Methylobacterium strains with the closely related compound dichloromethane (DCM), we find that chloride export does not limit growth on CM and, in general that the ability of a strain to grow on DCM is uncorrelated with its ability to grow on CM. This heterologous expression system allows us to investigate the components required for effective CM catabolism and the factors that limit effective catabolism after horizontal transfer.Chloromethane (CM) is an ozone-depleting gas, produced predominantly from natural sources, that provides an important carbon source for microbes capable of consuming it. CM catabolism has been difficult to study owing to the challenging genetics of its native microbial hosts. Since the pathways for CM catabolism show evidence of horizontal gene transfer, we reproduced this transfer process in the laboratory to generate new CM-catabolizing strains in tractable hosts. We demonstrate that six putative accessory genes improve CM catabolism, though heterologous expression of only one of the six is strictly necessary for growth on CM. In contrast to growth of  Chloromethane (CM) is the most abundant organohalide on earth, accounting for roughly 16% of tropospheric chlorine in 2012, and therefore contributes to chloride-catalyzed ozone depletion . SourcesMethylobacterium extorquens CM4 (hereafter ‘CM4’), an aerobic methylotrophic alpha-proteobacterium (12 cofactor on CmuA and then to tetrahydrofolate (H4F), with concomitant loss of HCl  -dependent pathway . Three additional enzymes not found in other strains of M. extorquens, MetF2, FolD, and PurU, are thought to convert methyl-H4F into formate  . In orde pathway . The formilation . Since C formate . Howevererminate .Hyphomicrobium sp. MC1 (hereafter ‘MC1’), have been sequenced (cmu pathway) are distributed around a large plasmid that also contains genes for cobalamin and folate metabolism. In MC1, the cmu genes form a putative operon. The other CM-utilizing strains contain cmu pathways with highly homologous enzymes, and their distribution and genetic organization strongly suggest that the pathway has been transferred by horizontal gene transfer . Catabolism of DCM requires a dedicated cytoplasmic dehalogenase (DcmA) that directly dechlorinates DCM to formaldehyde . In the case of DCM, expressing DcmA in a variety of other Methylobacterium strains initially led to little or no growth , an industrial solvent that differs from CM by only a single chlorine . It is ualdehyde . As witho growth . Effectio growth . Given tMethylobacterium strains, generating new CM-utilizing microbes. We demonstrate that these strains grow poorly on CM, indicating the need for post-transfer refinement. We find no correlation between a strain’s ability to grow with CM and DCM when provided with the corresponding heterologous catabolic pathway. Our heterologous expression system allows facile manipulation, allowing us to measure the fitness effect of accessory genes such as purU and folD. Finally, we show that growth on CM is not limited by chloride export, in contrast to growth on DCM.In this work, we have deliberately transferred the CM catabolic pathway into naïve Figure 1B). The cmu cluster from Hyphomicrobium sp. MC1 .To reproduce the process of HGT, we cloned the gene clusters implicated in CM catabolism into conjugative plasmids and transferred them into naïve recipient strains , while none of the pJM50 transconjugants reached a comparable optical density. Under these conditions, strain CM4 typically reaches an optical density of ~0.1. Control flasks, containing cells but no CM, did not exceed an OD600 of 0.01.Each of these plasmids was separately introduced into six different recipient strains unable to grow on CM: ains AM1 , PA1 . As expected, the fitness of the native CM-consuming strain CM4 was significantly higher than any of the transconjugants . However, each of the transconjugants had non-zero fitness, indicating that they grew with CM as the sole source of carbon and energy. For comparison, we also competed AM1, PA1, and DM4 against CM4 directly. These competitions confirmed that the transconjugant strains grow with CM, but at 23-44% of the fitness of CM4 .We previously measured the fitness of this same set of recipient strains during growth on DCM after introduction of a heterologous DCM catabolic pathway . Comparicmu gene cassette, metF2, purU, folD, paaE, hutI, and fmdB was individually deleted from pJM105, and the modified plasmids were introduced into AM1. We determined the fitness effect of each single-gene deletion by competing strains containing the modified plasmids against a strain containing the original plasmid during growth with CM . Only one gene, metF2 encoding a methylene H4F reductase , was essential for growth on CM, while the other deletions imposed fitness costs of 18–47%.Each of the six accessory genes in the Methylobacterium strains with DCM was limited by the need to export the chloride ions produced as a byproduct of dechlorination . Accordingly, we tested whether ClcA overexpression would increase the fitness of a Methylobacterium strain during growth with CM. We introduced the pJM105 plasmid into mutant strains of AM1 and PA1 that each overexpress ClcA. In both cases, the fitness of the ClcA overexpression strain was indistinguishable from an otherwise isogenic control .We previously showed that growth of transconjugant rination . MutatioFigure 2A). Despite these general similarities, the lack of correlation between fitness on CM and DCM suggests that the fitness-limiting factors are different for the catabolic pathways of these two chlorinated methanes. We assume in our interpretation of the competition experiments that the strains compete solely through consumption of the carbon source. Any other competitive interactions would likely have similar effects during growth both with CM and with DCM.As with growth on DCM, the ability to exploit this horizontally transferred pathway is common and all of the recipients were able to grow on CM. Consistent with our previous results, the phylogenetic relationships between recipients was not predictive of their fitness, though we might expect less of a phylogenetic effect since the heterologous pathway was transferred from outside the genus. Additionally, transfer of the pathway allowed only limited growth, ranging from 14 to 46% of the fitness of a natural isolate  and a plasmid-borne metF2 that is part of the cmu cluster and shares only 26% amino acid identity with the chromosomal homolog. Hyphomicrobium sp. MC1 also contains two metF homologs, metF with 65.2% amino acid identity to the CM4 metF and metF2 with 28.4% amino acid identity to the CM4 metF2. Since the native AM1 metF is unable to sustain growth with CM in the absence of metF2 from Hyphomicrobium sp. MC1, we conclude that the chromosomal metF is either misregulated during growth on CM or is incapable of catalyzing the oxidative reaction with the necessary flux.The MetF2 enzyme was essential for growth of AM1 with CM, as had previously been shown for CM4 . CM4 haspurU cmu gene cassette contrasts with previous findings showing that this gene was essential for growth of CM4 with CM , albeit at levels insufficient for the ΔfolD strain to match even the limited growth of AM1 with the intact cmu gene cluster. Indeed, previous work replacing MtdA and Fch with FolD in AM1 has shown that FolD is sufficient for growth on succinate, which requires relatively little flux through the H4F pathway, but is insufficient for growth on methanol, which requires a much higher flux : gene cmuC2 is only found in the CM4 cluster, while genes fmdB, paaE, and hutI were not included in the CM4 cluster that we cloned. Two of these genes, fmdB and hutI are immediately adjacent to cmuA in M. extorquens CM4 and the third gene, paaE, occurs on the native CM4 plasmid pCMU01 roughly equidistant between the two segments that we amplified . A simple multiplicative model based on our deletion studies, which assumes each mutation has a constant proportional effect on fitness regardless of the genetic background, would predict a fitness of ~20% for the triple deletion ΔfmdBΔhutIΔpaaE relative to the full pJM105 plasmid. If introduction of plasmid pJM50, which lacks fmdB, hutI, and paaE, allowed strains to grow with CM, but only 20% as effectively as with pJM105, we would not have been able to detect the growth of pJM50 transconjugants. Additionally, the cmu cluster that we cloned from Hyphomicrobium sp. MC1 has the native gene order and spacing, unlike the CM4 cluster, and this may favor heterologous cmu gene expression. In our single-gene deletion experiments, the start and stop codons of the deleted gene were preserved to minimize polar effects on the remainder of the gene cluster. However, even such conservative deletions may affect transcript stability, and we cannot rule out fitness effects due to disruption of undetected genes in the gene cluster or truncated polypeptides resulting from the deletions.These deletion experiments may also explain our inability to productively transfer the Methylobacterium strains . Therefore, the recipients may be misregulating their metabolic networks for the new demands placed upon them during growth with CM. Third, the transfer of a nine-gene pathway between microbial families may affect protein expression levels in the new genetic environment, with significant costs to pathway flux and organismal fitness  unless otherwise noted. s needed . For CM Hyphomicrobium sp. MC1 and M. extorquens CM4 were prepared using the Wizard Genomic DNA Purification Kit . To construct plasmid pJM50, two genomic regions were amplified from M. extorquens CM4 gDNA using the Q5 polymerase with the high GC enhancer   and transformed into chemically competent 10β E. coli (NEB). To construct plasmid pJM105, a single genomic region was amplified from Hyphomicrobium sp. MC1 gDNA as three overlapping amplicons of 2.6, 4.2, and 3.6 kb and assembled as for pJM50. Plasmids were mated from the 10β cloning E. coli strain into recipient Methylobacterium strains by tri-parental matings as described previously (Genomic DNA from eviously .E. coli, and mated into M. extorquens AM1 as described above.To construct the deletion plasmids, pJM107-112, plasmid pJM105 was amplified by inverse PCR with primers designed to overlap and create a clean deletion. The desired open reading frame was truncated, deleting the majority of the gene while leaving the start and stop codons intact and in-frame to minimize polar effects. The resulting amplicons were circularized using Gibson assembly, transformed into chemically competent Fitness assays were performed largely as described previously . In brieM. extorquens DM4 ΔdcmA Venus containing the appropriate plasmid (M. extorquens CM4 containing an empty tetracycline plasmid, pCM62 (M. extorquens AM1 Δcel Venus and competed against M. extorquens AM1 Δcel Cherry + pJM105. To measure the fitness of the chloride transport mutants, pJM105 was conjugated into M. extorquens PA1 Δcel mCherry clcAE1 and M. extorquens AM1 Δcel mCherry clcAE1 and competed against M. extorquens DM4 ΔdcmA Venus + pJM105 (To measure the fitness of the pJM50 and pJM105 transconjugants, strains were competed against  plasmid  or againd, pCM62 . To meas+ pJM105 .All authors contributed to the design of experiments, the interpretation of data, and drafting of the manuscript. Experiments were performed by JM.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


