Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations.

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6-9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3-5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments.

Exploring the Influence of Growth-Associated Host Genetics on the Initial Gut Microbiota in Horses.

The influences of diet and environmental factors on gut microbial profiles have been widely acknowledged; however, the specific roles of host genetics remain uncertain. To unravel host genetic effects, we raised 47 Jeju crossbred (Jeju × Thoroughbred) foals that exhibited higher genetic diversity. Foals were raised under identical environmental conditions and diets. Microbial composition revealed that Firmicutes, Bacteroidetes, and Spirochaetes were the predominant phyla. We identified 31 host-microbiome associations by utilizing 47,668 single nucleotide polymorphisms (SNPs) and 734 taxa with quantitative trait locus (QTL) information related to horse growth. The taxa involved in 31 host-microbiome associations were functionally linked to carbohydrate metabolism, energy metabolic processes, short-chain fatty acid (SCFA) production, and lactic acid production. Abundances of these taxa were affected by specific SNP genotypes. Most growth-associated SNPs are found between genes. The rs69057439 and rs69127732 SNPs are located within the introns of the VWA8 and MFSD6 genes, respectively. These genes are known to affect energy balance and metabolism. These discoveries emphasize the significant effect of host SNPs on the development of the intestinal microbiome during the initial phases of life and provide insights into the influence of gut microbial composition on horse growth.

Tackling the Catch-22 Situation of Optimizing a Sensor and a Transporter System in a Whole-Cell Microbial Biosensor Design for an Anthropogenic Small Molecule.

Whole-cell biosensors provide a convenient detection tool for the high-throughput screening of genetically engineered biocatalytic activity. But establishing a biosensor for an anthropogenic molecule requires both a custom transporter and a transcription factor. This results in an unavoidable "Catch-22" situation in which transporter activity cannot be easily confirmed without a biosensor and a biosensor cannot be established without a functional transporter in a host organism. We overcame this type of circular problem while developing an adipic acid (ADA) sensor. First, leveraging an established cis,cis-muconic acid (ccMA) sensor, an annotated ccMA transporter MucK, which is expected to be broadly responsive to dicarboxylates, was stably expressed in the genome of Pseudomonas putida to function as a transporter for ADA, and then a PcaR transcription factor (endogenous to the strain and naturally induced by ß-ketoadipic acid, BKA) was diversified and selected to detect the ADA molecule. While MucK expression is otherwise very unstable in P. putida under strong promoter expression, our optimized mucK expression was functional for over 70 generations without loss of function, and we selected an ADA sensor that showed a specificity switch of over 35-fold from BKA at low concentrations (typically <0.1 mM of inducers). Our ADA and BKA biosensors show high sensitivity (low detection concentration <10 µM) and dynamic range (∼50-fold) in an industrially relevant organism and will open new avenues for high throughput discovery and optimization of enzymes and metabolic pathways for the biomanufacture of these molecules. In particular, the novel ADA sensor will aid the discovery and evolution of efficient biocatalysts for the biological recycling of ADA from the degradation of nylon-6,6 waste.

Engineered pH-Sensitive Protein G/IgG Interaction.

While natural protein-protein interactions have evolved to be induced by complex stimuli, rational design of interactions that can be switched-on-demand still remain challenging in the protein design world. Here, we demonstrate that a computationally redesigned natural interface for improved binding affinity could further be mutated to adopt a pH switchable interaction. The redesigned interface of Protein G/human IgG Fc domain (referred to as PrG/hIgG), when incorporated with histidine and glutamic acid on PrG (PrG-EHHE), showed a switch in binding affinity by 50-fold when the pH was altered from mild acidic to mild basic. The wild-type (WT) interface showed a negligible switch. The overall binding affinity under mild acidic pH for PrG-EHHE outperformed the wild-type PrG (PrG-WT) interaction. The new reagent PrG-EHHE can be revolutionary in IgG purification, since the standard method of using an extreme acidic pH for elution can be circumvented.

