Research progress in mRNA drug modification and delivery systems / 浙江大学学报·医学版

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.

Establishment of a CFTR-based detection method for the second messenger cAMP in the cytoplasm / 中国应用生理学杂志

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.

Effect of dexamethasone on the expression of Dynein heavy chain and Dynactin in the cytoplasm of fetal rat cerebral cortical neurons cultured / 中国当代儿科杂志

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

O receptor aril de hidrocarbonetos (AhR) em melanomas: associação com mutações BRAF, com a progressão da doença e identificação de ligantes / The aryl hydrocarbon receptor (AhR) in melanomas: association with BRAF mutations, with disease progression and ligand identification

O melanoma é um tipo de câncer de pele geneticamente diverso, que surge diante das transformações em melanócitos. A mutação BRAFV600E está presente em mais de 90% de todas as mutações em BRAF, sendo assim ocorre em cerca de 50% dos casos registrados. As mutações em NRAS, ocupam o segundo lugar entre as mutações mais prevalentes, cerca de 20% dos casos. Informações sobre as assinaturas genéticas, permitiram o desenvolvimento de terapia alvo dirigida. O Vemurafenib, inibidor da quinase BRAFV600E, apresentou inicialmente resultados bastante satisfatórios, contudo existe registro de casos de recidiva e resistência. O receptor aril de hidrocarbonetos é expresso em vários componentes da pele, e assim está relacionado a homeostase e fisiopatologia da pele. Diante disso, a avaliação da expressão do receptor em um painel de linhagens mutadas para NRAS e BRAF, e BRAF resistentes, mostrou-se maior do que a encontrada em melanócitos. Também encontramos maior expressão de mRNA de AhR em linhagens de melanoma derivadas de sítio primário e metastático, mutadas para BRAFV600E, quando comparadas ao melanócito. Agregado a isto, a análise in silico no TCGA (The Cancer Genome Atlas) mostrou que há 18% de alteração genética em AhR, sendo em maior parte a alta regulação de mRNA. Também, a análise do banco público GSE12391, mostrou aumento de mRNA de AhR na fase de crescimento vertical do melanoma. Assim, concluímos que há maior expressão de mRNA e sua importância nas fases de desenvolvimento do melanoma, tanto nos processos iniciais quanto em processos de migração, invasão e metástase. Ainda, encontramos maior mRNA do receptor em linhagens resistentes ao Vemurafenib. Este resultado sustenta a hipótese de que AhR pode ser considerado um marcador de resistência em melanomas. O AhR, inicialmente no citoplasma, quando ativado pode atuar como fator de transcrição regulando vários genes que apresentam sequências definidas, participando de respostas carcinogênicas. Compostos halogenados e moléculas endógenas derivadas das vias de metabolização do triptofano são agonistas do receptor. Anteriormente, nosso grupo mostrou que linhagens de melanoma incubadas com triptamina e DMT exibiram menor clonogenicidade. Diante de uma literatura escassa sobre o papel do DMT no melanoma e com base nestes resultados, nosso objetivo foi avaliar o papel de AhR nesta interface DMT-melanoma. Para isto, nosso objetivo foi construir linhagem editada geneticamente para AhR através da ferramenta CRISPR-Cas9. Vários foram os esforços, sem sucesso, utilizados nas tentativas de comprovar a manutenção de células editadas na cultura. Atrelamos a este resultado a possibilidade de haver duas subpopulações editadas geneticamente pós CRISPR-Cas9, onde uma destas manteve o padrão de crescimento semelhante às células wild type. Devido a este crescimento diferencial, não obtivemos congruências nos ensaios e postulamos a perda do possível nocaute. A partir disso, realizamos ensaios de interactoma para avaliar a interação de DMT-AhR. Nosso resultado sugere a interação de DMT ao receptor sigma 1, e não ao receptor aril de hidrocarbonetos. Desta forma, o interactoma sustenta a hipótese de que DMT não é um ligante de AhR. Para certificar este resultado análises de docking associados a ensaios biológicos, avaliando o papel do receptor, devem ser realizados para averiguar a afinidade e seletividade de DMT como ligante do receptor na linhagem de melanoma

