An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins.

Many multi-spanning membrane proteins contain poorly hydrophobic transmembrane domains (pTMDs) protected from phospholipid in mature structure. Nascent pTMDs are difficult for translocon to recognize and insert. How pTMDs are discerned and packed into mature, muti-spanning configuration remains unclear. Here, we report that pTMD elicits a post-translational topogenesis pathway for its recognition and integration. Using six-spanning protein adenosine triphosphate-binding cassette transporter G2 (ABCG2) and cultured human cells as models, we show that ABCG2's pTMD2 can pass through translocon into the endoplasmic reticulum (ER) lumen, yielding an intermediate with inserted yet mis-oriented downstream TMDs. After translation, the intermediate recruits P5A-ATPase ATP13A1, which facilitates TMD re-orientation, allowing further folding and the integration of the remaining lumen-exposed pTMD2. Depleting ATP13A1 or disrupting pTMD-characteristic residues arrests intermediates with mis-oriented and exposed TMDs. Our results explain how a "difficult" pTMD is co-translationally skipped for insertion and post-translationally buried into the final correct structure at the late folding stage to avoid excessive lipid exposure.

Effects of multi-gradient equilibration during vitrification on oocyte survival and embryo development in mice.

Vitrification has been widely used for oocyte cryopreservation, but there is still a need for optimization to improve clinical outcomes. In this study, we compared the routine droplet merge protocol with modified multi-gradient equilibration vitrification for cryopreservation of mouse oocytes at metaphase II. Subsequently, the oocytes were thawed and subjected to intracytoplasmic sperm injection (ICSI). Oocyte survival and spindle status were evaluated by morphology and immunofluorescence staining. Moreover, the fertilization rates and blastocyst development were examined in vitro. The results showed that multi-gradient equilibration vitrification outperformed droplet merge vitrification in terms of oocyte survival, spindle morphology, blastocyst formation, and embryo quality. In contrast, droplet merge vitrification exhibited decreasing survival rates, a reduced proportion of oocytes with normal spindle morphology, and lower blastocyst rates as the number of loaded oocytes increased. Notably, when more than six oocytes were loaded, reduced oocyte survival rates, abnormal oocyte spindle morphology, and poor embryo quality were observed. These findings highlight that the vitrification of mouse metaphase II oocytes by the modified multi-gradient equilibration vitrification has the advantage of maintaining oocyte survival, spindle morphology, and subsequent embryonic development.

Evaluation of an automated dish preparation system for IVF and embryo culture using a mouse mode.

Manual dish preparation for IVF in human fertility clinics or animal laboratories heavily relies on embryologists' experience, which can lead to occupational illness due to long-term and monotonous operation. Therefore, introducing an automated technique to replace traditional methods is crucial for improving working efficiency and reducing work burden for embryologists. In the current study in the mouse, both manual and automated methods were used to prepare IVF or embryo culture dishes. A one-way analysis of variance was conducted to compare several factors, including preparation time, qualified rates, media osmolality of dishes, fertilization rates, and embryonic development to assess the efficiency and potential of automated preparation. The results showed that automation system significantly reduced the required time and increased the efficiencies and qualified rates of dish preparation, especially for embryo culture dishes, without significantly altering medium osmolalities. There were no significant differences between two preparations in fertilization rates and embryo development in mice. Thus, automated dish preparation can improve working efficiency and qualified rates while maintaining fertilization rates and subsequent embryonic development without compromising osmolality stability of medium. It presents a superior alternative to manual preparation, reducing the workload of embryologists and facilitating the standardization of operational procedures.

Phenolics and Terpenoids Profiling in Diverse Loquat Fruit Varieties and Systematic Assessment of Their Mitigation of Alcohol-Induced Oxidative Stress.

