Droplet-based microfluidic platform for high-throughput screening of Streptomyces.

Streptomyces are one of the most important industrial microorganisms for the production of proteins and small-molecule drugs. Previously reported flow cytometry-based screening methods can only screen spores or protoplasts released from mycelium, which do not represent the filamentous stationary phase Streptomyces used in industrial cultivation. Here we show a droplet-based microfluidic platform to facilitate more relevant, reliable and rapid screening of Streptomyces mycelium, and achieved an enrichment ratio of up to 334.2. Using this platform, we rapidly characterized a series of native and heterologous constitutive promoters in Streptomyces lividans 66 in droplets, and efficiently screened out a set of engineered promoter variants with desired strengths from two synthetic promoter libraries. We also successfully screened out several hyperproducers of cellulases from a random S. lividans 66 mutant library, which had 69.2-111.4% greater cellulase production than the wild type. Our method provides a fast, simple, and powerful solution for the industrial engineering and screening of Streptomyces in more industry-relevant conditions.

Antisense RNA Interference-Enhanced CRISPR/Cas9 Base Editing Method for Improving Base Editing Efficiency in Streptomyces lividans 66.

CRISPR/Cas9-mediated base editors, based on cytidine deaminase or adenosine deaminase, are emerging genetic technologies that facilitate genomic manipulation in many organisms. Since base editing is free from DNA double-strand breaks (DSBs), it has certain advantages, such as a lower toxicity, compared to the traditional DSB-based genome engineering technologies. In terms of Streptomyces, a base editing method has been successfully applied in several model and non-model species, such as Streptomyces coelicolor and Streptomyces griseofuscus. In this study, we first proved that BE2 (rAPOBEC1-dCas9-UGI) and BE3 (rAPOBEC1-nCas9-UGI) were functional base editing tools in Streptomyces lividans 66, albeit with a much lower editing efficiency compared to that of S. coelicolor. Uracil generated in deamination is a key intermediate in the base editing process, and it can be hydrolyzed by uracil DNA glycosidase (UDG) involved in the intracellular base excision repair, resulting in a low base editing efficiency. By knocking out two endogenous UDGs (UDG1 and UDG2), we managed to improve the base editing efficiency by 3.4-67.4-fold among different loci. However, the inactivation of UDG is detrimental to the genome stability and future application of engineered strains. Therefore, we finally developed antisense RNA interference-enhanced CRISPR/Cas9 Base Editing method (asRNA-BE) to transiently disrupt the expression of uracil DNA glycosidases during base editing, leading to a 2.8-65.8-fold enhanced editing efficiency and better genome stability. Our results demonstrate that asRNA-BE is a much better editing tool for base editing in S. lividans 66 and might be beneficial for improving the base editing efficiency and genome stability in other Streptomyces strains.

Prenatal diagnosis of mosaic trisomy 2 and literature review.

BACKGROUND: We presented two cases of mosaic trisomy 2 with high risk of maternal serum screening and non-invasive prenatal testing (NIPT). The invasive amniocentesis was performed and genetic tests including karyotype, single nucleotide polymorphism array(SNP-array), interphase fluorescence in situ hybridization (FISH) were employed to detect the chromosomal abnormality. RESULTS: Cytogentic analysis of the case 1 and 2 showed a mosaic karyotype consisting of two cell lines (mos 47,XY,+2[8]/46,XY[19] and mos 47,XX,+2[7]/46,XX[28], respectively). SNP-array using DNA extracted from uncultured amniotic fluid cells revealed a result of arr[GRCh38](2)x2~3, which indicated that chromosome 2 may be trisomy of mosaicism in both two cases. The results of interphases FISH confirmation test showed that three red signals of the CEP 2 specific probe in 14%(14/100) and 12%(12/100) of the two cases' cells, respectively, which indicated a mosaicism for trisomy 2 in the uncultured amniocytes. Fetal ultrasound of case 1 suggested that the long bone is smaller than the gestational age, while the case 2 showed that the biparietal diameter (BPD), head circumference (HC) and femur length (FL) were smaller than gestational age along with abnormal cardiac structure. CONCLUSIONS: We presented two cases with mosaic trisomy 2 and performed confirmatory genetic testing using cultured and uncultured amniocytes. When maternal serum screening and NIPT suggesting high risk, genetic counselor should be alert for increasing possibility of chromosomal anomalies if combined with abnormal ultrasound findings.

[Combined chromosomal microarray analysis and fluorescence in situ hybridization for prenatal diagnosis of two cases with Pallister-Killian syndrome].