Melanoma is a genetically diverse type of skin cancer, which arises from changes in melanocytes. The BRAFV600E mutation is present in more than 90% of all BRAF mutations, so it occurs in about 50% of registered cases. Mutations in NRAS occupy the second place among the most prevalent mutations, about 20% of cases. Information on genetic signatures allowed the development of targeted therapy. vemurafenib, kinase inhibitor BRAFV600E, initially presented very satisfactory results, however there is a record of cases of relapse and resistance. The aryl hydrocarbon receptor is expressed in several components of the skin and is thus related to homeostasis and skin pathophysiology. Therefore, the evaluation of receptor expression in a panel of strains mutated to NRAS and BRAF, and resistant BRAF, proved to be greater than that found in melanocytes. We also found main expression of AhR mRNA in melanoma strains derived from primary and metastatic site, mutated to BRAFV600E, when compared to melanocyte. Added to this, the in silico analysis in TCGA (The Cancer Genome Atlas) showed that there is 18% of genetic alteration in AhR, being mostly the high regulation of mRNA. Also, an analysis by the public bank GSE12391, showed an increase in AhR mRNA in the vertical growth phase of melanoma. Thus, it is concluded that there is greater expression of mRNA and its importance in the stages of development of melanoma, both in recent processes and in the processes of migration, invasion and metastasis. In addition, we found higher receptor mRNA in strains resistant to vemurafenib. This result supports the hypothesis that AhR can be considered a marker of resistance in melanomas. AhR, initially in the cytoplasm, when activated can act as a transcription factor regulating several genes that have defined sequences, participating in carcinogenic responses. Along with this, we show that along the tumor progression, there is an increase in AhR in the radial growth phase of melanoma. Halogenated compounds and endogenous molecules derived from the tryptophan metabolism pathways are receptor agonists. Previously, our group showed that melanoma strains incubated with tryptamine and DMT exhibited less clonogenicity. In view of a scarce literature on the role of DMT in melanoma and based on these results, our objective was to evaluate the role of AhR in this DMT-melanoma interface. For this, our goal was to build genetically edited strain for AhR using the CRISPR-Cas9 tool. Several efforts were unsuccessful in attempts to prove the maintenance of cells edited in the culture. We linked to this result the possibility of having two subpopulations genetically edited after CRISPR-Cas9, where one of them maintained the growth pattern like wild type cells. Due to this differential growth, we did not obtain congruence in the tests and postulated the loss of the possible knockout. From that, we performed interactome assays to evaluate the DMT-AhR interaction. Our result suggests the interaction of DMT with the sigma 1 receptor, and not the aryl hydrocarbon receptor. Thus, the interactome supports the hypothesis that DMT is not an AhR ligand. To certify this result, docking analyses associated with biological assays, evaluating the role of the receptor, should be performed to ascertain the affinity and selectivity of DMT as a ligand of the receptor in the melanoma lineage

The 40-91 aa sequence of porcine epidemic diarrhea virus ORF3 protein is the key structural domain controlling its location in cytoplasm / 生物工程学报

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.

Research progress of phase separation of intracellular biological macromolecules / 生物工程学报

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.

Characteristics of Renal Cell Carcinoma Harboring TPM3-ALK Fusion

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.

