Furthermore, only the 13 most informative markers were needed to achieve maximum differentiation. This simple, effective method provides robust and accurate genotypic data which allows for more efficient resource management , thus contributing to an increased knowledge on the genetic background of cacao worldwide. Notably, the described method can easily be integrated in other laboratories for a wide range of objectives and organisms.In this study, a double-mismatch allele-specific (DMAS) qPCR SNP genotyping method has been designed, tested and validated specifically for cacao, using 65 well annotated international cacao reference accessions retrieved from the Center for Forestry Research and Technology Transfer (CEFORTT) and the International Cocoa Quarantine Centre (ICQC). In total, 42 DMAS-qPCR SNP genotyping assays have been validated, with a 98.05% overall efficiency in calling the correct genotype. In addition, the test allowed for the identification of 15.38% off-types and two duplicates, highlighting the problem of mislabeling in cacao collections and the need for conclusive genotyping assays. The developed method showed on average a high genetic diversity (H Theobroma cacao L. (T. cacao) (2n = 20) are worldwide acknowledged for their application in the chocolate industry1. They are harvested in the tropical regions between 10 to 20 degrees north and south of the equator. Although the South-American Amazon region is recognized as the center of its origin, the three main cacao producing countries are Ivory Coast, Ghana and Ecuador3. The flavor characteristics of cacao are mainly influenced by genotype and origin, although growth conditions of the cacao tree and post-harvest processing factors have also shown to be of importance5. T. cacao consists of numerous morphologically distinct populations, mainly subdivided in Criollo, Trinitario and Forastero. They can all be crossed reciprocally, which is valuable for breeding and propagation. Such crop improvement programs primarily focus on cultivation of new, resistant and high-yielding cacao varieties using characterized genetic resources (germplasm)7. So far, up to 15 to 44% of the cacao accessions have been estimated to be mislabeled – called off types – as they show different genetic profiles than expected10. Therefore, more straightforward identification techniques based on genetic markers, also known as DNA fingerprinting methods, have been used.The beans of 15.Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) are mainly used for genotyping and classifying cacao varieties. Both have been employed successfully in cacao for genetic diversity studies, detecting mislabeling and establishing the genetic relationships within and between populations or individuals. Moreover, they proved to facilitate cacao conservation and domestication, and conjointly assisted in mapping useful genes and selecting breeding parents. In short, the use of genetic markers resulted in a reduction of inbreeding, mislabeling, unwanted progeny, and genetic drift, playing a main role in long-term breeding gains by enhancing efficient and substantiated cacao management17, progeny improvement16, parentage analysis18 and diversity assessments20. Since 2004, a set of fifteen SSRs has been recognized as the international standard for genotyping cacao21. In 2008, Motamayor et al. used these markers to classify Amazonian cacao germplasm (living genetic resources) more accurately into 10 major genetic clusters, namely Amelonado, Contamana, Criollo, Curaray, Guiana, Iquitos, Marañon, Nacional, Nanay and Purús8. This technique, in combination with the proposed classification, allows to reflect on the genetic diversity of cacao varieties worldwide. Unfortunately, many disadvantages are inherently linked to SSR. Besides the lack of associations with genes and traits22, this electrophoresis-based analysis often delivers ambiguous results making inter-lab comparisons difficult. Moreover, the technique is costly and requires specialized training and instrumentation, both usually limited in cacao producing countries and therefore often outsourced and unaffordable for small scale studies13.SSRs are the most commonly used type of marker for genotyping plants and animals. These tandem nucleotide repeats are highly polymorphic, abundantly present in the genome, co-dominant and suitable for automatization. Their efficiency in cacao genotyping has been demonstrated successfully for multiple applications such as off-type detection24. Approximately 1,560 SNP candidates from a wide range of cacao organs have already been published in Tropgenedb25. Recently, Livingstone et al. (2015) identified 330,000 SNPs by mapping RNA-seq data from 16 diverse cacao cultivars to the Matina 1–6 genome26. Despite the fact that SNPs are biallelic in nature and hence less polymorphic and informative than SSRs, they have shown to be 98% as efficient in calling off-types27. Furthermore, SNP-based genotyping is more robust and less ambiguous as it requires only a (quantitative) PCR instrument facilitating data publication in databases accessible worldwide, such as the International Cocoa Germplasm Database (ICGD).Alternative molecular markers such as SNPs have been proposed. They constitute the largest class of polymorphisms observed in plant genomes and can be found in coding regions, making associations with traits feasible6 and TaqMan-based quantitative PCR (qPCR) are the golden standard for SNP detection in cacao studies28. TaqMan-based genotyping has already been applied on-field and both methods can be used in multiplex and automated high-throughput contexts12. However, Maldi-TOF MS needs specialized equipment and training, whereas TaqMan-based qPCR assays depend on fluorescently labeled probes. This increases design complexity and cost in comparison to SYBR-green-based qPCR – especially for low throughput genotyping assays. Recently, a cost-effective qPCR-based method has been proposed for SNP genotyping purposes, coined DMAS qPCR. The method is based on straightforward readout of DNA-binding dye based qPCR technology. It entails two parallel qPCR reactions, each including a different allele-specific (AS) primer harboring an artificial mismatch to increase robustness and discriminating power. The advantage of DMAS-qPCR is its ease of use, high analytical sensitivity and specificity (matching TaqMan-based methods) without the need for expensive fluorescently labeled probes29. Moreover, a similar SNP genotyping method, known as AS Primer (ASP) PCR, was published in 2010 with promising results in genotyping rice30.Currently, Maldi-TOF mass spectrometry (MS)This paper describes the development of successful DMAS-qPCR SNP genotyping assays that are useable for cacao mislabeling and genetic diversity studies. Two main objectives can be distinguished, namely (1) the establishment of cacao specific DMAS-qPCR SNP assays, and (2) the testing and validation of resulting DMAS-qPCR assays on a subset of international cacao reference accessions. In general, the applicability, reliability and fast multi-sample and multi-locus SNP genotyping capacity of DMAS-qPCR will be demonstrated.th nucleotide of this end, and a common primer. Both combinations are used in parallel qPCR reactions and allows SNP detection by analysis of Cq values retrieved. Three genotyping models were proposed, all using the ΔCq – the difference in Cq between the REF and ALT allele reaction – from validated accessions to assign classify the SNP status. The first model (1) exploits setting manual thresholds around the heterozygous samples in the difference plots. Thresholds were based on critical assessment of the scatter and difference plots, found in Supplementary Each DMAS qPCR assay, consists of reference (REF) and alternative (ALT) allele specific primers, matching the SNP at their 3′ and containing an additional mismatch at the 4st model  exploitsst model  exploits31. DMAS-qPCR assay design was attempted for these 102 targets, for which 10 failed due to the limited design space and low genomic complexity at the associated regions. The remaining 92 DMAS-qPCR assays were first tested by qPCR on 6 to 10 cacao accessions for which a corresponding reference genotype status was available – through ICGD or SBG  of the 102 remaining SNP targets, genotype status of different cacao accessions has been published in ICGD, allowing comparison with DMAS-qPCR results later on. For the other 14 SNPs, genotype status was determined by means of in-house sequencing-based genotyping (SBG)The additional 55 international cacao accessions were genotyped using the 47 successful DMAS-qPCR assays (Supplementary HOM/HET distances (i.e. abs(ΔCqHOM − ΔCqHET) values) and thus increased discriminating power and more accurate genotype calling. When focusing on heterozygous calls, 92.9% (n = 39) assays showed an average ΔCq < 0.5 and 69.1% (n = 29) <0.25. For homozygous samples, ΔCqHOM/HET values >5 could be observed for 92.9% (n = 39) and 88.1% (n = 37) of the assays in reference and alternative samples, respectively, while 69.1% (n = 29) and 57.1% (n = 24) of the ΔCqHOM/HET distances exceeded 7.5. Overall, 78.6% of the assays generated ΔCqHOM/HET >5 , allowing straightforward genotype calling. However, an assay having lower ΔCqHOM/HET distances and very low SD for both the homozygous and heterozygous calls could still achieve similar (or even better) genotyping accuracy compared to an assay with perfect ΔCqHOM/HET distances.Performance of each of these assays was assessed using their average ΔCq value and its standard deviation (SD)  was overall high, from 89.97% for the second model (automated thresholds), over 91.51% for the third model (clustering), and up to 92.16% for the first model . In general, an overall average genotyping efficiency of 88.47% was observed when combining all models. By comparing models among themselves, similar trends could be observed with average genotype overlap reaching 97.60%. When taking the first model as a reference, the clustering-based model seems to outperform the automatic threshold-based model slightly . Overlap between the second and third model is substantially lower (97.75%)  – who considered cacao clones as dissimilar when they differed on at least two loci23 – and Padi et al. (2015) – who permitted only a single mismatch to account for genotyping errors9, we only classified samples as off-type if they differed in at least four loci from the reference accession, e.g. EET 95 and U45. CEFFORT carried a greater amount of off-types in contrast to ICQC, with 24% and 9.76% respectively.During these analyses, an overall 15.38% mislabeling was detected by comparing the multi-locus SNP profiles of the accessions with their reference status. The reference status of