Deleting specific residues from the HNH linkers creates a CRISPR-SpCas9 variant with high fidelity and efficiency.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems are immunological defenses used in archaea and bacteria to recognize and destroy DNA from external invaders. The CRISPR-SpCas9 system harnessed from Streptococcus pyogenes (SpCas9) has become the most widely utilized genome editing tool and shows promise for clinical application. However, the off-target effect is still the major challenge for the genome editing of CRISPR-SpCas9. Based on analysis of the structure and cleavage procedures, we proposed two strategies to modify the SpCas9 structure and reduce off-target effects. Shortening the HNH or REC3 linkers (Strategy #1) aimed to move the primary position of HNH or REC3 far away from the single-guide RNA (sgRNA)/DNA hybrid (hybrid), while elongating the helix around the sgRNA (Strategy #2) aimed to strengthen the contacts between SpCas9 and the sgRNA/DNA. We designed 11 SpCas9 variants (variant No.1- variant No.11) and verified their efficiencies on the classic genome site EMX1-1, EMX1-1-OT1, and EMX1-1-OT2. The top three effective SpCas9 variants, variant No.1, variant No.2, and variant No.5, were additionally validated on other genome sites. The further selected variant No.1 was compared with two previous SpCas9 variants, HypaCas9 (a hyper-accurate Cas9 variant released in 2017) and eSpCas9 (1.1) (an "enhanced specificity" SpCas9 variant released in 2016), on two genome sites, EMX1-1 and FANCF-1. The results revealed that the deletion of Thr769 and Gly906 could substantially decrease off-target effects, while maintaining robust on-target efficiency in most of the selected genome sites.

Outcomes of Endonasal Septoplasty and Extracorporeal Septal Reconstruction in Anterocaudal Septal Deviation.

OBJECTIVE: The aim of this study was to compare the functional and aesthetic outcomes of endonasal septoplasty (ES) and extracorporeal septal reconstruction (ESR) in anterocaudal septal deviation. METHODS: In this study, patients (n = 46) who underwent nasal septoplasty surgery due to anterocaudal septal deviation during February 2015 to August 2017 were analyzed; 23 patients underwent ES and the others (n = 23) underwent ESR. The decision of whether to use the ES or ESR was randomized by random number table method. Nasal obstruction symptoms evaluation (NOSE) scores, total nasal resistance (TNR), aesthetic visual analog scale (VAS), nasal anatomical angles, and incidence of complications were used to assess the patients in 2 groups. RESULTS: The NOSE scores, TNR, aesthetic VAS, tip deviation angle (TDA), nasolabial angle (NLA), nasofrontal angle (NFA) in the ESR group were significantly improved from preoperative to postoperative 1 year, whereas in the ES group, except aesthetic VAS, NLA, NFA, all other postoperative outcomes were improved from preoperative values. The objective and subjective postoperative results of ESR group were better than the ES group except TDA. The incidence of complications was not significantly different between the 2 groups. CONCLUSION: Our study have compared the nasal functional and aesthetic outcomes of 2 septoplasty techniques in a randomized controlled trial. The ESR technique is more effective than ES technique in correcting functional and aesthetic disorders caused by anterocaudal septal deviation.

Parameters Affecting the Antimicrobial Properties of Cold Atmospheric Plasma Jet.