Relationship between the maxillofacial skeletal pattern and the morphology of the mandibular symphysis: Structural equation modeling.

OBJECTIVE: The purpose of this study was to investigate the relationship between the facial skeletal patterns and the shape of the mandibular symphysis in adults with malocclusion by using a structural equation model (SEM). METHODS: Ninety adults who had malocclusion and had records of facial skeletal measurements performed using cone-beam computed tomography were selected for this study. The skeletal measurements were classified into three groups (vertical, anteroposterior, and transverse). Cross-sectional images of the mandibular symphysis were analyzed using generalized Procrustes and principal component (PC) analyses. A SEM was constructed after the factors were extracted via factor analysis. RESULTS: Two factors were extracted from the transverse, vertical, and anteroposterior skeletal measurements. Latent variables were extracted for each factor. PC1, PC2, and PC3 were selected to analyze the variations of the mandibular symphyseal shape. The SEM was constructed using the skeletal variables, PCs, and latent variables. The SEM showed that the vertical latent variable exerted the most influence on the mandibular symphyseal shape. CONCLUSIONS: The relationship between the skeletal pattern and the mandibular symphysis was analyzed using a SEM, which showed that the vertical facial skeletal pattern had the highest effect on the shape of the mandibular symphysis.

Analysis of the relationship between the morphology of the palate and facial skeletal patterns in Class III malocclusion using structural equation modelling.

INTRODUCTION: The present study investigated the relationship between facial skeletal patterns and morphology of the palate in adult patients with Class III malocclusion using structural equation modelling (SEM). SETTING AND SAMPLE POPULATION: One hundred cone beam computed tomography images of Class III adults were evaluated for skeletal measurements. MATERIALS AND METHODS: The skeletal measurements were classified into the vertical, anteroposterior and transverse group based on factor analysis. 3D scanning model of the maxilla was analysed by Generalized procrustes analysis (GPA) and principal component analysis (PCA). Structural equation modelling was used to analyse relationship among the skeletal and morphometric factors. RESULTS: According to the factor analysis, latent variables were extracted by each skeletal variable. First principal component (PC1) and PC2 of palatal morphology were used to analyse relationship with skeletal variables. As results of the structural equation model, the transverse latent variable had the most influence on PC1, followed by vertical and anteroposterior variables. This result means that as the facial width increases, the palate becomes narrower, deeper and longer. CONCLUSIONS: The relationship between the skeletal pattern with Class III malocclusion and palatal morphology was analysed through SEM. The transverse facial skeletal pattern showed the highest correlation with PC1 of palatal morphology.

Comparison of temporomandibular joint shape and size in patients with facial asymmetry.

OBJECTIVE: To examine the morphologic similarities and differences in mandibular condyle and glenoid fossa between the deviated and non-deviated sides in patients with facial asymmetry using statistical analysis. METHODS: One hundred eighty-four patients (95 men, 89 women; mean age, 22.30 ± 3.87 years) with facial asymmetry were examined with cone-beam computed tomography (CBCT). Shape analysis was performed to compare the shapes and sizes of the condyle and fossa between the deviated and non-deviated sides in the frontal and lateral aspects. Ordinary Procrustes analysis (OPA) was used to determine shape variations of the fossa and condyle. RESULTS: Shape variations derived from ordinary sum of squares (OSS) suggested that, in the frontal aspect, there was a difference between the two sides in both the fossa and condyle (P < 0.05). In the lateral aspect, there was no difference in fossa shape between the two sides (P > 0.05); however, the shape of condyle was different between the non-deviated and deviated sides (P < 0.05). Size comparison in OPA matching and centroid size (CS) in the frontal aspect demonstrated that the non-deviated side was larger than the deviated side. In the lateral aspect, fossa showed no difference in CS between the two sides (P > 0.05); however, the non-deviated side was larger than the deviated side for condyle (P < 0.05). CONCLUSIONS: Morphometric differences in condyle and fossa between the deviated and non-deviated sides were observed. Structures of the non-deviated side were relatively larger than those of the deviated side, except for fossa in the lateral aspect.

