The characterization of protein stability is essential for understanding the functions of proteins. Hydroxysteroid dehydrogenase is involved in the biosynthesis of steroid hormones and the ...detoxification of xenobiotic carbonyl compounds. However, the stability of hydroxysteroid dehydrogenases has not yet been characterized in detail. Here, we determined the changes in Gibbs free energy, enthalpy, entropy, and heat capacity of unfolding for 3α‐hydroxysteroid dehydrogenase/carbonyl reductase (3α‐HSD/CR) by varying the pH and urea concentration through differential scanning fluorimetry and presented pH‐dependent protein stability as a function of temperature. 3α‐HSD/CR shows the maximum stability of 30.79 kJ mol−1 at 26.4°C, pH 7.6 and decreases to 7.74 kJ mol−1 at 25.7°C, pH 4.5. The change of heat capacity of 30.25 ± 1.38 kJ mol−1 K−1 is obtained from the enthalpy of denaturation as a function of melting temperature at varied pH. Two proton uptakes are linked to protein unfolding from residues with differential pKa of 4.0 and 6.5 in the native and denatured states, respectively. The large positive heat capacity change indicated that hydrophobic interactions played an important role in the folding of 3α‐HSD/CR. These studies reveal the mechanism of protein unfolding in HSD and provide a convenient method to extract thermodynamic parameters for characterizing protein stability using differential scanning fluorimetry.
The Ion S5 (Thermo Fisher Scientific) and Miseq (Illumina) NGS systems are both widely used in the clinical laboratories conducting PGT-A. Each system employs discrepant library preparation steps, ...sequencing principles, and data processing algorithms. The automatic interpretation via Ion Reporter software (Thermo Fisher Scientific) and the manual interpretation via BlueFuse Multi software (Illumina) for chromosomal copy number variation (CNV) represent very different reporting approaches. Thus, it is intriguing to compare their ability of ploidy detection as PGT-A/NGS system. In the present study, four aneuploid cell lines were individually mixed with a diploid cell line at different aneuploid ratios of 0% (0:5), 10% (1:9), 20% (1:4), 40% (2:3), 50% (3:3), 60% (3:2), 80% (4:1) and 100% (5:0) to assess the sensitivity and specificity for whole chromosomal and segmental aneuploidy detection. The clinical biopsies of 107 blastocysts from 46 IVF/PGT-A cycles recruited between December 2019 and February 2020 were used to calculate the concordance. Initially, the pre-amplified products were divided into two aliquots for different library preparation procedures of each system. Applying the same calling criteria, automatic identification was achieved through the Ion Reporter, while well-trained technicians manually identified each sample through the BlueFuse Multi. The results displayed that both systems reliably distinguished chromosomal CNV of the mixtures with at least 10% aneuploidy from karyotypically normal samples (Ion S5 whole-chromosomal duplication: 2.14 vs. 2.05, p value = 0.009, segmental deletion: 1.88 vs. 2.05, p value = 0.003; Miseq whole-chromosomal duplication: 2.12 vs. 2.03, p value = 0.047, segmental deletion: 1.82 vs. 2.03, p value = 0.002). The sensitivity and specificity were comparable between the Ion S5 and Miseq (sensitivity 93% vs. 90%, p = 0.78; specificity 100% vs. 100%, p value = 1.0). In the 107 clinical biopsies, three displayed chaotic patterns (2.8%), which could not be interpreted for the ploidy. The ploidy concordance was 99.04% (103/104) per embryo and 99.47% (2265/2277) per chromosome pair. Since their ability of detection were proven to be similar, the automatic identification in Ion S5 system presents comparatively faster and more standardized performance.
Chromosomal mosaicism is observed as the presence of both euploid and aneuploid cells in a particular blastocyst. Recent studies have reported that the implantation rate of mosaic embryo transfer is ...remarkably lower than the euploid embryos. The superior capability of next-generation sequencing (NGS) to detect chromosomal mosaicism in preimplantation genetic screening (PGS) remains controversial, and several data displayed similar implantation and pregnancy rates using NGS or array comparative genomic hybridization (aCGH).
