miR-410 Regulates Helper T Cell Differentiation in Ovalbumin-Induced Asthma through the PI3K-AKT-VEGF Signaling Pathway.

INTRODUCTION: Asthma has been attributed to Th1/Th2 imbalance and inappropriate Th2 responses to environmental allergens. MicroRNAs (miRNAs), 21 to 23 RNA molecules, are first found in mammals and have been implicated in various biological activities. Our previous study found that miR-410 effectively ameliorates airway inflammation in the ovalbumin (OVA)-induced asthma murine model. However, the role of miR-410 in regulating helper T (Th) cell differentiation is not clear. In the present study, we aimed to explore the regulatory effects of miR-410 on the differentiation of Th cells through both in vivo and in vitro studies. METHODS: Dual-luciferase reporter assay was used to find if miR-410 has any direct binding position with VEGF mRNAs. PBMC and CD4+ T cells were isolated and stimulated with OVA. The miR-410 mimics and inhibitors were transfected into CD4+ T cells. The differentiation of Th cells was evaluated by enzyme-linked immunosorbent assay (ELISA) for the concentration of IL-4, IFN-γ, and TGF-ß levels in supernatants. Western Blot was used to detect protein expression and phosphorylation of PI3K and AKT. BALB/c mice were kept in a specific pathogen-free condition and received sterile OVA-free food and water. OVA-induced asthmatic mice model was established. ELISA was used to measure the bronchoalveolar lavage fluid (BALF) concentrations of IL-4, IFN-γ, TGF-ß, and VEGF. Hematoxylin and eosin staining and immunohistochemical staining were conducted to analyze inflammatory cell infiltration, pathological changes, and the expression of VEGF. RESULTS: Dual-luciferase reporter assay showed that miR-410 has no direct binding position with VEGF mRNAs. In the OVA-primed mononuclear cells compared to normal cells, IFN-γ and TGF-ß were decreased while IL-4 and VEGF were increased. This change was reversed while miRNA-410 mimics were transfected into CD4+ T cells. Besides, the OVA-primed CD4+ T cells treated with miR-410 decrease the proliferation of cytokine of Th2 cells as well as phosphorylation of PI3K, and AKT. In OVA-induced asthma mice, IFN-γ and TGF-ß were decreased in BALF while the IL-4 and VEGF were increased. OVA-induced mice with asthma treated with miR-410 mimics showed marked reductions in the infiltration of inflammatory cells as well as IL-4 and VEGF in BALF. The immunohistochemical staining of the expression of VEGF also decreased in OVA-induced asthma mice with the instillation of miR-410. CONCLUSIONS: In this study, we revealed that miR-410 could regulate the differentiation of Th cells via the PI3K-AKT-VEGF signaling pathway in asthma.

Extrusion puffing as a pretreatment method to change the surface structure, physicochemical properties and in vitro protein digestibility of distillers dried grains with solubles.

BACKGROUND: Distillers dried grains with solubles (DDGS) are rich in nutrition, and they are potential protein feed raw material. However, the existence of cellulose, hemicellulose and lignin hinders animals' digestion and absorption of DDGS. Making full use of unconventional feed resources such as DDGS can alleviate the shortage of feed resources to a certain extent. This research investigated the effects of twin-screw extrusion on the macromolecular composition, physical and chemical properties, surface structure and in vitro protein digestibility (IVPD) of DDGS. RESULTS: The findings showed that extrusion puffing significantly increased the protein solubility, bulk density, water holding capacity, and swelling capacity, while significantly decreased hemicellulose and crude protein content, particle size and zeta potential of DDGS. The structure damage of DDGS induced by the extrusion was characterized by scanning electron microscopy (SEM), Fourier-transform infrared (FITR) spectroscopy and X-ray diffraction (XRD) analysis. Interestingly, no random coil was observed in the analysis of the secondary structure, and extrusion promoted the transformation of α-helix and ß-turn to ß-sheet, which led to significant increases in protein solubility and IVPD of DDGS (P < 0.05). Additionally, correlation analysis revealed that IVPD and PS had a positive relationship. CONCLUSION: Extrusion puffing was an ideal pretreatment method for DDGS modification to improve in vitro protein digestibility. © 2023 Society of Chemical Industry.