An outbreak of acute flaccid paralysis among children in the United States during summer 2014 was tentatively associated with enterovirus D68 infection. This syndrome in a child in fall 2014 was associated with enterovirus C105 infection. The presence of this virus strain in North America may pose a diagnostic challenge. During the summer of 2014, the Centers for Disease Control and Prevention (CDC) reported an unusual increase in the frequency of acute flaccid myelitis among children in the United States , frontal headache, fatigue, and intermittent pain in the right ear and right axilla. The fever lasted only 1 day; the cough, fatigue, and headache improved over the next 2 days, but the patient continued to report right arm pain. On the day before seeking care, her parents observed that she had a right shoulder droop and difficulty using her right hand. No associated visual or mental status changes; difficulty with speech, swallowing, or respiration; or bowel/bladder disturbance were noted. Physical examination detected right upper extremity weakness; absent right biceps, triceps, and brachioradialis deep tendon reflexes; and a diminished right patellar reflex. Muscle strength was more severely affected in the proximal than in the distal right upper extremity. Sensation was intact. A diffuse papular rash was noted on the patient’s back.6 leukocytes/L (82% lymphocytes), 0.9 × 106 erythrocytes/L, 3 mmol glucose/L, and 280 mg protein/L. The IgG index R-value  was mildly elevated at 0.73, but oligoclonal bands were absent. Testing of a nasopharyngeal swab specimen with the xTAG Respiratory Viral Panel  produced positive results for picornavirus. PCR for enterovirus in CSF, performed by using the XpertEV test  was negative. Subsequent confirmatory testing at the Division of Consolidated Laboratories for the Commonwealth of Virginia also produced negative results. Subsequent testing of a nasopharyngeal swab specimen by sequence analysis of the viral protein 1 capsid gene revealed the presence of enterovirus C105  images of the spine revealed longitudinally extensive gray matter hyperintensity within the central cord at C3–7 and T11–12/L1 with associated edema . An MR iThe patient received intravenous immunoglobulin, 2 g/kg divided daily over 5 days, but did not respond. She continued to report intermittent pain in her right arm, low-back pain, and bilateral thigh pain when walking. During her hospital stay, the patient’s weakness remained stable . After discharge, her pain spontaneously resolved, and 8 months after illness onset (most recent follow-up visit), her proximal right arm weakness improved and the strength in her distal right arm had almost resolved.Enterovirus C105 was first detected in 2010 in patients from Peru and the Republic of Congo; subsequent reports suggest that the virus is circulating worldwide . The case definition for the outbreak is acute onset of focal limb weakness associated with a spinal cord lesion restricted to the gray matter (according to MR images) in a child <21 years of age. The patient reported here met this case definition. A detailed report of 88 of these children noted that most (81%) had experienced a preceding respiratory illness, similar to that described by the patient reported here (In the United States, the outbreak of acute flaccid myelitis that began in the summer of 2014 affected 118 children (EV-D68 is unusual in that, although it is a member of the enterovirus D species, this virus has phenotypic characteristics that are more consistent with rhinoviruses. The virus is acid labile and preferentially grows at 33°C, characteristics that have been used to classify picornaviruses as rhinoviruses (


Ictalurus punctatus). A total of 8.4 million candidate SNPs were identified by using next generation sequencing. On average, the channel catfish genome harbors one SNP per 116 bp. Approximately 6.6 million, 5.3 million, 4.9 million, 7.1 million and 6.7 million SNPs were detected in the Marion, Thompson, USDA103, Hatchery strain, and wild population, respectively. The allele frequencies of 407,861 SNPs differed significantly between the domestic and wild populations. With these SNPs, 23 genomic regions with putative selective sweeps were identified that included 11 genes. Although the function for the majority of the genes remain unknown in catfish, several genes with known function related to aquaculture performance traits were included in the regions with selective sweeps. These included hypoxia-inducible factor 1β· HIFιβ ¨ and the transporter gene ATP-binding cassette sub-family B member 5 (ABCB5). HIF1β· is important for response to hypoxia and tolerance to low oxygen levels is a critical aquaculture trait. The large numbers of SNPs identified from this study are valuable for the development of high-density SNP arrays for genetic and genomic studies of performance traits in catfish.Domestication and selection for important performance traits can impact the genome, which is most often reflected by reduced heterozygosity in and surrounding genes related to traits affected by selection. In this study, analysis of the genomic impact caused by domestication and artificial selection was conducted by investigating the signatures of selection using single nucleotide polymorphisms (SNPs) in channel catfish ( Strong selection can lead to significant allele frequency shifts with alleles directly affecting a trait reaching high frequencies. Due to genetic linkage, alleles nearby on the chromosome also change in frequency, leaving signatures of a selective sweep Gasterosteus aculeatus) Salmo salar) In contrast, hard selective sweeps can be readily detected as they result in an increase of a specific allele, and hence, are more useful for the detection of genes underlining the performance traits. Studies of selective sweeps due to domestication in teleost fish are limited because most selective breeding of fish has been in the last 50 years, and there are few active selection programs for fish. Selective sweeps have been identified from fish species such as three-spined stickleback (Oncorhynchus mykiss) Danio rerio) Lepomis gibbosus) Salmo trutta) et al.Ictalurus punctatus).Domestication is one kind of selection involving the removal of some selection pressure typical of natural environments but intensification of others relevant to farming conditions Genomic impact of domestication has not been well studied in fish species. Previous studies have shown morphological, behavioral and growth changes in channel catfish during domestication Molecular markers have been developed from catfish All procedures involving the handling and treatment of used fish during this study were approved by the Auburn University Institutional Animal Care and Use Committee (AU-IACUC) prior to initiation of the project. A total of 150 channel catfish, with 30 individuals from each of Marion, Thompson, USDA103, one outbred commercial strain (hereafter referred to as Hatchery), and one wild population were used for this study. The four aquaculture strains were from different geographic locations within the United States, which possess different production traits such as growth rate, disease resistance and feed conversion efficiency The fish were euthanized with tricaine methanesulfonate (MS 222) at 300 mg/l before blood collection. For each individual, 500 µl blood was collected for DNA isolation, placed into 5 ml lysis buffer immediately, and then into a water bath at 55°C for 12 h. Total DNA was isolated using the DNeasy Blood & Tissue Kit  following the manufacturer's protocol. Equal amounts of DNA (100 µg) from each individual were pooled for sequencing, one pool for each strain.http://www.ncbi.nlm.nih.gov/sra).Sequencing was conducted commercially at HudsonAlpha Genomic Services Lab . Genomic libraries were prepared with the Paired-end Sequencing Sample Preparation Kit  with 5 µg of genomic DNA for all strains, according to the manufacturer’s instructions. For each strain, the prepared DNA library was sequenced on one lane of the Illumina HiSeq 2000 platform for 100-bp paired-end reads. The short reads were deposited in the NCBI Sequence Read Archive (SRA) under Accession number SRA075234 (N = P(A)-P(Q), where A is the criterion of the minimal quality score. In this project, A = 20 (Phred score); Q is the Phred quality score of each base. This value would be negative for bases with quality scores below 20. For every base, the software calculated the running sum of this value. The part of the sequence not trimmed will be the region between the first positive value of the running sum and the highest value of the running sum. Everything before and after this region was trimmed.Sequence mapping was performed using CLC Genomics Workbench . Before mapping, raw sequence reads were trimmed to remove adaptor sequences, ambiguous nucleotides (N’s), extreme short reads (<30 bp) and low quality sequences  using CLC Genomics Workbench. The quality of each sequence was assessed as follows: First, convert Q  was converted to an error probability (P):The clean reads from each strain were then aligned with the preliminary catfish genome assembly (unpublished data). The mapping parameters were set as: mismatch cost of 2, deletion cost of 3 and insertion cost of 3. The highest scoring matches that shared ≥95% similarity with the reference sequence across ≥90% of their length were included in the alignment. The mapping output was converted into BAM format www.animalgenome.org/repository/pub/auburn2014.0530/).SNPs were identified from the pooled data from all the strains using the SAMtools (version 0.1.18) SNPs with significantly different allele frequency ratios were identified between domestic catfish strains and the wild population (hereafter referred to as significant SNPs). Two-tailed Fisher’s exact test was performed with the statistical significance level of false discovery rate corrected P value ≤0.01. Significant SNPs were categorized into three groups based on their location: 1) in the coding regions, 2) near the coding regions and 3) on non-coding regions. Near the coding regions means the SNP is located on non-coding regions but within 100 bp from the coding regions.Hp) score was calculated using the formula Hp = 2ΣnMAJΣnMIN/(ΣnMAJ + ΣnMIN)2nMAJ was the sum of the major allele reads, and ΣnMIN was the sum of the minor allele reads for all significant SNPs in one window. The Hp score was calculated based on 20 kb sliding window across the genome. Windows with less than five significant SNPs were not used for calculation. Putative selective sweeps were identified from windows with −log2(Hp) score ≥4.With the availability of significant SNPs, genomic regions with selective sweeps were identified from the four domestic strains by detecting the genome regions with extremely low heterozygosity. The pooled heterozygosity  not only affects the SNP applicability for future genetic studies because it directly determines the polymorphism information content of the SNP markers, but MAF also has an impact on the identification of quality SNPs. In general, the relationship curve can be arbitrarily divided into two phases: In the first phase, when minor allele counts were set as 2–4, the total number of SNPs was reduced sharply, while in the second phase, when minor allele reads were set as greater than 4, the total number of SNPs was also reduced, but at a much reduced rate, suggesting that minor allele reads of 4–6 may be appropriate for data in the present work . Thus, tIn addition to the initial assessment of these factors, the percentage of sequences that were included for SNP identification were examined. As shown in A total of more than 13 million potential single nucleotide variations were observed at the most relaxed set of criteria, i.e., minimum read depth of 20, maximum read depth is set as excluding the top 2% of all reads, and minor allele read counts of 2. At our selected set of criteria, a total of 8,395,720 (∼8.4 million) putative SNPs (hereafter referred to as SNPs) were identified .These 8.4 million SNPs were subsequently used for the assessment of the distribution of minor allele frequencies. The MAF of each identified SNP was estimated based on the reference number and variant allele reads observed in the reference mapping. Approximately 4 million SNPs have an estimated MAF ≤10% . Over 4.Putative SNPs identified from each of the five strains were shown in SNPs that were observed from only one strain were considered as putative strain-specific SNPs. SNPs that were polymorphic in all strains were considered as common SNPs. Approximately, 2.7 million common SNPs were identified. The number of strain-specific SNPs identified from each of the five strains varied from 66,487 to 143,126, accounting for 0.9%, 2.9%, 2.2%, 1.3%, and 1.7% of SNPs that were identified from that strain, respectively .As shown in A total of 237,655 (58.3%) significant SNPs were assigned to 29 tentative chromosomes based on the catfish linkage map pH) was calculated in 20-kb windows based on the major and minor alleles of significant SNPs, and were then log transformed. Most of the windows (73.5%) had the log-transformed pH scores between 1 and 1.5, indicating high levels of heterozygosity  had the most significant pH score, which was followed by ATP-binding cassette sub-family B member 5 (ABCB5).A total of 11 genes were found from these genomic regions with