Single Crossover Research Articles

Mitigation and diagnosis of pin-hole formation in polymer electrolyte membrane fuel cells

Membrane electrode assemblies (MEAs) are prone to process-induced pin-holes during the hot-press lamination step often required for gas diffusion electrode (GDE) based MEAs. Membrane pin-holes can result in the crossover of hydrogen (H2) fuel, which inevitably leads to fatal damage of the MEA and fuel cell failure. In this work, we implement a calendering technique to flatten stray fibers within the gas diffusion media, thereby mitigating pin-hole formation in the hot-pressed MEAs. We have investigated the influence of calendering on the long-term durability for several types of gas diffusion electrodes (GDEs) using a combined chemical and mechanical accelerated stress test (AST). The calendered MEAs demonstrate an average AST lifetime improvement of 77% relative to the as-fabricated MEAs. In addition, voltage transients measured throughout the AST were utilized to determine the onset of pin-hole formation and subsequent cell failure. Voltage transients have the advantage of application to fuel cell stacks in lieu of ubiquitous single cell H2 crossover measurements. The application of both voltage transients and calendering pre-treatment is of broad interest to those seeking improvements to the MEA process quality, since the use of GDEs in MEAs is universal across electrochemical energy conversion applications such as CO2 and water electrolysis.

Good clinical scores, no evidence of excessive anterior tibial translation, a high return to sport rate and a low re-injury rate is observed following anterior cruciate ligament reconstruction using autologous hamstrings augmented with suture tape.

Augmented anterior cruciate ligament reconstruction (ACLR) techniques have been proposed to reduce the high reported re-injury rates and low rates of return to sport (RTS). This study reports clinical outcomes, RTS and re-injury rates in patients undergoing ACLR using autologous hamstrings augmented with suture tape. A total of 53 patients were prospectively recruited, undergoing ACLR using hamstrings with suture tape augmentation, combined with a structured rehabilitation programme. Outcomes were collected to 24months, including patient-reported outcome measures (PROMs), KT-1000 measurements, peak isokinetic knee strength and a four hop test battery. Limb Symmetry Indices (LSIs) were calculated for performance measures, whilst RTS rates, re-tears and re-operations were presented. There were no significant side-to-side differences in anterior tibial translation between the operated and non-operated knees at 6months (p = 0.433), with no increase (p = 0.841) in side-to-side anterior tibial translation from 6 to 24months. At 24months, 98.0% of patients demonstrated normal (< 3mm) or near normal (3-5mm) side-to-side differences. LSIs for peak knee extensor torque (p < 0.0001) and the single (p = 0.001), triple (p = 0.001) and triple crossover (p < 0.0001) hop tests for distance significantly improved. All PROMs significantly improved (p < 0.0001), with 70.2% and 85.7% of patients actively participating in pivoting sports at 12 and 24months, respectively. Three patients underwent secondary procedures for meniscal symptoms. One patient suffered an ACL re-tear (17months), with no further ipsilateral or contralateral injuries. ACLR with suture tape augmentation demonstrated no evidence of excessive anterior tibial translation, high-scoring PROMs, sound performance scores, a high rate of RTS and low re-injury rate.

The Effect of a Formal Nonoperative Management Program Combining a Hip Injection With Structured Adjunctive Exercise Rehabilitation in Patients With Symptomatic Femoroacetabular Impingement Syndrome

