Identification of IFI44L as a new candidate molecular marker for systemic lupus erythematosus.

OBJECTIVES: We screened the type I interferon signal pathway factor involved in the pathogenesis of systemic lupus erythematosus (SLE) by whole-genome sequencing in SLE patients and initially analyse their potential functions. METHODS: Use high-throughput sequencing technology to sequence mRNAs on peripheral blood mononuclear cells from SLE patients and healthy controls,and screen out differentially expressed genes related to the type I interferon (IFN) pathway. Quantitative reverse transcription PCR (RT-qPCR) was utilised to verify the expression of the IFI44L gene in SLE patients and healthy controls, and the correlation between its expression level and clinical test indicators of SLE patients were analysed. The receiver operating characteristic (ROC) analyses were conducted to explore the value of IFI44L for SLE diagnosis. Cell counting kit-8 assay and flow cytometry were used to detect the effects of IFI44L on cell proliferation, apoptosis, and cell cycle. RESULTS: A total of 122 genes were significantly up-regulated and 34 genes were significantly down-regulated in the SLE group compared with the healthy control group in this research. The significantly up-regulated IFI44L in SLE patients was verified by RT-qPCR (p<0.01), furthermore, male SLE patients were significantly higher than that in female SLE patients (p<0.05). Moreover, ROC analyses proved IFI44L may have diagnostic value for SLE. Meanwhile, IFI44L expression level was significantly correlated with platelets, mean platelet volume, red blood cell distribution width to platelet ratio, complement component 3, and C-reactive protein (p<0.05). In addition, under the action of high interferon, IFI44L can resist the proapoptotic effect of IFN-α and improve the proliferation activity of cells. CONCLUSIONS: IFI44L may play an important role in SLE pathology through abnormal regulation of the type I interferon signalling pathway.

Comparative study of two different rat models of stress urinary incontinence.

INTRODUCTION AND HYPOTHESIS: The purpose of this study was to compare the success rate and urodynamic characteristics of the rat model of stress Urinary incontinence established by ovariectomy at different times after vaginal dilation. METHODS: Eighty female rats were randomly divided into three groups: the control group (n = 20), model group A (n = 30), i.e., the animals received colpectasis and bilateral oophorectomy within 1 day; model group B (n = 30), i.e., the animals received bilateral oophorectomy 1 week after colpectasis. The sneeze test and urodynamics were tested. RESULTS: There was no significant difference between the model group A and the model group B (p > 0.05) in the sneeze test. The leak point pressure (LPP) of model groups A and B was lower than that of the control group (p < 0.05). The LPP value of model group A was lower than that of model group B (p < 0.05). There was no significant difference in maximum bladder capacity (MBC) between model group A and the control group (p > 0.05); the MBC value of model group B was lower than that of the control group and model group A (p < 0.05). CONCLUSIONS: Models of stress urinary incontinence in rats can be successfully established by both modeling methods. In terms of LPP, model groups A and B had lower values than the control group, and model group A had lower values than model group B. In terms of MBC, there was no difference between model group A and the control group, and model group B had lower values than the control group and model group A.

Suppression of high-fat-diet-induced obesity in mice by dietary folic acid supplementation is linked to changes in gut microbiota.