ICS1(CEFORTT), LCTEEN62/S, IMC67, PA120, SCA9(CEFORTT), NA33, NA399 and SCA11 differed on more than 13 loci from our genotype calls and were thus classified as off-types. In contrast to Takrama 32 (Table mAF and MAF) were calculated, ranging from 0.046 and 0.945 to 0.492 and 0.508 with an average of 0.338 and 0.647, respectively. The large range of MAF suggests a good level of genetic diversity across the selected SNPs. This determines if a specific SNP is abundantly present in a population, making it useable as a selection criterion. From these frequencies expected and observed heterozygosity (He and Ho) was calculated the first represents the proportion of heterozygosity expected under random mating and is proportional with genetic variability within the population, which should be high in a diverse population as the one used here whereas the second is inversely proportional with inbreeding. The He ranged from 0.088 to 0.500  with an overall average of 0.416, while an average of 0.298 was observed for Ho, ranging from 0.062 to 0.462. For all markers, except TcSNP878, He was higher than Ho suggesting an isolate-breaking effect (the mixing of two previously isolated populations).For all validated assays, key descriptive analysis was computed using GenAIEx v6.532 Table . At leasI) – representing the informativeness of each marker – ranged from 0.187 (TcSNP786) to 0.693 for multiple loci, with a high average of 0.601. Fixation index (F) values approximating zero are expected under random mating, while substantial positive values indicate inbreeding. Negative F-values indicate excess of heterozygosity due to negative assortative mating or selection for heterozygotes32. Here, an F-value of 0.277 was observed, indicated random mating in the population. Further, the discriminating power of the SNP loci was computed through the probability of idenitity amongst siblings (PIDsib) over all analyzed loci and 65 cacao accessions counting for 3.4 × 10−22, indicating that the chance of finding two individuals with the same genotype in the population was close to zero. Identification of the previously detected off-types through pair-wise multi-locus analysis using all SNP data accessible in ICGD failed33 (data not shown). However, two duplicates could be identified, namely APA4 and SPEC41/6-18 – and – U45 and U70. Following ranking of the markers according to I, only 13 markers  were deemed necessary for differentiation with over 99.999% certainty as analyzed by matching and unique genotypes (data not shown). Furthermore, the validated assays were on average 98.05% efficient in calling the correct SNP genotype from unmislabeled cacao accessions with a 95% certainty . Different explanations for the high wet-lab failure rate can be proposed. Reaction conditions could be sub-optimal. Although the most critical factors known to affect PCR efficiency were taken into account during assay design, it is currently still difficult to accurately simulate reaction dynamics in silico. Another explanation includes unanticipated variation, such as polymorphisms, present in the primer sites. This could be confirmed for some failed assays – e.g. TcSNP90, TcSNP702 and TcSNP1439 – through targeted sequencing of the primer annealing sites. Variation in primer annealing sites has been shown to impede proper binding of a primer to its target, thus blocking or hampering efficient amplification, which could also be observed with the ASP-SNP genotyping method30. Since variation in T. cacao is less well characterized, this feature is most likely the major cause for some of the DMAS-qPCR failures.DMAS-qPCR design was attempted for 102 SNPs, which failed for 10 markers. These failures could be attributed to the setup of DMAS-qPCR requiring allele-specific primers to align with their 3′ terminal nucleotide on the SNP of interest. This leaves little room to reposition the oligo, thus only its length, desired annealing temperature and GC content can be adjusted. Secondly, the cacao genome is less well annotated, only two genome builds are currently availableHOM/HET ≥5) for most assays was sufficient to allow accurate genotyping. Evaluation of DMAS-qPCR-based genotyping accuracy was assessed by comparison with reference genotyping calls in corresponding accessions. Concordance of all tested genotyping models with reference calls was very high (88.47% on average), while overlap between the three models exceeded 97.60%. This shows genotyping efficiency and accuracy is independent of the classification method , allowing automated calling approaches to streamline and simplify genotype calling. Discrepancies between DMAS-qPCR calls and reference genotype status could be explained by the uncertainty and inaccuracy associated with any experimental method, including DMAS-qPCR, and potential errors that could have slipped in ICGD – we have already identified duplicates in this repository as have others in the same or different collections and in greater amounts40. The level of inaccuracy associated with DMAS-qPCR can only be tested by applying this method on a sufficiently large sample population, and comparing the calls with results generated using multiple different techniques . The DMAS-qPCR results have shown to be independent from technical variations however, specificity could be increased by optimizing the additional internal mismatch, as shown by Hirotsu et al.30.Quality assessment and comparison of genotype calls obtained with the final set of 42 DMAS-qPCR showed the high reproducibility and robustness of the method, having a 98.05% overall efficiency. Global standard deviation for all assays across all samples was low, while discriminating power . It appeared some of the ICGD calls were called on the reverse strand, while DMAS-qPCR results were called on the forward strand. This is most likely caused by the fact that the current ICGD data was generated using EST libraries, and ICGD genotype status thus depends on the strand from which the gene was transcribed. More extensive annotation  of genotype status in online repositories or adoption of common reporting guidelines  could prove helpful to prevent such discrepancies in future studies. As a start, the data from this study will be incorporated into the ICGD database to serve as an additional and independent source of genotype information for the corresponding samples.41. Mislabeling and the verification of genetic identity from introduced as well conserved germplasm is an acute problem, as can be seen in this study and different publications over the last ten years42. Despite the fact that different applications and strategies on tackling cacao mislabeling have been provided by Turnbull et al.43, it still seems challenging to deploy these into practice. This underscores the need for accessible genotyping methods and off-type detection such as the one described in this paper. Introducing these guidelines to minimize mislabeling would facilitate and substantiate cacao research, management and breeding, by preventing the spread of undesired traits and guiding the choice of potential parents to minimize inbreeding9. DMAS-qPCR would allow straightforward and cost-efficient genotyping in the country of origin in a more routinely manner, thereby contributing to a more profound insight into the genomic identity of the cultured cacao varieties. Although a limited set of genotyping markers would improve adoption of this method by laboratories in service of local farms, a larger set is bound to better reflect the genomic reality and improve genotyping accuracy. So it is important to find the sweet spot between the marker set size and genotyping power. More studies as the one presented here will allow for better management of any material held in plant collections. Nevertheless, it should be taken into account that this method does not allow multiplex analysis, in contrast to TaqMan based assays. Furthermore, successful design of DMAS qPCR assays does not guarantee successful genotype discrimination, we have tested 102 assays resulting in a 54% overall failure rate.Comparison of DMAS-qPCR results with publicly available status revealed 15.38% off-types in cacao accessions retrieved from CEFORTT and ICQC. This is undoubtedly less than the suspected level of mislabeling (15 to 44%) in germplasm collections worldwideet al. (2013) and Livingstone et al. (2011), 26 SNPs would be adequate to uniquely identify a cacao clone in a population of 84 accessions and 19 clones, respectively. By selecting the most informative markers and analyzing unique and matching genotypes, it could be shown that a limited set of 13 markers is sufficient to achieve maximum differentiation in this population (n = 65). To obtain a more global insight in the genetic diversity and informativeness of these SNPs, more genotypes of different origins should be analyzed simultaneously. To further validate the applicability of the described DMAS-qPCR SNP assay in genetic diversity studies, analysis using PCoA and STRUCTURE44 is recommended, next to descriptive analysis by means of GenAIEx v6.5 to gain insights in marker as well as population characteristics32.According to Ji In conclusion, we have developed a robust and accurate method for cacao genotype identification using a limited set of SNPs. It can be applied to a wide range of genetic diversity studies, either with or without the inclusion of additional markers. The ease of use and cost-efficiency of the method – without the need of specialized instruments – can contribute to the adoption of routine-based genotyping to prevent mislabeling in germplasm collections and select optimal breeding parents in cacao and other organisms. The described method can easily be implemented in any molecular biology lab in the context of genotyping, genetic diversity studies, parentage analysis, mutation detection and to facilitate gene mapping and marker-assisted selection for breeding purposes.et al.41 and seven Trinitario genotypes, were obtained from CEFORTT  and ICQC   was freeze-dried upon arrival  using the ALPHA 1–2 LDplus instrument (Christ) and further crushed with liquid nitrogen, before storage at −18 °C.Leaves from 65 international cacao reference accessions, representing the ten genetic groups of Motamayor ® Spin Plant Minikit (Stratec). The protocol was executed according to the suggestions of the manufacturer, with following adaptations: (1) the addition and incubation of RNase A  for 5 minutes at room temperature after transferring the lysis solution to the Prefilter before adding binding buffer A and (2) two consecutive elution steps of 50 µL instead of a single 100 µL elution. After extraction, DNA was qualitatively and quantitatively analyzed using the Nanodrop 1000 (Thermo Scientific) and Quantus™ Fluorometer (Promega), respectively. All samples were normalized to 0.5 ng/µL.Genomic DNA was isolated from lyophilized and crushed cacao leaf material with the Invisorb36, of which 12 have been associated with disease resistance27  was introduced to increase genotype discrimination power29 . In brief, each 6 µL reaction contained 1 µL common primer (1.25 µM), 1 µL AS primer  (1.25 µM), 3 µL SsoAdvanced SYBR Green Supermix (2×) (Bio-Rad) and 1 µL template DNA (0.5 ng/µL). The following cycling parameters were used for all assays: (1) 2 min at 95 °C, (2) 44 cycles of 5 s at 95 °C, 30 s at 60 °C and 1 s at 72 °C. After amplification, Cq values were collected from the LightCycler® 480 software to assess SNP genotype status by means of scatter and difference plots, as described by Lefever et al.29.DMAS-qPCR assays were designed for only 102 SNPs, as 13 failed to map, using the GCA_00403535.1 cacao genome build (Matina 1–6) and the in-house developed primerXL web toolFor SNPs without annotation in ICGD, a reference genotype status in the samples was determined by means of SBG using a MiSeq instrument (Illumina). In brief, primers surrounding the SNPs were designed using primerXL, followed by PCR in the samples of interest. After library preparation and sequencing, the reads were aligned to the Matina 1–6 genome using the Burrows-Wheeler aligner (BWA). Reads were sorted and indexed with SAMtools and duplicates removed using Picard. Finally, variants in the aligned reads were identified using the Genome Analysis ToolKit (GATK). Through variant allele frequency (VAF) analysis, the ratio of the number of reads harboring the variant and the total read count are calculated. Subsequently, homozygous (~100% or ~0% VAF) and heterozygous (~50% VAF) SNPs could be differentiated,32. Nucleotides were given the following aliases in the analyses: A = 1, C = 2, G = 3, T = 4 and missing = 0. The program allows the computation of the mAF, MAF (1 − mAF), I, Ho, He, F, PIDsib and the analysis of matching and unique genotypes, amongst others. Output of the analysis was used to determine the minimum SNP set required to achieve maximum genotype power, sensitivity and specificity. Through pair-wise multi-locus matching, duplicates  and off-types (homonymous mislabeling – accessions with different genotypes but same name) could be identified24. Three mismatches between DMAS-qPCR results and reference status were permitted to account for genotyping errors in each analyzed cacao accession.GenAIEx v6.5 was used to determine key descriptive statistics for the genetic markers tested on the available cacao populationSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Supplementary Dataset 4Supplementary Dataset 5Supplementary Dataset 6


Baoyuan decoction (BYD), a traditional representative formula, has a long usage history in the treatment of cardiovascular diseases. Since the hyperlipidemia-induced dysfunction of erythrocyte is one of the most important causes of cardiovascular diseases, the improving effects of BYD against high-fat diet (HFD) induced the physiological and physical function of the erythrocytic injury and the potential mechanisms were deeply researched in this study. After 6 weeks of drug treatment, all doses of BYD had significantly decreased the lipid peroxidation in plasma of HFD-induced ApoE−/− mice, even if it had not improved the lipid levels. Then, the erythrocyte-related experimental results showed that BYD had reduced erythrocyte osmotic fragility, stabilized erythrocyte membrane skeleton protein 4.2, and reformed the erythrocyte morphological changes by decreasing erythrocyte membrane lipid peroxidation levels. This study demonstrated that BYD may ameliorate the physiological and physical function of erythrocyte in hyperlipidemic mice through the antioxidant effect on erythrocyte membranes. Hyperlipidemia as a kind of abnormal lipid metabolism in the blood is one of the most common and important risk factors in the development of atherosclerosis and the resulting cardiovascular diseases . DysfuncBaoyuan decoction (BYD) is a traditional representative formula, which is composed of Panax ginseng C. A. Mey. (Chinese name: Renshen), Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao (Huangqi), Glycyrrhiza uralensis Fisch (Zhigancao), and Cinnamomum cassia Presl (Rougui). Our previous researches proved that the clarification of the chemical composition of BYD was very complex [−/− mice and to provide novel information for the clinical application and future studies on the mechanisms of BYD. complex –8. Up to complex , 10. How−/− mice on a C57BL/6J background (SPF grade), weighing 18~22 g, were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (license no. SCXK (Jing), 2016-0011). All mice were raised in the barrier environment (room temperature of 24 ± 1°C and relative humidity of 50 ± 1% with a 12 h day/night cycle) and adaptability fed 1 week. Then animals were randomly divided into 5 groups (n = 6~9): chow diet control group (CG), HFD-induced model group (HG), the low and high doses of BYD-treated group , and ezetimibe -treated group (EG). According to the daily dose of the adult, mice assigned to BYD-L, BYD-H, and EG groups were orally administrated 150 and 300 mg/kg/day of BYD and 10 mg/kg/day of ezetimibe. In addition, in the control group, mice were fed with chow diet; the other groups of mice were fed high-fat diet (HFD) to establish the model of hyperlipidemic mice. The HFD contained 89.8% chow diet, 0.2% cholesterol, and 10% fat. BYD was administered orally by gavage for 6 weeks while maintaining HFD. All animal experiments have followed the guidelines of the ethics committee of Beijing University of Chinese Medicine. And the experimental protocol was approved by the medical animal experiment ethics committee of Beijing University of Chinese Medicine.Male ApoEFour crude materials of BYD were purchased from Anguo TCM market  and were authenticated by Prof. Pengfei Tu. It was prepared by combining astragalus roots, ginseng, liquorice, and cinnamon with a ratio of 6 : 2 : 2 : 1. The extraction method and chemical profile were performed according to the previous researches [All mice underwent fasting for 6 hours before collecting blood samples. The blood samples were collected from the retro-orbital sinus and anticoagulated by heparin, centrifuged at 3000 g for 10 minutes at 4°C. According to the instructions of the kit  manufacturers, plasma levels of total cholesterol (TC) and triglyceride (TG) were measured using the GPO-PAP method. The polyethylene glycol (PEG) precipitation method was used for determining high-density lipoprotein cholesterol (HDLC). The plasma MDA levels were determined by the commercial assay kit .μL of anticoagulant whole blood was mixed with 200 μL of each dilution of phosphate buffered solution (PBS) and incubated for 1 hour at room temperature. After blood samples were centrifuged at 3000 g for 6 minutes, the optical density of supernatant was determined at 540 nm. The complete hemolytic erythrocyte sample, in distilled water, was used as the positive control, and the nonhemolytic erythrocyte samples under PBS of normal osmotic pressure were used as the negative control [2  control . The hemWhole blood samples were centrifuged at 3000 g for 10 minutes at 4°C to collect the lower erythrocyte, discard plasma, and buffy coats . Each saErythrocyte membrane lipids were extracted according to the previous studies . A certaThe quantified membrane protein was separated by SDS-PAGE and transferred to the polyvinylidene fluoride membrane . After being blocked with 5% skim milk for 1.5 hours at room temperature, blots were incubated overnight at 4°C with the following primary antibodies: anti-4-HNE  ; anti-SOD2 (superoxide dismutase2) ; anti-SOD3 (superoxide dismutase3) ; anti-4.1R ; anti-p55 CDC ; and anti-Na, K-ATPase . After that, the membranes were incubated with HRP-conjugated secondary antibody at 4°C for 4 hours. The protein bands were visualized using ECL reagents  and were quantified with ImageJ software.Fresh erythrocytes were fixed in the 2.5% glutaraldehyde solution at 4°C for 2 hours, rinsed with 0.1 M PBS 3 times. Then samples were dehydrated through graded ethanol , replaced with isoamyl acetate, and dripped onto the clean glass slide. After drying, carbon gold was sprayed to observe erythrocyte morphology under scanning electron microscopy (SEM) .t-test for two groups as appropriate. All statistical analyses were performed with Graphpad Prism 6.02 . P < 0.05 was considered statistically significant.The data were presented as means ± SEM. One-way ANOVA was used to compare the significant differences for multiple comparisons, followed by Student's −/− mice on HFD were treated with vehicle (0.5% CMCNa), ezetimibe (10 mg/kg/day), or BYD (150 or 300 mg/kg/day) for 6 weeks. Ezetimibe is an inhibitor of cholesterol absorption with cholesterol-lowering effect [P < 0.01) and non-HDLC (P < 0.01) levels almost 3 times (data not shown). After 6 weeks of drug treatment, no obvious toxicity of all doses of BYD and ezetimibe was observed. As for the lipid levels, the treatment of these hyperlipidemic mice with ezetimibe by oral administration resulted in a remarkable decrease in plasma TC, TG, HDLC, and non-HDLC levels  and VLDL (very low-density lipoprotein) may stimulate leukocytes to produce ROS and then overload the antioxidant system [Since erythrocyte membranes are rich in PUFA and hence are potential targets for oxidative modification, we researched the degree of lipid peroxidation in the following studies. Among the lipid peroxidation products, 4-HNE represents one of the most bioactive and well-studied lipid alkenals . Then wet system . The oxit system . SOD2 ist system . There wt system . Our stuIn the present study, the changes in erythrocyte morphology of hyperlipidemic mice were also worthy of our concern. Erythrocyte morphology is a crucial parameter in the field of rheology to maintain the body's homeostasis and vulnerability to many factors, including osmotic fragility, the composition of erythrocyte membrane skeleton protein, and lipid peroxidation induced by oxidative stress , 44. We −/− mice. Further study on BYD treatment had performed its improvement on erythrocyte osmotic fragility, membrane skeleton protein, and morphological changes by inhibiting lipid peroxidation of erythrocyte membranes. Therefore, we deemed that the potential mechanism of BYD in improving the physiological and physical function of erythrocyte is through its antioxidant effect.Since BYD has a curative effect and high safety in the treatment of heart failure, improvement of erythrocyte function was generally considered as one of the factors involved. Through the data accumulated from our research, we believed that hyperlipidemia may cause the abnormalities of erythrocyte in ApoE