selective sweeps . These gIn this study, next generation sequencing was conducted for multiple individuals from four aquaculture strains and one wild population to identify SNPs for determination of genomic impact of domestication. The large numbers of SNPs identified from this study will be useful for the development of high density SNP arrays for genetic and genomic analysis in catfish Pooled sequencing has been utilized as an efficient and reliable approach for detecting and genotyping SNPs from populations In that regards, reasonable criteria for SNP identification were set at a minimum read depth of 30, maximum read depth of 300, and minor allele count of 5, and 8.4 million putative SNPs were identified from five different catfish strains. On average, there was one SNP every 116 bp in channel catfish genome. Approximately, 66,000–143,000 SNPs were identified as strain-specific for each strain , which iet al.Liu SNPs with significant differences in allele frequency between domestic and wild catfish populations were identified to provide insight into genomic impact of domestication and selection. Compared with all the SNPs identified from channel catfish, significant SNPs were approximately 5% of the total SNPs, indicating that the vast majority of genomic regions have not been affected by domestication or selection. Additional analysis was conducted to determine the position and genes associated with significant SNPs. The vast majority of significant SNPs (87.2%) were located in the non-coding DNA sequences, while 12.8% of the significant SNPs were found in coding regions of catfish genes. This proportion of SNPs associated with genes is greater than the proportion of gene sequences from the whole genome sequences, suggesting that domestication and selection may have had a greater impact on genes than on intergenic regions.The significant SNPs were distributed on each of the catfish chromosomes . ChromospH scores) for the domestic populations using significant SNPs with the assumption that artificial selection by domestication tends to create runs of homozygosity A concern regarding the analysis of channel catfish was sampling since this species occupies a large geographical range, populations can be large and numerous domestic and wild populations exist. Assuming that all domesticated populations and a broad representation of wild populations can be achieved, significant SNPs between the domestic and wild populations could be used to reveal solid selective sweeps caused by domestication and selection. However, based on the nature of catfish industry, it is difficult to sequence large enough samples that can represent all genetic variations that exist in all domestic and wild strains. Therefore, we fully acknowledge the difficulties involved in the sampling of the domestic and wild populations for an aquatic species, however, analysis of putative selective sweeps should still provide insights into the potential impact of domestication on genome evolution. To identify hard type selective sweeps in domestic catfish caused by selective breeding, we analyzed the pooled heterozygosity  and ATP-Binding Cassette, Sub-Family B, Member 5 (ABCB5), were located in the first two strongest hard sweeps , mediates aryl hydrocarbon signaling and facilitates gene activation by dimerization with aryl hydrocarbon receptor (AHR) In the present study, we identified 11 genes from 9 of the 23 genomic regions with selective sweeps . Two gend sweeps . HIF-1β ABCB5 is a member of ATP-Binding Cassette transporter gene family and only exists in vertebrates ABCB5 was also significantly associated with tumor progression and recurrence, acting as an energy-dependent drug efflux transporter and function during the multidrug resistance process ABCB5 gene that was exclusively associated with childhood obesity ABCB5 was located in the second strongest selective sweep region Click here for additional data file.


Objective. To investigate oxidative stress (OS) and histological changes that occur in the periodontium of subjects with type 2 diabetes mellitus without signs of periodontal disease and to establish if oxidative stress is a possible link between diabetes mellitus and periodontal changes. Materials and Methods. Tissue samples from ten adult patients with type 2 diabetes mellitus (T2D) and eight healthy adults were harvested. The specimens were examined by microscope using standard hematoxylin-eosin stain, at various magnifications, and investigated for tissue levels of malondialdehyde (MDA) and glutathione (GSH). Results. Our results showed that periodontal tissues in patients with T2D present significant inflammation, affecting both epithelial and connective tissues. Mean MDA tissue levels were 3.578 ± 0.60 SD in diabetics versus 0.406 ± 0.27 SD in controls (P < 0.0001), while mean GSH tissue levels were 2.48 ± 1.02 SD in diabetics versus 9.7875 ± 2.42 SD in controls (P < 0.0001). Conclusion. Diabetic subjects had higher MDA levels in their periodontal tissues, suggesting an increased lipid peroxidation in T2D, and decreased GSH tissue levels, suggesting an alteration of the local antioxidant defense mechanism. These results are in concordance with the histological changes that we found in periodontal tissues of diabetic subjects, confirming the hypothesis of OS implication, as a correlation between periodontal disease incidence and T2D. In the recent years, an increasing number of scientists studied oxidative stress (OS) as a possible link between oral and systemic diseases. Many of these studies are related to periodontal disease, by far the most common link being oral infection .Oxidative stress plays a major role in the pathogenesis of many systemic and oral diseases . There iThe aim of this study was to investigate oxidative stress that occurs in the periodontium of subjects with type 2 diabetes mellitus without signs of periodontal disease and to establish a possible link between this systemic condition and the morphologic changes in periodontal structures.The present study was conducted on two groups of patients; one consisted of ten diabetic patients without signs of periodontal disease and the other one consisted of eight systemically and periodontally healthy subjects as controls. Diabetic subjects were recruited from the patients that addressed to the Department of Odontology and Periodontology, Faculty of Dental Medicine, UMF Tg-Mures, for different dental problems, and controls were selected from the patients that addressed to the Emergency Department, Faculty of Dental Medicine, UMF Tg-Mures, for the same reasons. All diabetic subjects included in the present study had records of at least 4 to 5 years of diagnosed T2D, were on medication with oral antidiabetic drugs, and all had well-controlled T2D; none was insulin treated and none used any antioxidant agent. Subjects aged 30 to 58, nonsmokers, without any inflammatory disease or use of anti-inflammatory drugs in the last three months prior to the study.This study was approved by the Ethical Committee of UMF Tg-Mures, and all the patients included in the study signed for informed consent.From each subject biopsy specimens were obtained during the extraction of irrecoverable teeth. Biopsy specimens were harvested from a dental-periodontal unit in the posterior region of dental arches. From the biopsy specimens harvested from all subjects, one fragment was sent for histopathology study, while the other was stored at −80°C until OS evaluation.Histopathological examination was performed using formalin-fixed, paraffin-embedded tissue fragments following standard protocols. The 4-5 micron thick tissue sections were stained with hematoxylin-eosin stain and also digitally archived using Zeiss MiraxScan system.From conserved tissue biopsies we determined the levels of malondialdehyde (MDA) as a marker for OS and glutathione (GSH) as a marker of defense antioxidant mechanism, using the fluorometric methods according to Conti et al.  and EllmP < 0.05 were considered statistically significant.Statistical analysis was performed using Microsoft Excel, Graph-Pad InStat, and NCSS 2007 software, and values of P = 0.8696).Mean age of patients in study group was 44.7 ± 7.66 and 44.125 ± 6.728 in controls, with a nonsignificant difference between groups , significantly higher than in controls, where the mean MDA value was 0.406 (0.21–0.9). Statistical comparison between the two groups yielded a < 0.0001 .P < 0.0001 , significantly lower than 9.788 (6.8–13.2) in controls, with < 0.0001 .Histological alterations in tissue sections obtained from diabetic patients were present in both the epithelium and the lamina propria of the gingival mucosa. The epithelium displayed variable amounts of acanthosis and parakeratosis, with reduced quantities of acute inflammatory infiltrate composed mostly of polymorphonuclear leucocytes (segmented granulocytes) throughout its thickness and in superficially located microabscesses. A diffuse polymorphous inflammatory infiltrate consisting of lymphocytes, plasma cells, and, to a lesser extent, granulocytes was present in the mildly fibrotic lamina propria, displacing collagen fibers and surrounding ectatic blood vessels and exteriorized erythrocytes Figures  and 4.The hypothesis of the study was that diabetes mellitus can increase OS at periodontal tissues level, contributing to periodontal disease development. Our results showed that tissue MDA and GSH levels differ significantly in diabetics without signs of periodontal disease compared to controls. In our study, tissue levels of MDA were higher in diabetics than in controls. As MDA  is considered a biomarker of OS , elevateThe microscopic changes are in concordance with previously published data  and with−), nitric oxide (NO), superoxide (O2−), hydrogen peroxide (H2O2), and peroxynitrite (ONOO−). ROS can react with different amino acids, generating a large range of products, from modified and less reactive enzymes to denatured, nonfunctional proteins. An important structural modification of protein molecules is nitrosylation, with peroxynitrite being responsible for this change. Tyrosine is an important amino acid involved in phosphorylation reactions and signal transduction pathways. Tyrosine nitration not only compromises protein function but may also have serious consequences in cell regulation. Enzymes such as superoxide dismutase (SOD) were identified as specific targets for nitrosylation.Oxidative stress (OS) can be defined as an imbalance between the production of some highly reactive molecule species and the antioxidant defense mechanism . ReactivSeveral studies have shown a decreased superoxide dismutase activity in the gingival tissue of parodontopathic patients compared to parodontopathic diabetic patients. This can be a compensatory mechanism derived from hyperglycemia. The diabetic with periodontal disease has a lower activity of the prooxidative enzyme myeloperoxidase in the gingival crevicular fluid, compared to nondiabetics .Reaction between mononuclear phagocytes and AGE (advanced glycation end products) induce the regulation of cytokine expression and OS . These pThe clinical relevance of our study is that MDA and GSH levels can predict the evolution of periodontal disease in subjects with type 2 diabetes mellitus.Limitations of the study are that we included only nonsmokers to minimize confounding variables. Further research should include smokers with T2D and periodontal disease, as smoking is a common habit in Romanian population and its effect on oral mucosa needs to be taken into consideration.One topic that remains to be explored is how antioxidant periodontal therapy can influence periodontal alterations in diabetic patients.Diabetic subjects have higher MDA levels in their periodontal tissues, suggesting an increased lipid peroxidation in case of diabetes mellitus. In periodontal tissues from diabetics, decreased levels of glutathione suggest an alteration of local antioxidant defense mechanism. These results are in concordance with the histological changes that we found in periodontal tissues of diabetic subjects. The results of our study confirm the hypothesis of OS implication, as a correlation between periodontal disease incidence and diabetes mellitus. These results can be a starting point for further research on the efficiency of different antioxidant agents for prevention and treatment of periodontal disease in diabetic patient.


