Strain Improvement Research Articles

Randomized Placebo-Controlled Trial to Evaluate Effects of Eplerenone on Myocardial Perfusion and Function Among Persons With Human Immunodeficiency Virus (HIV)-Results From the MIRACLE HIV Study.

Increased renin angiotensin aldosterone system (RAAS) activity may contribute to excess cardiovascular disease in people with HIV (PWH). We investigated how RAAS blockade may improve myocardial perfusion, injury, and function among well-treated PWH. Forty PWH, on stable ART, without known heart disease were randomized to eplerenone 50 mg PO BID (n = 20) or identical placebo (n = 20) for 12 months. The primary endpoints were (1) myocardial perfusion assessed by coronary flow reserve (CFR) on cardiac PET or stress myocardial blood flow (sMBF) on cardiac MRI or (2) myocardial inflammation by extracellular mass index (ECMi) on cardiac MRI. Beneficial effects on myocardial perfusion were seen for sMBF by cardiac MRI (mean [SD]: 0.09 [0.56] vs -0.53 [0.68] mL/min/g; P = .03) but not CFR by cardiac PET (0.01 [0.64] vs -0.07 [0.48]; P = .72, eplerenone vs placebo). Eplerenone improved parameters of myocardial function on cardiac MRI including left ventricular end diastolic volume (-13 [28] vs 10 [26] mL; P = .03) and global circumferential strain (GCS; median [interquartile range 25th-75th]: -1.3% [-2.9%-1.0%] vs 2.3% [-0.4%-4.1%]; P = .03), eplerenone versus placebo respectively. On cardiac MRI, improvement in sMBF related to improvement in global circumferential strain (ρ = -0.65, P = .057) among those treated with eplerenone. Selecting for those with impaired myocardial perfusion (CFR <2.5 and/or sMBF <1.8), there was a treatment effect of eplerenone versus placebo to improve CFR (0.28 [0.27] vs -0.05 [0.36]; P = .04). Eplerenone prevented a small increase in troponin (0.00 [-0.13-0.00] vs 0.00 [0.00-0.74] ng/L; P = .03) without effects on ECMi (0.9 [-2.3-4.3] vs -0.7 [-2.2--0.1] g/m2; P = .38). CD4+ T-cell count (127 [-38-286] vs -6 [-168-53] cells/μL; P = .02) increased in the eplerenone- versus placebo-treated groups. RAAS blockade with eplerenone benefitted key indices and prevented worsening of myocardial perfusion, injury, and function among PWH with subclinical cardiac disease when compared with placebo. NCT02740179 (https://clinicaltrials.gov/ct2/show/NCT02740179?term=NCT02740179&draw=2&rank=1).

Regularization-Based 2D Strain Tensor Imaging in Quasi-Static Ultrasound Elastography SAGE Publications.

Accurately estimating all strain components in quasi-static ultrasound elastography is crucial for the full analysis of biological media. In this study, 2D strain tensor imaging was investigated, focusing on the use of a regularization method to improve strain images. This method enforces the tissue property of (quasi-) incompressibility, while penalizing strong field variations, to smooth the displacement fields and reduce the noise in the strain components. The performance of the method was assessed with numerical simulations, phantoms, and in vivo breast tissues. For all the media examined, the results showed a significant improvement in both lateral displacement and strain, while axial fields were only slightly modified by the regularization. The introduction of penalty terms allowed us to obtain shear strain and rotation elastograms where the patterns around the inclusions/lesions were clearly visible. In phantom cases, the findings were consistent with the results obtained from the modeling of the experiments. Finally, the easier detectability of the inclusions/lesions in the final lateral strain images was associated with higher elastographic contrast-to-noise ratios (CNRs), with values in the range of [0.54-9.57] versus [0.08-0.38] before regularization.

