Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives.

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.

Author Correction: Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline.

A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity1-4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.

LexCHI: A quick lexical test for estimating language proficiency in Chinese.

A prominent methodological issue in cognitive research on bilingualism is the lack of consistency in measuring second language (L2) proficiency. To reduce the inconsistency in L2 proficiency measurements, brief and valid vocabulary tests have been developed as an objective measure of proficiency in a variety of languages (e.g., English, French, Spanish). Here, we present LexCHI, a valid lexical test to measure Chinese proficiency. This freely available short test consists of 60 two-character items presented in simplified Chinese. Although it only takes a few minutes to complete LexCHI, the LexCHI scores in two studies correlated significantly with L2 participants' performance in a translation task and a cloze test. We believe that LexCHI is a useful tool for researchers who need to objectively measure Chinese proficiency as part of their investigations.

[Research progress on chemical constituents from Kadsura genus and its pharmacological activities and clinical application].

The 29 plant species in the Kadsura genus of the Schisandraceae family are mainly distributed in eastern and southeas-tern Asia. Ten species of plants in this genus are distributed in China, some of which are folk medicinal plants with activating blood circulation, relieving pain, dispelling wind, and dehumidifying effects. Their main constituents are lignans and triterpenes. The current pharmacology and clinical studies have shown that their extracts and constituents have anti-rheumatoid arthritis, liver protection, antioxidation, anti-inflammatory, and other biological activities. The rheumatologic and liver diseases can also be treated with the plants in the clinic. The new chemical constituents reported in the last decade(2012 to date) from the plants of Kadsura genus in China, as well as their pharmacological effects and clinical applications in recent years were reviewed, so as to provide a theoretical basis for further research on the genus.

Prodigiosin as an antibiofilm agent against multidrug-resistant Staphylococcus aureus.

Staphylococcus aureus is known for forming bacterial biofilms that confer increased antimicrobial resistance. Combining antibiotics with antibiofilm agents is an alternative approach, but the antibiofilm ability of prodigiosin (PG), a potential antibiotic synergist, against antimicrobial-resistant (AMR) S. aureus remains to be understood. The antibiofilm activity of PG against 29 clinical AMR S. aureus strains was evaluated using crystal violet staining, and its synergistic effects with vancomycin (VAN) was confirmed using the checkerboard test. The viability and metabolic activity of biofilms and planktonic cells were also assessed. The results revealed that PG exhibited promising inhibitory activity against biofilm formation and synergistic activity with VAN. It effectively reduced the metabolic activity of biofilms and suppressed the production of exopolysaccharides, which might be attributed to the downregulation of biofilm-related genes such as sarA, agrA, and icaA. These findings suggest that PG could be used as a preventive coating or adjuvant against biofilms in clinical settings.

The Essential Role of H2S-ABA Crosstalk in Maize Thermotolerance through the ROS-Scavenging System.

Hydrogen sulfide (H2S) and abscisic acid (ABA), as a signaling molecule and stress hormone, their crosstalk-induced thermotolerance in maize seedlings and its underlying mechanism were elusive. In this paper, H2S and ABA crosstalk as well as the underlying mechanism of crosstalk-induced thermotolerance in maize seedlings were investigated. The data show that endogenous levels of H2S and ABA in maize seedlings could be mutually induced by regulating their metabolic enzyme activity and gene expression under non-heat stress (non-HS) and HS conditions. Furthermore, H2S and ABA alone or in combination significantly increase thermotolerance in maize seedlings by improving the survival rate (SR) and mitigating biomembrane damage. Similarly, the activity of the reactive oxygen species (ROS)-scavenging system, including enzymatic antioxidants catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and superoxide dismutase (SOD), as well as the non-enzymatic antioxidants reduced ascorbic acid (AsA), carotenoids (CAR), flavone (FLA), and total phenols (TP), was enhanced by H2S and ABA alone or in combination in maize seedlings. Conversely, the ROS level (mainly hydrogen peroxide and superoxide radical) was weakened by H2S and ABA alone or in combination in maize seedlings under non-HS and HS conditions. These data imply that the ROS-scavenging system played an essential role in H2S-ABA crosstalk-induced thermotolerance in maize seedlings.

LexMAL: A quick and reliable lexical test for Malay speakers.

