Programmable RNA 5-methylcytosine (m5C) modification of cellular RNAs by dCasRx conjugated methyltransferase and demethylase.

5-Methylcytosine (m5C), an abundant RNA modification, plays a crucial role in regulating RNA fate and gene expression. While recent progress has been made in understanding the biological roles of m5C, the inability to introduce m5C at specific sites within transcripts has hindered efforts to elucidate direct links between specific m5C and phenotypic outcomes. Here, we developed a CRISPR-Cas13d-based tool, named reengineered m5C modification system (termed 'RCMS'), for targeted m5C methylation and demethylation in specific transcripts. The RCMS editors consist of a nuclear-localized dCasRx conjugated to either a methyltransferase, NSUN2/NSUN6, or a demethylase, the catalytic domain of mouse Tet2 (ten-eleven translocation 2), enabling the manipulation of methylation events at precise m5C sites. We demonstrate that the RCMS editors can direct site-specific m5C incorporation and demethylation. Furthermore, we confirm their effectiveness in modulating m5C levels within transfer RNAs and their ability to induce changes in transcript abundance and cell proliferation through m5C-mediated mechanisms. These findings collectively establish RCMS editors as a focused epitranscriptome engineering tool, facilitating the identification of individual m5C alterations and their consequential effects.

Optimization of Internet of Things Remote Desktop Protocol for Low-Bandwidth Environments Using Convolutional Neural Networks.

This paper discusses optimizing desktop image quality and bandwidth consumption in remote IoT GUI desktop scenarios. Remote desktop tools, which are crucial for work efficiency, typically employ image compression techniques to manage bandwidth. Although JPEG is widely used for its efficiency in eliminating redundancy, it can introduce quality loss with increased compression. Recently, deep learning-based compression techniques have emerged, challenging traditional methods like JPEG. This study introduces an optimized RFB (Remote Frame Buffer) protocol based on a convolutional neural network (CNN) image compression algorithm, focusing on human visual perception in desktop image processing. The improved RFB protocol proposed in this paper, compared to the unoptimized RFB protocol, can save 30-80% of bandwidth consumption and enhances remote desktop image quality, as evidenced by improved PSNR and MS-SSIM values between the remote desktop image and the original image, thus providing superior desktop image transmission quality.

Targeted mutagenesis in mice via an engineered AsCas12f1 system.

SpCas9 and AsCas12a are widely utilized as genome editing tools in human cells, but their applications are largely limited by their bulky size. Recently, AsCas12f1 protein, with a small size (422 amino acids), has been demonstrated to be capable of cleaving double-stranded DNA protospacer adjacent motif (PAM). However, low editing efficiency and large differences in activity against different genomic loci have been a limitation in its application. Here, we show that engineered AsCas12f1 sgRNA has significantly improved the editing efficiency in human cells and mouse embryos. Moreover, we successfully generated three stable mouse mutant disease models using the engineered CRISPR-AsCas12f1 system in this study. Collectively, our work uncovers the engineered AsCas12f1 system expands mini CRISPR toolbox, providing a remarkable promise for therapeutic applications.

A strategy for Cas13 miniaturization based on the structure and AlphaFold.

The small size of the Cas nuclease fused with various effector domains enables a broad range of function. Although there are several ways of reducing the size of the Cas nuclease complex, no efficient or generalizable method has been demonstrated to achieve protein miniaturization. In this study, we establish an Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturization and generate five compact variants of Cas13 with full RNA binding and cleavage activity comparable the wild-type enzymes based on a combination of IDC strategy and AlphaFold2. In addition, we construct an RNA base editor, mini-Vx, and a single AAV (adeno-associated virus) carrying a mini-RfxCas13d and crRNA expression cassette, which individually shows efficient conversion rate and RNA-knockdown activity. In summary, these findings highlight a feasible strategy for generating downsized CRISPR/Cas13 systems based on structure predicted by AlphaFold2, enabling targeted degradation of RNAs and RNA editing for basic research and therapeutic applications.

Development of Woolly Hair and Hairlessness in a CRISPR-Engineered Mutant Mouse Model with KRT71 Mutations.

