Interfering RNA Expression Research Articles

Set1, Set2, and the Clock‐cycle complex are necessary to prevent age‐related retinal degeneration in Drosophila melanogaster

Age‐related eye disease is a prevalent problem that is exacerbated by the rising, aging population. Drosophila melanogaster serves as a powerful model organism for studying age‐related eye disease, as they share 60% of their genetic material with humans. Here, we seek to understand how changes in gene expression regulate photoreceptor neuron survival during aging. A preliminary RNA interference screen was carried out to identify genes necessary for cell survival throughout aging. Microscopy imaging and reverse transcriptase quantitative polymerase chain reaction (RT‐qPCR) were used to further investigate the role of histone methyltransferases Set1 and Set2 and the transcription factor Clock in photoreceptor neuron survival. Set1 and Set2 add methyl groups to lysine residues of histone H3, while Clock forms a heterodimer with the transcription factor cycle to activate transcription of genes involved in the circadian rhythm. Microscopy imaging was used to qualitatively and quantitatively assess loss of rhabdomeres, compartments of photoreceptor cells responsible for phototransduction, in flies lacking expression of either Set1, Set2, or a functional Clock‐cycle complex. Flies expressing RNA interference against Set1 and Set2 show retinal degeneration at or before the age of 30 days. Flies that fail to form a functional Clock‐cycle complex due to expression of a dominant negative form of Clock (ClkDN) show progressive retinal degeneration starting at the age of 5 days. Interestingly, this retinal degeneration is rescued when flies are raised in the dark. These phenotypes were validated by measuring target gene expression using RT‐qPCR. RNA interference against mCherry and overexpression of LacZ were used as controls for Set1 and Set2 RNA interference and overexpression of ClkDN, respectively. These findings suggest that histone methylation and Clock‐dependent gene expression play a role in age‐related eye disease in both Drosophila and humans.

RNAi-Based Gene Silencing of RXLR Effectors Protects Plants Against the Oomycete Pathogen Phytophthora capsici.

Phytophthora capsici is a broad-host range oomycete pathogen that can cause severe phytophthora blight disease of pepper and hundreds of other plant species worldwide. Natural resistance against P. capsici is inadequate, and it is very difficult to control by most of existing chemical fungicides. Therefore, it is urgent to develop alternative strategies to control this pathogen. Recently, host-induced or spray-induced gene silencing of essential or virulent pathogen genes provided an effective strategy for disease controls. Here, we demonstrate that P. capsici can effectively take up small interfering RNAs (siRNAs) from the environment. According to RNA-seq and quantitative reverse transcription PCR analysis, we identified four P. capsici RXLR effector genes that are significantly up-regulated during the infection stage. Transient overexpression and promote-infection assays indicated that RXLR1 and RXLR4 could promote pathogen infection. Using a virus-induced gene silencing system in pepper plants, we found that in planta-expressing RNA interference (RNAi) constructs that target RXLR1 or RXLR4 could significantly reduce pathogen infection, while co-interfering RXLR1 and RXLR4 could confer a more enhanced resistance to P. capsici. We also found that exogenously applying siRNAs that target RXLR1 or RXLR4 could restrict growth of P. capsici on the pepper and Nicotiana benthamiana leaves; when targeting RXLR1 and RXLR4 simultaneously, the control effect was more remarkable. These data suggested that RNAi-based gene silencing of RXLR effectors has great potential for application in crop improvement against P. capsici and also provides an important basis for the development of RNA-based antioomycete agents.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Functional analysis of <italic>StMAPK4</italic> in response to low temperature stress in potato