Pain is a prominent feature of acute dengue as well as a clinical criterion in World Health Organization guidelines in diagnosing dengue. We conducted a prospective cohort study to compare levels of pain during acute dengue between different ethnicities and dengue severity.Demographic, clinical and laboratory data were collected. Data on self-reported pain was collected using the 11-point Numerical Rating Scale. Generalized structural equation models were built to predict progression to severe disease.A total of 499 laboratory confirmed dengue patients were recruited in the Prospective Adult Dengue Study at Tan Tock Seng Hospital, Singapore. We found no statistically significant differences between pain score with age, gender, ethnicity or the presence of co-morbidity. Pain score was not predictive of dengue severity but highly correlated to patients’ day of illness. Prevalence of abdominal pain in our cohort was 19%. There was no difference in abdominal pain score between grades of dengue severity.Dengue is a painful disease. Patients suffer more pain at the earlier phase of illness. However, pain score cannot be used to predict a patient’s progression to severe disease. The World Health Organization (WHO) reported that over 40% of the global population is at risk of dengue and an estimated 390 million dengue infections happen worldwide every year.Dengue incidence has increased and its geographic range expanded.Pain is an important component of dengue diagnostic criteria. In WHO 1997 guideline, probable dengue requires the presence of fever with any two of headache, retro-orbital pain, mylagia, arthralgia, rash, positive tourniquet test or or hemarroghic manifestation. Higher prevalence of abdominal pain, backache, headache and myalgia was noted in adult dengue patients.Ethical approval was provided by the Domain Specific Review Board of the National Healthcare Group, Singapore (DSRB/E/2009/432). Written informed consent was obtained from all subjects.Acutely febrile patients above the age of 18 were recruited prospectively from January 2010 to September 2012 at the Communicable Disease Center, Tan Tock Seng Hospital, Singapore, a tertiary referral infectious disease centre. All dengue diagnostic testing was performed by the Enviromental Health Institute, a WHO Collaborating Center for Reference and Research on Arbovirus and its Associated Vectors in Singapore. Patients were classified as having laboratory-confirmed dengue according to WHO standards if they were RT-PCR or NS1 positive using the dengue NS1 antigen (Ag) strip .The Numeric Rating Scale (NRS) is a widely used tool to assess patients’ pain level in chronic and acute illnesses with good responsiveness and sensitivity.The primary outcome was the severity of dengue: 1: Dengue fever, 2: Dengue hemorrhagic fever  Ordinal logistic regression was used to determine how pain score was associated with age, gender, co-morbidity and ethnicity . Generalized structural equation model (GSEM) was applied as a more confirmatory analysis to analyze the data accounting for temporal data structure and the complex dependence among pain score, fever and the outcome.Two sets of analyses were carried out, with the outcome ascertained on day 2 and day 3 of enrolment. In both analyses, the number of fever days reported by patients preceded fever at presentation which in turn preceded days 2 and 3 of enrolment see  and 2.The first GSEM model to predict dengue severity on the second day of enrolment (day 2) involved baseline covariates such as age, gender, ethnicity, Charlson’s co-morbidity scoreEstimated with maximum likelihood, the models were analysed with the multinomial distribution and logit link. Analyzed with Stata MP 13 , all statistical tests were conducted with 95% confidence intervals.Of the 850 patients recruited, 499 tested positive for RT-PCR or NS1 were included in the analysis. There were 111 DHF I–II and 13 DHF III–IV(DSS) cases according to WHO 1997 classification. Twenty were severe dengue cases in accordance with the WHO 2009 classification. The median duration of fever was 6 (3–9) days. One patient died owing to myocarditis. One patient was diagnosed with both DSS and SD. In total, 32 patients fulfilled the severe disease classification.th–95th percentile, 21–51 years). Male patients comprised 79% and 77.6% of the cohort were Chinese. Medical co-morbidity, mainly myocardial infarction, diabetes and cerebrovascular dieseases was present in 1.8%. The majority (76.3%) of patients had fever on the first visit. The median pain score was similar in DF, DHF I–II and severe disease patients . (The median age of the cohort was 34 years (5 = 0.7).  There weFor detailed sample sizes for each day, refer to Pain score on study days 1 and 2, number of fever days, ethnicity and co-morbidity were not significantly associated with dengue severity . The GSESimilarly, pain score on study day 2 was not predictive but age and fever on day 3 of study were predictive of dengue severity. The risk of age on severe disease largely remained the same as the day before. In addition, fever on day 3 increased a patient’s risk of severe disease. Notably, patients who developed severe disease on day 3 were more likely to report higher pain scores .th–95th percentile, 2–9) and 4  for non-severe disease (p = 0.38).Ninety five patients (19%) experienced abdominal pain. Four of them had SD while the other 3 had DSS. Of these 7 cases, 3 of them experienced the worst abdominal pain on the same day as they were diagnosed with severe disease or after progressing on to severe disease. The median abdominal pain score for patients with severe disease was 5 .(DOC)Click here for additional data file.


Arabidopsis arose from gene duplication events during the evolution of the Brassicales. Analyses of 46 gene pairs duplicated by an ancient whole-genome duplication  indicated that many imprinted genes show an accelerated rate of amino acid changes compared with their paralogs. Analyses of microarray expression data from 63 organ types and developmental stages indicated that many imprinted genes have expression patterns restricted to flowers and/or seeds in contrast to their broadly expressed paralogs. Assays of expression in orthologs from outgroup species revealed that some imprinted genes have acquired an organ-specific expression pattern restricted to flowers and/or seeds. The changes in expression pattern and the accelerated sequence evolution in the imprinted genes suggest that some of them may have undergone neofunctionalization. The imprinted genes MPC, HOMEODOMAIN GLABROUS6 (HDG6), and HDG3 are particularly interesting cases that have different functions from their paralogs. This study indicates that a large number of imprinted genes in Arabidopsis are evolutionarily recent duplicates and that many of them show changes in expression profiles and accelerated sequence evolution. Acquisition of imprinting is a mode of duplicate gene divergence in plants that is more common than previously thought.Eukaryotic genomes have large numbers of duplicated genes that can evolve new functions or expression patterns by changes in coding and regulatory sequences, referred to as neofunctionalization. In flowering plants, some duplicated genes are imprinted in the endosperm, where only one allele is expressed depending on its parental origin. We found that 125 imprinted genes in  Arabidopsis lineage has experienced two WGD events after the divergence from a common ancestor with Carica (papaya) in the order Brassicales , which doubles the entire genome. WGDs at various evolutionary time scales have been shown across eukaryotes including yeasts, animals, and plants . Orthologs of imprinted genes and their paralogs were identified according to the gene-tree topology and used for sequence rate analyses and expression assays. Imprinted genes that arose from the alpha WGD were identified with their paralogs according to Imprinted genes were selected for subsequent analyses if they had recent paralogs that are specific to the Brassicales after the Brassicaceae family diverged from the Caricaceae family , where X indicates twice likelihood ratio, ln 1 indicates the likelihood estimate from Model I, and ln 2 indicates the likelihood estimate from Model II was compared against a χ2 distribution with one degree of freedom (df). A significant difference (P < 0.05) indicates that the duplicated pair has asymmetric sequence rate evolution. In a gene pair showing asymmetric evolution, whether the imprinted gene evolves faster or slower than its paralog was determined by comparison of the branch lengths estimated from Model I. For each gene pair, three separate tests were applied using different outgroup sequences as references, Carica, Vitis, and Ricinus, respectively. Thus, asymmetric evolution was determined according to the majority outcome (two or three out of three) from the three tests.To detect whether there has been an asymmetric sequence evolution in each duplicated pair, we followed the method described in Arabidopsis in general, when comparing sequence evolution rates between A. thaliana and A. lyrata . Data from 63 different organ types and developmental stages were included  is the highest log2 transformed expression value for gene i across the n organ types. A gene with expression limited to one or a few organ types or developmental stages would demonstrate a high organ specificity, whereas broadly expressed genes with similar expression levels in most organ types and developmental stages would have a low organ specificity value.included  and wereincluded . A gene MPC/PAB8, FWA (HOMEODOMAIN GLABROUS6 [HDG6])/HDG7, HDG3/HDG2, HDG9/HDG10, and SUVH7/SUVH8, additional selection analyses were performed. For each gene pair, sequences from A. thaliana, C. papaya, V. vinifera, P. trichocarpa, R. communis, and M. esculenta were aligned using MUSCLE with the default settings  described in A. thaliana (ecotype Col-0), C. papaya (cultivar Sun-Up), and V. vinifera (cultivar Pinot Noir), which were grown together in a greenhouse. For each species, five organ types were used for the RNA extraction: Roots, stems, leaves (rosette leaves in Arabidopsis), flowers, and seeds (whole siliques in Arabidopsis). Fresh plant materials were collected and frozen in liquid nitrogen. Total RNA of each sample was extracted as in supplementary table S1, Supplementary Material online). Primers were checked against the genome of each species, using BLASTn, to check for specificity to the target gene. For PCR analyses, the cycling programs were 94 °C for 3 min; 30–35 cycles of 94°C for 30 s, 55–58 °C for 30 s, 72 °C for 30 s, and 72 °C for 7 min. PCR products were checked on 1.2% agarose gels.Total RNA was extracted from Arabidopsis, we analyzed imprinted genes identified by two studies that used Illumina transcriptome sequencing in polymorphic F1 seeds of A. thaliana . We found that 125 out of the 183 imprinted genes (68%) originated