Value of CT in targeted CT-guided epidural blood patching: Predictors for successful epidural punctures.

BACKGROUND AND PURPOSE: Differentiating epidural from intrathecal punctures before computed tomography (CT)-guided epidural blood patching (EBP) is subjective, relying on operator experience. This study aimed to investigate CT findings for epidural and intrathecal punctures and identify reliable predictors for successful epidural punctures before targeted CT-guided EBP. MATERIALS AND METHODS: We included 65 patients with low-cerebrospinal fluid (CSF)-pressure headache receiving targeted CT-guided EBP between January 2021 and October 2022 in this retrospective study. We analyzed clinical data, technical information, and CT features before EBP. Fisher's exact test was used for discrete variables, while Mann-Whitney U test was used for continuous variables. Positive (PLR) and negative likelihood ratios (NLR) were calculated to identify predictors for confirming epidural punctures. RESULTS: We confirmed 43 patients as epidural punctures and 22 patients as intrathecal punctures. Before contrast injection, epidural fat at the needle tip in the epidural group was higher than the intrathecal group (37.2 % [16/43] vs. 4.5 % [1/22], p = 0.006). After contrast injection, the "contrast-needle tip connection" sign was mostly observed in the epidural group than the intrathecal group (95.3 % [41/43] vs. 9.1 % [2/22], p < 0.001). Additionally, the epidural group had significantly higher boomerang-shaped contrast morphology than the intrathecal group (65.1 % [28/43] vs. 9.1 % [2/22], p < 0.001). The "contrast-needle tip connection" sign had the highest PLR (10.49) and lowest NLR (0.05). CONCLUSION: Identifying epidural fat at the needle tip, "contrast-needle tip connection" sign, and boomerang-shaped contrast morphology on CT scans are useful for confirming proper placement of the needle tip within the epidural space.

Efficacy and reliability of three-dimensional fusion guidance for fluoroscopic navigation in transarterial embolization for refractory musculoskeletal pain.

Background: This study aimed to evaluate the efficacy and reliability of three-dimensional (3D) fusion guidance in roadmapping for fluoroscopic navigation during trans-arterial embolization for refractory musculoskeletal pain (TAE-MSK pain) in the extremities. Methods: The included research patients were divided into two groups: group A-TAE-MSK pain performed without the use of 3D fusion guidance; group B-TAE-MSK pain performed with the use of 3D fusion guidance for fluoroscopic navigation. We compared the procedure time, radiation dose, visual analogue scale for pain scores, and adverse effects (before and 3 months after TAE-MSK pain) among the two groups. In the group B, we determined the reliability of ideal branch angle for pre-operative non-contrast 3D magnetic resonance angiography (MRA) and intra-operative 3D cone beam computed tomography (CBCT) angiography. Results: We recruited 65 patients, including 23 males and 42 females (average age 58.20±12.58 years), with 38 and 27 patients in groups A and B. A total of 247 vessels were defined as target branch vessels. Significant changes were observed in the fluoroscopy time which was 32.31±12.39 and 14.33±3.06 minutes, in group A and group B (P<0.001), respectively; procedure time, which was 46.45±17.06 in group A and 24.67±9.78 in group B (P<0.001); and radiation exposure dose, determined as 0.71±0.64 and 0.34±0.29 mSv (P<0.01) in group A and group B, respectively. Furthermore, the number of target branch vessels, that underwent successful catheterization were 107 (97%) in group B as compared to 96 (70%) in group A, which was also significant (P<0.001). The study also showed that the ideal branch-angle has a similarly high consistency in pre-operative and intra-operative angiography based on the intra-class correlation coefficient (ICC) (0.994; 0.990, respectively). Conclusions: 3D fusion guidance for fluoroscopic navigation not only is a reliable process, but also effectively reduces the operation time and radiation dose of TAE-MSK pain.

Genome-wide profiling of rice Double-stranded RNA-Binding Protein 1-associated RNAs by targeted RNA editing.