<p id="C3">Potato is vulnerable to low temperatures resulting in reduced yield production. <italic>MAPK</italic> gene is widely involved in a variety of environmental stress, and it has been detected to be involved in low temperature regulation. In this present study, to explore <italic>StMAPK4</italic> function in response to low temperature stress, potato cultivar ‘Atlantic’ as the experimental material, the expression characteristics of <italic>StMAPK4</italic> gene were analyzed in potato root, stem, and leaf at the different time under low temperature (4℃) stress. <italic>StMAPK4</italic> gene was analyzed using bioinformatics and its encoded protein subcellular localization was assayed. <italic>StMAPK4</italic> overexpression and RNA interference expression vectors were constructed and obtained transgenic potato plants. The activities of superoxide dismutase (SOD) and peroxidase (POD), and the contents of proline (Pro) and malondialdehyde (MDA) in non-transgenic (NT), overexpressed and RNAi interfered transgenic plants were analyzed under 4℃. The results showed that the isoelectric point (pI) of StMAPK4 was 4.97 and it was acidic protein localized in the nucleus and cell membrane. The relative expression levels of <italic>StMAPK4</italic> in roots, stems, and leaves significantly increased under low temperature stress. Compared with non-transgenic plants, the activities of SOD and POD and the content of proline in <italic>StMAPK4</italic> overexpressed plants were significantly increased, while the content of MDA was significantly decreased. Compared with NT plants, the activities of SOD and POD, and the content of Pro in <italic>StMAPK4</italic> overexpression plants were significantly decreased, while the content of MDA was significantly increased. Phenotypic observation revealed that the leaves of non-transgenic and RNAi interfered expression plants wilted seriously, while the leaves of overexpressed plants were less affected. In summary, the overexpression of <italic>StMAPK4</italic> gene can enhance the tolerance of low temperature stress in potato plants.

The Roles of microRNAs in Cancer Multidrug Resistance.

Simple SummaryThe resistance of neoplastic cells to multiple drugs is a serious problem in cancer chemotherapy. The molecular causes of multidrug resistance in cancer are largely known, but less is known about the mechanisms by which cells deliver phenotypic changes that resist the attack of anticancer drugs. The findings of RNA interference based on microRNAs represented a breakthrough in biology and pointed to the possibility of sensitive and targeted regulation of gene expression at the post-transcriptional level. Such regulation is also involved in the development of multidrug resistance in cancer. The aim of the current paper is to summarize the available knowledge on the role of microRNAs in resistance to multiple cancer drugs.Cancer chemotherapy may induce a multidrug resistance (MDR) phenotype. The development of MDR is based on various molecular causes, of which the following are very common: induction of ABC transporter expression; induction/activation of drug-metabolizing enzymes; alteration of the expression/function of apoptosis-related proteins; changes in cell cycle checkpoints; elevated DNA repair mechanisms. Although these mechanisms of MDR are well described, information on their molecular interaction in overall multidrug resistance is still lacking. MicroRNA (miRNA) expression and subsequent RNA interference are candidates that could be important players in the interplay of MDR mechanisms. The regulation of post-transcriptional processes in the proteosynthetic pathway is considered to be a major function of miRNAs. Due to their complementarity, they are able to bind to target mRNAs, which prevents the mRNAs from interacting effectively with the ribosome, and subsequent degradation of the mRNAs can occur. The aim of this paper is to provide an overview of the possible role of miRNAs in the molecular mechanisms that lead to MDR. The possibility of considering miRNAs as either specific effectors or interesting targets for cancer therapy is also analyzed.

KIFC1 Regulates the Trajectory of Neuronal Migration

During neuronal migration, forces generated by cytoplasmic dynein yank on microtubules extending from the centrosome into the leading process and move the nucleus along microtubules that extend behind the centrosome. Scaffolds, such as radial glia, guide neuronal migration outward from the ventricles, but little is known about the internal machinery that ensures that the soma migrates along its proper path rather than moving backward or off the path. Here we report that depletion of KIFC1, a minus-end-directed kinesin called HSET in humans, causes neurons to migrate off their appropriate path, suggesting that this molecular motor is what ensures fidelity of the trajectory of migration. For these studies, we used rat migratory neurons in vitro and developing mouse brain in vivo, together with RNA interference and ectopic expression of mutant forms of KIFC1. We found that crosslinking of microtubules into a nonsliding mode by KIFC1 is necessary for dynein-driven forces to achieve sufficient traction to thrust the soma forward. Asymmetric bouts of microtubule sliding driven by KIFC1 thereby enable the soma to tilt in one direction or another, thus providing midcourse corrections that keep the neuron on its appropriate trajectory. KIFC1-driven sliding of microtubules further assists neurons in remaining on their appropriate path by allowing the nucleus to rotate directionally as it moves, which is consistent with how we found that KIFC1 contributes to interkinetic nuclear migration at an earlier stage of neuronal development.SIGNIFICANCE STATEMENT Resolving the mechanisms of neuronal migration is medically important because many developmental disorders of the brain involve flaws in neuronal migration and because deployment of newly born neurons may be important in the adult for cognition and memory. Drugs that inhibit KIFC1 are candidates for chemotherapy and therefore should be used with caution if they are allowed to enter the brain.