Novel variants in ZP1, ZP2 and ZP3 associated with empty follicle syndrome and abnormal zona pellucida.

RESEARCH QUESTION: Which genetic variants might explain the causes of empty follicle syndrome (EFS) and abnormal zona pellucida (ZP) and affect the success of treatment with assisted reproductive technologies (ART)? DESIGN: Whole-exome sequencing was performed in probands with EFS and abnormal ZP. Sanger sequencing was used for variant validation. Using HEK-293T cells, the effects of ZP1 and ZP2 variants on protein expression were explored by western blotting, and the effect of the ZP1 variant on protein location was investigated via immunofluorescence. The protein structure was also analysed to investigate the pathogenicity of variants. RESULTS: A homozygous nonsense variant in ZP1 (c.874C>T, p.Gln292*) was detected in a patient with EFS. A novel homozygous frameshift variant in ZP2 (c.836_837delAG, p.Glu279Valfs*6) and a novel heterozygous missense variant in ZP3 (c.1159G>A, p.Val387Met) were identified in two patients with ZP morphological abnormalities, respectively. Western blotting and immunofluorescence analysis showed that the ZP1 variant results in a premature stop codon, leading to the truncated ZP1 protein. The ZP2 variant, which is situated in the N-terminus, triggers the degradation of a premature termination protein. Additionally, the patient with the ZP3 variant achieved clinical pregnancy following intracytoplasmic sperm injection treatment. CONCLUSIONS: These findings expand the mutational spectrum of ZP1, ZP2 and ZP3, and provide new evidence for genetic diagnosis of female infertility. The targeted genetic diagnosis of ZP genes is recommended to choose appropriate fertilization methods and improve success rates of treatment with ART.

Genetic screening and analysis of TUBB8 variants in females seeking ART.

RESEARCH QUESTION: More than 100 variants have been identified in the TUBB8 gene, which account for approximately 30% of infertile women with oocyte maturation defects. But what is the correlation between the highly phenotypic diversity and genetic variability? Are there other variants in TUBB8 related to female infertility? DESIGN: TUBB8 resequencing was performed in 80 female subjects who were experiencing infertility and were seeking treatment with assisted reproductive technologies (ART), or had ever experienced ART failure due to oocyte maturation defects. All variants were evaluated with pedigree analysis, population frequency, in-silico analysis and molecular modelling. The effects of the variants on oocytes/arrested embryos were assessed by morphological observations, immunostaining, embryo biopsies and chromosome euploidy analysis. RESULTS: Nine missense variants and two frameshift variants from an additional 15 families were identified, including four novel variants and seven previously reported recurrent variants. These TUBB8 variants were related to highly variable phenotypes, including abnormalities in oocyte maturation or morphology, fertilization failure, embryonic development abnormalities and implantation failure. Also further clarified were the incomplete penetrance of heterozygous p.E108K, the likely benign significance of heterozygous p.A313V and the clinical effect of a novel variant of p.R380C. CONCLUSIONS: This study significantly expands the variant spectrum of the TUBB8 gene and, together with the available findings on TUBB8 variants and female infertility, will potentially facilitate the genetic counselling of infertile women in future.

Blocker displacement amplification-based genetic diagnosis for autosomal dominant polycystic kidney disease and the clinical outcomes of preimplantation genetic testing.

OBJECTIVE: Given that the molecular diagnosis of autosomal dominant polycystic kidney disease (ADPKD) is complicated, we aim to apply blocker displacement amplification (BDA) on the mutational screening of PKD1 and PKD2. METHODS: A total of 35 unrelated families with ADPKD were recruited from the Center for Reproductive Medicine, Women and Children's Hospital of Chongqing Medical University (Chongqing, China), from October 2018 to October 2021. Long-range PCR followed by next-generation sequencing were applied for resequencing of PKD1 and PKD2, and the putatively disease-causative variants were verified with BDA. The effects of ADPKD on male and female infertility and the factors influencing the clinical outcomes of preimplantation genetic testing (PGT) for ADPKD were investigated. RESULTS: A total of 26 PKD1 variants and 5 PKD2 variants were identified, of which 13 were newly discovered. The BDA system worked effectively for eliminating the interference of pseudogenes in genetic testing of PKD1 (1-33 exons) with different concentrations of genome DNA. The females with ADPKD have no specific infertility factors, while 68.2% of the affected men were with abnormal sperm concentration and/or motility with an indefinite genotype-phenotype relationship. As for PGT, the fertilization rate of couples with the male partner having ADPKD was relatively lower compared to those with the female partner being affected. The ADPKD patients receiving PGT usually achieved high rates of live births. CONCLUSION: These findings expanded the variant spectrum of PKD genes and emphasized the application prospect of blocker displacement amplification on PKD1-related genetic diagnosis.