Correction of dyssynchrony by conduction system pacing: results of LEVEL-AT trial

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Catalan Society of Cardiology; Asociación del Ritmo Cardiaco (Spanish Society of Cardiology). Background Correction of dyssynchrony measured with echo strain, in conduction system pacing (CSP) vs biventricular pacing (BIVP) has not been reported. Objectives Our purpose was to compare echocardiographic dyssynchrony correction and strain improvement during follow-up between CSP and BIVP. Methods A treatment received analysis was performed in patients (n=29 CSP; BIVP 40). Patients were evaluated at baseline (ON and OFF programming) and at 6 and 12 months. All echocardiograms were completed by operators blinded to the type of device implanted. Intraventricular (septal flash), interventricular (difference between left and right ventricular outflow times), and auriculoventricular (diastolic filling time) dyssynchrony, as well as strain parameters (septal rebound, global longitudinal strain (GLS), and Risum pattern (typical left bundle branch block contraction pattern)) were analyzed. Results 32% (22/69) were women; baseline LVEF was 27.5% (7% SD) and left ventricular end-systolic volume (LVESV) 138ml (77ml SD) without differences between groups. At the 12-month follow-up, there were no differences in CSP vs. BIVP in LVEF (45.8% (10% SD) vs. 42.7% (12% SD), p=0.25) and LVESV (81.5 ml (41 ml SD) vs. 101.7 ml (56 ml SD), p=0.08). Longitudinal analyses showed that the improvement of all echocardiographic dyssynchrony parameters over time was similar between groups (p&amp;lt;0.001) (Table 1 and Fig 1A). GLS acute correction significantly correlated with LVEF and LVESV at the 12-month follow-up (Fig 1B). Conclusions CSP and BIVP provided a similar dyssynchrony and strain correction over time. Acute strain correction predicted ventricular remodeling at 12 months follow-up.

Towards the optimization of genetic polymorphism with EMS-induced mutagenesis in Phaeodactylum tricornutum

Microalgae remain an exciting target for biotechnology as they offer a largely unexploited reservoir of novel and valuable bioactive compounds. Strain improvement programs are an expanding research field aiming to multiply microalgal potential. This study evaluates the genetic diversity created in populations of the diatom Phaeodactylum tricornutum subjected to random mutagenesis. We explored the genetic diversity using genotyping-by-sequencing (GBS) to estimate and compare the impact of the most common chemical mutagen (ethyl methanesulfonate, EMS). Five microalga populations obtained following EMS treatment had survival rates between 1 and 98 %. High genetic diversity was obtained for only one of these P. tricornutum populations, with a survival rate close to 30 %.

Evolutionary engineering and chemical mutagenesis of Propionibacterium acidipropionici for improved propionic acid production from sugarcane-derived saccharides

Propionic acid (PA) is a 3-carbon carboxylic acid widely used in the chemical, food and pharmaceutical industries. Current production of PA is primarily via petrochemical routes and alternative sources using renewable feedstocks are desirable. The goal of the present study was to develop bacterial strains for efficient PA production by fermentation of sugarcane-derived saccharides in inverted sugarcane syrup. Adaptive evolution and random chemical mutagenesis of Propionibacterium acidipropionici were individually evaluated. Although evolutionary engineering proved to be a suitable strategy for producing strains tolerant to acid stress, chemical mutagenesis coupled with a novel screening methodology proved to be a superior strategy for strain improvement. The selected strain presented high PA productivity (0.55 g L−1 h−1) and titer (40 g L−1) in relation to the parental strain (0.38 g L−1 h−1 and 27 g L−1, respectively), in fermentations using inverted sugarcane syrup.

Efficient Production of Glucaric Acid by Engineered Saccharomyces cerevisiae.

