Liquiritin targeting Th17 cells differentiation and abnormal proliferation of keratinocytes alleviates psoriasis via NF-κB and AP-1 pathway.

Psoriasis is a common immune-mediated inflammatory skin disease, caused by disturbed interactions between keratinocytes and immune cells. Chinese medicine shows potential clinical application for its treatment. Liquiritin is a flavone compound extracted from licorice and shows potential antitussive, antioxidant and antiinflammatory effects, and therefore may have potential as a psoriasis therapeutic. The aim of this work was to examine the possible roles that liquiritin may have in treating psoriasis. HaCaT cells were stimulated by TNF-α with or without liquiritin, harvested for analysis by western blots and RT-qPCR, and the cellular supernatants were collected and analyzed by ELISA for cytokines. In addition, 4 groups of mice were examined: Normal, Vehicle, LQ-L and LQ-H. The mice were sacrificed after 6 days and analyzed using IHC, ELISA, RT-qPCR and flow cytometry. The results showed that liquiritin could significantly inhibit the progression of psoriasis both in vitro and in vivo. Liquiritin strongly suppressed the proliferation of HaCaT keratinocytes but did not affect cell viability. Moreover, liquiritin alleviated imiquimod-induced psoriasis-like skin inflammation and accumulation of Th17 cells and DCs in vivo. In TNF-α-induced HaCaT keratinocytes, both protein and mRNA expression levels of inflammatory cytokines were sharply decreased. In imiquimod-induced mice, the activation of NF-κB and AP-1 was reduced after treatment with liquiritin. Collectively, our results show that liquiritin might act as a pivotal regulator of psoriasis via modulating NF-κB and AP-1 signal pathways.

Nuclear localization signal peptides enhance genetic transformation of Dunaliella salina.

BACKGROUND: Dunaliella salina (D. salina) expression system shows a very attractive application prospect, but it currently has a technical bottleneck, namely the low or unstable expression of recombinant proteins. Given the characteristics of cell-penetrating peptides or/and nuclear localization signal (NLS) peptides, this study is the first attempt to improve the transformation rate of foreign gene with trans-activating transcriptional (TAT) protein or/and NLS peptides. METHODS AND RESULTS: Using salt gradient method, exogenous plasmids were transferred into D. salina cells with TAT or TAT/NLS complexes simultaneously. The ß-glucuronidase gene expression was identified by means of histochemical stain and RT-qPCR detection. Through observation with light microscope, TAT-mediating cells exhibit an apparent cytotoxicity even at ratios of 0.5, no significant toxicity was noted in the TAT/plasmid/NLS complex group. It is obvious that with the addition of peptides the toxicity decreases significantly. Histochemical staining showed that the transformants presented blue color under light microscope, but the negative control and blank control are not. Furthermore, based on a TAT/plasmids ratio of 4 with 10 µg NLS peptides mediation, RT-qPCR results demonstrated that the transcripts of target gene were increased by 269 times than that of control group. CONCLUSIONS: This study demonstrated that combination of TAT and NLS peptides can significantly improve the transformation rate and expression level of foreign gene in D. salina system. It offers a promising way for promoting the application and development of D. salina bioreactor.

The value of gene Xpert MTB / RIF, ADA, TB-DNA in the early diagnosis of TB meningitis.