Objective language proficiency measures have been found to provide better and more consistent estimates of bilinguals' language processing than self-rated proficiency (e.g., Tomoschuk et al., 2019; Wen & van Heuven, 2017a). However, objectively measuring language proficiency is often not possible because of a lack of quick and freely available language proficiency tests (Park et al., 2022). Therefore, quick valid vocabulary tests, such as LexTALE (Lemhöfer & Broersma, 2012) and its extensions (e.g., LexITA: Amenta et al., 2020; LEXTALE-FR: Brysbaert, 2013; LexPT: Zhou & Li, 2022) have been developed to reliably assess language proficiency of speakers of various languages. The present study introduces a Lexical Test for Malay Speakers (LexMAL), which estimates language proficiency for Malay first language (L1) and second language (L2) speakers. An initial 180-item LexMAL prototype was evaluated using 60 Malay L1 and 60 L2 speakers in Experiment 1. Sixty words and 30 nonwords with the highest discriminative power that span across the full difficulty range were selected for the final LexMAL based on point-biserial correlations and an item response theory analysis. The validity of LexMAL was demonstrated through a reliable discrimination between L1 and L2 speakers, significant correlations between LexMAL scores and performance on other Malay language tasks (i.e., translation accuracy and cloze test scores), and LexMAL outperforming self-rated proficiency. A validation study (Experiment 2) with the 90-item final LexMAL tested with a different group of Malay L1 (N = 61) and L2 speakers (N = 61) replicated the findings of Experiment 1. LexMAL is freely available for researchers at www.lexmal.org .

Translation norms for Malay and English words: The effects of word class, semantic variability, lexical characteristics, and language proficiency on translation.

Translation equivalents are widely used in bilingual research concerning word processing (e.g., Eddington & Tokowicz, 2013; Jouravlev & Jared, 2020) and second-language vocabulary learning (e.g., Bracken et al., 2017; Degani et al., 2014). Although translation norms exist in several languages, to date there are no Malay-English translation norms. This study presents the first Malay-English translation norms collected with highly proficient Malay-English bilinguals. Furthermore, the study investigates the impact of lexical characteristics on translation ambiguity. The forward translation (FT) task (N = 30) collected English translations for 1004 Malay words selected from the Malay Lexicon Project (Yap et al., 2010), and subsequently the backward translation (BT) task (N = 30) gathered Malay translations for 845 English words obtained from the FT phase. The data revealed a high prevalence of translation ambiguity in both translation directions. Specifically, verbs, adjectives, and class-ambiguous words were more translation-ambiguous than nouns. Furthermore, within-language semantic variability and word length were positively correlated with translation ambiguity, whereas word frequency only correlated with translation ambiguity in FT. Word length and word frequency of the source words and their translations were positively correlated. Intriguingly, only in FT were bilinguals with higher Malay proficiency more likely to provide accurate and dominant translations for the Malay words. These findings contrast with those reported in translation norming studies involving other language pairs. The translation norms provide a useful resource for bilingual language studies involving Malay-English bilinguals.

Eriodictyol ameliorates cognitive dysfunction in APP/PS1 mice by inhibiting ferroptosis via vitamin D receptor-mediated Nrf2 activation.