from gene duplication events in the Brassicaceae lineage after it diverged from the Caricaceae lineage within the Brassicales . Among the 125 imprinted genes, 54 genes were derived from the alpha WGD , which are specific to the Brassicaceae family, and 44 genes by tandem duplication. Other kinds of gene duplication events also have contributed to the formation of the imprinted genes. Most of the paralogs of those imprinted genes are not reported as imprinted . In contrast, of the imprinted genes from supplementary table S4, Supplementary Material online). Thus, the imprinted genes often showed a faster rate of sequence evolution than their duplicated partners, especially among the genes identified in JMJ15 . Thus, imprinted genes and their paralogs have a higher frequency of accelerated sequence evolution than gene pairs as a whole that were duplicated by the alpha WGD. In addition, among 267 alpha WGD pairs analyzed in Arabidopsis ATH1 microarray data from 63 different organ types and developmental stages . Calculating the expression specificity in different organ types, 13 out of 24 imprinted genes (54%) have higher expression organ specificity than the paralogs; nine pairs overlapped in the two analyses. A similar trend is more apparent with imprinted genes from supplementary table S5, Supplementary Material online). The imprinted genes with limited expression patterns also have higher expression levels in reproductive organs. Overall, the reproductive organ-specific expression is a relatively common feature of imprinted genes, which contrasts to the broadly expressed paralogs.For imprinted genes from When comparing the results from the expression analysis and asymmetric sequence rate analysis, we observed that many imprinted genes that evolved faster than the paralogs also show a restricted expression pattern. In Carica and Vitis to infer the preduplication ancestral expression pattern. We examined six duplicated pairs, which have imprinted genes with reproductive organ restricted expression patterns and paralogous partners with broad or nonreproductive organ restricted expression patterns.We tested the hypothesis that the imprinted genes with reproductive organ-restricted expression might result from the loss of expression in vegetative organs by investigating the expression pattern of orthologs from Arabidopsis  and VEL2 , along with the paternally expressed genes TAR1 , PKR2 , and AT3G50720 (a protein kinase). In contrast, the alpha WGD paralogs all have broad expression in both vegetative and reproductive organs , and HDG3 were among the initial imprinted genes to be discovered in A. thaliana before the high-throughput sequencing studies in 2011. All three genes were derived from the Brassicaceae-specific alpha WGD . The PAB8 paralog of MPC encodes a poly(A) binding protein that plays a role in mRNA stability and translation, whereas maternally expressed MPC encodes a truncated protein that only has one-fourth the length of regular PAB proteins at the C-terminal domain . In contrast, HDG7 is expressed only in young vegetative tissues, such as root and stem tips (a). The FWA/HDG7 orthologs in Carica and Vitis had a similar expression pattern to HDG7 (a) suggesting that HDG7 has the ancestral state of expression and the expression pattern of FWA in siliques was derived after gene duplication.siliques , whereasnd seeds a. In contem tips , and protem tips a. The FW to HDG7 a suggestMPC and PAB8, FWA and HDG7, and HDG3 and HDG2 using a free-ratio analysis in PAML to calculate the Ka/Ks ratio of imprinted genes and their paralogs. We found that MPC, FWA, and HDG3 all experienced accelerated sequence rate evolution  compared with their paralogs (b). The Ka/Ks ratio of MPC is approximately twice that of PAB8, and the Ka/Ks ratio of FWA is approximately three times that of HDG7. HDG3 has a Ka/Ks ratio approximately seven times that of HDG2. Branch site-specific positive selection analysis indicated that the HD-ZIP domain of FWA has several amino acid sites that show evidence for positive selection (c). It is noteworthy that of four potential DNA sequence-specific contact residues of Arabidopsis HDG proteins that are conserved across the HDG proteins (c). Together, the detection of positive selection and the evolution of a new expression profile in FWA suggest neofunctionalization of FWA.We further characterized sequence rate evolution in paralogs b. The Kaelection c. It is proteins , two of proteins c. TogethHDG9 and HDG10 that are members of the HD-ZIP IV gene family, along with SUVH7 and SUVH8 that encode SET domain proteins. Described as imprinted by HDG9 is a meg with exclusively maternal expression, and its alpha WG duplicate, HDG8 is a primarily maternally expressed gene. In contrast, HDG10, which was derived from HDG9, was identified by SU(VAR)3-9 HOMOLOG 7 (SUVH7) which is a peg, and SUVH8 which is a meg, both of which were identified in SUVH3, as well as another duplicate SUVH1 both of which to appear to be nonimprinted . In addition to having a new limited expression pattern (a), MPC has a different function as a truncated protein (b) and a new restricted expression pattern in reproductive organs (a), MPC has acquired a new function through the loss of N-terminus (5′-end on corresponding mRNA) mRNA binding domains.Imprinted  pattern a, MPC ha protein . PAB pro protein . Thus, i protein b and a ne organs a, MPC haFWA (HDG6) and HDG7 are HD-ZIP class IV proteins, which are characterized by the homeodomain helix III followed by the leucine zipper-loop-zipper motif . FWA expression is female gametophyte- and endosperm specific, and the epigenetic FWA mutant with ectopic FWA expression has a late-flowering phenotype (FWA in seeds is not yet known. The accelerated sequence evolution and site-specific positive selection in FWA (b and c) may have been involved in FWA gaining a new function that is different from HDG7.Both er motif . They arpression . However species a. FWA exhenotype . Althougn in FWA b and c mHDG3 and HDG2 are another paralogous pair in the HDG family, and their most closely related paralogs in HDG family are ATML1 and PDF2 , which may be random events, not directional . TEs ared dosage .Supplementary figures S1 and S2 and tables S1–S5 are available at Genome Biology and Evolution online .


Surface water, sub-surface water, and sediment were sampled from 22 locations in the lake, including areas frequently used by carp. In water, areas of high carp use had a higher rate of detection and concentration of eDNA, but there was no effect of fish use on sediment eDNA. The detection rate and concentration of eDNA in surface and sub-surface water were not significantly different (p≥0.5), indicating that eDNA did not accumulate in surface water. The detection rate followed the trend: high-use water > low-use water > sediment. The concentration of eDNA in sediment samples that were above the limit of detection were several orders of magnitude greater than water on a per mass basis, but a poor limit of detection led to low detection rates. The patchy distribution of eDNA in the water of our study lake suggests that the mechanisms that remove eDNA from the water column, such as decay and sedimentation, are rapid. Taken together, these results indicate that effective eDNA sampling methods should be informed by fish distribution, as eDNA concentration was shown to vary dramatically between samples taken less than 100 m apart.Although environmental DNA (eDNA) has been used to infer the presence of rare aquatic species, many facets of this technique remain unresolved. In particular, the relationship between eDNA and fish distribution is not known. We examined the relationship between the distribution of fish and their eDNA (detection rate and concentration) in a lake. A quantitative PCR (qPCR) assay for a region within the cytochrome  Hypophthalmichthys spp.) in the Chicago Area Waterway System and the Mississippi River Methods to quantify the abundance of fish populations, such as mark-recapture and electrofishing, are costly and time-consuming. In addition, fish are often difficult to capture and detect at low densities, and capture methods themselves can lead to behavioral changes of the target species Cyprinus carpio, hereafter ‘carp’) was positively correlated with water temperature and was spatially heterogeneous. The cause of this pattern, and in particular whether it was due to the distribution of carp or higher metabolic activity of fish in warmer waters, was not examined as the distribution of carp was not measured. From the few studies that address the question of eDNA distribution in water, it is clear that it varies among habitat types, and more conclusive explanations of eDNA distribution patterns are needed. Also of interest is the distribution of eDNA in sediments, as sediments likely retain eDNA for long periods of time Despite the immense potential for eDNA technology to revolutionize monitoring programs for fish and other aquatic species, little is known about the production, fate, and distribution of eDNA in the natural environment. The distribution of eDNA is of particular importance for development of effective monitoring methods C. carpio eDNA was developed and validated in the lab. Next, since eDNA is often assumed to accumulate in surface water and sediment, surface, sub-surface, and sediment samples were taken throughout the lake. Finally, the concentration and detection rate of eDNA was compared between areas of low- and high-fish use identified from radiotelemetry data. Results of this study provide insights into optimal eDNA sampling methods for small lakes as well as information on how eDNA is distributed in aquatic systems in relation to the distribution of target organisms.To determine whether fish distribution affects eDNA concentration and detection rate in lake water and sediment, we examined the distribution of carp eDNA in a small, shallow lake and compared it to known patterns of carp distribution, which had been monitored for several years. We were interested both in detection rate (percentage of samples in which eDNA levels were present above detection threshold) as well as concentration, because the former is commonly used to assess the likely distribution of invasive Bigheaded carps while the latter measure, if understood, might add more resolution and value to the technique. First, a qPCR assay specific for C. carpio qPCR assays had been developed prior to this study b, (2) mitochondrial gene cytochrome c oxidase subunit 1, (3) mitochondrial gene control (D-loop) region, and (4) the nuclear gene recombination activating gene 1 (RAG1). Candidate primer sets were identified by NCBI Primer-BLAST using sequences under GenBank accession number X61010.1 q) value of less than 40 cycles within the linear range of the standard curve.Although two Candidate markers were screened for specificity for carp by testing for amplification of 15 ng of fin clip DNA from carp and 34 native and non-native fish species . Fin cliCarassius auratus) (50 g), while the third tank was stocked with five fish of both species. These stocking levels corresponded to a biomass of 0, 438, and 875 mg/L of carp. Fish were fed once daily ad libitum a combination of flake feed  and 2.5 mm pellet feed  that did not contain target genetic markers. After 6 days, 4 1 L water samples were collected from each tank, immediately stored at 4°C, and filtered within 4 h. Molecular analyses followed protocols described below. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol for care and holding of laboratory fish was approved by the University of Minnesota’s Institutional Animal Care and Use Committee (IACUC) (Protocol: 1407-31659A). No anesthesia or euthanasia was required as part of this study.Next, we tested markers using aqueous samples. Three 340 L flow-through tanks were set up to confirm the ability of the marker to detect carp eDNA. Prior to this experiment, all tanks were treated with 10% bleach for 30 minutes to remove all traces of DNA. The flow through rate was set at 600 mL/min, and temperature was maintained at 18°C. The first tank was stocked with 10 carp (35 g), and the second tank was stocked with 10 goldfish (Nymphaea odorata), which covers less than 10% of the lake area.The study site was Lake Staring, a small freshwater lake located in the Upper Mississippi River Basin . Lake Staring is a small, shallow lake that experiences frequent mixing due to wind and is typical of high carp density lakes in this region Carp population abundance in Lake Staring was estimated in 2011 using a mark-recapture analysis Since 2011, the distribution of carp has been regularly assessed by locating 10–20 carp equipped with internal radio tags . Carp location was determined by identification of signal directionality (bearing) with a hand-held antenna and a compass while positioned within 200 m of the radiotagged fish. Two bearings were measured for each fish, each from a different location, and their intersection calculated  to estimate fish location. Mean measurement error (30 m) was estimated using dummy tags.Using previously determined carp locations, the pattern of carp habitat usage was examined for the warm season (June–October) when carp maintain stable summertime distributions 2 was used as the cut-off between high- and low-use areas. As such, areas with lower values were considered to be low-use areas, whereas areas with higher densities were classified as high-use areas. The value of 800 radiotagged carp/km2 corresponded to approximately 1,248 carp/ha. This cut-off value was chosen because lower densities were associated with relatively isolated radiotagged carp observations. The high-use areas were located within or near the patches of lilies, which the carp most likely use for cover as the lake lacks other physical structure. In addition, all high-use areas were within 200 m of the shoreline and less than 2 m deep.Carp showed well-defined areas of habitat usage in Lake Staring . A densiN. odorata where we knew carp were generally found. At each sampling site, surface water, sub-surface (0.5 m depth) water, and sediment were sampled. A surface water sample was also taken in both the inflow and outflow of the lake.Field sampling took place on 8 October 2013. Average wind speed was 20 km/h from the S Water samples were collected in 1-L HDPE bottles  that had been previously soaked in 10% bleach for at least 30 min to remove all traces of DNA. Bottles were subsequently rinsed with distilled water to remove residual bleach. Surface water samples were taken by partially submerging a sample bottle to collect water from the top few cm. Sub-surface samples were taken using a stainless steel Van Dorn sampler . Sediment samples were collected using a stainless steel Petite Ponar grab sampler . Sediment was transferred to a sterile Whirl-Pak bag  using a sterile polystyrene spatula . Once collected, samples were immediately placed on ice. Water and sediment samples were stored at 4°C and were filtered within 24 h. No specific permissions were required for access to the study site or collection of samples as part of this study. No animals were collected as part of this study.Water samples were filtered through Whatman 934-AH 1.5 µm glass microfiber filters  using a polyphenylsulfone filter funnel . Filter funnels and forceps were soaked in 10% bleach and rinsed in distilled water prior to use and between samples. For tank samples, 1 L of water was filtered per sample. For field samples, only 200 mL could be filtered per sample due to clogging from the high amount of suspended solids. Filters were stored at −80°C until DNA extraction.Sediment samples were homogenized, and a 0.1 g subsample was stored at −80°C for DNA extraction. Preliminary experiments showed that extraction of greater than 0.1 g of sediment lead to reduction in eDNA yield and inhibition of qPCR, regardless of post-extraction inhibitor removal protocols or inclusion of PCR adjuvants. For moisture content analysis, a 10 g subsample of sediment was weighed and then dried at 100°C for 24 h.DNA was extracted using the QIAamp DNA Stool Mini Kit  using the human DNA analysis protocol. Frozen filters were sliced into 1 mm×5 mm fragments with a sterile razor blade and then transferred to extraction tubes. For sediment samples, extraction buffer was directly added to frozen sediment. Before extraction, 50 ng of UltraPure Salmon Sperm Solution  was added to adjust for extraction efficiency of DNA as previously described b and the extraction control targets, and the oligonucleotide concentrations were optimized for this study . Reactions contained 12.5 µL mastermix, 10 µg bovine serum albumin  primers and probe, and water or sediment DNA in a final reaction volume of 25 µL. The volume of water and sediment DNA added to the qPCR reaction was adjusted by testing a dilution series of a subset of 5 samples from water and sediment to confirm that inhibition was not present. For sediment samples, 2.5 µL of DNA extract was added to the reaction, and for water samples 5 µL of DNA extract was added. Reaction conditions consisted of an initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 15 s and an annealing and extension step at 60°C for 1 min. Each qPCR run contained triplicate reactions of standards, non-transcript controls, and samples. Amplifications were performed using the StepOnePlus Real-Time PCR System , and CDetection rate of eDNA was defined as the proportion of samples that were above the qPCR assay LOD. To analyze the effects of water sample depth , carp usage , and matrix type  on eDNA detection rate, the number of detections and non-detections were statistically compared using Fisher’s exact test. Fisher’s exact test was used due to low expected values (<5) in some cells.Concentration of eDNA in water was analyzed using a three-way Analysis of Variance (ANOVA). Main effects and 2-way interactions of carp usage , water sample depth , and lake depth (m) were examined. Lake depth was included to determine the potential for suspended sediment to affect water column eDNA concentration, with shallower depths more likely to be affected by sediment mixing into the water column. The 3-way ANOVA was restricted to sites greater than 0.5 m and less than 2 m depth  because of a partial confound . Finally, student’s t-test was used to determine whether there was a significant difference between eDNA concentration of sites included in the ANOVA and those excluded for the low-use areas. Since only 2 sites within the high-use area were excluded, no statistical comparison was done.10 transformed to achieve normal data distribution. Values below the LOD were given a value of half the LOD prior to analysis in order to reduce skewing of data. For graphical representation of eDNA concentrations across sampling points, data above the LOD were divided into four equal percentile categories. Percentiles were determined independently for the two sample types: (1) surface and sub-surface water and (2) sediment samples. All statistical tests were conducted in JMP, Version 10 .For all parametric descriptive analyses and statistical tests, eDNA concentrations were logb) was developed for a 149 bp region in the cytochrome b gene . The sample LOD varied slightly for individual samples depending on extraction efficiency. Further details regarding qPCR calibration curves can be found in 5 copies per reaction, the highest standard tested. No amplification of non-transcript controls was observed. The assay did not amplify DNA from a selection of 34 native and non-native fish species, including Bigheaded carps and other related Cyprinids  than that of carp alone, indicating that the presence of more individuals yielded more eDNA. On average, individual carp contributed 3.1×1011 copies of CarpCytb in the mixed species tank and 4.4×1011 copies per individual in the carp only tank.In validation tests using laboratory tanks which were stocked with combinations of carp and goldfish, no CarpCytb in water samples was 75% . However, detection rate was significantly higher in water samples collected in high-use areas , a difference of nearly 40% . The detection rate of CarpCytb in low-use areas in the sediment was approximately 30% less than for the average value for water samples, however, there was no difference between the detection rate . The detection rate of eDNA in sediment within high-use areas was nearly 60% less than water within high-use areas, and the difference was statistically significant .The overall detection rate of CarpCytonly 36% . Similaronly 36% . The detb concentration in water ranged from below the LOD (2.0×104 copies/L) to 1.7×106 copies/L .Three-way ANOVA showed that carp use pattern had a significant (main) effect on eDNA concentration . There w5 copies/g) to 5.4×105 copies/g . On a per mass basis, the lowest measureable sediment concentration of CarpCytb was nearly two orders of magnitude greater than the water sample with the highest concentration of CarpCytb.For sediment samples, the concentration of eDNA ranged from below the LOD  were below the LOD, we were unable to reliably calculate a mean sediment eDNA concentration. The accumulation of eDNA in sediment has been suspected based on the high concentration of microbial DNA in sediment Regardless of the low detection rate of eDNA in sediment, its importance as a reservoir of carp eDNA cannot be disregarded. The concentration of CarpCytSampling design has been previously identified as one of the four critical aspects that must be optimized in a DNA-based monitoring program Table S1List of fish species tested for marker specificity.(DOCX)Click here for additional data file.Table S2Quantitative PCR calibration data.(DOCX)Click here for additional data file.Table S3Coordinates, lake depth, fish use, and eDNA concentration at sampling sites.(DOCX)Click here for additional data file.