PURPOSE: To investigate whether the effects of dietary folic acid supplementation on body weight gain are mediated by gut microbiota in obesity. METHODS: Male C57 BL/6J conventional (CV) and germ-free (GF) mice both aged three to four weeks were fed a high-fat diet (HD), folic acid-deficient HD (FD-HD), folic acid-supplement HD (FS-HD) and a normal-fat diet (ND) for 25 weeks. Faecal microbiota were analyzed by 16S rRNA high-throughput sequencing, and the mRNA expression of genes was determined by the real-time RT-PCR. Short-chain fatty acids (SCFAs) in faeces and plasma were measured using gas chromatography-mass spectrometry. RESULTS: In CV mice, HD-induced body weight gain was inhibited by FS-HD, accompanied by declined energy intake, smaller white adipocyte size, and less whitening of brown adipose tissue. Meanwhile, the HD-induced disturbance in the expression of fat and energy metabolism-associated genes (Fas, Atgl, Hsl, Ppar-α, adiponectin, resistin, Ucp2, etc.) in epididymal fat was diminished, and the dysbiosis in faecal microbiota was lessened, by FS-HD. However, in GF mice with HD feeding, dietary folic acid supplementation had almost no effect on body weight gain and the expression of fat- and energy-associated genes. Faecal or plasma SCFA concentrations in CV and GF mice were not altered by either FD-HD or FS-HD feeding. CONCLUSION: Dietary folic acid supplementation differently affected body weight gain and associated genes' expression under HD feeding between CV and GF mice, suggesting that gut bacteria might partially share the responsibility for beneficial effects of dietary folate on obesity.

Effect of electroacupuncture on the degradation of collagen in pelvic floor supporting tissue of stress urinary incontinence rats.

INTRODUCTION AND HYPOTHESIS: To examine the changes induced by electroacupuncture in stress urinary incontinence (SUI) rats, including the urodynamics and collagen degradation-related cytokine molecular biological expression changes, and to explore the effect and mechanism of EA treatment in SUI. METHODS: Female SPF Sprague-Dawley rats were randomly assigned to five groups (n = 10): sham, model, electroacupuncture control, electroacupuncture, and blocker. The leak point pressure (LPP) and maximum bladder capacity (MBC) were measured for each group of rats, and collagen I, collagen III, matrix metalloproteinases (MMPs), and tissue inhibitor of metalloproteinase (TIMPs) in the anterior vaginal wall of rats in each group were determined by reverse transcription-polymerase chain reaction and western blotting. The data were analyzed by one-way analysis of variance or Kruskal-Wallis test. RESULTS: Electroacupuncture Shenshu (BL23) and Huiyang (BL35) increased the LPP and MBC in SUI rats (P < 0.05). Electroacupuncture treatment significantly increased the protein expression of collagen I and collagen III in the anterior vaginal wall of SUI rats (P < 0.05) and significantly reduced the protein expression of MMP1, MMP2, and MMP9 (P < 0.05). CONCLUSIONS: Electroacupuncture stimulation can alleviate the signs of SUI, and its mechanism is related to the degradation of collagen in the anterior vaginal wall.

An insight into the exploration of proliferation of antibiotic resistance genes in high-fat diet induced obesity mice.

Using mice as an animal model, we first demonstrated the significant proliferation of ARGs and the change of mobile genetic elements (MGEs) in high-fat diet induced obesity (DIO) mice, which the ermB and tnpA-03 genes mostly increased, illuminating that DIO could enrich the abundance of ARGs. Additionally, Lactobacillus sharply increased in the DIO mice and might contribute to the proliferation of ARGs and dramatical change of MGEs in the HFD groups. Finally, procrustes analysis showed the explanatory variables of the MGEs, the metabolites, and the microbial communities for the ARGs accounted for 94.3%, 53.4%, and 68.1%, respectively, and implying that MGEs might be the most direct factor affecting ARGs, and microbiota could be the main driver of the proliferation of ARGs in the DIO mice.

Associations Between Cytochrome P450 (CYP) Gene Single-Nucleotide Polymorphisms and Second-to-Fourth Digit Ratio in Chinese University Students.

