CRISPR-array-mediated imaging of non-repetitive and multiplex genomic loci in living cells.

Dynamic imaging of genomic loci is key for understanding gene regulation, but methods for imaging genomes, in particular non-repetitive DNAs, are limited. We developed CRISPRdelight, a DNA-labeling system based on endonuclease-deficient CRISPR-Cas12a (dCas12a), with an engineered CRISPR array to track DNA location and motion. CRISPRdelight enables robust imaging of all examined 12 non-repetitive genomic loci in different cell lines. We revealed the confined movement of the CCAT1 locus (chr8q24) at the nuclear periphery for repressed expression and active motion in the interior nucleus for transcription. We uncovered the selective repositioning of HSP gene loci to nuclear speckles, including a remarkable relocation of HSPH1 (chr13q12) for elevated transcription during stresses. Combining CRISPR-dCas12a and RNA aptamers allowed multiplex imaging of four types of satellite DNA loci with a single array, revealing their spatial proximity to the nucleolus-associated domain. CRISPRdelight is a user-friendly and robust system for imaging and tracking genomic dynamics and regulation.

Dual-recognition colorimetric platform based on porous Au@Pt nanozymes for highly sensitive washing-free detection of Staphylococcus aureus.

A dual-recognition strategy is reported to construct a one-step washing and highly efficient signal-transduction tag system for high-sensitivity colorimetric detection of Staphylococcus aureus (S. aureus). The porous (gold core)@(platinum shell) nanozymes (Au@PtNEs) as the signal labels show highly efficient peroxidase mimetic activity and are robust. For the sake of simplicity the detection involved the use of a vancomycin-immobilized magnetic bead (MB) and aptamer-functionalized Au@PtNEs for dual-recognition detection in the presence of S. aureus. In addition, we designed a magnetic plate to fit the 96-well microplate to ensure consistent magnetic properties of each well, which can quickly remove unreacted Au@PtNEs and sample matrix while avoiding tedious washing steps. Subsequently, Au@PtNEs catalyze hydrogen peroxide (H2O2) to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) generating a color signal. Finally, the developed Au@PtNEs-based dual-recognition washing-free colorimetric assay displayed a response in the range of S. aureus of 5 × 101-5 × 105 CFU/mL, and the detection limit was 40 CFU/mL within 1.5 h. In addition, S. aureus-fortified samples were analyzed to further evaluate the performance of the proposed method, which yielded average recoveries ranging from 93.66 to 112.44% and coefficients of variation (CVs) within the range 2.72-9.01%. These results furnish a novel horizon for the exploitation of a different mode of recognition and inexpensive enzyme-free assay platforms as an alternative to traditional enzyme-based immunoassays for the detection of other Gram-positive pathogenic bacteria.

Discovery and engineering of ChCas12b for precise genome editing.

Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b (CRISPR-Cas12b) nucleases have been computationally identified, yet their potential for genome editing remains largely unexplored. In this study, we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing, identifying five active candidates. Candidatus hydrogenedentes Cas12b (ChCas12b) was found to recognize a straightforward WTN (W = T or A) proto-spacer adjacent motif (PAM), thereby dramatically expanding the targeting scope. Upon optimization of the single guide RNA (sgRNA) scaffold, ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci. Additionally, we identified nine mutations enhancing ChCas12b specificity. More importantly, we demonstrated that both ChCas12b and its high-fidelity variant, ChCas12b-D496A, enabled allele-specific disruption of genes harboring single nucleotide polymorphisms (SNPs). These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.

USP36 inhibits apoptosis by deubiquitinating cIAP1 and survivin in colorectal cancer cells.

