Vehicle Trajectory Prediction Method for Task Offloading in Vehicular Edge Computing.

Real-time computation tasks in vehicular edge computing (VEC) provide convenience for vehicle users. However, the efficiency of task offloading seriously affects the quality of service (QoS). The predictive-mode task offloading is limited by computation resources, storage resources and the timeliness of vehicle trajectory data. Meanwhile, machine learning is difficult to deploy on edge servers. In this paper, we propose a vehicle trajectory prediction method based on the vehicle frequent pattern for task offloading in VEC. First, in the initialization stage, a T-pattern prediction tree (TPPT) is constructed based on the historical vehicle trajectory data. Secondly, when predicting the vehicle trajectory, the vehicle frequent itemset with the largest vehicle trajectory support is found in the vehicle frequent itemset of the TPPT. Finally, in the update stage, the TPPT is updated in real time with the predicted vehicle trajectory results. Meanwhile, based on the proposed prediction method, the strategies of task offloading and optimization algorithm are designed to minimize energy consumption with time constraints. The experiments are carried out on real-vehicle datasets and the Capital Bikeshare datasets. The results show that compared with the baseline T-pattern method, the accuracy of the prediction method is improved by more than 10% and the prediction efficiency is improved by more than 6.5 times. The vehicle trajectory prediction method based on the vehicle frequent pattern has high accuracy and prediction efficiency, which can solve the problem of vehicle trajectory prediction for task offloading.

SnRK1.1-mediated resistance of Arabidopsis thaliana to clubroot disease is inhibited by the novel Plasmodiophora brassicae effector PBZF1.

Plants have evolved a series of strategies to combat pathogen infection. Plant SnRK1 is probably involved in shifting carbon and energy use from growth-associated processes to survival and defence upon pathogen attack, enhancing the resistance to many plant pathogens. The present study demonstrated that SnRK1.1 enhanced the resistance of Arabidopsis thaliana to clubroot disease caused by the plant-pathogenic protozoan Plasmodiophora brassicae. Through a yeast two-hybrid assay, glutathione S-transferase pull-down assay, and bimolecular fluorescence complementation assay, a P. brassicae RxLR effector, PBZF1, was shown to interact with SnRK1.1. Further expression level analysis of SnRK1.1-regulated genes showed that PBZF1 inhibited the biological function of SnRK1.1 as indicated by the disequilibration of the expression level of SnRK1.1-regulated genes in heterogeneous PBZF1-expressing A. thaliana. Moreover, heterogeneous expression of PBZF1 in A. thaliana promoted plant susceptibility to clubroot disease. In addition, PBZF1 was found to be P. brassicae-specific and conserved. This gene was significantly highly expressed in resting spores. Taken together, our results provide new insights into how the plant-pathogenic protist P. brassicae employs an effector to overcome plant resistance, and they offer new insights into the genetic improvement of plant resistance against clubroot disease.

SUFU mediates EMT and Wnt/ß-catenin signaling pathway activation promoted by miRNA-324-5p in human gastric cancer.

The poor prognosis of late gastric carcinomas (GC) underscores the necessity to identify novel biomarkers for earlier diagnosis and effective therapeutic targets. MiRNA-324-5p has been shown to be over-expressed in GC, however the biological function of miRNA-324-5p implicated in gastric cancer and its downstream targets were not well understood. Wnt/ß-catenin signaling pathway is aberrantly regulated in GC. We sought to explore if miRNA-324-5p promotes oncogenesis through modulating Wnt signaling and EMT. MiRNA-324-5p is highly expressed in GC based on qRT-PCR and TCGA data. In addition, in vitro cell proliferation, cell migration assays and in vivo animal exenograft were executed to show that miRNA-324-5p is an oncogenic miRNA in GC. MiRNA-324-5p activates Wnt signaling and induces EMT in GC. Further, SUFU was identified as a target of miRNA-324-5p confirmed by western blotting and luciferase assays. Spearson analysis and TCGA data indicate that the expression of SUFU is negatively associated with the expression of miRNA-324-5p. Rescue experiments were performed to determine if SUFU mediates the Wnt activation, EMT and oncogenic function of miRNA-324-5p. MiRNA-324-5p inhibitors plus SUFU siRNAs rescue partially the inhibitory effect on Wnt signaling and EMT caused by miRNA-324-5p inhibitors. Finally, the suppression of cell proliferation, migration, and colony formation ability induced by miRNA-324-5p inhibitors is alleviated by addition of SUFU siRNAs. In summary, miRNA-324-5p is overexpressed in vivo and exerts cell growth and migration-promoting effects through activating Wnt signaling and EMT by targeting SUFU in GC. It represents a potential miRNA with an oncogenic role in human gastric cancer.