Correlation between the cross-sectional morphology of the mandible and the three-dimensional facial skeletal pattern: A structural equation modeling approach.

OBJECTIVE: To clarify the relationship between the cross-sectional morphology of the mandible and vertical, transverse, and anteroposterior facial skeletal patterns using statistical shape analysis and structural equation modeling (SEM). MATERIALS AND METHODS: We used 150 cone beam computed tomography (CBCT) images to obtain three-dimensional (3D) facial landmarks and cross-sectional images of the mandible. The morphology of the inner and outer cortices of the mandible was analyzed using statistical shape analysis, including generalized Procrustes analysis and principal component analysis (PCA). Factor analysis was performed to determine factors pertaining to the skeletal measurements and shape variations for the inner and outer cortices, following which a structural equation model was constructed. RESULTS: Using factor analysis, characteristics of the vertical, transverse, and anteroposterior facial skeletal patterns were determined. PCA of the cross-sectional morphology of the mandible revealed 70% of the cumulative proportion by PC1 and PC2 after generalized Procrustes superimpositions. SEM showed complex relationships between the facial skeletal patterns and variations in the cross-sectional morphology of the mandibular cortices. The influence of the transverse factors on the outer cortex as a latent variable was relatively significant ( P = .057). However, the influence of the vertical factors on the outer and inner cortices was not significant. CONCLUSIONS: The transverse skeletal pattern is associated with the morphology of the outer cortex of the mandible.

Antioxidant and skin-whitening effects of aerial part of Euphorbia supina Raf. Extract.

BACKGROUND: Euphorbia supina (ES) has been widely used in folk medicine owing to its antibacterial, hemostatic, and anti-inflammatory properties. The aim of this study was to evaluate the antioxidant and skin-whitening effects of a 70% ethanol extract of ES. METHODS: The aerial parts of ES plant were extracted with 70% ethanol. The viability of B16F10 cells was evaluated by MTT assay to determine the non-toxic doses for further experiments. The tyrosinase and cellular tyrosinase activities were then measured using an enzyme-substrate assay. In addition, the expression of whitening-related proteins was measured using western blot. RESULTS: The antioxidant activity of the ES samples increased in a dose-dependent manner, as confirmed by their radical scavenging activities in the 2,2-diphenyl-1-1-picrylhydrazyl and 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) assays. The ES extract significantly reduced tyrosinase activity and melanin content in a dose-dependent manner. Furthermore, it decreased α-melanocyte stimulating hormone (MSH)-induced protein expression of tyrosinase and microphthalmia-associated transcription factor (MITF). CONCLUSIONS: Our results indicate that the ES extract attenuated α-MSH-stimulated melanin synthesis by modulating tyrosinase and MITF expression. Therefore, the ES extract could be a promising therapeutic agent to treat hyperpigmentation and as an ingredient for skin-whitening cosmetics.

Genome-wide analyses of the Jeju, Thoroughbred, and Jeju crossbred horse populations using the high density SNP array.

The Jeju horse is an indigenous Korean horse breed that is currently registered with the Food and Agriculture Organization of the United Nations. However, there is severe lack of genomic studies on Jeju horse. This study was conducted to investigate genetic characteristics of horses including Jeju horse, Thoroughbred and Jeju crossbred (Jeju × Thoroughbred) populations. We compared the genomes of three horse populations using the Equine SNP70 Beadchip array. Short-range Linkage disequilibrium was the highest in Thoroughbred, whereas r2 values were lowest in Jeju horse. Expected heterozygosity was the highest in Jeju crossbred (0.351), followed by the Thoroughbred (0.337) and Jeju horse (0.311). The level of inbreeding was slightly higher in Thoroughbred (- 0.009) than in Jeju crossbred (- 0.035) and Jeju horse (- 0.038). FST value was the highest between Jeju horse and Thoroughbred (0.113), whereas Jeju crossbred and Thoroughbred showed the lowest value (0.031). The genetic relationship was further assessed by principal component analysis, suggesting that Jeju crossbred is more genetically similar to Thoroughbred than Jeju horse population. Additionally, we detected potential selection signatures, for example, in loci located on LCORL/NCAPG and PROP1 genes that are known to influence body. Genome-wide analyses of the three horse populations showed that all the breeds had somewhat a low level of inbreeding within each population. In the population structure analysis, we found that Jeju crossbred was genetically closer to Thoroughbred than Jeju horse. Furthermore, we identified several signatures of selection which might be associated with traits of interest. To our current knowledge, this study is the first genomic research, analyzing genetic relationships of Jeju horse, Thoroughbred and Jeju crossbred.