Thromboxane Mobilizes Insect Blood Cells to Infection Foci

Innate immune responses are effective for insect survival to defend against entomopathogens including a fungal pathogen, Metarhizium rileyi, that infects a lepidopteran Spodoptera exigua. In particular, the fungal virulence was attenuated by cellular immune responses, in which the conidia were phagocytosed by hemocytes (insect blood cells) and hyphal growth was inhibited by hemocyte encapsulation. However, the chemokine signal to drive hemocytes to the infection foci was little understood. The hemocyte behaviors appeared to be guided by a Ca2+ signal stimulating cell aggregation to the infection foci. The induction of the Ca2+ signal was significantly inhibited by the cyclooxygenase (COX) inhibitor. Under the inhibitory condition, the addition of thromboxane A2 or B2 (TXA2 or TXB2) among COX products was the most effective to recover the Ca2+ signal and hemocyte aggregation. TXB2 alone induced a microaggregation behavior of hemocytes under in vitro conditions. Indeed, TXB2 titer was significantly increased in the plasma of the infected larvae. The elevated TXB2 level was further supported by the induction of phospholipase A2 (PLA2) activity in the hemocytes and subsequent up-regulation of COX-like peroxinectins (SePOX-F and SePOX-H) in response to the fungal infection. Finally, the expression of a thromboxane synthase (Se-TXAS) gene was highly expressed in the hemocytes. RNA interference (RNAi) of Se-TXAS expression inhibited the Ca2+ signal and hemocyte aggregation around fungal hyphae, which were rescued by the addition of TXB2. Without any ortholog to mammalian thromboxane receptors, a prostaglandin receptor was essential to mediate TXB2 signal to elevate the Ca2+ signal and mediate hemocyte aggregation behavior. Specific inhibitor assays suggest that the downstream signal after binding TXB2 to the receptor follows the Ca2+-induced Ca2+ release pathway from the endoplasmic reticulum of the hemocytes. These results suggest that hemocyte aggregation induced by the fungal infection is triggered by TXB2 via a Ca2+ signal through a PG receptor.

Identification and Functional Characterization of Antifreeze Protein and Its Mutants in Dendroctonus armandi (Coleoptera: Curculionidae: Scolytinae) Larvae Under Cold Stress.

Dendroctonus armandi (Tsai and Li) (Coleoptera: Curculionidae: Scolytinae) is considered to be the most destructive forest pest in the Qinling and Bashan Mountains of China. Low winter temperatures limit insect's populations, distribution, activity, and development. Insects have developed different strategies such as freeze-tolerance and freeze-avoidance to survive in low temperature conditions. In the present study, we used gene cloning, real-time polymerase chain reaction (PCR), RNA interference (RNAi), and heterologous expression to study the function of the D. armandi antifreeze protein gene (DaAFP). We cloned the 800 bp full-length cDNA encoding 228 amino acids of DaAFP and analyzed its structure using bioinformatics analysis. The DaAFP amino acid sequence exhibited 24-86% similarity with other insect species. The expression of DaAFP was high in January and in the larvae, head, and midgut of D. armandi. In addition, the expression of DaAFP increased with decreasing temperature and increasing exposure time. RNAi analysis also demonstrated that AFP plays an important role in the cold tolerance of overwintering larvae. The thermal hysteresis and antifreeze activity assay of DaAFP and its mutants indicated that the more regular the DaAFP threonine-cystine-threonine (TXT) motif, the stronger the antifreeze activity. These results suggest that DaAFP plays an essential role as a biological cryoprotectant in overwintering D. armandi larvae and provides a theoretical basis for new pest control methods.

The key regulator LcERF056 enhances salt tolerance by modulating reactive oxygen species-related genes in Lotus corniculatus