OBJECTIVE: To carry out prenatal diagnosis for two cases of Pallister-Killian syndrome (PKS) using combined chromosomal microarray analysis (CMA) and fluorescence in situ hybridization (FISH). METHODS: Umbilical cord blood was sampled from the two fetuses and subjected to G-banding chromosomal karyotyping, CMA and FISH assay. RESULTS: Chromosomal karyotyping showed that the two fetuses were mos 47,XX,+i(12)(p10)[3]/46,XX[197] and mos 47,XY,+i(12)(p10)[5]/46,XY[95], respectively. CMA showed that both had carried duplication of 12p. The results of interphase FISH confirmed mosaicism of 12p tetrasomy. Combined with ultrasonographic findings, both fetuses were diagnosed as PKS. CONCLUSION: Prenatal ultrasound examination, karyotype analysis of umbilical cord blood, G-banded chromosomal analysis, CMA and FISH may be used in conjunct for the prenatal diagnosis of PKS.

Landscape ecological security assessment based on projection pursuit in Pearl River Delta.

Regional landscape ecological security is an important issue for ecological security, and has a great influence on national security and social sustainable development. The Pearl River Delta (PRD) in southern China has experienced rapid economic development and intensive human activities in recent years. This study, based on landscape analysis, provides a method to discover the alteration of character among different landscape types and to understand the landscape ecological security status. Based on remotely sensed products of the Landsat 5 TM images in 1990 and the Landsat 7 ETM+ images in 2005, landscape classification maps of nine cities in the PRD were compiled by implementing Remote Sensing and Geographic Information System technology. Several indices, including aggregation, crush index, landscape shape index, Shannon's diversity index, landscape fragile index, and landscape security adjacent index, were applied to analyze spatial-temporal characteristics of landscape patterns in the PRD. A landscape ecological security index based on these outcomes was calculated by projection pursuit using genetic algorithm. The landscape ecological security of nine cities in the PRD was thus evaluated. The main results of this research are listed as follows: (1) from 1990 to 2005, the aggregation index, crush index, landscape shape index, and Shannon's diversity index of nine cities changed little in the PRD, while the landscape fragile index and landscape security adjacent index changed obviously. The landscape fragile index of nine cities showed a decreasing trend; however, the landscape security adjacent index has been increasing; (2) from 1990 to 2005, landscape ecology of the cities of Zhuhai and Huizhou maintained a good security situation. However, there was a relatively low value of ecological security in the cities of Dongguan and Foshan. Except for Foshan and Guangzhou, whose landscape ecological security situation were slightly improved, the cities had reduced values in landscape ecological security, with the most decreased number 0.52 in Zhaoqing. Results of this study offer important information for regional eco-construction and natural resource exploitation.

A novel route for the production of chitosan/poly(lactide-co-glycolide) graft copolymers for electrospinning.

Both chitosan and polylactide/polyglycolide have good biocompatibility and can be used to produce tissue engineering scaffolds for cultured cells. However the synthetic scaffolds lack groups that would facilitate their modification, whereas chitosan has extensive active amide and hydroxyl groups which would allow it to be subsequently modified for the attachment of peptides, proteins and drugs. Also chitosan is very hydrophilic, whereas PLGA is relatively hydrophobic. Accordingly there are many situations where it would be ideal to have a copolymer of both, especially one that could be electrospun to provide a versatile range of scaffolds for tissue engineering. Our aim was to develop a novel route of chitosan-g-PLGA preparation and evaluate the copolymers in terms of their chemical characterization, their performance on electrospinning and their ability to support the culture of fibroblasts as an initial biological evaluation of these scaffolds. Chitosan was first modified with trimethylsilyl chloride, and catalyzed by dimethylamino pyridine. PLGA-grafted chitosan copolymers were prepared by reaction with end-carboxyl PLGA (PLGA-COOH). FT-IR and(1)H-NMR characterized the copolymer molecular structure as being substantially different to that of the chitosan or PLGA on their own. Elemental analysis showed an average 18 pyranose unit intervals when PLGA-COOH was grafted into the chitosan molecular chain. Differential scanning calorimetry results showed that the copolymers had different thermal properties from PLGA and chitosan respectively. Contact angle measurements demonstrated that copolymers became more hydrophilic than PLGA. The chitosan-g-PLGA copolymers were electrospun to produce either nano- or microfibers as desired. A 3D fibrous scaffold of the copolymers gave good fibroblast adhesion and proliferation which did not differ significantly from the performance of the cells on the chitosan or PLGA electrospun scaffolds. In summary this work presents a methodology for making a hybrid material of natural and synthetic polymers which can be electrospun and reacts well as a substrate for cell culture.