Quantitative Real-time PCR Analysis Research Articles

Food odors elicit feeding behavior through the activation of the olfactory system in blunt snout bream (Megalobrama amblycephala)

Olfaction plays an important role in the survival and reproduction of various organisms. Aside from being a nutrient, in many fish species, amino acids also function as attractants to induce feeding behaviors. As the herbivorous fish, blunt snout bream (Megalobrama amblycephala) commonly consumes an aquatic plant, Hydrilla verticillata. Here, we studied the effect of food odors on the feeding behavior of blunt snout bream using a mixture of amino acids and H. verticillata extract. The behavioral and electro-olfactogram tests demonstrated that the mixture of amino acids and H. verticillata extract induced an attractive reaction and olfactory response in blunt snout bream, respectively. Through Western blotting and immunohistochemical analyses, we confirmed that ciliated and microvillus neurons are distributed on the olfactory placode. The results of phospho-extracellular regulated protein kinases (pERK) showed that a large number of microvillus and ciliated neurons were activated under the stimulation of 10−5 mol/L amino acid mixture and 0.005× H. verticillata extract. The calcium signal imagery demonstrated that the nerve signals of the olfactory bulb were activated by the food odors. The quantitative real-time PCR (qPCR) analysis revealed significant changes in the expression of representative olfactory receptor genes in the olfactory bulb and brain of blunt snout bream upon stimulation with food odor. Collectively, these results provided evidence and preliminary explanations for the mediating role of olfaction in the feeding behavior of fish.

Comparative proteomic analysis of resistant and susceptible cotton genotypes in response to leaf hopper infestation

The cotton leaf hopper is a major pest in cotton, causing a hopper burn in leaves. In this study, a comparative proteomic analysis of NDLH2010 (Resistant) and LRA5166 (Susceptible), infected with leaf hopper, was employed using a nano LC-MS/MS approach. A total of 1402 proteins varied significantly between leaf hopper-infected and control plants. The resistant and susceptible genotypes had differentially expressed proteins (DEPs) of 743 and 659, respectively. Functional annotation of DEPs revealed that the DEPs were primarily associated with stress response, hormone synthesis, photosynthesis, cell wall, and secondary metabolites. Notably, DEPs such as polyphenol oxidase, carboxypeptidase, heat shock proteins, protein BTR1-like isoform X2, chaperone protein ClpB1, and β glucosidase factors associated with environmental stress response were also detected. Quantitative real-time PCR (qRT-PCR) analysis confirmed a positive correlation between protein abundances and transcripts for all genes. Collectively, this study provides the molecular mechanisms associated with cotton defense responses against leaf hopper. Significance statementCotton, a natural fiber, assumes a pivotal role as a raw material for textile industries, thereby bearing significant importance in the global economy. The cotton production sector is considerably affected by both biotic and abiotic stresses. The cotton leaf hopper (Amrasca biguttula biguttula (Ishida)) stands as a polyphagous insect, emerging as a dominant sap-feeding pest of the cotton crop. The continuous onslaught of sap-feeding insects on cotton plants has a detrimental impact, with leaf hoppers potentially causing yield reductions of up to 50%. Therefore, comprehending the molecular interplay between cotton and leaf hopper, elucidated at the proteome level, holds promise for more effective pest management strategies. This approach holds the potential to offer insights that contribute to the development of leaf hopper-resistant cotton varieties.

Screening of Reference Genes for Quantitative Real-Time PCR Analysis in Tissues and during Testis Development, and Application to Analyze the Expression of kifc1 in Hemibarbus labeo (Teleostei, Cypriniformes, Cyprinidae).

The selection of proper reference genes is vital for ensuring precise quantitative real-time PCR (qPCR) assays. This study evaluates the stability of the expression of nine candidate reference genes in different tissues and during testicular development in H. labeo. The results show that eef1a is recommended as a reference gene for qPCR analysis in tissues and during testicular development. Furthermore, we evaluated the optimal number of reference genes needed when calculating gene expression levels using the geomean method, revealing that two reference genes are sufficient. Specifically, eef1a and rps27 are recommended for analysis of gene expression in tissues, whereas eef1a and actb are advised for evaluating gene expression during testicular development. In addition, we examined the expression pattern of kifc1, a kinesin involved in the reshaping of spermatids. We detected peak expression levels of kifc1 in testes, with its expression initially increasing before decreasing throughout testicular development. The highest expression of kifc1 was observed in stage IV testes, the active period of spermiogenesis, suggesting a possible role for kifc1 in the regulation of the reshaping of spermatids and hence testicular development. This study represents the first investigation of reference genes for H. labeo, providing a foundation for studying gene expression patterns and investigating gene expression regulation during testicular development.