Yersinia secrete virulence factors that invade eukaryotic cells via type III secretion system. One particular virulence member, Yersinia outer protein E (YopE), targets Rho family of small GTPases by mimicking regulator GAP protein activity, and its secretion mainly induces cytoskeletal disruption and depolymerization of actin stress fibers within the host cell. In this work, potent drug-like inhibitors of YopE are investigated with virtual screening approaches. More than 500,000 unique small molecules from ZINC database were screened with a five-point pharmacophore, comprising three hydrogen acceptors, one hydrogen donor, and one ring, and derived from different salicylidene acylhydrazides. Binding modes and features of these molecules were investigated with a multistep molecular docking approach using Glide software. Virtual screening hits were further analyzed based on their docking score, chemical similarity, pharmacokinetic properties, and the key Arg144 interaction along with other active site residue interactions with the receptor. As a final outcome, a diverse set of ligands with inhibitory potential were proposed.Gram-negative bacteria  The Rho family of small 20–40 kDa) GTPases, which are monomeric G-proteins, belong to the Ras superfamily of GTPases containing more than 150 proteins 0–40 kDa , 4. Up tSimilar to other G-proteins and GTPases, Rho family proteins can serve as molecular switches, by binding to either GDP or GTP. GTPases are active and are capable of transmitting cell signals to downstream proteins when they are bound to GTP and inactive when they are bound to GDP , 11. SinBurkholderia, Chlamydia, Salmonella, Shigella, and Yersinia [Owing to the important role of Rho GTPases in cell signaling events and several cellular functions, they are favored by many bacterial pathogens as targets to deliver cytotoxins . BacteriYersinia . These bYersinia . BacteriYersinia , 22. As Yersinia . YopE alYersinia . Yersinia outer protein E (YopE), which has been found to exert GAP activity towards RhoA, Rac1, and Cdc42 of GTPases in vitro, is investigated. [α-helix bundle, four small helices, and two β-strands [Pseudomonas aeruginosa and SptP of Salmonella spp. (22% sequence identity with ExoSGAP and 29% sequence identity with SptPGAP) [In this work, the bacterial protein toxin tigated. –27. YopEtigated. –31. YopE-strands . C-termiSptPGAP) . YopE inSptPGAP) . ResidueSptPGAP) .Yersinia [Yersinia pseudotuberculosis proteins Tpx and WrbA, which take role in YopE secretion,were recognized as targets of these compounds, [Up to date, no cocrystallized or experimentally discovered ligands of YopE have been reported. However, inhibition of type III secretion system has been investigated by several studies –37, and Yersinia . AlthougYersinia , 40, 41.mpounds, . NeverthHere, we represent a hybrid virtual screening approach to identify molecules with inhibition potential against YopE, using computational drug discovery tools. Pharmacophore building was carried out via Phase, based on the three-dimensional structures of 23 salicylidene acylhydrazides . Small mAll virtual screening applications were performed using Schrödinger Suite 2011  on Linux platform using HP xw6600 Workstation. The following Schrödinger modules were used: Protein Preparation Wizard –44, LigPhttp://dx.doi.org/10.1155/2013/640518) were prepared via LigPrep and ConfGen scripts and merged using Phase Database Generation module. The parameters used in the preparation step are the following: (a) possible ionization and tautomer states were generated at pH 7.0 ± 2.0; (b) chiralities were obtained from the 3D geometry of the structures; (c) for each compound, maximum 4 low energy isomers, 1 ring conformation, and 10 conformations per rotatable bond were generated; (d) 50 steps of energy minimization were carried out for the conformers with the truncated Newton (TCNG) method using the OPLS_2005 force field and a distance dependent dielectric constant of 4; (e) conformers with high-energy ionization/tautomer states were automatically removed and the number of conformers was limited to 100 per compound, which is the default value for conformer generation in Phase Database Generation module; (f) a built-in QikProp script predicted the ADME and druglikeness properties of the molecules; (g) molecules that violated Lipinski's rule  or had reactive functional groups were removed. Database preparation yielded a total of 25.8 million conformations from the initial 500000 molecules.The 3D structures of the small molecules from the big vendors of ZINC database  were obAfter preparation, receptor grids were generated with Glide by specifying the binding site with a 3D cubic box. SiteMap was used to estimate the location of the active site by searching regions near the protein surface, generating hydrophobic and hydrophilic contour maps of the protein, and calculating energy potentials. Enclosing box of 14 Å side lengths was placed by centering the critical Arg144 residue, depending on the SiteMap prediction . Based upon the fact that the binding site is not shallow, the nonpolar atoms were slightly scaled back, by choosing the van der Waals radius scaling factor of 0.75 for nonpolar parts, so that nonnative ligands would dock to the receptor better. Rotation of all receptor hydroxyl and thiol groups within the grid was allowed.Pharmacophore model was developed using the 23 experimentally determined inhibitors of YopE secretion  via PhasMolecules obtained by filtering were used for multistep receptor docking workflow. Glide standard precision (SP) docking was performed with these molecules, and hits above 4 kcal/mol based on docking score were redocked to YopE in XP mode, keeping all docking parameters as default. No bonding constraints were given during docking calculations. Using Monte Carlo random search algorithm, ligand poses were generated for each input molecule, and binding affinity of these molecules to YopE was predicted in terms of Glide docking score. Potential energies of the docked molecules were also predicted with empirical E-model scoring function. Postdocking minimization was performed with OPLS_2005 force field, and one pose per ligand was saved. Strain energies of bound and free forms of ligands were calculated, and hits with more than 4 kcal/mol energy difference between the two forms received a penalty equal to the quarter of their strain energy difference, which is added to the docking score.Investigation of novel inhibitors targeting the bacterial YopE was performed with the help of computational drug design tools. Database generation, pharmacophore modeling and screening, and molecular docking and scoring were carried out to propose a set of biochemically active molecules with inhibition potential against YopE.23 chemically synthesized compounds belonging to a class of acylated hydrazones of salicylidene acylhydrazides that inhibit YopE secretion in vitro were utilized (Table S2) for pharmacophore development. The ability of these salicylidene acylhydrazides to inhibit YopE and to make important interactions upon direct contact, similar to Tpx and WrbA, was sought. For this purpose, prior to pharmacophore development, extra precision Glide docking was carried out with these 23 compounds, where docking scores were observed to be between −2.0 and −5.9 kcal/mol (data not shown). The binding orientations of these compounds were found to be in close proximity to the critical arginine residue, occupying the cleft between Arg144 and a bulge formed by residues Thr183-Gly185. The most common interactions between YopE and 23 inhibitors were observed in residues Arg144, Gln151, and Thr183. The compounds also interacted with other important residues reported upon GTPase contact, some of which are Thr138, Gly139, Thr148, Ser179, Gln180, Gly182, Ile184, and Gly185. The binding modes and numerous interactions between YopE and the salicylidene acylhydrazides supported the choice of using these compounds for pharmacophore development. Six out of 23 compounds that have the critical Arg144 interaction and docking scores above −4 kcal/mol were selected . It was The pharmacophore model was developed based on the structures of the six selected compounds using Phase. After hypotheses were constructed, they were scored and ranked according to their coordination to the ligands, which can be seen in 2) arbitrary ligand molecules would match the hypothesis, regardless of their activity value. Therefore, higher selectivity is favored, since it implies uniqueness to the ligand set. However, site, vector and, volume scores are underlined in the literature due to their geometric significance [The quality of each hypothesis was measured by its survival score, which is a weighted combination of site, vector, volume, and selectivity scores. Site score indicates root mean squared deviation of compounds from the hypothesis site positions, whereas vector score is the average cosine of the angles formed by corresponding pairs of site features in the aligned structures and volume score measures how a compound overlays with pharmacophore sites based on van der Waals spheres. Site, volume, and vector scores range between 0 and 1, and high values indicate better alignment of ligands on hypothesis. Selectivity is an empirical prediction defined on log scale, which calculates the fraction of molecules that could match the hypothesis, whether it is an active or inactive ligand. For example, a selectivity value of 2 means that 1 molecule out of 100  docking was first conducted with 18230 hits using the 3D structure of YopE prepared for docking calculations, as described in the receptor protein preparation and receptor grid generation sections. Top 30% hits of Glide SP in terms of docking score, which comprises 5604 molecules, were redocked to YopE in Glide extra precision (XP) mode with the identical internal docking parameters. Docking scores varied between −7.2 and −0.5 kcal/mol in Glide XP mode. A pose filter was carried out and virtual screening hits that interact with the critical Arg144 residue were determined. This filter yielded 2605 hits out of the initial 5604. The number of remaining hits was further reduced to 185 by filtering with a docking score threshold of −4 kcal/mol. Structures of the remaining molecules were analyzed and clustered by their 2D structural similarities via ChemMine Web Tool . HierarcMolecules were sorted according to their docking scores in Pharmacokinetic properties, druglikeness as well as other significant descriptors, such as molecular weight, H-bond donors, H-bond acceptors, solvent accessible surface area, log HERG (blockage of K+ channels), log S (aqueous solubility), log P (octanol/water), and human oral absorption, for the selected hits were determined by QikProp . DruglikSuperposition of the proposed molecules YopE showed that their binding orientations are similar . The intThe hits were visually inspected and their similarity to the salicylidene acylhydrazides used in pharmacophore development was determined. The nine selected hits satisfied all of the pharmacophore sites with a 2 Å tolerance, suggesting that the pharmacophore building and docking results converged. The superposition of the selected hits on the pharmacophore hypothesis AAADR.21 is given in Figure S3. The hits from the third cluster  were fouP. aeruginosa cytotoxins ExoSGAP [S. enterica SptPGAP  [α coordinates, the root mean square deviations between the Cα coordinates of YopE with ExoSGAP and with SptPGAP are 1.26 Å and 1.36 Å, respectively [ExoSGAP and Arg209SptPGAP. Binding selectivity of selected hits to ExoSGAP and SptPGAP was investigated. Their 3D structures were taken from the Protein Data Bank (Pdb ID: 1g4u for SptPGAP and Pdb id: 1he1 for ExoSGAP) prepared in Maestro workspace, and their binding site grids were generated for all-atom docking calculations, as described in receptor protein preparation and receptor grid generation sections. By centering the key arginine residues and keeping the previous docking parameters identical, Glide XP docking was performed on ExoSGAP and SptPGAP. The results reveal that the selected nine hits have higher average ligand strain and lower binding affinity toward ExoSGAP and SptPGAP in terms of docking scores  were clustered based on their structural similarity using a Tanimoto coefficient threshold of 0.5. The three top scoring representative members from the most populated clusters were pooled and further analyzed. One cluster was found to be structurally similar to the salicylidene acylhydrazides, which can inhibit the bacterial activity of type III secretion systems. The cluster with erythritol substructure is analogous to THI (2-acetyl-4-tetrahydroxybutylimidazole). THI acts as an immunosuppressant inhibitor of Sphingosine-1-phosphate lyase (S1PL), whose reduced activity is targeted for autoimmune disorder treatment . The othGAP and SptPGAP. Docking results showed that these hits have lower binding affinity toward the homologous proteins, and the critical arginine bonding was not observed in the majority of hits. The proposed set of ligands has shown a promising inhibitory potential toward YopE in silico and hence, can be used for further scientific studies, and the results can be extended to experimental validation.The aim of this virtual screening study was to find potent YopE inhibitors that would hinder the interaction between their GAP domain and GTPases. The binding site was selected such that the inhibitors would occlude the vicinity of the Arg144 residues that contact the switch regions of GTPases. Indeed, the molecules made contacts with the critical arginine finger as well as the other residues that were reported to interact with both Switch I and Switch II regions. The binding modes of the hits showed that the molecules occupied the cleft formed in the vicinity of Arg144 of YopE. Selectivity against YopE was verified by docking the nine hits to the known YopE homologs, namely, ExoSThe list of vendors used in small molecule database (Table S1), 2D structures of small compounds found in literature (Table S2), pharmacophore site measurements (Table S3), detailed docking results (Table S4 and Table S5), detailed ADME and pharmacokinetic property estimates (Table S6), SiteMap and ChemMine outputs  and pharmacophore-hit superimpositions  are provided.