BACKGROUND Cytochrome P450 (CYP) genes are necessary for the production or metabolism of fetal sex hormones during pregnancy. The second-to-fourth digit ratio (2D: 4D) is formed in the early stage of human fetal development and considered an indicator reflecting prenatal sex steroids levels. We explored the association between 2D: 4D and single-nucleotide polymorphisms (SNPs) of CYP. MATERIAL AND METHODS Correlation analysis between 2D: 4D and 8 SNPs, rs2687133 (CPY3A7), rs7173655 (CYP11A1), rs1004467, rs17115149, and rs2486758 (CYP17A1), and rs4646, rs2255192, rs4275794 (CYP19A1), was performed using data from 426 female and 412 male Chinese university students. SNP genotyping was conducted using PCR. Digit lengths were photographed and measured by image processing software. RESULTS rs2486758 (CYP17A1) correlated with left hand 2D: 4D in men (P=0.026), and rs1004467 (CYP17A1) correlated with right hand 2D: 4D in men (P=0.008) and the whole population (P=0.032). In men, allele G rs1004467 decreased right hand 2D: 4D, while allele C of rs2486758 increased left hand 2D: 4D. In women, left hand 2D: 4D was higher in genotypes with allele A of SNP rs4646 (CYP19A1) under the dominant genetic model; female DR-L was higher in genotypes with allele T of rs17115149 (CYP11A1). SNPs rs2687133 (CYP3A7) and rs1004467 (CYP17A1) were significantly correlated with right hand 2D: 4D (P=0.0107). CONCLUSIONS SNPs rs1004467 and rs2486758 of CYP17A1 are significant in the relationship between 2D: 4D and CYP gene polymorphisms under different conditions. SNP interactions between CYP genes probably impact 2D: 4D. The correlation between 2D: 4D and some sex hormone-related diseases may be due to the effect of CYP variants on the 2 phenotypes.

Lignocellulose pretreatment in a fungus-cultivating termite.

Depolymerizing lignin, the complex phenolic polymer fortifying plant cell walls, is an essential but challenging starting point for the lignocellulosics industries. The variety of ether- and carbon-carbon interunit linkages produced via radical coupling during lignification limit chemical and biological depolymerization efficiency. In an ancient fungus-cultivating termite system, we reveal unprecedentedly rapid lignin depolymerization and degradation by combining laboratory feeding experiments, lignocellulosic compositional measurements, electron microscopy, 2D-NMR, and thermochemolysis. In a gut transit time of under 3.5 h, in young worker termites, poplar lignin sidechains are extensively cleaved and the polymer is significantly depleted, leaving a residue almost completely devoid of various condensed units that are traditionally recognized to be the most recalcitrant. Subsequently, the fungus-comb microbiome preferentially uses xylose and cleaves polysaccharides, thus facilitating final utilization of easily digestible oligosaccharides by old worker termites. This complementary symbiotic pretreatment process in the fungus-growing termite symbiosis reveals a previously unappreciated natural system for efficient lignocellulose degradation.

Efficacy of caesarean scar defect repair in improving postmenstrual bleeding and factors associated with poor effect.

Caesarean scar defect (CSD) can cause postmenstrual bleeding. Defect repair is an effective technique to improve this symptom, but there are still a few patients getting little improvement. This retrospective study evaluates the efficacy of scar repair and explores the factors associated with poor effect. In total, 123 patients were involved in the final analysis. All of them complained about menstruation period >7 days due to postmenstrual bleeding. Before surgery, 87.8% of patients had a menstruation period more than 10 days and 20.3% had a period more than 15 days. After surgery, a normal menstruation period (< =7 days) was achieved in 46.3% (95%CI 37.3%-55.6%) of patients and a menstruation period lasting no more than 10 days was achieved in 74.8% (95%CI 66.2%-82.2%). Through multivariate logistic analysis, four factors were found dependently associated with poor effect (defined as menstruation period >10 days after surgery): repeated caesarean section (OR 9.75, 95%CI 2.30-41.36, 0.002) was a risk factor, while defect volume >600 mm3 (OR 0.14, 95%CI 0.03-0.56, 0.006), interval from caesarean section to symptom emerging >3 months (OR 0.25, 95%CI 0.07-0.94, 0.041) and straight or retroflexed uterus (OR 0.19, 95%CI 0.05-0.79, 0.022) were protective factors. Impact statement What is already known on this subject? Caesarean scar defect can cause postmenstrual bleeding. Defect repair can improve this symptom, but there are still a few patients getting little improvement after surgery. What do the results of this study add? Defect volume >600 mm3, interval from caesarean section to symptom emerging >3 months and straight or retroflexed uterus are protective factors of poor effect (defined as menstruation period >10 days after surgery), and repeated caesarean section is a risk factor. What are the implications of these findings for clinical practice and/or further research? These findings may help in counselling the patients and in medical decision. Further researches are needed to explore other factors associated with surgical effect and build prediction models.

