RESUMEN
Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 µg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 µM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.
Asunto(s)
Antibacterianos/farmacología , Chlamydomonas reinhardtii/metabolismo , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Penaeidae/metabolismo , Proteínas Citotóxicas Formadoras de Poros/farmacología , Animales , Antibacterianos/biosíntesis , Chlamydomonas reinhardtii/genética , Regulación Bacteriana de la Expresión Génica , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/crecimiento & desarrollo , Proteínas Citotóxicas Formadoras de Poros/biosíntesis , Proteínas Citotóxicas Formadoras de Poros/genéticaRESUMEN
Anti-lipopolysaccharide factors (ALFs) are important host-defense molecules of crustaceans. They all contain a lipopolysaccharide-binding domain (LBD) and some ALFs exhibit strong antimicrobial activity. In this research, a Group G ALF from Penaeus monodon (ALFPm11) was studied. It is an anionic peptide specifically having a cationic and highly amphipathic LBD, with five positively charged residues separated by aromatic residues. It was abundantly expressed in the hepatopancreas of P. monodon normally but the expression level in other tissues was relatively low or undetectable. However, in the shrimps challenged by Vibrio, expression of ALFPm11 could be detected in all tissues. Chemically synthesized ALFPm11-LBD displayed high inhibitory activity (minimum inhibition concentration≤ 4⯵M) against various bacteria, e.g. Exiguobacterium sp. L33, Bacillus sp. T2, and Acinetobacter sp. L32. It also displayed apparent activity in the agar well diffusion assay. Furthermore, it could efficiently induce agglutination of both Gram-positive and Gram-negative bacteria and cause significant membrane permeabilization of the bacteria. As a comparative study, ALFPm11-LBD showed a better or equal antimicrobial function to ALFPm3-LBD which was reported to possess strong antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. Thus, this research found a new effective ALF in P. monodon and demonstrated its antimicrobial mechanism, suggesting its potential applications in the future.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/inmunología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Inmunidad Innata/genética , Penaeidae/genética , Penaeidae/inmunología , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Secuencia de Bases , Perfilación de la Expresión Génica , Pruebas de Sensibilidad Microbiana , Alineación de SecuenciaRESUMEN
Antimicrobial peptides (AMPs) are an essential component of innate immunity of invertebrates. Anti-lipopolysaccharide factor (ALF), as a main type of AMPs in crustaceans, attends in the disease prevention in general. In this research, a novel Group D ALF was identified and characterized from Penaeus monodon, named PenmonALF8. It was an anionic peptide, with both the full-length peptide and lipopolysaccharide binding domain (LBD) a low isoelectric point. PenmonALF8, composed of a signal peptide of 26 amino acids and a mature peptide of 98 amino acids, probably contained three alpha helixes and four beta sheets. Moreover, PenmonALF8 was detected in all tested tissues of P. monodon, and the expression level in hemocyte and intestine was relatively high. When challenged by Vibrio parahaemolyticus, PenmonALF8 showed 30-100 times higher expression level in all the tissues except in hemocyte and intestine, indicating that PenmonALF8 played a very important role in the immune response of P. monodon. By fusing to a SUMO protein, PenmonALF8 was successfully over-expressed in E. coli and purified by affinity chromatography. Additionally, the reconstituted PenmonALF8 and its LBD region displayed modest antimicrobial activity. This is the first research about the Group D ALF in P. monodon, which provides more information for humoral immunity study of shrimps.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Penaeidae/genética , Penaeidae/inmunología , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/química , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Perfilación de la Expresión Génica , Filogenia , Alineación de Secuencia , Vibrio/inmunologíaRESUMEN
Haematococcus pluvialis is widely distributed in the world and well known as the richest natural source of astaxanthin that is a strong antioxidant with excellent commercial value. The pathway of astaxanthin biosynthesis in H. pluvialis has been documented as an enzymatic reaction. Several enzymes have been reported, but their isoforms or homologs have not been investigated genome-wide. To better understand the astaxanthin biosynthesis pathway in H. pluvialis, eight candidates of the geranylgeranyl pyrophosphate synthase gene (HpGGPPS) predicted from Iso-seq data were isolated in this study. The length of coding region of these candidates varied from 960 bp to 1272 bp, composing of 7-9 exons. The putative amino acids of all candidates composed the signature domain of GGPPS gene. However, the motifs in the domain region are varied, indicating different bio-functions. Phylogenetic analysis revealed eight candidates can be clustered into three groups. Only two candidates in Group1 encode the synthase participating in the astaxanthin formation. The yield of astaxanthin from these two candidates, 7.1 mg/g (DW) and 6.5 mg/g (DW) respectively, is significant higher than that from CrtE (2.4 mg/g DW), a GGPPS gene from Pantoea ananatis. This study provides a potential productive pathway for astaxanthin synthesis.