Hsa_circ_0004776 regulates the retina neovascularization in progression of diabetic retinopathy via hsa-miR-382-5p/BDNF axis

The aim of this work was to identify the regulatory function of hsa_circ_0004776 in the progression of diabetic retinopathy (DR). The direct interactions between hsa_circ_0004776 and hsa-miR-382-5p and between hsa-miR-382-5p and BDNF, were confirmed via dual-luciferase reporter assays. Quantitative Real-Time PCR analysis indicated that hsa_circ_0004776 was highly expressed in aqueous humour samples of DR patients and human retinal microvascular epithelial cells (hRECs) under a high-glucose environment, whereas hsa-miR-382-5p showed the opposite trend. Overexpressed hsa_circ_0004776 significantly enhanced DNA synthesis, proliferation, migration, and tube formation in hRECs in hyperglycaemia, while hsa-miR-382-5p mimics reversed these changes. Additionally, in a streptozotocin-induced Sprague-Dawley rat model of DR, vitreous microinjection of rno-miR-382-5p agomir reversed the pathologic features in the progression of DR, including retinal vascular leakage, capillary decellularization, loss of pericytes, fibrosis, and gliosis. Our results indicated that under hyperglycaemic conditions, hsa_circ_0004776 influences the progression of DR via hsa-miR-382-5p and thus represents a potential therapeutic target.

Hsa_circ_0004776 regulates the retina neovascularization in progression of diabetic retinopathy via hsa-miR-382-5p/BDNF axis

The aim of this work was to identify the regulatory function of hsa_circ_0004776 in the progression of diabetic retinopathy (DR). The direct interactions between hsa_circ_0004776 and hsa-miR-382-5p and between hsa-miR-382-5p and BDNF, were confirmed via dual-luciferase reporter assays. Quantitative Real-Time PCR analysis indicated that hsa_circ_0004776 was highly expressed in aqueous humour samples of DR patients and human retinal microvascular epithelial cells (hRECs) under a high-glucose environment, whereas hsa-miR-382-5p showed the opposite trend. Overexpressed hsa_circ_0004776 significantly enhanced DNA synthesis, proliferation, migration, and tube formation in hRECs in hyperglycaemia, while hsa-miR-382-5p mimics reversed these changes. Additionally, in a streptozotocin-induced Sprague-Dawley rat model of DR, vitreous microinjection of rno-miR-382-5p agomir reversed the pathologic features in the progression of DR, including retinal vascular leakage, capillary decellularization, loss of pericytes, fibrosis, and gliosis. Our results indicated that under hyperglycaemic conditions, hsa_circ_0004776 influences the progression of DR via hsa-miR-382-5p and thus represents a potential therapeutic target.

Analysis of the relationship between intestinal segmented filamentous bacteria and rotavirus infection in children

