Summary• Using pharmacological and biochemical approaches, the role of cadmium (Cd)-induced carbon monoxide (CO) release and the relationship between CO and oxidative stress conferred by Cd exposure in the root tissues of alfalfa ( Medicago sativa ) plants were investigated.• Cd treatments showed a dose-dependent enhancement in lipid peroxidation. Both 100 and 200 µ M CdCl 2 treatments caused the increase of CO release, which is consistent with the changes in the activity of the CO synthetic enzyme heme oxygenase (HO) and its HO-1 transcript.• A 100 µ M CdCl 2 exposure enhanced the formation of nonprotein thiols (NPT), and reduced glutathione (GSH) to oxidized glutathione (GSSG), which was potentiated by the pretreatment of CO scavenger hemoglobin (Hb). Plants pretreated for 6 h with 50% CO-saturated aqueous solution, which induced the rapid endogenous CO release followed by a gradual decrease when subsequently exposed to 100 µ M CdCl 2 for 72 h, effectively decreased oxidative damage. Meanwhile, CO pretreatment modulated several enzymes responsible for GSH metabolism, thus resulting in the partial restoration of GSH : GSSG ratio, which was significantly blocked by Hb.• These results are suggestive of a role for CO release as a signal element for the alleviation of Cd-induced oxidative damage by modulating glutathione metabolism.

Bacterial CRISPR–Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR–Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR–Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.

Life sciences have been revolutionized by genome editing (GE) tools, including zinc finger nucleases, transcription activator‐Like effector nucleases, and CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas (CRISPR‐associated) systems, which make the targeted modification of genomic DNA of all organisms possible. CRISPR/Cas systems are being widely used because of their accuracy, efficiency, and cost‐effectiveness. Various classes of CRISPR/Cas systems have been developed, but their extensive use may be hindered by off‐target effects. Efforts are being made to reduce the off‐target effects of CRISPR/Cas9 by generating various CRISPR/Cas systems with high fidelity and accuracy. Several approaches have been applied to detect and evaluate the off‐target effects. Here, the current GE tools, the off‐target effects generated by GE technology, types of off‐target effects, mechanisms of off‐target effects, major concerns, and outcomes of off‐target effects in plants and animals are summarized. The methods to detect off‐target effects, tools for single‐guide RNA (sgRNA) design, evaluation and prediction of off‐target effects, and strategies to increase the on‐target efficiency and mitigate the off‐target impact on intended genome‐editing outcomes are summarized.

SummaryThe base‐editing technique using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, a novel G. hirsutum‐Base Editor 3 (GhBE3) base‐editing system has been developed to create single‐base mutations in the allotetraploid genome of cotton (Gossypium hirsutum). A cytidine deaminase sequence (APOBEC) fused with nCas9 and uracil glycosylase inhibitor (UGI) was inserted into our CRISPR/Cas9 plasmid (pRGEB32‐GhU6.7). Three target sites were chosen for two target genes, GhCLA and GhPEBP, to test the efficiency and accuracy of GhBE3. The editing efficiency ranged from 26.67 to 57.78% at the three target sites. Targeted deep sequencing revealed that the C→T substitution efficiency within an ‘editing window’, approximately six‐nucleotide windows of −17 to −12 bp from the PAM sequence, was up to 18.63% of the total sequences. The 27 most likely off‐target sites predicted by CRISPR‐P and Cas‐OFFinder tools were analysed by targeted deep sequencing, and it was found that rare C→T substitutions (average < 0.1%) were detected in the editing windows of these sites. Furthermore, whole‐genome sequencing analyses on two GhCLA‐edited and one wild‐type plants with about 100× depth showed that no bona fide off‐target mutations were detectable from 1500 predicted potential off‐target sites across the genome. In addition, the edited bases were inherited to T1 progeny. These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton.