Salmonella and Campylobacter are important gastroenteric pathogens. Arcobacter butzleri is an emerging enteric pathogen. Data on the frequencies of these poultry-associated pathogens on meat products sold in sub-Saharan Africa are scarce. This study aimed to analyze the frequency of Salmonella, Campylobacter, and Arcobacter antibiotic resistance and underlying mechanisms of resistance to fluoroquinolones in locally produced and imported poultry sold in urban Ghana. Chicken meat was collected and cultured on standard media. Bacterial strains were identified by biochemical methods and by mass spectrometry. Antibiotic susceptibility was tested by disk diffusion. Ciprofloxacin-resistant strains were assessed for molecular mechanisms of resistance. Among 200 samples, comprising 34% (n = 68) from the Ghanaian poultry industry and 66% (n = 132) from imports, 9% (n = 17) contained Salmonella, 11% (n = 22) Campylobacter, and 26.5% (n = 53) A. butzleri. Higher overall contamination frequencies were found in local meat. Most common Salmonella serovars identified were Kentucky  and Poona . Campylobacter were C. coli  and C. jejuni . Resistance to fluoroquinolones was high with 63% (n = 10), 75% (n = 15), and 52% (n = 25) in Salmonella, Campylobacter, and Arcobacter, respectively. A link between Salmonella Kentucky [sequence type (ST) 198] and a ciprofloxacin minimum inhibitory concentration of 16 μg/mL was found. Salmonella Poona-ST308 revealed transferable qnrB2 fluoroquinolone resistance genes. Markedly high frequencies of resistant Salmonella, Campylobacter, and Arcobacter predominant in locally produced meat represent a probable transmission reservoir for human infections. These findings highlight the need for implementation of surveillance systems that focus on food hygiene, use of antibiotics in animal husbandry, and continuous monitoring of the quality of meat products from imports. The steadily increasing global trade of meat products between developing and industrialized countries bears the risk of spreading the animal-associated pathogens across different countries and continents. This threat is mainly a problem in countries without surveillance systems where pathogens can be imported or exported undetected. A high risk is posed to SSA regions, where data on the frequency of the bacteria in both locally produced and imported poultry are scarce and poorly monitored.Globally, 1 Salmonella and Campylobacter isolated from humans and other animals with reduced ciprofloxacin susceptibility and multidrug-resistance profiles have been rapidly emerging in recent years, with considerably high reported prevalences from Asia, Europe, and Africa  and from imports (frozen meat) were collected in Kumasi, the capital of the Ashanti region of Ghana. Sampling was based on availability. In total, 75 supermarkets were identified for sample collection of imported frozen meat, which was kept in freezers. Each supermarket was sampled once and the origin of the meat from imports was retrieved from packaging. If meat from different countries was available, one piece from each country was sampled per supermarket. Chickens originating from small local farms were purchased live from 19 open markets. Each merchant was visited on one occasion only. The chickens, which had been kept in cages, were slaughtered on purchase. For each sample (fresh and frozen), ∼15 g of leg, including skin, was collected and placed into a sterile homogenizing plastic bag and returned to the laboratory at 4–8°C in a cool box.Salmonella , Campylobacter , and Arcobacter . The selenite broth was incubated with loose tops at 35–37°C for 18–24 h in normal atmosphere. Preston- and Arcobacter enrichment broths were incubated at 35–37°C for 18–24 h with loose tops under microaerophilic conditions . The selenite broth was subsequently cultured on Xylose Lysine Deoxycholate agar (Oxoid) and incubated at 35–37°C for 18–24 h in normal atmosphere. Salmonella strains were identified by a latex agglutination test (Oxoid) and by the analytical profile index test . Preston No. 2 broth was further cultured on selective Karmali agar (Oxoid), using a filter technique as described by Atabay et al.  with 5% sheep blood was used for culture of the broth using the filter technique as described for Campylobacter. Plates were incubated for 18–24 h at 30°C in microaerophilic conditions  and if negative, for 4 more days. Preliminary confirmation of Campylobacter and Arcobacter was done by oxidase test and Gram staining. All bacterial strains were sent to Germany on dry ice where species identification was confirmed by mass spectrometry . Salmonella serotyping was performed following the White–Kauffmann–Le Minor scheme . Quality control of susceptibility testing was performed according to EUCAST (QC table v.5). Salmonella were tested for ampicillin, amoxicillin/clavulanic acid, tetracycline, chloramphenicol, cefotaxime, ceftazidime, trimethoprim/sulfamethoxazole, and ciprofloxacin. Salmonella isolates exhibiting resistance to ampicillin, trimethoprim/sulfamethoxazole, and chloramphenicol were defined as multidrug resistant  and interpreted as ciprofloxacin resistant with an MIC >0.06 μg/mL. Salmonella isolates were screened for extended-spectrum beta-lactamase production (ESBL) using cefotaxime and ceftazidime disks.Antibiotic susceptibility was tested using locally available antibiotics by the disk diffusion method and interpreted following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines  following the manufacturer's instructions. Salmonella exhibiting ciprofloxacin resistance were further screened for plasmid-located genes , contributing to aminoglycoside and fluoroquinolone resistance  were screened for point mutations  were determined by multilocus sequence typing according to a previously published protocol (http://enterobase.warwick.ac.uk).Campylobacter were assessed for point mutations in the gyrA gene as described by Tang et al. (A. butzleri isolates could be grown. All isolates were examined for point mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene using the amplification and sequencing primers described by Abdelbaqi et al.  along with their 95% confidence intervals (CIs) were calculated to show associations between dichotomous variables. Missing values were excluded from the analysis; hence, in some calculations the denominator differs. All analyses were conducted using Stata Statistical Software 14 .n = 4; 11%) and the United States  was also collected from open markets. The majority of imported meat products originated from the Netherlands , the United States , and Brazil . Other countries included Belgium , Germany , Poland , Ireland , and Turkey . Countries of origin were not traceable for 11% (n/N = 15/132) of the samples.In total, 200 chicken meat samples, comprising 68 (34%) samples from the local Ghanaian poultry industry and 132 (66%) from imports, were collected within Kumasi. The Ghanaian meat was exclusively sold at open markets, whereas imported meat was primarily sold in supermarkets/cold stores. Only frozen meat from the Netherlands (n = 17) were positive for Salmonella, 11% (n = 22) for Campylobacter, and 27% (n = 53) for Arcobacter. In local meat, Salmonella was detected in 19% (n/N = 13/68), Campylobacter in 28% (n/N = 19/68), and Arcobacter in 62% (n/N = 42/68) of the samples. In contrast, in imported meat, Salmonella, Campylobacter, and Arcobacter were found in 3% (n/N = 4/132), 2% (n/N = 3/132), and 8% (n/N = 11/132), respectively. Accordingly, there was a significant difference between locally sourced compared with imported meat products with higher frequencies in local meat for Salmonella , Campylobacter , and Arcobacter . Multiple pathogens (pathogens under investigation found simultaneously) were primarily observed in local meat wherein 18 of 68 (26%) samples were concurrently positive with two different bacteria and 3 of 68 (4%) meat samples contained three bacterial genera. The country of origin of bacterial isolates from imported meat (considering countries with at least two samples) is summarized in Salmonella serovars identified included Kentucky , Poona , Agama , Enteritidis , Kaapstad , and Ajiobo . One serovar could not be identified due to loss of the isolate during culturing. Campylobacter comprised the species C. coli  and C. jejuni . Three Campylobacter spp. were lost during culturing, the species could not be identified. All Arcobacter spp.  were A. butzleri.Isolates: of all collected samples, 9%  followed by ciprofloxacin  and ampicillin . Three ST198 Salmonella Kentucky isolates with a ciprofloxacin MIC of 16 μg/mL showed several mutations, including double mutations in gyrA and in parC  and tetracycline . Multidrug resistance was detected in four strains  isolates were observed with no measurable zones of inhibition (ø < 6 mm) for ciprofloxacin. Although only single Thr86IIe gyrA mutations were detected in C. coli, two C. jejuni isolates revealed double or triple point mutations  and encompassing 82% among the samples from imports. Although low temperatures seem to have no significant effect on the viability of certain bacterial genera such as, for example, on Escherichia as outlined in a study by Eibach et al.  are the main countries for poultry exports, having contributed to 66% of poultry imports to Ghana in 2016 . Salmonella Kentucky ST198 first emerged in Egypt and has since been associated with high-level ciprofloxacin resistance (Raufu et al., et al., Salmonella Kentucky strain but the ST was not investigated (Andoh et al., Salmonella Poona ST308 with plasmid-mediated resistance to ciprofloxacin. One isolate of this ST was recently reported from a human infection in Ghana (Kudirkiene et al., There was a significant contamination of Ghanaian poultry with the Salmonella, Campylobacter, and Arcobacter predominantly in locally produced meat represent a potential transmission reservoir for human infections. Also, the observed overall high level of resistance seen in locally available antibiotics, in particular, to fluoroquinolones is concerning. This level of resistance urges the implementation of strict surveillance systems that focus on food hygiene as well as on the use of antibiotics in animal farming. Furthermore, health authorities need to inform the public about emerging resistant strains in the communities such as the highly ciprofloxacin-resistant Salmonella Kentucky-ST198 and Salmonella Poona ST308; molecular investigations are the essential precondition for this. Surveillance programs also need to be coordinated with controlling the microbiological quality of imported meat and the food production end consumer chain by increasing awareness on food hygiene in the population.