In this study, the main inconsistency of aneuploidy detection and clinical performance between the NGS and aCGH were assessed. The phase I consisted of a parallel comparison in 182 blastocysts from 45 selected PGS patients for both the NGS and aCGH platforms. The phase II retrospectively compared the clinical outcomes of 90 patients with NGS-screened euploid embryo transfer to that of 129 patients with aCGH-screened euploid embryo transfer. The parallel comparison showed that the inconsistency of embryo euploidy was 11.8% (
= 0.01). Chromosomal mosaicism (10.7% with NGS
3.9% with aCGH) and segmental aneuploidy (10.7% with NGS
6.7% with aCGH) contributed to the discrepancy mainly. The chromosomally mosaic embryos (20%-50% of aneuploidy) and several embryos with segmental aneuploidy (≥10 Mbp) were hard to distinguish using the aCGH platform, but could be clearly identified using the NGS platform. After the first euploid embryo cryotransfer, the β-HCG(+) rate and implantation rate significantly increased in the PGS/NGS patients (HCG+ rate: 73.3% in PGS/NGS
60.5% in PGS/aCGH,
= 0.048; implantation rate: 53.2% in PGS/NGS
45.0% in PGS/aCGH,
= 0.043). The clinical and ongoing pregnancy rates appeared higher in the NGS group, but did not reached statistical significance.
The results demonstrated that the NGS platform can identify embryos with chromosomal mosaicism and segmental aneuploidy more precisely than the aCGH platform, and the following clinical performance of NGS was more favorable.
Enzymes are powerful biological catalysts for natural substrates but they have low catalytic efficiency for non-natural substrates. Protein engineering can be used to optimize enzymes for catalysis ...and stability. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidoreduction reaction of NAD+ with androsterone. Based on the structure and catalytic mechanism, we mutated the residues of T11, I13, D41, A70, and I112 and they interacted with different portions of NAD+ to switch cofactor specificity to biomimetic cofactor nicotinamide mononucleotide (NMN+). Compared to wild-type 3α-HSD/CR, the catalytic efficiency of these mutants for NAD+ decreased significantly except for the T11 mutants but changed slightly for NMN+ except for the A70K mutant. The A70K mutant increased the catalytic efficiency for NMN+ by 8.7-fold, concomitant with a significant decrease in NAD+ by 1.4 × 104-fold, resulting in 9.6 × 104-fold cofactor specificity switch toward NMN+ over NAD+. Meanwhile, the I112K variant increased the thermal stability and changed to a three-state transition from a two-state transition of thermal unfolding of wild-type 3α-HSD/CR by differential scanning fluorimetry. Molecular docking analysis indicated that mutations on these residues affect the position and conformation of the docked NAD+ and NMN+, thereby affecting their activity. A70K variant sterically blocks the binding with NAD+, restores the H-bonding interactions of catalytic residues of Y155 and K159 with NMN+, and enhances the catalytic efficiency for NMN+.
To evaluate the effectiveness of transvaginal ultrasound aspiration and ethanol sclerotherapy in patients with recurrent ovarian endometriomas.
Retrospective study.
Teaching hospital affiliated with ...Chang Gung University, Taipei.
Patients (n = 108) with recurrent ovarian endometriomas >or=3 cm.
Preoperative evaluation of previous pathology, midcycle serum CA-125 level, and color Doppler ultrasonography to exclude possibility of malignancies. After aspiration, sclerotherapy with 95% ethanol irrigation of the cystic cavity was performed (group 1, n = 78, 0-10 minutes of retention; group 2, n = 30, ethanol left in situ retention).
Ultrasonography was performed at 3, 6, 9, and 12 months to determine persistence and size of cysts and the number of antral follicles. Pelvic pain score was also determined at those time points.
The 1-year recurrence rate for group 2 patients was significantly lower than for group 1 patients (13.3% vs. 32.1%). Antral follicle count was increased and pain score was decreased in both groups to a similar level. No significant change in CA-125 was observed.
Ultrasound-guided sclerotherapy with 95% ethanol is an effective therapy for ovarian endometriomas. Retention of ethanol is more effective than irrigation only.
To investigate whether the mitochondrial DNA (mtDNA) content of a single biopsy at trophoblast correlates with the developmental potential and reproductive outcomes of blastocyst.