Asunto(s)
Antioxidantes/aislamiento & purificación , Chlorophyceae/química , Geranilgeranil-Difosfato Geranilgeraniltransferasa/genética , Genoma , Filogenia , Xantófilas/aislamiento & purificaciónRESUMEN
Background: The residual stone fragment is a tremendous issue after ureteroscopic lithotripsy and requires urologists to evaluate the condition of patients comprehensively. Our study aimed to construct a nomogram to make a personalized prediction of postoperative residual stone rate (RSR). Methods: We implemented a retrospective cohort study in the Department of Urology, Shanghai General Hospital. A total of 277 patients undergoing ureteroscopy (URS) were enrolled in our study. Among them, 186 patients were included in the training group and the remaining 91 patients comprised the testing group. We utilized stepwise forward algorithm and logistic regression analysis to build predictive models and selected the best model based on Akaike's information criterion (AIC). The model was assessed by receiver operating characteristic (ROC) curves and the Hosmer-Lemeshow (HL) test. We also conducted decision curve analysis (DCA) to demonstrate the net benefit of the model. The independent testing group was used to validate the practicability of the nomogram. Results: The severity of hydronephrosis, stone location, the transverse diameter of stone, hypertension, and white blood cell (WBC) were found to be significant predictive variables for RSR after URS. The area under the curve (AUC) of the training group was 0.7203 and that of the testing group was 0.7280. Besides, the nomogram also presented great calibration and accepted net benefit in a wide range of probabilities. Conclusions: Our study achieved a predictive nomogram with excellent application value for urologists to assess RSR and make personalized treatment decisions.
RESUMEN
BACKGROUND: Bladder cancer is a common malignant type in the world, and over 90% are transitional cell carcinoma. While the impact of inflammatory response on cancer progression has been reported, the role of inflammatory response-associated genes (IRAGs) in transitional bladder cancer still needs to be understood. METHODS: In this study, IRAGs were download from Molecular Signature Database (MSigDB). The transcriptional expression and matched clinicopathological data were separately obtained from public databases. The TCGA-BLCA cohort was used to identify the differentially expressed IRAGs, and prognostic IRAGs were filtrated by univariate survival analysis. The intersection between them was displayed by Venn diagram. Based on least absolute shrinkage and selection operator (LASSO) regression analysis method, the TCGA-BLCA cohort was used to construct a risk signature. Survival analysis was conducted to calculate the overall survival (OS) in TCGA and GSE13507 cohort between two groups. We then conducted univariate and multivariate survival analyses to identify independently significant indicators for prognosis. Relationships between the risk scores and age, grade, stage, immune cell infiltration, immune function, and drug sensitivity were demonstrated by correlation analysis. The expression level of prognostic genes in vivo and in vitro were determined by qRT-PCR assay. RESULTS: Comparing with normal tissues, there were 49 differentially expressed IRAGs in cancer tissues, and 12 of them were markedly related to the prognosis in TCGA cohort for transitional bladder cancer patients. Based on LASSO regression analysis, a risk model consists of 10 IRAGs was established. Comparing with high-risk groups, survival analysis showed that patients in low-risk groups were more likely to have a better survival time in TCGA and GSE13507 cohorts. Besides, the accuracy of the model in predicting prognosis is acceptable, which is demonstrated by receiver operating characteristic curve (ROC) analysis. Age, stage, and risk scores variables were identified as the independently significant indicators for survival in transitional bladder cancer. Correlation analysis represented that the risk score was identified to be significantly related to the above variables except gender variable. Moreover, the expression level of prognostic genes in vivo and in vitro was markedly upregulated for transitional bladder cancer. CONCLUSIONS: A novel model based on the 10 IRAGs that can be used to predict survival time for transitional bladder cancer. In addition, this study may provide treatment strategies according to the drug sensitivity in the future.