BackgroundThe APETALA2/ethylene response factor (AP2/ERF) family are important regulatory factors involved in plants’ response to environmental stimuli. However, their roles in salt tolerance in Lotus corniculatus remain unclear.ResultsHere, the key salt-responsive transcription factor LcERF056 was cloned and characterised. LcERF056 belonging to the B3–1 (IX) subfamily of ERFs was considerably upregulated by salt treatment. LcERF056-fused GFP was exclusively localised to nuclei. Furthermore, LcERF056- overexpression (OE) transgenic Arabidopsis and L. corniculatus lines exhibited significantly high tolerance to salt treatment compared with wild-type (WT) or RNA interference expression (RNAi) transgenic lines at the phenotypic and physiological levels. Transcriptome analysis of OE, RNAi, and WT lines showed that LcERF056 regulated the downstream genes involved in several metabolic pathways. Chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) and yeast one-hybrid (Y1H) assay demonstrated that LcERF056 could bind to cis-element GCC box or DRE of reactive oxygen species (ROS)-related genes such as lipid-transfer protein, peroxidase and ribosomal protein.ConclusionOur results suggested that the key regulator LcERF056 plays important roles in salt tolerance in L. corniculatus by modulating ROS-related genes. Therefore, it may be a useful target for engineering salt-tolerant L. corniculatus or other crops.

The relationship between EuSRPP1 gene expression and rubber biosynthesis in Eucommia ulmoides Oliver (Du-zhong)

Because of the unique properties, trans-polyisoprene rubber (TPI) has gradually realized that it has great development potential. Eucommia ulmoides Oliver (E. ulmoides Oliver), also named Du-zhong, is one of the few woody plants producing TPI. The significant expansion of small rubber particle protein and rubber elongation factor (SRPP/REF) genes in E. ulmoides Oliver may be associated with the producing of TPI. To understand the relationship between EuSRPP1 gene expression and rubber biosynthesis in E. ulmoides Oliver, in this study, an overexpression transgenic (OT) vector of EuSRPP1 driven by the act promoter and an RNA interference (RNAi) expression vector of EuSRPP1 were constructed. Then, Agrobacterium-mediated genetic transformation of E. ulmoides Oliver was conducted to obtain the OT and RNAi seedlings. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) showed that the EuSRPP1 expression levels in OT seedlings significantly increased, and were 2.53 × and 3.68 × higher in OT-1 and OT-2 seedlings, respectively, than the wild-type (WT) seedlings, Meanwhile, the expression levels of E. ulmoides Isopentenyl Diphosphate Isomerase (EuIPPS) and E. ulmoides Farnesyl Diphosphate Synthase (EuFPS) genes in OT plants, which are related to rubber biosynthesis synthesis, were also up-regulated. To the contrary, EuSRPP1 gene expression levels in the RNAi-1 and RNAi-2 seedlings significantly decreased, which were 3.92% and 42.38% of the WT seedlings, respectively. EuIPPS and EuFPS genes expression levels in the RNAi seedlings were also down-regulated. The TPI content analysis revealed that the TPI content in the overexpression EuSRPP1 callus increased to 1.85 × that of the WT callus, whereas that in the RNAi callus decreased to only 52% of the WT callus. Molecular weights of rubber were significantly different in OT callus (2.31 × 106 Da) and RNAi callus (0.36 × 106 Da). Observation of the rubber particles revealed that the rubber particle electron density was higher in OT seedlings, and the immunogold localization results showed that EuSRPP1 was localized to rubber particles. These results indicate that the EuSRPP1 expression level influences the chain elongation, particle development, and content of rubber in E. ulmoides Oliver.

Toxicity after AAV delivery of RNAi expression constructs into nonhuman primate brain.

RNA interference (RNAi) for spinocerebellar ataxia type 1 can prevent and reverse behavioral deficits and neuropathological readouts in mouse models, with safety and benefit lasting over many months. The RNAi trigger, expressed from adeno-associated virus vectors (AAV.miS1), also corrected misregulated microRNAs (miRNA) such as miR150. Subsequently, we showed that the delivery method was scalable, and that AAV.miS1 was safe in short-term pilot nonhuman primate (NHP) studies. To advance the technology to patients, investigational new drug (IND)-enabling studies in NHPs were initiated. After AAV.miS1 delivery to deep cerebellar nuclei, we unexpectedly observed cerebellar toxicity. Both small-RNA-seq and studies using AAVs devoid of miRNAs showed that this was not a result of saturation of the endogenous miRNA processing machinery. RNA-seq together with sequencing of the AAV product showed that, despite limited amounts of cross-packaged material, there was substantial inverted terminal repeat (ITR) promoter activity that correlated with neuropathologies. ITR promoter activity was reduced by altering the miS1 expression context. The surprising contrast between our rodent and NHP findings highlight the need for extended safety studies in multiple species when assessing new therapeutics for human application.

Identification and Characterization of 24-Dehydrocholesterol Reductase (DHCR24) in the Two-Spotted Cricket, Gryllus bimaculatus.

