RESUMO
Valuable polysaccharides are usually produced using wild-type or metabolically-engineered host microbial strains through fermentation. These hosts act as cell factories that convert carbohydrates, such as monosaccharides or starch, into bioactive polysaccharides. It is desirable to develop effective in vivo high-throughput approaches to screen cells that display high-level synthesis of the desired polysaccharides. Uses of single or dual fluorophore labeling, fluorescence quenching, or biosensors are effective strategies for cell sorting of a library that can be applied during the domestication of industrial engineered strains and metabolic pathway optimization of polysaccharide synthesis in engineered cells. Meanwhile, high-throughput screening strategies using each individual whole cell as a sorting section are playing growing roles in the discovery and directed evolution of enzymes involved in polysaccharide biosynthesis, such as glycosyltransferases. These enzymes and their mutants are in high demand as tool catalysts for synthesis of saccharides in vitro and in vivo. This review provides an introduction to the methodologies of using cell-based high-throughput screening for desired polysaccharide-biosynthesizing cells, followed by a brief discussion of potential applications of these approaches in glycoengineering.
Assuntos
Bactérias/metabolismo , Ensaios de Triagem em Larga Escala , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos/biossíntese , Bactérias/genética , Técnicas Biossensoriais , Evolução Molecular Direcionada , Fluorescência , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Engenharia Metabólica , Monossacarídeos/metabolismoRESUMO
Interventional therapies are increasingly used in clinical trials for hepatocellular carcinoma (HCC). Sorafenib is the front-line remedy for HCC, however, chemoresistance occurs immutably and affects the effectiveness of treatment. In a previous study, a norcantharidin liposome emulsion hybrid (NLEH) delivery system for HCC was developed. This study aims to examine the therapeutic effects of the combination of intratumoral injection of NLEH and sorafenib in treating HCC. Sorafenib combined with NLEH activated the apoptosis pathway by synergistically upregulating caspase-9, promoting cytotoxicity, apoptosis (64.57%), and G2/M cell cycle arrest (48.96%). Norcantharidin could alleviate sorafenib resistance by counteracting sorafenib-induced phosphorylation of Akt. Additionally, intratumoral injection of NLEH exhibited a sustained accumulation in the tumor within 24 h and didn't distribute to other major organs. Intratumoral injection of NLEH in combination with oral sorafenib displayed the most potent tumor growth inhibitory effect (77.91%) in vivo. H&E staining results and the indicators of the renal and liver function tests demonstrated the safety of this combination therapy. Overall, these results showed that intratumoral injection of NLEH in combination with oral sorafenib treatment represented a rational potential therapeutic option for HCC.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Lipossomos/farmacologia , Neoplasias Hepáticas/patologia , Emulsões/farmacologia , Injeções Intralesionais , Linhagem Celular Tumoral , Apoptose , Proliferação de CélulasRESUMO
The introduction of unnatural chemical moieties into glycosaminoglycans (GAGs) has enormous potential to facilitate studies of the mechanism and application of these critical, widespread molecules. Unnatural N-acetylhexosamine analogs were metabolically incorporated into the capsule polysaccharides of Escherichia coli and Bacillus subtilis via bacterial metabolism. Targeted metabolic labeled hyaluronan and the precursors of heparin and chondroitin sulfate were obtained. The azido-labeled polysaccharides (purified or in capsules) were reacted with dyes, via bioorthogonal chemistry, to enable detection and imaging. Site-specific introduction of fluorophores directly onto cell surfaces affords another choice for observing and quantifying bacteria in vivo and in vitro. Furthermore, azido-polysaccharides retain similar biological properties to their natural analogs, and reliable and predictable introduction of functionalities, such as fluorophores, onto azido-N-hexosamines in the disaccharide repeat units provides chemical tools for imaging and metabolic analysis of GAGs in vivo and in vitro.
