CCDC6-RET fusion protein regulates Ras/MAPK signaling through the fusion- GRB2-SHC1 signal niche.

Rearranged during transfection (RET) rearrangement oncoprotein-mediated Ras/MAPK signaling cascade is constitutively activated in cancers. Here, we demonstrate a unique signal niche. The niche is a ternary complex based on the chimeric RET liquid-liquid phase separation. The complex comprises the rearranged kinase (RET fusion); the adaptor (GRB2), and the effector (SHC1). Together, they orchestrate the Ras/MAPK signal cascade, which is dependent on tyrosine kinase. CCDC6-RET fusion undergoes LLPS requiring its kinase domain and its fusion partner. The CCDC6-RET fusion LLPS promotes the autophosphorylation of RET fusion, with enhanced kinase activity, which is necessary for the formation of the signaling niche. Within the signal niche, the interactions among the constituent components are reinforced, and the signal transduction efficiency is amplified. The specific RET fusion-related signal niche elucidates the mechanism of the constitutive activation of the Ras/MAPK signaling pathway. Beyond just focusing on RET fusion itself, exploration of the ternary complex potentially unveils a promising avenue for devising therapeutic strategies aimed at treating RET fusion-driven diseases.

Fluorescence immunosensor based on a specific monoclonal antibody for highly sensitive and rapid detection of gizzerosine in feed.

Gizzerosine is a biogenic amine produced in fish meal drying process and posted higher mortality due to gizzard erosion in poultry than histamine. However, it is difficult to obtain gizzerosine and achieve sensitive practical detection due to its simple structure. Herein, a monoclonal antibody (mAb) specific to gizzerosine was generated based on the new structural design and a fluorescence immunosensor for sensitive and on-site detection of gizzerosine in feed was first established. Molecular modeling of the three-dimensional (3D) structure and surface electrostatic potential of gizzerosine indicated that the carbonyl group of gizzerosine hapten might affect the important sites of antigen-antibody interactions. The proposed structure was used to obtain the sensitive and specific mAb with IC50 of 3.88 ng/mL in indirect competitive ELISA which was approximately 100-fold lower than that of direct competitive ELISA. Considering the practical application scenarios, a fluorescence immunosensor based on microporous dry method integrated with independent quality control line was established to improve detection stability. Under the optimum conditions, the proposed immunosensor showed a good linear relationship from 1.10 to 19.78 ng/mL and provided a low detection limit of 50 ng/g which was approximately 80-fold lower than the maximum recommended amount (0.4 mg/kg) of gizzerosine in feed. The recoveries of 6 kinds of feed ranged from 83.1 % to 114.3 %, which was in good consistence with that of UHPLC-MS/MS. Overall, this work provides a fast, cost-effective and reliable on-site tool for rapid screening of gizzerosine residues in feed samples.

[Relationship between viral burden in urine and hearing loss in neonates with cytomegalovirus infection].

OBJECTIVE: To determine the relationship between viral burden in urine and hearing loss in neonates with cytomegalovirus (CMV) infection. METHODS: Twenty-two neonates with CMV infection between April 2006 and January 2010 were enrolled. Their viral burden in urine and hearing loss information were studied. The receiver operating characteristic curve (ROC) was constructed and the cutoff was determined based on their medical information. The hearing levels were evaluated by brain stem auditory evoked potential (BAEP) during the age of 3 to 6 months in 20 patients. RESULTS: The viral burden in urine in neonates with abnormal BAEP was higher than that in neonates with normal BAEP (5.06 ± 1.50 vs 3.73 ± 0.86, P<0.05). Hearing loss was predicted with a sensitivity of 0.545 and a specificity of 1.0 by using ROC at the cutoff point of 5.1 which were defined after logarithmic conversion at 1.27×10(5) copies/mL of CMV burden in urine. The incidence of hearing loss during the age of 3 to 6 months was strikingly higher in high viral burden group than that in low viral load group (P<0.05). CONCLUSIONS: The viral burden in urine can predict the possibility of hearing loss in neonates with CMV infection. Hearing loss is likely to be developed when viral burden in urine ≥1.27×10(5) copies/mL in neonates with CMV infection.