Simple SummaryDHCR24 (24-dehydrocholesterol reductase) is a key enzyme for producing cholesterol from desmosterol and that is also involved in the conversion of plant sterols to cholesterol in most plant-feeding insects. This study extensively examined the possibility of DHCR24 involved in the sterol conversion in omnivorous insects, which feed on multiple food origins. Homologs of DHCR24 (GbDHCR24-1 and -2) were identified and characterized by using the two-spotted cricket, Gryllus bimaculatus, as an experimental model. The quantitative expression analyses and RNA interference experiments revealed that GbDHCR24-1 rather than GbDHCR24-2 facilitates the desmosterol-to-cholesterol conversion in crickets. Our data suggested that the omnivorous species produced cholesterol from desmosterol in the same manner as the plant-feeding species do.Arthropods, including insects, convert sterols into cholesterol due to the inability to synthesise cholesterol de novo. 24-dehydrocholesterol reductase (DHCR24) plays an important role in the conversion. Not only involving the cholesterol biosynthesis in vertebrates, DHCR24 is required for the conversion of desmosterol into cholesterol in phytophagous insects. The current study extensively examined DHCR24 in omnivorous insects, which feed on both plants and animals, using Gryllus bimaculatus as the experimental model. We identified cDNAs encoding two homologues of DHCR24 from G. bimaculatus, which were designated as GbDHCR24-1 and GbDHCR24-2. Both homologues contained the flavin adenine dinucleotide binding domain, which is a feature of DHCR24. Quantitative polymerase chain reaction revealed that among tissues of adult crickets, fat body and anterior midgut expressed high levels of GbDHCR24s. Both fat body and anterior midgut demonstrated DHCR24 activities in which one of the functions is the conversion of desmosterol into cholesterol in vitro. Knockdown of GbDHCR24-1 significantly reduced the conversion activity in the anterior midgut while knockdown of the GbDHCR24-2 did not. Additionally, the accumulation of desmosterol was detected in a feeding experiment with a specific DHCR24 inhibitor, azacosterol. We finally concluded that GbDHCR24-1 is the major enzyme that facilitates the desmosterol-to-cholesterol-conversion in crickets.

PGE2 mediates hemocyte-spreading behavior by activating aquaporin via cAMP and rearranging actin cytoskeleton via Ca2+

Spreading behavior of hemocytes (= insect blood cells) is essential for cellular immune responses against various microbial pathogens. It is activated by prostaglandin E2 (PGE2) via its membrane receptor associated with secondary messenger, cAMP, in insects. This study observed an increase of calcium ion (Ca2+) level after an acute increase of cAMP induced by PGE2 treatment and clarified the intracellular signals underlying the hemocyte-spreading behavior. Inhibition of Ca2+ flux significantly impaired the hemocyte-spreading and subsequent cellular immune response, phagocytosis. The up-regulation of intracellular Ca2+ in response to PGE2 was dependent on cAMP because RNA interference (RNAi) of PGE2 receptor expression or inhibiting adenylate cyclase prevented Ca2+ mobilization. The up-regulation of Ca2+ was induced by inositol triphosphate (IP3) via its specific IP3 receptor. Furthermore, inhibition of ryanodine receptor impaired Ca2+ mobilization, suggesting Ca2+-induced Ca2+ release. However, the effective spreading behavior of hemocytes was dependent on both secondary messengers. Ca2+ signal stimulated by cAMP was required for activating small G proteins because RNAi treatments of small G proteins such as Rac1, RhoA, and Cdc42 failed to stimulate hemocyte-spreading. In contrast, aquaporin was activated by cAMP. Its activity was necessary for changing cell volume during hemocyte-spreading. These results indicate that PGE2 mediates hemocyte-spreading via cAMP signal to activate aquaporin and via Ca2+ signal to activate actin cytoskeletal rearrangement.

Expression of 20-hydroxyecdysone-related genes during gonadal development of Teleogryllus emma (Orthoptera: Gryllidae).