Objective: To investigate the association between intestinal colonization of segmented filamentous bacteria (SFB) and the risk of rotavirus infection, and the possible mechanisms by which SFB resist rotavirus infection. Methods: This case-control study enrolled 50 children aged 0 to 5 years who present to the outpatient Department of Children's Hospital, Zhejiang University School of Medicine with diarrhea and positive stool tests for rotavirus. The children were divided into rotavirus enteritis group and control group consisting of 55 children with non-gastrointestinal and non-infectious surgical diseases.The age and sex composition of the two groups was matched. The DNA of the fecal flora was extracted and SFB was detected by real-time fluorescence quantitative PCR analysis. The children in the rotavirus enteritis group and the control group were subgrouped by age and sex to analyze the differences in SFB positivity rates between different groups, and further compare and analyze the differences in SFB positivity rates between these two groups of children in the ≤2 years old subgroup and the >2-5 years old subgroup. Neutralization test was performed with p3340 protein and rotavirus to determine the relationship between rotavirus infection rate and p3340 concentration in Vero cells. χ2 test or Fisher's exact probability method was used for comparison between the two groups. Results: There were 50 children in the rotavirus enteritis group with an age of (1.7±0.9) years, and 55 children in the control group with an age of (1.8±1.1) years. The positive rate of SFB in children with rotavirus enteritis showed a declining trend across ages groups, with the highest rate of 10/14 in the ≤1 year old group, followed by 67% (14/21) in the >1-2 years old group, 9/15 in the >2-5 years old group, and there was no statistically significant difference (P=0.867). The positive rate of SFB in the control group was 12/15 in the ≤1 year old group, 95% (19/20) in the >1-2 years old group, 50% (10/20) in the >2-5 years old group, with statistical significance (P=0.004). The positive rate of SFB in children with rotavirus enteritis was 74% (20/27) in males and 56% (13/23) in females (χ2=1.71, P=0.192). In the control group, it was 79% (22/28) in males and 70% (19/27) in females (χ2=0.49, P=0.485). The positive rate of SFB was 66% (33/50) in the rotavirus enteritis group and 75% (41/55) in the control group, with no statistically significant (χ2=0.56, P=0.454). In the children ≤2 years old, the SFB positivity rate was 69% (24/35) in the rotavirus enteritis group and 89% (31/35) in the control group, with a statistically significant difference (χ2=4.16, P=0.041). However, in the children >2-5 years old, no statistically significant difference was observed, with the positive rate of SFB being 9/15 in the rotavirus enteritis group and 50% (10/20) in the control group (P=0.734). Pearson correlation analysis revealed a negative correlation between rotavirus infection and SFB positivity (r=-0.87,P<0.001). As the concentration of the p3340 specific protein increased, the luminescence intensity of the luciferase in the Vero cells, which were suitable for cultivating rotavirus, exhibited a decreasing trend (F=4.17, P=0.001). Conclusions: SFB colonization in infants less than 2 years old is associated with a reduced risk of rotavirus infection. Cloning of specific SFB functional protein p3340 neutralizes rotavirus infection of Vero cells, and this mechanism of targeting rotavirus infection differs from the common antiviral mechanism.

Intervention of epithelial mesenchymal transition against colon cancer cell growth and metastasis based on SOX21/POU4F2/Hedgehog signaling axis

AimsColon cancer poses a major threat to human health and a heavy burden on the national economy. As a member of the SOX transcription factor family, SRY-box transcription factor 21 (SOX21) is associated with various cancers, but its mechanism of action in colon cancer remains unclear. This study focused on the molecular mechanisms of transcription factor SOX21 in proliferation and metastasis of colon cancer cells. Main methodsWe analyzed SOX21 expression level and its impact on survival in colon cancer patients by bioinformatics analysis. We used public databases for gene correlation, GSEA enrichment analysis. Cell function experiments (colony formation assay, wound healing assay, Transwell migration and invasion assay) were utilized to determine the impact of SOX21 silencing and over-expression on cell proliferation and metastasis. The luciferase reporter assay, CUT&RUN-qPCR assay and Methylation Specific PCR were used to explore SOX21-POU class 4 homeobox 2 (POU4F2) molecular interactions. The molecular mechanisms were verified by Quantitative real-time PCR and Western blot analysis. Key findingsSOX21 is highly expressed and affects the overall survival of colon cancer patients. SOX21 can attenuates POU4F2 methylation state by binding with it. In addition, this interaction facilitate its transcriptional activation of Hedgehog pathway, mediates epithelial-mesenchymal transition (EMT), consequently promoting the proliferation and metastasis of colon cancer cells. SignificanceOur study reveals that SOX21 is an oncogenic molecule and suggests its regulatory role in colon carcinogenesis and progression, providing new insights into the treatment of this disease.