Glucaric acid is a valuable chemical with applications in the detergent, polymer, pharmaceutical and food industries. In this study, two key enzymes for glucaric acid biosynthesis, MIOX4 (myo-inositol oxygenase) and Udh (uronate dehydrogenase), were fused and expressed with different peptide linkers. It was found that a strain harboring the fusion protein MIOX4-Udh linked by the peptide (EA3K)3 produced the highest glucaric acid titer and thereby resulted in glucaric acid production that was 5.7-fold higher than that of the free enzymes. Next, the fusion protein MIOX4-Udh linked by (EA3K)3 was integrated into delta sequence sites of the Saccharomyces cerevisiae opi1 mutant, and a strain, GA16, that produced a glucaric acid titer of 4.9 g/L in a shake flask fermentation was identified by a high-throughput screening method using an Escherichia coli glucaric acid biosensor. Strain improvement by further engineering was performed to regulate the metabolic flux of myo-inositol to increase the supply of glucaric acid precursors. The downregulation of ZWF1 and the overexpression of INM1 and ITR1 increased glucaric acid production significantly, and glucaric acid production was increased to 8.49 g/L in the final strain GA-ZII in a shake flask fermentation. Finally, in a 5-L bioreactor, GA-ZII produced a glucaric acid titer of 15.6 g/L through fed-batch fermentation. IMPORTANCE Glucaric acid is a value-added dicarboxylic acid that was synthesized mainly through the oxidation of glucose chemically. Due to the problems of the low selectivity, by-products, and highly polluting waste of this process, producing glucaric acid biologically has attracted great attention. The activity of key enzymes and the intracellular myo-inositol level were both rate-limiting factors for glucaric acid biosynthesis. To increase glucaric acid production, this work improved the activity of the key enzymes in the glucaric acid biosynthetic pathway through the expression of a fusion of Arabidopsis thaliana MIOX4 and Pseudomonas syringae Udh as well as a delta sequence-based integration. Furthermore, intracellular myo-inositol flux was optimized by a series of metabolic strategies to increase the myo-inositol supply, which improved glucaric acid production to a higher level. This study provided a way for constructing a glucaric acid-producing strain with good synthetic performance, making glucaric acid production biologically in yeast cells much more competitive.

Regulation of bimodal structure and mechanical properties of powder-thixoformed GO/ZK60 magnesium matrix composite through adjusting GO content

Although remarkable strength enhancements can be achieved in graphene oxide (GO)/ graphene nanoplatelets (GNPs) reinforced Mg matrix composites by using the available techniques, their ductility is always quite poor due to the difficultly avoided strength-ductility trade-off. To conquer this dilemma, GO/ZK60 composites with bimodal-grain structure were fabricated using powder thixoforming in this work. The results indicate that the grain size and volume fraction of coarse grains (CGs) first decrease as the GO content increases to 0.2 wt.% and then increase again as the content increases to 0.3 wt.%, while the grain size in the fine grains (FGs) almost does not change. Consequently, the strength of the composites is improved with increasing GO content and reaches the peak values at the content of 0.2 wt.%. The composite with 0.1 wt.% GO content exhibits significantly increased tensile yield strength up to 177 ± 2 MPa while maintaining a high elongation of 23.1% ± 2.5%, being equivalent to that of the ZK60 matrix alloy. The increased FGs volume fraction, together with the promoted dislocation accumulation and storage via GO and grain refinement of large-sized CGs lead to the improvement of strain hardening ability, thus rendering the composite an excellent ductility. Furthermore, the deformation of the GO/ZK60 composites occurs progressively from the FGs to the CGs, which is opposite to the status of the milled ZK60 matrix alloy. In view of the microstructure characteristics of the composites, a new complex calculation model was proposed and it could well predict the strength of the bimodal GO/ZK60 composites. This study provides a new insight into the microstructure design and fabrication technology of GO/GNPs reinforced metal-based composites with high strength and ductility.

Lignocellulose-Derived Arabinose for Energy and Chemicals Synthesis through Microbial Cell Factories: A Review

The exploration of natural substrates for microbial conversion to synthesize industrial platform and fuel chemicals seems to be inevitable within a circular bioeconomy context. Hemicellulose is a natural carbohydrate polymer consisting of a variety of pentose (C5) sugar monomers such as arabinose, mannose, erythrose, and xylose. Among the C5 sugars, L-arabinose (L-Ara) is the second-most-abundant pentose sugar in the lignocellulosic biomass after xylose. L-Ara has been used as an industrial carbon source to produce several value-added chemicals such as putrescine, which is used to synthesize polymers in the textile industry; sugar alcohols that are used as sweeteners in diet foods; and amino acids such as L-lysine, L-glutamate, L-arginine, and L-ornithine, which are used in nutritional supplements, fertilizers, and other products in the food and beverage industries. L-Ara, a natural non-caloric sweetener, is used as a substitute in the food and beverage industry, when the risk of blood sugar and lipid levels could be reduced. Major use of L-Ara is also found in the medical and pharmaceutical sectors to treat several conditions, including mineral absorption disorder, constipation, and diabetes, among others. In recent years, there has been a rising interest in synthesizing various sugar alcohols and derivatives, including arabitol, xylitol, and 2,3-butanediol, through the modification of producer organisms either genetically or metabolically to produce value-added products. Understanding the current demand and the need to increase the diversified production of industrial green chemicals with the reduced waste of useful lignocellulosic resources, this review focuses on the background of L-Ara and its various sources, microbes that utilize L-Ara to produce high-value-added products, and the future prospects for strain improvements to increase the yield of high-value-added products.