RESUMEN
BACKGROUND: The unicellular alga Haematococcus pluvialis has achieved considerable interests for its capacity to accumulate large amounts of triacylglycerol and astaxanthin under various environmental stresses. To our knowledge, studies focusing on transcriptome research of H. pluvialis under exogenous hormones together with physical stresses are rare. In the present study, the change patterns at transcriptome level were analyzed to distinguish the multiple defensive systems of astaxanthin and fatty acid metabolism against exogenous salicylic acid and high light (SAHL) stresses. RESULTS: Based on RNA-seq data, a total of 112,463 unigenes and 61,191 genes were annotated in six databases, including NR, KEGG, Swiss-Prot, PFAM, COG and GO. Analysis of differentially expressed genes (DEGs) in KEGG identified many transcripts that associated with the biosynthesis of primary and secondary metabolites, photosynthesis, and immune system responses. Furthermore, 705 unigenes predicted as putative transcription factors (TFs) were identified, and the most abundant TFs families were likely to be associated with the biosynthesis of astaxanthin and fatty acid in H. pluvialis upon exposure to SAHL stresses. Additionally, majority of the fifteen key genes involved in astaxanthin and fatty acid biosynthesis pathways presented the same expression pattern, resulting in increased accumulation of astaxanthin and fatty acids in single celled H. pluvialis, in which astaxanthin content increased from 0.56 ± 0.05 mg·L-1 at stage Control to 0.89 ± 0.12 mg·L-1 at stage SAHL_48. And positive correlations were observed among these studied genes by Pearson Correlation (PC) analysis, indicating the coordination between astaxanthin and fatty acid biosynthesis. In addition, protein-protein interaction (PPI) network analysis also demonstrated that this coordination might be at transcriptional level. CONCLUSION: The results in this study provided valuable information to illustrate the molecular mechanisms of coordinate relations between astaxanthin and fatty acid biosynthesis. And salicylic acid might play a role in self-protection processes of cells, helping adaption of H. pluvialis to high light stress.
RESUMEN
NIR-II fluorophores have shown great promise for biomedical applications with superior in vivo optical properties. To date, few small-molecule NIR-II fluorophores have been discovered with donor-acceptor-donor (D-A-D) or symmetrical structures, and upconversion-mitochondria-targeted NIR-II dyes have not been reported. Herein, we report development of D-A type thiopyrylium-based NIR-II fluorophores with frequency upconversion luminescence (FUCL) at ~580 nm upon excitation at ~850 nm. H4-PEG-PT can not only quickly and effectively image mitochondria in live or fixed osteosarcoma cells with subcellular resolution at 1 nM, but also efficiently convert optical energy into heat, achieving mitochondria-targeted photothermal cancer therapy without ROS effects. H4-PEG-PT has been further evaluated in vivo and exhibited strong tumor uptake, specific NIR-II signals with high spatial and temporal resolution, and remarkable NIR-II image-guided photothermal therapy. This report presents the first D-A type thiopyrylium NIR-II theranostics for synchronous upconversion-mitochondria-targeted cell imaging, in vivo NIR-II osteosarcoma imaging and excellent photothermal efficiency.
