Messenger RNA (mRNA) has shown tremendous potential in disease prevention and therapy. The clinical application requires mRNA with enhanced stability and high translation efficiency, ensuring it not to be degraded by nucleases and targeting to specific tissues and cells. mRNA immunogenicity can be reduced by nucleotide modification, and translation efficiency can be enhanced by codon optimization. The 5´ capping structure and 3´ poly A increase mRNA stability, and the addition of 5' and 3' non-translational regions regulate mRNA translation initiation and protein production. Nanoparticle delivery system protects mRNA from degradation by ubiquitous nucleases, enhances mRNA concentration in circulation and assists it cytoplasmic entrance for the purpose of treatment and prevention. Here, we review the recent advances of mRNA technology, discuss the methods and principles to enhance mRNA stability and translation efficiency; summarize the requirements involved in designing mRNA delivery systems with the potential for industrial translation and biomedical application. Furthermore, we provide insights into future directions of mRNA therapeutics to meet the needs for personalized precision medicine.
RNA, Messenger/genetics* ; Cytoplasm ; Nanoparticles ; Precision Medicine

Objective: To establish a detection method based on Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that can sensitively detect the second messenger cyclic AMP (cAMP) in the cytoplasm. Methods: The eukaryotic expression vectors of CFTR and YFP-H148Q / I152L were constructed respectively. FRT cells co-expressing CFTR and YFP-H148Q / I152L were obtained by liposome transfection. The expression of CFTR and YFP-H148Q / I152L in FRT cells was observed by an inverted fluorescence microscopy, and flow cytometry was used to detect the purity of cells; The cell model was identified by the fluorescence quenching kinetics test. The validation of the cell model which could screen CFTR modulators was verified by the fluorescence quenching kinetics experiments. The radioimmunoassay was used to detect the cAMP concentration in cytoplasm after adding CFTR activator. Results: The results of the inverted fluorescence microscope showed that CFTR was expressed in the cell membrane and YFP-H148Q / I152L was expressed in the cytoplasm of FRT cells. The FRT cell model stably co-expressing ANO1 and YFP-H148Q / I152L was successfully constructed. The model could screen CFTR modulators, and the slope of fluorescence change and the concentration of CFTR modulators were in a dose-dependent manner. The slope of the fluorescence could reflect the cAMP concentration in the cytoplasm. The cell model could sensitively detect the intracellular cAMP concentration. Conclusion: The cell model could efficiently and sensitively detect the second messenger cAMP concentration in the cytoplasm, and it provided a simple and efficient method for the study of other targets associated cAMP signal.
Cyclic AMP ; Cystic Fibrosis Transmembrane Conductance Regulator ; Cytoplasm ; Second Messenger Systems

OBJECTIVE: To study the effect of dexamethasone (DEX) on the expression of Dynein heavy chain (DHC) and Dynactin in the cytoplasm of fetal rat cerebral cortical neurons cultured METHODS: Primary cerebral cortical neurons of fetal rats were cultured RESULTS: There was no significant difference in the mRNA expression levels of DHC and Dynactin among the three groups at all time points ( CONCLUSIONS DEX affects the protein expression of DHC and Dynactin in the fetal rat cerebral cortical neurons cultured
Animals ; Cytoplasm ; Dexamethasone/pharmacology* ; Dynactin Complex/genetics* ; Dyneins ; Neurons ; Rats

The World Health Organization 2016 edition assigned anaplastic lymphoma kinase (ALK) rearrangement-associated renal cell carcinoma (ALK-RCC) as an emerging renal tumor entity. Identifying ALK-RCC is important because ALK inhibitors have been shown to be effective in treatment. Here, we report the case of a 14-year-old young man with ALK-RCC. Computed tomography revealed a well-demarcated 5.3-cm enhancing mass at the upper pole of the left kidney. There was no further history or symptoms of the sickle-cell trait. The patient underwent left radical nephrectomy. Pathologically, the mass was diagnosed as an unclassified RCC. Targeted next-generation sequencing identified a TPM3-ALK fusion gene. The present report and literature review demonstrate that TPM3-ALK RCC may be associated with distinct clinicopathological features. Microscopically, the tumors showed diffuse growth and tubulocystic changes with inflammatory cell infiltration. Tumor cells were dis-cohesive and epithelioid with abundant eosinophilic cytoplasm and cytoplasmic vacuoles. If morphological features and TFE3 expression are present in adolescent and young patients, molecular tests for ALK translocation should be performed. This awareness is critically important, because ALK rearrangement confers sensitivity to ALK inhibitors.
Adolescent ; Carcinoma, Renal Cell ; Cytoplasm ; Eosinophils ; Gene Rearrangement ; Humans ; Kidney ; Lymphoma ; Nephrectomy ; Phosphotransferases ; Vacuoles ; World Health Organization