Mutagenesis and Selection Strategies of SCO and Carotenoid Producing Microorganisms

Biotechnologies of microorganisms for producing various industrially important products, such as single-cell oil (SCO), single-cell protein (SCP), carotenoid, enzymes, etc., have been studied since the last century and are of current actuality to the present day. The SCO and carotenoids are an alternative to oil of plant and animal origin and to pigment of plant and synthetic origin, respectively. These cellular components of microorganisms can be used in the food, aquaculture and livestock feed, pharmaceutical, and cosmetic industries. However, microorganisms-based technologies have not found ubiquitous practice due to a number of limitations. One of them is the threshold of the oil and carotenoids content in the biomass of microorganisms due to their nature. Therefore, the development of the fast-growing strains with a high level of these product accumulation is required. Random mutagenesis, adaptive laboratory evolution (ALE), and genetic engineering are used for strain improvement. This paper reviews random mutagenesis as a simple, cost-effective tool for improving single-cell oil and carotenoid synthesis in microorganisms, followed by the selecting of mutants with preferable characteristics. Nevertheless, it should be considered that random mutagenesis accelerates naturally occurring nonspecific mutations by exposure to a physical or chemical mutagenic agent. Despite the result of a large variety of created mutants, the characteristics of the mutants can be unstable and reversible. Therefore, different cultivation strategies for developing traits of interest and testing their persistence are reviewed in this study. Choosing effective mutagenesis techniques, screening, and selection methods is essential for creating suitable mutant strains. Various strengths and drawbacks of such tools are discussed in this review, and the main directions for further development are highlighted.

Heterologous Expression of Inulinase Gene in Bacillus licheniformis 24 for 2,3-Butanediol Production from Inulin

Inulin is a renewable and cheap carbon source used in microbial fermentations. Bacillus licheniformis 24 is known as an excellent 2,3-butanediol (2,3-BD) producer from fructose; therefore, the cloning and expression of a robust heterologous inulinase could enhance its 2,3-BD production from inulin. The inu gene of Lacticaseibacillus paracasei DSM 23505 encoding fructan-β-fructosidase (EC 3.2.1.80) was chosen for the purpose. PCR fragments containing the complete inu (3.6 kb) and its truncated variant inu-tr (2.2 kb, lacking Big3 cell wall attachment domains) were cloned into Escherichia coli StellarTM and B. licheniformis 24. The high quality of the recombinant constructs was confirmed by restriction analysis, PCR, sequencing, and phenotypic tests. The results showed that the inulinase activity of B. licheniformis cells harboring the full-length inu variant (T26) was eightfold higher compared to the wild type, retaining cell wall attachment in the B. licheniformis host. In contrast, the truncated variant inu-tr (T14) showed mostly extracellular but weak activity, thus suggesting that the Big3 domains are also important for the enzyme’s function. During flask-batch fermentation of 100 g/L raw chicory flour (containing 90% inulin), T26 produced acetoin and 2,3-BD from inulin. Contrariwise, T14 and the wild type formed products only from the mono- and disaccharides naturally found in the chicory flour. In the fermenter, from 200 g/L of raw chicory flour, the recombinant T26 degraded approximately 140 g/L of the inulin. However, the final concentrations of the produced 2,3-BD and acetoin were 18.5 g/L and 8.2 g/L, respectively, because of the accumulation of unconverted sucrose. To conclude, further strain improvement is necessary to make the process efficient for obtaining 2,3-BD from inulin by simultaneous saccharification and fermentation (SSF).