Estimation of genetic parameters for temperament in Jeju crossbred horses.

OBJECTIVE: Temperament can be defined as a type of behavioral tendency that appears in a relatively stable manner in responses to various external stimuli over time. The aim of this study was to estimate genetic parameters for the records of temperament testing that are used to improve the temperament of Jeju crossbred (Jeju×Thoroughbred) horses. METHODS: This study was conducted using 205 horses (101 females and 104 males) produced between 2010 and 2015. The experimental animals were imprinted and tamed according to the Manual for Horse Taming and Evaluation for Therapeutic Riding Horses and evaluated according to the categories for temperament testing (gentleness, patience, aggressiveness, sensitivity, and friendliness) between 15 months and 18 months of age. Each category was scored on a five-point linear scale. Genetic parameters for the test categories were analyzed using a multi-trait mixed model with repeated records. The ASReml program was used to analyze the data. RESULTS: The heritability of gentleness, patience, aggressiveness, sensitivity and friendliness ranged from 0.08 to 0.53. The standard errors of estimated heritability ranged from 0.13 to 0.17. The test categories showed high genetic correlations with each other, ranging from 0.96 to 0.99 and high repeatability, ranging from 0.70 to 0.73. CONCLUSION: The results of this study showed that the test categories had moderate heritability and high genetic correlations, but additional studies may be necessary to use the results for the improvement programs of the temperament of Jeju crossbred horses.

Morphometric analysis for evaluating the relation between incisal guidance angle, occlusal plane angle, and functional temporomandibular joint shape variation.

OBJECTIVE: The correlations between morphology of the temporomandibular joint structure, the anterior guidance angle, and occlusal plane were investigated. MATERIALS AND METHODS: A cone beam computed tomography analysis was performed in 158 patients (86 women and 72 men). 3D software was employed to obtain the coordinates of the shape of the incisal guidance angle, occlusal guidance angle, articular fossa, and mandibular condyle. Generalized Procrustes analysis including principal components analysis (PCA) were performed and produced principal components (PCs) scores of each shape and their centroid size (CS). RESULTS: A significant Pearson correlation coefficient of 0.3451 (p < .001) was observed between the incisal guidance angle and occlusal plane. The CS also showed a correlation with the incisal guidance angle, but not with the occlusal plane angle. The PCA results revealed that there were no significant correlations between the temporomandibular joint structure (TMJ) shape (fossa and condyle) and the incisal guidance angle. CONCLUSIONS: Incisor guidance angle and occlusal plane angle were correlated. In addition, there was a correlation between CS and incisal guidance angle. In the PCA, It can be concluded that the size is more related to the incisor guidance angle than the morphological factors of the constituent components of the TMJ.

Methylglyoxal synthase regulates cell elongation via alterations of cellular methylglyoxal and spermidine content in Bacillus subtilis.