Inhibitory Effect of Rosae Multiflorae Fructus Extracts on the Receptor Activator of NF-κB Ligand-Induced Osteoclastogenesis through Modulation of P38- and Ca2⁺-Mediated Nuclear Factor of Activated T-Cells Cytoplasmic 1 Expression

BACKGROUND: Rosae Multiflorae fructus (RMF), known to have anti-inflammatory and antioxidant properties, has been used as a traditional remedy for inflammatory diseases such as arthritis in Eastern Asia. However, its effect on osteoclasts, which play a crucial role in resorptive inflammatory bone diseases, is yet to be elucidated.METHODS: The effect of extract of RMF (RMF-E) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining, real-time polymerase chain reaction and western blot analysis. In addition, RANKL-induced Ca2⁺-oscillation was also investigated.RESULTS: RMF-E remarkably inhibited TRAP+-osteoclast and resorptive pit formation in a dose-dependent manner. In addition, the expression of c-Fos and nuclear factor of activated T-cells cytoplasmic, known as pivotal transcription factors for osteoclast formation in vitro and in vivo, and that of the osteoclast differentiation markers such as Acp5, Oscar, CtsK, Atp6v0d2, Tm7sf4, and Nfatc1 were significantly decreased by RMF-E treatment during osteoclastogenesis. The inhibitory effect of RMF-E on RANKL-induced osteoclastogenesis was caused by the suppression of p38 mitogen-activated protein kinase activation, and RANKL-induced Ca2⁺-oscillation removal via inactivation of Bruton's tyrosine kinase (BTK), and subsequently phospholipase C-γ2.CONCLUSIONS: RMF-E negatively regulates osteoclast differentiation and formation. These findings suggest the possibility of RMF-E as a traditional therapeutic agent against osteoclast-related bone disorders such as osteoporosis, rheumatoid arthritis, and periodontitis.

Subcellular localization of GTPase of immunity-associated protein 2 / 北京大学学报(医学版)

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.

Role of cytoplasmic lncRNAs in regulating cancer signaling pathways / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Cancer remains a serious healthcare problem despite significant improvements in early detection and treatment approaches in the past few decades. Novel biomarkers for diagnosis and therapeutic strategies are urgently needed. In recent years, long noncoding RNAs (lncRNAs) have been reported to be aberrantly expressed in tumors and show crosstalk with key cancer-related signaling pathways. In this review, we summarized the current progress of research on cytoplasmic lncRNAs and their roles in regulating cancer signaling and tumor progression, further characterization of which may lead to effective approaches for cancer prevention and therapy.

Langerhans Cell Sarcoma of the Scalp with Concomitant Multiple Cervical Lymph Node and Adjacent Soft Tissue Involvement: A Case Report / 대한피부과학회지

Currently, the World Health Organization classifies Langerhans cell tumors into Langerhans cell histiocytosis and Langerhans cell sarcoma (LCS). LCS is a neoplastic proliferation of Langerhans cells showing malignant cytological features and aggressive clinical behavior with grave prognosis. Only a few cases have been reported in the available literature; therefore, to date, no definitive treatment has been established. A 64-year-old woman presented with a 1-year history of an asymptomatic, slow-growing erythematous nodule measuring 0.7 cm on her scalp. The patient also reported a 3-month history of a painful swelling on the right side of her neck. Histopathological examination of a scalp biopsy specimen revealed sheets of atypical cells with hyperchromatic nucleoli and clear cytoplasm. Immunohistochemical studies revealed malignant cells positive for CD1a, CD31, CD68, and S-100 expression. Additionally, positron emission tomography–computed tomography and fine-needle aspiration revealed LCS of the cervical lymph nodes and surrounding soft tissue. We recommended surgical excision and adjunctive chemotherapy; however, the patient refused treatment and died of the disease 28 months later.

Tetrabromobisphenol A Promotes the Osteoclastogenesis of RAW264.7 Cells Induced by Receptor Activator of NF-kappa B Ligand In Vitro