Background: Although the initial treatment recommendations for femoroacetabular impingement syndrome (FAIS) may include nonsurgical therapies such as injections and rehabilitation, many patients undergo isolated injections or a rudimentary exercise regimen. Purpose: To investigate the benefit of an intra-articular hip injection and concomitant structured exercise rehabilitation program in patients with symptomatic FAIS ≥6 months. Study Design: Case series; Level of evidence, 4. Methods: For this study we recruited 49 patients with a mean age of 32.8 years (range, 16-56 years) and symptoms ≥6 months (range, 6-250 months) associated with FAIS, as confirmed via magnetic resonance imaging and radiography. After a guided intra-articular injection of corticosteroid and local anesthetic, patients underwent a structured 12-week rehabilitation program. Patients were assessed before and after injection (8 weeks and 4, 6, 12, and 24 months) with a range of patient-reported outcome measures (PROMs), including the 33-item International Hip Outcome Tool, Hip Outcome Score, modified Harris Hip Score, the Tegner Activity Score, a visual analog scale assessing the frequency and severity of hip pain, and a Global Rating of Change scale. Range of motion, peak isometric hip strength, and hop tests were assessed. Absolute scores and limb symmetry indices were calculated. The percentage of patients transitioning toward surgery over the period was evaluated. Results: Of the 44 patients who underwent the injection and completed the initial 8-week rehabilitation component, 14 (31.8%) progressed toward surgical intervention over the 24-month postinjection period owing to dissatisfaction and/or symptom recurrence. Patients who progressed toward surgery, as compared with those who did not, reported significantly worse (P < .05) PROMs presurgery and more pain within the first 4 weeks after injection. In the nonoperative cohort, a significant improvement (P < .05) in all PROMs was observed, with 93% of these patients satisfied overall. The Global Rating of Change did not improve, although this was measured only after the injection, reflecting no further significant perceived global change from 8 weeks to 24 months. A significant increase (P < .05) in all hip range of motion and most isometric strength measures was observed at 8 weeks after injection, with these improvements largely sustained until 24 months. Bilateral improvements in hop capacity were observed, with hop test LSIs significantly improving for the single (p = 0.009), triple (p = 0.029) and triple crossover (p = 0.005) hop tests for distance. Conclusion: Although 32% of patients progressed toward surgery, significant improvement in hip pain, symptoms, and physical function was observed in the majority of patients with symptomatic FAIS as a result of a targeted nonoperative management pathway consisting of an intra-articular injection and a structured exercise program.

Video-based marker-free tracking and multi-scale analysis of mouse locomotor activity and behavioral aspects in an open field arena: A perspective approach to the quantification of complex gait disturbances associated with Alzheimer's disease.

Complex gait disturbances represent one of the prominent manifestations of various neurophysiological conditions, including widespread neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Therefore, instrumental measurement techniques and automatic computerized analysis appears essential for the differential diagnostics, as well as for the assessment of treatment effectiveness from experimental animal models to clinical settings. Here we present a marker-free instrumental approach to the analysis of gait disturbances in animal models. Our approach is based on the analysis of video recordings obtained with a camera placed underneath an open field arena with transparent floor using the DeeperCut algorithm capable of online tracking of individual animal body parts, such as the snout, the paws and the tail. The extracted trajectories of animal body parts are next analyzed using an original computerized methodology that relies upon a generalized scalable model based on fractional Brownian motion with parameters identified by detrended partial cross-correlation analysis. We have shown that in a mouse model representative movement patterns are characterized by two asymptotic regimes characterized by integrated 1/f noise at small scales and nearly random displacements at large scales separated by a single crossover. More detailed analysis of gait disturbances revealed that the detrended cross-correlations between the movements of the snout, paws and tail relative to the animal body midpoint exhibit statistically significant discrepancies in the Alzheimer's disease mouse model compared to the control group at scales around the location of the crossover. We expect that the proposed approach, due to its universality, robustness and clear physical interpretation, is a promising direction for the design of applied analysis tools for the diagnostics of various gait disturbances and behavioral aspects in animal models. We further believe that the suggested mathematical models could be relevant as a complementary tool in clinical diagnostics of various neurophysiological conditions associated with movement disorders.

SENSITIVITY ANALYSIS AND OPTIMIZATION OF WAVY FIN CIRCULAR TUBE HEAT EXCHANGER

The present study shows the strong ability of a genetic algorithm (GA) in the thermal design and optimization of a herringbone wavy fin and circular tube compact heat exchanger (FTHE). The influences of various design parameters on friction factor, Colburn factor, and minimum total annual cost (TAC) are presented. Mainly, three design objectives are considered, such as minimum friction factor, maximum Colburn factor, and minimum TAC. For optimization, the Optimization toolbox of Matlab is used. In the GA routine, tournament selection, single point crossover, and constraint dependent mutation are adopted. The multiobjective optimization reveals a couple of sets of optimal solutions by optimizing the two objectives, i.e., minimum friction factor and maximum Colburn factor. By minimizing the TAC, a significant amount of cost is found to be reduced as compared to previous works in the literature with some optimum geometrical parameters.

