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mRNA vaccines against SARS-CoV-2 have revolutionized vaccine development, but their immunological mechanisms are not fully understood. Here, we investigate injection site responses of mRNA vaccines by generating a comprehensive single-cell transcriptome profile upon lipid nanoparticle (LNP) or LNP-mRNA challenge in female BALB/c mice. We show that LNP-induced stromal pro-inflammatory responses and mRNA-elicited type I interferon responses dominate the initial injection site responses. By tracking the fate of delivered mRNA, we discover that injection site fibroblasts are highly enriched with the delivered mRNA and that they express IFN-ß specifically in response to the mRNA component, not to the LNP component of mRNA vaccines. Moreover, the mRNA-LNP, but not LNP alone, induces migratory dendritic cells highly expressing IFN-stimulated genes (mDC_ISGs) at the injection site and draining lymph nodes. When co-injected with LNP-subunit vaccine, IFN-ß induces mDC_ISGs at the injection site, and importantly, it substantially enhances antigen-specific cellular immune responses. Furthermore, blocking IFN-ß signaling at the injection site significantly decreases mRNA vaccine-induced cellular immune responses. Collectively, these data highlight the importance of injection site fibroblasts and IFN-ß signaling during early immune responses against the mRNA vaccine and provide detailed information on the initial chain of immune reactions elicited by mRNA vaccine injection.
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Células Dendríticas , Fibroblastos , Inmunidad Celular , Inmunidad Innata , Interferón beta , Ratones Endogámicos BALB C , Nanopartículas , Vacunas de ARNm , Animales , Interferón beta/inmunología , Interferón beta/metabolismo , Femenino , Inmunidad Innata/inmunología , Inmunidad Innata/efectos de los fármacos , Ratones , Vacunas de ARNm/inmunología , Células Dendríticas/inmunología , Nanopartículas/química , Fibroblastos/inmunología , Fibroblastos/metabolismo , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , SARS-CoV-2/inmunología , Lípidos/química , COVID-19/prevención & control , COVID-19/inmunología , ARN Mensajero/metabolismo , ARN Mensajero/genética , Análisis de la Célula Individual , LiposomasRESUMEN
For several decades, a plant-based expression system has been proposed as an alternative platform for the production of biopharmaceuticals including therapeutic monoclonal antibodies (mAbs), but the immunogenicity concerns associated with plant-specific N-glycans attached in plant-based biopharmaceuticals has not been completely solved. To eliminate all plant-specific N-glycan structure, eight genes involved in plant-specific N-glycosylation were mutated in rice (Oryza sativa) using the CRISPR/Cas9 system. The glycoengineered cell lines, PhytoRice®, contained a predominant GnGn (G0) glycoform. The gene for codon-optimized trastuzumab (TMab) was then introduced into PhytoRice® through Agrobacterium co-cultivation. Selected cell lines were suspension cultured, and TMab secreted from cells was purified from the cultured media. The amino acid sequence of the TMab produced by PhytoRice® (P-TMab) was identical to that of TMab. The inhibitory effect of P-TMab on the proliferation of the BT-474 cancer cell line was significantly enhanced at concentrations above 1 µg/mL (****P < 0.0001). P-TMab bound to a FcγRIIIa variant, FcγRIIIa-F158, more than 2.7 times more effectively than TMab. The ADCC efficacy of P-TMab against Jurkat cells was 2.6 times higher than that of TMab in an in vitro ADCC assay. Furthermore, P-TMab demonstrated efficient tumour uptake with less liver uptake compared to TMab in a xenograft assay using the BT-474 mouse model. These results suggest that the glycoengineered PhytoRice® could be an alternative platform for mAb production compared to current CHO cells, and P-TMab has a novel and enhanced efficacy compared to TMab.
