Antiviral type III CRISPR signalling via conjugation of ATP and SAM.

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements1,2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against mobile genetic elements either directly, via the integral histidine-aspartate (HD) nuclease domain3-5 or indirectly, via synthesis of cyclic oligoadenylate second messengers to activate diverse ancillary effectors6-9. A subset of type III CRISPR systems encode an uncharacterized CorA-family membrane protein and an associated NrN family phosphodiesterase that are predicted to function in antiviral defence. Here we demonstrate that the CorA-associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against mobile genetic elements when expressed in Escherichia coli. However, B. fragilis Cmr does not synthesize cyclic oligoadenylate species on activation, instead generating S-adenosyl methionine (SAM)-AMP (SAM is also known as AdoMet) by conjugating ATP to SAM via a phosphodiester bond. Once synthesized, SAM-AMP binds to the CorA effector, presumably leading to cell dormancy or death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR-associated phosphodiesterases or a SAM-AMP lyase, potentially providing an 'off switch' analogous to cyclic oligoadenylate-specific ring nucleases10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.

Antiviral signalling by a cyclic nucleotide activated CRISPR protease.

CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA3-5. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates6,7, a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family7 fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system.

RNA processing by the CRISPR-associated NYN ribonuclease.

CRISPR-Cas systems confer adaptive immunity in prokaryotes, facilitating the recognition and destruction of invasive nucleic acids. Type III CRISPR systems comprise large, multisubunit ribonucleoprotein complexes with a catalytic Cas10 subunit. When activated by the detection of foreign RNA, Cas10 generates nucleotide signalling molecules that elicit an immune response by activating ancillary effector proteins. Among these systems, the Bacteroides fragilis type III CRISPR system was recently shown to produce a novel signal molecule, SAM-AMP, by conjugating ATP and SAM. SAM-AMP regulates a membrane effector of the CorA family to provide immunity. Here, we focus on NYN, a ribonuclease encoded within this system, probing its potential involvement in crRNA maturation. Structural modelling and in vitro ribonuclease assays reveal that NYN displays robust sequence-nonspecific, Mn2+-dependent ssRNA-cleavage activity. Our findings suggest a role for NYN in trimming crRNA intermediates into mature crRNAs, which is necessary for type III CRISPR antiviral defence. This study sheds light on the functional relevance of CRISPR-associated NYN proteins and highlights the complexity of CRISPR-mediated defence strategies in bacteria.

Two Cryptic Self-Resistance Mechanisms in Streptomyces tenebrarius Reveal Insights into the Biosynthesis of Apramycin.

Apramycin is a clinically promising aminoglycoside antibiotic (AGA). To date, mechanisms underlying the biosynthesis and self-resistance of apramycin remain largely unknown. Here we report that apramycin biosynthesis proceeds through unexpected phosphorylation, deacetylation, and dephosphorylation steps, in which a novel aminoglycoside phosphotransferase (AprU), a putative creatinine amidohydrolase (AprP), and an alkaline phosphatase (AprZ) are involved. Biochemical characterization revealed that AprU specifically phosphorylates 5-OH of a pseudotrisaccharide intermediate, whose N-7' acetyl group is subsequently hydrolyzed by AprP. AprZ is located extracellularly where it removes the phosphate group from a pseudotetrasaccharide intermediate, leading to the maturation of apramycin. Intriguingly, 7'-N-acetylated and 5-O-phosphorylated apramycin that were accumulated in ΔaprU and ΔaprZ respectively exhibited significantly reduced antibacterial activities, implying Streptomyces tenebrarius employs C-5 phosphorylation and N-7' acetylation as two strategies to avoid auto-toxicity. Significantly, this study provides insight into the design of new generation AGAs to circumvent the emergence of drug-resistant pathogens.

Retinal fundus image super-resolution based on generative adversarial network guided with vascular structure prior.

Many ophthalmic and systemic diseases can be screened by analyzing retinal fundus images. The clarity and resolution of retinal fundus images directly determine the effectiveness of clinical diagnosis. Deep learning methods based on generative adversarial networks are used in various research fields due to their powerful generative capabilities, especially image super-resolution. Although Real-ESRGAN is a recently proposed method that excels in processing real-world degraded images, it suffers from structural distortions when super-resolving retinal fundus images are rich in structural information. To address this shortcoming, we first process the input image using a pre-trained U-Net model to obtain a structural segmentation map of the retinal vessels and use the segmentation map as the structural prior. The spatial feature transform layer is then used to better integrate the structural prior into the generation process of the generator. In addition, we introduce channel and spatial attention modules into the skip connections of the discriminator to emphasize meaningful features and accordingly enhance the discriminative power of the discriminator. Based on the original loss functions, we introduce the L1 loss function to measure the pixel-level differences between the segmentation maps of retinal vascular structures in the high-resolution images and the super-resolution images to further constrain the super-resolution images. Simulation results on retinal image datasets show that our improved algorithm results have a better visual performance by suppressing structural distortions in the super-resolution images.

Perception-oriented generative adversarial network for retinal fundus image super-resolution.

