and chitin biosynthesis.

Stable Introduction of Plant-Virus-Inhibiting Wolbachia into Planthoppers for Rice Protection.

Progress has been made in developing the maternally inherited endosymbiotic bacterium Wolbachia as a tool for protecting humans from mosquito-borne diseases. In contrast, Wolbachia-based approaches have not yet been developed for the protection of plants from insect pests and their associated diseases, with a major challenge being the establishment of artificial Wolbachia infections expressing desired characteristics in the hemipterans that transmit the majority of plant viruses. Here, we report stable introduction of Wolbachia into the brown planthopper, Nilaparvata lugens, the most destructive rice pest that annually destroys millions of hectares of staple crops. The Wolbachia strain wStri from the small brown planthopper, Laodelphax striatellus, was transferred to this new host, where it showed high levels of cytoplasmic incompatibility, enabling rapid invasion of laboratory populations. Furthermore, wStri inhibited infection and transmission of Rice ragged stunt virus and mitigated virus-induced symptoms in rice plants, opening up the development of Wolbachia-based strategies against major agricultural pests and their transmitted pathogens. VIDEO ABSTRACT.

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.

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.

The insect ecdysone receptor is a good potential target for RNAi-based pest control.

RNA interference (RNAi) has great potential for use in insect pest control. However, some significant challenges must be overcome before RNAi-based pest control can become a reality. One challenge is the proper selection of a good target gene for RNAi. Here, we report that the insect ecdysone receptor (EcR) is a good potential target for RNAi-based pest control in the brown planthopper Nilaparvata lugens, a serious insect pest of rice plants. We demonstrated that the use of a 360 bp fragment (NlEcR-c) that is common between NlEcR-A and NlEcR-B for feeding RNAi experiments significantly decreased the relative mRNA expression levels of NlEcR compared with those in the dsGFP control. Feeding RNAi also resulted in a significant reduction in the number of offspring per pair of N. lugens. Consequently, a transgenic rice line expressing NlEcR dsRNA was constructed by Agrobacterium- mediated transformation. The results of qRT-PCR showed that the total copy number of the target gene in all transgenic rice lines was 2. Northern blot analysis showed that the small RNA of the hairpin dsNlEcR-c was successfully expressed in the transgenic rice lines. After newly hatched nymphs of N. lugens fed on the transgenic rice lines, effective RNAi was observed. The NlEcR expression levels in all lines examined were decreased significantly compared with the control. In all lines, the survival rate of the nymphs was nearly 90%, and the average number of offspring per pair in the treated groups was significantly less than that observed in the control, with a decrease of 44.18-66.27%. These findings support an RNAi-based pest control strategy and are also important for the management of rice insect pests.

Conserved microRNAs miR-8-5p and miR-2a-3p modulate chitin biosynthesis in response to 20-hydroxyecdysone signaling in the brown planthopper, Nilaparvata lugens.

Molting is an important developmental process in insects, usually along with synthesis and degradation of chitin. 20-hydroxyecdysone (20E), an insect hormone, has been reported to contribute to many processes including molting. However, little is known about the link between the chitin biosynthesis pathway and 20E signaling. Here, we report that conserved miR-8-5p (miR-8-5p) and miR-2a-3p and their new target genes are critical for ecdysone-induced chitin biosynthesis in a hemipteran insect Nilaparvata lugens. We found that membrane-bound trehalase (Tre-2) and phosphoacetylglucosamine mutase (PAGM) in the chitin biosynthesis pathway were targets of miR-8-5p and miR-2a-3p, respectively, through bioinformatic analysis and experimental verification. The levels of miR-8-5p and miR-2a-3p were reduced, whereas the levels of Tre-2 and PAGM were up-regulated in response to 20E. In addition, miR-8-5p and miR-2a-3p were transcriptionally repressed by an early-response gene, the Broad-Complex (BR-C), in the 20E signaling pathway. Moreover, the overexpression of miR-8-5p and miR-2a-3p led to a significant reduction in the survival rate along with a molting obstacles defect phenotype caused by miR-2a-3p mimics feeding, and the chitin content of N. lugens was simultaneously reduced. Thus, miR-8-5p and miR-2a-3p act as molecular link that tune the chitin biosynthesis pathway in response to 20E signaling.

Optimizing conditions of polysaccharide extraction from Shiitake mushroom using response surface methodology and its regulating lipid metabolism.

Process conditions (extraction time, extraction temperature, water/solid ratio and incubation time) of Shiitake mushroom polysaccharides (SMP) were optimized by conducting experiments at three different levels using the response surface method (RSM). A second-order polynomial response surface equation was developed indicating the effect of variables on polysaccharides yield. Contour maps generated using the response surface equation showed that all the experimental variables significantly affected the yield. The effect of SMP on oxidative damage in mice fed by high cholesterol diet (HCD) was done in vivo. Results showed that SMP can decreased serum total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-c) levels, and increased high density lipoprotein (HDL-c) levels in HCD mice. Treatment with SMP reduced blood, liver lipid peroxidation level and increased antioxidant enzymes activities. Thus it can be concluded that SMP can improve lipid metabolism and decreased oxidative damage in HCD mice.

Effects of antireflux treatment on bronchial hyper-responsiveness and lung function in asthmatic patients with gastroesophageal reflux disease.

AIM: To investigate the effects of antireflux treatment on bronchial hyper-responsiveness and lung function in asthmatic patients with gastroesophageal reflux disease (GERD). METHODS: Thirty asthmatic patients with GERD were randomly divided into two groups (group A and group B). Patients in group A (n=15) only received asthma medication including inhaled salbutamol 200 microg four times a day and budesonide 400 microg twice a day for 6 weeks. Patients in Group B (n=15) received the same medication as group A, and also antireflux therapy including oral omeprazole 20 mg once a day and domperidone 10 mg three times a day for 6 weeks. Pulmonary function tests and histamine bronchoprovocation test were performed before and after the study. RESULTS: There was no significant difference in the baseline values of pulmonary function and histamine PC(20-FEV1) between the two groups. At the end of the study, the mean values for VC, VC%, FVC, FVC%, FEV(1), FEV(1)%, PEF, PEF%, PC(20-FEV1) were all significantly improved in group B, compared with group A. CONCLUSION: Antireflux therapy may improve pulmonary function and inhibit bronchial hyper-responsiveness in asthmatic patients with GERD.