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The complexity of the tumor microenvironment poses significant challenges in cancer therapy. Here, to comprehensively investigate the tumor-normal ecosystems, we perform an integrative analysis of 4.9 million single-cell transcriptomes from 1070 tumor and 493 normal samples in combination with pan-cancer 137 spatial transcriptomics, 8887 TCGA, and 1261 checkpoint inhibitor-treated bulk tumors. We define a myriad of cell states constituting the tumor-normal ecosystems and also identify hallmark gene signatures across different cell types and organs. Our atlas characterizes distinctions between inflammatory fibroblasts marked by AKR1C1 or WNT5A in terms of cellular interactions and spatial co-localization patterns. Co-occurrence analysis reveals interferon-enriched community states including tertiary lymphoid structure (TLS) components, which exhibit differential rewiring between tumor, adjacent normal, and healthy normal tissues. The favorable response of interferon-enriched community states to immunotherapy is validated using immunotherapy-treated cancers (n = 1261) including our lung cancer cohort (n = 497). Deconvolution of spatial transcriptomes discriminates TLS-enriched from non-enriched cell types among immunotherapy-favorable components. Our systematic dissection of tumor-normal ecosystems provides a deeper understanding of inter- and intra-tumoral heterogeneity.
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Neoplasias , Análisis de la Célula Individual , Transcriptoma , Microambiente Tumoral , Humanos , Microambiente Tumoral/inmunología , Microambiente Tumoral/genética , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Regulación Neoplásica de la Expresión Génica , Inmunoterapia/métodos , Perfilación de la Expresión Génica , Interferones/metabolismoRESUMEN
BACKGROUND: Recent studies using single-cell transcriptomic analysis have reported several distinct clusters of neoplastic epithelial cells and cancer-associated fibroblasts in the pancreatic cancer tumor microenvironment. However, their molecular characteristics and biological significance have not been clearly elucidated due to intra- and inter-tumoral heterogeneity. METHODS: We performed single-cell RNA sequencing using enriched non-immune cell populations from 17 pancreatic tumor tissues (16 pancreatic cancer and one high-grade dysplasia) and generated paired spatial transcriptomic data from seven patient samples. RESULTS: We identified five distinct functional subclusters of pancreatic cancer cells and six distinct cancer-associated fibroblast subclusters. We deeply profiled their characteristics, and we found that these subclusters successfully deconvoluted most of the features suggested in bulk transcriptome analysis of pancreatic cancer. Among those subclusters, we identified a novel cancer cell subcluster, Ep_VGLL1, showing intermediate characteristics between the extremities of basal-like and classical dichotomy, despite its prognostic value. Molecular features of Ep_VGLL1 suggest its transitional properties between basal-like and classical subtypes, which is supported by spatial transcriptomic data. CONCLUSIONS: This integrative analysis not only provides a comprehensive landscape of pancreatic cancer and fibroblast population, but also suggests a novel insight to the dynamic states of pancreatic cancer cells and unveils potential therapeutic targets.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Transcriptoma , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Perfilación de la Expresión Génica , Pronóstico , Microambiente Tumoral/genética , Análisis de la Célula Individual , Proteínas de Unión al ADN/genética , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Management of early-stage gallbladder cancer is becoming more important as the rate of early detection is increasing. Although there have been many studies about the clinical implication of the invasion depth or peritoneal/hepatic location of gallbladder cancers, there is no study on the clinical implication of the geometric location of cancer along the longitudinal length of the gallbladder. METHODS: The location of gallbladder cancer was defined as the geometric center of the primary site of a tumour, which lies on the longitudinal diameter of the surgical specimens. We compared the oncologic outcomes following surgery between gallbladder cancers located on the fundal end and those located on the cystic ductal end. We also analysed patients with stage 1 gallbladder cancer who recurred after surgery. RESULTS: A total of 575 patients with gallbladder cancer were included in this study. Patients with gallbladder cancer on the cystic ductal end had significantly lower rates of recurrence-free survival (P = 0.016) and overall survival (P = 0.023) compared to those with gallbladder cancer on the fundal end. Among 90 patients with stage 1 gallbladder cancer, three patients had a recurrence, all of whom had cystic ductal end gallbladder cancer and showed cystic duct invasion or concomitant xanthogranulomatous cholecystitis in permanent pathology. CONCLUSIONS: Gallbladder cancers on the cystic ductal end had worse postoperative oncologic outcomes compared with those on the fundal end.