This study was to explore the expression and correlation between gene Xpert MTB / RIF, ADA, and TB-DNA in TB meningitis. For this purpose, we selected 102 patients in the TB meningitis progression diagnosed and treated in our hospital from January 2019 to December 2020, and another 100 patients in the non-TB meningitis group were selected for the control experiment. Two sets of CSF samples were taken to analyze the gene Xpert MTB / RIF positive rate and the correlation between the expression and the progression of tuberculous meningitis by testing the levels of ADA and TB-DNA in the patient body using an automatic biochemical analyzer. Research indicated that The levels of gene Xpert MTB / RIF, ADA, and TB-DNA in the non-tuberculous meningitis group were lower than those in the tuberculous meningitis group (P<0.05; Levels of gene Xpert MTB / RIF, ADA, and TB-DNA were higher (P<0.05) in patients with group III tuberculous meningitis compared with those under grades I-II tuberculous meningitis, and levels of gene Xpert MTB / RIF, ADA, and TB-DNA were higher (P<0.05) in patients with group VI tuberculous meningitis compared with group III tuberculous meningitis; Gene Xpert MTB / RIF, ADA, TB-DNA) as factors occurring in TB meningitis progression, and all three were associated (P<0.05) with TB meningitis progression; Gene Xpert MTB / RIF, ADA showed a positive correlation (r = 0.296, P = 0.002); Gene Xpert MTB / RIF, TB-DNA showed a positive correlation (r = 0.422, P = 0.001); ADA, TB-DNA showed a positive correlation (r = 0.366, P = 0.001). It was concluded that Gene X-Pert MTB / RIF, ADA, and TB-DNA showed high levels in TB meningitis progression, and as the disease worsened, all three showed a positive association in TB meningitis progression.

Current and prospective strategies for advancing the targeted delivery of CRISPR/Cas system via extracellular vesicles.

Extracellular vesicles (EVs) have emerged as a promising platform for gene delivery owing to their natural properties and phenomenal functions, being able to circumvent the significant challenges associated with toxicity, problematic biocompatibility, and immunogenicity of the standard approaches. These features are of particularly interest for targeted delivery of the emerging clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems. However, the current efficiency of EV-meditated transport of CRISPR/Cas components remains insufficient due to numerous exogenous and endogenous barriers. Here, we comprehensively reviewed the current status of EV-based CRISPR/Cas delivery systems. In particular, we explored various strategies and methodologies available to potentially improve the loading capacity, safety, stability, targeting, and tracking for EV-based CRISPR/Cas system delivery. Additionally, we hypothesise the future avenues for the development of EV-based delivery systems that could pave the way for novel clinically valuable gene delivery approaches, and may potentially bridge the gap between gene editing technologies and the laboratory/clinical application of gene therapies.

A potential paradigm in CRISPR/Cas systems delivery: at the crossroad of microalgal gene editing and algal-mediated nanoparticles.

Microalgae as the photosynthetic organisms offer enormous promise in a variety of industries, such as the generation of high-value byproducts, biofuels, pharmaceuticals, environmental remediation, and others. With the rapid advancement of gene editing technology, CRISPR/Cas system has evolved into an effective tool that revolutionised the genetic engineering of microalgae due to its robustness, high target specificity, and programmability. However, due to the lack of robust delivery system, the efficacy of gene editing is significantly impaired, limiting its application in microalgae. Nanomaterials have become a potential delivery platform for CRISPR/Cas systems due to their advantages of precise targeting, high stability, safety, and improved immune system. Notably, algal-mediated nanoparticles (AMNPs), especially the microalgae-derived nanoparticles, are appealing as a sustainable delivery platform because of their biocompatibility and low toxicity in a homologous relationship. In addition, living microalgae demonstrated effective and regulated distribution into specified areas as the biohybrid microrobots. This review extensively summarised the uses of CRISPR/Cas systems in microalgae and the recent developments of nanoparticle-based CRISPR/Cas delivery systems. A systematic description of the properties and uses of AMNPs, microalgae-derived nanoparticles, and microalgae microrobots has also been discussed. Finally, this review highlights the challenges and future research directions for the development of gene-edited microalgae.

Benzoylaconitine Alleviates Progression of Psoriasis via Suppressing STAT3 Phosphorylation in Keratinocytes.