Recombinant human protein TCFL5-activated NRSN2-AS1 promotes esophageal cancer progression via the microRNA-874-5p/RELT regulatory axis

The incidence of esophageal cancer continues to increase worldwide. Current therapeutic approaches have limited efficacy, so in order to search for better markers of the disease, it is necessary to further elucidate its molecular pathogenesis. Regulation of gene expression by long non-coding Rnas plays a role in many diseases, however the role in esophageal cancer is unclear. The aim of this study was to elucidate the role and regulatory mechanism of long non-coding RNA NRSN2-AS1 in the progression of esophageal cancer. By real-time quantitative PCR, immunohistochemistry, RNA interference, western blotting, and double luciferase reporter gene analysis, we found that NRSN2-AS1 was up-regulated in esophageal cancer tissues and cell lines, and was closely related to disease stage and prognosis. Functional studies have shown that the silencing of NRSN2-AS1 inhibits the proliferation of esophageal cancer cells, induces apoptosis, and prevents cell migration and invasion. In mouse models, NRSN2-AS1 also promoted tumor growth. The transcription factor TCFL5 upregulates the transcription of NRSN2-AS1, which acts as a sponge for microRNA(miR)-874–5p, thereby upregulating the expression of the oncogene RELT. Activation of the NRSN2-AS1/miR-874-5p/RELT regulatory axis was validated in vivo.

Genomic Identification of Callose Synthase (CalS) Gene Family in Sorghum (Sorghum bicolor) and Comparative In Silico Expression Analysis under Aphid (Melanaphis sacchari) Infestation

Callose is widely present in higher plants and plays a significant role in plant growth, development, and response to various stresses. Although numerous studies have highlighted the importance of the callose synthase (CalS) genes, their role in the resistance of sorghum (Sorghum bicolor) to aphids (Melanaphis sacchari) remains limitedly understood. This study identified 11 sorghum callose synthase genes (SbCalS), unevenly distributed across four chromosomes of sorghum. All SbCalS proteins contain glucan synthase and Fks1 domains, with segmental duplication playing a major role in gene diversification. Cis-element prediction revealed the presence of numerous stress-responsive elements, indicating that this gene family is primarily involved in stress resistance. Using published RNA-seq data, we discovered the differential expression of the SbCalS5 gene between resistant and susceptible sorghum varieties. Real-time quantitative PCR (qPCR) analysis confirmed the relative expression levels of all SbCalS members under aphid stress. To further verify the role of callose in sorghum, we measured the callose content in both resistant and susceptible sorghum varieties. The results indicated that callose plays a critical role in aphid resistance in sorghum, particularly the SbCalS5 gene. This study provides a reference for further investigation into the role of callose synthase genes in sorghum aphid resistance.

Erythroid Differentiation Regulator 1 as a Regulator of Neuronal GSH Synthesis

Erythroid differentiation regulator 1 (Erdr1) is a cytokine known to play important roles in cell survival under stressful conditions, maintenance of cellular growth homeostasis, and activation of the immune system. However, the impact of Erdr1 on neurons remains undefined. In this study, we present novel evidence that Erdr1 plays a role in regulating glutathione (GSH) synthesis via glutamate transporter-associated protein 3-18 (GTRAP3-18), an anchor protein in the endoplasmic reticulum that holds excitatory amino acid carrier 1 (EAAC1) in neurons. Both DNA microarray and quantitative real-time PCR analyses revealed an approximately 2-fold increase in Erdr1 levels in the hippocampus of GTRAP3-18-deficient mice compared to those of wild-type mice. Knockdown of Erdr1 in vitro resulted in a decrease in GTRAP3-18 levels, leading to an increase in EAAC1 expression and intracellular GSH levels, and subsequently, cytoprotective effects against oxidative stress. Our findings shed light on the regulatory mechanisms involving Erdr1, GTRAP3-18, EAAC1, and GSH in the context of neuronal defense against oxidative stress. Understanding the intricate interplay among these molecules may pave the way for the development of promising therapeutic strategies for neurodegenerative disorders.

KIAA1429 promotes gastric cancer progression by destabilizing RASD1 mRNA in an m6A-YTHDF2-dependent manner