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Neoplasias de la Vesícula Biliar , Recurrencia Local de Neoplasia , Estadificación de Neoplasias , Humanos , Neoplasias de la Vesícula Biliar/cirugía , Neoplasias de la Vesícula Biliar/patología , Neoplasias de la Vesícula Biliar/mortalidad , Femenino , Masculino , Persona de Mediana Edad , Anciano , Estudios Retrospectivos , Invasividad Neoplásica , Conducto Cístico/cirugía , Conducto Cístico/patología , Colecistectomía/métodos , Vesícula Biliar/patología , Vesícula Biliar/cirugía , Adulto , Anciano de 80 o más Años , Supervivencia sin EnfermedadRESUMEN
ß1,3-galactose is the component of outer-chain elongation of complex N-glycans that, together with α1,4-fucose, forms Lewis a structures in plants. Previous studies have revealed that N-glycan maturation is mediated by sequential attachment of ß1,3-galactose and α1,4-fucose by individual ß1,3-galactosyltransferase (GalT) and α1,4-fucosyltransferase (1,4-FucT), respectively. Although GalT from several species has been studied, little information about GalT from rice is available. I therefore characterized three GalT candidate genes on different chromosomes in Oryza sativa. Seeds of rice lines that had T-DNA insertions in regions corresponding to individual putative GalT genes were obtained from a Rice Functional Genomic Express Database and plants grown until maturity. Homozygotes were selected from the next generation by genotyping PCR, and used for callus induction. Callus extracts of two independent T-DNA mutant rice which have T-DNA insertions at the same gene on chromosome 6 but in different exons showed highly reduced band intensity on a western blots using an anti-Lewis a antibody. Cell extracts and cultured media from suspension culture of the one of these mutant rice were further analysed by N-glycan profiling using matrix-associated laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). Identified N-glycan species containing ß1,3-galactose from both cell extracts and cultured media of knock-out mutant were less than 0.5% of total N-glycans while that of WT cells were 9.8% and 49.1%, respectively. This suggests that GalT located on rice chromosome 6 plays a major role in N-glycan galactosylation, and mutations within it lead to blockage of Lewis a epitope formation.
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Oryza , Humanos , Oryza/genética , Cromosomas Humanos Par 6 , Fucosa , Galactosa , Extractos Celulares , Polisacáridos/genética , Galactosiltransferasas/genéticaRESUMEN
The current production of the Japanese encephalitis virus (JEV) vaccine is based on animal cells, where various risk factors for human health should be resolved. This study used a transient expression system to express the chimeric protein composed of antigenic epitopes from the JEV envelope (E) protein in Nicotiana benthamiana. JEV multi-epitope peptide (MEP) sequences fused with FLAG-tag or 6× His-tag at the C- or N-terminus for the purification were introduced into plant expression vectors and used for transient expression. Among the constructs, vector pSK480, which expresses MEP fused with a FLAG-tag at the C-terminus, showed the highest level of expression and yield in purification. Optimization of transient expression procedures further improved the target protein yield. The purified MEP protein was applied to an ICR mouse and successfully induced an antibody against JEV, which demonstrates the potential of the plant-produced JEV MEP as an alternative vaccine candidate.