Retinal fundus imaging is a crucial diagnostic tool in ophthalmology, enabling the early detection and monitoring of various ocular diseases. However, capturing high-resolution fundus images often presents challenges due to factors such as defocusing and diffraction in the digital imaging process, limited shutter speed, sensor unit density, and random noise in the image sensor or during image transmission. Super-resolution techniques offer a promising solution to overcome these limitations and enhance the visual details in retinal fundus images. Since the retina has rich texture details, the super-resolution images often introduce artifacts into texture details and lose some fine retinal vessel structures. To improve the perceptual quality of the retinal fundus image, a generative adversarial network that consists of a generator and a discriminator is proposed. The proposed generator mainly comprises 23 multi-scale feature extraction blocks, an image segmentation network, and 23 residual-in-residual dense blocks. These components are employed to extract features at different scales, acquire the retinal vessel grayscale image, and extract retinal vascular features, respectively. The generator has two branches that are mainly responsible for extracting global features and vascular features, respectively. The extracted features from the two branches are fused to better restore the super-resolution image. The proposed generator can restore more details and more accurate fine vessel structures in retinal images. The improved discriminator is proposed by introducing our designed attention modules to help the generator yield clearer super-resolution images. Additionally, an artifact loss function is also introduced to enhance the generative adversarial network, enabling more accurate measurement of the disparity between the high-resolution image and the restored image. Experimental results show that the generated images obtained by our proposed method have a better perceptual quality than the state-of-the-art image super-resolution methods.

[Wrist arthroscopic treatment of Die-punch fracture of distal radius].

OBJECTIVE: To evaluate the effect of open reduction assisted by wrist arthroscopy in the treatment of Diepunch fracture of the distal radius. METHODS: The clinical data of 50 patients with die punch fracture of distal radius from December 2015 to May 2017 were analyzed retrospectively, including 31 males and 19 females, aged 20 to 45 (34.12±2.56) years. All patients were treated with open reduction and internal fixation of volar plate through volar approach under the assistance of wrist arthroscope. The range of wrist movement and Cooney wrist function score before and after treatment were compared. RESULTS: All patients were followed up with an average of 18 months. DR scan showed that all fractures healed and no shortening of radial axis. Three cases of incision infection occurred and disappeared after treatment. At 18 months after operation, the range of wrist movement was significantly larger than that before operation (P<0.05) . At 18 months after operation, Cooney wrist function score was higher than that before operation (P<0.05) , excellent in 33 cases, good in 13 cases, fair in 3 cases and poor in 1 case. CONCLUSION: The treatment of die punch fracture of the distal radius with open reduction assisted by arthroscopy can restore the flatness of the joint surface, promote the recovery of the function of the wrist joint quickly, and has high safety, which is worth popularizing.

[Genomics approaches decode natural products synthesis].

Novel natural products have always been the most important sources for discovery of new drugs. Since the end of the 20th century, advances in genomics technology have contributed to decode and analyze numerous genomes, revealing remarkable potential for production of new natural products in organisms. However, this potential is hampered by laboratory culture conditions. Therefore, the integration of all these new advances is necessary to unveil these treasures, addressing the rise in resistance to antibiotics. In this review, we discuss the strategies of genome mining, inducing the expression of silent biosynthetic gene clusters and construction of biological chassis.

Engineering and modification of microbial chassis for systems and synthetic biology.

Engineering and modifying synthetic microbial chassis is one of the best ways not only to unravel the fundamental principles of life but also to enhance applications in the health, medicine, agricultural, veterinary, and food industries. The two primary strategies for constructing a microbial chassis are the top-down approach (genome reduction) and the bottom-up approach (genome synthesis). Research programs on this topic have been funded in several countries. The 'Minimum genome factory' (MGF) project was launched in 2001 in Japan with the goal of constructing microorganisms with smaller genomes for industrial use. One of the best examples of the results of this project is E. coli MGF-01, which has a reduced-genome size and exhibits better growth and higher threonine production characteristics than the parental strain [1]. The 'cell factory' project was carried out from 1998 to 2002 in the Fifth Framework Program of the EU (European Union), which tried to comprehensively understand microorganisms used in the application field. One of the outstanding results of this project was the elucidation of proteins secreted by Bacillus subtilis, which was summarized as the 'secretome' [2]. The GTL (Genomes to Life) program began in 2002 in the United States. In this program, researchers aimed to create artificial cells both in silico and in vitro, such as the successful design and synthesis of a minimal bacterial genome by John Craig Venter's group [3]. This review provides an update on recent advances in engineering, modification and application of synthetic microbial chassis, with particular emphasis on the value of learning about chassis as a way to better understand life and improve applications.

Tissue-engineered nerve graft with tetramethylpyrazine for repair of sciatic nerve defects in rats.

A tissue-engineered nerve with tetramethylpyrazine (TMP) was repaired for sciatic nerve defects in rats. A total of 55 adult Sprague Dawley (SD) rats were classified into 4 groups, with 15 rats in each of groups A, B, and C as well as 10 rats in group D. About 1.5cm of a sciatic nerve of the right hind limb located 0.5cm below the inferior margin of the piriformis was resected to form the defects. Four types of nerve grafts used for bridging nerve defects in the SD rats corresponded to the 4 groups: tissue-engineered nerves with TMP in group A, tissue-engineered nerves without TMP in group B, acellular nerve grafts (ANGs) in group C, and autologous nerves in group D. Twelve weeks post-surgery, the sciatic functional index, nerve conduction velocity, and gastrocnemius wet weight of groups A and D were higher than those of groups B and C (P<0.05). Results of fluorescence microscopy and histological staining indicated that group A performed better than groups B and C (P<0.05). Similarly, the number of horseradish peroxidase-labeled positive cells was significantly larger in group A than in groups B and C. Regenerative nerve fibers were abundant in group A and consisted mainly of myelinated nerve fibers, which were better than those in groups B and C (P<0.05). The study demonstrated that tissue-engineered nerves constructed by ANGs seeded with neural stem cells and combined with TMP can effectively repair sciatic nerve defects in rats.