[Retinoic acid protects neonatal rats from hyperoxia-induced lung injury through down-regulating connective tissue growth factor].

OBJECTIVE: To explore the mechanism of retinoic acid (RA)　protection　against hyperoxia-induced lung injury. METHODS: Ninety Sprague-Dawley rats were randomly assigned into three groups (n=30 each): air control group (exposed to air) and hyperoxia groups (exposed to 85% oxygen) with and without RA treatment. The RA-treated hyperoxia group received an intraperitoneal injection of RA (500 µg/kg) daily. Lungs were removed by tnoracotomy 4, 7 and 14 days after exposure. Radical alveolar counts (RAC) were observed by hematoxylin and eosin staining under a light microscope. The mRNA level of CTGF in lungs was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of CTGF protein in lungs was detected by immunohistochemistry. RESULTS: With the prolonged hyperoxia exposure, the lungs developed inflammatory cell infiltration, alveolar structure disorders, a decrease in the number of alveoli, and alveolar interstitial thickening in the hyperoxia groups with and without RA treatment. Pathological changes in the RA-treated hyperoxia group were less severe than the untreated hyperoxia group. The CTGF mRNA and protein expression were up-regulated in the hyperoxia groups with and without RA treatment 7 and 14 days after exposure compared with the air control group. Significantly decreased CTGF mRNA and protein expression were noted in the RA-treated hyperoxia group compared with the untreated hyperoxia group 14 days after exposure. CONCLUSIONS: The expression of CTGF mRNA and protein increases in neonatal rats with hyperoxia-induced lung injury. RA may provide protections against the lung injury possibly through down-regulating CTGF expression.

Transcriptome sequencing of a toxic dinoflagellate, Karenia mikimotoi subjected to stress from solar ultraviolet radiation.

Solar ultraviolet radiation (UVR) is a stress factor in aquatic environments and may act directly or indirectly on orgnisms in the upper layers of the water column. However, UVR effects are usually species-specific and difficult to extrapolate. Here we use the HAB-forming, toxic dinoflagellate Karenia mikimotoi (which was found to be relatively resistant in previous studies) to investigate its transcriptional responses to a one-week UVR exposure. For this, batch cultures of K. mikimotoi were grown with and without UVR, and their transcriptomes (generated via RNAseq technology) were compared. RNA-seq generated 45.31 million reads, which were further assembled to 202600 unigenes (>300bp). Among these, ca. 61% were annotated with NCBI, NR, GO, KOG, PFAM, Swiss-Prot, and KEGG database. Transcriptomic analysis revealed 722 differentially expressed unigenes (DEGs, defined as being within a |log2 fold change| ≥ 2 and padj < 0.05) responding to solar UVR, which were only 0.36% of all unigenes. 716 unigenes were down-regulated, and only 6 unigenes were up-regulated in the UVR compared to non-UVR treatment. KEGG pathway further analysis revealed DEGs were involved in the different pathway; genes involved in the ribosome, endocytosis and steroid biosynthesis pathways were highly down-regulated, but this was not the case for those involved in the energy metabolisms (including photosynthesis, oxidative phosphorylation) which may contribute to the sustainable growth observed in UVR treatment. The up-regulated expression of both zinc-finger proteins (ZFPs) and ribosomal protein L11 (RPL11) may be one of the acclimated mechanisms against UVR. In addition, this work identified down-regulated genes involved in fatty acid degradation and the hydrophobic branched chain amino acids (e.g., Valine, leucine, and isoleucine), which act as structural components of cell membranes modulating lipid homeostasis or turnover. In conclusion, the present study suggests that the toxic dinoflagellate K. mikimotoi has limited transcriptomic regulation but confirms that it appears as a tolerant species in response to solar UVR. These findings expand current knowledge of gene expression in HAB-forming species in response to natural environment factors such as solar radiation.