Using the Taguchi method to narrow experimental parameters, the antimicrobial efficiency of a cold atmospheric plasma jet (CAPJ) treatment was investigated. An L9 array with four parameters of CAPJ treatments, including the application voltage, CAPJ-sample distance, argon (Ar) gas flow rate, and CAPJ treatment time, were applied to examine the antimicrobial activity against Escherichia coli (E. coli). CAPJ treatment time was found to be the most influential parameter in its antimicrobial ability by evaluation of signal to noise ratios and analysis of variance. 100% bactericidal activity was achieved under the optimal bactericidal activity parameters including the application voltage of 8.5 kV, CAPJ-sample distance of 10 mm, Ar gas flow rate of 500 sccm, and CAPJ treatment time of 300 s, which confirms the efficacy of the Taguchi method in this design. In terms of the mechanism of CAPJ's antimicrobial ability, the intensity of hydroxyl radical produced by CAPJ positively correlated to its antimicrobial efficiency. The CAPJ antimicrobial efficiency was further evaluated by both DNA double-strand breaks analysis and scanning electron microscopy examination of CAPJ treated bacteria. CAPJ destroyed the cell wall of E. coli and further damaged its DNA structure, thus leading to successful killing of bacteria. This study suggests that optimal conditions of CPAJ can provide effective antimicrobial activity and may be grounds for a novel approach for eradicating bacterial infections.

Repression of Rgg But Not Upregulation of LacD.1 in emm1-type covS Mutant Mediates the SpeB Repression in Group A Streptococcus.

CovR/CovS is an important two-component regulatory system in human pathogen group A Streptococcus (GAS). Epidemiological studies have shown that inactivation of the sensor kinase CovS is correlated with invasive clinical manifestations. The phosphorylation level of response regulator CovR decreases dramatically in the absence of CovS, resulting in the derepression of virulence factor expression and an increase in bacterial invasiveness. Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease and is negatively regulated by CovR; however, the expression of SpeB is almost completely repressed in the covS mutant. The present study found that in the emm1-type A20 strain, non-phosphorylated CovR acts as a transcriptional repressor for SpeB-positive regulator Rgg. In addition, the expression of Rgg-negative regulator LacD.1 is upregulated in the covS mutant. These results suggest that inactivation of Rgg in the covS mutant would directly mediate speB repression. The current study showed that overexpression of rgg but not inactivation of lacD.1 in the covS mutant partially restores speB expression, indicating that only rgg repression, but not lacD.1 upregulation, contributes to the speB repression in the covS mutant.

A controllable bacterial lysis system to enhance biological safety of live attenuated Vibrio anguillarum vaccine.

Bacterial strains used as backbone for the generation of vaccine prototypes should exhibit an adequate and stable safety profile. Given the fact that live attenuated vaccines often contain some potential risks in virulence recovery and spread infections, new approaches are greatly needed to improve their biological safety. Here, a critically iron-regulated promoter PviuA was screened from Vibrio anguillarum, which was demonstrated to respond to iron-limitation signal both in vitro and in vivo. By using PviuA as a regulatory switch to control the expression of phage P22 lysis cassette 13-19-15, a novel in vivo inducible bacterial lysis system was established in V. anguillarum. This system was proved to be activated by iron-limitation signals and then effectively lyse V. anguillarum both in vitro and in vivo. Further, this controllable bacterial lysis system, after being transformed into a live attenuated V. anguillarum vaccine strain MVAV6203, was confirmed to significantly improve biological safety of the live attenuated vaccine without impairing its immune protection efficacy.

In Vivo Programmed Gene Expression Based on Artificial Quorum Networks.

The quorum sensing (QS) system, as a well-functioning population-dependent gene switch, has been widely applied in many gene circuits in synthetic biology. In our work, an efficient cell density-controlled expression system (QS) was established via engineering of the Vibrio fischeri luxI-luxR quorum sensing system. In order to achieve in vivo programmed gene expression, a synthetic binary regulation circuit (araQS) was constructed by assembling multiple genetic components, including the quorum quenching protein AiiA and the arabinose promoter ParaBAD, into the QS system. In vitro expression assays verified that the araQS system was initiated only in the absence of arabinose in the medium at a high cell density. In vivo expression assays confirmed that the araQS system presented an in vivo-triggered and cell density-dependent expression pattern. Furthermore, the araQS system was demonstrated to function well in different bacteria, indicating a wide range of bacterial hosts for use. To explore its potential applications in vivo, the araQS system was used to control the production of a heterologous protective antigen in an attenuated Edwardsiella tarda strain, which successfully evoked efficient immune protection in a fish model. This work suggested that the araQS system could program bacterial expression in vivo and might have potential uses, including, but not limited to, bacterial vector vaccines.