miRNA-192 and -215 activate Wnt/ß-catenin signaling pathway in gastric cancer via APC.

Although great progress has been made in surgical techniques, traditional radiotherapy, and chemotherapy, gastric cancer (GC) is still the most common malignant tumor and has a high mortality, which highlights the importance of novel diagnostic markers. Emerging studies suggest that different microRNAs (miRNAs) are involved in tumorigenesis of GC. In this study, we found that miRNA-192 and -215 are significantly upregulated in GC and promote cell proliferation and migration. Adenomatous polyposis coli (APC), a well-known negative regulator in Wnt signaling, has been proved to be a target of miRNA-192 and -215. Inhibition of miRNA-192 or -215 reduced the Topflash activities and repressed the expression of Wnt signaling pathway proteins, while APC small interfering RNAs reversed the inhibitory effects, suggesting that miRNA-192 and -215 activate Wnt signaling via APC. In addition, APC mediates the cell proliferation and migration regulated by miRNA-192 and -215. Furthermore, APC is downregulated in GC tissues and negatively correlated with the expression of miRNA-192 and -215. In summary, miRNA-192 and -215 target APC and function as oncogenic miRNAs by activating Wnt signaling in GC, revealing to be potential therapeutic targets.

CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish.

BACKGROUND: Gene targeting by homology-directed repair (HDR) can precisely edit the genome and is a versatile tool for biomedical research. However, the efficiency of HDR-based modification is still low in many model organisms including zebrafish. Recently, long single-stranded DNA (lssDNA) molecules have been developed as efficient alternative donor templates to mediate HDR for the generation of conditional mouse alleles. Here we report a method, zLOST (zebrafish long single-stranded DNA template), which utilises HDR with a long single-stranded DNA template to produce more efficient and precise mutations in zebrafish. RESULTS: The efficiency of knock-ins was assessed by phenotypic rescue at the tyrosinase (tyr) locus and confirmed by sequencing. zLOST was found to be a successful optimised rescue strategy: using zLOST containing a tyr repair site, we restored pigmentation in at least one melanocyte in close to 98% of albino tyr25del/25del embryos, although more than half of the larvae had only a small number of pigmented cells. Sequence analysis showed that there was precise HDR dependent repair of the tyr locus in these rescued pigmented embryos. Furthermore, quantification of zLOST knock-in efficiency at the rps14, nop56 and th loci by next generation sequencing demonstrated that zLOST showed a clear improvement. We utilised the HDR efficiency of zLOST to precisely model specific human disease mutations in zebrafish with ease. Finally, we determined that this method can achieve a germline transmission rate of up to 31.8%. CONCLUSIONS: In summary, these results show that zLOST is a useful method of zebrafish genome editing, particularly for generating desired mutations by targeted DNA knock-in through HDR.

Loss of rps9 in Zebrafish Leads to p53-Dependent Anemia.

Ribosome is a vital molecular machine for protein translation in the cell. Defects in several ribosomal proteins including RPS19, RPL11 and RPS14 have been observed in two types of anemia: Diamond Blackfan Anemia and 5q- syndrome. In zebrafish, deficiency of these ribosomal proteins shows similar anemic phenotype. It remains to be determined if any other ribosome proteins are similarly involved in regulating erythropoiesis. Here we generated mutations in zebrafish rps9, a rarely studied ribosomal protein gene, and investigated its function. Analysis of this mutant demonstrates that rps9 disruption leads to impairment of erythrocyte maturation, resulting in anemia. In addition, the overall phenotype including the anemic state is p53-dependent in rps9 mutants. Furthermore, this anemic state can be partially relieved by the treatment of L-leucine, and dexamethasone, which have been previously used in rescuing the phenotype of other ribosomal protein mutants. Finally, by comparing the phenotype, we show that there are considerable differences in morphology, cytomorphology, and hemoglobin levels for four ribosomal protein mutants in zebrafish. Based on the observed difference, we suggest that the level of anemic severity correlates with the delayed status of erythrocyte maturation in zebrafish models.