Chemotherapeutic agents for treating colorectal cancer (CRC) primarily induce apoptosis in tumor cells. The ubiquitin-proteasome system is critical for apoptosis regulation. Deubiquitinating enzymes (DUBs) remove ubiquitin from substrates to reverse ubiquitination. Although over 100 DUB members have been discovered, the biological functions of only a small proportion of DUBs have been characterized. Here, we aimed to systematically identify the DUBs that contribute to the development of CRC. Among the DUBs, ubiquitin-specific protease 36 (USP36) is upregulated in CRC. We showed that the knockdown of USP36 induces intrinsic and extrinsic apoptosis. Through gene silencing and coimmunoprecipitation techniques, we identified survivin and cIAP1 as USP36 targets. Mechanistically, USP36 binds and removes lysine-11-linked ubiquitin chains from cIAP1 and lysine-48-linked ubiquitin chains from survivin to abolish protein degradation. Overexpression of USP36 disrupts the formation of the XIAP-second mitochondria-derived activator of caspase complex and promotes receptor-interacting protein kinase 1 ubiquitination, validating USP36 as an inhibitor to intrinsic and extrinsic apoptosis through deubiquitinating survivin and cIAP1. Therefore, our results suggest that USP36 is involved in CRC progression and is a potential therapeutic target.

Engineering of a high-fidelity Cas12a nuclease variant capable of allele-specific editing.

CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new Cas12a nucleases for mammalian genome editing, successfully identifying 9 active ones. Notably, these Cas12a nucleases prefer pyrimidine-rich PAMs. Among these nucleases, we extensively characterized Mb4Cas12a obtained from Moraxella bovis CCUG 2133, which recognizes a YYN PAM (Y = C or T). Our biochemical analysis demonstrates that Mb4Cas12a can cleave double-strand DNA across a wide temperature range. To improve specificity, we constructed a SWISS-MODEL of Mb4Cas12a based on the FnCas12a crystal structure and identified 8 amino acids potentially forming hydrogen bonds at the target DNA-crRNA interface. By replacing these amino acids with alanine to disrupt the hydrogen bond, we tested the influence of each mutation on Mb4Cas12a specificity. Interestingly, the F370A mutation improved specificity with minimal influence on activity. Further study showed that Mb4Cas12a-F370A is capable of discriminating single-nucleotide polymorphisms. These new Cas12a orthologs and high-fidelity variants hold substantial promise for therapeutic applications.

Direct de/carboxylation of cannabidiolic acid (CBDA) and cannabidiol (CBD) from hemp plant material under supercritical CO2.

Many organic reactions rely on CO2 sources to generate important structural units and valuable chemicals. In this study, we compared the effects of cannabidiol (CBD) and cannabidiolic acid (CBDA) on the supercritical CO2 (scCO2)-induced de/carboxylation reaction. The results showed that CBD was directly carboxylated in the ortho-position to form CBDA with up to 62% conversion. Meanwhile, CBDA decarboxylation occurred on hemp plant material via varying composition. Mechanistic studies revealed that CBD carboxylation was influenced not only by the physical properties of scCO2, but also by the vegetable matrix.

An immunochromatographic test for serological diagnosis of scrub typhus.

A fluorescent immunochromatographic test (FM-ICT) was developed for rapid detection of anti-Orientia tsutsugamushi antibodies in serum samples. The FM-ICT was constructed based on the dual-antigen sandwich method. Truncated 56 kDa outer membrane protein of O. tsutsugamushi strain SJ, was expressed in E. coli and mixed with those of Ptan and Gillam strains. A thin line of the protein mixture was precisely sprayed across a nitrocellulose membrane making this the "Test" line. Polyclonal antibodies (pAbs) to O.tsutsugamushi were sprayed in another line across the membrane making this the "Control" line. Fluorescent microspheres conjugated 56 kDa proteins reacting with sample serum will be captured on the "Test" line if the sample contains antibodies to O.tsutsugamushi. Several experimental parameters were optimized. After optimizing the reaction procedure, the results are visible, within 6 min, with the naked eye under ultraviolet light. The limit of detection (LOD) was determined to be 7.63 ng/mL with prepared polyclonal antibodies. No cross-reaction was observed with sera samples from other febrile diseases. In clinical evaluations, the strips showed 94.92% sensitivity (106/112) and 93.75% specificity (56/60). The FM-ICT we developed will provide a new tool for on-site diagnosis of scrub typhus.

Effect of two different doses of nalbuphine for postoperative analgesia in children with cleft palate: a randomized controlled trial.