Mechanical properties of calcium silicate hydrate under uniaxial and biaxial strain conditions: a molecular dynamics study.

Calcium silicate hydrate (C-S-H) is the main hydration product of cementitious materials, often experiencing complex stress conditions in practical applications. Therefore, reactive molecular dynamics methods were used to investigate the mechanical response of the atomistic structure of C-S-H under various uniaxial and biaxial strain conditions. The results of uniaxial simulations show that C-S-H exhibits mechanical anisotropy and tension-compression asymmetry due to its layered atomistic structure. By fitting the stress-strain data, a stress-strain relationship that accurately represents the elastoplasticity of C-S-H was developed. The biaxial yield surface obtained from biaxial simulations was ellipsoidal, again reflecting the anisotropy and asymmetry of C-S-H. Four yield criteria (von Mises, Drucker-Prager, Hill, and Liu-Huang-Stout) were further investigated, and it was found that the Liu-Huang-Stout criterion can effectively capture all the major features of the yield surface. During a uniaxial tensile process in the z direction, multi-crack propagation was observed, which was aggravated and weakened by y direction tensile and compressive strains respectively. The results of chemical bond analyses revealed that, for different strain conditions, the CaW-OS and CaS-OS bonds play different roles in resisting deformation.

Trophectoderm biopsy protocols may impact the rate of mosaic blastocysts in cycles with pre-implantation genetic testing for aneuploidy.

PURPOSE: This study aimed to analyze the impact of different biopsy protocols on the rate of mosaic blastocysts. METHODS: This is a retrospective cohort study which included 115 cycles with pre-implantation genetic testing for aneuploidy (PGT-A). Two groups were allocated based on the biopsy protocols: method 1 group, the zona pellucida (ZP) was drilled on day 3 embryos followed by trophectoderm (TE) biopsy; and method 2 group, the ZP was opened on day 5 or 6 blastocysts followed by TE biopsy. All biopsy samples were assessed using next-generation sequencing (NGS) at a single reference laboratory. The euploid, aneuploid, and mosaic blastocyst rates and clinical outcomes were compared. RESULTS: The mosaicism rate in the method 1 group was 19.58%, significantly higher than the method 2 group (8.12%; P < 0.05). No statistically significant difference was observed in euploid, aneuploid blastocyst rates, and clinical pregnancy rates between the two groups. Logistic regression analysis indicated that the biopsy protocols were independently associated with the mosaicism rates among all the variables. CONCLUSIONS: The present study showed that different biopsy protocols may have an impact on the mosaic blastocyst rate. ZP opening on day 3 combined with TE biopsy might increase the incidence of mosaic blastocysts.

LC-high-resolution-MS/MS analysis of chemical compounds in rat plasma after oral administration of Nao-Mai-Tong and its individual herbs.

Nao-Mai-Tong (NMT), a Chinese herbal formula, is used for treating ischemia cerebral apoplexy. To discover the components of NMT with potential in vivo bioactivity and investigate the differences between NMT and its single herb components, this study rigorously compared plasma samples collected from rats dosed with NMT and single-herb extracts at different times through LC-high resolution-MS/MS and data processing (Metworks). The plasma of the NMT group contained a total of 66 identified compounds (25 prototypes and 41 metabolites), including anthraquinones, triterpenoid saponins, isoflavones and phthalides. Additionally, glucuronidation, sulfation and cysteine conjugation were the major reactions through which the compounds in NMT were metabolized. The comparison of the groups revealed two metabolites that were only detected in the plasma from the NMT-dosed group, whereas seven prototype ingredients (chrysophanol-8-O-glucoside, ginsenoside Rf, Rg2, Rh1, F1, F2 and chikusetsusaponin IVa) and 12 metabolites (two novel triterpenoid saponins) were only discovered in the plasma samples from the single-herb-dosed groups. Moreover, the trends in the chemical compounds detected presented marked differences between NMT-dosed rat plasma and plasma samples from the single-herb-dosed groups. The above data indicate that prescription compatibility affects the assimilation and elimination of ingredients and provides useful information for further pharmacokinetic studies.