A novel crossover operator for genetic algorithm: Stas crossover

The genetic algorithm (GA) is a natural selection-inspired optimization algorithm. It is a population-based search algorithm that utilizes the concept of survival of the fittest. This study creates a new crossover operator called “Stas Crossover” that is a combination of four crossover operators, including Single point crossover, Two points crossover, Arithmetic crossover, and Scattered crossover, and then presents the performance of this crossover operator. The area size and probability of Stas crossover can be adjusted.GA is used to find the optimal solution for this multi-product and multi-period aggregate production planning (APP) problem, which was used to test the algorithm, which provides optimal levels of inventory, backorders, overtime and regular production rates, and other controllable variables. According to the findings of this study, the benefit of stable crossover is that it allows for more variety in the way offspring are created and increases the opportunity for offspring to obtain good genetic information directly.

Understanding the complex interplay of persistent and antipersistent regimes in animal movement trajectories as a prominent characteristic of their behavioral pattern profiles: Towards an automated and robust model based quantification of anxiety test data

Rapid advancement in computer vision technologies provides increasing opportunities for the quantitative characterization of animal behavior, although reduction of their analysis to several scalar metrics appears a common limitation for the representation of complex behavioral patterns. Here we suggest an alternative approach to the quantitative assessment of animal behavioral patterns by parameterization of a generalized scalable model based on fractional Brownian motion using detrended fluctuation analysis of the observational movement trajectories and validate it using novel tank test data. In a zebrafish model representative movement patterns are characterized by two asymptotic regimes, with persistent increments at small scales and antipersistent increments at large scales. A single crossover between these asymptotic regimes that appears a single free parameter of the animal movement model acts as a complementary behavioral indicator leading to a more explicit characterization of both stimulative and sedative effects. We show explicitly that the model can be also used for a robust estimation of interpretable scalar metrics commonly used in behavioral analysis leading to the emphasized differences between experimental groups. We believe that this approach, due to its universality, robustness and clear physical interpretation, is a perspective tool for the analysis of animal behavior complexity under various experimental and natural conditions.

An Approach for Autonomous Feeding Robot Path Planning in Poultry Smart Farm.

In order to solve the problems of poor feeding environment, untimely feeding and high labor demand in poultry smart farms, the development of feeding robots is imminent, while the research on path planning algorithms is an important part of developing feeding robots. The energy consumption of the feeding robot is one of the important elements of concern in the process of path planning. In this study, the shortest path does not mean that the feeding robot consumes the least energy, because the total mass of the feeding robot keeps changing during the feeding process. It is necessary to find the most suitable path so that the feeding robot consumes the lowest amount of energy during the feeding process. A branch and bound algorithm to calculate the minimum energy consumption travel path for small-scale buckets lacking feed is proposed. The lower bound of the branch and bound on the energy consumption is obtained by the approach of preferred selection of the set of shortest edges combined with the sequence inequality, and the upper bound could be obtained based on Christofides's Heuristic algorithm. A double-crossover operator genetic algorithm based on an upper bound on energy consumption for large-scale buckets lacking feed is proposed, and different crossover operations are performed according to the relationship between the fitness value and the upper bound of energy consumption in order to find a better path. The experiment results show that the approach proposed in this study is efficient; for small-scale buckets lacking feed, a branch and bound algorithm could calculate the minimum energy consumption path of 17 points in 300 s, and for large-scale buckets lacking feed, a double-crossover operator genetic algorithm based on an upper bound on energy consumption could calculate the minimum energy consumption travel path within 30 points in 60 s. The result is more accurate compared to the genetic algorithm with a single crossover operator.

Acute postprandial gut hormone, leptin, glucose and insulin responses to resistant starch in obese children: a single blind crossover study

IntroductionResistant starch (RS) has beneficial effects on postprandial glucose metabolism in both animals and adults. Hitherto, there have been no studies in children of the acute metabolic and hormonal effects...