BACKGROUND: Cleft palate repair surgery may result in severe pain in the immediate postoperative period. The aim of this study is to compare the effects of different doses of nalbuphine for postoperative analgesia in children with cleft palate. METHODS: From November 2019 to June 2021, 90 children (45 males and 45 females, age 9-20 months old, ASA class I-II) were selected for palatoplasty. They were randomly divided into three groups: the control group (Group C), the N1 group (postoperative analgesia with 0.05 mg/kg/h nalbuphine) and the N2 group (postoperative analgesia with 0.075 mg/kg/h nalbuphine). Each group had 30 cases. Nalbuphine was not continuously infused in Group C but was continuously infused in Groups N1 and N2 at rates of 0.05 mg/kg/h and 0.075 mg/kg/h, respectively, for 24 h for postoperative analgesia. The FLACC analgesia score and Ramsay Sedation score were recorded at 10 min (T1), 30 min (T2), 2 h (T3), 12 h (T4) and 24 h (T5) after the operation. Adverse reactions such as nausea, vomiting and respiratory depression were observed and recorded. RESULTS: Compared with those in Group C, the FLACC scores in the N1 and N2 groups decreased significantly at T1-T5 (p < 0.05); the Ramsay Sedation score in the N1 group was significantly higher at T3 and T4 (p < 0.05), and that in the N2 group was significantly higher at T1-T5 (p < 0.05). Compared with that in the N1 group, the FLACC score in the N2 group was not significantly different, and the Ramsay Sedation score increased significantly at T5 (p < 0.05). CONCLUSION: Using 0.05 mg/kg/h Nalbuphine continuously for 24 h for postoperative analgesia in children with cleft palate has a better effect and fewer adverse reactions. TRIAL REGISTRATION: This study was registered at ChiCTR1900027385 (11/11/2019).

Design and application of the transformer base editor in mammalian cells and mice.

Fusing apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like cytidine deaminase with catalytically impaired Cas proteins (e.g., nCas9 or dCas9) provides a novel gene-editing technology, base editing, that grants targeted base substitutions with high efficiency. However, genome-wide and transcriptome-wide off-target mutations are observed in base editing, which raises safety concerns regarding therapeutic applications. Previously, we developed a new base editing system, the transformer base editor (tBE), to induce efficient editing with no observable genome-wide or transcriptome-wide off-target mutations both in mammalian cells and in mice. Here we describe a detailed protocol for the design and application of the tBE. Steps for designing single-guide RNA (sgRNA) and helper sgRNA pairs, making constructs, determining the genome-wide and transcriptome-wide off-target mutations, producing the tBE-containing adeno-associated viruses, delivering adeno-associated viruses into mice and examining the in vivo editing effects are included in this protocol. High-precision base editing by the tBE can be completed within 2-3 weeks (in mammalian cells) or within 6-8 weeks (in mice), with sgRNA-helper sgRNA pairs. The whole process can be collaboratively accomplished by researchers using standard techniques from molecular biology, bioinformatics and mouse husbandry.

Air embolism after CT-guided localization of pulmonary ground-glass nodules.

OBJECTIVE: To investigate the incidence of air embolism (AE) related to CT-guided localization of pulmonary ground-glass nodules (GGNs) prior to video-assisted thoracoscopic surgery (VATS). METHODS: The data of all patients who received CT-guided localization of GGNs before VATS from May 2020 to October 2021 were retrospectively analyzed. RESULTS: A total of 1395 consecutive patients with 1553 GGNs were enrolled. AEs occurred in seven patients (0.5%). In four of the seven patients with AE, the embolism was detected before the patients left the CT table and emergency treatments were carried out. Among them, one patient had chest tightness and unilateral limb dyskinesia, one patient had convulsions and transient loss of consciousness, and two patients had no definite clinical symptoms. After a short-term high-flow oxygen inhalation, the clinical symptoms of two patients with symptomatic AE disappeared and two patients with asymptomatic AE did not show any symptoms. In the remaining three patients with AE, the embolism were detected retrospectively when evaluating the images in the PACS for this study. Fortunately, these three patients never developed clinical symptoms related to AE. All seven patients with AE underwent VATS on the day of localization and all GGNs were successfully removed under the guidance of markers. CONCLUSION: The incidence of AE related to CT-guided localization of GGNs was 0.5%, which was significantly higher than expected. Post-localization whole thoracic CT should be performed and observed carefully so as to avoid missed AE and delayed treatment. ADVANCES IN KNOWLEDGE: The incidence of AE related to CT-guided localization of GGNs was 0.5%. In order to timely detect AE, whole thoracic CT scan rather than local CT in the lesion area should be performed after localization. A small amount of AE may be missed if the post- localization CT images are not carefully observed.