Assuntos
Escherichia coli , Glicosaminoglicanos , Glicosaminoglicanos/química , Escherichia coli/metabolismo , Polissacarídeos , Heparina , Sulfatos de Condroitina , Polissacarídeos BacterianosRESUMO
Pseudomonas plecoglossicida is a rod-shaped, gram-negative bacterium with flagella. It causes visceral white spot disease and high mortality in Larimichthys crocea during culture, resulting in serious economic loss. Analysis of transcriptome and quantitative real-time polymerase chain reaction (PCR) data showed that dksA gene expression was significantly up-regulated after 48 h of infection with Epinephelus coioides (log 2FC=3.12, P<0.001). RNAi of five shRNAs significantly reduced the expression of dksA in P. plecoglossicida, and the optimal silencing efficiency was 96.23%. Compared with wild-type strains, the symptoms of visceral white spot disease in L. crocea infected with RNAi strains were reduced, with time of death delayed by 48 h and mortality reduced by 25%. The dksA silencing led to a substantial down-regulation in cellular component-, flagellum-, and ribosome assembly-related genes in P. plecoglossicida, and the significant up-regulation of fliC may be a way in which virulence is maintained in P. plecoglossicida. The GO and KEGG results showed that RNAi strain infection in L. crocea led to the down-regulation of inflammatory factor genes in immune-related pathways, which were associated with multiple immune response processes. Results also showed that dksA was a virulence gene in P. plecoglossicida. Compared with the wild-type strains, RNAi strain infection induced a weaker immune response in L. crocea.
Assuntos
Proteínas de Bactérias/genética , Doenças dos Peixes/imunologia , Perciformes , Infecções por Pseudomonas/veterinária , Pseudomonas/fisiologia , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/metabolismo , Doenças dos Peixes/microbiologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , RNA Bacteriano/análise , RNA-Seq/veterinária , Fatores de Virulência/metabolismoRESUMO
AIM: To present clinical characteristics, diagnosis and treatment strategies in elderly patients with biliary diseases. METHODS: A total of 289 elderly patients with biliary diseases were enrolled in this study. The clinical data relating to these patients were collected in our hospital from June 2013 to May 2016. Patient age, disease type, coexisting diseases, laboratory examinations, surgical methods, postoperative complications and therapeutic outcomes were analyzed. RESULTS: The average age of the 289 patients with biliary diseases was 73.9 ± 8.5 years (range, 60-102 years). One hundred and thirty-one patients (45.3%) had one of 10 different biliary diseases, such as gallbladder stones, common bile duct stones, and cholangiocarcinoma. The remaining patients (54.7%) had two types of biliary diseases. One hundred and seventy-nine patients underwent 9 different surgical treatments, including pancreaticoduodenectomy, radical resection of hilar cholangiocarcinoma and laparoscopic cholecystectomy. Ten postoperative complications occurred with an incidence of 39.3% (68/173), and hypopotassemia showed the highest incidence (33.8%, 23/68). One hundred and sixteen patients underwent non-surgical treatments, including anti-infection, symptomatic and supportive treatments. The cure rate was 97.1% (168/173) in the surgical group and 87.1% (101/116) in the non-surgical group. The difference between these two groups was statistically significant (χ2 = 17.227, P < 0.05). CONCLUSION: Active treatment of coexisting diseases, management of indications and surgical opportunities, appropriate selection of surgical procedures, improvements in perioperative therapy, and timely management of postoperative complications are key factors in enhancing therapeutic efficacy in elderly patients with biliary diseases.