BACKGROUND: Tetrabromobisphenol A (TBBPA), one of the most widely used brominated flame-retardants, is a representative persistent organic pollutants group. Studies on TBBPA toxicity have been conducted using various target cells; however, few studies have investigated TBBPA toxicity in bone cells. Therefore, this study investigated the in vitro effects of TBBPA on osteoclasts, a cell type involved in bone metabolism. METHODS: RAW264.7 cells were cultured in medium containing 50 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL) and varying concentrations of TBBPA. To evaluate the effects of TBBPA on the differentiation and function of osteoclasts, osteoclast-specific gene expression, tartrate-resistant acid phosphatase (TRAP) activity, bone resorbing activity, mitochondrial membrane potential (MMP) and mitochondrial superoxide were measured. RESULTS: The presence of 20 μM TBBPA significantly increased TRAP activity in RANKL-stimulated RAW264.7 cells, the bone resorbing activity of osteoclasts, and the gene expression of Akt2, nuclear factor of activated T-cells cytoplasmic 1, and chloride channel voltage-sensitive 7. However, TBBPA treatment caused no change in the expression of carbonic anhydrase II, cathepsin K, osteopetrosis-associated transmembrane protein 1, Src, extracellular signal-related kinase, GAB2, c-Fos, or matrix metalloproteinase 9. Furthermore, 20 μM TBBPA caused a significant decrease in MMP and a significant increase in mitochondrial superoxide production. CONCLUSION: This study suggests that TBBPA promotes osteoclast differentiation and activity. The mechanism of TBBPA-stimulated osteoclastogenesis might include increased expression of several genes involved in osteoclast differentiation and reactive oxygen species production.

Primary Peripheral Gamma Delta T-Cell Lymphoma of the Central Nervous System: Report of a Case Involving the Intramedullary Spinal Cord and Presenting with Myelopathy

Primary central nervous system lymphoma of T-cell origin (T-PCNSL) is rare, and its clinicopathological features remain unclear. Peripheral T-cell lymphoma of γδ T-cell origin is an aggressive lymphoma mainly involving extranodal sites. Here, we report a case of γδ T-PCNSL involving the intramedullary spinal cord and presenting with paraplegia. A 75-year-old Korean woman visited the hospital complaining of back pain and lower extremity weakness. Magnetic resonance imaging revealed multifocal enhancing intramedullary nodular lesions in the thoracic and lumbar spinal cord. An enhancing nodular lesion was observed in the periventricular white matter of the lateral ventricle in the brain. There were no other abnormalities in systemic organs or skin. Laminectomy and tumor removal were performed. The tumor consisted of monomorphic, medium-to-large atypical lymphocytes with pale-to-eosinophilic cytoplasm. Immunohistochemically, the tumor cells were CD3(+), TCRβF1(-), TCRγ(+), CD30(-), CD4(-), CD8(-), CD56(+), TIA1(+), granzyme B(+), and CD103(+). Epstein-Barr virus in situ was negative. This case represents a unique T-PCNSL of γδ T-cell origin involving the spinal cord.

Cytopathologic Features of Secretory Carcinoma of Salivary Gland: Report of Two Cases

Secretory carcinoma of the salivary gland (SC) is a newly introduced rare salivary gland tumor that shares histological, immunohistochemical, and genetic characteristics with secretory carcinoma of the breast. Here, we report the cytologic features of two cases of SC confirmed by surgical resection. In these two cases, SC was incidentally detected in a 64-year-old female and a 56-yearold male. Fine needle aspiration cytology revealed nests of tumor cells with a papillary or glandular structure floating in mucinous secretions. The tumor cells demonstrated uniform, round, smooth nuclear contours and distinct nucleoli. Multiple characteristic cytoplasmic vacuoles were revealed. Singly scattered tumor cells frequently showed variable sized cytoplasmic vacuoles. The cytopathologic diagnosis of SC should be considered when characteristic cytological findings are revealed. Further immunohistochemistry and gene analyses are helpful to diagnose SC.