Psoriasis is a chronic and multifactorial skin disease which is caused by inflammatory infiltrates, keratinocyte hyperproliferation, and accumulation of immune cells. As part of the Aconitum species, Benzoylaconitine (BAC) shows potential antiviral, anti-tumor, and anti-inflammatory effects. In this study, we investigated the effects and mechanisms of BAC on tumor necrosis factor-alpha (TNF-α)/LPS-induced HaCaT keratinocytes in a imiquimod(IMQ)-induced mice model. The results showed that BAC could relieve the symptoms of psoriasis by inhibiting cell proliferation, the release of inflammatory factors, and the accumulation of Th17 cells, while no obvious effect on cell viability and safety was observed both in vitro and in vivo. Additionally, BAC can markedly inhibit the protein and mRNA levels of inflammatory cytokines in TNF-α/LPS-induced HaCaT keratinocytes by inhibiting the phosphorylation of STAT3. In brief, our data indicated that BAC could alleviate the progression of psoriasis and may be a potential therapeutic agent for treating psoriasis in clinical practice.

Identification and characteristics of a novel aminoglycoside phosphotransferase, APH(3')-IId, from an MDR clinical isolate of Brucella intermedia.

OBJECTIVES: To describe a novel chromosomal aminoglycoside phosphotransferase named APH(3')-IId identified in an MDR Brucella intermedia ZJ499 isolate from a cancer patient. METHODS: Species identity was determined by PCR and MALDI-TOF MS analysis. WGS was performed to determine the genetic elements conferring antimicrobial resistance. Gene cloning, transcriptional analysis and targeted gene deletion, as well as protein purification and kinetic analysis, were performed to investigate the mechanism of resistance. RESULTS: APH(3')-IId consists of 266 amino acids and shares the highest identity (48.25%) with the previously known APH(3')-IIb. Expression of aph(3')-IId in Escherichia coli decreased susceptibility to kanamycin, neomycin, paromomycin and ribostamycin. The aph(3')-IId gene in ZJ499 was transcriptionally active under laboratory conditions and the relative abundance of this transcript was unaffected by treatment with the above four antibiotics. However, deletion of aph(3')-IId in ZJ499 results in decreased MICs of these drugs. The purified APH(3')-IId showed phosphotransferase activity against kanamycin, neomycin, paromomycin and ribostamycin, with catalytic efficiencies (kcat/Km) ranging from ∼105 to 107 M-1 s-1. Genetic environment and comparative genomic analyses suggested that aph(3')-IId is probably a ubiquitous gene in Brucella, with no mobile genetic elements detected in its surrounding region. CONCLUSIONS: APH(3')-IId is a novel chromosomal aminoglycoside phosphotransferase and plays an important role in the resistance of B. intermedia ZJ499 to kanamycin, neomycin, paromomycin and ribostamycin. To the best of our knowledge, APH(3')-IId represents the fourth characterized example of an APH(3')-II enzyme.

CRISPR/Cas technology promotes the various application of Dunaliella salina system.

Dunaliella salina (D. salina) has been widely applied in various fields because of its inherent advantages, such as the study of halotolerant mechanism, wastewater treatment, recombinant proteins expression, biofuel production, preparation of natural materials, and others. However, owing to the existence of low yield or in the laboratory exploration stage, D. salina system has been greatly restricted for practical production of various components. In past decade, significant progresses have been achieved for research of D. salina in these fields. Among them, D. salina as a novel expression system demonstrated a bright prospect, especially for large-scale production of foreign proteins, like the vaccines, antibodies, and other therapeutic proteins. Due to the low efficiency, application of traditional regulation tools is also greatly limited for exploration of D. salina system. The emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system offers a precise editing tool to overcome the obstacles of D. salina system. This review not only comprehensively summarizes the recent progresses of D. salina in domain of gene engineering but also gives a deep analysis of problems and deficiencies in different fields of D. salina. Moreover, further prospects of CRISPR/Cas system and its significant challenges have been discussed in various aspects of D. salina. It provides a great referencing value for speeding up the maturity of D. salina system, and also supplies practical guiding significance to expand the new application fields for D. salina. KEY POINTS: • The review provides recent research progresses of various applications of D. salina. • The problems and deficiencies in different fields of D. salina were deeply analyzed. • The further prospects of CRISPR/Cas technology in D. salina system were predicted. • CRISPR/Cas system will promote the new application fields and maturity for D. salina.