Insect gonads develop under endocrine signals. In this study, we assessed the characters of partial complementary DNAs encoding the Teleogryllus emma orthologs of 20-hydroxyecdysone (20E)-related genes (RXR, E75, HR3, Hsc70, and Hsp90) and analyzed their expression patterns in both nymph and adult crickets. 20E treatment suppressed expression of TeEcR, TeRXR, TeE75, TeHR3, TeHsc70, and TeHsp90. Temporal expression analysis demonstrated that TeERR and 20E-related genes were expressed in four stages of gonadal development from the fourth-instar nymph stage to the adult stage. The expression pattern of these genes differed in testicular and ovarian development. TeRXR, HR3, TeHsc70, and TeHsp90 were irregularly expressed in gonads of the same developmental stages, while mRNAs encoding TeERR, TeEcR, and TeE75 accumulated in higher levels in ovaries than in testes. RNA interference (RNAi) of TeEcR expression led to decrease of the expression levels of TeEcR, TeRXR, TeHR3, and TeHsc70, while it enhanced TeE75 and TeHsp90 expressions. These results demonstrate that the TeERR and 20E-related genes help regulate gonadal development, while TeEcR appears to inhibit TeE75 expression, TeE75 inhibits HR3 expression. Hsc70 indirectly regulated the expression of the primary and secondary response genes E74A, E75B, and HR3. Hsp90 regulated Usp expression with no direct regulatory relationship with EcR.

Vitellogenin regulates estrogen-related receptor expression by crosstalk with the JH and IIS-TOR signaling pathway in Polyrhachis vicina Roger (Hymenoptera, Formicidae)

The Estrogen-related receptor (ERR) can regulate the growth and development, metabolism, reproduction, and other physiological activities of insects, but its specific mechanism of action is still unclear. The aim of this study was to explore the relationship between expression of ERR and Vitellogenins (Vg) and the juvenile hormone (JH) and insulin/insulin-like growth factor/target of rapamycin (IIS/TOR) signaling pathways in Polyrhachis vicina Roger. P. vicina was used as the experimental model to clone the PvVg gene, perform double-stranded RNA synthesis and delivery and observe the effects of pharmacological treatments. The full-length PvVg cDNA product is 5586 bp. Higher PvVg mRNA expression was seen in the pupa and adults, and varying levels were seen in the different body parts of three different castes. RNA interference of PvVg expression led to disturbed development, an abnormal phenotype, and high mortality. PvVg RNAi also led to a reduction in mRNA levels of PvERR, ultraspiracle (PvUSP), forkhead box protein O (PvFOXO) and PvTOR genes in fourth instar larval, but a significant increase was seen in pupa and females. No significant change was seen in workers and males. After PvVg knockdown, application of exogenous JHIII reduced the expression of these genes in pupa and females, increased expression in workers, and decreased PvUSP mRNA expression in males. Both protein and mRNA expression levels of PvFOXO were affected by PvVg RNAi. PvERR RNAi increased PvVg expression in pupa and females and Kruppel-homolog 1 (PvKr-h1) and PvFOXO expression in males. The results of this study suggest that there is an interaction between PvERR and PvVg, and that crosstalk with the JH and IIS/TOR signaling pathways can affect development and reproduction. This effect is caste and developmental stage specific. We also speculate that the FOXO/USP complex participates in JH regulation of PvVg in P. vicina.

Discovering antiviral restriction factors and pathways using genetic screens.

Viral infections activate the powerful interferon (IFN) response that induces the expression of several hundred IFN stimulated genes (ISGs). The principal role of this extensive response is to create an unfavourable environment for virus replication and to limit spread; however, untangling the biological consequences of this large response is complicated. In addition to a seemingly high degree of redundancy, several ISGs are usually required in combination to limit infection as individual ISGs often have low to moderate antiviral activity. Furthermore, what ISG or combination of ISGs are antiviral for a given virus is usually not known. For these reasons, and since the function(s) of many ISGs remains unexplored, genome-wide approaches are well placed to investigate what aspects of this response result in an appropriate, virus-specific phenotype. This review discusses the advances screening approaches have provided for the study of host defence mechanisms, including clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9), ISG expression libraries and RNA interference (RNAi) technologies.