Identification and Characterization of Plasmodiophora brassicae Primary Infection Effector Candidates that Suppress or Induce Cell Death in Host and Nonhost Plants.

Clubroot caused by Plasmodiophora brassicaeis one of the most important diseases in cruciferous crops. The recognition of P. brassicae by host plants is thought to occur at the primary infection stage, but the underlying mechanism remains unclear. Secretory proteins as effector candidates play critical roles in the recognition of pathogens and the interactions between pathogens and hosts. In this study, 33 P. brassicae secretory proteins expressed during primary infection were identified through transcriptome, secretory protein prediction, and yeast signal sequence trap analyses. Furthermore, the proteins that could suppress or induce cell death were screened through an Agrobacterium-mediated plant virus transient expression system and a protoplast transient expression system. Two secretory proteins, PBCN_002550 and PBCN_005499, were found to be capable of inducing cell death associated with H2O2 accumulation and electrolyte leakage in Nicotiana benthamiana. Moreover, PBCN_002550 could also induce cell death in Chinese cabbage. In addition, 24 of the remaining 31 tested secretory proteins could suppress mouse Bcl-2-associated X protein-induced cell death, and 28 proteins could suppress PBCN_002550-induced cell death.

Inhibition of miR­194 suppresses the Wnt/ß­catenin signalling pathway in gastric cancer.

A mounting body of evidence has revealed that microRNAs (miRs) serve pivotal roles in various developmental processes, and in tumourigenesis, by binding to target genes and subsequently regulating gene expression. Continued activation of the Wnt/ß­catenin signalling is positively associated with human malignancy. In addition, miR­194 dysregulation has been implicated in gastric cancer (GC); however, the molecular mechanisms underlying the effects of miR­194 on GC carcinogenesis remain to be elucidated. The present study demonstrated that miR­194 was upregulated in GC tissues and SUFU negative regulator of Ηedgehog signaling (SUFU) was downregulated in GC cell lines. Subsequently, inhibition of miR­194 attenuated nuclear accumulation of ß­catenin, which consequently blocked Wnt/ß­catenin signalling. In addition, the cytoplasmic translocation of ß­catenin induced by miR­194 inhibition was mediated by SUFU. Furthermore, genes associated with the Wnt/ß­catenin signalling pathway were revealed to be downregulated following inhibition of the Wnt signalling pathway by miR­194 suppression. Finally, the results indicated that cell apoptosis was markedly increased in response to miR­194 inhibition, strongly suggesting the carcinogenic effects of miR­194 in GC. Taken together, these findings demonstrated that miR­194 may promote gastric carcinogenesis through activation of the Wnt/ß­catenin signalling pathway, making it a potential therapeutic target for GC.

Inhibition of the miR-192/215-Rab11-FIP2 axis suppresses human gastric cancer progression.

Less than a century ago, gastric cancer (GC) was the most common cancer throughout the world. Despite advances in surgical, chemotherapeutic, and radiotherapeutic treatment, GC remains the number 3 cancer killer worldwide. This fact highlights the need for better diagnostic biomarkers and more effective therapeutic targets. RAB11-FIP2, a member of the Rab11 family of interacting proteins, exhibits potential tumor suppressor function. However, involvement of RAB11-FIP2 in gastric carcinogenesis is yet to be elucidated. In this study, we demonstrated that RAB11-FIP2 was downregulated in GC tissues and constituted a target of the known onco-miRs, miR-192/215. We also showed that functionally, Rab11-FIP2 regulation by miR-192/215 is involved in GC-related biological activities. Finally, RAB11-FIP2 inhibition by miR-192/215 affected the establishment of cell polarity and tight junction formation in GC cells. In summary, this miR-192/215-Rab11-FIP2 axis appears to represent a new molecular mechanism underlying GC progression, while supplying a promising avenue of further research into diagnosis and therapy of GC.