Neuromuscular Training following Anterior Cruciate Ligament reconstruction – Pain, Function, Strength, Power &amp; Quality of Life Perspective: A Randomized Control Trial

To determine the effectiveness of neuromuscular physical Therapy as compared to strength training following anterior cruciate ligament reconstruction in terms of pain, function, quality of life, strength and power of participants. A randomized clinical trial was conducted at Kanaan Physiotherapy & Spine Clinic, Lahore, Pakistan from July 2020 to December 2020. Seventy-six patients were selected by non-probability convenience sampling technique and randomly divided into either neuromuscular training or strength training group. Sample included 20-40 years aged adults with unilateral anterior cruciate ligament injury who had undergone surgical reconstruction of ACL two months ago using hamstring graft. Patients were assessed using the Cincinnati Knee Score for function, Numeric Pain Rating Scale (NPRS) for pain, SF-36 for quality of life, and Single Leg Hop, Triple Hop, Crossover Hop and 6-meter Hop test for power and strength. Data was analyzed using SPSS Version-21. A t-test was used to assess difference between groups. P<0.05 was considered significant. Results revealed that neuromuscular training is statistically significant in reducing pain (p<0.001) and improving function (p<0.001), power & strength (p<0.001) and quality of life (p=0.001). Study concludes that compared to strength training, neuromuscular training was significantly more effective in reducing pain; improving function, quality of life, strength and power.

Drug-Drug Interaction Study to Evaluate the Pharmacokinetics, Safety, and Tolerability of Ipatasertib in Combination with Darolutamide in Patients with Advanced Prostate Cancer.

Ipatasertib is a selective, small molecule Akt inhibitor that is currently being developed for the treatment of metastatic castration-resistant prostate cancer. Darolutamide is an androgen receptor (AR) inhibitor that is approved for the treatment of non-metastatic castration-resistant prostate cancer. Ipatasertib is metabolized by CYP3A4 to form a less active metabolite M1 (G-037720). Ipatasertib is also a weak time-dependent CYP3A4 inhibitor. Darolutamide is a mild CYP3A4 inducer and is metabolized into an active keto-darolutamide metabolite via CYP3A4. In this Phase 1b open-label, single sequence crossover study, ipatasertib pharmacokinetics safety and tolerability were evaluated in combination with darolutamide in metastatic castration-resistant prostate cancer (n = 15 patients). Specifically, the effect of 600 mg BID of darolutamide on 400 mg QD ipatasertib was evaluated in this study. Based on pharmacokinetic analysis, a mild reduction in ipatasertib AUC0–24 h,ss and Cmax,ss exposures was observed (~8% and ~21%, respectively) when administered in combination with darolutamide, which is considered not clinically meaningful. M1 exposures were similar with and without darolutamide administration. Darolutamide and keto-darolutamide exposures in combination with ipatasertib were similar to previously reported exposures for single agent darolutamide. Overall, the combination appears to be well-tolerated in the metastatic castration-resistant prostate cancer indication with very few AEs.

Identification and Predictors of Age-Relevant and Activity-Relevant Hop Test Targets in Young Athletes After Anterior Cruciate Ligament Reconstruction.