Tweety-homolog (Ttyh) Family Encodes the Pore-forming Subunits of the Swelling-dependent Volume-regulated Anion Channel (VRAC(swell)) in the Brain

In the brain, a reduction in extracellular osmolality causes water-influx and swelling, which subsequently triggers Cl⁻- and osmolytes-efflux via volume-regulated anion channel (VRAC). Although LRRC8 family has been recently proposed as the pore-forming VRAC which is activated by low cytoplasmic ionic strength but not by swelling, the molecular identity of the pore-forming swelling-dependent VRAC (VRAC(swell)) remains unclear. Here we identify and characterize Tweety-homologs (TTYH1, TTYH2, TTYH3) as the major VRAC(swell) in astrocytes. Gene-silencing of all Ttyh1/2/3 eliminated hypo-osmotic-solution-induced Cl⁻ conductance (I(Cl,swell)) in cultured and hippocampal astrocytes. When heterologously expressed in HEK293T or CHO-K1 cells, each TTYH isoform showed a significant I(Cl,swell) with similar aquaporin-4 dependency, pharmacological properties and glutamate permeability as I(Cl,swell) observed in native astrocytes. Mutagenesis-based structure-activity analysis revealed that positively charged arginine residue at 165 in TTYH1 and 164 in TTYH2 is critical for the formation of the channel-pore. Our results demonstrate that TTYH family confers the bona fide VRAC(swell) in the brain.

Physical and Functional Interaction between 5-HT₆ Receptor and Nova-1

5-HT₆ receptor (5-HT₆R) is implicated in cognitive dysfunction, mood disorder, psychosis, and eating disorders. However, despite its significant role in regulating the brain functions, regulation of 5-HT₆R at the molecular level is poorly understood. Here, using yeast two-hybrid assay, we found that human 5-HT₆R directly binds to neuro-oncological ventral antigen 1 (Nova-1), a brain-enriched splicing regulator. The interaction between 5-HT₆R and Nova-1 was confirmed using GST pull-down and co-immunoprecipitation assays in cell lines and rat brain. The splicing activity of Nova-1 was decreased upon overexpression of 5-HT₆R, which was examined by detecting the spliced intermediates of gonadotropin-releasing hormone (GnRH), a known pre-mRNA target of Nova-1, using RT-PCR. In addition, overexpression of 5-HT₆R induced the translocation of Nova-1 from the nucleus to cytoplasm, resulting in the reduced splicing activity of Nova-1. In contrast, overexpression of Nova-1 reduced the activity and the total protein levels of 5-HT₆R. Taken together, these results indicate that when the expression levels of 5-HT₆R or Nova-1 protein are not properly regulated, it may also deteriorate the function of the other.

An Autopsy Proven Case of CSF1R-mutant Adult-onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP) with Premature Ovarian Failure

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a progressive degenerative white matter disorder caused by mutations in the tyrosine kinase domain of the CSF1R gene. ALSP is often misdiagnosed as other diseases due to its rarity and various clinical presentations such as Parkinsonism, pyramidal signs, cognitive impairment and/or psychiatric symptoms. We describe an autopsy case of ALSP with a CSF1R mutation. A 61-year-old woman presented insidious-onset gait difficulty for 12 years since her age of 49, and premature ovarian failure since her age of 35. At initial hospital visit, brain magnetic resonance imaging revealed hydrocephalus. Initially, Parkinson's syndrome was diagnosed, and she was prescribed L-dopa/carbidopa because of spasticity and rigidity of extremities, which had worsened. Subsequently, severe neuropsychiatric symptoms and cognitive impairment developed and radiologically, features of leukoencephalopathy or leukodystrophy were detected. She showed a down-hill course and died, 12 years after initial diagnosis. At autopsy, the brain showed severe symmetric atrophy of bilateral white matter, paper-thin corpus callosum, thin internal capsule, and marked hydrocephalus. Microscopically, diffuse loss of white matter, relatively preserved subcortical U-fibers, and many eosinophilic bulbous neuroaxonal spheroids were noted, but there was no calcification. Pigmented glia with brown cytoplasmic pigmentation were readily found in the white matter, which were positive for Periodic acid-Schiff, p62, and CD163 stains, but almost negative for CD68. Whole-exome and Sanger sequencing revealed a CSF1R mutation (c.2539G>A, p.Glu847Lys) which was reported in prior one ALSP case. This example demonstrates that ALSP could be associated with premature ovarian failure.