Meta-analysis of antiviral protection of white spot syndrome virus vaccine to the shrimp.

Currently, white spot syndrome virus (WSSV) is one of the most serious pathogens that impacts shrimp farming around the world. A WSSV vaccine provides a significant protective benefit to the host shrimp. Although various types of vaccines against WSSV have emerged, the immune effects among them were not compared, and it remains unclear which type of vaccine has the strongest protective effect. Meanwhile, due to the lack of effective routes of administration and immunization programs, WSSV vaccines have been greatly limited in the actual shrimp farming. To answer these questions, this study conducted a comprehensive meta-analysis over dozens of studies and compared all types WSSV vaccines, which include sub-unit protein vaccines, whole virus inactivated vaccines, DNA vaccines and RNA-based vaccines. The results showed that the RNA-based vaccine had the highest protection rate over the other three types of vaccines. Among the various sub-unit protein vaccines, VP26 vaccine had the best protective effects than other sub-unit protein vaccines. Moreover, this study demonstrated that vaccines expressed in eukaryotic hosts had higher protection rates than that of prokaryotic systems. Among the three immunization modes (oral administration, immersion and injection) used in monovalent protein vaccines, oral administration had the highest protection rate. In natural conditions, shrimp are mostly infected by the virus orally. These results provide a guide for exploration of a novel WSSV vaccine and help facilitate the application of WSSV vaccines in shrimp farming.

Recent progress in the development of white spot syndrome virus vaccines for protecting shrimp against viral infection.

White spot syndrome virus (WSSV) has been extensively investigated since the white spot disease outbreak in shrimp in 1990. These investigations have mainly focused on determining the function of viral structural proteins, and on utilizing the envelope proteins as subunit vaccines to protect the host. These studies have shown that a WSSV vaccine can be a practical and effective approach for controlling WSSV infection. The development of such a vaccine has become an intense focus of research in the field. This research has resulted in significant progress in the development of WSSV vaccines. However, because of the progress of this work, periodic summaries are needed to facilitate further research on the development of effective WSSV vaccines. This paper provides a comprehensive summary on the status of WSSV vaccines with regard to the following aspects: subunit vaccines, inactivated whole virus vaccines, DNA vaccines, protective antibodies, and the application of RNAi technology. Current limitations in this area of research are also described, as well as prospects for the development and application of improved WSSV vaccines in the future, and for investigating other important aquatic diseases.

Removal of orbital-maxillary sinus-pterygopalatine fossa foreign body with external and endoscopic combined approach.

Craniofacial trauma involving the pterygopalatine fossa region is reported to be rare. We present a case of a foreign body involving the orbit, maxillary sinus, and pterygopalatine fossa in a 4-year-old boy. The object was a reed shaft. Three-dimensional computed tomographic scans and magnetic resonance imaging were done to make a correct diagnosis and to apply the best surgical treatment. The Caldwell-Luc approach combined with endoscopic approach was applied to remove all the fragments of the foreign body, which had been decayed in the human body. One month later, the patient showed satisfactory aesthetic and functional results.

A rapid, accurate, and low-cost method for detecting Mycobacterium tuberculosis and its drug-resistant genes in pulmonary tuberculosis: Applications of MassARRAY DNA mass spectrometry.