High Sensitivity Determination of TNF-α for Early Diagnosis of Neonatal Infections with a Novel and Reusable Electrochemical Sensor.

Early diagnosis is vital for the reduction of mortality caused by neonatal infections. Since TNF-α can be used as a marker for the early diagnosis, the detection of TNF-α with high sensitivity and specificity has great clinical significance. Herein, a highly sensitive and reusable electrochemical sensor was fabricated. Due to the high specificity of aptamers, TNF-α could be accurately detected from five similar cytokines, even from serum samples. In addition, Au nanoparticles (AuNPs) with a high surface area were able to combine a large number of doxorubicin hydrochloride (DOXh), which made the sensor have a high sensitivity. The sensor had a good linear relationship with TNF-α concentration in the range from 1 to 1 × 104 pg/mL and the lowest detection limit is 0.7 pg/mL. More important was that the sensor could be reused 6 times by a crafty use of chain replacement reaction. Meanwhile, the detection time and cost were greatly reduced. Thus, we believe that these advantages of higher specificity and sensitivity, lower cost, and shorter detection time will provide a stronger potential for early diagnosis of neonatal infections in clinical applications.

[Risk factors for the failure of the InSure method in very preterm infants with respiratory distress syndrome].

OBJECTIVE: To study the risk factors for the failure of the InSure method in very preterm infants with respiratory distress syndrome (RDS). METHODS: Seventy-one very preterm infants with RDS treated with InSure method were enrolled. These infants were categorized into two groups: InSure success (42 cases) and InSure failure (29 cases). The differences in basic information were compared between the two groups, and logistic regression analysis was used to identify the risk factors for InSure failure. RESULTS: The failure rate of the InSure method was 41%. The failure group were much lower in the birth weight, the antenatal steroids utilization rate and the vaginal delivery rate than the success group (P<0.05). The incidence of patent ductus arteriosus in the failure group was significantly higher than in the success group (P<0.05). PaO2, PaO2/FiO2 and PaO2/PAO2 in the failure group were significantly lower than in the success group (P<0.05). PaCO2 in the failure group was much higher than in the success group (P<0.05). Further logistic regression analysis showed that birth weight <1 150 g (OR=22.240 95%CI=2.124-232.901), PaCO2>54 mm Hg(OR=9.360, 95%CI=1.958-44.741, and PaO2/FiO2 <195 (OR=6.570, 95%CI=1.027-42.003), were the independmend risk factors for InSure failure. Furthermore, the duration of oxygen therapy, the total time of hospitalization and the incidence of BPD in the failure group were much longer and higher than in the success group (P<0.05). CONCLUSIONS: Low birth weight, elevated PaCO2 and low PaO2/PiO2 ratio are the risk factors for the failure of the InSure method in very preterm infants.

Source analysis of chlorine in municipal solid waste under waste classification: a case study of Hangzhou, China.

Inorganic chlorine is susceptible to water and soil salinization due to its non-degradability and high mobility. To clarify the environmental risks associated with the active inorganic chlorine in municipal solid waste (MSW), the specific characteristics and contributions of inorganic chlorine in different MSW categories were investigated in this study. MSW samples were collected from eight representative waste classification residential areas in Hangzhou, China. It was found that the inorganic chlorine content in different MSW categories varied significantly (0-113 mg/g). Perishable waste, paper, and plastic were found to be the main sources of inorganic chlorine in MSW. A four-category classification system was used to quantify the contribution of inorganic chlorine from each waste category. It was found that the misclassification of inorganic chlorine contributions from perishable waste and other waste accounted for 51.96% and 48.04%, respectively. However, when correctly classified into the four-category system, their contributions were reduced to 67.14% and 30.65%, respectively. Therefore, MSW classification showed a significant reduction in the overall contribution of inorganic chlorine. The misclassification reduces the contribution of inorganic chlorine to 48.04%, while correct classification increases the reduction to 69.35%.