Strain improvement, artificial intelligence optimization, and sensitivity analysis of asparaginase-mediated acrylamide reduction in sweet potato chips.

The online version contains supplementary material available at 10.1007/s13197-023-05757-5.

PO-03-008 CORRECTION OF DYSSYNCHRONY BY CONDUCTION SYSTEM PACING: RESULTS OF LEVEL-AT TRIAL

Correction of dyssynchrony measured with echo strain in conduction system pacing (CSP) vs biventricular pacing (BIVP) has not been reported. Our purpose was to compare echocardiographic dyssynchrony correction and strain improvement during follow-up between CSP and BIVP. A treatment received analysis was performed in patients included in the LEVEL-AT trial (NCT04054895) (n=29 CSP; BIVP 40). Patients were evaluated at baseline (ON and OFF programming) and at 6 and 12 months. All echocardiograms were completed by operators blinded to the type of device implanted. Intraventricular (septal flash), interventricular (difference between left and right ventricular outflow times), and auriculoventricular (diastolic filling time) dyssynchrony, as well as strain parameters (septal rebound, global longitudinal strain (GLS), and Risum pattern (typical left bundle branch contraction pattern)) were analyzed. 32% (22/69) were women; baseline LVEF was 27.5 ± 7% and left ventricular end-systolic volume (LVESV) 138 ± 77ml without differences between groups. At the 12-month follow-up, there were no differences in CSP vs. BIVP in LVEF (45.8 ± 10% vs. 42.7 ± 12%, p=0.25) and LVESV (81.5 ± 41 ml vs. 101.7 ± 56 ml, p=0.08). Longitudinal analyses showed that the improvement of all echocardiographic dyssynchrony parameters over time (p<0.001) was similar between groups (Table 1 and Fig 1A). A representative example of GLS at baseline and follow-up is shown in Figure 1B. GLS acute correction significantly correlated with LVEF (Fig 1C) and LVESV at the 12-month follow-up. CSP and BIVP provided similar dyssynchrony and strain correction over time. Acute strain correction predicted ventricular remodeling at 12 months follow-up.Tabled 1Table 1. (PO-03-008)Conduction system pacingBiventricular pacingEffects15 days OFF12 Months15 days OFF12 MonthsGroupTimeInteraction(Group*Time)Categorical variablesAdjusted prevalence +/- SEAdjusted prevalence +/- SEp-valueSeptal rebound0.769 +/- 0.0830.150 +/- 0.080.763 +/- 0.070.312 +/- 0.0820.13<0.0010.77Septal flash0.759 +/- 0.0790.310 +/- 0.0860.750 +/- 0.0680.108 +/- 0.0510.10<0.0010.49Risum pattern0.919 +/- 0.0730.0150 +/- 0.0210.84 +/- 0.1000.070 +/- 0.0590.81<0.0010.48Quantitative variablesAdjusted mean +/- SEAdjusted mean +/- SEp-valueGlobal longitudinal strain, %-9.62 +/- 0.68-11.28 +/- 0.73-8.69 +/- 0.57-11.28 +/- 0.590.32<0.0010.55Diastolic filling time, %47.6 +/- 1.5753.2 +/- 1.5746.0 +/- 1.3448.8 +/- 1.370.10<0.0010.57Difference in left and right ventricular outflow times, ms45.7 +/- 3.4816.6 +/- 3.4835.5 +/- 2.9621.4 +/- 3.110.76<0.0010.06Treatment received longitudinal analysis comparing CSP and BiVP in LEVEL-AT trial. Treatment effect over time but not different between groups. SE: standard error. Open table in a new tab

Effects of Edge Heating and Strain Gradient on Stretch Flange Deformation Limit of Steel Sheet