Assuntos
Doenças dos Ductos Biliares/terapia , Idoso , Idoso de 80 Anos ou mais , Doenças dos Ductos Biliares/epidemiologia , Procedimentos Cirúrgicos do Sistema Biliar , China/epidemiologia , Comorbidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/epidemiologia , Resultado do TratamentoRESUMO
A 48-year-old woman was admitted with 15-mo history of abdominal pain, diarrhea and hematochezia, and 5-mo history of defecation difficulty. She had been successively admitted to nine hospitals, with an initial diagnosis of inflammatory bowel disease with stenotic sigmoid colon. Findings from computed tomography virtual colonoscopy, radiography with meglumine diatrizoate, endoscopic balloon dilatation, metallic stent implantation and later overall colonoscopy, coupled with the newfound knowledge of compound Qingdai pill-taking, led to a subsequent diagnosis of ischemic or toxic bowel disease with sigmoid colon stenosis. The patient was successfully treated by laparoscopic sigmoid colectomy, and postoperative pathological examination revealed ischemic or toxic injury of the sigmoid colon, providing a final diagnosis of drug-induced sigmoid colon stenosis. This case highlights that adequate awareness of drug-induced colon stenosis has a decisive role in avoiding misdiagnosis and mistreatment. The diagnostic and therapeutic experiences learnt from this case suggest that endoscopic balloon expansion and colonic metallic stent implantation as bridge treatments were demonstrated as crucial for the differential diagnosis of benign colonic stenosis. Skillful surgical technique and appropriate perioperative management helped to ensure the safety of our patient in subsequent surgery after long-term use of glucocorticoids.
Assuntos
Colo Sigmoide/efeitos dos fármacos , Constrição Patológica/diagnóstico , Diarreia/diagnóstico , Medicamentos de Ervas Chinesas/efeitos adversos , Doenças Inflamatórias Intestinais/diagnóstico , Obstrução Intestinal/diagnóstico , Pitiríase Rósea/tratamento farmacológico , Dor Abdominal/etiologia , Dor Abdominal/terapia , Antibacterianos/uso terapêutico , Biópsia , Colectomia/métodos , Colo Sigmoide/diagnóstico por imagem , Colo Sigmoide/patologia , Colo Sigmoide/cirurgia , Colonografia Tomográfica Computadorizada , Colonoscopia/instrumentação , Colonoscopia/métodos , Constipação Intestinal/etiologia , Constrição Patológica/induzido quimicamente , Constrição Patológica/complicações , Constrição Patológica/terapia , Meios de Contraste/administração & dosagem , Diagnóstico Diferencial , Diarreia/etiologia , Diarreia/microbiologia , Diatrizoato de Meglumina/administração & dosagem , Dilatação/métodos , Feminino , Hidratação , Hemorragia Gastrointestinal/etiologia , Hemorragia Gastrointestinal/terapia , Humanos , Obstrução Intestinal/induzido quimicamente , Obstrução Intestinal/complicações , Obstrução Intestinal/terapia , Laparoscopia/métodos , Levofloxacino/uso terapêutico , Pessoa de Meia-Idade , Stents Metálicos AutoexpansíveisRESUMO
Erythromycin is a kind of antibiotic drugs with certain biological toxicity. In order to investigate the inhibitory effect of erythromycin on methanogens and its acclimation capacity, Anaerobic Toxicity Assay (ATA) and continuous experiment were conducted in anaerobic bottles and the Up-flow Anaerobic Sludge Blanket Reactor (UASB), respectively, to determine the accumulated methane production, ratio of methane production rate, COD removal efficiency, and methane content. The results showed that the methane production ratio was reduced to 56.1% in the presence of 150 mg x L(-1) of erythromycin and it was reduced by 99% when the erythromycin reached 250 mg x L(-1), indicating that the activity was completely inhibited. Keeping the erythromycin at an concentration of 20 mg x L(-1) in the process of continuous operation for 60d, the COD removal efficiency and methane content reached up to 81.4% and 64.2%, respectively. The results suggested that erythromycin had an inhibitory effect on methane bacteria, and the half inhibitory concentration was 150 mg x L(-1) (IC50:150 mg x L(-1)). The COD removal efficiency and methane content were increased by 15.13% and 22.05%, respectively, after domestication for 60 d.