A retrospective ...analysis applied the dataset of 1,675 embryos with preimplantation genetic testing for aneuploidy (PGT-A) from 1,305 individuals, and 1,383 embryos involved cryotransfers of single euploid embryo between January 2015 and December 2019. The studied cohort was divided for algorithm establishment on the NGS platform (n=40), correlation of biological features (n=1,635), and correlation of reproductive outcomes (n=1,340). Of the algorithm derived from the NGS platform, the reliability and repeatability were validated
qPCR assay and inter-run controls, respectively. Of the correlation across biological features, stratification analyses were applied to evaluate the effect from a single contributor. Eventually, the correlation between the mtDNA ratios and reproductive outcomes was adjusted according to the significant effector(s).
The mtDNA ratios showed statistically different between embryos with different days of blastocyst formation (Day 5: 1.06 vs. Day 6: 0.66, p=0.021), and between embryos with different expansion stages (Expansion 5: 1.05 vs. Expansion 6: 0.49, p=0.012). None or weakly correlated with the maternal age, morphology, ploidy, and gender. Analyzed by the different days of blastocyst formation with fixed expansion score as 5 in the euploid single embryo transfers (eSET), the day 6 eSET showed significantly lower reduced mtDNA ratio (n=139) in failure groups of fetal heartbeat (p=0.004), ongoing pregnancy (p=0.007), and live birth (p=0.01); however, no correlation between mtDNA ratios and pregnancy outcomes was observed in the day 5 eSET (n=1,201).
The study first demonstrated that mtDNA ratio was dependent on the days of blastocyst formation while expansion stage was fixed. Lower mtDNA ratios were observed in the day 6 eSET with adverse outcomes. The present stratification analyses reveal that the timeline of embryo is an important covariate to the mtDNA content.
Supply chain network design is one of the most important strategic decisions that need to be optimized for long-term efficiency. Critical decisions include facility location, inventory, and ...transportation issues. This study proposes that with a dual-channel supply chain network design model, the traditional location-inventory problem should be extended to consider the vast amount of online customers at the strategic level, since the problem usually involves multiple and conflicting objectives. Therefore, a multi-objective dual-channel supply chain network model involving three conflicting objectives is initially proposed to allow a comprehensive trade-off evaluation. In addition to the typical costs associated with facility operation and transportation, we explicitly consider the pivotal online customer service rate between the distribution centers (DCs) and their assigned customers. This study proposes a heuristic solution scheme to resolve this multi-objective programming problem, by integrating genetic algorithms, a clustering analysis, a Non-dominated Sorting Genetic Algorithm II (NSGA-II), and a Technique for Order Preference by Similarity to Ideal Solution (TOPSIS). Several experiments are simulated to demonstrate the possibility and efficacy of the proposed approach. A scenario analysis is conducted to understand the model’s performance.
To evaluate the efficiency of transvaginal aspiration accompanied by ethanol sclerotherapy for treating cyst recurrence in patients who have previously undergone surgery to treat endometriosis and to ...analyze various factors that influence success rates using a data mining system.
Retrospective cohort study (Canadian Task Force classification II-3).
Teaching hospital affiliated with Chang Gung University, Taoyuan, Taiwan.
One hundred ninety-six patients with endometrioma recurrence.
A total of 274 transvaginal aspirations followed by sclerotherapy with 95% ethanol. Treatment times varied from immediate removal (0-10 minutes) to in situ retention. Patients were followed up at 3, 6, and 12 months to detect complications, determine the size and persistence of cysts, obtain the pelvic pain score, and assess for pregnancy or the need for repeat surgical intervention. A decision tree was used to determine factors from the collected data that most influenced the success of treatment.
Cyst size was consistently reduced until 6 months after ethanol sclerotherapy. The mean (SD) cyst reduction rate was 37.2% (42.2%), and the pain score reduction rate was 20.5% (71.5%). The antral follicle count was simultaneously increased by 36.4%. Sixty-three patients (23%) required repeated surgery during the observation period and were treated with either repeat aspiration (13.5%) or major laparoscopic or open laparotomic interventions (8.4%). Eighteen of 101 infertile patients (17.8%) achieved pregnancy. The total recovery rate (pregnancy or no persistence of symptoms or cyst) was significantly higher in patients in the groups that received longer treatment (7-10 minutes and retention) than in the groups with shorter treatment (0-6 minute) (47.0% vs 28.7%; p < .005). The highest recovery rate was observed in patients with longer treatment time, smaller cysts (≤5.05 cm), lower CA 125 level (≤62.03 IU/mL), and fewer cysts (≤3 cm) (35 of 49 71.4%). In patients with larger cysts and cysts with clear contents, better success can be achieved with longer treatment. The use of postoperative ovarian suppression, traditional Chinese medicine, or no therapy for 6 months before the study was not significant among groups.