Hypotrichosis simplex (HS) and woolly hair (WH) are rare and monogenic disorders of hair loss. HS, characterized by a diffuse loss of hair, usually begins in early childhood and progresses into adulthood. WH displays strong coiled hair involving a localized area of the scalp or covering the entire side. Mutations in the keratin K71(KRT71) gene have been reported to underlie HS and WH. Here, we report the generation of a mouse model of HS and WH by the co-injection of Cas9 mRNA and sgRNA, targeting exon6 into mouse zygotes. The Krt71-knockout (KO) mice displayed the typical phenotypes, including Krt71 protein expression deletion and curly hair in their full body. Moreover, we found that mice in 3-5 weeks showed a new phenomenon of the complete shedding of hair, which was similar to nude mice. However, we discovered that the mice exhibited no immune deficiency, which was a typical feature of nude mice. To our knowledge, this novel mouse model generated by the CRISPR/Cas9 system mimicked woolly hair and could be valuable for hair disorder studies.

Clinical study of Tongdu Shujin decoction in the treatment of ankylosing spondylitis with cold-dampness obstruction type: Study protocol for a randomized controlled trial.

BACKGROUND: Ankylosing spondylitis (AS) has a high incidence, and severe cases can lead to spinal deformity and even joint fusion, which causes a huge burden on patients life, work and psychology. Tongdu Shujin decoction (TDSJ) has a definite effect in the treatment of ankylosing spondylitis, so we designed a randomized controlled trial to observe the efficacy of TDSJ in the treatment of AS, and to evaluate its safety. METHODS: In this randomized controlled trial, 80 eligible patients were randomly assigned in a 1:1 ratio to a treatment group TDSJ and a control group (celecoxib capsules in combination with thalidomide tablets) for 8 weeks. Visual analogue scale, bath ankylosing spondylitis disease activity index, bath ankylosing spondylitis functional index, and traditional Chinese medicine syndrome scores will be used as primary indicators. Erythrocyte sedimentation rate, C-reactive protein, spinal mobility (figure-ground distance, occipital tubercle-wall distance, Schober test) will be used as secondary indicators. Vital signs (respiration, heart rate, body temperature, blood pressure, electrocardiogram), blood routine, urine routine, stool routine, liver function, and renal function will be used as safety indicators. The primary and secondary indicators will be detected at 0th and 8th week, while safety indicators at 0th, 4th, and 8th week. DISCUSSION: This study will provide high-quality clinical evidence for the efficacy and safety of TDSJ in the treatment of AS.

A comparison study between hybrid surgery and anterior cervical discectomy and fusion for the treatment of multilevel cervical spondylosis.

AIMS: Whether to perform hybrid surgery (HS) in contrast to anterior cervical discectomy and fusion (ACDF) when treating patients with multilevel cervical disc degeneration remains a controversial subject. To resolve this we have undertaken a meta-analysis comparing the outcomes from HS with ACDF in this condition. METHODS: Seven databases were searched for studies of HS and ACDF from inception of the study to 1 September 2019. Both random-effects and fixed-effects models were used to evaluate the overall effect of the C2-C7 range of motion (ROM), ROM of superior/inferior adjacent levels, adjacent segment degeneration (ASD), heterotopic ossification (HO), complications, neck disability index (NDI) score, visual analogue scale (VAS) score, Japanese Orthopaedic Association (JOA) score, Odom's criteria, blood loss, and operating and hospitalization time. To obtain more credible results contour-enhanced funnel plots, Egger's and Begg's tests, meta-regression, and sensitivity analyses were performed. RESULTS: In total, 17 studies involving 861 patients were included in the analysis. HS was found to be superior to ACDF in maintaining C2-C7 ROM and ROM of superior/inferior adjacent levels, but HS did not reduce the incidence of associated level ASD. Also, HS did not cause a higher rate of HO than ACDF. The frequency of complications was similar between the two techniques. HS failed to achieve more favourable outcomes than ACDF using the NDI, VAS, JOA, and Odom's scores. HS did not show any more advantages in operating or hospitalization time but did show reduction in blood loss. CONCLUSION: Although HS maintained cervical kinetics, it failed to reduce the incidence of ASD. This finding differs from previous reports. Moreover, patients did not show more benefits from HS with respect to symptom improvement, prevention of complications, and clinical outcomes. Cite this article: Bone Joint J 2020;102-B(8):981-996.

Correction to: Is cell transplantation a reliable therapeutic strategy for spinal cord injury in clinical practice? A systematic review and meta-analysis from 22 clinical controlled trials.