To compare postoperative astigmatic correction between femtosecond lenticule extraction (FLEx) and small-incision lenticule extraction (SMILE) in eyes with myopic astigmatism.We examined 26 eyes of 26 patients undergoing FLEx and 26 eyes of 26 patients undergoing SMILE to correct myopic astigmatism (manifest astigmatism of 1 diopter (D) or more). Visual acuity, cylindrical refraction, the predictability of the astigmatic correction, and the astigmatic vector components using Alpin’s method, were compared between the two groups 3 months postoperatively.We found no statistically significant difference in manifest cylindrical refraction (p=0.74) or in the percentage of eyes within ± 0.50 D of their refraction (p=0.47) after the two surgical procedures. Moreover, no statistically significant difference was detected between the groups in astigmatic vector components, namely, surgically induced astigmatism (0.80), target induced astigmatism (p=0.87), astigmatic correction index (p=0.77), angle of error (p=0.24), difference vector (p=0.76), index of success (p=0.91), flattening effect (p=0.79), and flattening index (p=0.84).Both FLEx and SMILE procedures are essentially equivalent in correcting myopic astigmatism using vector analysis, suggesting that the lifting or non-lifting of the flap does not significantly affect astigmatic outcomes after these surgical procedures. The femtosecond laser is one of the most revolutionary inventions in recent medical technology that has been used mainly in ophthalmology for laser in situ keratomileusis (LASIK). It is employed as an alternative to the mechanical microkeratome for precisely and reproducibly creating corneal flaps. A recent breakthrough in this technology is refractive lenticule extraction (ReLEx), which requires neither a microkeratome nor an excimer laser, but uses only the femtosecond laser system as an all-in-one device for flap and lenticule processing. The first clinical results were obtained in highly myopic eyes , and in 2, and no history of ocular surgery, severe dry eye, progressive corneal degeneration, cataract or uveitis. Eyes with keratoconus were excluded from the study by using the keratoconus screening test that employs Placido disk videokeratography . Written informed consent was obtained from all patients after explanation of the nature and possible consequences of the study. The study was approved by the Institutional Review Board of Kitasato University and followed the tenets of the Declaration of Helsinki. The author’s Institutional Review Board waived the requirement for informed consent for this retrospective study.Twenty-six eyes of 26 consecutive patients (10 men and 16 women) who underwent FLEx and 26 eyes of 26 consecutive patients (9 men and 17 women) who underwent SMILE, for the correction of myopic astigmatism were included in this experimental study. One eye from each patient was chosen randomly for the measurement. Some of the subjects were those in our preceding report on visual and refractive outcomes after FLEx and SMILE [Both FLEx and SMILE were performed under topical anesthesia  using the VisuMax femtosecond laser system  with a 500 kHz repetition rate. The laser was visually centered on the pupil. A small curved interface cone was used in all cases. The main refractive and non-refractive femtosecond incisions were performed in the following automated sequence: the posterior surface of the lenticule , then the anterior surface of the lenticule , followed by a side cut of the flap. The femtosecond laser parameters were as follows: 120 μm flap thickness, 7.5 mm flap diameter, 6.5 mm lenticule diameter, 140 nJ power for lenticule and flap, and a 310-degree side cut (superior hinge) with side cut angles of 90 degrees for FLEx; and 120 μm cap thickness, 7.5 mm cap diameter, 6.5 mm lenticule diameter, 140 nJ power for lenticule and cap and a 50-degree side cut for access to the lenticule with angles of 90 degrees for SMILE. After the suction was released, the patient was moved toward the observation position under the VisuMax integrated surgical microscope. For FLEx, after completion of the laser sequence, a Siebel spatula was inserted under the flap near the hinge and the flap was lifted; and the refractive lenticule was next grasped with forceps and extracted. The flap was then repositioned. For SMILE, a thin spatula was inserted through the side cut over the roof of the refractive lenticule dissecting this plane, followed by the back surface of the lenticule. The lenticule was subsequently grasped with modified serrated McPherson forceps  and removed. Then, the intrastromal space was flushed using a standard LASIK irrigating cannula. After surgery, steroidal medication  and antibiotic medication  were topically administered 4 times daily for 2 weeks, after which the frequency was steadily reduced.We determined the logarithm of the minimum angle of resolution (logMAR) of uncorrected distance visual acuity (UDVA), the logMAR of corrected distance visual acuity (CDVA), and the manifest spherical equivalent or cylindrical refraction before and 3 months after surgery. Corneal astigmatism was measured using an autokeratometer .Manifest refraction was converted to the corneal plane value before astigmatic vector analysis was done by Alpin’s method . In bothU test was used to compare the visual acuity, the refraction, and the vector components between FLEx and SMILE and the Fisher’s exact test, to compare patient sex and the percentage of eyes within ± 0.50 D or ± 1.00 D of the manifest refractive cylinder correction between the two surgical techniques. The results are expressed as mean ± SD, and a value of p<0.05 was considered statistically significant.All statistical analyses were performed using SPSS . Sample size calculation was performed using PASS 2008 software . The Mann-Whitney Preoperative patient demographics are summarized in U-test), CDVA (p = 0.52), manifest spherical equivalent (p = 0.60), or manifest cylinder (p = 0.74) after surgery between the two groups. The postoperative refractive and visual acuity outcomes at 3 months are shown in r p = 0.7 after suThere was also no statistically significant difference 3 months postoperatively in the corneal astigmatism between the two groups (p = 0.50).The attempted versus the archived manifest cylindrical corrections made 3 months postoperatively, and the preoperative and postoperative manifest cylinder are shown in The vector analysis results using 3-month refractive data are shown in In the current study, we found no significant difference in the percentage of eyes within ± 0.50 D or ± 1.00 D between FLEx and SMILE techniques, indicating that both FLEx and SMILE are able to correct low to moderate astigmatism to similar degrees using Alpin’s method of vector analysis, and that the step of flap lifting in the FLEx procedure does not seem to induce astigmatism to the same significant extent as surgery does.Although we present a relatively small sample size, this is to our knowledge the first published study comparing the astigmatic outcomes of FLEx and SMILE using Alpin’s vector analysis method. Every parameter used in this vector analysis has clinical relevance pertinent to the treatment of an eye after refractive surgery or to its outcome. This method has clinical benefits in the comparison of astigmatic correction between techniques, since it uses several parameters and provides a more realistic picture than simple numerical analysis of the treatment efficacy of a refractive procedure for astigmatism. There are a few studies available to validate the astigmatic correction of FLEx or SMILE –14. WhenIt has been reported that the IntraLase flaps created with the femtosecond laser have better visual acuity and induce less astigmatism than the flaps created with the mechanical microkeratome in the LASIK procedure (0.22 D vs. 0.32 D), which indicates that the method of flap creation might dominate the surgically induced astigmatism, rather than the step of flap lifting –17. AlthPossible reasons for the residual cylindrical errors observed in both FLEx and SMILE techniques could be cyclotorsion or astigmatic nomogram of the femtosecond laser system itself. Prakash et al.  reportedThere were at least two limitations to this study. Firstly, preoperatively, the study did not completely match the items in the patient backgrounds, such as age, gender, manifest spherical equivalent, and manifest astigmatism. These differences in the patient backgrounds may affect the astigmatic outcomes after ReLEx. Secondly, we determined the postoperative astigmatism 3 months postoperatively, when the corneal shape was considered to have been stabilized, taking into account the wound-healing responses of the cornea. A prospective randomized controlled study with a longer follow-up is necessary to confirm the authenticity of the results.In conclusion, our results demonstrated that FLEx and SMILE were essentially equivalent in correcting astigmatism equal to or more than -1.00 D in terms of cylindrical refraction, the predictability of astigmatic correction, and astigmatic vector analysis components without vision-threatening complications occurring throughout the 3-month follow-up period. As far as we can judge, the presence or absence of flap lifting does not significantly affect the astigmatic outcomes after ReLEx. These novel surgical techniques may prove to be promising alternatives to excimer laser-based corneal refractive procedures for myopic astigmatism correction.