The phenomenon of phase separation of intracellular biological macromolecules is an emerging research field that has received great attention in recent years. As an aggregation and compartment mechanism of cell biochemical reactions, it widely exists in nature and participates in important physiological processes such as gene transcription and regulation, as well as influences organism's response to external stimuli. Disequilibrium of phase separation may lead to the occurrence of some major diseases. Researchers in cross-cutting fields are trying to examine dementia and other related diseases from a new perspective of phase separation, exploring its molecular mechanism and the potential possibility of intervention and treatment. This review intends to introduce the latest research progress in this field, summarize the major research directions, biochemical basis, its relationship with disease occurrence, and giving a future perspective of key problems to focus on.
Animals ; Chemistry Techniques, Analytical ; trends ; Cytoplasm ; chemistry ; metabolism ; Humans ; Macromolecular Substances ; isolation & purification ; Research ; trends

ORF3 protein, the single accessory protein encoded by porcine epidemic diarrhea virus (PEDV), is related to viral pathogenicity. In order to determine the cytoplasmic location signal of PEDV ORF3, we constructed a series of recombinant plasmids carrying full-length or truncated segments of PEDV DR13 ORF3 protein. When the acquired plasmids were transfected into Vero cells, expression and distribution of the EGFP-fused full-length ORF3 protein and its truncated forms in the cells were observed by laser confocal microscopy. The results showed that ORF3 protein or their truncated forms containing 40-91 aa segment including two transmembrane domains were localized in the cytoplasm, whereas ORF3 truncated peptides without the 40-91 aa segment were distributed in the whole cell (in both cytoplasm and nucleus). This suggests that the 40-91 aa is the key structural domain determining cytoplasmic location of PEDV ORF3 protein. The discovery provides reference for further clarifying intracellular transport and biological function of PEDV ORF3 protein.
Amino Acid Sequence ; Animals ; Chlorocebus aethiops ; Coronavirus Infections ; virology ; Cytoplasm ; virology ; Porcine epidemic diarrhea virus ; genetics ; Protein Domains ; Swine ; Vero Cells ; Viral Proteins ; chemistry ; metabolism

OBJECTIVE: To analyze the subcellular localization of GTPase of immunity-associated protein 2 (GIMAP2) for the further functional study. METHODS: In the study, we first obtained the protein sequences of GTPase of immunity-associated protein 2 (GIMAP2) from National Center for Biotechnology Information (NCBI) database, and then performed a prediction analysis of its transmembrane structure, nuclear localization signal (NLS), nuclear export signal (NES) and subcellular localization through bioinformatics online tools. GIMAP2 gene amplified by PCR was inserted into the expression vector pQCXIP-mCherry-N1 and positive clones were selected by ampicillin resistance. After using methods to extract and purify, the sequenced recombinant plasmid pQCXIP-GIMAP2-mCherry, together with the retroviral packaging plasmids VSVG and Gag/pol, was transferred into HEK293FT cells by liposomes for virus packaging. The virus supernatant was collected 48 h after transfection and directly infected the human breast cancer cell line MDA-MB-436. Immunofluorescence staining was constructed to detect the localization of endogenous and exogenous GIMAP2 in MDA-MB-436 cells. Meanwhile, green fluorescent chemical dyes were used to label mitochondria, endoplasmic reticulum, and lipid droplets in living MDA-MB-436 cells stably expressing the GIMAP2-mCherry fusion protein. Images for the three dye-labeled organelles and GIMAP2-mCherry fusion protein were captured by super-resolution microscope N-SIM. RESULTS: Bioinformatics analysis data showed that GIMAP2 protein composed of 337 amino acids might contain two transmembrane helix (TM) structures at the carboxyl terminus, of which TMs were estimated to contain 40-41 expected amino acids, followed by the residual protein structures toward the cytoplasmic side. NES was located at the 279-281 amino acids of the carboxyl terminus whereas NLS was not found. GIMAP2 might locate in the lumen of the endoplasmic reticulum. Sequencing results indicated that the expression vector pQCXIP-GIMAP2-mCherry was successfully constructed. Fluorescent staining confirmed that GIMAP2-mCherry fusion protein, co-localized well with endogenous GIMAP2, expressed successfully in the endoplasmic reticulum and on the surface of lipid droplets in MDA-MB-436 cells. CONCLUSION GIMAP2 localizes in the endoplasmic reticulum and on the surface of LDs, suggesting potential involvement of GIMAP2 in lipid metabolism.
Amino Acid Sequence ; Cytoplasm ; GTP Phosphohydrolases ; Humans ; Membrane Proteins ; Nuclear Export Signals ; Nuclear Localization Signals ; Recombinant Fusion Proteins ; Transfection