Comparison of Radiological and Histological Findings of Lung Parenchyma in Patients with Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

PURPOSE: The present study investigated chest computed tomography (CT) patterns and lung histological features, as well as the consistency between radiological and histological features among patients with microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and eosinophilic GPA (EGPA). MATERIALS AND METHODS: The medical records of 74 antineutrophil cytoplasmic antibody-associated vasculitis (AAV) patients with radiological lung parenchymal lesions were reviewed along with the histological results for 28 of them. Chest CT patterns were divided according 12 items mostly suggested by radiologists and histological features were divided according to necrotising granuloma, necrotising vasculitis, eosinophilic infiltration, and hemosiderin laden macrophages as defined by a pathologist. RESULTS: The mean age was 57.1 years (22 men). The most common clinical manifestation other than lung manifestation was renal manifestation (62.2%), and the most common chest CT pattern was lung involvement of vasculitis (35.1%). In MPA patients, the major histological features were hemosiderin-laden macrophages in the alveolar space and vasculitis. In GPA patients, the major histological features were necrotizing vasculitis and necrotizing granuloma, while in EGPA patients, the major histological feature was only necrotising vasculitis. The consistency rate in GPA patients was the highest (100%), followed by that in MPA patients (66.7%) and EGPA patients (50.0%). CONCLUSION: When lung involvement of AAV is suspected on chest CT, lung biopsy should be recommended for the proper classification of AAV, due to the discordance rate between radiological and histological findings in MPA and EGPA patients, but not GPA patients.

Different expression of GSK3β and pS9GSK3β depending on phenotype of cervical cancer: possible association of GSK3β with squamous cell carcinoma and pS9GSK3β with adenocarcinoma

OBJECTIVE: This study aimed to analyze the expression pattern of glycogen synthase kinase 3β (GSK3β) and its phosphorylated forms, GSK3β phosphorylated at Ser9 (pS9GSK3β), and GSK3β phosphorylated at Tyr216 (pY216GSK3β), in cervical squamous cell carcinoma (SCC) and adenocarcinoma (AC). METHODS: We performed immunohistochemical staining for GSK3β, pS9GSK3β, and pY216GSK3β in 64 SCC and 20 AC cases and compared their expression patterns between the 2 tumor types. RESULTS: Increased GSK3β and pS9GSK3β expression but decreased pY216GSK3β expression compared with that in the normal cervix were observed in both SCC and AC specimens. Specifically, the levels of GSK3β and pS9GSK3β were significantly increased in SCC and AC, respectively. GSK3β was localized in the nucleus and/or cytoplasm of SCC and AC cells. However, pS9GSK3β was predominantly localized in the membrane of AC cells, whereas it was present in the nucleus and/or cytoplasm of SCC cells. CONCLUSION: The results suggest that the phosphorylation status of GSK3β changes during cervical cancer development and the different expression levels and patterns of GSK3β and pS9GSK3β are associated with the specific histologic phenotype of cervical cancer.

Primary Histiocytic Sarcoma Presenting as a Breast Mass: A Case Report / 한국유방암학회지

Histiocytic sarcoma is a rare hematologic malignancy, with very few cases of primary histiocytic sarcoma of the breast described in English scientific literature. Herein, we describe a case of primary histiocytic sarcoma of the breast in a 75-year-old woman, with no clinical history of malignant tumors, who presented with a palpable solitary breast mass. Microscopically, the resected breast mass showed large pleomorphic cells, some multinucleated giant cells, and admixed inflammatory components. The pleomorphic tumor cells further showed a diffuse, noncohesive growth pattern, an abundant eosinophilic cytoplasm, and strong and diffuse immunoreactivity for cluster of differentiation (CD) 68 and CD163. Furthermore, a whole-body positron-emission tomography/computed tomography using deoxy-2-[¹⁸F]fluoro-D-glucose performed after surgery showed no other masses or lesions. After surgical excision, the patient was followed up, and no evidence of tumor recurrence or metastasis was noted.