Selection and Validation of Reference Genes for RT-qPCR Normalization in Bradysia odoriphaga (Diptera: Sciaridae) Under Insecticides Stress

Bradysia odoriphaga (Diptera: Sciaridae) is the most serious root maggot pest which causes substantial damage to the Chinese chive. Organophosphate (OP) and neonicotinoid insecticides are widely used chemical pesticides and play important roles in controlling B. odoriphaga. However, a strong selection pressure following repeated pesticide applications has led to the development of resistant populations of this insect. To understand the insecticide resistance mechanism in B. odoriphaga, gene expression analysis might be required. Appropriate reference gene selection is a critical prerequisite for gene expression studies, as the expression stability of reference genes can be affected by experimental conditions, resulting in biased or erroneous results. The present study shows the expression profile of nine commonly used reference genes [elongation factor 1α (EF-1α), actin2 (ACT), elongation factor 2α (EF-2α), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), ubiquitin-conjugating enzyme (UBC), and α-tubulin (TUB)] was systematically analyzed under insecticide stress. Moreover, we also evaluated their expression stability in other experimental conditions, including developmental stages, sexes, and tissues. Five programs (NormFinder, geNorm, BestKeeper, RefFinder, and ΔCt) were used to validate the suitability of candidate reference genes. The results revealed that the most appropriate sets of reference genes were RPL10 and ACT across phoxim; ACT and TUB across chlorpyrifos and chlorfluazuron; EF1α and TUB across imidacloprid; EF1α and EF2α across developmental stages; RPL10 and TUB across larvae; EF1α and ACT across tissues, and ACT and G6PDH across sex. These results will facilitate the standardization of RT-qPCR and contribute to further research on B. odoriphaga gene function under insecticides stress.

The Bacterial Cytoskeleton based on Bacterial Translation Elongation Factor EF-Tu: Novel Insights

Bacteria possess an EF-Tu-based cytoskeleton.This article presents a short review. A number of questions which are not discussed in the former publications can be asked, such as: all bacteria possess a ribosomal protein synthesis system and, hence, also EF-Tu. EF-Tu is produced in an amount that is higher than the need for a function as translation elogation factor in ribsomal protein synthesis. This article tries to answer the question regarding the surplus of EF-Tu: formation of a "cell-wide web" by self-assembly as a feafure that stabilizes cell integrity. An additional question can be asked: what is the origin of this bacterial cytoskeleton? This article contains a speculation on this topic. A third question regards the'ntteructjon of ribosomes in the process of protemsynthesis: does the EF-Tu protein move to the ribosome, or does the ribosome move to the EF-Tu intergated in a fibril of the bacterial cytoskeleton? The former publication depicts electron micrographs which show colocalizatton of botth entities. EF-Tu is an example for aprotein with two independent functions: participation in the ribosomal protein synthesis as a kanslation elongation factor, and component of a bacterial cytoskeleton. This situation can open up a discussion ofthe sequence of events and states of early cells during evolution.

Evaluation of qPCR reference genes for taimen (Hucho taimen) under heat stress

As a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquaculture research. Understanding the biology of taimen (Hucho taimen) has drawn increasing interest because of its ecological and economic value. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimized for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder. The expression levels of the 10 genes were detected using 240 samples from 48 experimental groups consisting of 40 individuals treated under four heat-stress conditions (18, 20, 22, and 24 °C) for 24 h and 26 °C for 4, 24, 48, and 72 h. Six tissues (blood, heart, brain, gill, skin, and liver) were collected from each individual. Ribosomal protein S29 (RPS29) and ribosomal protein L19 (RPL19) were the most stable genes among all of the samples, whereas 28S ribosomal RNA (28S rRNA), attachment region binding protein (ARBP), and 18S ribosomal RNA (18S rRNA) were the least stable. These results were verified by an expression analysis of taimen heat-stress genes (heat shock protein 60, hsp60, and heat shock protein 70, hsp70). In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions. These results allow the reliable study of gene expression in taimen.

Mesothelin-Specific T Cell Cytotoxicity Against Triple Negative Breast Cancer Is Enhanced By 40s Ribosomal Protein Subunit 3-Treated Self-Differentiated Dendritic Cells

Purpose Triple negative breast cancer (TNBC) is deficient in targeted treatment resulting in poor prognosis. Targeting overexpressed mesothelin (MSLN) using MSLN-specific T cells is an attractive treatment approach.Methods The immunohistochemistry of MSLN in TNBC tissues were performed. A lentiviral vector harboring granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and MSLN cDNAs was constructed to generate self-differentiated myeloid-derived antigen-presenting-cells reactive against tumor expressing MSLN dendritic cells (MSLN-SmartDC) for MSLN-specific T cell activation. The antigen specificity and cancer killing of activated T cells were accessed.Results The high expression of MSLN was found in 32.8% all breast cancer subtypes and 57% in TNBC. High MSLN was significantly associated with the TNBC subtype and the absence of ER, PR and HER2. MSLN-SmartDC exhibited comparable phenotype to DC generated by exogenous cytokine treatment; addition of 40s ribosomal protein subunit 3 (RPS3), a toll-like receptor 4 ligand, enhanced DC maturation and function by upregulation of CD40, CD80 and CD83 expression and IL-12p70 secretion. MSLN-specific CD8+CD69+ IFN-γ+ T cells were detected in T cells activated by both MSLN-SmartDC and RPS3-MSLN-SmartDC. MSLN-specific T cells activated by these DCs showed more specific killing capability against naturally expressed MSLN-HCC70 and artificially MSLN-overexpressing MDA-MB-231 compared to parental MDA-MB-231 in both 2 dimensional (2D)- and 3D-culture systems. Conclusion High MSLN was observed in TNBC patients, a potential target for TNBC treatment. MSLN-SmartDC could promote MSLN-specific T cell response against TNBC and RPS3 can enhance the cytolytic activity of these T cells providing an alternative treatment approach for TNBC patients.

RSL1D1 promotes the progression of colorectal cancer through RAN-mediated autophagy suppression

RSL1D1 (ribosomal L1 domain containing 1), a member of the universal ribosomal protein uL1 family, was suggested to be a new candidate target for colorectal cancer (CRC). However, the role of RSL1D1 in cancer, including CRC, remains largely elusive. Here, we demonstrated that RSL1D1 expression was significantly elevated in tumors from CRC patients and that high expression of RSL1D1 was correlated with poorer survival of CRC patients. Functionally, RSL1D1 promoted the proliferation, invasion, and metastasis of CRC cells by suppressing autophagy. Interestingly, RSL1D1 interacted with RAN and inhibited its deacetylation by competitively binding with Sirt7. By affecting the acetylation of RAN, RSL1D1 inhibited the accumulation of nuclear STAT3 and the STAT3-regulated autophagic program. Taken together, our study uncovered the key role of the RSL1D1/RAN/STAT3 regulatory axis in autophagy and tumor progression in CRC, providing a new candidate target for CRC treatment.