A renal oncocytoma was diagnosed in an 8-year-old female Maltese dog with a history of renal cysts. Tumor cells were not detected until six months after observation of renal cysts. Nephrectomy was performed to treat the neoplasia. Tumor-like masses with numerous nodules were observed in the inner surface of cysts present in the caudal part of the left kidney. Histologically, the tumor consisted of cells with abundant eosinophilic cytoplasm. The diagnosis was based on histological features, periodic acid-Schiff reaction, and immunohistochemical cytokeratin staining. Based on a literature review, this is the first canine renal oncocytoma case reported in Korea.
Adenoma, Oxyphilic ; Animals ; Child ; Cytoplasm ; Diagnosis ; Dogs ; Eosinophils ; Female ; Humans ; Keratins ; Kidney ; Korea ; Nephrectomy ; Periodic Acid-Schiff Reaction

Surface immunoglobulin light-chain restriction is evidence of clonality in mature B-cell neoplasms. An aberrant pattern of surface light-chain expression can also be considered evidence of clonality. However, because this result could occur due to nonspecific staining or failure to stain, careful interpretation is required for accurate diagnosis. According to a previous study, flow cytometric analysis of the cytoplasmic pattern of light-chain expression in mature B-cell neoplasms is a viable approach to confirming clonality. Herein, we report a case, in which clonality could not be proven by surface light-chain analysis, but was demonstrated by cytoplasmic light-chain analysis. The case was in a patient with B-cell lymphoma showing non-specific surface expression of light-chains. This case support consideration of flow cytometric analysis of cytoplasmic light-chain expression patterns when aberrant surface light chain expression is observed, to confirm clonality of mature B-cell neoplasms.
B-Lymphocytes ; Cytoplasm ; Diagnosis ; Flow Cytometry ; Humans ; Immunoglobulin Light Chains ; Lymphoma, B-Cell

Endometrial glassy cell carcinoma (EGCC) is a rare neoplasm, accounting for 0.5% of the carcinomas in the endometrium, composed of cells with granular eosinophilic or amphophilic cytoplasm, giving it a ground glass appearance. Till date, only 14 cases of this carcinoma have been reported. In this report, we have described a case of EGCC to help define standard diagnostic criteria and better understand the course, ideal treatment, and accurate prognosis of this disease. We report a case of a 64-year-old woman diagnosed with EGCC after an abnormal pap smear. She underwent a hysteroscopy, which led to the histological diagnosis. Laparotomic total hysterectomy and bilateral salpingo-oophorectomy were performed with pelvic lymphadenectomy and peritoneal and omental biopsies. Final pathological examination confirmed the initial diagnosis. Pelvic nodes removed during surgery and peritoneal and omental biopsies were negative for tumor cells. Treatment was considered appropriate and the patient did not require additional therapies. She was subsequently assigned to clinical follow-up and is alive, with no evidence of the disease.
Biopsy ; Cytoplasm ; Diagnosis ; Endometrial Neoplasms ; Endometrium ; Eosinophils ; Female ; Follow-Up Studies ; Glass ; Humans ; Hysterectomy ; Hysteroscopy ; Lymph Node Excision ; Middle Aged ; Papanicolaou Test ; Prognosis ; Uterine Neoplasms