A quorum sensing-based in vivo expression system and its application in multivalent bacterial vaccine.

BACKGROUND: Delivery of antigens by live bacterial carriers can elicit effective humoral and cellular responses and may be an attractive strategy for live bacterial vaccine production through introduction of a vector that expresses an exogenous protective antigen. To overcome the instability and metabolic burden associated with plasmid introduction, alternative strategies, such as the use of in vivo-inducible promoters, have been proposed. However, screening an ideal in vivo-activated promoter with high efficiency and low leak expression in a particular strain poses great challenges to many researchers. RESULTS: In this work, we constructed an in vivo antigen-expressing vector suitable for Edwardsiella tarda, an enteric Gram-negative invasive intracellular pathogen of both animals and humans. By combining quorum sensing genes from Vibrio fischeri with iron uptake regulons, a synthetic binary regulation system (ironQS) for E. tarda was designed. In vitro expression assay demonstrated that the ironQS system is only initiated in the absence of Fe2+ in the medium when the cell density reaches its threshold. The ironQS system was further confirmed in vivo to present an in vivo-triggered and cell density-dependent expression pattern in larvae and adult zebrafish. A recombinant E. tarda vector vaccine candidate WED(ironQS-G) was established by introducing gapA34, which encodes the protective antigen glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the fish pathogen Aeromonas hydrophila LSA34 into ironQS system, and the immune protection afforded by this vaccine was assessed in turbot (Scophtalmus maximus). Most of the vaccinated fish survived under the challenge with A. hydrophila LSA34 (RPS=67.0%) or E. tarda EIB202 (RPS=72.3%). CONCLUSIONS: Quorum sensing system has been extensively used in various gene structures in synthetic biology as a well-functioning and population-dependent gene circuit. In this work, the in vivo expression system, ironQS, maintained the high expression efficiency of the quorum sensing circuit and achieved excellent expression regulation of the Fur box. The ironQS system has great potential in applications requiring in vivo protein expression, such as vector vaccines. Considering its high compatibility, ironQS system could function as a universal expression platform for a variety of bacterial hosts.

[Fire behavior of Quercus mongolica leaf litter fuelbed under zero-slope and no-wind conditions. II. Analysis and modelling of fireline intensity, fuel consumption, and combustion efficiency].

Mongolian oak (Quercus mongolica) is an important constructive and accompanying species in mixed broadleaf-conifer forest in Northeast China, In this paper, a laboratory burning experiment was conducted under zero-slope and no-wind conditions to study the effects of fuel moisture content, loading, and thickness on the fireline intensity, fuel consumption, and combustion efficiency of the Mongolian oak leaf litter fuelbed. The fuel moisture content, loading, and thickness all had significant effects on the three fire behavior indices, and there existed interactions between these three affecting factors. Among the known models, the Byram model could be suitable for the prediction of local leaf litter fire intensity only after re-parameterization. The re-estimated alpha and beta parameters of the re-parameterized Byram model were 98.009 and 1.099, with an adjusted determination coefficient of 0.745, the rooted mean square error (RMSE) of 8.676 kW x m(-1), and the mean relative error (MRE) of 21%, respectively (R2 = 0.745). The re-estimated a and b by the burning efficiency method proposed by Albini were 0.069 and 0.169, and the re-estimated values were all higher than 93%, being mostly overestimated. The Consume model had a stronger suitability for the fuel. The R2 of the general linear models established for fireline intensity, fuel consumption, and burning efficiency was 0.82, 0.73 and 0.53, and the RMSE was 8.266 kW x m(-1) 0.081 kg x m(-2), and 0.203, respectively. In low intensity surface fires, the fine fuels could not be completely consumed, and thus, to consider the leaf litter and fine fuel in some forest ecosystems being completely consumed would overestimate the carbon release from forest fires.