RNA-binding proteins (RBPs) play essential roles in regulating gene expression. However, the RNA ligands of RBPs are poorly understood in plants, not least due to the lack of efficient tools for genome-wide identification of RBP-bound RNAs. An RBP-fused adenosine deaminase acting on RNA (ADAR) can edit RBP-bound RNAs, which allows efficient identification of RNA ligands of RBPs in vivo. Here, we report the RNA editing activities of the ADAR deaminase domain (ADARdd) in plants. Protoplast experiments indicated that RBP-ADARdd fusions efficiently edited adenosines within 41 nucleotides (nt) of their binding sites. We then engineered ADARdd to profile the RNA ligands of rice (Oryza sativa) Double-stranded RNA-Binding Protein 1 (OsDRB1). Overexpressing the OsDRB1-ADARdd fusion protein in rice introduced thousands of A-to-G and T-to-C RNA‒DNA variants (RDVs). We developed a stringent bioinformatic approach to identify A-to-I RNA edits from RDVs, which removed 99.7% to 100% of background single-nucleotide variants in RNA-seq data. This pipeline identified a total of 1,798 high-confidence RNA editing (HiCE) sites, which marked 799 transcripts as OsDRB1-binding RNAs, from the leaf and root samples of OsDRB1-ADARdd-overexpressing plants. These HiCE sites were predominantly located in repetitive elements, 3'-UTRs, and introns. Small RNA sequencing also identified 191 A-to-I RNA edits in miRNAs and other sRNAs, confirming that OsDRB1 is involved in sRNA biogenesis or function. Our study presents a valuable tool for genome-wide profiling of RNA ligands of RBPs in plants and provides a global view of OsDRB1-binding RNAs.

Fine-tuning OsCPK18/OsCPK4 activity via genome editing of phosphorylation motif improves rice yield and immunity.

Plants have evolved complex signalling networks to regulate growth and defence responses under an ever-changing environment. However, the molecular mechanisms underlying the growth-defence tradeoff are largely unclear. We previously reported that rice CALCIUM-DEPENDENT PROTEIN KINASE 18 (OsCPK18) and MITOGEN-ACTIVATED PROTEIN KINASE 5 (OsMPK5) mutually phosphorylate each other and that OsCPK18 phosphorylates and positively regulates OsMPK5 to suppress rice immunity. In this study, we found that OsCPK18 and its paralog OsCPK4 positively regulate plant height and yield-related traits. Further analysis reveals that OsCPK18 and OsMPK5 synergistically regulate defence-related genes but differentially regulate development-related genes. In vitro and in vivo kinase assays demonstrated that OsMPK5 phosphorylates C-terminal threonine (T505) and serine (S512) residues of OsCPK18 and OsCPK4, respectively. The kinase activity of OsCPK18T505D , in which T505 was replaced by aspartic acid to mimic T505 phosphorylation, displayed less calcium sensitivity than that of wild-type OsCPK18. Interestingly, editing the MAPK phosphorylation motif in OsCPK18 and its paralog OsCPK4, which deprives OsMPK5-mediated phosphorylation but retains calcium-dependent activation of kinase activity, simultaneously increases rice yields and immunity. This editing event also changed the last seven amino acid residues of OsCPK18 and attenuated its binding with OsMPK5. This study presents a new regulatory circuit that fine tunes the growth-defence tradeoff by modulating OsCPK18/4 activity and suggests that CRISPR/Cas9-mediated engineering phosphorylation pathways could simultaneously improve crop yield and immunity.

Simultaneous Patent Blue Dye Injections Aid in the Preoperative CT-Guided Localization of Multiple Pulmonary Nodules.