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Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Animales , Ratones , Humanos , Virus de la Encefalitis Japonesa (Especie)/genética , Encefalitis Japonesa/prevención & control , Epítopos/genética , Nicotiana/genética , Anticuerpos Antivirales , Ratones Endogámicos ICR , Péptidos/genética , Ratones Endogámicos BALB C , Proteínas del Envoltorio Viral/genéticaRESUMEN
BACKGROUND: Despite the lack of high-level evidence, laparoscopic distal pancreatectomy (LDP) is frequently performed in patients with pancreatic ductal adenocarcinoma (PDAC) owing to advancements in surgical techniques. The aim of this study was to investigate the long-term oncologic outcomes of LDP in patients with PDAC via propensity score matching (PSM) analysis using data from a large-scale national database. METHODS: A total of 1202 patients who were treated for PDAC via distal pancreatectomy across 16 hospitals were included in the Korean Tumor Registry System-Biliary Pancreas. The 5-year overall (5YOSR) and disease-free (5YDFSR) survival rates were compared between LDP and open DP (ODP). RESULTS: ODP and LDP were performed in 846 and 356 patients, respectively. The ODP group included more aggressive surgeries with higher pathologic stage, R0 resection rate, and number of retrieved lymph nodes. After PSM, the 5YOSRs for ODP and LDP were 37.3% and 41.4% (p = 0.150), while the 5YDFSRs were 23.4% and 27.2% (p = 0.332), respectively. Prognostic factors for 5YOSR included R status, T stage, N stage, differentiation, and lymphovascular invasion. CONCLUSION: LDP was performed in a selected group of patients with PDAC. Within this group, long-term oncologic outcomes were comparable to those observed following ODP.
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Carcinoma Ductal Pancreático , Laparoscopía , Neoplasias Pancreáticas , Humanos , Pancreatectomía/efectos adversos , Pancreatectomía/métodos , Sesgo de Selección , Estudios Retrospectivos , Neoplasias Pancreáticas/patología , Laparoscopía/efectos adversos , Laparoscopía/métodos , Neoplasias PancreáticasRESUMEN
BACKGROUNDS/AIMS: Endoscopic ultrasonography-guided ethanol lavage and Taxol injection (EUS-ELTI) for pancreatic cystic lesions have been recently performed in some medical centers. The aim of this study was to optimize patient selection and analyze outcomes of patients who underwent surgeries after EUS-ELTI for pancreatic cystic lesions. METHODS: Among 310 patients who underwent EUS-ELTI between January 2007 and December 2014, 23 underwent surgeries after EUS-ELTI owing to incomplete treatment and/or adverse events. Surgical outcomes of patients who underwent surgeries after EUSELTI were evaluated. Clinical outcomes of patients who underwent surgeries after EUS-ELTI were then retrospectively compared with those of patients who underwent upfront surgery for left-sided pancreatic lesions without an EUS-ELTI procedure. RESULTS: The pathology revealed degenerated cysts in 12 patients, mucinous cyst neoplasms in five, neuroendocrine tumors in two, intraductal papillary mucinous neoplasm (IPMN) in one, solid pseudopapillary tumor in one, pancreatic ductal adenocarcinoma arising from an IPMN in one, and hepatoid carcinoma in one. Twelve patients underwent laparoscopic distal pancreatectomy and five patients underwent open distal pancreatectomy. When clinical outcomes were retrospectively compared between patients who underwent laparoscopic distal pancreatectomy after EUS-ELTI and those who did not receive an EUS-ELTI procedure, the spleen-preserving rate was 0% in the EUS-ELTI group and 61.7% (365/592) in the control group (p < 0.001). CONCLUSIONS: Surgical outcomes are compromised after EUS-ELTI for cystic tumor of the pancreas. Further studies are needed to investigate the efficacy and safety of the EUS-ELTI procedure.