To improve the stretch flange deformation limit of ultrahigh-strength steels, a partial edge heating method was investigated. The effect of the strain gradient on the stretch flange deformation limit when a partial edge heating method was applied was investigated by the hole expansion test and FEM analysis. The stretch flange formability was improved by application of a partial edge heating method because of the recovery of the shearing microstructure and the reduction of the hardness difference of microstructure. It was found that the improvement of the deformation limit strain for the strain gradient with edge heating depends on the strain gradient region. In the low strain gradient region, the improvement margin of the deformation limit strain with edge heating was small. On the other hand, in the high strain gradient region, the deformation limit strain was markedly improved compared with that without heating. It was confirmed that the strain gradient in the direction perpendicular to the maximum principal strain could be used for the prediction of the deformation limit and also the determination of the stretch flange fracture by the FEM analysis of actual parts even under edge heating conditions.

Development of high cell density Limosilactobacillus reuteri KUB-AC5 for cell factory using oxidative stress reduction approach

BackgroundExpression systems for lactic acid bacteria have been developed for metabolic engineering applications as well as for food-grade recombinant protein production. But the industrial applications of lactic acid bacteria as cell factories have been limited due to low biomass formation resulted in low efficiency of biomanufacturing process. Limosilactobacillus reuteri KUB-AC5 is a safe probiotic lactic acid bacterium that has been proven as a gut health enhancer, which could be developed as a mucosal delivery vehicle for vaccines or therapeutic proteins, or as expression host for cell factory applications. Similar to many lactic acid bacteria, its oxygen sensitivity is a key factor that limits cell growth and causes low biomass production. The aim of this study is to overcome the oxidative stress in L. reuteri KUB-AC5. Several genes involved in oxidative and anti-oxidative stress were investigated, and strain improvement for higher cell densities despite oxidative stress was performed using genetic engineering.ResultsAn in-silico study showed that L. reuteri KUB-AC5 genome possesses an incomplete respiratory chain lacking four menaquinone biosynthesis genes as well as a complete biosynthesis pathway for the production of the precursor. The presence of an oxygen consuming enzyme, NADH oxidase (Nox), leads to high ROS formation in aerobic cultivation, resulting in strong growth reduction to approximately 25% compared to anaerobic cultivation. Recombinant strains expressing the ROS scavenging enzymes Mn-catalase and Mn-superoxide dismutase were successfully constructed using the pSIP expression system. The Mn-catalase and Mn-SOD-expressing strains produced activities of 873 U/ml and 1213 U/ml and could minimize the ROS formation in the cell, resulting in fourfold and sevenfold higher biomass formation, respectively.ConclusionsExpression of Mn-catalase and Mn-SOD in L. reuteri KUB-AC5 successfully reduced oxidative stress and enhanced growth. This finding could be applied for other lactic acid bacteria that are subject to oxidative stress and will be beneficial for applications of lactic acid bacteria for cell factory applications.

Processing and characterizing repulping reject of liquid package board

AbstractLiquid package board (LPB) packages typically have the smallest global warming potential compared to other package solutions, but recycling of the integrated plastic and aluminum fractions of the packages is still limited. In the fiber recycling process the plastic and aluminum fractions with some residual fibers are concentrated to the repulping reject (Polyal). This study investigates composition and properties of repulping rejects from recycling of separately collected LPB packages. The rejects were modified in different ways, hot pressed, grinded and finally compounded with a twin screw extruder. Compositions of the compounds were characterized with Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) and their properties were studied with tensile testing and melt flow rate (MFR) measurements. All studied compounds had relatively good processability and mechanical properties. Properties of compounds were improved by extraction of residual fibers and melt filtering of polyethylene terephthalate (PET) flakes. Especially melt filtering offered a significant improvement in strain at break from 11% to 33%. It was also found that high density polyethylene (HDPE) should be extracted from the reject and recycled separately since the extraction is relatively easy, does not cause drastic changes in the compound properties and recycled HDPE has more potential applications than the Polyal compounds.