Performance symmetry between limbs (limb symmetry index [LSI] ≥ 90%) on a battery of single-leg hop tests is recommended to inform return-to-sport (RTS) decisions after anterior cruciate ligament (ACL) reconstruction (ACLR). Achieving current hop test symmetry values has not been associated with future clinical outcomes. The identification of age-relevant and activity-relevant target values to benchmark the hop test performance of young athletes post-ACLR may provide greater specificity and clinical relevance for interpretation of hop test data. To identify single-leg hop test-target values for individual-limb performance and symmetry between limbs for athletes without a history of ACL injury and evaluate the proportion of young athletes post-ACLR who met the newly derived target values at the time of RTS clearance. The secondary objective was to test the hypothesis that better function and strength would be associated with achieving the newly derived hop test target values. Cross-sectional study. Pediatric medical center and academic medical center. A total of 159 young athletes (age = 16.9 ± 2.2 years) at the time of RTS clearance after primary, unilateral ACLR and 47 uninjured control athletes (age = 17.0 ± 2.3 years). All participants completed a single-leg hop test battery (single hop, triple hop, and crossover hop for distance [cm], and 6-m timed hop [seconds]). Raw distance values were normalized by body height, and LSI (%) was calculated for each hop test. Target values were defined as the lower bound of the 95% CI for each hop test, using control group data. Participants with ACLR also completed the Knee injury and Osteoarthritis Outcome Score subscales and a quadriceps femoris strength (newton meters/kilogram) assessment. Logistic regression determined predictors of achieving hop test target values in the ACLR group among injury, function, and strength data (P < .05). In the ACLR group, 79% to 84% of participants met the 90% LSI threshold on each hop test. They achieved the target values for surgical-limb performance in the following proportions (% participants): single hop = 29%, triple hop = 24%, crossover hop = 30%, 6-m timed hop = 18%, all hops= 12%. Also, they met the target values for LSI in the following proportions: single hop = 43%, triple hop = 48%, crossover hop = 50%, 6-m timed hop = 69%, all hops = 25%. The only predictor of achieving all hop test targets for surgical-limb performance was greater surgical-limb quadriceps femoris strength (odds ratio = 4.10, P = .007). We noted a trend toward quadriceps femoris strength LSI ≥ 90% (odds ratio = 2.44, P = .058) as a predictor for meeting all hop test symmetry targets. At the time of RTS post-ACLR, only a small proportion of young athletes achieved the age-relevant and activity-relevant single-leg hop test targets for surgical-limb performance or symmetry between limbs, even though a majority met the traditionally recommended 90% LSI threshold on hop tests.

Unit Commitment Problem with Emission Cost Constraints by Using Genetic Algorithm

A power system’s operating cost needs to be minimized by satisfying varying load demand while taking into account the prevailing constraints in a multiple unit electrical power system. In this study, by using genetic algorithms (GA), a short-term thermal unit commitment problem was solved and an economical generating unit schedule was made with the solution obtained. Taking into account the negative effects of emissions due to the use of fossil fuels, emission costs were added to the objective function together with fuel and start-up costs. The GA chromosome structure was formed by binary encoding, new generations were selected by roulette wheel selection mechanism and single point crossover was applied. The representation, formulation and the simulation results of the problem for a 5-unit test system during the scheduling hours of the period are presented. The number and the operating hours of the generating units to be committed were determined by satisfying the prevailing constraints. During the planning period, 13360 MW of power demand was met by 755 MW of spinning reserve. Total operating cost was calculated as $430330. Of the total operating cost, 32% consists of emission costs.

Douching cessation and molecular bacterial vaginosis: a reanalysis of archived specimens

ObjectivesObservational studies demonstrate an association between vaginal douching and bacterial vaginosis (BV) characterised by Gram stain. We sought to describe the effect of a douching cessation intervention on the composition...

A Return to Sport Test Battery does not Predict Second Injury After Anterior Cruciate Ligament Reconstruction in Adolescent Athletes

