scholarly journals The Aflibercept-Induced MicroRNA Profile in the Vitreous of Proliferative Diabetic Retinopathy Patients Detected by Next-Generation Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Ju Guo ◽  
Pengyi Zhou ◽  
Zhenhui Liu ◽  
Fangfang Dai ◽  
Meng Pan ◽  
...  
Keyword(s):  
Diabetic Retinopathy ◽  
Next Generation Sequencing ◽  
Treatment Group ◽  
Proliferative Diabetic Retinopathy ◽  
Function Analysis ◽  
Group Versus ◽  
Next Generation ◽  
Target Mrnas ◽  
Intravitreal Aflibercept ◽  
Generation Sequencing

Purpose: Vascular endothelial growth factor-A (VEGF-A) is an important pathogenic factor in proliferative diabetic retinopathy (PDR), and aflibercept (Eylea) is one of the widely used anti-VEGF agents. This study investigated the microRNA (miRNA) profiles in the vitreous of 5 idiopathic macular hole patients (non-diabetic controls), 5 untreated PDR patients (no-treatment group), and 5 PDR patients treated with intravitreal aflibercept injection (treatment group).Methods: Next-generation sequencing was performed to determine the miRNA profiles. Deregulated miRNAs were validated with quantitative real-time PCR (qRT-PCR) in another cohort. The mRNA profile data (GSE160310) of PDR patients were retrieved from the Gene Expression Omnibus (GEO) database. The function of differentially expressed miRNAs and mRNAs was annotated by bioinformatic analysis and literature study.Results: Twenty-nine miRNAs were significantly dysregulated in the three groups, of which 19,984 target mRNAs were predicted. Hsa-miR-3184-3p, hsa-miR-24-3p, and hsa-miR-197-3p were validated to be remarkably upregulated in no-treatment group versus controls, and significantly downregulated in treatment group versus no-treatment group. In the GSE160310 profile, 204 deregulated protein-coding mRNAs were identified, and finally 179 overlapped mRNAs between the 19,984 target mRNAs and 204 deregulated mRNAs were included for further analysis. Function analysis provided several roles of aflibercept-induced miRNAs, promoting the alternation of drug sensitivity or resistance-related mRNAs, and regulating critical mRNAs involved in angiogenesis and retinal fibrosis.Conclusion: Hsa-miR-3184-3p, hsa-miR-24-3p, and hsa-miR-197-3p were highly expressed in PDR patients, and intravitreal aflibercept injection could reverse this alteration. Intravitreal aflibercept injection may involve in regulating cell sensitivity or resistance to drug, angiogenesis, and retinal fibrosis.

scholarly journals A Transposon Mutagenesis System forBifidobacterium longumsubsp.longumBased on an IS3Family Insertion Sequence, ISBlo11

2018 ◽  
Vol 84 (17) ◽  
Author(s):  
Mikiyasu Sakanaka ◽  
Shingo Nakakawaji ◽  
Shin Nakajima ◽  
Satoru Fukiya ◽  
Arisa Abe ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Insertion Sequence ◽  
Function Analysis ◽  
Bacterial Species ◽  
Bifidobacterium Longum ◽  
Transposon Mutagenesis ◽  
Insertion Mutant ◽  
Next Generation ◽  
Content Type ◽  
Generation Sequencing

ABSTRACTBifidobacteria are a major component of the intestinal microbiota in humans, particularly breast-fed infants. Therefore, elucidation of the mechanisms by which these bacteria colonize the intestine is desired. One approach is transposon mutagenesis, a technique currently attracting much attention because, in combination with next-generation sequencing, it enables exhaustive identification of genes that contribute to microbial fitness. We now describe a transposon mutagenesis system forBifidobacterium longumsubsp.longum105-A (JCM 31944) based on ISBlo11, a native IS3family insertion sequence. To build this system, xylose-inducible or constitutive bifidobacterial promoters were tested to drive the expression of full-length or a truncated form at the N terminus of the ISBlo11transposase. An artificial transposon plasmid, pBFS12, in which ISBlo11terminal inverted repeats are separated by a 3-bp spacer, was also constructed to mimic the transposition intermediate of IS3elements. The introduction of this plasmid into a strain expressing transposase resulted in the insertion of the plasmid with an efficiency of >103CFU/μg DNA. The plasmid targets random 3- to 4-bp sequences, but with a preference for noncoding regions. This mutagenesis system also worked at least inB. longumNCC2705. Characterization of a transposon insertion mutant revealed that a putative α-glucosidase mediates palatinose and trehalose assimilation, demonstrating the suitability of transposon mutagenesis for loss-of-function analysis. We anticipate that this approach will accelerate functional genomic studies ofB. longumsubsp.longum.IMPORTANCESeveral hundred species of bacteria colonize the mammalian intestine. However, the genes that enable such bacteria to colonize and thrive in the intestine remain largely unexplored. Transposon mutagenesis, combined with next-generation sequencing, is a promising tool to comprehensively identify these genes but has so far been applied only to a small number of intestinal bacterial species. In this study, a transposon mutagenesis system was established forBifidobacterium longumsubsp.longum, a representative health-promotingBifidobacteriumspecies. The system enables the identification of genes that promote colonization and survival in the intestine and should help illuminate the physiology of this species.

Labordiagnostik bei gastrointestinalen Tumoren

2020 ◽  
Vol 11 (05) ◽  
pp. 232-238
Author(s):  
Marcus Kleber
Keyword(s):  
Next Generation Sequencing ◽  
Kolorektales Karzinom ◽  
Next Generation ◽  
Generation Sequencing

ZUSAMMENFASSUNGDas kolorektale Karzinom (KRK) ist einer der häufigsten malignen Tumoren in Deutschland. Einer frühzeitigen Diagnostik kommt große Bedeutung zu. Goldstandard ist hier die Koloskopie. Die aktuelle S3-Leitlinie Kolorektales Karzinom empfiehlt zum KRK-Screening den fäkalen okkulten Bluttest. Für das Monitoring von Patienten vor und nach Tumorresektion werden die Messung des Carcinoembryonalen Antigens (CEA) und der Mikrosatellitenstabilität empfohlen. Für die Auswahl der korrekten Chemotherapie scheint derzeit eine Überprüfung des Mutationsstatus, mindestens des KRAS-Gens und des BRAF-Gens, sinnvoll zu sein. Eine Reihe an neuartigen Tumormarkern befindet sich momentan in der Entwicklung, hat jedoch noch nicht die Reife für eine mögliche Anwendung in der Routinediagnostik erreicht. Den schnellsten Weg in die breite Anwendung können Next-Generation-Sequencing-basierte genetische Tests finden.

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