Age polyethism drives community structure of the bacterial gut microbiota in the fungus-cultivating termite Odontotermes formosanus.

Fungus-cultivating termites (Macrotermitinae) possess an elaborate strategy of lignocellulose digestion. It involves a lignocellulose-degrading fungal symbiont (genus Termitomyces), a diverse gut microbiota and a characteristic labour division in food processing. In this study, using pyrotag sequencing and electron microscopy, we analysed the bacterial microbiota in the hindgut of Odontotermes formosanus and its fungus comb to investigate the spatial organization, establishment and temporal succession of the bacterial communities colonizing specific microhabitats. Our results document strong differences between the communities at the hindgut epithelium and the luminal fluid of newly moulted, young and old worker termites. The differences in community structure were consistent with the density, morphology and spatial distribution of bacterial cells and the pools of microbial metabolites in the hindgut compartment, underlining that both gut development and the age-specific changes in diet affect the composition and functional role of their gut microbiota. These findings provide strong support for the concept that changes in diet and gut environment are important determinants of community structure because they create new niches for microbial symbionts.

Investigation of Age Polyethism in Food Processing of the Fungus-Growing Termite Odontotermes formosanus (Blattodea: Termitidae) Using a Laboratory Artificial Rearing System.

Laboratory rearing systems are useful models for studying Rhinotermitid behavior. Information on the biology of fungus-growing termites, however, is limited because of the difficulty of rearing colonies in the laboratory settings. The physical structure of termite nests makes it impossible to photograph or to observe colonies in the field. In this study, an artificial rearing system for field-collected colonies of the fungus-growing termite Odontotermes formosanus (Shiraki) was developed to facilitate observation in the laboratory. We recorded colony activity within the artificial rearing system and documented a variety of social behaviors that occurred throughout the food processing of the colony. This complex miniature ecosystem was cooperatively organized via division of labor in the foraging and processing of plant materials, and the observed patterns largely resembled the caste and age-based principles present in Macrotermes colonies. This work extends our insights into polyethism in the subfamily Macrotermitinae.

Dietary Folic Acid Supplementation Inhibits HighFat DietInduced Body Weight Gain through Gut Microbiota-Associated Branched-Chain Amino Acids and Mitochondria in Mice.

The effects of folic acid on body weight gain in obesity and gut microbiota-associated branched-chain amino acids (BCAAs) and mitochondrial function were investigated. Three- to four-wk-old male C57BL/6J conventional (CV) and germ-free (GF) mice were fed a high-fat diet (HD), folic acid-supplemented HD (FSHD) and a normal-fat diet (ND) for 25 wk. In CV mice, the HD-induced increases in body weight and plasma BCAA concentrations, downregulated expression of genes related to BCAA catabolism (Bcat2, Bckdha, or Ppm1k), mitochondrial biogenesis (Pgc-1α, Cox1, Nd1 or Nd6), fusion (Mfn1, Mfn2 or Opa1) and mitophagy (Pink1 or Park2), and upregulated expression of the fission-associated gene Drp1 in epididymal fat were reversely corrected with FSHD feeding. In contrast, the expression of these genes in the liver was the opposite under HD feeding or folic acid supplementation. In GF mice, plasma BCAA concentrations were much less affected by HD feeding and were reduced by FSHD feeding, with almost no alterations in the expression of genes associated with BCAA catabolism and mitochondrial function. Further analysis indicated a correlation between adipose and hepatic Mt C/N and plasma BCAA concentrations, and the latter had a close association with specific gut bacteria. Therefore, dietary folic acid supplementation differentially affected body weight gain, BCAA catabolism, and mitochondrial dynamics and metabolism under HD feeding between CV and GF mice, suggesting that gut bacteria-altered BCAAs and mitochondria might partially share the responsibility for the beneficial effects of dietary folic acid on obesity.