Dystroglycanopathies, which are caused by reduced glycosylation of alpha-dystroglycan, are a heterogeneous group of neurodegenerative disorders characterized by variable brain and skeletal muscle involvement. Muscle-eye-brain disease (or muscular dystrophy-dystroglycanopathy type 3 A) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities, and lissencephaly.POMGNT1 gene (transcript NM_001243766.1): c.1539 + 1G > A and c.385C > T.We report clinical and genetic characteristics of a 6-year-old boy affected by muscular dystrophy-dystroglycanopathy. He has severe a delay in psychomotor and speech development, muscle hypotony, congenital myopia, partial atrophy of the optic nerve disc, increased level of creatine kinase, primary-muscle lesion, polymicrogyria, ventriculomegaly, hypoplasia of the corpus callosum, cysts of the cerebellum. Exome sequencing revealed compound heterozygous mutations in The present case report shows diagnostic algorithm step by step and helps better understand the clinical and genetic features of congenital muscular dystrophy. Muscle biopsies in patients with CMDs show considerable variability but always exhibit a characteristic pattern of dystrophic lesions. Disease typically manifests at birth or during the first year of life. The incidence of CMD in populations is not sufficiently known, there are small number of studies reporting incidence of CMD in several countries and regions: western Sweden 6.3 × 1005, NorthProgress in the field of molecular genetics allowed to determine genes whose mutated forms are responsible for CMDs. Identification of genetic cause and accurate diagnosis improve supportive therapy and family planning. But currently there is no specific treatment for this group of diseases. The most common CMDs are Ulrich congenital muscular dystrophy  due to the pathology of collagen VI (mutations are found in three genes) .POMT1, POMT2, POMGNT1, FKTN, FKRP, LARGE, DPM2, DPM3, DOLK, ISPD, GTDC2, TMEM5, B3GALNT2, B3GNT1, GMPPB, DPM1 [Dystroglycanopathies are inherited in an autosomal recessive manner and characterized by brain and eye anomalies . ClinicaPB, DPM1 . There iPB, DPM1 . But thi: linkage between the extracellular matrix and the membrane cytoskeleton in muscle fibers [Dystroglycan is composed of two subunits (α and β) and is an essential component of the dystrophin-glycoprotein complexe fibers . Destruce fibers .Clinical findings of dystroglycanopathy include hypotonia starting in early infancy, marked mental and motor retardation, seizures, eye abnormalities . The following CNS abnormalities are observed: pachygyria, polymicrogyria, lissencephaly, hydrocephalus, ventriculomegaly, agenesis of the corpus callosum, hypoplasia of the brain stem, hypoplasia, dysplasia or cysts of cerebellum . The mosCMD diagnosis can be challenging, and in some cases patients with muscular dystrophy received therapy to treat perinatal CNS damage or other neurological diseases. Genetic testing allows to confirm CMD diagnosis and identify the disease at an early stage . In thisA 6-year-old boy was born of the second pregnancy and first childbirth to healthy nonconsanguineous parents. The first pregnancy ended in an early miscarriage. Family history is not burdened.Pregnancy was with threatened spontaneous interruption. He was delivered by scheduled Cesarean section due to high myopia. His birth weight was 3650 g and height was 52 cm. Apgar scores were 8 and 9 at 1 and 5 min respectively. He was noted to have hypotonia at birth.His motor milestones were delayed: held head by 7 months, rolled from back to side at 8 months, put into sitting position by 24 months, walked with support at 2,5 years and walked independently at 4 years. His speech development was delayed: at the age of 6 years he had no words.At 8 month he was hospitalized to Scientific and Practical Centre of Pediatric psychoneurology. Brain computed tomography revealed signs of leukodystrophy and cortical atrophy of frontal, parietal and temporal areas. Radiological studies (neuroimaging) have never been performed until the age of 8 months.At 13 months he was admitted to the National Medical Research Centre for Children’s Health and was monitored for the next 5 years.At admission in 13 months brain MRI revealed signs of leukodystrophy, lissencephaly of left occipital lobe, polymicrogyria of both frontal lobes, secondary ventriculomegaly, brain atrophic changes with enlargement of sub-arachnoid spaces. Biochemical analysis showed creatine kinase (CK) level to be elevated to 2024 U/L , alanine aminotransferase (ALT) to 59 U/L , aspartate aminotransferase (AST) to 82 U/L  and lactate dehydrogenase (LDH) level to 318 U/L . On electromyographic examination, signs of primary muscle lesion have been identified.At admission in 6 years weight was 21 kg, height was 113 cm, head circumference was 53 cm. Convergent strabismus, insufficiency of convergence and accommodation were noted. He had hypotonia, distal more than proximal. Trunk ataxia, walking on his toes with an atactic component, flat valgus foot were noted. Patient reacted favorably to examination. He can maintain short-term eye contact; speech is misunderstood. The face is hypomimic. In active speech there are long vocalized sounds, rarely - simple babbling syllables, no words. Behavioral features are manifested by periodic fading with subsequent rapidity of breathing and stereotyped swings of hands , expansion of retrocerebellar cistern, symmetrical expansion of the ventricular system, pachygyria-polymicrogyria of cerebral hemispheres with preservation of the occipital lobe. Increased intensity of MR signal was observed in T2 and T2 FLAIR images. Decreased intensity of MR signal was observed in T1 image. In the cerebellum multiple small cysts, hypoplasia of the corpus callosum and pons with the extension of the anterior and covering cisterns are revealed , ALT to 70,4 U/L , AST to 62 U/L  and LDH level to 503 U/L . Other blood and urine indicators were within normal limits. Patient level of CK was increased 10–15 times and for two years decreased from 3126 (3 years 4 months) to 1986 (at 5 years 2 months). The level of CK-MB was also increased - 61.1 .On electroneuromyographic examination (ENMG), signs of primary muscle lesion have been identified. Motor unit potentials (MUP) were short in duration and low in amplitude. The number of polyphasic MUP has increased. Spontaneous activity in each test muscle was determined.Electroencephalography (EEG) did not reveal epileptiform activity.The electrocardiogram (ECG) showed a vertical position of the electrical heart axis, mild bradycardia and arrhythmia, nonspecific intraventricular block, early repolarization syndrome of the ventricles.A standard karyotype was normal 46, XY. Tandem mass spectrometry did not reveal any changes in the plasma level of amino acids and acylcarnitines.Genotek Ltd. did clinical exome sequencing. Genomic DNA from peripheral blood sample was extracted using QIAamp DNA Mini Kit (Qiagen) according to manufacturerʼs protocol. DNA libraries were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs) with adapters for sequencing on Illumina platform according to manufacturer’s protocol. We used SureSelect XT2 (Agilent Technologies) for target enrichment. Enriched samples were sequenced on Illumina HiSeq 2500 using pair-end 100 base pairs reads. After sequencing, we trimmed 3′-nucleotides with read quality below 10 using Cutadapt . Raw reaPOMGNT1 pathogenic variants identified by exome sequencing. For labeling amplicons with fluorescent labels, BigDye Terminator Cycle Sequencing Kit v3.1 (Thermo Fisher Scientific) was used. Sanger sequencing was performed on ABI PRISM 3500 Genetic Analyzer (Applied Biosystems) according to manufacturerʼs protocol.Sanger sequencing confirmed After exome sequencing, patient was commenced on muscle relaxants and anticonvulsants. His condition is stable.POMGNT1 gene were found by NGS analysis and confirmed by Sanger sequencing. First mutation c.1539 + 1G > A present in the father and second mutation c.385C > T present in the mother  is a substitution in intron 17, which altered the conserved GT splicing donor sequence to AT. This mutation caused read-through of intronic sequences, resulting in the introduction of a premature termination codon. The mutation also caused skipping of the upstream exon 17, resulting in the deletion of 42 amino acids (p.Leu472_His513del) [Mutation c.385C > T (chr1:46661719) is a missense mutation results in arginine substitution for tryptophan at position 129 (p.Arg129Trp).FKTN gene . According to the UCSC Genome Browser [FKTN are the main cause of the Fukuyama congenital muscular dystrophy. For all of these reasons, we defined this mutation as non-pathogenic.Exome sequencing also revealed heterozygous deletion in the first intron of  Browser , this deDiagnostic algorithm for the detection of CMDs contains following steps: assessment of presence or absence of CNS, muscle and vision damage, as well as evaluation of phenotypic features and the plasma level of CK. In the case of CNS involvement, brain MRI is performed. The final step is a molecular study to confirm the diagnosis .Ulrich’s disease is the most common form of CMD in Europe but does not cause CNS anomalies . In our Dystroglycanopathy is associated with an abnormal α-dystroglycan function. α-dystroglycan is one of the subunits of dystroglycan and central component responsible for the stability of the myocyte membrane . α-dystrSecondary dystroglycanopathies include MEB, WWS syndrome and syndrome FCMD. Genetic analysis was conducted to establish an accurate diagnosis among the variants described.FKTN are the main cause of the Fukuyama congenital muscular dystrophy. FKTN is located on chromosome 9q31 and encodes membrane protein that is believed to have a function similar to glycosyltransferase and may be involved in protein modification after the phosphorylation process [FKTN was analyzed by NGS. A nonpathogenic heterozygous deletion in the first intron of this gene was detected . Based on clinical signs of the patient  and absence of pathogenic mutations in the FKTN gene, the diagnosis of Fukuyama syndrome was excluded.FCMD syndrome (OMIM 253800) is an autosomal recessive disease characterized by severe muscular dystrophy in combination with CNS lesions  . Clinica process . The codPOMGNT1. All described POMGNT1 mutations lead to complete loss of enzymatic activity of the POMGNT1 