Methylglyoxal regulates cell division and differentiation through its interaction with polyamines. Loss of their biosynthesizing enzyme causes physiological impairment and cell elongation in eukaryotes. However, the reciprocal effects of methylglyoxal and polyamine production and its regulatory metabolic switches on morphological changes in prokaryotes have not been addressed. Here, Bacillus subtilis methylglyoxal synthase (mgsA) and polyamine biosynthesizing genes encoding arginine decarboxylase (SpeA), agmatinase (SpeB), and spermidine synthase (SpeE), were disrupted or overexpressed. Treatment of 0.2mM methylglyoxal and 1mM spermidine led to the elongation and shortening of B. subtilis wild-type cells to 12.38±3.21µm (P<0.05) and 3.24±0.73µm (P<0.01), respectively, compared to untreated cells (5.72±0.68µm). mgsA-deficient (mgsA-) and -overexpressing (mgsAOE) mutants also demonstrated cell shortening and elongation, similar to speB- and speE-deficient (speB- and speE-) and -overexpressing (speBOE and speEOE) mutants. Importantly, both mgsA-depleted speBOE and speEOE mutants (speBOE/mgsA- and speEOE/mgsA-) were drastically shortened to 24.5% and 23.8% of parental speBOE and speEOE mutants, respectively. These phenotypes were associated with reciprocal alterations of mgsA and polyamine transcripts governed by the contents of methylglyoxal and spermidine, which are involved in enzymatic or genetic metabolite-control mechanisms. Additionally, biophysically detected methylglyoxal-spermidine Schiff bases did not affect morphogenesis. Taken together, the findings indicate that methylglyoxal triggers cell elongation. Furthermore, cells with methylglyoxal accumulation commonly exhibit an elongated rod-shaped morphology through upregulation of mgsA, polyamine genes, and the global regulator spx, as well as repression of the cell division and shape regulator, FtsZ.

Qualitative correlation between postoperatively increased vertical dimension and mandibular position in skeletal class III using partial-least-square path modeling.

BACKGROUND: This study constructed a partial-least-square path-modeling (PLS-PM) model and found the pathway by which the postsurgical vertical dimension (VD) affects the extent of the final mandibular setback on the B point at the posttreatment stage for the skeletal class III surgery-first approach (SFA). METHODS: This study re-analyzed the data from the retrospective study by Lee et al. on 40 patients with skeletal class III bimaxillary SFA. Variables were obtained from cone beam computed tomography (CBCT)-generated cephalograms. Authors investigated all variables at each time point to build a PLS-PM model to verify the effect of the VD on the final setback of the mandible. RESULTS: From PLS-PM, an increase in VD10 was found to decrease the absolute value of the final setback amount of the mandible, which reflects the postsurgical physiological responses to both surgery and orthodontic treatment, which, in turn, can be interpreted as an increase in postoperative mandibular changes. CONCLUSIONS: To resolve the issue of collinear cephalometric data, the present study adopted PLS-PM to assess the orthodontic treatment. From PLS-PM, it was able to summarize the effect of increased postsurgery occlusal vertical dimension on the increased changeability of the B point position at the posttreatment stage.

The estimation of skeletal maturity of patients with cleft lip and palate using statistical shape analysis: a preliminary study.

OBJECTIVES: To propose a skeletal maturity assessment method by developing a statistical regression analysis model through the integration of lateral and axial images of the cervical vertebrae of patients with cleft lip and palate obtained through CBCT. METHODS: 49 patients with cleft lip and palate (28 females, 21 males; age range, 4-16 years) underwent CBCT examination, and the hand-wrist radiographic data were selected. With coordinates of landmarks from lateral and axial images of the cervical vertebrae, Procrustes analysis and principal component (PC) analysis yielded PC scores of each cervical vertebra, with the centroid size as the size factor. The meaningful PC scores from these were used for multiple regression models. RESULTS: When both axial and lateral cervical vertebrae were used together, there was a 6.7% increase in the Sempé maturation level explanatory power for skeletal maturation estimation in females and an 11.4% increase in males compared with that when only the chronological age was used. CONCLUSIONS: This study improved the estimating regression models using statistical shape analysis with lateral and axial cervical vertebral shapes. The obtained models had improved explanatory power for skeletal maturity estimation than previous studies with healthy people.

Genome scan linkage analysis identifies a major quantitative trait loci for fatty acid composition in longissimus dorsi muscle in an F2 intercross between Landrace and Korean native pigs.