BackgroundKIAA1429, a regulatory subunit of the N6-methyladenosine (m6A) methyltransferase complex, has been implicated in the progression of various cancers. However, the role of KIAA1429 in gastric cancer (GC) and its underlying mechanisms remain elusive. This study aimed to investigate the role of KIAA1429 in GC and to elucidate the underlying mechanisms.MethodsThe expression patterns and clinical relevance of KIAA1429 in GC were assessed using quantitative real-time PCR (qRT-PCR), Western blotting, immunohistochemistry (IHC), and bioinformatic analysis. In vitro and in vivo loss- and gain-of-function assays, m6A dot blot assays, methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA-seq, MeRIP-qPCR, dual luciferase reporter assays, RNA stability assays, RNA immunoprecipitation (RIP) assays, and RNA pull-down assays were performed to investigate the biological functions and underlying molecular mechanisms of KIAA1429 in GC.ResultsBoth the mRNA and protein expression of KIAA1429 were greater in GC tissues than in normal gastric tissues. High KIAA1429 expression correlated positively with poor prognosis in GC patients. KIAA1429 not only promoted GC cell proliferation, colony formation, G2/M cell cycle transition, migration, and invasion in vitro but also enhanced GC tumor growth and metastasis in vivo. Mechanistically, KIAA1429 increased the m6A level of RASD1 mRNA and enhanced its stability in an m6A-YTHDF2-dependent manner, thereby upregulating its expression. RASD1 knockdown partially rescued the KIAA1429 knockdown-induced impairment of pro‑oncogenic ability in GC cells. The expression levels of KIAA1429 and RASD1 were negatively correlated in GC tissues.ConclusionsKIAA1429 plays a pro‑oncogenic role in GC by downregulating RASD1 expression through destabilizing RASD1 mRNA in an m6A-YTHDF2-dependent manner. KIAA1429 may serve as a prognostic biomarker and therapeutic target for GC.

Involvement of ArlI, ArlJ, and CirA in archaeal type IV pilin-mediated motility regulation.

Many prokaryotes use swimming motility to move toward favorable conditions and escape adverse surroundings. Regulatory mechanisms governing bacterial flagella-driven motility are well-established; however, little is yet known about the regulation underlying swimming motility propelled by the archaeal cell surface structure, the archaella. Previous research showed that the deletion of the adhesion pilins (PilA1-6), subunits of the type IV pili cell surface structure, renders the model archaeon Haloferax volcanii non-motile. In this study, we used ethyl methanesulfonate mutagenesis and a motility assay to identify motile suppressors of the ∆pilA[1-6] strain. Of the eight suppressors identified, six contain missense mutations in archaella biosynthesis genes, arlI and arlJ. In trans expression of arlI and arlJ mutant constructs in the respective multi-deletion strains ∆pilA[1-6]∆arlI and ∆pilA[1-6]∆arlJ confirmed their role in suppressing the ∆pilA[1-6] motility defect. Additionally, three suppressors harbor co-occurring disruptive missense and nonsense mutations in cirA, a gene encoding a proposed regulatory protein. A deletion of cirA resulted in hypermotility, while cirA expression in trans in wild-type cells led to decreased motility. Moreover, quantitative real-time PCR analysis revealed that in wild-type cells, higher expression levels of arlI, arlJ, and the archaellin gene arlA1 were observed in motile early-log phase rod-shaped cells compared to non-motile mid-log phase disk-shaped cells. Conversely, ∆cirA cells, which form rods during both early- and mid-log phases, exhibited similar expression levels of arl genes in both growth phases. Our findings contribute to a deeper understanding of the mechanisms governing archaeal motility, highlighting the involvement of ArlI, ArlJ, and CirA in pilin-mediated motility regulation.IMPORTANCEArchaea are close relatives of eukaryotes and play crucial ecological roles. Certain behaviors, such as swimming motility, are thought to be important for archaeal environmental adaptation. Archaella, the archaeal motility appendages, are evolutionarily distinct from bacterial flagella, and the regulatory mechanisms driving archaeal motility are largely unknown. Previous research has linked the loss of type IV pili subunits to archaeal motility suppression. This study reveals three Haloferax volcanii proteins involved in pilin-mediated motility regulation, offering a deeper understanding of motility regulation in this understudied domain while also paving the way for uncovering novel mechanisms that govern archaeal motility. Understanding archaeal cellular processes will help elucidate the ecological roles of archaea as well as the evolution of these processes across domains.