Introduction: Mycobacterium tuberculosis (MTB) identification and drug resistance diagnosis are very important for treatment of drug-resistant tuberculosis (DR-TB). Therefore, high throughput, accurate and low-cost molecular detection techniques are urgently needed. This study aimed to evaluate the clinical application value of MassARRAY in tuberculosis diagnosis and drug resistance screening. Methods: The limit of detection (LOD) and clinical application value of MassARRAY were evaluated using reference strains and clinical isolates. MTB in bronchoalveolar lavage fluid (BALF) and sputum samples were detected using MassARRAY, quantitative real-time polymerase chain reaction (qPCR) and MGIT960 liquid culture (culture). Using culture as the standard, the efficacy of MassARRAY and qPCR for the detection of TB was analyzed. Mutation of drug resistance genes in MTB clinical isolates was tested using MassARRAY, high-resolution melting curve (HRM), and Sanger sequencing. Using sequencing as the standard, the efficacy of MassARRAY, and HRM for the detection of each drug resistance site of MTB was analyzed. Simultaneously, the mutation of drug resistance genes by the MassARRAY method was compared with the results of drug susceptibility testing (DST), and the genotype-phenotype relationship was analyzed. The ability of MassARRAY to discriminate mixed infections was detected using mixtures of standard strains (M. tuberculosis H37Rv) and drug-resistant clinical isolates and mixtures of wild-type and mutant plasmids. Results: In MassARRAY, 20 related gene mutations could be detected by two PCR systems. All genes could be accurately detected when the bacterial load was 104 CFU/mL. When the load of wild-type and drug-resistant MTB mixture was 105 CFU/mL (respectively reached 104 CFU/mL), variants and wild-type genes could be detected simultaneously. The sensitivity of MassARRAY (96.9%) for identification was higher than that of qPCR (87.5%) (p < 0.001). The sensitivity and specificity of MassARRAY for all drug resistance gene mutations were 100.0%, with higher accuracy and consistency than HRM (sensitivity = 89.3% and specificity = 96.9%, p = 0.001). Analyzing the relationship between MassARRAY genotype and DST phenotype, the accuracy of katG_315, rpoB_531, rpsL_43, rpsL_88, and rrs_513 sites was 100.0%, and embB_306 and rpoB_526 were inconsistent with the DST results when the base changes were different. Discussion: MassARRAY can obtain base mutation information and identify heteroresistance infections simultaneously when the mutant proportion was at least 5-25%. It has good application prospects in the diagnosis of DR-TB with high throughput, accurate and low-cost.

ANT(9)-Ic, a Novel Chromosomally Encoded Aminoglycoside Nucleotidyltransferase from Brucella intermedia.

Aminoglycoside-modifying enzymes are among the most important mechanisms of resistance to aminoglycoside antibiotics, typically conferring high-level resistance by enzymatic drug inactivation. Previously, we isolated a multidrug-resistant Brucella intermedia strain ZJ499 from a cancer patient, and whole-genome sequencing revealed several putative novel aminoglycoside-modifying enzyme genes in this strain. Here, we report the characterization of one of them that encodes an intrinsic, chromosomal aminoglycoside nucleotidyltransferase designated ANT(9)-Ic, which shares only 33.05% to 47.44% amino acid identity with the most closely related ANT(9)-I enzymes. When expressed in Escherichia coli, ANT(9)-Ic conferred resistance only to spectinomycin and not to any other aminoglycosides tested, indicating a substrate profile typical of ANT(9)-I enzymes. Consistent with this, deletion of ant(9)-Ic in ZJ499 resulted in a specific and significant decrease in MIC of spectinomycin. Furthermore, the purified ANT(9)-Ic protein showed stringent substrate specificity for spectinomycin with a Km value of 44.83 µM and a kcat/Km of 2.8 × 104 M-1 s-1, echoing the above observations of susceptibility testing. In addition, comparative genomic analysis revealed that the genetic context of ant(9)-Ic was conserved in Brucella, with no mobile genetic elements found within its 20-kb surrounding region. Overall, our results demonstrate that ANT(9)-Ic is a novel member of the ANT(9)-I lineage, contributing to the intrinsic spectinomycin resistance of ZJ499. IMPORTANCE The emergence, evolution, and worldwide spread of antibiotic resistance present a significant global public health crisis. For aminoglycoside antibiotics, enzymatic drug modification is the most common mechanism of resistance. We identify a novel chromosomal aminoglycoside nucleotidyltransferase from B. intermedia, called ANT(9)-Ic, which shares the highest identity (47.44%) with the previously known ANT(9)-Ia and plays an important role in spectinomycin resistance of the host strain. Analysis of the genetic environment and origin of ant(9)-Ic shows that the gene and its surrounding region are widely conserved in Brucella, and no mobile elements are detected, indicating that ANT(9)-Ic may be broadly important in the natural resistance to spectinomycin of Brucella species.