Drimane-type sesquiterpenoids and their anti-inflammatory evaluation from Pyrrhoderma noxium HNNU0524.

Chemical investigation of a culture broth from the marine-derived fungus Pyrrhoderma noxium HNNU0524 yielded two new compounds including a drimane-type sesquiterpenoid named pyrrnoxin A (1) and a benzoic acid derivative, pyrrnoxin B (5), together with three related known analogues (2-4). The chemical structures of 1 and 5 were determined by detailed analysis of spectroscopic data, single-crystal X-ray crystallography, quantum mechanics-based DP4+ and ECD calculations. Compounds 2 and 3 moderately inhibited NO production of lipopolysaccharide-induced microglia cells BV2 with IC50 values of 26.6 and 60.5 µM, respectively.

Novel PLCZ1 mutation caused polyspermy during in vitro fertilization.

Failure of oocyte activation, including polyspermy and defects in pronuclear (PN) formation, triggers early embryonic developmental arrest. Many studies have shown that phospholipase C zeta 1 ( PLCZ1 ) mutations cause failure of PN formation following intracytoplasmic sperm injection (ICSI); however, whether PLCZ1 mutation is associated with polyspermy during in vitro fertilization (IVF) remains unknown. Whole-exome sequencing (WES) was performed to identify candidate mutations in couples with primary infertility. Sanger sequencing was used to validate the mutations. Multiple PLCZ1 -mutated sperm were injected into human and mouse oocytes to explore whether PN formation was induced. Assisted oocyte activation (AOA) after ICSI was performed to overcome the failure of oocyte activation. We identified three PLCZ1 mutations in three patients who experienced polyspermy during IVF cycles, including a novel missense mutation c.1154C>T, p.R385Q. PN formation failure was observed during the ICSI cycle. However, injection of multiple PLCZ1- mutated sperm induced PN formation, suggesting that the Ca 2+ oscillations induced by the sperm exceeded the necessary threshold for PN formation. AOA after ICSI enabled normal fertilization, and all patients achieved successful pregnancies. These findings expand the mutational spectrum of PLCZ1 and suggest an important role for PLCZ1 in terms of blocking polyspermy. Furthermore, this study may benefit genetic diagnoses in cases of abnormal fertilization and provide potential appropriate therapeutic measures for these patients with sperm-derived polyspermy.

Risk prediction of bronchopulmonary dysplasia in preterm infants by the nomogram model.

Backgrounds and Aims: Bronchopulmonary dysplasia (BPD) has serious immediate and long-term sequelae as well as morbidity and mortality. The objective of this study is to develop a predictive model of BPD for premature infants using clinical maternal and neonatal parameters. Methods: This single-center retrospective study enrolled 237 cases of premature infants with gestational age less than 32 weeks. The research collected demographic, clinical and laboratory parameters. Univariate logistic regression analysis was carried out to screen the potential risk factors of BPD. Multivariate and LASSO logistic regression analysis was performed to further select variables for the establishment of nomogram models. The discrimination of the model was assessed by C-index. The Hosmer-Lemeshow test was used to assess the calibration of the model. Results: Multivariate analysis identified maternal age, delivery option, neonatal weight and age, invasive ventilation, and hemoglobin as risk predictors. LASSO analysis selected delivery option, neonatal weight and age, invasive ventilation, hemoglobin and albumin as the risk predictors. Both multivariate (AUC = 0.9051; HL P = 0.6920; C-index = 0.910) and LASSO (AUC = 0.8935; HL P = 0.7796; C-index = 0.899) - based nomograms exhibited ideal discrimination and calibration as confirmed by validation dataset. Conclusions: The probability of BPD in a premature infant could be effectively predicted by the nomogram model based on the clinical maternal and neonatal parameters. However, the model required external validation using larger samples from multiple medical centers.

Congenital pulmonary hypoplasia combined with congenital cardiac disease and ectopic kidney: a case report.