BACKGROUND: Alzheimer's disease (AD) is the most common type of neurodegenerative disease in the contemporary era, and it is still clinically incurable. Eriodictyol, a natural flavonoid compound that is mainly present in citrus fruits and some Chinese herbal medicines, has been reported to exert anti-inflammatory, antioxidant, anticancer and neuroprotective effects. However, few studies have examined the anti-AD effect and molecular mechanism of eriodictyol. METHODS: APP/PS1 mice were treated with eriodictyol and the cognitive function of mice was assessed using behavioral tests. The level of amyloid-ß (Aß) aggregation and hyperphosphorylation of Tau in the mouse brain were detected by preforming a histological analysis and Western blotting. HT-22 cells induced by amyloid-ß peptide (1-42) (Aß1-42) oligomers were treated with eriodictyol, after which cell viability was determined and the production of p-Tau was tested using Western blotting. Then, the characteristics of ferroptosis, including iron aggregation, lipid peroxidation and the expression of glutathione peroxidase type 4 (GPX4), were determined both in vivo and in vitro using Fe straining, Western blotting and qPCR assays. Additionally, the expression level of vitamin D receptor (VDR) and the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway were tested using Western blotting and qPCR assays. Afterward, HT-22 cells with VDR knockout were used to explore the potential mechanisms, and the relationship between VDR and Nrf2 was further assessed by performing a coimmunoprecipitation assay and bioinformatics analysis. RESULTS: Eriodictyol obviously ameliorated cognitive deficits in APP/PS1 mice, and suppressed Aß aggregation and Tau phosphorylation in the brains of APP/PS1 mice. Moreover, eriodictyol inhibited Tau hyperphosphorylation and neurotoxicity in HT-22 cells induced by Aß1-42 oligomer. Furthermore, eriodictyol exerted an antiferroptosis effect both in vivo and in vitro, and its mechanism may be associated with the activation of the Nrf2/HO-1 signaling pathway. Additionally, further experiments explained that the activation of Nrf2/HO-1 signaling pathway by eriodictyol treatment mediated by VDR. CONCLUSIONS: Eriodictyol alleviated memory impairment and AD-like pathological changes by activating the Nrf2/HO-1 signaling pathway through a mechanism mediated by VDR, which provides a new possibility for the treatment of AD.

SARS-CoV-2 productively infects human brain microvascular endothelial cells.

BACKGROUND: The emergence of the novel, pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global health emergency. SARS-CoV-2 is highly contagious and has a high mortality rate in severe patients. However, there is very limited information on the effect of SARS-CoV-2 infection on the integrity of the blood-brain barrier (BBB). METHODS: RNA-sequencing profiling was performed to analyze the transcriptomic changes in human brain microvascular endothelial cells (hBMECs) after SARS-CoV-2 infection. Bioinformatic tools were used for differential analysis. Immunofluorescence, real-time quantitative PCR, and Western blotting analysis were used to explore biological phenotypes. RESULTS: A total of 927 differentially expressed genes were identified, 610 of which were significantly upregulated while the remaining 317 were downregulated. We verified the significant induction of cytokines, chemokines, and adhesion molecules in hBMECs by SARS-CoV-2, suggesting an activation of the vascular endothelium in brain. Moreover, we demonstrated that SARS-CoV-2 infection could increase the BBB permeability, by downregulating as well as remodeling the intercellular tight junction proteins. CONCLUSIONS: Our findings demonstrated that SARS-CoV-2 infection can cause BBB dysfunction, providing novel insights into the understanding of SARS-CoV-2 neuropathogenesis. Moreover, this finding shall constitute a new approach for future prevention and treatment of SARS-CoV-2-induced CNS infection.

Transcriptional landscape of human neuroblastoma cells in response to SARS-CoV-2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious, and the neurological symptoms of SARS-CoV-2 infection have already been reported. However, the mechanisms underlying the effect of SARS-CoV-2 infection on patients with central nervous system injuries remain unclear. METHODS: The high-throughput RNA sequencing was applied to analyze the transcriptomic changes in SK-N-SH cells after SARS-CoV-2 infection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to identify the functions of differentially expressed genes and related pathways. RESULTS: A total of 820 mRNAs were significantly altered, including 671 upregulated and 149 downregulated mRNAs (showing an increase of ≥ 2-fold or decrease to ≤ 0.5-fold, respectively; p ≤ 0.05). Moreover, we verified the significant induction of cytokines, chemokines, and their receptors, as well as the activation of NF-κB, p38, and Akt signaling pathways, in SK-N-SH by SARS-CoV-2. CONCLUSIONS: To our knowledge, this is the first time the transcriptional profiles of the host mRNAs involved in SARS-CoV-2 infection of SK-N-SH cells have been reported. These findings provide novel insight into the pathogenic mechanism of SARS-CoV-2 and might constitute a new approach for future prevention and treatment of SARS-CoV-2-induced central nervous system infection.

FLT3 inhibition upregulates HDAC8 via FOXO to inactivate p53 and promote maintenance of FLT3-ITD+ acute myeloid leukemia.