Can egg yolk antibodies terminate the CSBV infection in apiculture?

Chinese sacbrood virus (CSBV) is the most severe pathogen of Apis cerana, which leads to serious fatal diseases in bee colonies and eventual catastrophe for the Chinese beekeeping industry. Additionally, CSBV can potentially infect Apis mellifera by bridging the species barrier and significantly affect the productivity of the honey industry. Although several approaches, such as feeding royal jelly, traditional Chinese medicine, and double-stranded RNA treatments, have been employed to suppress CSBV infection, their practical applicabilities are constrained due to their poor effectiveness. In recent years, specific egg yolk antibodies (EYA) have been increasingly utilized in passive immunotherapy for infectious diseases without any side effects. According to both laboratory research and practical use, EYA have demonstrated superior protection for bees against CSBV infection. This review provided an in-depth analysis of the issues and drawbacks in this field in addition to provide a thorough summary of current advancements in CSBV studies. Some promising strategies for the synergistic study of EYA against CSBV, including the exploitation of novel antibody drugs, novel TCM monomer/formula determination, and development of nucleotide drugs, are also proposed in this review. Furthermore, the prospects for the future perspectives of EYA research and applications are presented. Collectively, EYA would terminate CSBV infection soon, as well as will provide scientific guidance and references to control and manage other viral infections in apiculture.

[Metabolic detoxification of bakuchiol is mediated by cytochrome P450 enzymes in human liver microsomes].

OBJECTIVE: To analyze cytochrome P450 (CYP) phenotyping for bakuchiol metabolism and study the mechanism of detoxification of bakuchiol by human liver microsomes (HLM) in vitro. METHODS: The CYP phenotyping for bakuchiol metabolism was determined using HLM combined with CYP specific inhibitors and recombinant human CYP isoforms. The relative activities of CYP isoforms were determined by analyzing the formation of the substrate metabolites using HPLC-MS/MS, in presence or absence of 1-aminobenzotriazole (ABT) which was CYP enzymes' broad spectrum inhibitor. The residual concentrations of bakuchiol in microsomal incubates were determined using HPLC to investigate ABT's effect on the metabolism of bakuchiol. The effects of CYP enzymes on the nephrotoxicity of bakuchiol were investigated using human kidney-2(HK-2) by MTT assay, in presence or absence of ABT. RESULTS: CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in HLM were involved in bakuchiol metabolism, among which CYP2C19 showed the highest metabolic rate. Co-incubation with ABT (2.5 mmol/L) could inhibit more than 90% of the enzyme activities for CYP1A2, CYP2C9, CYP2C19 and CYP3A4. ABT (2.5 mmol/L) could inhibit the HLM metabolism of bakuchiol with inhibition ratio 83.24%±2.13%. When preincubated with ABT, the metabolic detoxification of bakuchiol by HLM was significantly reduced (P<0.05). CONCLUSION: The mechanism of metabolic detoxification of bakuchiol by HLM is associated with bakuchiol metabolism by CYP enzymes to form non toxic or lower toxic metabolites. The broad spectrum inhibitor of CYP could inverse the detoxification of HLM.

Enhancement of antiviral activity of egg yolk antibodies against Chinese sacbrood virus.

Chinese sacbrood virus (CSBV) poses a serious threat to the apiculture of China. Although several approaches have been attempted to control CSBV infection, their applications have been greatly limited in practical breeding of honeybees due to poor effectiveness. Egg yolk antibodies (EYA) have shown a promising protection for bees against CSBV infection. This study was conducted to produce high titer EYA and then further improve their antiviral effect. Among three vaccination groups, the EYA titer in graphene oxide-chitosan group was highest (1.591 ± 0.145), in Freund's group was modest (1.195 ± 0.040), and in white oil group was lowest (1.058 ± 0.056). After three injections of each vaccine in hens, EYA were produced at the highest level with a 14-day period. After application of EYA for more than two years in actual bee breeding, prevention and treatment assays showed that EYA confered 98.9 to 100% protection from CSBV infection. The mortality of the control group reached to a range of 91.2 to 100%. This study demonstrated that the high titer EYA have been successfully prepared with significant anti-CSBV activity and that these antibodies may feasibly be used for CSBV treatment to meet the practical needs of apiculture.