Multiple strategies for the development of multienzyme complex for one-pot reactions

The main intention in the modern era is to makelife and activities on earth more comfortable by adding necessary products through biological machinery. Millions of tons of biological raw materials and lignocellulosic biomass are wasted by burning each year without providing benefits to living organisms. Instead of being the cause of disturbing the natural environment by increasing global warming and pollutants worldwide, now, it is the need of the hour to develop an advanced strategy to utilize these biological raw materials to produce renewable energy resources to meet the energy crisis. The review presents the idea of multiple enzymes in one step to hydrolyze complex biomaterials into useful products. The paper discusses how multiple enzymes are arranged in a cascade for complete hydrolysis of raw material in one-pot to prevent multistep, time consuming, and expensive methods. Furthermore, there was the immobilization of multiple enzymes in a cascade system with in vitro and in vivo conditions for reusability of enzymes. The role of genetic engineering, metabolic engineering, and random mutation techniques is described for the development of multiple enzyme cascades. Techniques that are involved in the improvement of native strain to recombinant strain for the enhancement of hydrolytic capacity were used. The preparative steps, before enzymatic hydrolysis like acid, and base treatment methods are more effective for improving the hydrolysis of biomass by multiple enzymes in a one-pot system. Finally, the applications of one-pot multienzyme complexes in biofuel production from lignocellulosic biomass, biosensor production, medicine, food industry, and the conversion of biopolymers into useful products are described.

Transcriptome Analysis Reveals an Eicosapentaenoic Acid Accumulation Mechanism in a Schizochytrium sp. Mutant.

Eicosapentaenoic acid (EPA) is an omega-3 long-chain polyunsaturated fatty acid (PUFA) essential for human health. Schizochytrium is a marine eukaryote that has been widely utilized for the synthesis of PUFAs. The current low potency and performance of EPA production by fermentation of Schizochytrium spp. limits its prospect in commercial production of EPA. Since the synthesis pathway of EPA in Schizochytrium spp. is still unclear, mutagenesis combined with efficient screening methods are still desirable. In this study, a novel screening strategy was developed based on a two-step progressive mutagenesis method based on atmospheric and room temperature plasma (ARTP) and diethyl sulfate (DES) after multiple stresses (sethoxydim, triclosan and 2,2'-bipyridine) compound screening. Finally, the mutant strain DBT-64 with increased lipid (1.57-fold, 31.71 g/L) and EPA (5.64-fold, 1.86 g/L) production was screened from wild-type (W) strains; the docosahexaenoic acid (DHA) content of mutant DBT-64 (M) was 11.41% lower than that of wild-type strains. Comparative transcriptomic analysis showed that the expression of genes related to the polyketide synthase, fatty acid prolongation, and triglyceride synthesis pathways was significantly upregulated in the mutant strain, while the expression of genes involved in the β-oxidation pathway and fatty acid degradation pathway was downregulated in favor of EPA biosynthesis in Schizochytrium. This study provides an effective strain improvement method to enhance EPA accumulation in Schizochytrium spp. IMPORTANCE Schizochytrium, a marine eukaryotic microorganism, has emerged as a candidate for the commercial production of PUFAs. EPA is an omega-3 PUFA with preventive and therapeutic effects against cardiovascular diseases, schizophrenia, and other disorders. Currently, the low potency and performance of EPA production by Schizochytrium spp. limits its commercialization. In this study, we performed two-step progressive mutagenesis based on ARTP and DES and screened multiple stresses (sethoxydim, triclosan, and 2,2'-bipyridine) to obtain the EPA-high-yielding Schizochytrium mutant. In addition, high expression of the polyketide synthase pathway, fatty acid elongation pathway, and triglyceride synthesis pathway in the mutants was confirmed by transcriptomic analysis. Therefore, the multistress screening platform established in this study is important for breeding EPA-producing Schizochytrium spp. and provides valuable information for regulating the proportion of EPA in microalgal lipids by means of genetic engineering.

Strain Analysis in Patients with Frequent Premature Ventricular Complexes and Preserved Left Ventricular Function Undergoing Ablation.