Background: Congenital pulmonary hypoplasia (CPH) is a rare pulmonary disease featured by incomplete development of pulmonary tissues. Its diagnosis is still a challenge as patients are usually misdiagnosed as atelectasis. Case presentation: A female neonate was admitted to our hospital due to post-birth jaundice for 12 hrs. Physical examination showed accelerated breathing. There was no respiratory sound in the left lung. Chest film indicated decline of lucency in the left lung. Chest CT scan indicated absence of left lung and primary bronchus of the left lung. The boundary between left mediastinum was not clearly displayed. Three-dimensional CT scan indicated absence of left lung and left principal bronchus. Cardiac ultrasonography confirmed congenital heart disease. She showed ectopic kidney. Finally, she was diagnosed with CPH concurrent with congenital heart disease and ectopic kidney. Conclusions: On 17-month follow-up visit, the patient is still survived, but she presents with obstruction in ventilation function.

Dietary ethylenediamine dihydroiodide improves intestinal health in Cherry Valley ducks.

This study investigated the effect of ethylenediamine dihydroiodide (EDDI) on the growth performance, thyroid function, immune function, intestinal development, intestinal permeability, intestinal barrier functions and microbial characteristics of Cherry Valley ducks. The results showed that the addition of EDDI significantly increased body weight, average daily gain, serum level of lymphocytes, basophils, triiodothyronine, thyroxine and thyrotropin, villus height, and villus height-to-crypt depth ratio, and significantly decreased crypt depth, diamine oxidase, serum D-Lactic acid of ducks (P < 0.05). EDDI also significantly up-regulated the mRNA expression of zonula occludens-1, zonula occludens-2, zonula occludens-3, mucin 2, secretory immunoglobulin A, interleukin-10 and avian ß-defensin 2 in the jejunum and ileum (P < 0.05), and down-regulated the mRNA expression of occludin and interleukin-6 in the jejunum and ileum. Additionally, the addition of EDDI significantly increased cecal level of acetic acid, propionic acid, butyric acid (P < 0.05). Cecal microbiome analysis indicated that the addition of EDDI significantly increased the relative abundance of these microorganisms that can produce short-chain fatty acids, mainly including Actinobacteria, Verrucomicrobia, Clostridiales and Lactobacillales, and decreased the relative abundance of pathogenic bacteria Deferribactere. Interestingly, triiodothyronine and thyroxine levels were highly positively correlated with the relative abundance of Actinobacteria. These results revealed that the addition of EDDI could promote the growth and development of meat ducks by improving their thyroid function, immune function, intestinal development and intestinal barrier functions of ducks.

Change of intestinal microbiota in mice model of bronchopulmonary dysplasia.

Background: Gut microbiota has been proposed to be related to the pathogenesis of pulmonary diseases such as asthma and lung cancer, according to the gut-lung axis. However, little is known about the roles of gut microbiota in the pathogenesis of bronchopulmonary dysplasia (BPD). This study was designed to investigate the changes of gut microbiota in neonatal mice with BPD. Methods: BPD model was induced through exposure to high concentration of oxygen. Hematoxylin and eosin (H&E) staining was utilized to determine the modeling efficiency. Stool samples were collected from the distal colon for the sequencing of V3-V4 regions of 16S rRNA, in order to analyze the gut microbiota diversity. Results: Alpha diversity indicated that there were no statistical differences in the richness of gut microbiota between BPD model group and control group on day 7, 14 and 21. Beta diversity analysis showed that there were statistical differences in the gut microbiota on day 14 (R = 0.368, p = 0.021). Linear discriminant analysis effect size (LEfSe) showed that there were 22 markers with statistical differences on day 14 (p < 0.05), while those on day 7 and 21 were 3 and 4, respectively. Functional prediction analysis showed that the top three metabolic pathways were signal transduction (PFDR = 0.037), glycan biosynthesis and metabolism (PFDR = 0.032), and metabolism of terpenoids and polyketides (PFDR = 0.049). Conclusions: BPD mice showed disorder of gut microbiota, which may involve specific metabolic pathways in the early stage. With the progression of neonatal maturity, the differences of the gut microbiota between the two groups would gradually disappear.

Effect of air and water on the release of chlorine from semi-aerobic landfill.