Background and Objectives: Clinically, a major challenge of multiple nodule localization is puncture-related pneumothorax, which may hamper the successful localization. This study aims to investigate and compare the efficacy and safety of the simultaneous and sequential patent blue dye (PBD) injections for identifying multiple pulmonary nodules during preoperative CT-guided localization. Materials and Methods: Sixty-one consecutive patients with multiple pulmonary nodules who underwent preoperative CT-guided localization with PBD injections between January 2020 and December 2020 were retrospectively enrolled. Of these patients, 31 patients with 64 nodules who underwent simultaneous injections were designated as the simultaneous group; the remaining 30 patients with 63 nodules who underwent sequential punctures were designated as the sequential group. The clinical and radiological features, technical information, pathological results, and procedure-related variables and complications of the two groups were reviewed and analyzed. Results: The localization success rate of the simultaneous group was higher than that of the sequential group (100% [64/64] vs. 93.7% [59/63], p = 0.041). The incidences of pneumothorax (32.3 vs. 33.3%, p = 0.929) and pulmonary hemorrhage (6.3 vs. 3.0%, p = 1) were not significantly different between the two groups, and all cases were minor, which did not require further intervention. Additionally, a significantly lower radiation dose (2.7 vs. 3.5 mSv, p = 0.001) and a shorter procedure time (20.95 vs. 25.28 min, p = 0.001) were observed in the simultaneous group than in the sequential group. Conclusions: Compared with the sequential method, simultaneous PBD injections may improve the localization success rate with a shorter procedure time and less radiation exposure if the patient with multiple pulmonary nodules can be approached in a single position. Further prospective studies are needed to validate these results.

A FLASH pipeline for arrayed CRISPR library construction and the gene function discovery of rice receptor-like kinases.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated gene editing is revolutionizing plant research and crop breeding. Here, we present an effective and streamlined pipeline for arrayed CRISPR library construction and demonstrate it is suitable for small- to large-scale genome editing in plants. This pipeline introduces artificial PCR fragment-length markers for distinguishing guide RNAs (gRNAs) (FLASH), and a group of 12 constructs harboring different FLASH tags are co-transformed into plants each time. The identities of gRNAs in Agrobacterium mixtures and transgenic plants can therefore be read out by detecting the FLASH tags, a process that requires only conventional PCR and gel electrophoresis rather than sequencing. We generated an arrayed CRISPR library targeting all 1,072 members of the receptor-like kinase (RLK) family in rice. One-shot transformation generated a mutant population that covers gRNAs targeting 955 RLKs, and 74.3% (710/955) of the target genes had three or more independent T0 lines. Our results indicate that the FLASH tags act as bona fide surrogates for the gRNAs and are tightly (92.1%) associated with frameshift mutations in the target genes. In addition, the FLASH pipeline allows for rapid identification of unintended editing events without corresponding T-DNA integrations and generates high-order mutants of closely related RLK genes. Furthermore, we showed that the RLK mutant library enables rapid discovery of defense-related RLK genes. This study introduces an effective pipeline for arrayed CRISPR library construction and provides genome-wide rice RLK mutant resources for functional genomics.

A versatile nanoluciferase toolkit and optimized in-gel detection method for protein analysis in plants.

Dissection of gene function requires sophisticated tools to monitor gene expression. Gene tagging with epitope peptides and fluorescent protein tags is a routine method to investigate protein expression using tag-specific antibodies and western blotting with tedious blotting and immunodetection steps. Nanoluciferase (NanoLuc) exhibits extremely bright bioluminescence and is engineered as a sensitive genetic reporter. Due to its small size and high bioluminescent activity, NanoLuc could be engineered to function as a novel protein tag that permits direct detection of tagged protein in the gel matrix (in-gel detection). In this study, we developed Gateway compatible vectors to tag proteins with NanoLuc in plants. We also tailored the in-gel detection conditions which can detect NanoLuc-tagged MPK3 from as low as 200 pg of total protein extracts. Compared to FLAG tag and western blotting-based detection, NanoLuc tag and optimized in-gel detection exhibit increased detection sensitivity but omit the blotting and immunodetection steps. We also demonstrated versatile applications of the NanoLuc-based in-gel detection method for protein expression analysis, probing protein-protein interactions by coimmunoprecipitation, and in vivo protein phosphorylation detection with Phos-tag gel electrophoresis. Finally, NanoLuc was used to tag the gene at its endogenous locus using the wheat dwarf virus replicon and CRISPR/Cas9-mediated gene targeting. Our data suggest that NanoLuc tag and in-gel detection permit fast detection of tagged protein with high sensitivity. The versatile NanoLuc toolkit and convenient in-gel detection method are expected to facilitate in vitro and in vivo protein analysis for plant functional genomics. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01210-7.