Nickel-Catalyzed Metathesis between Carboxylic Acids and Thioesters: A Direct Access to Thioesters.

We describe a unique strategy for generating thioesters from carboxylic acids and thioesters. This transformation features operational simplicity and high step-economy, wherein the -SR moiety of thioesters was smoothly transferred to carboxylic acid from thioacetates as the starting material. Various substrates with different levels of electronic nature were all applicable to this reaction, furnishing thioesters in moderate to outstanding yields. According to the preliminary mechanistic studies, the anhydride intermediates may be involved in the present reaction.

Prime editor-mediated functional reshaping of ACE2 prevents the entry of multiple human coronaviruses, including SARS-CoV-2 variants.

The spike protein of SARS-CoV-2 hijacks the host angiotensin converting enzyme 2 (ACE2) to meditate its entry and is the primary target for vaccine development. Nevertheless, SARS-CoV-2 keeps evolving and the latest Omicron subvariants BQ.1 and XBB have gained exceptional immune evasion potential through mutations in their spike proteins, leading to sharply reduced efficacy of current spike-focused vaccines and therapeutics. Compared with the fast-evolving spike protein, targeting host ACE2 offers an alternative antiviral strategy that is more resistant to viral evolution and can even provide broad prevention against SARS-CoV and HCoV-NL63. Here, we use prime editor (PE) to precisely edit ACE2 at structurally selected sites. We demonstrated that residue changes at Q24/D30/K31 and/or K353 of ACE2 could completely ablate the binding of tested viruses while maintaining its physiological role in host angiotensin II conversion. PE-mediated ACE2 editing at these sites suppressed the entry of pseudotyped SARS-CoV-2 major variants of concern and even SARS-CoV or HCoV-NL63. Moreover, it significantly inhibited the replication of the Delta variant live virus. Our work investigated the unexplored application potential of prime editing in high-risk infectious disease control and demonstrated that such gene editing-based host factor reshaping strategy can provide broad-spectrum antiviral activity and a high barrier to viral escape or resistance.

CRISPR-dCas13-tracing reveals transcriptional memory and limited mRNA export in developing zebrafish embryos.

BACKGROUND: Understanding gene transcription and mRNA-protein (mRNP) dynamics in single cells in a multicellular organism has been challenging. The catalytically dead CRISPR-Cas13 (dCas13) system has been used to visualize RNAs in live cells without genetic manipulation. We optimize this system to track developmentally expressed mRNAs in zebrafish embryos and to understand features of endogenous transcription kinetics and mRNP export. RESULTS: We report that zygotic microinjection of purified CRISPR-dCas13-fluorescent proteins and modified guide RNAs allows single- and dual-color tracking of developmentally expressed mRNAs in zebrafish embryos from zygotic genome activation (ZGA) until early segmentation period without genetic manipulation. Using this approach, we uncover non-synchronized de novo transcription between inter-alleles, synchronized post-mitotic re-activation in pairs of alleles, and transcriptional memory as an extrinsic noise that potentially contributes to synchronized post-mitotic re-activation. We also reveal rapid dCas13-engaged mRNP movement in the nucleus with a corralled and diffusive motion, but a wide varying range of rate-limiting mRNP export, which can be shortened by Alyref and Nxf1 overexpression. CONCLUSIONS: This optimized dCas13-based toolkit enables robust spatial-temporal tracking of endogenous mRNAs and uncovers features of transcription and mRNP motion, providing a powerful toolkit for endogenous RNA visualization in a multicellular developmental organism.