Identification and Clinical Correlation Analysis of IFI44 in Systemic Lupus Erythematosus Combined with Bioinformatics and Immune Infiltration Analysis.

Purpose: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can cause systemic damage to multiple organs. This study aims to analyze the value and function of IFI44 in the diagnosis and pathology of SLE by bioinformatics and immune infiltration analysis. Patients and Methods: GSE49454 and GSE65391 of SLE were obtained from the GEO dataset, and R software was employed to identify DEGs and investigate their functions. The PPI network was utilized to identify hub genes associated with SLE. CIBERSORT was used to assess differences in immune cell infiltration in SLE patients and controls. ROC curve analysis was performed to evaluate the diagnostic value of IFI44 in SLE. The expression of IFI44 in PBMCs was detected by RT-qPCR, and the correlation between IFI44 expression and SLE-related clinical indicators was analyzed. Results: A total of 65 DEGs were identified from the GSE49454 and GSE65391 databases. Through PPI analysis, IFI44 and RSAD2 were identified as significantly aberrantly expressed in SLE patients. SLE patients and controls showed a significant difference in the proportion of immune cell infiltration. IFI44 expression was positively correlated with activated DCs, monocytes, PCs, neutrophils, and activated memory CD4+T cells, while negatively correlated with M0 and CD8+T cells. The expression of IFI44 was significantly higher in SLE patients (P<0.01), especially in male patients (P=0.0376). ROC curve analysis demonstrated that IFI44 had a high diagnostic value for SLE. Correlation analysis indicated that IFI44 expression was correlated with levels of RBC, HGB, HCT, IgA, ESR, UPRO, C3, C4, and ENA in SLE patients. Conclusion: IFI44 may play a role in the pathogenesis of SLE by influencing the immune microenvironment of SLE patients, and thus has the potential to serve as a diagnostic marker and therapeutic target for SLE.

Molecular insights into the evolution of woody plant decay in the gut of termites.

Plant cell walls represent the most abundant pool of organic carbon in terrestrial ecosystems but are highly recalcitrant to utilization by microbes and herbivores owing to the physical and chemical barrier provided by lignin biopolymers. Termites are a paradigmatic example of an organism's having evolved the ability to substantially degrade lignified woody plants, yet atomic-scale characterization of lignin depolymerization by termites remains elusive. We report that the phylogenetically derived termite Nasutitermes sp. efficiently degrades lignin via substantial depletion of major interunit linkages and methoxyls by combining isotope-labeled feeding experiments and solution-state and solid-state nuclear magnetic resonance spectroscopy. Exploring the evolutionary origin of lignin depolymerization in termites, we reveal that the early-diverging woodroach Cryptocercus darwini has limited capability in degrading lignocellulose, leaving most polysaccharides intact. Conversely, the phylogenetically basal lineages of "lower" termites are able to disrupt the lignin-polysaccharide inter- and intramolecular bonding while leaving lignin largely intact. These findings advance knowledge on the elusive but efficient delignification in natural systems with implications for next-generation ligninolytic agents.

Simultaneous identification and quantification of canrenone and 11-α-hydroxy-canrenone by LC-MS and HPLC-UVD.