Ckip-1 3’UTR alleviates prolonged sleep deprivation induced cardiac dysfunction by activating CaMKK2/AMPK/cTNI pathway

Sleep deprivation (SD) has emerged as a critical concern impacting human health, leading to significant damage to the cardiovascular system. However, the underlying mechanisms are still unclear, and the development of targeted drugs is lagging. Here, we used mice to explore the effects of prolonged SD on cardiac structure and function. Echocardiography analysis revealed that cardiac function was significantly decreased in mice after five weeks of SD. Real-time quantitative PCR (RT-q-PCR) and Masson staining analysis showed that cardiac remodeling marker gene Anp (atrial natriuretic peptide) and fibrosis were increased, Elisa assay of serum showed that the levels of creatine kinase (CK), creatine kinase-MB (CK-MB), ANP, brain natriuretic peptide (BNP) and cardiac troponin T (cTn-T) were increased after SD, suggesting that cardiac remodeling and injury occurred. Transcript sequencing analysis indicated that genes involved in the regulation of calcium signaling pathway, dilated cardiomyopathy, and cardiac muscle contraction were changed after SD. Accordingly, Western blotting analysis demonstrated that the cardiac-contraction associated CaMKK2/AMPK/cTNI pathway was inhibited. Since our preliminary research has confirmed the vital role of Casein Kinase-2 -Interacting Protein-1 (CKIP-1, also known as PLEKHO1) in cardiac remodeling regulation. Here, we found the levels of the 3’ untranslated region of Ckip-1 (Ckip-1 3’UTR) decreased, while the coding sequence of Ckip-1 (Ckip-1 CDS) remained unchanged after SD. Significantly, adenovirus-mediated overexpression of Ckip-1 3’UTR alleviated SD-induced cardiac dysfunction and remodeling by activating CaMKK2/AMPK/cTNI pathway, which proposed the therapeutic potential of Ckip-1 3’UTR in treating SD-induced heart disease.

A comparative analysis of real-time quantitative PCR and metabarcoding methods for eDNA-based detection of the toxic dinophyte Alexandrium tamiyavanichii (Dinophyceae)

The marine dinophyte Alexandrium tamiyavanichii is a toxigenic species that produces a group of neurotoxins that is responsible for paralytic shellfish poisoning in humans. Early detection of the species is essential for efficient monitoring. Harmful microalgal monitoring systems have evolved over the years with the advent of environmental DNA (eDNA)-based species detection techniques. In this study, eDNA samples were collected from a large-scale sampling covering the southern South China Sea. The sensitivity and specificity of metabarcoding of the V4 and V9 18S ribosomal DNA barcodes by high-throughput sequencing (HTS) were compared to the species-specific real-time qPCR targeting the A. tamiyavanichii ITS2 region. Environmental samples were screened for A. tamiyavanichii by qPCR (n = 43) and analyzed with metabarcoding (n = 30). Our results revealed a high occupancy profile across samples for both methods; 88% by qPCR, and 80–83% by HTS. When comparing the consistency between the two approaches, only two samples out of 30 were discordant. The V4 and V9 molecular units detected in each sample were positively correlated with the qPCR ITS2 gene copies (V4, rs = 0.67, p < 0.0001; V9, rs = 0.65, p < 0.0001), indicating that metabarcoding could be used as a useful tool for early detection of the species. Our results also revealed that the estimation of A. tamiyavanichii cell abundances based on the HTS read abundances was comparable to that of the qPCR quantification. For long-term monitoring, metabarcoding could serve as a cost-effective screening of detecting not only single HAB species but also simultaneously detecting a multitude of potentially harmful species, which is valuable in informing the subsequent implementation of species-specific monitoring strategies.

RNAi-mediated knockdown of papilin gene affects the egg hatching in Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens, is one of the most destructive pests of rice. Owing to the rapid adaptation of BPH to many pesticides and resistant varieties, identifying putative gene targets for developing RNA interference (RNAi)-based pest management strategies has received much attention for this pest. The glucoprotein papilin is the most abundant component in the basement membranes of many organisms, and its function is closely linked to development. In this study, we identified a papilin homologous gene in BPH (NlPpn). Quantitative Real-time PCR analysis showed that the transcript of NlPpn was highly accumulated in the egg stage. RNAi of NlPpn in newly emerged BPH females caused nonhatching phenotypes of their eggs, which may be a consequence of the maldevelopment of their embryos. Moreover, the transcriptomic analysis identified 583 differentially expressed genes between eggs from the dsGFP- and dsNlPpn-treated insects. Among them, the 'structural constituent of cuticle' cluster ranked first among the top 15 enriched GO terms. Consistently, ultrastructural analysis unveiled that dsNlPpn-treated eggs displayed a discrete and distorted serosal endocuticle lamellar structure. Furthermore, the hatchability of BPH eggs was also successfully reduced by the topical application of NlPpn-dsRNA-layered double hydroxide nanosheets onto the adults. Our findings demonstrate that NlPpn is essential to maintaining the regular structure of the serosal cuticle and the embryonic development in BPH, indicating NlPpn could be a potential target for pest control during the egg stage. © 2024 Society of Chemical Industry.