Frequent premature ventricular complexes (PVCs) can cause PVC-induced cardiomyopathy. The value of PVC ablation in patients with preserved left ventricular function in the low-normal range (ejection fraction: 50-55%) is not established. Strain analysis has been used to estimate changes in left ventricular function beyond assessment of the ejection fraction (EF). Longitudinal strain has been proposed as a method to detect changes over time in the setting of frequent asymptomatic premature ventricular complexes and preserved left ventricular (LV) function. A decrease in strain may be evidence of PVC-induced cardiomyopathy. In this study, we assessed the role of PVC ablation in patients with low-normal EF and the effect on EF and myocardial strain before and after PVC ablation. A total of 70 consecutive patients with either low-normal EF (0.5-<0.55, n = 35) or high-normal EF (≥0.55; n = 35), using available imaging and Holter data, were referred for ablation due to frequent PVCs. EF and longitudinal strain were assessed pre- and post-ablation. There was a significant increase in EF (53.2 ± 0.4% to 58.3 ± 0.5%, p < 0.001) and improvement in longitudinal strain (-15.2 ± 3.3 to -16.6 ± 3, p = 0.007) post-ablation in patients with low-normal EF and successful ablation. There was no change in EF or longitudinal strain in patients with high-normal EF and a successful ablation pre- vs. post-ablation. Patients with frequent PVCs and low-normal LV EF compared to patients with frequent PVCs and high-normal LV EF have evidence of PVC-induced cardiomyopathy and may benefit from ablation despite a preserved left ventricular EF.

Characterization of a Riboflavin-Producing Mutant of Bacillus subtilis Isolated by Droplet-Based Microfluidics Screening.

Bacillus subtilis is one of the commonly used industrial strains for riboflavin production. High-throughput screening is useful in biotechnology, but there are still an insufficient number of articles focusing on improving the riboflavin production of B. subtilis by this powerful tool. With droplet-based microfluidics technology, single cells can be encapsulated in droplets. The screening can be carried out by detecting the fluorescence intensity of secreted riboflavin. Thus, an efficient and high-throughput screening method suitable for riboflavin production strain improvement could be established. In this study, droplet-based microfluidics screening was applied, and a more competitive riboflavin producer U3 was selected from the random mutation library of strain S1. The riboflavin production and biomass of U3 were higher than that of S1 in flask fermentation. In addition, the results of fed-batch fermentation showed that the riboflavin production of U3 was 24.3 g/L, an 18% increase compared with the parent strain S1 (20.6 g/L), and the yield (g riboflavin/100 g glucose) increased by 19%, from 7.3 (S1) to 8.7 (U3). Two mutations of U3 (sinRG89R and icdD28E) were identified through whole genome sequencing and comparison. Then they were introduced into BS168DR (parent of S1) for further analysis, which also caused riboflavin production to increase. This paper provides protocols for screening riboflavin-producing B. subtilis with droplet-based microfluidics technology and reveals mutations in riboflavin overproduction strains.

Enhanced Pentostatin Production in Actinomadura sp. by Combining ARTP Mutagenesis, Ribosome Engineering and Subsequent Fermentation Optimization

The special structure of pentostatin causes it to possess a wide spectrum of biological and pharmacological properties, and it has been extensively employed to treat malignant tumors and is the first-line treatment for hairy cell leukemia. Pentostatin is mainly distributed in several actinomycetes and fungi species. However, its low titer in microbes is not able to meet medical needs. Here, we report a strain improvement strategy based on combined atmospheric and room-temperature plasma (ARTP) mutagenesis and ribosome engineering screening, as well as fermentation optimization, for enhanced pentostatin production. The original strain, Actinomadura sp. ATCC 39365, was treated with ARTP and screened by ribosome engineering to obtain one stable pentostatin high-yield mutant Actinomadura sp. S-15, which produced 86.35 mg/L pentostatin, representing a 33.79% increase compared to Actinomadura sp. ATCC 39365. qRT-PCR analysis revealed that pentostatin biosynthesis-related gene expression was significantly upregulated in Actinomadura sp. S-15. Then, to further enhance pentostatin production, the fermentation medium was optimized in flask culture and the pentostatin production of Actinomadura sp. S-15 reached 152.06 mg/L, which is the highest pentostatin production reported so far. These results demonstrate the effectiveness of combined ARTP mutation, ribosome engineering screening, and medium optimization for the enhancement of pentostatin production, and provide a methodology enabling the sustainable production of pentostatin on an industrial scale.