Biology Open 

About: Biology Open is an academic journal published by The Company of Biologists. The journal publishes majorly in the area(s): Biology & Medicine. It has an ISSN identifier of 2046-6390. It is also open access. Over the lifetime, 2005 publications have been published receiving 32726 citations. The journal is also known as: Biol Open & BIO.

Nrf2 impacts cellular bioenergetics by controlling substrate availability for mitochondrial respiration

Abstract: Transcription factor Nrf2 and its repressor Keap1 regulate a network of cytoprotective genes involving more than 1% of the genome, their best known targets being drug-metabolizing and antioxidant genes. Here we demonstrate a novel role for this pathway in directly regulating mitochondrial bioenergetics in murine neurons and embryonic fibroblasts. Loss of Nrf2 leads to mitochondrial depolarisation, decreased ATP levels and impaired respiration, whereas genetic activation of Nrf2 increases the mitochondrial membrane potential and ATP levels, the rate of respiration and the efficiency of oxidative phosphorylation. We further show that Nrf2-deficient cells have increased production of ATP in glycolysis, which is then used by the F1Fo-ATPase for maintenance of the mitochondrial membrane potential. While the levels and in vitro activities of the respiratory complexes are unaffected by Nrf2 deletion, their activities in isolated mitochondria and intact live cells are substantially impaired. In addition, the rate of regeneration of NADH after inhibition of respiration is much slower in Nrf2-knockout cells than in their wild-type counterparts. Taken together, these results show that Nrf2 directly regulates cellular energy metabolism through modulating the availability of substrates for mitochondrial respiration. Our findings highlight the importance of efficient energy metabolism in Nrf2-mediated cytoprotection.

3D-structured illumination microscopy provides novel insight into architecture of human centrosomes.

Abstract: Centrioles are essential for the formation of cilia and flagella. They also form the core of the centrosome, which organizes microtubule arrays important for cell shape, polarity, motility and division. Here, we have used super-resolution 3D-structured illumination microscopy to analyse the spatial relationship of 18 centriole and pericentriolar matrix (PCM) components of human centrosomes at different cell cycle stages. During mitosis, PCM proteins formed extended networks with interspersed γ-Tubulin. During interphase, most proteins were arranged at specific distances from the walls of centrioles, resulting in ring staining, often with discernible density masses. Through use of site-specific antibodies, we found the C-terminus of Cep152 to be closer to centrioles than the N-terminus, illustrating the power of 3D-SIM to study protein disposition. Appendage proteins showed rings with multiple density masses, and the number of these masses was strongly reduced during mitosis. At the proximal end of centrioles, Sas-6 formed a dot at the site of daughter centriole assembly, consistent with its role in cartwheel formation. Plk4 and STIL co-localized with Sas-6, but Cep135 was associated mostly with mother centrioles. Remarkably, Plk4 formed a dot on the surface of the mother centriole before Sas-6 staining became detectable, indicating that Plk4 constitutes an early marker for the site of nascent centriole formation. Our study provides novel insights into the architecture of human centrosomes and illustrates the power of super-resolution microscopy in revealing the relative localization of centriole and PCM proteins in unprecedented detail.

Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation

Abstract: Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate.

Targeted mutagenesis using CRISPR/Cas system in medaka

Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-based RNA-guided endonuclease (RGEN) has recently emerged as a simple and efficient tool for targeted genome editing. In this study, we showed successful targeted mutagenesis using RGENs in medaka, Oryzias latipes. Somatic and heritable mutations were induced with high efficiency at the targeted genomic sequence on the DJ-1 gene in embryos that had been injected with the single guide RNA (sgRNA) transcribed by a T7 promoter and capped RNA encoding a Cas9 nuclease. The sgRNAs that were designed for the target genomic sequences without the 59 end of GG required by the T7 promoter induced the targeted mutations. This suggests that the RGEN can target any sequence adjacent to an NGG protospacer adjacent motif (PAM) sequence, which occurs once every 8 bp. The offtarget alterations at 2 genomic loci harboring double mismatches in the 18-bp targeting sequences were induced in the RGEN-injected embryos. However, we also found that the off-target effects could be reduced by lower dosages of sgRNA. Taken together, our results suggest that CRISPR/Cas-mediated RGENs may be an efficient and flexible tool for genome editing in medaka.

Effects of drought stress on photosynthesis and photosynthetic electron transport chain in young apple tree leaves.

Abstract: In our study, the effects of water stress on photosynthesis and photosynthetic electron transport chain (PETC) were studied in several ways, including monitoring the change of gas exchange parameters, modulated chlorophyll fluorescence, rapid fluorescence induction kinetics, reactive oxygen species (ROS), antioxidant enzyme activities and D1 protein levels in apple leaves. Our results show that when leaf water potential ( ψ w ) is above –1.5 MPa, the stomatal limitation should be the main reason for a drop of photosynthesis. In this period, photosynthetic rate ( P N ), stomatal conductance ( G s ), transpiration rate ( E ) and intercellular CO 2 concentration ( C i ) all showed a strong positive correlation with ψ w . Modulated chlorophyll fluorescence parameters related to photosynthetic biochemistry activity including maximum photochemical efficiency (F v /F m ), actual photochemical efficiency of PSII (Φ PSII ), photochemical quenching coefficient ( q P ) and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae ( q L ) also showed a strong positive correlation as ψ w gradually decreased. On the other hand, in this period, Stern-Volmer type non-photochemical quenching coefficient (NPQ) and quantum yield of light-induced non-photochemical fluorescence quenching [ Y (NPQ) ] kept going up, which shows an attempt to dissipate excess energy to avoid damage to plants. When ψ w was below –1.5 MPa, P N continued to decrease linearly, while C i increased and a ‘V’ model presents the correlation between C i and ψ w by polynomial regression. This implies that, in this period, the drop in photosynthesis activity might be caused by non-stomatal limitation. F v /F m , Φ PSII , q P and q L in apple leaves treated with water stress were much lower than in control, while NPQ and Y (NPQ) started to go down. This demonstrates that excess energy might exceed the tolerance ability of apple leaves. Consistent with changes of these parameters, excess energy led to an increase in the production of ROS including H 2 O 2 and O 2 • − . Although the activities of antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) increased dramatically and ascorbate peroxidase (APX) decreased in apple leaves with drought stress, it was still not sufficient to scavenge ROS. Consequently, the accumulation of ROS triggered a reduction of net D1 protein content, a core protein in the PSII reaction center. As D1 is responsible for the photosynthetic electron transport from plastoquinone A (Q A ) to plastoquinone B (Q B ), the capacity of PETC between Q A and Q B was considerably downregulated. The decline of photosynthesis and activity of PETC may result in the shortage of adenosine triphosphate (ATP) and limitation the regeneration of RuBP ( J max ), a key enzyme in CO 2 assimilation. These are all non-stomatal factors and together contributed to decreased CO 2 assimilation under severe water stress.