Repat33 Acts as a Downstream Component of Eicosanoid Signaling Pathway Mediating Immune Responses of Spodoptera exigua, a Lepidopteran Insect

Simple SummaryEicosanoids are oxygenated polyunsaturated fatty acids containing 20 carbons and subdivided into prostaglandin, leukotriene, and epoxyeicosatrienoic acid. They mediate immune responses in insects as well as mammals. Excessive eicosanoids cause severe inflammatory diseases in humans. However, a lack of eicosanoids induces immunosuppressive conditions in insects, which are highly susceptible to various entomopathogens. Despite the physiological significance of eicosanoids, their molecular action was not clearly understood in insects. This study was focused on Repat (Response to Pathogen) gene family, which might be regulated by eicosanoids. Among 44 members of Repat family genes, Repat33 was highly inducible to Gram-negative bacteria. Its expression was dependent on eicosanoids. Loss of function of Repat33 by RNA interference (RNAi) caused significant immunosuppression of a lepidopteran insect, Spodoptera exigua. Larvae of S. exigua treated by RNAi did not exhibit efficient cellular immune responses. They also failed to express antimicrobial peptide genes at high inducible levels in response to bacterial challenges. These results suggest that Repat33 is a downstream component of eicosanoid immune mediation.Repat (=response to pathogen) is proposed for an immune-associated gene family from Spodoptera exigua, a lepidopteran insect. In this gene family, 46 members (Repat1–Repat46) have been identified. They show marked variations in their inducible expression patterns in response to infections by different microbial pathogens. However, their physiological functions in specific immune responses and their interactions with other immune signaling pathways remain unclear. Repat33 is a gene highly inducible by bacterial infections. The objective of this study was to analyze the physiological functions of Repat33 in mediating cellular and humoral immune responses. Results showed that Repat33 was expressed in all developmental stages and induced in immune-associated tissues such as hemocytes and the fat body. RNA interference (RNAi) of Repat33 expression inhibited the hemocyte-spreading behavior which impaired nodule formation of hemocytes against bacterial infections. Such RNAi treatment also down-regulated expression levels of some antimicrobial genes. Interestingly, Repat33 expression was controlled by eicosanoids. Inhibition of eicosanoid biosynthesis by RNAi against a phospholipase A2 (PLA2) gene suppressed Repat33 expression while an addition of arachidonic acid (a catalytic product of PLA2) to RNAi treatment recovered such suppression of Repat33 expression. These results suggest that Repat33 is a downstream component of eicosanoids in mediating immune responses of S. exigua.

A Meloidogyne graminicola Pectate Lyase Is Involved in Virulence and Activation of Host Defense Responses.

Plant-parasitic nematodes secrete an array of cell-wall-degrading enzymes to overcome the physical barrier formed by the plant cell wall. Here, we describe a novel pectate lyase gene Mg-PEL1 from M. graminicola. Quantitative real-time PCR assay showed that the highest transcriptional expression level of Mg-PEL1 occurred in pre-parasitic second-stage juveniles, and it was still detected during the early parasitic stage. Using in situ hybridization, we showed that Mg-PEL1 was expressed exclusively within the subventral esophageal gland cells of M. graminicola. The yeast signal sequence trap system revealed that it possessed an N-terminal signal peptide with secretion function. Recombinant Mg-PEL1 exhibited hydrolytic activity toward polygalacturonic acid. Rice plants expressing RNA interference vectors targeting Mg-PEL1 showed an increased resistance to M. graminicola. In addition, using an Agrobacterium-mediated transient expression system and plant immune response assays, we demonstrated that the cell wall localization of Mg-PEL1 was required for the activation of plant defense responses, including programmed plant cell death, reactive oxygen species (ROS) accumulation and expression of defense-related genes. Taken together, our results indicated that Mg-PEL1 could enhance the pathogenicity of M. graminicola and induce plant immune responses during nematode invasion into plants or migration in plants. This provides a new insight into the function of pectate lyases in plants-nematodes interaction.

Long non-coding RNA UPK1A-AS1 promotes glycolysis in hepatocellular carcinoma cells via stabilization of HIF-1α