Asunto(s)
Colorantes Fluorescentes/química , Rayos Infrarrojos , Mitocondrias/metabolismo , Neoplasias/diagnóstico por imagen , Imagen Óptica , Terapia Fototérmica , Animales , Muerte Celular , Durapatita/química , Colorantes Fluorescentes/síntesis química , Luminiscencia , Ratones Desnudos , Conformación Molecular , Polietilenglicoles/química , Espectrometría de Fluorescencia , Resultado del TratamientoRESUMEN
Osteosarcoma is an aggressive tumor of mesenchymal origin that is more likely to spread to the lung than others, with a major impact on patients' prognosis. The optimal imaging method that can reliably detect or exclude pulmonary metastases from osteosarcoma is still scarce. Herein, two homologous types of fluorescent probes CH1055-PEG-PT and CH1055-PEG-Affibody, which show highly promising results for targeting imaging of osteosarcoma and its lung metastasis, respectively, are designed and synthesized. It is found that the NIR-II imaging quality of CH1055-PEG-PT is far superior to that of computed tomography for the early in vivo 143B tumor imaging, and this probe-guided surgery for accurate resection of 143B tumor is further performed. The high-resolution visualization of primary and micrometastatic lung lesions of osteosarcoma by using CH1055-PEG-Affibody is also demonstrated. Therefore, the attractive imaging properties of CH1055-PEG-PT and CH1055-PEG-Affibody, including high levels of uptakes, and high spatial and temporal resolution, open up opportunities for molecular imaging and clinical translation of osteosarcoma and its lung metastasis in the unique second near-infrared window.
Asunto(s)
Neoplasias Óseas/diagnóstico por imagen , Colorantes Fluorescentes/química , Neoplasias Pulmonares/diagnóstico por imagen , Imagen Óptica/métodos , Osteosarcoma/diagnóstico por imagen , Animales , Neoplasias Óseas/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Colorantes Fluorescentes/metabolismo , Colorantes Fluorescentes/farmacología , Humanos , Neoplasias Pulmonares/secundario , Ratones , Ratones Desnudos , Osteosarcoma/patología , Fenilpropionatos/química , Polietilenglicoles/química , Proteínas Recombinantes de Fusión/química , Espectroscopía Infrarroja Corta , Tiadiazoles/química , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Small-molecule subcellular organelle-targeting theranostic probes are crucial for early disease diagnosis and treatment. The imaging window of these molecules is mainly focused on the visible and near-infrared region (below â¼900 nm) which limits the tissue penetration depth and therapeutic effects. Herein, a novel NIR-II small-molecule probe H4-PEG-Glu with a thiopyrylium cation was synthesized. H4-PEG-Glu not only can quickly and effectively image mitochondria in acute myeloid leukemia (AML) cells, and induce G0/G1 phase arrest by the intrinsic mitochondrial apoptosis pathway w/o irradiation, but also exhibit moderate cytotoxicity against AML cancer cells in a dose dependent-manner without laser irradiation. The THP-1 cells treated with H4-PEG-Glu upon NIR laser irradiation showed enhanced chemo- and photothermal therapy (CPTT) with 93.07% ± 6.43 apoptosis by Annexin V staining. Meanwhile, H4-PEG-Glu displayed high synergistic CPTT effects in vivo, as well as specific NIR-II tumor imaging in AML patient derived PDX mouse models for the first time. Our work lays down a solid foundation for designing small-molecule NIR-II mitochondria-selective theranostic probes.