A procedure for simultaneous identification and quantification of canrenone and its biotransformed product 11-α-hydroxy-canrenone by high-performance liquid chromatography with ultraviolet detector (HPLC-UVD) and mass spectrometry (LC-MS) methods was proposed. The optimal determination variables on the HPLC-UVD or LC-MS coupled with a ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 µm) were set as follows: detection wavelength of 280 nm, mobile phase of water and methanol gradient elution, temperature for the chromatographic column of 30°C, flow rate of mobile phase of 0.8 mL/min, sample injection volume of 5 µL, and elution time of 40 min. The MS conditions were set as follows: the flow rate of sheath gas, aux gas, and sweep gas were kept at 35 arb, 5 arb, and 0 arb, respectively. The temperature of capillary was held at 300°C, and capillary voltage was set at 30.00 V. Tube lens were performed at 100.00 V. The proposed method was validated by linearity (r² ≥ 0.9910), average recovery (94.93%, RSD1.21%), precision (RSD ≤ 1.31%), limit of detection, and limit of quantification (LOD 0.1~0.12 mg/L, LOQ 0.5~0.67 mg/L), which proved to be affordable for simultaneously determining canrenone and its bio-transformed product 11-α-hydroxy-canrenone.

The E3 Ubiquitin Ligase TRIM65 Negatively Regulates Inflammasome Activation Through Promoting Ubiquitination of NLRP3.

The dysregulation of NLRP3 inflammasome plays a critical role in pathogenesis of various human inflammatory diseases, thus NLRP3 inflammasome activation must be tightly controlled at multiple levels. However, the underlying mechanism regulating NLRP3 inflammasome activation remains unclear. Herein, the effects of Tripartite motif-containing protein 65 (TRIM65) on NLRP3 inflammasome activation and the underlying molecular mechanism were investigated in vitro and in vivo. Inhibition or deletion of Trim65 could significantly strengthen agonist induced NLRP3 inflammasome activation in THP-1 cells and BMDMs, indicated by increased caspase-1 activation and interleukin-1ß secretion. However, TRIM65 had no effect on poly (dA: dT)-induced AIM2 inflammasome activation or flagellin-induced IPAF inflammasome activation. Mechanistically, immunoprecipitation assays demonstrated that TRIM65 binds to NACHT domain of NLRP3, promotes lys48- and lys63- linked ubiquitination of NLRP3 and restrains the NEK7-NLRP3 interaction, thereby inhibiting NLRP3 inflammasome assembly, caspase-1 activation, and IL-1ß secretion. In vivo, three models of inflammatory diseases were used to confirm the suppression role of TRIM65 in NLRP3 inflammasome activation. TRIM65-deficient mice had a higher production of IL-1ß induced by lipopolysaccharide in sera, and more IL-1ß secretion and neutrophil migration in the ascites, and more severity of joint swelling and associated IL-1ß production induced by monosodium urate, suggesting that TRIM65 deficiency was susceptible to inflammation. Therefore, the data elucidate a TRIM65-dependent negative regulation mechanism of NLRP3 inflammasome activation and provide potential therapeutic strategies for the treatment of NLRP3 inflammasome-related diseases.

The Efficacy and Safety of Acupuncture for Prophylaxis of Vestibular Migraine: A Study Protocol for a Randomized Controlled Trial.

Introduction: With a high incidence rate and low diagnosis rate, vestibular migraine (VM) can seriously affect the quality of life of patients, but it remains difficult to manage by current treatment options. Acupuncture may be a potential treatment option for VM prophylaxis, but the currently available evidence is still uncertain. Therefore, this trial aims to evaluate the efficacy and safety of acupuncture for VM prophylaxis. Methods: This is a 28-week parallel, randomized, controlled clinical trial including 4 weeks of baseline, 8 weeks of treatment, and 16 weeks of follow-up. A total of 72 participants will be randomly assigned to two groups. The participants will receive acupuncture in the experimental group, while the participants in the control group will be treated with venlafaxine. The primary outcome measures are change in vertigo/migraine days and vertigo/migraine attacks, vertigo severity, and migraine intensity per 4 weeks from baseline. The secondary outcome measures are change in doses of rescue medication, anxiety level, depression level, and quality of life per 4 weeks from baseline. Adverse events will be recorded for safety evaluation. Discussion: This study will investigate the efficacy and safety of acupuncture for VM prophylaxis. The results will contribute to determining whether acupuncture can serve as an optional treatment strategy for treating VM. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT0464088.