Targeting USP7 to induce ubiquitination degradation of oncogenic ATAD2 and effects on cholangiocarcinoma growth.

e16215 Background: Cholangiocarcinoma (CCA) is a highly fatal disease due to limited therapeutic targets. Deubiquitinase USP7 is an oncogene and has been suggested to be deeply involved in regulating tumor initiation and progression, but its abundance and role in CCA still remain unclear. Methods: USP7 mRNA expression was performed using TCGA and GEO database. Survival analysis in CCA patients with high- and low-USP7 mRNA expression was performed using NODE database. CCK8, colony formation, flow cytometry and shRNA interference assay were used to evaluate the biological function of USP7-ATAD2 axis on CCA cancer cells in vitro. 4D label-free quantification proteomics was used to analyze the targets and networks regulated by USP7. The molecular mechanism of USP7 and ATAD2 in CCA was determined by conducting Western blot, co-immunoprecipitation, IHC and quantitative real-time PCR (qPCR) analyses. The CCA PDX model was used to evaluate the efficacy of targeting USP7 in the treatment of cholangiocarcinoma. Results: Here, we found USP7 is highly expressed in CAA, and is associated with poor outcomes of CCA patients. We show that USP7 depletion induces S phase arrest, and suppresses CCA tumor cells proliferation. Next, we provide a comprehensive understanding of targets and networks affected by USP7 depletion by performing a 4D label-free quantification proteomics analysis. We show that ATAD2 is a USP7 target, and USP7 interacts with and deubiquitinates ATAD2 to stabilize ATAD2 protein level. Ectopic expression of ATAD2 in USP7-depleted cells rescues the cell proliferation phenotype. Further, pharmacological blocking the activity of USP7 by P5091 inhibits tumor growth in CCA patient-derived xenograft (PDX) mouse model. Conclusions: This study uncovers the role of USP7-ATAD2 axis in regulating tumor growth, and targeting USP7 may provide a novel therapeutic strategy for CCA.

Targeting USP7 to induce ubiquitination degradation of oncogenic ATAD2 and effects on cholangiocarcinoma growth.

e16215 Background: Cholangiocarcinoma (CCA) is a highly fatal disease due to limited therapeutic targets. Deubiquitinase USP7 is an oncogene and has been suggested to be deeply involved in regulating tumor initiation and progression, but its abundance and role in CCA still remain unclear. Methods: USP7 mRNA expression was performed using TCGA and GEO database. Survival analysis in CCA patients with high- and low-USP7 mRNA expression was performed using NODE database. CCK8, colony formation, flow cytometry and shRNA interference assay were used to evaluate the biological function of USP7-ATAD2 axis on CCA cancer cells in vitro. 4D label-free quantification proteomics was used to analyze the targets and networks regulated by USP7. The molecular mechanism of USP7 and ATAD2 in CCA was determined by conducting Western blot, co-immunoprecipitation, IHC and quantitative real-time PCR (qPCR) analyses. The CCA PDX model was used to evaluate the efficacy of targeting USP7 in the treatment of cholangiocarcinoma. Results: Here, we found USP7 is highly expressed in CAA, and is associated with poor outcomes of CCA patients. We show that USP7 depletion induces S phase arrest, and suppresses CCA tumor cells proliferation. Next, we provide a comprehensive understanding of targets and networks affected by USP7 depletion by performing a 4D label-free quantification proteomics analysis. We show that ATAD2 is a USP7 target, and USP7 interacts with and deubiquitinates ATAD2 to stabilize ATAD2 protein level. Ectopic expression of ATAD2 in USP7-depleted cells rescues the cell proliferation phenotype. Further, pharmacological blocking the activity of USP7 by P5091 inhibits tumor growth in CCA patient-derived xenograft (PDX) mouse model. Conclusions: This study uncovers the role of USP7-ATAD2 axis in regulating tumor growth, and targeting USP7 may provide a novel therapeutic strategy for CCA.