[Fire behavior of ground surface fuels in Pinus koraiensis and Quercus mongolica mixed forest under no wind and zero slope condition: a prediction with extended Rothermel model].

A laboratory burning experiment was conducted to measure the fire spread speed, residual time, reaction intensity, fireline intensity, and flame length of the ground surface fuels collected from a Korean pine (Pinus koraiensis) and Mongolian oak (Quercus mongolica) mixed stand in Maoer Mountains of Northeast China under the conditions of no wind, zero slope, and different moisture content, load, and mixture ratio of the fuels. The results measured were compared with those predicted by the extended Rothermel model to test the performance of the model, especially for the effects of two different weighting methods on the fire behavior modeling of the mixed fuels. With the prediction of the model, the mean absolute errors of the fire spread speed and reaction intensity of the fuels were 0.04 m X min(-1) and 77 kW X m(-2), their mean relative errors were 16% and 22%, while the mean absolute errors of residual time, fireline intensity and flame length were 15.5 s, 17.3 kW X m(-1), and 9.7 cm, and their mean relative errors were 55.5%, 48.7%, and 24%, respectively, indicating that the predicted values of residual time, fireline intensity, and flame length were lower than the observed ones. These errors could be regarded as the lower limits for the application of the extended Rothermel model in predicting the fire behavior of similar fuel types, and provide valuable information for using the model to predict the fire behavior under the similar field conditions. As a whole, the two different weighting methods did not show significant difference in predicting the fire behavior of the mixed fuels by extended Rothermel model. When the proportion of Korean pine fuels was lower, the predicted values of spread speed and reaction intensity obtained by surface area weighting method and those of fireline intensity and flame length obtained by load weighting method were higher; when the proportion of Korean pine needles was higher, the contrary results were obtained.

[Fire behavior of Mongolian oak leaves fuel-bed under no-wind and zero-slope conditions. I. Factors affecting fire spread rate and modeling].

Aimed to understand the fire behavior of Mongolian oak leaves fuel-bed under field condition, the leaves of a secondary Mongolian oak forest in Northeast Forestry University experimental forest farm were collected and brought into laboratory to construct fuel-beds with varied loading, height, and moisture content, and a total of 100 experimental fires were burned under no-wind and zero-slope conditions. It was observed that the fire spread rate of the fuel-beds was less than 0.5 m x min(-1). Fuel-bed loading, height, and moisture contents all had significant effects on the fire spread rate. The effect of fuel-bed moisture content on the fire spread had no significant correlations with fuel-bed loading and height, but the effect of fuel-bed height was related to the fuel-bed loading. The packing ratio of fuel-beds had less effect on the fire spread rate. Taking the fuel-bed loading, height, and moisture content as predictive variables, a prediction model for the fire spread rate of Mongolian oak leaves fuel-bed was established, which could explain 83% of the variance of the fire spread rate, with a mean absolute error 0.04 m x min(-1) and a mean relative error less than 17%.

[Fire behavior of Mongolian oak leaves fuel bed under no-wind and zero-slope conditions. II. Analysis of the factors affecting flame length and residence time and related prediction models].

Taking fuel moisture content, fuel loading, and fuel bed depth as controlling factors, the fuel beds of Mongolian oak leaves in Maoershan region of Northeast China in field were simulated, and a total of one hundred experimental burnings under no-wind and zero-slope conditions were conducted in laboratory, with the effects of the fuel moisture content, fuel loading, and fuel bed depth on the flame length and its residence time analyzed and the multivariate linear prediction models constructed. The results indicated that fuel moisture content had a significant negative liner correlation with flame length, but less correlation with flame residence time. Both the fuel loading and the fuel bed depth were significantly positively correlated with flame length and its residence time. The interactions of fuel bed depth with fuel moisture content and fuel loading had significant effects on the flame length, while the interactions of fuel moisture content with fuel loading and fuel bed depth affected the flame residence time significantly. The prediction model of flame length had better prediction effect, which could explain 83.3% of variance, with a mean absolute error of 7.8 cm and a mean relative error of 16.2%, while the prediction model of flame residence time was not good enough, which could only explain 54% of variance, with a mean absolute error of 9.2 s and a mean relative error of 18.6%.