Ultrasound-guided sclerotherapy with 95% ethanol retention is an effective alternative therapy for recurrent ovarian endometrioma, in particular in selected patient groups.
In PGS, does chromosomal constitution differ among trophectoderm (TE) biopsy sites and between them and the inner cell mass (ICM)?
The ploidy concordance between ICM and TE was independent of whether ...the biopsy site in the TE was near to or far from the ICM.
TE biopsies are considered less harmful to developing embryos than blastomere biopsies. Removal of multi-cellular samples permits high-resolution next-generation sequencing (Veriseq NGS) to detect aneuploidy present in a minority of cells (mosaicism of diploid and aneuploid cells). However, the prevalence of ploidy discrepancies between different TE biopsy sites and the ICM, as well as confined mosaicism (aneuploidy only in a particular area), has not been established.
Biopsies were taken from a site opposite to the ICM (TE1), near the ICM (TE2) and within the ICM of the same embryo in 33 donated blastocysts obtained from 12 volunteer patients. The samples were analyzed by the Veriseq NGS to assess ploidy concordance.
The mean age of the patients was 34.4 years, and samples from all three biopsy sites were achieved in 29 frozen thawed blastocysts. The aneuploid percentage in each sample was interpreted by Veriseq NGS at the finest resolution involving the number of reads after filtering, sample overall noise score, and average quality/alignment scores according to the Veriseq quality control assessment. Ploidy concordance was then assessed between different TE fractions, and between the TE and ICM.
The euploid rates were similar in the TEs and ICM, and no preferential allocation of euploid lineage within a blastocyst was demonstrated. Whether the biopsy site in the TE was near to or far from the ICM, the chromosomal consistency rate was similar TE1-to-ICM, 86.2% (25/29) versus TE2-to-ICM, 89.7% (26/29); P = 1.0, suggesting that the cells with different chromosomal components may spread randomly throughout the TE. The following two types of inconsistent PGS conclusions between TE and ICM due to confined mosaicism were observed: (i) euploid TE with mosaic ICM (3%) (1/29); and (ii) mosaic TE with euploid ICM (3%) (1/29) or with aneuploid ICM (7%) (2/29). Thus, the overall rate of confined mosaicism was 14% (4/29).
N/A.
The approach used in the present study was affected by biopsy manipulation limitations involving possible cell contamination and the technical challenge of comprehensive chromosomal screening (CCS) procedures.
The rate of confined mosaicism in the blastocysts was estimated in this preliminary study, thus, specifying the incidence of biological sampling biases. The results also verified the random distribution of different cell lineages, and the representative value of a single biopsied sample from the TE.
No external funding was obtained; all the authors declare no conflicts of interest regarding this study.
► Vendor-managed inventory (VMI) is one of the emerging solutions for improving the supply chain efficiency. ► This article presented a Multi-Objective Location–Inventory Problem (MOLIP) model. ► The ...potential of the proposed approach is demonstrated by comparing to a well-known multi-objective evolutionary algorithm. ► Computational results have presented promise solutions for different sizes of VMI problems.
Vendor-managed inventory (VMI) is one of the emerging solutions for improving the supply chain efficiency. It gives the supplier the responsibility to monitor and decide the inventory replenishments of their customers. In this paper, an integrated location–inventory distribution network problem which integrates the effects of facility location, distribution, and inventory issues is formulated under the VMI setup. We presented a Multi-Objective Location–Inventory Problem (MOLIP) model and investigated the possibility of a multi-objective evolutionary algorithm based on the Non-dominated Sorting Genetic Algorithm (NSGA2) for solving MOLIP. To assess the performance of our approach, we conduct computational experiments with certain criteria. The potential of the proposed approach is demonstrated by comparing to a well-known multi-objective evolutionary algorithm. Computational results have presented promise solutions for different sizes of problems and proved to be an innovative and efficient approach for many difficult-to-solve problems.
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