Background:There is mixed evidence on the effectiveness of return to sport test (RTST) batteries and their ability to predict second Anterior Cruciate Ligament (ACL) injury. The majority of tests in the literature include objective measures of strength, hop test distance, knee range of motion and laxity, and measures of psychological readiness to return. Few RTST batteries include movement quality assessments.Purpose:To determine the effectiveness of a RTST test battery, including qualitative movement assessments, to predict second ACL injury in adolescent athletes after primary Anterior Cruciate Ligament Reconstruction (ACLR).Methods:A retrospective chart review of 77 adolescent athletes who underwent primary ACLR between 2014 and 2018 (55 males, age at surgery 16 + 2 years, BMI 25 ± 5) was conducted. All athletes completed a RTST battery consisting of psychological assessment with the Tampa Scale of Kinesiophobia (TSK-11), lateral step down test (LSDT), Tuck jump assessment (TJA), single leg hop testing (SLHT) including the single, triple, and triple crossover hop for distance, and an evaluation of cutting performance. The LSDT, SLHT’s and cutting task were evaluated using a qualitative movement assessment checklist, which awarded a point for each movement fault identified. Athletes passed the RTST if they scored <19 on the TSK-11, <2 on the LSDT, <3 on all SLHT’s with a Limb Symmetry index > 95%, < 5 on the TJA and, < 2 on the cutting assessment. All variables were analyzed descriptively. For continuous variables, median and interquartile ranges (IQR) were reported and Wilcoxon rank sum test was used to assess the differences. For categorical variables, frequency and proportion were reported and chi-square or Fisher’s exact test was used to assess differences between “no second ACL injury” and “second ACL injury” groups.Results:Overall, 14.2% of athletes (N=11) suffered a second ACL injury (81.8% males). Median Tegner score in both groups was 7 (IQR: 7-9). Median time to RTS clearance was significantly different between the no second ACL injury and second ACL injury groups (10 [IQR: 8-12] vs 8[IQR:8-9] months; p=0.017). A total of 66.2% of athletes (N= 51) passed the RTST. Of those who suffered a second ACL injury, 63.6% passed the RTS. There was no statistically significant difference between those who passed the RTST and those that did not on the occurrence of second ACL injury.Conclusion:A RTST battery including qualitative movement assessments does not predict second ACL injury in adolescent athletes after ACLR.

Personalized Research on Diet in Ulcerative Colitis and Crohn's Disease: A Series of N-of-1 Diet Trials.

Evidence about specific carbohydrate diet (SCD) for inflammatory bowel disease (IBD) is limited. We conducted 54 single-subject, double-crossover N-of-1 trials comparing SCD with a modified SCD (MSCD) and comparing each with the participant's baseline, usual diet (UD). Across 19 sites, we recruited patients aged 7-18 years with IBD and active inflammation. Following a 2-week baseline (UD), patients were randomized to 1 of 2 sequences of 4 alternating 8-week SCD and MSCD periods. Outcomes included fecal calprotectin and patient-reported symptoms. We report posterior probabilities from Bayesian models comparing diets. Twenty-one (39%) participants completed the trial, 9 (17%) completed a single crossover, and 24 (44%) withdrew. Withdrawal or early completion occurred commonly (lack of response [n = 11], adverse events [n = 11], and not desiring to continue [n = 6]). SCD and MSCD performed similarly for most individuals. On average, there was <1% probability of a clinically meaningful difference in IBD symptoms between SCD and MSCD. The average treatment difference was -0.3 (95% credible interval -1.2, 0.75). There was no significant difference in the ratio of fecal calprotectin geometric means comparing SCD and MSCD (0.77, 95% credible interval 0.51, 1.10). Some individuals had improvement in symptoms and fecal calprotectin compared with their UD, whereas others did not. SCD and MSCD did not consistently improve symptoms or inflammation, although some individuals may have benefited. However, there are inherent difficulties in examining dietary changes that complicate study design and ultimately conclusions regarding effectiveness.

Influence of ice formation on the dynamic and thermodynamic properties of aqueous solutions

Water dynamics in solutions with biological or non-biological solutes has been intensely studied when both components (solvent and solute) are amorphous. Here, we apply broadband dielectric spectroscopy combined with calorimetric measurements to analyze the dynamics of the aqueous solutions tri-propylene glycol (3PG) and ε-poly (lysine) (ε-PLL), after their water becomes semi-crystalline. Various crystallization levels were explored by conducting experiments with different annealing times at temperatures above the glass transition temperature (Tg). We find that the amount of ice depends on both the time and temperature of the annealing, and that this, in turn, affects Tg and dynamics of the amorphous part of the samples. However, it should be noted that the observed differences are relatively small for the degrees of crystallinity we have studied (up to about 26 wt% of the water). This also implies that the dynamic crossover of the water relaxation from a high temperature non-Arrhenius behavior to a low temperature Arrhenius dependence is unaffected by the partial crystallization and still occurs as a single crossover at the calorimetric Tg. Thus, we cannot detect two different crossovers, as commonly observed for other types of two-component systems, such as two glass formers.