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Biomarcadores de Tumor/genética , Quimioterapia Adyuvante/mortalidad , Regulación Neoplásica de la Expresión Génica , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , ARN Largo no Codificante/genética , Neoplasias Gástricas/patología , Estudios de Seguimiento , Humanos , Pronóstico , Estudios Retrospectivos , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Tasa de SupervivenciaRESUMEN
The microphthalmia-associated transcription factor family (MiT family) proteins are evolutionarily conserved transcription factors that perform many essential biological functions. In mammals, the MiT family consists of MITF (microphthalmia-associated transcription factor or melanocyte-inducing transcription factor), TFEB (transcription factor EB), TFE3 (transcription factor E3), and TFEC (transcription factor EC). These transcriptional factors belong to the basic helix-loop-helix-leucine zipper (bHLH-LZ) transcription factor family and bind the E-box DNA motifs in the promoter regions of target genes to enhance transcription. The best studied functions of MiT proteins include lysosome biogenesis and autophagy induction. In addition, they modulate cellular metabolism, mitochondria dynamics, and various stress responses. The control of nuclear localization via phosphorylation and dephosphorylation serves as the primary regulatory mechanism for MiT family proteins, and several kinases and phosphatases have been identified to directly determine the transcriptional activities of MiT proteins. In different immune cell types, each MiT family member is shown to play distinct or redundant roles and we expect that there is far more to learn about their functions and regulatory mechanisms in host defense and inflammatory responses.
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Fosforilación/inmunología , Factores de Transcripción/inmunología , Activación Transcripcional/inmunología , Secuencia de Aminoácidos , HumanosRESUMEN
Background/Aims: Several prediction models for evaluating the prognosis of nonmetastatic resected pancreatic ductal adenocarcinoma (PDAC) have been developed, and their performances were reported to be superior to that of the 8th edition of the American Joint Committee on Cancer (AJCC) staging system. We developed a prediction model to evaluate the prognosis of resected PDAC and externally validated it with data from a nationwide Korean database. Methods: Data from the Surveillance, Epidemiology and End Results (SEER) database were utilized for model development, and data from the Korea Tumor Registry System-Biliary Pancreas (KOTUS-BP) database were used for external validation. Potential candidate variables for model development were age, sex, histologic differentiation, tumor location, adjuvant chemotherapy, and the AJCC 8th staging system T and N stages. For external validation, the concordance index (C-index) and time-dependent area under the receiver operating characteristic curve (AUC) were evaluated. Results: Between 2004 and 2016, data from 9,624 patients were utilized for model development, and data from 3,282 patients were used for external validation. In the multivariate Cox proportional hazard model, age, sex, tumor location, T and N stages, histologic differentiation, and adjuvant chemotherapy were independent prognostic factors for resected PDAC. After an exhaustive search and 10-fold cross validation, the best model was finally developed, which included all prognostic variables. The C-index, 1-year, 2-year, 3-year, and 5-year time-dependent AUCs were 0.628, 0.650, 0.665, 0.675, and 0.686, respectively. Conclusions: The survival prediction model for resected PDAC could provide quantitative survival probabilities with reliable performance. External validation studies with other nationwide databases are needed to evaluate the performance of this model.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/patología , Humanos , Estadificación de Neoplasias , Páncreas/patología , Neoplasias Pancreáticas/epidemiología , Neoplasias Pancreáticas/patología , Pronóstico , Sistema de Registros , República de Corea/epidemiologíaRESUMEN
Ubiquitination is an important environmental stress response, and E3 ubiquitin ligases play a major role in the process. T-DNA insertion mutants of rice, Oscbe1-1, and Oscbe1-2, were identified through the screening of cold stress tolerance at seedling stage. Oscbe1 mutants showed a significantly higher cold stress tolerance in the fresh weight, chlorophyll content, and photosynthetic efficiency than wild type. Molecular prediction showed that OsCBE1 (Oryza sativa Cullin4-Based E3 ubiquitin ligase1) encoded a novel substrate receptor of Cullin4-based E3 ubiquitin ligase complex (C4E3). Whereas Oscbe1 mutants had fewer panicles and grains than wild type in the paddy field, the overexpression lines of OsCBE1 had more panicles and grains, suggesting that OsCBE1 is involved in the regulation of both abiotic stress response and development. Oscbe1 mutants also showed ABA hypersensitivity during seed germination, suggesting OsCBE1 function for the stress response via ABA signaling. In silico analysis of OsCBE1 activity predicted a CCCH-type transcription factor, OsC3H32, as a putative substrate. Co-IP (Co-immunoprecipitation) study showed that OsCBE1 interacts with OsDDB1, an expected binding component of OsCBE1 and OsC3H32. Additionally, expression of OsOLE16, OsOLE18, and OsBURP5 were negatively related with expression of OsCBE1. These results suggest that OsCBE1 functions as a regulator of the abiotic stress response via CCCH as a member of the C4E3.