RESUMEN
Articular cartilage (AC) is a complex water-bearing tissue consisting of chondrocytes, proteoglycans, and collagen. AC degeneration, which occurs in the early stage and throughout the entire course of osteoarthritis (OA), is one of the main pathological changes of OA. However, current clinical approaches are unable to detect AC degradation during the early stage of OA. Herein, a novel NIR-II probe, CH1055-WL, was developed with an organic fluorophore (CH1055) and type II collagen-binding peptide (WYRGRL) for AC targeting and degeneration imaging. In vitro and in vivo imaging studies demonstrated that CH1055-WL specifically bound to AC and permitted sensitive detection of age-related or surgically induced AC degeneration in living mice. In vitro imaging of cartilage samples from pig knee joint and in vivo imaging of live mice with the probe administered via local injection in joint cavities demonstrated that CH1055-WL specifically and efficiently bound to AC. Further evaluation of CH1055-WL revealed sensitive detection of age-related AC degeneration and surgically induced AC degeneration in living mice. Our results indicated that the cartilage-targeting probe CH1055-WL allowed visual monitoring of AC degeneration in living subjects, thus displaying promise for early OA detection.
Asunto(s)
Colorantes Fluorescentes/química , Osteoartritis de la Rodilla/diagnóstico , Péptidos/química , Fenilpropionatos/química , Tiadiazoles/química , Animales , Cartílago Articular/diagnóstico por imagen , Cartílago Articular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Osteoartritis de la Rodilla/diagnóstico por imagen , Péptidos/metabolismo , Fenilpropionatos/síntesis química , Fenilpropionatos/farmacología , Espectroscopía Infrarroja Corta , Tiadiazoles/síntesis química , Tiadiazoles/farmacologíaRESUMEN
Traditional luminescent materials including fluorescent probes suffer from notorious aggregation-caused quenching (ACQ) in aqueous solutions. Although several approaches such as the aggregation-induced emission (AIE) effect have been developed, it remains a significant challenge to identify an effective and efficient strategy to resolve this issue. Herein, quaternary ammonium salts Q8PBn and Q8PNap as a novel class of bright near infrared window II (NIR-II, 1,000 - 1,700 nm) probes were designed and synthesized, and the twisted intramolecular charge transfer (TICT) formation at the excited state can be effectively suppressed for the newly designed probes. Furthermore, Q8PNap complexation with fetal bovine serum (Q8PNap/FBS) significantly increased the quantum yield by ~ 32-fold compared with PEGylated tertiary amine Q8P, and Q8PNap/FBS was successfully used to achieve high spatial and temporal resolution imaging of hind limb vasculature, lymphatic system, and small tumor metastasis, as well as precise NIR-II imaging-guided tumor and lymph node surgery in small animal models for the first time.
RESUMEN
Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates proliferation, differentiation, and function of hematopoietic progenitor cells. Aside from expansion of hematopoietic cells, GM-CSF has shown efficacy in other diseases, including Crohn's disease. While GM-CSF being clinically used in humans, the ability to perform mechanistic studies in murine models is difficult due to the limited availability and rapid clearance of murine GM-CSF in the peripheral blood. To address these issues, we efficiently expressed murine GM-CSF under the control of the AOX1 gene promoter in Pichia pastoris using the Mut(S) strain KM71H. We describe the unique conditions that are required for efficient production by high-density fermentation and purification of mGM-CSF protein. Recombinant mGM-CSF protein was purified by tangential flow ultrafiltration and preparative reverse phase chromatography. To address limited half life or rapid clearance in mice, recombinant murine GM-CSF was modified by lysine-directed polyethylene glycol conjugation (PEGylation). PEG-modified and unmodified proteins were characterized by amino terminus sequence analysis and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Under the mild reaction conditions, the recombinant protein is efficiently modified by PEGylation on an average of 2-3 sites per molecule. In vivo treatment of mice with PEGylated mGM-CSF, but not the unmodified recombinant mGM-CSF, reproduces the potent colony stimulating effects of human GM-CSF in patients on myeloid progenitor populations, as assessed by FACs analysis. This simplified approach for the expression, purification, and modification of a biologically potent form of murine GM-CSF should facilitate the study of central mechanisms of action in murine disease models.