Expression, purification, and characterization of protective MPT64 antigen protein and identification of its multimers isolated from nontoxic Mycobacterium tuberculosis H37Ra.

MPT64, a secreted protein of Mycobacterium tuberculosis (MTB), stimulates the immune reactions within cells and is a protective antigen that is lost by the bacilli Calmette-Guérin (BCG) vaccine during propagation. To minimize the toxicity caused by MTB, we used the MPT64 gene encoded by nontoxic H37Ra MTB to carry out genetic expansion via polymerase chain reaction and gene clone MPT64. The plasmid DNA encoded MPT64 was expressed at 20°C for 22 H, and a large quantity of MPT64 was obtained. In the absence of urea, MPT64 multimers with subunits being covalently connected via disulfide bonds were detected by Western blot showing strong protein-protein interactions, as evidenced by the formation of MPT64 tetramers. Finally, with urea of decreasing concentrations, we refolded MPT64 purified in the presence of urea and determined its secondary structures using circular dichroism. MPT64 was found to contain 2.2% α-helix, 50.9% ß-sheet, 19.5% turn, and 27.4% random coil. The molecular weight of MPT64 was determined by a matrix-assisted laser desorption ionization-time of flight mass spectrometer and found to be 23,497 Da, very close to the theoretical molecular weight of MPT64. The results presented here provide a sound basis for future biochemical and biophysical studies of MPT64 or any other proteins encoded by nontoxic H37Ra MTB.

Cloning and molecular characterization of lycC1INH genes in large yellow croaker (Pseudosciaena crocea).

Complement component 1 inhibitor (C1INH) is a crucial protein in controlling activation of many plasma mediator pathways and can directly interact with Gram negative bacteria. The full-length cDNA of lycC1INH gene was identified from the large yellow croaker. It is of 2046 nucleotides (nt) encoding a protein of 599 amino acids, with a 5'-untranslated region of 99 nt and a 3'-untranslated region of 147 nt including the poly (A) tail. The deduced protein contains a C-terminal serpin (serine protease inhibitor) domain, and two N-terminus immunoglobulin domains without significant homology to other species. Western blot analysis of the protein expression showed that the expression of lycC1INH was obviously up-regulated in liver, spleen and head kidney of the fish challenged by attenuated live Vibrio anguillarum strain. This indicated that lycC1INH might be involved in the immune response of large yellow croaker to bacterial challenge.

Treatment persistence in and cost of therapy for patients with chronic hepatitis C: Peginterferon alfa-2a plus ribavirin versus peginterferon alfa-2b plus ribavirin.