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Proteínas Cullin/metabolismo , Regulación de la Expresión Génica de las Plantas , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Estrés Fisiológico/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Cullin/genética , Oryza/genética , Oryza/crecimiento & desarrollo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
KEY MESSAGE: CRISPR/Cas9-mediated OsXylT and OsFucT mutation caused the elimination of plant-specific ß1,2-xylose and α1,3-fucose residues on glycoproteins in rice, which is the first report of OsXylT/OsFucT double KO mutation in rice. N-glycosylation pathway is the one of post-translational mechanism and is known as highly conserved in eukaryotes. However, the process for complex-N-glycan modification is different between mammals and plants. In plant-specific manner, ß1,2-xylose and α1,3-fucose residues are transferred to N-glycan core structure on glycoproteins by ß1,2-xylosyltransferase (ß1,2-XylT) and α1,3-fucosyltransferase (α1,3-FucT), respectively. As an effort to use plants as a platform to produce biopharmaceuticals, the plant-specific N-glycan genes of rice (Oryza sativa), ß1,2-xylT (OsXylT) and α1,3-FucT (OsFucT), were knocked out using multiplex CRISPR/Cas9 technology. The double knock-out lines were found to have frameshift mutations by INDELs. Both ß1,2-xylose and α1,3-fucose residues in the lines were not detected in Western blot analysis. Consistently, there was no peak corresponding to the N-glycans in MALDI-TOF/MS analysis. Although α1,3-fucose and ß1,2-xylose residues were not detected in the line, other plant-specific residues of ß1,3-galactose and α1,4-fucose were detected. Thus, we suggest that each enzymes working on the process for complex N-glycan biosynthesis might independently act in rice, hence the double knock-out of both OsXylT and OsFucT might be not enough to humanize N-glycan structure in rice.
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Sistemas CRISPR-Cas , Fucosiltransferasas/genética , Oryza/genética , Pentosiltransferasa/genética , Polisacáridos/metabolismo , Epítopos/genética , Edición Génica/métodos , Silenciador del Gen , Mutación , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Polisacáridos/genética , Polisacáridos/inmunología , UDP Xilosa Proteína XilosiltransferasaRESUMEN
Approximately 80% of pancreatic cancer is diagnosed at an advanced stage, due to lack of or vague symptoms when the cancer is still localized, leading to a high mortality rate. Known risk factors for developing pancreatic cancer are family history, obesity, type 2 diabetes, and alcohol and tobacco use. There has been a remarkable development in diagnosis modalities and molecular testing, but early detection is still infrequent. The majority of clinical trials have not shown significant efficacy in pancreatic cancer, and treatment strategy remains limited. Additional prognostic factors should be highlighted to obtain appropriate treatment options, including precision medicine, and improve survival outcomes. After the PRODIGE study in 2011 and the MPAC trial in 2013, a new drug (liposomal irinotecan; Onivyde ®) appeared in the strategy, especially after failure of gemcitabine-based treatment. In 2016, the NAPOLI-1 trial showed evidence of the efficacy of the liposomal irinotecan combination (liposomal irinotecan +5-fluorouracile + folinic acid); now, it is considered the standard treatment for relapsing patients. Since NAPOLI-1, real-world data have provided similar results. Herein, we report the story of a 61-year-old woman who was treated with liposomal irinotecan combination (nal-IRI/5-FU/LV) for 8 months with good surgical response, but treatment was discontinued due to economic burden. After the start of treatment (or 1? cycle of liposomal irinotecan treatment), the patient was in a better condition. The liver metastases had disappeared. The combination with liposomal irinotecan was re-administered with patient's approval. Upon rechallenge with the liposomal irinotecan combination, she showed a partial response, and the treatment was given for 7 months. In this report, we tried to identify the prognostic factors leading to the efficacy of the liposomal irinotecan combination.