Influence of the temperature-dependent dielectric constant on the photoacoustic effect of gold nanospheres.

Photoacoustic imaging techniques with gold nanoparticles as contrast agents have received a great deal of attention. The photoacoustic response of gold nanoparticles strongly depends on the far-field optical properties, which essentially depend on the dielectric constant of the material. The dielectric constant of gold not only varies with wavelength but is also affected by temperature. However, the effect of the temperature dependence of the dielectric constant on gold nanoparticles' photoacoustic response has not been fully investigated. In this work, the Drude-Lorentz model and Mie theory are used to calculate the dielectric constant and absorption efficiency of gold nanospheres in aqueous solution, respectively. Then, the finite element method is used to simulate the heat transfer process of gold nanospheres and surrounding water. Finally, the one-dimensional velocity-stress equation is solved by the finite-difference time-domain method to obtain the photoacoustic response of gold nanospheres. The results show that under the irradiation of a high-fluence nanosecond pulse laser, ignoring the temperature dependence of the dielectric constant will lead to large errors in the photothermal response and the nonlinear photoacoustic signals (it can even exceed 20% and 30%). The relative error of the photothermal and photoacoustic response caused by ignoring the temperature-dependent dielectric constant is determined from both the temperature dependence of absorption efficiency and the maximum temperature increase of gold nanospheres. This work provides a new perspective for the photothermal and photoacoustic effects of gold nanospheres, which is meaningful for the development of high-resolution photoacoustic detectors and nano/microscale temperature measurement techniques.

Palladium-Catalyzed Thiocarbonylation of Aryl Iodides with S-Aryl Thioformates via Thioester Transfer.

Herein we reported a novel approach to synthesize thioesters with S-aryl thioformates as thioester sources. The reaction proceeded at ambient temperature using widely available starting ingredients, wherein the thioester moiety was smoothly transferred to aryl iodides from S-aryl thioformates. A variety of substrates with various electronic natures were all tolerated under the reaction conditions to furnish desirable thioesters in ranges from moderate to excellent yields. The gram-scale reaction was also conducted, and there was virtually little change in chemical yield, indicating that large-scale synthesis of thioesters may be viable using this method.

Efficacy and safety of remimazolam besylate in bronchoscopy for adults: A multicenter, randomized, double-blind, positive-controlled clinical study.

Background: With the development of fiberoptic bronchoscopy in the diagnosis and treatment of various pulmonary diseases, the anesthesia/sedation requirements are becoming more demanding, posing great challenges for patient safety while ensuring a smooth examination/surgery process. Remimazolam, a brand-new ultra-short-acting anesthetic, may compensate for the shortcomings of current anesthetic/sedation strategies in bronchoscopy. Methods: This study was a prospective, multicenter, randomized, double-blind, parallel positive controlled phase 3 clinical trial. Subjects were randomized to receive 0.2 mg/kg remimazolam besylate or 2 mg/kg propofol during bronchoscopy to evaluate the efficacy and safety of remimazolam. Results: A total of 154 subjects were successfully sedated in both the remimazolam group and the propofol group, with a success rate of 99.4% (95%CI of the adjusted difference -6.7 × 10%-6% to -5.1 × 10%-6%). The sedative effect of remimazolam was noninferior to that of propofol based on the prespecified noninferiority margin of -5%. Compared with the propofol group, the time of loss of consciousness in the remimazolam group (median 61 vs. 48s, p < 0.001), the time from the end of study drug administration to complete awakening (median 17.60 vs. 12.80 min, p < 0.001), the time from the end of bronchoscopy to complete awakening (median 11.00 vs. 7.00 min, p < 0.001), the time from the end of study drug administration to removal of monitoring (median 19.50 vs. 14.50 min, p < 0.001), and the time from the end of bronchoscopy to removal of monitoring (median 12.70 vs. 8.60 min, p < 0.001) were slightly longer. The incidence of Adverse Events in the remimazolam group and the propofol group (74.8% vs. 77.4%, p = 0.59) was not statistically significant, and none of them had Serious Adverse Events. The incidence of hypotension (13.5% vs. 29.7%, p < 0.001), hypotension requiring treatment (1.9% vs. 7.7%, p = 0.017), and injection pain (0.6% vs. 16.8%, p < 0.001) were significantly lower in the remimazolam group than in the propofol group. Conclusion: Moderate sedation with 0.2 mg/kg remimazolam besylate is effective and safe during bronchoscopy. The incidence of hypotension and injection pain was less than with propofol, but the time to loss of consciousness and recovery were slightly longer. Clinical Trial Registration: clinicaltrials.gov, ChiCTR2000039753.