protein, responsible for the modification of α-dystroglycan after the phosphorylation process. Normally, it catalyzes the addition of the N-acetylglucosamine residue to O-linked mannose [POMGNT1 was analyzed by NGS. Two mutations were found, in intron 17 (c.1539 + 1G > A) and in exon 5 (c.385C > T). Mutation c.1539 + 1G > A is found in splice site and is registered in the databases as pathogenic (https://www.ncbi.nlm.nih.gov/clinvar/variation/56582/). Mutation c.385C > T in the compound heterozygous state is registered with unknown effect (http://databases.lovd.nl/whole_genome/variants/0000067353#00014727).MEB disease  is an autosomal recessive disease characterized by congenital muscular dystrophy, visual impairment and defects of brain development . The average life expectancy of patients with MEB is 10 to 30 years . The mos mannose . The codPOMGNT1 can lead not only to MEB disease but also to the development of Walker-Warburg syndrome associated with congenital muscular dystrophy, violation of neuronal migration and various visual disorders [Mutations in the isorders , 52. Howisorders , 54. BasThus, based on clinical features , increased level of CK, ENMG data (primary-muscle lesion), MRI data  and results of molecular genetic analysis patient was diagnosed: сongenital muscular dystrophy-dystroglycanopathy, type 3A .Identification of disease-causing mutations can provide a proper molecular diagnosis of dystroglycanopathies, which will help to consider more reliable therapeutic approaches and develop information about prognosis.Currently, there is no specific treatment for dystroglycanopathies. Treatment of such patients focuses on decreasing the severity of symptoms. However, with the limited available therapeutic option for most of the patients, molecular diagnosis might enable geneticists and pediatricians to provide informative genetic counseling, perform prenatal diagnosis, and implement prevention measures for such patients. Therefore, genetic counseling should be recommended to all individuals with dystroglycanopathies and families for their next pregnancies.POMGNT1 gene were identified in a compound heterozygous boy with muscular dystrophy-dystroglycanopathy. We described detailed algorithm for correct differential diagnosis of dystroglycanopathies: MEB syndrome, WWS syndrome and FCMD. Our study may help to establish an appropriate genetic counseling and prenatal diagnosis for individuals at the high risk of dystroglycanopathy.In summary, two mutations of the


In anaerobic treatment of complex wastewater containing fat, oils, and grease, high long-chain fatty acid (LCFA) concentrations may inhibit microbial communities, particularly those of methanogens. Here, we investigated if anaerobic degradation of LCFAs can proceed when methanogens are inhibited and in the absence of typical external electron acceptors, such as nitrate, iron, or sulfate. Inhibition studies were performed with the methanogenic inhibitor 2-bromoethanesulfonate (BrES). We noticed that, after autoclaving, BrES underwent partial hydrolysis and turned out to be a mixture of two sulfonates (BrES and isethionate). We found out that LCFA conversion proceeded faster in the assays where methanogenesis was inhibited, and that it was dependent on the utilization of isethionate. In this study, we report LCFA degradation coupled to desulfonation. Our results also showed that BrES can be utilized by anaerobic bacteria. 18:1 LCFA)-degrading anaerobic enrichment cultures, one methanogenic (ME) and another in which methanogenesis was inhibited (IE). Inhibition of methanogens was attained by adding a solution of 2-bromoethanesulfonate (BrES), which turned out to consist of a mixture of BrES and isethionate. Approximately 5 times faster oleate degradation was accomplished by the IE culture compared with the ME culture. A bacterium closely related to Syntrophomonas zehnderi (99% 16S rRNA gene identity) was the main oleate degrader in both enrichments, in syntrophic relationship with hydrogenotrophic methanogens from the genera Methanobacterium and Methanoculleus (in ME culture) or with a bacterium closely related to Desulfovibrio aminophilus (in IE culture). A Desulfovibrio species was isolated, and its ability to utilize hydrogen was confirmed. This bacterium converted isethionate to acetate and sulfide, with or without hydrogen as electron donor. This bacterium also utilized BrES but only after 3 months of incubation. Our study shows that syntrophic oleate degradation can be coupled to desulfonation.Degradation of long-chain fatty acids (LCFAs) in methanogenic environments is a syntrophic process involving the activity of LCFA-degrading bacteria and hydrogen-utilizing methanogens. If methanogens are inhibited, other hydrogen scavengers are needed to achieve complete LCFA degradation. In this work, we developed two different oleate  concentrations may inhibit microbial communities, particularly those of methanogens. Here, we investigated if anaerobic degradation of LCFAs can proceed when methanogens are inhibited and in the absence of typical external electron acceptors, such as nitrate, iron, or sulfate. Inhibition studies were performed with the methanogenic inhibitor 2-bromoethanesulfonate (BrES). We noticed that, after autoclaving, BrES underwent partial hydrolysis and turned out to be a mixture of two sulfonates (BrES and isethionate). We found out that LCFA conversion proceeded faster in the assays where methanogenesis was inhibited, and that it was dependent on the utilization of isethionate. In this study, we report LCFA degradation coupled to desulfonation. Our results also showed that BrES can be utilized by anaerobic bacteria. Desulfovibrio, Desulfomicrobium, and Desulforhabdus species) are able to outcompete methanogens for hydrogen (Long-chain fatty acids (LCFAs) are found in several types of waste and wastewater, and they can be converted to methane in anaerobic bioreactors , 2. Conv3–hydrogen .Syntrophomonas species, although usually found in low abundance in these ecosystems, have been identified as key players in the conversion of LCFA, in syntrophy with hydrogenotrophic methanogens or sulfate-reducing bacteria  were converted to the corresponding saturated LCFA with two fewer carbon atoms , which was not further degraded (Conversion of LCFAs was reported in continuous bioreactors where methanogenesis was inhibited by 2-bromoethanesulfonate (BrES) and in the absence of external inorganic electron acceptors . In thatdegraded . MotivatBrES is the best-known methanogenic inhibitor and is used in several applied and fundamental studies in which methanogenic inhibition is required –20. BrES–23–To get further insight on LCFA degradation when methanogenesis is inhibited, we developed long-term oleate-degrading microbial enrichments with and without BrES, which later turned out to be a mixture of BrES and isethionate. We hypothesize that by inhibition of methanogens, oleate degradation can be coupled to homoacetogenesis (forming acetate from hydrogen and carbon dioxide) or to sulfonate reduction, since some sulfate-reducing bacteria are able to utilize sulfonates as electron donors and/or electron acceptors –31. Diff–Oleate-degrading enrichment cultures, one methanogenic (ME) and another in which methanogenesis was inhibited (IE), were obtained after five successive transfers. The two enrichments exhibited different oleate degradation rates and product profiles. The concentrations of oleate and the accumulation of the degradation products show that oleate conversion was faster in IE culture than in ME culture. Complete conversion of oleate in ME culture was achieved in approximately 113 days, while in IE culture, the same amount of oleate was degraded in 17 days .Methanobacterium beijingense, Methanobacterium formicicum, and Methanoculleus bourgensis, which are hydrogenotrophic. Typical acetoclastic methanogens  were not detected by cloning and sequencing analysis  and approximately 3 mM methane , which canalysis .The IE culture produced acetate (10.4 ± 0.4 mM) and residual amounts of hexanoate and butyrate (<1 mM) from oleate degradation . PalmitaSyntrophomonas (sharing 99% identity to the 16S rRNA gene of Syntrophomonas zehnderi strain OL-4) and Desulfovibrio (98% identity to Desulfovibrio aminophilus strain ALA-3) were present in both enrichments (Denaturing gradient gel electrophoresis (DGGE) analysis showed that bacterial diversity decreased greatly from the inoculum sludge to the final enrichment cultures . Syntropichments .Thermodynamically, complete syntrophic oleate degradation can only occur if the electrons released by oleate-degrading bacteria are captured by another organism. In the ME culture, methanogens consumed the hydrogen, but in IE culture, the final electron acceptor was not known. To investigate the possibility that BrES could serve as electron acceptor, or that homoacetogenesis took place in IE culture, attempts were made to isolate hydrogen-utilizing microorganisms in anaerobic medium containing BrES.2-CH2-SO3−) is a sulfonate with the same structure as BrES (Br-CH2-CH2-SO3−), but with the bromide ion replaced by a hydroxide ion. After one cycle of sterilization by autoclavation, 16% ± 1.5% of the bromide is released from BrES, meaning that circa 16% of BrES was hydrolyzed to isethionate (Table S1). Both sulfonates could serve as electron acceptors for the conversion of oleate with sulfide production, and the sulfate-reducing bacteria identified in the IE culture    could prDesulfovibrio was isolated after several transfers and serial dilutions of the IE culture, performed in anaerobic medium containing H2/CO2 and autoclaved BrES. To get a more reliable taxonomic identification of the isolate (here designated Desulf-BrES), the nearly complete 16S rRNA gene  was sequenced and aligned with 16S rRNA sequences from the NCBI nucleotide collection database and from the Ribosomal Database Project (RDP) database. From the 90 clones obtained, 13 different clone sequences were retrieved, which shared a minimum of 99.12% and a maximum of 99.93% identity with each other . Desulfovibrio aminophilus strain ALA-3 shared the highest identity (98.58% ± 0.14%) of the 16S rRNA gene with Desulf-BrES . Desulf-BrES probably represents a new Desulfovibrio species, since the percentage of identity is lower than 98.7%, which is the threshold to classify microorganisms as the same species .The bacterium belonging to the