Internal tandem duplication (ITD) mutations within the FMS-like receptor tyrosine kinase-3 (FLT3) can be found in up to 25% to 30% of acute myeloid leukemia (AML) patients and confer a poor prognosis. Although FLT3 tyrosine kinase inhibitors (TKIs) have shown clinical responses, they cannot eliminate primitive FLT3-ITD+ AML cells, which are potential sources of relapse. Therefore, elucidating the mechanisms underlying FLT3-ITD+ AML maintenance and drug resistance is essential to develop novel effective treatment strategies. Here, we demonstrate that FLT3 inhibition induces histone deacetylase 8 (HDAC8) upregulation through FOXO1- and FOXO3-mediated transactivation in FLT3-ITD+ AML cells. Upregulated HDAC8 deacetylates and inactivates p53, leading to leukemia maintenance and drug resistance upon TKI treatment. Genetic or pharmacological inhibition of HDAC8 reactivates p53, abrogates leukemia maintenance, and significantly enhances TKI-mediated elimination of FLT3-ITD+ AML cells. Importantly, in FLT3-ITD+ AML patient-derived xenograft models, the combination of FLT3 TKI (AC220) and an HDAC8 inhibitor (22d) significantly inhibits leukemia progression and effectively reduces primitive FLT3-ITD+ AML cells. Moreover, we extend these findings to an AML subtype harboring another tyrosine kinase-activating mutation. In conclusion, our study demonstrates that HDAC8 upregulation is an important mechanism to resist TKIs and promote leukemia maintenance and suggests that combining HDAC8 inhibition with TKI treatment could be a promising strategy to treat FLT3-ITD+ AML and other tyrosine kinase mutation-harboring leukemias.

Preoperative CA125 as a risk factor for symptom recurrence of adenomyosis after ultrasound-guided high-intensity focused ultrasound ablation surgery.

OBJECTIVES: To investigate the relationship between preoperative CA125 and symptom recurrence in adenomyosis after ultrasound-guided high-intensity focused ultrasound ablation surgery (FUAS). METHODS: A total of 502 adenomyosis patients after FUAS in Affiliated Nanchong Central Hospital of North Sichuan Medical College from June 2017 to March 2021 were reviewed. Factors associated with symptom recurrence of adenomyosis were analyzed by binary logistic regression model. ROC was used to determine the optimal cutpoint. Magnitude of preoperative CA125 relating to timing of symptom recurrence was measured by cox regression and Kaplan-Meier (K-M) curves. Besides, multiple liner regression model was used to identify the impacting factors for preoperative CA125. RESULTS: Multiple binary logistic analysis showed preoperative CA125 was related to symptom recurrence (OR = 1.002, 95%: 1.000～1.004, p = 0.043). The ROC of preoperative CA125 for recurrence validated 35 U/ml had a high sensitivity (82.5%). Preoperative CA125 was related to timing of symptom recurrence (HR = 2.255, 95%: 1.387-3.667, p = 0.001). K-M curves showed medium recurrence time in preoperative CA125 level >35 U/ml group (38.5 months) was shorter than that in CA125 level ≤35 U/ml group (44.5 months) (p = 0.001). Multiple liner regression analyses showed uterus volume and adenomyotic lesions volume positively correlated to preoperative CA125 level, while age negatively correlated to preoperative CA125 level. CONCLUSION: The higher level of preoperative CA125 was related to an earlier onset of symptom recurrence after FUAS.

Effects of Different Degrees of Carotid Artery Stenosis on the Expression of XIAP and Smac in the Ischemic Penumbra of Rats with Cerebral Ischemia-Reperfusion.

OBJECTIVE: To investigate the effects of different degrees of carotid artery stenosis (CAS) on the expression of XIAP and Smac in ischemic penumbra of rats with cerebral ischemia-reperfusion (I/R). MATERIALS AND METHODS: Samples were collected at 12 h and 24 h after reperfusion, and then the treated groups were divided into the NC-12 group, NC-24 group, MIS-12 group, MIS-24 group, MOS-12 group, MOS-24 group, SES-12 group and SES-24 group. HE staining was used to observe the pathological changes of the brain tissue. TUNEL assay was used to detect the apoptosis in the ischemic penumbra. IHC and RT-qPCR were used to detect the expression of XIAP and Smac in the brain tissue. RESULTS: By observing the pathological sections of brain tissue, the rats in MIS, MOS and SES groups showed loose brain tissue on the infarcted side and neuronal pyknosis in the ischemic penumbra. And with the aggravation and prolongation of the degree of stenosis, the degree of brain injury deepened. It was further found that the TUNEL positive rate was significantly increased in the ischemic penumbra in the SES and MOS groups compared with that in the normal control (NC) group. The results of IHC and RT-qPCR showed that the mRNA expression of XIAP and Smac in the ischemic penumbra was significantly up-regulated in the MIS, MOS and SES groups compared with that in the NC group. CONCLUSIONS: CAS may activate XIAP/Smac signaling pathway to induce neuronal apoptosis and promote the injury in the ischemic penumbra caused by cerebral I/R.