Beneficial Changes in Growth Performance, Antioxidant Capacity, Immune Response, Hepatic Health, and Flesh Quality of Trachinotus ovatus Fed With Oedocladium carolinianum.

The purpose of this study is to assess the feasibility of astaxanthin-rich Oedocladium carolinianum as an immunostimulant in the diet for Trachinotus ovatus. Three experimental diets containing 0% (OC0), 1% (OC1), and 5% (OC5) O. carolinianum powder were formulated for 6-week feeding trials. The results indicated that the OC5 diet boosted the growth performance through decreasing the feed conversion ratio and increasing digestive enzyme activities and intestinal villus length. Meanwhile, fish fed with the OC5 diet promoted antioxidant ability via stimulating the Nrf2-ARE signal pathway and enhancing antioxidant enzyme activities. Furthermore, the OC5 diet exerted hepatoprotective effects by suppressing the lipid deposition and inflammation response and enhancing the transport capacity of cholesterol. Besides, the OC5 diet improved the non-specific immunity by activating the lysozyme and complement system and increasing the nitric oxide content and total nitric oxide synthase activity. Dietary O. carolinianum supplementation promoted the deposition of astaxanthin in the whole body. Therefore, a diet supplemented with 5% O. carolinianum is recommended to boost the growth, antioxidant capacity, immune response, and flesh quality of T. ovatus.

Effects of Barranca yajiagengensis Powder in the Diet of Trachinotus ovatus on the Growth Performance, Antioxidant Capacity, Immunity and Morphology of the Liver and Intestine.

Barranca yajiagengensis, a novel filamentous microalga, can accumulate lutein under high-light and low-nitrogen conditions. It is well known that lutein has antioxidant, anti-inflammatory and immune-modulating properties. The purpose of this study is to evaluate the effects of including lutein-rich B. yajiagengensis powder in the diet of Trachinotus ovatus on the growth performance, antioxidant capacity, immunity, liver, and intestinal morphology. For this aim, three experimental diets containing 0% (BY0), 1% (BY1), and 5% (BY5) B. yajiagengensis powder were formulated for six-week feeding trials. The results indicated that growth performance, feed utilization, and intestinal morphology were not affected by different diet treatments. Fish fed with the BY5 diet promoted antioxidant ability by activating the Nrf2-ARE signal pathway and enhancing antioxidant enzymes activities. Furthermore, the BY5 diet improved non-specific immunity and antibacterial ability by activating lysozymes and the complement system and increasing the nitric oxide (NO) content and total nitric oxide synthase activity. Dietary B. yajiagengensis supplementation improved the liver morphology and exerted hepatoprotective effects. Therefore, as a natural source of lutein, B. yajiagengensis has the potential as a safe and non-toxic immunostimulant for T. ovatus. A diet supplemented with 5% B. yajiagengensis is recommended to improve the growth, antioxidant capacity, immune response, and liver health of T. ovatus.

Amorphous poly-N-vinylcarbazole polymer as a novel matrix for the determination of low molecular weight compounds by MALDI-TOF MS.

Traditional matrices for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) are usually crystalline small molecules. The heterogeneous co-crystallization of the analyte and the matrix creates a sweet spot effect and reduces point-to-point reproducibility. In this study, an amorphous poly-N-vinylcarbazole polymer (PVK) was studied as a novel matrix for MALDI-TOF MS to detect various low molecular weight compounds (LMWCs) in the negative ion mode. The PVK achieved excellent matrix action and showed high sensitivity, good salt tolerance, and reproducibility. These results significantly broaden the design rules for new and efficient polymeric MALDI matrices.