Landfill leachate has a high chloride (Cl-) content. Because it is highly mobile, and cannot be sorbed or transformed bio-chemically, it is important to have detailed information about how it migrates in landfill sites. In this study, we set up four lab-scale simulated landfills, including an anaerobic landfill (AL), an anaerobic landfill with leachate recirculation (RAL), an anaerobic/semi-anaerobic landfill with leachate recirculation (RASL), and an anaerobic/semi-aerobic landfill (ASL), to explore how, when regulated, moisture and air affected the migration of chlorine. We found that water and air had a strong influence on the release of Cl-. Leachate obviously promoted Cl- dissolution in refuse when recirculated. When air was introduced into landfill, thereby changing it from anaerobic to semi-aerobic, the leachate Cl- concentration increased sharply from around 4-9 g L-1 (RASL) and 18 g L-1 (ASL), respectively. In principle, Cl- is released continuously when leachate is recirculated in landfills (RAL and RASL), but it can also be found a terminal when the leachate recirculation stops (AL and ASL). Cumulative amounts of 64, 66, 27, and 53 g of Cl- were released from the AL, RAL, RASL, and ASL, respectively. Lower COD/Cl and NH4+-N/Cl ratios in ASL and RASL after day 175 indicated that lower Cl- pollution risk than that in AL and RAL.

Accumulation of Cleavage-Stage Embryos by Vitrification may Compromise Embryonic Developmental Potential in Preimplantation Genetic Testing.

It was suggested that the embryo pooling was an alternative for patients with insufficient number of embryos for preimplantation genetic testing (PGT) in a single ovarian stimulation cycle. However, limited study noticed whether it is an efficient strategy to pool cleavage-stage embryos by vitrification. This study included 71 cycles with vitrified-warmed and fresh embryos simultaneously for PGT between May 2016 and May 2021. The embryos from the same patients were split into two groups based on the origin: warming group and fresh group. Embryo development, sequencing results, clinical and neonatal outcomes were compared. The results showed that the rate of high-quality embryos in the warming group was significantly higher than that in the fresh group (64.53% versus 52.61%, P = 0.011); however, the available blastocyst rate in this group was significantly lower than that in the fresh group (47.29% versus 57.83%, P = 0.026). There were 96 and 144 blastocysts that underwent trophectoderm (TE) biopsy in warming and fresh groups, respectively. The high-quality blastocyst rate was significantly lower in the warming group compared to the fresh group (57.29% versus 70.14%, P = 0.041). The rates of genetic transferable blastocyst were comparable between the two groups (P = 0.956). There were no statistical differences in terms of embryo implantation, clinical pregnancy, miscarriage rates, and neonatal outcomes between the two groups. In conclusion, this study demonstrated that the cleavage-stage embryo pooling strategy might be unfavorable for the maintenance of embryonic development potential. If not necessary, it is not recommended to pool cleavage-stage embryos for PGT.

Case Report: Preimplantation Genetic Testing for Meckel Syndrome Induced by Novel Compound Heterozygous Mutations of MKS1.

Meckel syndrome (MKS), also known as the Meckel-Gruber syndrome, is a severe pleiotropic autosomal recessive developmental disorder caused by dysfunction of the primary cilia during early embryogenesis. The diagnostic criteria are based on clinical variability and genetic heterogeneity. Mutations in the MKS1 gene constitute approximately 7% of all MKS cases. Herein, we present a non-consanguineous couple with three abnormal pregnancies as the fetuses showed MKS-related phenotypes of the central nervous system malformation and postaxial polydactyly. Whole-exome sequencing identified two novel heterozygous mutations of MKS1: c.350C>A and c.1408-14A>G. The nonsense mutation c.350C>A produced a premature stop codon and induced the truncation of the MKS1 protein (p.S117*). Reverse-transcription polymerase chain reaction (RT-PCR) showed that c.1408-14A>G skipped exon 16 and encoded the mutant MKS1 p.E471Lfs*92. Functional studies showed that these two mutations disrupted the B9-C2 domain of the MKS1 protein and attenuated the interactions with B9D2, the essential component of the ciliary transition zone. The couple finally got a healthy baby through preimplantation genetic testing for monogenic disorder (PGT-M) with haplotype linkage analysis. Thus, this study expanded the mutation spectrum of MKS1 and elucidated the genetic heterogeneity of MKS1 in clinical cases.