[Mechanism of Huanglian Wendan Decoction in improving impaired glucose tolerance based on skeletal muscle NLRP3/caspase-1/IL-1ß, IL-18 pathway].

This study investigated the mechanism of improving impaired glucose tolerance(IGT) of rats by Huanglian Wendan Decoction from the perspective of the skeletal muscle Nod-like receptor protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1)/interleukin-1ß(IL-1ß), interleukin-18(IL-18) pathway. Healthy male SD rats were fed with the diet containing 45% fat for 20 weeks, accompanied by a high-temperature and high-humidity environment and an inactive lifestyle, for the establishment of the IGT rat model. The rats were divided into the blank control group, model control group, metformin hydrochloride group(positive drug group, 0.05 g·kg~(-1)·d~(-1)) and Huanglian Wendan Decoction group(7.8 g·kg~(-1)·d~(-1)). After continuous intragastric administration for 4 weeks, the obesity and glycemic indexes of all the rats were measured. The fasting serum insulin(FINS) level was determined by ELISA, with the insulin sensitivity index(ISI) and insulin resistance index(IRI) calculated. The mRNA and protein expression le-vels of nuclear factor kappaB(NF-κB), NLRP3, caspase-1, IL-1ß and IL-18 in skeletal muscle tissue were detected by real-time polymerase chain reaction(PCR), Western blot and immunofluorescence. Compared with the blank control group, the model control group witnessed significantly increased mRNA and protein expression of NF-κB, NLRP3, caspase-1, IL-1ß and IL-18. As revealed by the comparison with the model control group, Huanglian Wendan Decoction could effectively regulate the obesity status, reduce body weight, correct the abnormal levels of 2-hour plasma glucose(2 hPG), insulin resistance index(IRI), insulin sensitivity index(ISI), and lower the mRNA and protein expression of NF-κB, NLRP3, caspase-1, IL-1ß and IL-18 in the skeletal muscle tissue of IGT rats. Combined with previous studies, the above results showed that the occurrence and development of IGT was closely related to inflammatory response and the classic pyroptosis pathway in skeletal muscle, such as NLRP3/caspase-1/IL-1ß, IL-18. It is inferred that the mechanism of Huanglian Wendan Decoction was to alleviate insulin resistance(IR) and then reverse the course of IGT lies in the regulation of the abnormal insulin receptor signaling pathway based on the NLRP3 inflammasome pathway.

Boron-based ternary Rb6Be2B6 cluster featuring unique sandwich geometry and a naked hexagonal boron ring.

Computational evidence is reported on a boron-based ternary Rb6Be2B6 cluster as the "Big Mac" sandwich on a subnanoscale with thickness of 0.58 nm. The core hexagonal B6 ring, occurring in the naked form due to double 6π/6σ aromaticity, is capped by two tetrahedral BeRb3 ligands. Such a B6 motif is scarce in boron clusters. The sandwich cluster has four-fold 2σ/6π/6σ/2σ aromaticity and its tetrahedral BeRb3 ligand is the simplest case of three-dimensional aromaticity (or spherical aromaticity). The sandwich can be formulated as a charge-transfer complex, [Rb3Be]3+[B6]6-[BeRb3]3+, whose components are held together by robust electrostatics, facilitating dual-mode dynamic fluxionality.

Enhancing the decolorization activity of Bacillus pumilus W3 CotA-laccase to Reactive Black 5 by site-saturation mutagenesis.