To investigate the effect of long non-coding RNA UPK1A-AS1 on glycolysis of hepatocellular carcinoma cells and its underlying molecular mechanisms. A hepatocellular carcinoma (HCC) cell line with lentivirus-mediated stable UPK1A-AS1 overexpression and the cells infected with a negative control lentiviral vector were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 h. The effect of UPK1A-AS1 overexpression on glycolysis of the HCC cells was examined. The expressions of glycolysis-related genes HIF1A, GLUT1, HK1, HK2 and PGK1 were detected by qRTPCR, and the effect of UPK1A-AS1 overexpression on HRE activity was detected by dual luciferase report assay. The HCC cells were treated with cycloheximide to detect the effect of UPK1A-AS1 overexpression on the stability of HIF-1α protein. Immunoprecipitation assay was used to analyze the changes in ubiquitin modification of HIF-1α protein in response to UPK1A-AS1 overexpression. The effects of UPK1A-AS1 overexpression and RNA interference of HIF-1α expression on glucose consumption, lactate production and expressions of HRE activity and glycolysis-related genes (HK1, HK2 and PGK1) were examined in the HCC cells. Compared with the control group, overexpression of UPK1A-AS1 significantly promoted glucose consumption and lactate production in HCC cells under both normoxic and hypoxic conditions (P < 0.05). Overexpression of UPK1A-AS1 significantly increased the expression of glycolysis-related genes including HIF1A, GLUT1, HK1, HK2 and PGK1. Upregulation of UPK1A-AS1 obviously promoted the transcriptional activity of HRE (P < 0.05). Western blotting showed that UPK1A-AS1 overexpression obviously increased the stability of HIF-1α protein and significantly reduced ubiquitin modification of HIF-1α. Downregulation of HIF-1α obviously reversed the effect of UPK1A-AS1 overexpression in promoting glucose consumption, lactate production and HRE luciferase activity. Silencing HIF-1α also suppressed the upregulation of glycolysis-related gene expressions induced by UPK1A-AS1 overexpression (P < 0.05). The long noncoding RNA UPK1A-AS1 upregulates the expression of glycolysis-related genes by stabilizing the expression of HIF-1α, thereby promoting glycolysis level in HCC cells.

Immunomodulatory Agents Combat Multidrug-Resistant Tuberculosis by Improving Antimicrobial Immunity.

BackgroundMultidrug-resistant (MDR) tuberculosis has low treatment success rates, and new treatment strategies are needed. We explored whether treatment with active vitamin D3 (vitD) and phenylbutyrate (PBA) could improve conventional chemotherapy by enhancing immune-mediated eradication of Mycobacterium tuberculosis.MethodsA clinically relevant model was used consisting of human macrophages infected with M. tuberculosis isolates (n = 15) with different antibiotic resistance profiles. The antimicrobial effect of vitD+PBA, was tested together with rifampicin or isoniazid. Methods included colony-forming units (intracellular bacterial growth), messenger RNA expression analyses (LL-37, β-defensin, nitric oxide synthase, and dual oxidase 2), RNA interference (LL-37-silencing in primary macrophages), and Western blot analysis and confocal microscopy (LL-37 and LC3 protein expression).ResultsVitD+PBA inhibited growth of clinical MDR tuberculosis strains in human macrophages and strengthened intracellular growth inhibition of rifampicin and isoniazid via induction of the antimicrobial peptide LL-37 and LC3-dependent autophagy. Gene silencing of LL-37 expression enhanced MDR tuberculosis growth in vitD+PBA–treated macrophages. The combination of vitD+PBA and isoniazid were as effective in reducing intracellular MDR tuberculosis growth as a >125-fold higher dose of isoniazid alone, suggesting potent additive effects of vitD+PBA with isoniazid.ConclusionsImmunomodulatory agents that trigger multiple immune pathways can strengthen standard MDR tuberculosis treatment and contribute to next-generation individualized treatment options for patients with difficult-to-treat pulmonary tuberculosis.

SOX2 enhances cell survival and induces resistance to apoptosis under serum starvation conditions through the AKT/GSK-3β signaling pathway in esophageal squamous cell carcinoma.

The human SOX2 gene was recently identified as a novel major oncogene, recurrently amplified and overexpressed in esophageal squamous cell carcinoma (ESCC). However, the role and molecular mechanism of SOX2 in the carcinogenesis of ESCC remain to be elucidated. The present study investigated the effect of SOX2 on ESCC cell survival and resistance to apoptosis under serum starvation conditions. An adenoviral vector-mediated expression system and RNA interference were used to study the effect of SOX2. The present results revealed that SOX2 promoted ESCC cell survival and enhanced resistance to apoptosis under serum starvation conditions, but not in culture conditions with serum. Mechanistically, SOX2 increased the expression levels of phosphorylated AKT and glycogen synthase kinase-3β (GSK-3β), a downstream factor of AKT, under serum starvation conditions, leading to the promotion of ESCC cell survival. Additionally, SOX2 activated AKT through the PTEN/PI3K/phosphoinositide-dependent protein kinase 1 and mammalian target of rapamycin complex 2 signaling pathways. Therefore, SOX2 may facilitate the survival of ESCC cells under poor nutrient conditions by activating the AKT/GSK-3β signaling pathway.