Crystal structures of arginine sensor CASTOR1 in arginine-bound and ligand free states

The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a master regulator of metabolism and cell growth. Among the numerous extracellular and intracellular signals, certain amino acids activate mTORC1 in a Rag-dependent manner. Arginine can stimulate mTORC1 activity by releasing the inhibitor CASTOR1 (Cellular Arginine Sensor of mTORC1) from GATOR2, a positive regulator of mTORC1 which interacts with GATOR1, the GAP for RagA/B. Three groups have resolved the structures of arginine-CASTOR1 complex, shedding a new light on molecular basis of the regulation of mTORC1 activity by arginine. However, lacking the apo structure of CASTOR1 prelimited the molecular understanding of mechanism underlying mTORC1 regulation. Here, we report crystal structures of arginine sensor CASTOR1 in arginine-bound and ligand free states at 2.05?Å and 2.8?Å, respectively. Structural comparison of CASTOR1 between two states reveals near identical conformations, except in two loop regions. It indicates CASTOR1 does not undergo large conformational change during arginine binding. Therefore, we conclude a detailed structural interpretation of arginine sensing by CASTOR1 in mTORC1 pathway.     

Fertilisation practice changes rhizosphere microbial community structure in the agroecosystem

Rhizosphere microbial community is important for the acquisition of soil nutrients and closely related to plant species. Fertilisation practice changed soil quality. With the hypothesis of stronger rhizosphere effect of plant on rhizosphere microbial community than fertilisation management, we designed this research based on a long‐term field experiment (1982–present). This study consists of no fertilisation (NF), mineral fertilisers (NPK), mineral fertilisers plus 7,500 kg/ha of wheat straw addition (WS) and mineral fertilisers plus 30,000 kg/ha of cow manure (CM). After analysing, we found that fertilisation management not only elevated crop yield but also affected crop rhizosphere microbial community structure. The influence of fertilisation practice on wheat rhizosphere microbial structure was stronger than that of wheat. For wheat rhizosphere bacterial community, it was significantly affected by soil water content (SWC), nitrogen (TN), phosphorus (TP), pH, available phosphorus (AVP) and nitrogen (AVN), dissolved organic nitrogen (DON) and carbon (DOC). Besides SWC, pH, AVP, AVN, TN, TP and DOC, the wheat rhizosphere fungi community was also significantly affected by soil organic matter (SOM) and available potassium (AVK). Moreover, compared to rhizosphere bacterial community, the influences of soil physiochemical properties on rhizosphere fungal community was stronger. In conclusion, fertilisation practice was the primary factor structuring rhizosphere microbial community by changing soil nutrients availabilities in the agroecosystem.     

Front Cover Image,Volume 116, Number 11, November 2019

CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome ( www.ibiodesign.net/gBIG ).     

Trophoblast–endothelium signaling involves angiogenesis and apoptosis in a dynamic bioprinted placenta model

Trophoblast invasion and remodeling of the maternal spiral arteries are required for pregnancy success. Aberrant endothelium–trophoblast crosstalk may lead to preeclampsia, a pregnancy complication that has serious effects on both the mother and the baby. However, our understanding of the mechanisms involved in this pathology remains elementary because the current in vitro models cannot describe trophoblast–endothelium interactions under dynamic culture. In this study, we developed a dynamic three-dimensional (3D) placenta model by bioprinting trophoblasts and an endothelialized lumen in a perfusion bioreactor. We found the 3D printed perfusion bioreactor system significantly augmented responses of endothelial cells by encouraging network formations and expressions of angiogenic markers, cluster of differentiation 31 (CD31), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor A (VEGFA). Bioprinting favored colocalization of trophoblasts with endothelial cells, similar to in vivo observations. Additional analysis revealed that trophoblasts reduced the angiogenic responses by reducing network formation and motility rates while inducing apoptosis of endothelial cells. Moreover, the presence of endothelial cells appeared to inhibit trophoblast invasion rates. These results clearly demonstrated the utility and potential of bioprinting and perfusion bioreactor system to model trophoblast–endothelium interactions in vitro. Our bioprinted placenta model represents a crucial step to develop advanced research approach that will expand our understanding and treatment options of preeclampsia and other pregnancy-related pathologies.     

De novo phenol bioproduction from glucose using biosensor-assisted microbial coculture engineering

Microbial biosynthesis has been extensively adapted for the production of commodity chemicals using renewable feedstocks. This study integrated metabolite biosensors into rationally designed microbial cocultures to achieve high-efficiency bioproduction of phenol from simple carbon substrate glucose. Specifically, two sets of E. coli–E. coli cocultures were first constructed for accommodation of two independent phenol biosynthesis pathways via 4-hydroxybenzoate (4HB) and tyrosine (TYR), respectively. Biosensor-assisted microbial cell selection mechanisms were subsequently incorporated into the coculture systems to address the insufficient pathway intermediate provision that limited the overall bioproduction. For the 4HB- and TYR-dependent pathways, this approach improved the phenol production by 2.3- and 3.9-fold, respectively, compared to the monoculture controls. Notably, the use of biosensor-assisted cell selection strategy in monocultures resulted in reduced phenol production, highlighting the advantage of coculture engineering for coupling with biosensing. After stepwise optimization, the phenol bioproduction yield of the engineered coculture's reached 0.057 g/g glucose. Furthermore, the coculture biosynthesis was successfully scaled up at both shake flask and bioreactor levels. Overall, the findings of this study demonstrate the outstanding potential of coupling biosensing and modular coculture engineering for advancing microbial biosynthesis of valuable molecules from renewable carbon substrates.     