genus  species . Also, a2/CO2 and autoclaved BrES, the culture optical density increased to 0.140 ± 0.005, and 4.1 ± 0.1 mM acetate and 4.8 ± 0.3 mM sulfide were produced, together with the consumption of 13 ± 0.5 mM hydrogen  were utilized as electron acceptors by  acetate . However acetate . AdditioControl assays with hydrogen and carbon dioxide, but without sulfonates, did not result in growth or acetate production, indicating that culture Desulf-BrES has no homoacetogenic activity. The ability of culture Desulf-BrES to degrade oleate was tested in both the presence and absence of sulfonates, and no degradation occurred, even after prolonged incubation (data not shown).Incubation of culture Desulf-BrES with increasing concentrations of sulfonates  showed that the amount of acetate formed was directly proportional to sulfonate concentration, and that this activity was linked to microbial growth . However, only approximately 20% of the sulfonate mixture was converted to acetate and sulfide. Additional incubations with isethionate (98%) as the only carbon and energy source revealed that this compound was completely utilized by culture Desulf-BrES as electron donor and as electron acceptor . The proIn incubations with BrES sterilized by filtering (which causes no transformation of BrES to isethionate) and hydrogen , acetate was produced (up to 3.4 mM), and free bromide ion accumulated (up to 7.7 mM) in the medium . HoweverSyntrophomonas zehnderi, which is an obligate syntrophic bacterium that degrades LCFAs  than the conversion of oleate coupled to hydrogenotrophic methanogenesis (ΔG0ʹ= −182.57 kJ/reaction)  resulted in the enrichment of es LCFAs . In the radation . Indeed,S. zehnderi and Desulfovibrio sp. relationship was dependent on the simultaneous utilization of isethionate (which was present in the BrES solution) as electron acceptor by the Desulfovibrio sp. (Desulfovibrio sp. (culture Desulf-BrES) showed that hydrogen utilization only occurred concomitantly with isethionate consumption. Therefore, oleate and isethionate (the carbon moiety) served as electron donors for Syntrophomonas and Desulfovibrio species, respectively, and isethionate (the sulfonate moiety) was the final electron acceptor .The Desulfovibrio sp. with isethionate as the sole carbon and energy source showed that it was converted to acetate, sulfate, and sulfide , while D. thiosulfatigenes strain GKNTAU was reported to grow only in taurine (Desulfovibrio strain isolated in this study is the second microorganism described to utilize isethionate as electron donor and acceptor. Assays with BrES (and lactate as electron donor) were performed with Desulfovibrio desulfuricans strain IC1, but no BrES utilization was reported . ComparThe enrichment strategy allowed us to uncover the role of specific microorganisms within microbial communities degrading oleate. Under anaerobic conditions, oleate was converted to methane by syntrophic communities of acetogenic bacteria and methanogens, but when methanogenesis was inhibited, another syntrophic relationship took place. In the presence of isethionate, oleate could be completely converted to acetate by the synergistic activity of fatty acid-degrading bacteria and sulfate-reducing bacteria capable of sulfonate metabolism. Since there are several microorganisms that use sulfonates as electron acceptors, syntrophic LCFA degradation coupled to sulfonate reduction may be widespread.−1 · day−1 and a hydraulic retention time of 1 day were applied. Once acclimated, the biomass was washed with anaerobic medium and incubated in batch at 37°C over 25 days, until all of the accumulated substrate was consumed. This sludge was then used as inoculum for the development of two distinct enrichments, one methanogenic (ME) and another in which methanogenesis was inhibited (IE) . Inhibition of methanogenesis was achieved by adding 2-bromoethanesulfonate (BrES)  at a final concentration of 20 mM. BrES was sterilized by autoclaving . Incubations were done aseptically and under strict anaerobic conditions, as described elsewhere  at 1.7 × 105 Pa, and the medium was sterilized by autoclaving . Before inoculation, the medium was supplemented with salts and vitamins as described by Stams et al.  was acclimatized to LCFA in a mesophilic (37°C) anaerobic bioreactor (2.8 liters) working in continuous mode during 15 days. The feeding was a mixture of LCFA  composed of 41% oleate, 44% stearate, 14% palmitate, and 1% myristate, supplemented with macronutrients, micronutrients, and sodium bicarbonate, as described elsewhere . An orgalsewhere . The analsewhere , the heas et al.  and reduStable enrichments, ME and IE cultures, were obtained after 5 successive transfers , over a period of 1 year. LCFA, VFA, and methane were monitored during the time course of oleate degradation. Total RNA was isolated for taxonomic characterization of the active fraction of the microbial communities. The microbial communities’ dynamics were followed by 16S rRNA gene PCR-DGGE fingerprinting, and microbial composition was obtained by cloning and sequencing of 16S rRNA genes. All assays were done in triplicate.2/CO2  plus 20 mM sulfonates (approximately 84% of BrES and 16% of isethionate) . Isethionate has no known effect on methanogenic activity. It is a sulfonate that appears in the composition of some cosmetics and detergents and that can therefore end up in wastewater treatment plants. A total of 24 transfers and 3 sequential serial dilutions were performed in order to reduce microbial diversity and isolate hydrogen-consuming microorganisms. Composition of the stable enrichment culture, designated Desulf-BrES, was determined by direct sequencing of amplified partial 16S rRNA genes , and confirmed by sequencing the nearly complete 16S rRNA genes , obtained by cloning and Sanger sequencing. The purity of this culture was confirmed by microscopic observation of only one cell morphotype and by Illumina 16S rRNA sequencing, from which all sequences retrieved were assigned to Desulfovibrio sp. .A new enrichment series was set up by incubating IE culture with H2/CO2 were performed. Additional experiments were performed with increasing concentrations of sulfonates (ranging from 5 to 50 mM), maintaining as constant the concentration of hydrogen/carbon dioxide, to investigate the relationship between sulfonate concentration and the amount of acetate produced. Growth in phosphate-buffered medium containing no bicarbonate nor carbon dioxide, with sulfonates as the sole carbon and energy sources, was also tested. Incubations with oleate, with and without sulfonates, were performed to investigate the ability of the hydrogen scavengers to degrade oleate. All incubations were done in triplicate assays.Microbial growth was determined by measuring the optical density (wavelength at 600 nm). Hydrogen, VFA, and sulfide concentrations were monitored during the incubations. Control assays without HIncubations of culture Desulf-BrES with sulfonates were performed under the following conditions: (i) with 20 mM BrES sterilized by autoclaving (composed of BrES plus isethionate) and with hydrogen (55 mM) as additional electron donor; (ii) with 4 mM sodium isethionate  and with hydrogen as an additional electron donor; (iii) with 4 mM isethionate as the sole electron donor and electron acceptor; and (iv) with 20 mM BrES sterilized by filtering  and with hydrogen as additional electron donor . The optical density (600 nm) of the cultures and the concentrations of VFA, hydrogen, sulfide, bromide, and isethionate were followed over time.−1) was the carrier gas, and injector, detector, and column temperatures were set at 100, 130, and 35°C, respectively. Methane was analyzed with a gas chromatograph (GC) (Chrompack 9000) equipped with a flame ionization detector (FID) and a 2 m × 1/8 in. Chromosorb 101 (80 to 120 mesh) column. Nitrogen was the carrier gas (30 ml · min−1), and column, injector, and detector temperatures were set at 35, 110, and 220°C, respectively.Hydrogen was measured by gas chromatography by using a MolSieve column  connected to a thermal conductivity detector Bruker Scion 456 chromatograph . Argon (30 ml · minLong-chain fatty acids (LCFAs) were first extracted with dichloromethane and esterified with 2-propanol prior to separation and quantification by gas chromatograph-flame ionization detector (GC-FID), as described by Neves et al. .2) at 60°C and sulfuric acid (0.005 mol · liter−1) as mobile phase at a flow rate of 0.9 ml · min−1. VFA detection was done using a UV detector at 210 nm. Sulfide measurements were obtained using standard kits . Bromide ion concentration was determined by the phenol red colorimetric method described in the Standard Methods for the Examination of Water and Wastewater  prior to VFA, bromide, isethionate, and sulfate determination. VFA concentrations were determined by high-performance liquid chromatography  using a Chrompack column . RNA was extracted by using the commercial kit FastRNA Pro Soil-Direct kit  and following the manufacturer’s instructions. cDNA was synthetized from RNA by SuperScript III reverse transcriptase  using random primers.Taq DNA polymerase (recombinant) , for subsequent denaturing gradient gel electrophoresis (DGGE) analysis and cloning. Primer sets U968-f/1401-r (Bacteria and A109(T)-f/515-r  cells by using the Promega pGEM-T Easy vector system , as previously described (https://blast.ncbi.nlm.nih.gov), and phylogenetic assignment was confirmed with the Ribosomal Database Project (RDP) classifier . Gels containing 8% (wt/vol) polyacrylamide (37.5:1 acrylamide:bis-acrylamide) were used with a linear denaturing gradient , with 100% of denaturant, corresponding to 7 M urea and 40% (vol/vol) formamide. Electrophoresis was performed for 16 h at 85 V and 60°C in a 0.5× Tris-acetate-EDTA buffer, and DGGE gels were stained with silver nitrate . PCR proescribed . Clones assifier . NucleotLT991962 to LT991974, associated with the BioProject study accession number PRJEB25655.Samples from culture Desulf-BrES were centrifuged and frozen at −20°C in phosphate-buffered saline (PBS) buffer. Total DNA was isolated with the commercial FastDNA SPIN kit for soil  and submitted to cloning and sequencing, following the procedure described for oleate enrichment cultures but using the primer set Bac27f/Uni1492r  to ampliERX2452693 (BioProject accession number PRJEB25655).Sequencing of 16S rRNA genes by Illumina MiSeq was performed to check the purity of the culture. Clone libraries, sequencing and data analysis were performed at the Research and Testing Laboratory . The primer set 28F/388R , 50 was Desulfovibrio sp. strain (Desulf-BrES) was deposited in Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under accession number DSM 108261. Nucleotide sequences described in this study have been submitted to the European Nucleotide Archive under accession numbers LT992923 to LT992943 (BioProject study accession number PRJEB25834) and accession numbers LT991962 to LT991974 (BioProject study accession number PRJEB25655). FASTQ files were submitted to the European Nucleotide Archive under the study accession number ERX2452693 (BioProject accession number PRJEB25655).The