Jasmonic Acid-Mediated Aliphatic Glucosinolate Metabolism Is Involved in Clubroot Disease Development in Brassica napus L.

Glucosinolate (GSL) is associated with clubroot disease, which is caused by the obligate biotrophic protist Plasmodiophora brassicae. Due to the complicated composition of GSLs, their exact role in clubroot disease development remains unclear. By investigating clubroot disease resistance in cruciferous plants and characterizing the GSL content in seeds, we can determine if clubroot disease development is related to the components of GSLs. The difference in the infection process between Matthiola incana L. (resistant) and Brassica napus L. (susceptible) was determined. Root hair infection was definitely observed in both resistant and susceptible hosts, but no infection was observed during the cortical infection stage in resistant roots; this finding was verified by molecular detection of P. brassicae via PCR amplification at various times after inoculation. Based on the time course detection of the contents and compositions of GSLs after P. brassicae inoculation, susceptible roots exhibited increased accumulation of aliphatic, indolic, and aromatic GSLs in B. napus, but only aromatic GSLs were significantly increased in M. incana. Gluconapin, which was the main aliphatic GSL in B. napus and present only in B. napus, was significantly increased during the secondary infection stage. Quantification of the internal jasmonic acid (JA) concentration showed that both resistant and susceptible plants exhibited an enhanced level of JA, particularly in susceptible roots. The exogenous JA treatment induced aliphatic GSLs in B. napus and aromatic GSLs in M. incana. JA-induced aromatic GSLs may be involved in the defense against P. brassicae, whereas aliphatic GSLs induced by JA in B. napus likely play a role during the secondary infection stage. Three candidate MYB28 genes regulate the content of aliphatic GSLs identified in B. napus; one such gene was BnMYB28.1, which was significantly increased following both the treatment with exogenous JA and P. brassicae inoculation. In summary, the increased content of JA during the secondary infection stage may induce the expression of BnMYB28.1, which caused the accumulation of aliphatic GSLs in clubroot disease development.

SMG-1 inhibition by miR-192/-215 causes epithelial-mesenchymal transition in gastric carcinogenesis via activation of Wnt signaling.

SMG-1,a member of the phosphoinositide kinase-like kinase family, functioned as a tumor suppressor gene. However, the role of SMG-1 in GC remain uncharacterized. In this study, regulation of SMG-1 by miR-192 and-215, along with the biological effects of this modulation, were studied in GC. We used gene microarrays to screening and luciferase reporter assays were to verify the potential targets of miR-192 and-215. Tissue microarrays analyses were applied to measure the levels of SMG-1 in GC tissues. Western blot assays were used to assess the signaling pathway of SMG-1 regulated by miR-192 and-215 in GC. SMG-1 was significantly downregulated in GC tissues.The proliferative and invasive properties of GC cells were decreased by inhibition of miR-192 and-215, whereas an SMG-1siRNA rescued the inhibitory effects. Finally, SMG-1 inhibition by miR-192 and-215 primed Wnt signaling and induced EMT. Wnt signaling pathway proteins were decreased markedly by inhibitors of miR-192 and-215, while SMG-1 siRNA reversed the inhibition apparently. Meanwhile, miR-192 and-215 inhitibtors increased E-cadherin expression and decreased N-cadherin and cotransfection of SMG-1 siRNA reversed these effects. In summary, these findings illustrate that SMG-1 is suppressed by miR-192 and-215 and functions as a tumor suppressor in GC by inactivating Wnt signaling and suppressing EMT.

MiRNA-194 activates the Wnt/ß-catenin signaling pathway in gastric cancer by targeting the negative Wnt regulator, SUFU.