l-Theanine and NEP1-40 promote nerve regeneration and functional recovery after brachial plexus root avulsion

Brachial plexus root avulsion causes severe sequelae Treatments and prognosis face many problems, including inflammatory reaction, oxidative damage, and myelin related inhibitory effect. l-Theanine has anti-inflammatory, anti-oxidative, and neuroprotective effects. NEP1-40 competitively inhibits Nogo-66 receptor (NgR1) promotes axonal regeneration. Forty-eight Sprague-Dawley rats were randomly assigned into four groups to establish an animal model of brachial plexus root avulsion. Inflammation and oxidative damage were evaluated by spectrophotometry and motor function of the upper limbs was assessed via Terzis grooming test after modeling. Immunofluorescence and hematoxylin and eosin staining were utilized to determine the content of reactive oxygen species, activation of microglial cells, neuroprotection, and nerve regeneration. Compared with the control group, the L-Theanine + NEP1-40 group had significantly decreased myeloperoxidase, malondialdehyde, interleukin-6, reactive oxygen species, and microglial cells, significantly increased score on the Terzis grooming test, increased motor neuron content, and thickened muscle fibers, increased area, and appearance of large and clear motor endplate structures. The results of this study suggest that l-Theanine combined with NEP1-40significantly promoted nerve regeneration after brachial plexus root avulsion, and may be a potential treatment for promoting nerve regeneration. Possible mechanisms underlying these results are alleviation of oxidative damage and inflammatory responses in the injured area and antagonism of myelin inhibition.     

Extracellular αB-crystallin modulates the inflammatory responses

Neuroinflammation is considered a challenging clinical problem. Chronic inflammatory responses play important roles in the onset and progression of various neurodegenerative diseases, including multiple sclerosis (MS). Previous studies have shown that astrocytes express small heat shock protein αB-crystallin (CRYAB) which is capable of inhibiting inflammatory responses in astrocytes per se. However, the underlying mechanisms of CRYAB-induced modulation of neuroinflammation are still not fully understood. In the present study, we investigated the role of extracellular CRYAB in the interaction between microglia and astrocytes in the context of MS-associated neuroinflammation. We found that the expression of CRYAB was profoundly increased in EAE mice. CRYAB was preferentially expressed in astrocytes and could be secreted via exosomes. Levels of exosomal CRYAB secreted from astrocytes were markedly increased under stress conditions. Furthermore, incubation of immortalized astrocytes or microglia cell lines with CRYAB remarkably suppressed astrocytes and microglia-mediated inflammatory responses in both autocrine and paracrine manners. Our results reveal a novel function for extracellular CRYAB in the regulation of neuroinflammation. Targeting extracellular CRYAB-modulated neuroinflammation is a potential therapeutic intervention for MS.     

Estrogen inhibits osteoclasts formation and bone resorption via microRNA-27a targeting PPARγ and APC

Inhibition of osteoclasts formation and bone resorption by estrogen is very important in the etiology of postmenopausal osteoporosis. The mechanisms of this process are still not fully understood. Recent studies implicated an important role of microRNAs in estrogen-mediated responses in various cellular processes, including cell differentiation and proliferation. Thus, we hypothesized that these regulatory molecules might be implicated in the process of estrogen-decreased osteoclasts formation and bone resorption. Western blot, quantitative real-time polymerase chain reaction, tartrate-resistant acid phosphatase staining, pit formation assay and luciferase assay were used to investigate the role of microRNAs in estrogen-inhibited osteoclast differentiation and bone resorption. We found that estrogen could directly suppress receptor activator of nuclear factor B ligand/macrophage colony-stimulating factor-induced differentiation of bone marrow-derived macrophages into osteoclasts in the absence of stromal cell. MicroRNA-27a was significantly increased during the process of estrogen-decreased osteoclast differentiation. Overexpressing of microRNA-27a remarkably enhanced the inhibitory effect of estrogen on osteoclast differentiation and bone resorption, whereas which were alleviated by microRNA-27a depletion. Mechanistic studies showed that microRNA-27a inhibited peroxisome proliferator-activated receptor gamma (PPARγ) and adenomatous polyposis coli (APC) expression in osteoclasts through a microRNA-27a binding site within the 3′-untranslational region of PPARγ and APC. PPARγ and APC respectively contributed to microRNA-27a-decreased osteoclast differentiation and bone resorption. Taken together, these results showed that microRNA-27a may play a significant role in the process of estrogen-inhibited osteoclast differentiation and function.     

Non-invasive estimation of root zone soil moisture from coarse root reflections in ground-penetrating radar images

Plant and Soil - Root zone soil moisture is an important component in water cycling through the soil-plant-atmosphere continuum. However, its measurement in the field remains a challenge,...