OBJECTIVE: This study was conducted to locate quantitative trait loci (QTL) influencing fatty acid (FA) composition in a large F2 intercross between Landrace and Korean native pigs. METHODS: Eighteen FA composition traits were measured in more than 960 F2 progeny. All experimental animals were genotyped with 165 microsatellite markers located throughout the pig autosomes. RESULTS: We detected 112 QTLs for the FA composition; Forty seven QTLs reached the genome-wide significant threshold. In particular, we identified a cluster of highly significant QTLs for FA composition on SSC12. QTL for polyunsaturated fatty acid on pig chromosome 12 (F-value = 97.2 under additive and dominance model, nominal p-value 3.6×10-39) accounted for 16.9% of phenotypic variance. In addition, four more QTLs for C18:1, C18:2, C20:4, and monounsaturated fatty acids on the similar position explained more than 10% of phenotypic variance. CONCLUSION: Our findings of a major QTL for FA composition presented here could provide helpful information to locate causative variants to improve meat quality traits in pigs.

Preliminary Study to Determine the Reference Plane of Patients With a Unilateral Cleft Lip and Palate.

OBJECTIVE: The objective of this study was to determine the three-dimensional midsagittal reference planes for unilateral cleft lip and palate (UCLP) patients that can be easily applied in a clinical setting. DESIGN: This was a retrospective analysis. PATIENTS: There were 35 UCLP patients (25 men, 10 women; 28.1 ± 6.9 years old) in this study. METHODS: With landmark's three-dimensional coordinates obtained from cone-beam computed tomography, the symmetric midsagittal reference planes were calculated by applying the ordinary Procrustes superimposition method using the original and mirror images. Procrustes analysis was also used to find the closest landmarks to the calculated symmetric midsagittal reference plane and test its compatibility with the symmetrical midsagittal reference plane. MAIN OUTCOME MEASURE: The three nearest landmarks to the symmetric midsagittal reference plane were Opisthion, Basion, and Nasion. RESULTS: The averages of the sums of the squared Euclidean distance and squared Procrustes distance differences between the two configurations and shapes fabricated by the symmetrical and landmark-based midsagittal reference planes, respectively, were calculated as 1.836 ± 3.295 and 1.519 × 10-5 ± 2.351 × 10-5. CONCLUSION: It was confirmed that the midsagittal reference planes from these selected landmarks for UCLP patients were compatible with symmetric midsagittal reference planes from the Procrustes analysis and the asymmetric measurements.

Statistical shape analysis-based determination of optimal midsagittal reference plane for evaluation of facial asymmetry.

INTRODUCTION: The purpose of this study was to determine, by statistical shape analysis of original and mirrored skeletal landmarks, the optimal landmark-based midsagittal reference plane for evaluation of facial asymmetry. METHODS: The study sample comprised 69 patients with facial asymmetry (36 men, 33 women; mean age, 23.0 ± 4.1 years). All landmarks were obtained with cone-beam computed tomography using a 3-dimensional coordinate system. For identifying the landmark-based midsagittal reference plane, the 3 landmarks nearest to the symmetric midsagittal reference plane were selected by ordinary and generalized Procrustes analyses. To verify the 3-landmark-based midsagittal reference plane's compatibility with the symmetric midsagittal reference plane, asymmetry measurements were calculated and tested for each. RESULTS: The 3 nearest landmarks (nasion, anterior nasal spine, and posterior nasal spine) were selected for the 3-landmark-based midsagittal reference plane. The averages of the sums of the squared Euclidean distance and the squared Procrustes distance differences between the 2 configurations and shapes fabricated by the symmetric and landmark-based midsagittal reference planes, respectively, were calculated as 0.121 ± 0.241 mm and 1.69 × 10(-6) ± 3.25 × 10(-6). The testing results for the symmetric and landmark-based midsagittal reference planes were almost the same. CONCLUSION: The results indicated that a 3-dimensional midsagittal reference plane constructed of nasion, anterior nasal spine, and posterior nasal spine could be a valuable tool for the evaluation of patients with facial asymmetry.