Increasing interest in microincision cataract surgery has led to the use of more flexible intraocular lens (IOL). Flexible IOL may cause more IOL deformation and refractive error when capsule contraction syndrome (CCS) occurred. In this retrospective observational case series study, the aim was to report four cases of hyperopic shift caused by CCS after phacoemulsification with microincision foldable intraocular lens implantation.All of four patients underwent phacoemulsification and in-the-bag implantation of an Akreos MI60 (Bausch and Lomb) IOL from 2010 to 2016 in our clinic. These patients had been diagnosed with CCS and had undergone Nd:YAG laser anterior capsulotomy. The mean age of the patients with CCS was 66.8 ± 6.7 years and the mean time for development of CCS after the cataract surgery was 9.3 ± 6.9 months. The mean spherical equivalent (SE) value at the time of the CCS diagnosis was 0.88 ± 0.91 D, which had shown a hyperopic shift compared to the SE value of − 0.91 ± 1.29 D after cataract surgery. The mean SE decreased by − 0.47 ± 1.14 D after Nd:YAG laser anterior capsulotomy. The mean age, axial length, anterior chamber depth, and preoperative SE were not significantly different between the patient with CCS and the patients without CCS.In the case of IOL implantation with flexible materials in microincision cataract surgery, CCS can cause a hyperopic shift. Refractive error caused by CCS can be effectively corrected by Nd:YAG laser anterior capsulotomy. Capsule contraction syndrome (CCS) is a common postoperative complication of cataract surgery. CCS is caused by lens epithelial cell proliferation and fibrosis leading to capsule shrinkage and contraction of the capsulorhexis opening as the anterior lens capsule becomes thicker and turbid. CCS is known to occur frequently in patients with weak zonules, diabetes, retinitis pigmentosa, pseudoexfoliation syndrome, uveitis, and high myopia –4. SurgiThe occurrence of CCS has been reported with the use of various IOL materials such as polymethylmethacrylate, silicone, and acrylic and there have been a few reports that the material and design of IOLs have a strong influence on the occurrence of CCS , 7–20. HIn this study, we reviewed patients who underwent phacoemulsification with in-the-bag implantation of a single-piece hydrophilic IOL  on demographics, refractive change according to CCS development, and reported 4 cases of hyperopic shift caused by CCS that were effectively treated with Nd:YAG laser anterior capsulotomy. In this study, CCS was defined as the exaggerated extreme reduction in diameter and fibrosis of anterior capsulorhexis opening following cataract removal. Anterior capsulorhexis opening with capsular phimosis was identified on slit-lamp examination.This retrospective clinical study includes 299 eyes (238 patients) who underwent cataract surgeries with in-the-bag implantation of an Akreos MI60  IOL, at our clinic between January 2010 and December 2016.One surgeon (S.W.M) had performed the surgical procedure under 0.5% proparacaine hydrochloride topical anesthesia. The procedure was performed after creating a self-sealing 2.80 mm superior clear corneal incision adjacent to the limbus. The size of the continuous curvilinear capsulorhexis was approximately 5.5–6.0 mm in diameter. Phacoemulsification was performed with the phaco chop technique using the AMO Sovereign Compact-WhiteStar System  followed by in-the-bag implantation of a single-piece hydrophilic IOL (Akreos MI60).Mann-Whitney U-tests were used to compare age, axial length, anterior chamber depth, and pre- and postoperative refractive spherical equivalent (SE) between patients with and without CCS. We also compared the refractive changes, UCVA, and BCVA of patients with CCS and those without CCS.n = 4) and patients without CCS (n = 295)Table . The demA 60-year-old woman presented with decreased visual acuity in the right eye. She was on regular follow-up at our clinic due to non-proliferative diabetic retinopathy and hypertension. She had undergone microincision phacoemulsification and an in-the-bag implantation of an Akreos MI60 IOL at our clinic 11 months ago.At 1 month after the cataract surgery, her UCVA and BCVA was 20/20 with a SE of − 0.125 D. At the time of presentation, her UCVA was 20/32 and BCVA was 20/25, in the right eye. In the refraction test, the SE showed a hyperopic shift of + 1.375 D. Her intraocular pressure (IOP) was within the normal limit. A slit lamp examination after pupil dilation revealed anterior capsule contraction syndrome with a markedly thickened anterior capsule  implantation in his right eye 18 months ago. At 1 month after the cataract surgery, his UCVA was 20/63 and his BCVA was 20/20 with an SE of − 2.00 D. At the time of presentation, his UCVA and BCVA were 20/250 and 20/100, respectively, and his SE was + 0.375 D. The IOP and anterior ocular surface did not show any abnormality. Slit lamp examination after dilation revealed marked shrinking of the anterior capsular opening. An Nd:YAG laser anterior capsulotomy was performed. One month after the Nd:YAG laser treatment, his UCVA and BCVA improved to 20/100 and 20/20, respectively, and his SE value returned to – 1.375, which was similar to the value after the cataract surgery. Twelve months later, his BCVA was 20/20 in the right eye, without recurrence.A 66-year-old woman with no systemic disease visited our clinic due to decreased visual acuity in her right eye. She underwent phacoemulsification surgery with IOL (Akreos MI60) implantation 2 months ago. Her postoperative UCVA and BCVA were 20/100 and 20/25, respectively, and her SE was − 2.00 D. At the time of presentation, her UCVA and BCVA were 20/100 and 20/50 respectively. On a refraction test, her SE was − 0.125 D, which showed a hyperoptic shift compared to the value immediately after her cataract surgery. Dilated slit lamp examination revealed phimosis of the anterior capsule with posterior vaulting of the IOL optic. Laser anterior capsulotomy was performed with an Nd:YAG laser in the right eye. One month after the Nd:YAG treatment, her UCVA and BCVA improved to 20/63 and 20/32, respectively. On a refraction test, the SE was − 1.50 D. Eight months later, her UCVA and BCVA were 20/63 and 20/32, respectively, in the right eye, without any complications.A 76-year-old woman who had phacoemulsification cataract extraction 6 months ago was referred to our clinic due to decreased visual acuity in the right eye. She had controlled hypertension and diabetes. At the time of the cataract surgery, a Akreos MI-60 IOL was implanted in the capsular bag. Postoperative UCVA and BCVA were 20/32 and 20/20, respectively. The postoperative refraction test showed that her SE was + 0.50 D. At the time of presentation, UCVA and BCVA were 20/50 and 20/25, respectively, and the SE showed a hyperoptic shift of + 1.875 D. Dilated slit lamp examination revealed 360 degrees of anterior capsular phimosis. Nd:YAG laser anterior capsulotomy was used to create a radial opening in the capsular phimosis. One month after the Nd:YAG treatment, her UCVA and BCVA improved to 20/25 and 20/20. The refraction test showed an SE of + 0.75 D. Twelve months later, her UCVA and BCVA were 20/20 in the right eye. There was no sign of anterior capsular contraction in the right eye.CCS can affect vision not only because of opacification of the visual axis but also because of IOL decentration such as tilting, deformation of the IOL, hypotony, and retinal detachment , 18, 21.CCS is caused by lens epithelial cell proliferation. Spang et al.  reportedCCS can be provoked by a number of contributing factors such as capsulorhexis size, high myopia, retinitis pigmentosa, uveitis, diabetes, and IOL material and design.1–4 In this study, capsulorhexis size did not likely have a large impact on CCS incidence because the 4 patients included in our study had a similar capsulorhexis size (range: 5.5–6.0 mm). Additionally, all procedures were performed by a single surgeon. Therefore, it is presumed that the surgical factors did not have a significant influence on the occurrence of CCS in our study. The CCS patients did not have high myopia or ocular inflammatory disease. Two patients had diabetes, which may have been a risk factor, but CCS did not occur earlier than in the patients without diabetes. There were no differences in the axial length, anterior chamber depth, or preoperative and postoperative SE between the affected eyes in the 4 patients with CCS and the 295 eyes without CCS , and forceps enlargement of the phimosis with IOL scleral fixation , 8–20. ITo our knowledge, there are few studies on refractive changes after CCS treatment. Qatarneh et al.  reportedThis study was limited by its small sample size. Therefore, our study findings cannot be generalized. Another limitation is that the anterior chamber depth of the patient with CCS was estimated by an anterior photograph or a medical record of slit lamp examination. Different angles of the incident slit beam can affect anterior chamber depth estimation. Further studies that include a larger number of patients and measure anterior chamber depth through anterior segment optical coherence tomography or ultrasound biomicroscopy are needed to confirm our results. However, it is meaningful that eyes with CCS and flexible IOLs tended to be vulnerable to hyperopic refractive shift.In conclusion, when microincision cataract surgery was performed with with Akreos MI60 IOL implantation, IOL deformation due to capsular bag contraction during CCS may cause hyperopic shift. Nd:YAG laser anterior capsulotomy can effectively restore vision and correct the refractive error in cases with CCS.