Inactivated vaccine injection and immunoglobulin G levels related to severe coronavirus disease 2019 (Delta) pneumonia in Xi'an, China: A single-centered, retrospective, observational study.

The World Health Organization declared a public health emergency of international concern in January 2020. The Delta variant became the main epidemic strain on 11 May 2021. Vaccines were proven highly effective in controlling hospitalization and deaths associated with severe acute respiratory syndrome coronavirus 2 infections. Real data on vaccine efficacy against B.1.617.2 infection in the Chinese population were currently limited. This study aimed to evaluate the protective effect of inactivated vaccine injection and immunoglobulin (Ig) G levels in coronavirus disease 2019 (COVID-19) severity. This retrospective study included patients with COVID-19 in Xi'an Chest Hospital from December 2021 to January 2022. The protective effect of inactivated vaccine injection and IgG levels on COVID-19 severity was analyzed using multiple logistic regressions. A total of 580 patients were included in the study, of whom 158 (27.24%) were mild, 412 (71.03%) were moderate, 5 (0.9%) were severe, and 5 (0.86%) were critical. Severe case (including severe and critical) rates were 1.72% (10/580). Compared with the unvaccinated group, the vac+IgG- group had a 0.21 (0.02-2.05)-fold risk of suffering from severe cases, and the vac+IgG+ group had a 0.05 (0-0.63)-fold risk of suffering from severe cases. Of the 10 severe cases, 8 were older than 60 years, 8 were men, 8 had underlying diseases, 6 were in the unvaccinated group, and 2 were in the vac+IgG- group. Vaccination and sufficient IgG antibody production can protect patients with COVID-19 from severe cases. Booster vaccine injection can produce a stronger immune response and protection.

Anion complexation and sensing using modified urea and thiourea-based receptors.

This critical review highlights recent advances in the structurally modified (thio)urea-based receptors for anion complexation and sensing. Modifications of the (thio)urea structure are aimed at a better anion binding in terms of higher binding constant, anion selectivity and feasibility. Major (thio)urea receptors are reviewed as N-alkyl, N-aryl and N-amido/N-amino (thio)ureas. Hints for designing (thio)urea-based receptors for anions are discussed (102 references).

CRISPR/Cas9-induced ß-carotene hydroxylase mutation in Dunaliella salina CCAP19/18.

Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, ß-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the ß-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the ß-carotene levels of mutants showed a significant increase, nearly up to 1.4 µg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.

Strategies for High-Efficiency Mutation Using the CRISPR/Cas System.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems have revolutionized traditional gene-editing tools and are a significant tool for ameliorating gene defects. Characterized by high target specificity, extraordinary efficiency, and cost-effectiveness, CRISPR/Cas systems have displayed tremendous potential for genetic manipulation in almost any organism and cell type. Despite their numerous advantages, however, CRISPR/Cas systems have some inherent limitations, such as off-target effects, unsatisfactory efficiency of delivery, and unwanted adverse effects, thereby resulting in a desire to explore approaches to address these issues. Strategies for improving the efficiency of CRISPR/Cas-induced mutations, such as reducing off-target effects, improving the design and modification of sgRNA, optimizing the editing time and the temperature, choice of delivery system, and enrichment of sgRNA, are comprehensively described in this review. Additionally, several newly emerging approaches, including the use of Cas variants, anti-CRISPR proteins, and mutant enrichment, are discussed in detail. Furthermore, the authors provide a deep analysis of the current challenges in the utilization of CRISPR/Cas systems and the future applications of CRISPR/Cas systems in various scenarios. This review not only serves as a reference for improving the maturity of CRISPR/Cas systems but also supplies practical guidance for expanding the applicability of this technology.