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Rice (Oryza sativa L.), a staple crop plant that is a major source of calories for approximately 50% of the human population, exhibits various physiological responses against temperature stress. These responses are known mechanisms of flexible adaptation through crosstalk with the intrinsic circadian clock. However, the molecular regulatory network underlining this crosstalk remains poorly understood. Therefore, we performed systematic transcriptome data analyses to identify the genes involved in both cold stress responses and diurnal rhythmic patterns. Here, we first identified cold-regulated genes and then identified diurnal rhythmic genes from those (119 cold-upregulated and 346 cold-downregulated genes). We defined cold-responsive diurnal rhythmic genes as CD genes. We further analyzed the functional features of these CD genes through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses and performed a literature search to identify functionally characterized CD genes. Subsequently, we found that light-harvesting complex proteins involved in photosynthesis strongly associate with the crosstalk. Furthermore, we constructed a protein-protein interaction network encompassing four hub genes and analyzed the roles of the Stay-Green (SGR) gene in regulating crosstalk with sgr mutants. We predict that these findings will provide new insights in understanding the environmental stress response of crop plants against climate change.
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Ritmo Circadiano/fisiología , Respuesta al Choque por Frío/fisiología , Bases de Datos de Ácidos Nucleicos , Regulación de la Expresión Génica de las Plantas/fisiología , Oryza , Transcriptoma/fisiología , Oryza/genética , Oryza/metabolismoRESUMEN
Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-CSF) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-CSF, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codon-optimized hG-CSF sequence, hG-CSF polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella.
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Chlorella vulgaris/crecimiento & desarrollo , Factor Estimulante de Colonias de Granulocitos/metabolismo , Nitrógeno/metabolismo , Regiones Promotoras Genéticas , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Chlorella vulgaris/genética , Chlorella vulgaris/metabolismo , Factor Estimulante de Colonias de Granulocitos/genética , Humanos , Organismos Modificados Genéticamente/crecimiento & desarrollo , Organismos Modificados Genéticamente/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/metabolismoRESUMEN
N-linked glycosylation is one of the key post-translational modifications. α1,3-Fucosyltransferase (OsFucT) is responsible for transferring α1,3-linked fucose residues to the glycoprotein N-glycan in plants. We characterized an Osfuct mutant that displayed pleiotropic developmental defects, such as impaired anther and pollen development, diminished growth, shorter plant height, fewer tillers, and shorter panicle length and internodes under field conditions. In addition, the anthers were curved, the pollen grains were shriveled, and pollen viability and pollen number per anther decreased dramatically in the mutant. Matrix-assisted laser desorption/ionization time-of-flight analyses of the N-glycans revealed that α1,3-fucose was lacking in the N-glycan structure of the mutant. Mutant complementation revealed that the phenotype was caused by loss of Osfuct function. Transcriptome profiling also showed that several genes essential for plant developmental processes were significantly altered in the mutant, including protein kinases, transcription factors, genes involved in metabolism, genes related to protein synthesis, and hypothetical proteins. Moreover, the mutant exhibited sensitivity to an increased concentration of salt. This study facilitates a further understanding of the function of genes mediating N-glycan modification and anther and pollen development in rice.
