[Characteristics of Phytoplankton Communities and Key Impact Factors in Three Types of Lakes in Wuhan].

To reveal the characteristics and key impact factors of phytoplankton communities in different types of lakes, sampling surveys for phytoplankton and water quality parameters were conducted at 174 sampling sites in a total of 24 lakes covering urban, countryside, and ecological conservation areas of Wuhan in spring, summer, autumn, and winter 2018. The results showed that a total of 365 species of phytoplankton from nine phyla and 159 genera were identified in the three types of lakes. The main species were green algae, cyanobacteria, and diatoms, accounting for 55.34%, 15.89%, and 15.07% of the total number of species, respectively. The phytoplankton cell density varied from 3.60×106-421.99×106 cell·L-1, chlorophyll-a content varied from 15.60-240.50 µg·L-1, biomass varied from 27.71-379.79 mg·L-1, and the Shannon-Wiener diversity index varied from 0.29-2.86. In the three lake types, cell density, Chla, and biomass were lower in EL and UL, whereas the opposite was true for the Shannon-Wiener diversity index. NMDS and ANOSIM analysis showed differences in phytoplankton community structure (Stress=0.13, R=0.048, P=0.2298). In addition, the phytoplankton community structure of the three lake types had significant seasonal characteristics, with chlorophyll-a content and biomass being significantly higher in summer than in winter (P<0.05). Spearman correlation analysis showed that phytoplankton biomass decreased with increasing N:P in UL and CL, whereas the opposite was true for EL. Redundancy analysis (RDA) showed that WT, pH, NO3-, EC, and N:P were the key factors that significantly affected the variability in phytoplankton community structure in the three types of lakes in Wuhan (P<0.05).

Reactivation of γ-globin expression using a minicircle DNA system to treat ß-thalassemia.

Reactivation of fetal hemoglobin by editing the B-cell lymphoma/leukemia 11A (BCL11A) erythroid enhancer is an effective gene therapy for ß-thalassemia. Using the CRISPR/Cas9 system, fetal γ-globin expression can be robustly reactivated to mitigate the clinical course of ß-thalassemia. In our study, we found that the transfection efficiencies of CD34+ hematopoietic stem/progenitor cells (HSPCs) were significantly and negatively correlated with the length of plasmids and greatly affected by the linearization of plasmids. Furthermore, the transgene expression of minicircles (MC) without plasmid backbone sequences was better both in vitro and in vivo compared with conventional plasmids. Thus, MC DNA was used to deliver the cassette of Staphylococcus aureus Cas9 (SaCas9) into HSPCs, and a single-guide RNA targeting the erythroid enhancer region of BCL11A was selected. After electroporation with MC DNA, an evident efficiency of gene editing and reactivation of γ-globin expression in erythroblasts derived from unsorted HSPCs was acquired. No significant off-target effects were found by deep sequencing. Furthermore, fragments derived from lentiviral vectors, but not MC DNA, were highly enriched in promoter, exon, intron, distal-intergenic, and cancer-associated genes, indicating that MC DNA provided a relatively safe and efficient vector for delivering transgenes. The developed MC DNA vector provided a potential approach for the delivery of SaCas9 cassette and the reactivation of γ-globin expression for ameliorating syndromes of ß-thalassemia.

Effects of BmKIT 3 R gene transfer on pupal development of Bombyx mori Linnaeus using a Gal4/UAS binary transgenic system.

The pupal stage of the silkworm Bombyx mori Linnaeus lasts for approximately two weeks. However, prolongation of pupal duration would reduce the labor required to process and dry fresh cocoons. This study investigated the effects of BmKIT(3)(R) gene (from the Chinese scorpion Buthus martensii Karsch) transfer on the pupal development of B. mori using a Gal4/UAS binary transgenic system. Gal4 driven by a pupa-specific promoter BmWCP4 (from a B. mori wing-cuticle protein gene) or PDP (from a B. mori cocoonase gene), and BmKIT(3)(R) driven by a UAS cis-acting element were used to construct novel piggyBac-derived plasmids containing a neomycin-resistance gene (neo) controlled by the Bombyx mori nucleopolyhedrovirus (BmNPV) ie-1 (immediate-early gene) promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin 3 (A3) promoter. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes, and gene transfer was verified by polymerase chain reaction and dot-blot hybridization. The larval development of the hybrid progeny of Gal4- and UAS-transgenic silkworms was similar to that of normal silkworms, but some pupae failed to metamorphose into moths, and the development of surviving pupae was arrested as a result of BmKIT(3)(R) expression. Moreover, Gal4 driven by the BmWCP4 promoter delayed pupal development more effectively than that driven by the PDP promoter in the Gal4/UAS binary transgenic system. Pupal durations of hybrid transgenic silkworm progeny with BmWCP4 and PDP promoters were approximately 5, 2, and 4 days longer, respectively, compared to corresponding normal silkworms, BmWCP4/Gal4, and UAS/BmKIT(3)(R) transgenic silkworms, respectively. These results suggest new avenues of research for prolonging the pupal duration of silkworms.