Eigenstate entanglement scaling for critical interacting spin chains

With increasing subsystem size and energy, bipartite entanglement entropies of energy eigenstates cross over from the groundstate scaling to a volume law. In previous work, we pointed out that, when strong or weak eigenstate thermalization (ETH) applies, the entanglement entropies of all or, respectively, almost all eigenstates follow a single crossover function. The crossover functions are determined by the subsystem entropy of thermal states and assume universal scaling forms in quantum-critical regimes. This was demonstrated by field-theoretical arguments and the analysis of large systems of non-interacting fermions and bosons. Here, we substantiate such scaling properties for integrable and non-integrable interacting spin-1/2 chains at criticality using exact diagonalization. In particular, we analyze XXZ and transverse-field Ising models with and without next-nearest-neighbor interactions. Indeed, the crossover of thermal subsystem entropies can be described by a universal scaling function following from conformal field theory. Furthermore, we analyze the validity of ETH for entanglement in these models. Even for the relatively small system sizes that can be simulated, the distributions of eigenstate entanglement entropies are sharply peaked around the subsystem entropies of the corresponding thermal ensembles.

Single Crossover to Inactivate Target Gene in Cyanobacteria.

Anabaena sp. PCC 7120 (hereafter Anabaena 7120) is a model cyanobacterium for studying pathways such as photosynthesis and nitrogen fixation along with many other metabolic pathways common to plants. In addition, since Anabaena 7120 forms specialized N2-fixing cells, called heterocysts, to perform uniquely solar-powered, oxic nitrogen fixation under fixed-nitrogen depleted conditions, this cyanobacterium provides the unique opportunity to study cellular differentiation in bacteria. Since more than 155,810 sequenced prokaryotic genomes are currently available (Zhang et al., Microbiome 8(1):134, 2020), target gene inactivation, combined with analyses of the corresponding mutant's phenotype, has become a powerful tool to assess gene function through detecting a loss-of-function in the knockout mutant. In the method described here, a single crossover approach is used to knockout a target gene in Anabaena 7120. The method requires inserting an internal fragment of the target gene into the cyanobacterial integration vector pZR606 to create a knockout plasmid, and then is introduced to Anabaena 7120 via conjugative transformation. A single crossover, occurring via homologous recombination, disrupts the target gene, creating 3'- and 5'-deleted fragments (Fig. 1). The mutant containing the inactivated gene can then be studied to determine any loss of function, thereby defining the gene's function. This gene inactivation approach is based on an integrative vector pZR606 (Chen et al., Appl Microbiol Biotechnol 99:1779-1793, 2015), which may be broadly applied to gene inactivation in other cyanobacterial species as well as other prokaryotic organisms.

Creation of Markerless Genome Modifications in a Nonmodel Bacterium by Fluorescence-Aided Recombineering.

Metabolic engineering of nonmodel bacteria is often challenging because of the paucity of genetic tools for iterative genome modification necessary to equip bacteria with pathways to produce high-value products. Here, we outline a homologous recombination-based method developed to delete or add genes to the genome of a nonmodel bacterium, Zymomonas mobilis, at the desired locus using a suicide plasmid that contains gfp as a fluorescence marker to track its presence in cells. The suicide plasmid is engineered to contain two 500bp regions homologous to the DNA sequence immediately flanking the target locus. A single crossover event at one of the two homologous regions facilitates insertion of the plasmid into the genome and subsequent homologous recombination events excise the plasmid from the genome, leaving either the original genotype or the desired modified genotype. A key feature of this plasmid is that Green Fluorescent Protein (GFP) expressed from the suicide plasmid allows easy identification and sorting of cells that have lost the plasmid by use of a fluorescence activated cell sorter. Subsequent PCR amplification of genomic DNA from strains lacking GFP allows rapid identification of the desired genotype, which is confirmed by DNA sequencing. This method provides an efficient and flexible platform for improved genetic engineering of Z. mobilis, which can be easily adapted to other nonmodel bacteria.