Zhao-he and Sun-qingling are the co-first authors for this manuscript in the initial submission. Because of author's negligence and fault, this information was not shown clearly in the originally published article.

Is cell transplantation a reliable therapeutic strategy for spinal cord injury in clinical practice? A systematic review and meta-analysis from 22 clinical controlled trials.

PURPOSE: It is an open question whether cell transplantation can provide safety and effective outcome to spinal cord injury (SCI) patient which has remained controversial for almost 40 years. This study aimed to evaluate the safety and efficacy of cell transplantation in SCI patients. METHOD: Studies of the cell transplantation for SCI were retrieved from PubMed, Embase, Medline, Cochrane Library and analyzed quantitative data by Review Manager 5.3. RESULTS: Twenty-one clinical controlled studies with 973 patients were included. The pooled results suggested that cell transplantation significantly improved ASIA score, ASIA motor score, ASIA sensory score, Barthel Index score, residual urine volume, rehabilitative time of automatic micturition. Furthermore, subgroup analysis indicated that the stem cells exhibited more potent than the non-stem cells in spinal cord repair. Cell transplantation at more than 14 days after injury showed more significant improvements than that within 14 days from injury. The dosage of cell transplantation between 1-5 × 107 and 10-20 × 107 was the potent quantity for the patient with SCI. Intrathecal injection and intravenous + intrathecal injection showed more superior to the other method. The top 5 adverse events were febrile reaction (11.5%), neurologic pain (11.3%), headache (2.6%), neurologic deterioration (2.4%), and rigidity or spasticity (1.6%). CONCLUSION: Cell transplantation appears to be a safe therapeutic strategy possessing substantial beneficial effects in the patients with SCI in clinic. Moreover, treating SCI with stem cell, the dosage of cells between 1-5 × 107 and 10-20 × 107, in intermediate or chronic phase, minimally invasive techniques, may bring more advantage to SCI patient. These slides can be retrieved under Electronic Supplementary Material.

[Application of perfusion bone cement screw in lumbar degenerative disease with osteoporosis].

OBJECTIVE: To observe the early clinical effect of perfusion bone cement screw for lumbar degenerative diseases with osteoporosis. METHODS: The clinical data of 28 patients with lumbar degenerative diseases combined with moderate to severe osteoporosis treated by posterior lateral graft fusion with perfusion of bone cement screws from June 2015 to June 2017 were retrospectively analyzed. There were 9 males and 19 females, aged from 55 to 86 years old with an average of 76 years. Anteroposterior, oblique, and dynamic radiography were performed before operation, and the diagnosis was confirmed by CT, MRI and dual energy X-ray absorptionmetry(DXA). All the patients had moderate to severe lumbar spinal stenosis, including 16 cases with degenerative scoliosis, 12 cases with degenerative lumbar spondylolisthesis, and 16 cases with lumbar disc herniation. According to Jikei grade of osteoporosis, 9 cases were grade II and 19 cases were grade III. Visual analogue scale (VAS) and Japanese Orthopaedic Association (JOA) score were used to assess the improvement of lumbar leg pain, and neurological function. Imaging data were used to observe the circumstance of pedicle loosening, prolapse, breakage and bone cement leakage, and comprehensively evaluate the fusion. RESULTS: The hospital stay was from 10 to 14 days with an average of 12 days; the operative time was 100 to 150 min with an average of 120 min;the blood loss was 200 to 600 ml with an average of 350 ml (for operations more than 3 vertebral segments, blood filtration recovery was intraoperatively used);the postoperative drainage volume was 150 to 600 ml with an average of 300 ml, no allogeneic blood was used in all the patients. Bone cement of 2 to 3 ml were injected into each vertebral body, and bone cement leakage occurred in 2 cases during injection, both of which were paravertebral vessel leakage, and there was no evidence of intravertebral leakage. The injection of bone cement was terminated in a timely manner without serious complications such as nerve injury, bone cement toxicity, and vascular embolization and pulmonary embolism. According to the fusion criteria by X-ray, 18 cases achieved strong bone fusion, and 10 cases were inaccurate fusion, but no pseudarthrosis occurred in the fusion segment. No screw loosening, prolapse or fracture were found, and postoperative VAS, JOA scores were significantly improved. CONCLUSIONS: Perfusion bone cement screw technique can obtain satisfactory effect in treating lumbar degenerative diseases with osteoporosis.