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Fucosiltransferasas/genética , Genes de Plantas , Oryza/enzimología , Oryza/genética , Polen/enzimología , Polen/crecimiento & desarrollo , Supervivencia Tisular/fisiología , Alelos , ADN Bacteriano/genética , Fucosiltransferasas/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Mutagénesis Insercional , Mutación/genética , Oryza/anatomía & histología , Oryza/efectos de los fármacos , Fenotipo , Plantas Modificadas Genéticamente , Polen/anatomía & histología , Polen/efectos de los fármacos , Cloruro de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Supervivencia Tisular/efectos de los fármacosRESUMEN
Post-operative pancreatic fistula (POPF) following pancreatic resection is a life-threatening surgical complication. Cell sheets were prepared and harvested using temperature-responsive culture dishes and transplanted as patches to seal POPF. Two different mesenchymal stem cell (MSC) sheets were compared in terms of the preventative ability for pancreatic leakage in a rat model. Both rat adipose-derived stem cell (rADSC) and bone marrow-derived stem cell (rBMSC) sheets were transplanted. Those rADSC and rBMSC sheets are created without enzymes and thus maintained their cell-cell junctions and adhesion proteins with intact fibronectin on the basal side, as well as characteristics of MSCs. The rats with post-pancreatectomy rADSC- or rBMSC-sheet patches had significantly decreased abdominal fluid leakage compared with the control group, demonstrated by MR image analysis and measurement of the volume of abdominal fluid. Amylase level was significantly lower in the rats with rADSC-sheet and rBMSC-sheet patches compared with the control groups. The rADSC sheet patches had increased adhesive and immune-cytokine profiles (ICAM-1, L-selectin, TIMP-1), and the rBMSC sheets had reduced immune reactions compared to the control. This is first project looking at the feasibility of tissue engineering therapy using MSC-sheets as tissue patches preventing leakage of abdominal fluid caused by POPF.
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Células Madre Mesenquimatosas , Fístula Pancreática/etiología , Fístula Pancreática/terapia , Complicaciones Posoperatorias , Ingeniería de Tejidos , Amilasas/metabolismo , Animales , Biomarcadores , Modelos Animales de Enfermedad , Inmunohistoquímica , Imagen por Resonancia Magnética , Células Madre Mesenquimatosas/citología , Fístula Pancreática/diagnóstico por imagen , Fístula Pancreática/patología , Ratas , Ratas Transgénicas , Resultado del TratamientoRESUMEN
Cold stress is very detrimental to crop production. However, only a few genes in rice have been identified with known functions related to cold tolerance. To meet this agronomic challenge more effectively, researchers must take global approaches to select useful candidate genes and find the major regulatory factors. We used five Gene expression omnibus series data series of Affymetrix array data, produced with cold stress-treated samples from the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), and identified 502 cold-inducible genes common to both japonica and indica rice cultivars. From them, we confirmed that the expression of two randomly chosen genes was increased by cold stress in planta. In addition, overexpression of OsWRKY71 enhanced cold tolerance in 'Dongjin,' the tested japonica cultivar. Comparisons between japonica and indica rice, based on calculations of plant survival rates and chlorophyll fluorescence, confirmed that the japonica rice was more cold-tolerant. Gene Ontology enrichment analysis indicate that the 'L-phenylalanine catabolic process,' within the Biological Process category, was the most highly overrepresented under cold-stress conditions, implying its significance in that response in rice. MapMan analysis classified 'Major Metabolic' processes and 'Regulatory Gene Modules' as two other major determinants of the cold-stress response and suggested several key cis-regulatory elements. Based on these results, we proposed a model that includes a pathway for cold stress-responsive signaling. Results from our functional analysis of the main signal transduction and transcription regulation factors identified in that pathway will provide insight into novel regulatory metabolism(s), as well as a foundation by which we can develop crop plants with enhanced cold tolerance.