PURPOSE: Treatment persistence and cost of therapy for patients with chronic hepatitis C (CHC) treated with peginterferon alfa-2a plus ribavirin and peginterferon alfa-2b plus ribavirin were evaluated. METHODS: This retrospective database analysis used eligibility, pharmacy, and medical claims data from a large U.S. health plan for patients with CHC treated with peginterferon alfa-2a plus ribavirin and peginterferon alfa-2b plus ribavirin from January 2002 through June 2006. For the purposes of this analysis, the study population included all hepatitis C virus (HCV) genotypes. Comparable groups for assessment of outcomes were constructed using propensity score matching to reduce the effect of known sources of bias. Outcome variables included treatment persistence and annualized overall and HCV-attributable health care costs. RESULTS: A total of 1783 matched pairs were analyzed. Compared with patients receiving peginterferon alfa-2a plus ribavirin, patients receiving peginterferon alfa-2b plus ribavirin were 18% less likely to be persistent with therapy at week 48 (p = 0.013). During the first six months of follow-up, mean all-cause costs (p = 0.0368) and HCV-attributable costs (p < 0.0001) were significantly lower for peginterferon alfa-2a plus ribavirin than for peginterferon alfa-2b plus ribavirin. Mean annualized all-cause costs (p = 0.0060) and HCV-attributable costs (p = 0.0167) over the entire follow-up period were significantly lower for patients treated with peginterferon alfa-2a plus ribavirin versus peginterferon alfa-2b plus ribavirin. CONCLUSION: Analysis of information from a health care claims database suggests that treating CHC with peginterferon alfa-2a plus ribavirin may improve treatment persistence and help reduce the health care costs imposed by CHC compared with treatment with peginterferon alfa-2b plus ribavirin.

Maintenance fluconazole therapy for recurrent vulvovaginal candidiasis.

BACKGROUND: No safe and convenient regimen has proved to be effective for the management of recurrent vulvovaginal candidiasis. METHODS: After inducing clinical remission with open-label fluconazole given in three 150-mg doses at 72-hour intervals, we randomly assigned 387 women with recurrent vulvovaginal candidiasis to receive treatment with fluconazole (150 mg) or placebo weekly for six months, followed by six months of observation without therapy. The primary outcome measure was the proportion of women in clinical remission at the end of the first six-month period. Secondary efficacy measures were the clinical outcome at 12 months, vaginal mycologic status, and time to recurrence on the basis of Kaplan-Meier analysis. RESULTS: Weekly treatment with fluconazole was effective in preventing symptomatic vulvovaginal candidiasis. The proportions of women who remained disease-free at 6, 9, and 12 months in the fluconazole group were 90.8 percent, 73.2 percent, and 42.9 percent, as compared with 35.9 percent, 27.8 percent, and 21.9 percent, respectively, in the placebo group (P< 0.001). The median time to clinical recurrence in the fluconazole group was 10.2 months, as compared with 4.0 months in the placebo group (P<0.001). There was no evidence of fluconazole resistance in isolates of Candida albicans or of superinfection with C. glabrata. Fluconazole was discontinued in one patient because of headache. CONCLUSIONS: Long-term weekly treatment with fluconazole can reduce the rate of recurrence of symptomatic vulvovaginal candidiasis. However, a long-term cure remains difficult to achieve.

A randomized and blinded multicenter trial of high-dose fluconazole plus placebo versus fluconazole plus amphotericin B as therapy for candidemia and its consequences in nonneutropenic subjects.

A randomized, blinded, multicenter trial was conducted to compare fluconazole (800 mg per day) plus placebo with fluconazole plus amphotericin B (AmB) deoxycholate (0.7 mg/kg per day, with the placebo/AmB component given only for the first 5-6 days) as therapy for candidemia due to species other than Candida krusei in adults without neutropenia. A total of 219 patients met criteria for a modified intent-to-treat analysis. The groups were similar except that those who were treated with fluconazole plus placebo had a higher mean (+/- standard error) Acute Physiology and Chronic Health Evaluation II score (16.8+/-0.6 vs. 15.0+/-0.7; P=.039). Success rates on study day 30 by Kaplan-Meier time-to-failure analysis were 57% for fluconazole plus placebo and 69% for fluconazole plus AmB (P=.08). Overall success rates were 56% (60 of 107 patients) and 69% (77 of 112 patients; P=.043), respectively; the bloodstream infection failed to clear in 17% and 6% of subjects, respectively (P=.02). In nonneutropenic subjects, the combination of fluconazole plus AmB was not antagonistic compared with fluconazole alone, and the combination trended toward improved success and more-rapid clearance from the bloodstream.