Reactive Black 5 (RB5) is a typical refractory azo dye. Widespread utilization of RB5 has caused a variety of environmental and health problems. The enzymatic degradation of RB5 can be a promising solution due to its superiority as an eco-friendly and cost-competitive process. Bacterial CotA-laccase shows great application prospect to eliminate hazardous dyes from wastewater. However, efficient decolorization of RB5 CotA-laccase generally requires the participation of costly, toxic mediators. In the present study, we modified the amino acids Thr415 and Thr418 near the type 1 copper site and the amino acid Gln442 at the entrance of the substrate-binding pocket of Bacillus pumilus W3 CotA-laccase to boost its RB5 decolorization activity based on molecular docking analysis and site-saturation mutagenesis. Through the strategies, two double site mutants T415D/Q442A and T418K/Q442A obtained demonstrated 43.94 and 52.64% RB5 decolorization rates in the absence of a mediator at pH 10.0, respectively, which were about 3.70- and 4.43-fold higher compared with the wild-type CotA-laccase. Unexpectedly, the catalytic efficiency of the T418K/Q442A to ABTS was enhanced by 5.33-fold compared with the wild-type CotA-laccase. The mechanisms of conferring enhanced activity to the mutants were proposed by structural analysis. In summary, the mutants T415D/Q442A and T418K/Q442A have good application potentials for the biodegradation of RB5. KEY POINTS: • Three amino acids of CotA-laccase were manipulated by site-saturation mutagenesis. • Decolorization rate of two mutants to RB5 was enhanced 3.70- and 4.43-fold, respectively. • The mechanisms of awarding enhanced activity to the mutants were supposed.

Extracellular expression of mutant CotA-laccase SF in Escherichia coli and its degradation of malachite green.

In this study, mutant CotA-laccase SF was successfully expressed in Escherichia coli by co-expression with phospholipase C. The optimized extracellular expression of CotA-laccase SF was 1257.22 U/L. Extracellularly expressed CotA-laccase SF exhibits enzymatic properties similar to intracellular CotA-laccase SF. CotA-laccase SF could decolorize malachite green (MG) under neutral and alkaline conditions. The Km and kcat values of CotA-laccase SF to MG were 39.6 mM and 18.36 s-1. LC-MS analysis of degradation products showed that MG was finally transformed into 4-aminobenzophenone and 4-aminophenol by CotA-laccase. The toxicity experiment of garlic root tip cell showed that the toxicity of MG metabolites decreased. In summary, CotA-laccase SF had a good application prospect for degrading malachite green.

Accurate Clinical Diagnosis of Liver Cancer Based on Simultaneous Detection of Ternary Specific Antigens by Magnetic Induced Mixing Surface-Enhanced Raman Scattering Emissions.

Establishing an accurate, simple, and rapid serodiagnosis method aiming for specific cancer antigens is critically important for the clinical diagnosis, therapy, and prognostication of cancer. Currently, surface-enhanced Raman scattering (SERS) readout techniques challenge fluorescent-based detection methods in terms of both optical stability and more importantly multiple detection capability, which become more desirable for clinical diagnostics. We thus started using an interference-free mixing SERS emission (m-SERS) readout to simultaneously indicate, for the first time, three specific liver cancer antigens, including α-fetoprotein (AFP), carcinoembryonic antigen (CEA), and ferritin (FER), even in one clinical serum sample. Here, three triple bonds (C≡N and C≡C) coded SERS tags contribute separate SERS emissions located at 2105, 2159, and 2227 cm-1, respectively; must have one-to-one correspondence from AFP, to FER, to CEA, In the process of detection, the mature double antibody sandwich allows the formation of microscale core-satellite assembly structure between a magnetic bead (MB) and single SERS tags, and therefore a pure and single SERS emission can be observed under the routine excitation laser spot. Because of the action of magnetic force, the uniform 3D packing of SERS tags absorbed MBs will in contrast generate a so-called m-SERS signals. With the help of enrichment and separation by MBs, the proposed m-SERS immunoassay provides an extremely rapid, sensitive, and accurate solution for multiplex detection of antigens or other biomarkers. Herein, the limit of detection (LOD) for simultaneous m-SERS detection of AFP, CEA, and FER was 0.15, 20, and 4 pg/mL, respectively. As expected for 39 clinical serum samples, simultaneous detection of ternary specific antigens can significantly improve the accuracy of liver cancer diagnosis.