Identification and expression analysis of XIP gene family members in rice.

Xylanase inhibitor proteins (XIP) are widely distributed in the plant kingdom, and also exist in rice. However, a systematic bioinformatics analysis of this gene family in rice (OsXIP) has not been conducted to date. In this study, we identified 32 members of the OsXIP gene family and analyzed their physicochemical properties, chromosomal localization, gene structure, protein structure, expression profiles, and interaction networks. Our results indicated that OsXIP genes exhibit an uneven distribution across eight rice chromosomes. These genes generally feature a low number of introns or are intronless, all family members, except for OsXIP20, contain two highly conserved motifs, namely Motif 8 and Motif 9. In addition, it is worth noting that the promoter regions of OsXIP gene family members feature a widespread presence of abscisic acid response elements (ABRE) and gibberellin response elements (GARE-motif and TATC-box). Quantitative Real-time PCR (qRT-PCR) analysis unveiled that the expression of OsXIP genes exhibited higher levels in leaves and roots, with considerable variation in the expression of each gene in these tissues both prior to and following treatments with abscisic acid (ABA) and gibberellin (GA3). Protein interaction studies and microRNA (miRNA) target prediction showed that OsXIP engages with key elements within the hormone-responsive and drought signaling pathways. The qRT-PCR suggested osa-miR2927 as a potential key regulator in the rice responding to drought stress, functioning as tissue-specific and temporally regulation. This study provides a theoretical foundation for further analysis of the functions within the OsXIP gene family.

Tyrosinase deficiency impairs social novelty preference in mice.

Tyrosinase is a rate-limiting enzyme for the biosynthesis of melanin pigment in peripheral tissues, such as skin and the retina. We recently reported the expression and enzymatic activity of tyrosinase as well as its protective effects against oxidative stress-induced protein damage in the mouse brain. The functional role of tyrosinase in the central nervous system, however, remains largely unknown. In the present study, we investigated the involvement of tyrosinase in social behavior in mice. Pigmented C57BL/10JMsHir (B10) and tyrosinase-deficient albino B10.C-Tyrc/Hir (B10-c) mice were subjected to the three-chamber sociability test to assess sociability and social novelty preference. In addition, we measured the mRNA expression of genes involved in catecholamine metabolism in the hippocampus by real-time quantitative PCR analysis. The results obtained showed that tyrosinase deficiency impaired social novelty preference, but not sociability in mice. We also found that the hippocampal expression of genes involved in catecholamine metabolism, such as monoamine oxidase A and catechol-O-methyltransferase, were significantly decreased in tyrosinase-deficient B10-c mice. These results suggest that tyrosinase activity is functionally involved in the phenotypic expression of social behavior, particularly social novelty preference, in mice. The present study will advance our understanding of the functional role of tyrosinase in the central nervous system.

Establishment of canine mammary gland tumor cell lines harboring PI3K/Akt activation as a therapeutic target

Canine mammary gland tumors (MGT) have a poor prognosis in intact female canines, posing a clinical challenge. This study aimed to establish novel canine mammary cancer cell lines from primary tumors and characterize their cellular and molecular features to find potential therapeutic drugs. The MGT cell lines demonstrated rapid cell proliferation and colony formation in an anchorage-independent manner. Vimentin and α-SMA levels were significantly elevated in MGT cell lines compared to normal canine kidney (MDCK) cells, while CDH1 expression was either significantly lower or not detected at all, based on quantitative real-time PCR (qRT-PCR) analysis. Functional annotation and enrichment analysis revealed that epithelial-mesenchymal transition (EMT) phenotypes and tumor-associated pathways, particularly the PI3K/Akt signaling pathway, were upregulated in MGT cells. BYL719 (Alpelisib), a PI3K inhibitor, was also examined for cytotoxicity on the MGT cell lines. The results show that BYL719 can significantly inhibit the proliferation of MGT cell lines in vitro. Overall, our findings suggest that the MGT cell lines may be valuable for future studies on the development, progression, metastasis, and management of tumors.