Emerging evidence has shown that miRNA-194 is aberrantly upregulated in gastric cancer (GC); however, the biological mechanisms underlying its involvement are largely unknown. Wnt/ß-catenin signaling has been implicated in gastric tumorigenesis; we therefore hypothesized that miRNA-194 promotes gastric carcinogenesis by activating Wnt/ß-catenin signaling. MiRNA-194 was found to be overexpressed in GC cell lines and 43 paired GC tissues. Overexpression of miRNA-194 promoted cell proliferation and migration, while inhibition of miRNA-194 blocked these processes. Inhibition of miRNA-194 decreased tumor volumes in nude mice. Furthermore, miRNA-194 inhibitors promoted cytoplasmic localization of ß-catenin, leading to repression of Wnt signaling. We also discovered that SUFU, a known negative regulator of Hedgehog and Wnt signaling, was a target of miRNA-194. Anti-SUFU siRNAs rescued the inhibitory effects of miRNA-194 antagonists on cell proliferation and migration and on colony formation. We also found that SUFU expression was downregulated in GC tissues and cell lines and negatively correlated with miRNA-194 expression in primary GC tissues. Moreover, SUFU expression was negatively correlated with tumor stage, supporting its potential as a diagnostic or prognostic marker in GC. Taken together, these findings suggest that miRNA-194 is oncogenic and promotes GC cell proliferation and migration by activating Wnt signaling, at least in part, via suppression of SUFU.

Nom1 mediates pancreas development by regulating ribosome biogenesis in zebrafish.

Ribosome biogenesis is an important biological process for proper cellular function and development. Defects leading to improper ribosome biogenesis can cause diseases such as Diamond-Blackfan anemia and Shwachman-Bodian-Diamond syndrome. Nucleolar proteins are a large family of proteins and are involved in many cellular processes, including the regulation of ribosome biogenesis. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish line carrying mutation in nucleolar protein with MIF4G domain 1 (nom1), which encodes a conserved nulceolar protein with a role in pre-rRNA processing. Zebrafish nom1 mutants exhibit major defects in endoderm development, especially in exocrine pancreas. Further studies revealed that impaired proliferation of ptf1a-expressing pancreatic progenitor cells mainly contributed to the phenotype. RNA-seq and molecular analysis showed that ribosome biogenesis and pre-mRNA splicing were both affected in the mutant embryos. Several defects of ribosome assembly have been shown to have a p53-dependent mechanism. In the nom1 mutant, loss of p53 did not rescue the pancreatic defect, suggesting a p53-independent role. Further studies indicate that protein phosphatase 1 alpha, an interacting protein to Nom1, could partially rescue the pancreatic defect in nom1 morphants if a human nucleolar localization signal sequence was artificially added. This suggests that targeting Pp1α into the nucleolus by Nom1 is important for pancreatic proliferation. Altogether, our studies revealed a new mechanism involving Nom1 in controlling vertebrate exocrine pancreas formation.

A novel anti-tumor inhibitor identified by virtual screen with PLK1 structure and zebrafish assay.

Polo-like kinase 1 (PLK1), one of the key regulators of mitosis, is a target for cancer therapy due to its abnormally high activity in several tumors. Plk1 is highly conserved and shares a nearly identical 3-D structure between zebrafish and humans. The initial 10 mitoses of zebrafish embryonic cleavages occur every∼30 minutes, and therefore provide a rapid assay to evaluate mitosis inhibitors including those targeting Plk1. To increase efficiency and specificity, we first performed a computational virtual screen of∼60000 compounds against the human Plk1 3-D structure docked to both its kinase and Polo box domain. 370 candidates with the top free-energy scores were subjected to zebrafish assay and 3 were shown to inhibit cell division. Compared to general screen for compounds inhibiting zebrafish embryonic cleavage, computation increased the efficiency by 11 folds. One of the 3 compounds, named I2, was further demonstrated to effectively inhibit multiple tumor cell proliferation in vitro and PC3 prostate cancer growth in Xenograft mouse model in vivo. Furthermore, I2 inhibited Plk1 enzyme activity in a dose dependent manner. The IC50 values of I2 in these assays are compatible to those of ON-01910, a Plk1 inhibitor currently in Phase III clinic trials. Our studies demonstrate that zebrafish assays coupled with computational screening significantly improves the efficiency of identifying specific regulators of biological targets. The PLK1 inhibitor I2, and its analogs, may have potential in cancer therapeutics.