Nonspecific interactions between SpCas9 and dsDNA sites located downstream of the PAM mediate facilitated diffusion to accelerate target search.

RNA-guided Streptococcus pyogenes Cas9 (SpCas9) is a sequence-specific DNA endonuclease that works as one of the most powerful genetic editing tools. However, how Cas9 locates its target among huge amounts of dsDNAs remains elusive. Here, combining biochemical and single-molecule fluorescence assays, we revealed that Cas9 uses both three-dimensional and one-dimensional diffusion to find its target with high efficiency. We further observed surprising apparent asymmetric target search regions flanking PAM sites on dsDNA under physiological salt conditions, which accelerates the target search efficiency of Cas9 by ∼10-fold. Illustrated by a cryo-EM structure of the Cas9/sgRNA/dsDNA dimer, non-specific interactions between DNA ∼8 bp downstream of the PAM site and lysines within residues 1151-1156 of Cas9, especially lys1153, are the key elements to mediate the one-dimensional diffusion of Cas9 and cause asymmetric target search regions flanking the PAM. Disrupting these non-specific interactions, such as mutating these lysines to alanines, diminishes the contribution of one-dimensional diffusion and reduces the target search rate by several times. In addition, low ionic concentrations or mutations on PAM recognition residues that modulate interactions between Cas9 and dsDNA alter apparent asymmetric target search behaviors. Together, our results reveal a unique searching mechanism of Cas9 under physiological salt conditions, and provide important guidance for both in vitro and in vivo applications of Cas9.

Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for investigating the dynamic properties of biomacromolecules. However, the success of protein smFRET relies on the precise and efficient labeling of two or more fluorophores on the protein of interest (POI), which has remained highly challenging, particularly for large membrane protein complexes. Here, we demonstrate the site-selective incorporation of a novel unnatural amino acid (2-amino-3-(4-hydroselenophenyl) propanoic acid, SeF) through genetic expansion followed by a Se-click reaction to conjugate the Bodipy593 fluorophore on calmodulin (CaM) and ß-arrestin-1 (ßarr1). Using this strategy, we monitored the subtle but functionally important conformational change of ßarr1 upon activation by the G-protein coupled receptor (GPCR) through smFRET for the first time. Our new method has broad applications for the site-specific labeling and smFRET measurement of membrane protein complexes, and the elucidation of their dynamic properties such as transducer protein selection.

Correction: Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction.

[This corrects the article DOI: 10.1039/D1SC02653D.].

Conformational Dynamics and Cleavage Sites of Cas12a Are Modulated by Complementarity between crRNA and DNA.

Cas12a is an RNA-guided endonuclease, which displays great potentials and several advantages over the well-known Cas9 in genome editing and engineering. Here, we established a quantitative kinetic scheme to describe the conformational dynamics of Cas12a/crRNA/dsDNA ternary complexes. The highly dynamic nature of Cas12a complexes, including their reversible formation, disassembly, and transition between different conformational states, is likely to be one of the key aspects contributing to their high specificity. The non-target strand is cleaved when its cleavage sites are released from DNA duplex after DNase activation of Cas12a. Cleaved non-target strand stabilizes target strand pre-cleavage states to permit subsequent cleavage and to ensure two DNA strands cleaved in a well-defined order. The extent of complementarity between crRNA and DNA modulates the relative stabilities of target strand pre-cleavage states targeting different cleavage sites. Our discoveries provide insights to fully elucidate the working mechanisms of Cas12a and to optimize it for genome engineering.