Oxidative stress is a major pathogenic mechanism in Parkinson's disease (PD). As an important cellular antioxidant, glutathione (GSH) balances the production and incorporation of free radicals to protect neurons from oxidative damage. GSH level is decreased in the brains of PD patients. Hence, clarifying the molecular mechanism of GSH deficiency may help deepen our knowledge of PD pathogenesis. Here we report that the astrocytic dopamine D2 receptor (DRD2) regulates GSH synthesis via PKM2-mediated Nrf2 transactivation. In addition we find that pyridoxine can dimerize PKM2 to promote GSH biosynthesis. Further experiments show that pyridoxine supplementation increases the resistance of nigral dopaminergic neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in wild-type mice as well as in astrocytic Drd2 conditional knockout mice. We conclude that dimerizing PKM2 may be a potential target for PD treatment.

It has long been observed that rare earth elements (REEs) regulate multiple facets of plant growth and development. However, the underlying mechanisms remain largely unclear. Here, using electron microscopic autoradiography, we show the life cycle of a light REE (lanthanum) and a heavy REE (terbium) in horseradish leaf cells. Our data indicate that REEs were first anchored on the plasma membrane in the form of nanoscale particles, and then entered the cells by endocytosis. Consistently, REEs activated endocytosis in plant cells, which may be the cellular basis of REE actions in plants. Moreover, we discovered that a portion of REEs was successively released into the cytoplasm, self-assembled to form nanoscale clusters, and finally deposited in horseradish leaf cells. Taken together, our data reveal the life cycle of REEs and their cellular behaviors in plant cells, which shed light on the cellular mechanisms of REE actions in living organisms.activation | endocytic vesicle S ome trace elements, such as rare earth elements (REEs), have been observed for a long time to be beneficial to plant growth (1, 2). REEs are f-block elements in the periodic table, including 15 lanthanides, plus scandium and yttrium (2). The beneficial effect of REEs on plants was first reported in 1917, when it was found that cerium, an REE, improved physiological activities of water-floss (Spirogyra) (3). Since the 1970s, REEs have been widely used in agriculture as plant growth stimulants (4). Currently, more than 100 of crop species have been applied with REEs, which has increased crop yields by 5-15% (4). In addition, REEs also serve to protect plants against certain plant diseases and some stresses, such as drought, cold, acid rain, or heavy metals (2). The wide use of REEs has caused an overaccumulation of REEs in the ecosystem, including soil (5), atmosphere (6), and water (7). Moreover, it was shown that REEs have dual effects on plant growth: low concentrations of REEs exert positive effects on growth and yield of crops, whereas high concentrations of REEs seem to be harmful for plants, indicating that REEs are not absolutely good for plants (2). Therefore, it is imperative to elucidate how REEs act on plant cells to ensure food safety.Despite of many advances achieved in recent years, the action mechanisms of REEs on plant cells still remain poorly understood (2). Several explanations, which are mainly focused on the physiological roles of REEs in plant cells, have been proposed (2). These mechanisms include the possible changes of photosynthesis, mineral nutrient uptake and metabolism, catalytic activities of some enzymes, hormonal balance, and so forth (2). However, these hypotheses are not connected to any cellular or molecular principles and are therefore far from explaining the action mechanisms of REEs in plants (2,8). In addition, little consensus has yet been reached regarding the life cycle of REEs in plant cells. Some studies report that REEs cannot enter the plant cells but only be localized in apoplast (8-14); other st...

No abstract

To assess the impact of sanitation of a living environment on gut microbiota and development of the immune system, we raised BALB/c mice under three distinct environmental conditions: a specific pathogen-free animal room (SPF), a general animal room (XZ) and a farmhouse (JD). All other variables like diet, age, genetic background, physiological status and original gut microbiota were controlled for in the three groups. Using high-throughput sequencing of the 16S rRNA gene, we found that each mouse group had a specific structure of the gut microbial community. Groups JD and XZ harboured a significantly more diverse and richer gut microbiota than did group SPF. Bacteroidetes were significantly more abundant in groups XZ and JD than in group SPF, whereas Firmicutes showed the inverse pattern. Total serum immunoglobulin E (IgE) levels were significantly lower in groups XZ and JD than in group SPF. There were no significant differences in gut microbiota diversity and serum IgE concentration between groups JD and XZ, but we found higher abundance of dominant genera in the gut microflora of group JD. We conclude that exposure to soil, house dust and decaying plant material enhances gut microbial diversity and innate immunity. Our results seem to provide new evidence supporting the hygiene hypothesis.