To compare and investigate the phenolic compounds in the peel and flesh of loquat (Eriobotrya japonica) and evaluate their ability to protect against alcohol-induced liver oxidative stress, we employed a combination of ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry (HRMS) to qualitatively and quantitatively analyze 22 phenolics and 2 terpenoid compounds in loquat peel and flesh extracts (extraction with 95% ethanol). Among these, six compounds were identified for the first time in loquat, revealing distinct distribution patterns based on variety and tissue. Various chemical models, such as DPPH, FRAP, ORAC, and ABTS, were used to assess free radical scavenging and metal ion reduction capabilities. The results indicate that peel extracts exhibited higher antioxidant capacity compared with flesh extracts. Using a normal mouse liver cell line, AML-12, we explored the protective effects of loquat extracts and individual compounds against ethanol-induced oxidative stress. The findings demonstrate the enhanced cell viability and the induction of antioxidant enzyme activity through the modulation of Nrf2 and Keap1 gene expression. In a C57/BL6 mouse model of alcohol-induced liver damage, loquat extract was found to alleviate liver injury induced by alcohol. The restoration of perturbed serum liver health indicators underscored the efficacy of loquat extract in reclaiming equilibrium. The culmination of these findings significantly bolsters the foundational knowledge necessary to explore the utilization of loquat fruit extract in the creation of health-focused products.

4D Printing of Biocompatible Scaffolds via In Situ Photo-crosslinking from Shape Memory Copolyesters.

The complexity of surgical treatments for large-area soft tissue injuries makes placing large implants into injury sites challenging. Aliphatic polyesters are often used for scaffold preparation in tissue engineering owing to their excellent biodegradability and biocompatibility. Scaffolds with shape-memory effect (SME) can also avoid large-volume trauma during the implantation. However, the complexity and diversity of diseases require more adaptable and precise processing methods. Four-dimensional (4D) printing, a booming smart material additive manufacturing technology, provides a new opportunity for developing shape memory scaffolds. With the aim of personalized or patient-adaptable soft tissues such as blood vessels, we developed a feasible strategy for fabricating scaffolds with fine architectures using 4D printing crosslinkable shape memory linear copolyesters using fused deposition modeling (FDM). To overcome the weak bonding strength of each printed layer during FDM, a catalyst-free photo-crosslinkable functional group derived from biocompatible cinnamic acid was embedded into the linear copolyesters as in situ crosslinking points during FDM printing. Under ultraviolet-assisted irradiation, the resulting 4D scaffold models demonstrated excellent SME, desirable mechanical performance, and good stability in a water environment owing to the chemical bonding between each layer. Moreover, the excellent biocompatibility of the scaffold was evaluated in vitro and in vivo. The developed composite scaffolds could be used for minimally invasive soft tissue repair.

Shape Memory Polyester Scaffold Promotes Bone Defect Repair through Enhanced Osteogenic Ability and Mechanical Stability.

Bone tissue engineering involving scaffolds is recognized as the ideal approach for bone defect repair. However, scaffold materials exhibit several limitations, such as low bioactivity, less osseointegration, and poor processability, for developing bone tissue engineering. Herein, a bioactive and shape memory bone scaffold was fabricated using the biodegradable polyester copolymer's four-dimensional fused deposition modeling. The poly(ε-caprolactone) segment with a transition temperature near body temperature was selected as the molecular switch to realize the shape memory effect. Another copolymer segment, i.e., poly(propylene fumarate), was introduced for post-cross-linking and improving the regulation effect of the resulting bioadaptable scaffold on osteogenesis. To mimic the porous structures and mechanical properties of the native spongy bone, the pore size of the printed scaffold was set as ∼300 µm, and a comparable compression modulus was achieved after photo-cross-linking. Compared with the pristine poly(ε-caprolactone), the scaffold made from fumarate-functionalized copolymer considerably enhanced the adhesion and osteogenic differentiation of MC3T3-E1 cells in vitro. In vivo experiments indicated that the bioactive shape memory scaffold could quickly adapt to the defect geometry during implantation via shape change, and bone regeneration at the defect site was remarkably promoted, providing a promising strategy to treat bone defects in the clinic, substantial bone defects with irregular geometry.

UBE4A catalyzes NRF1 ubiquitination and facilitates DDI2-mediated NRF1 cleavage.

The transcription factor nuclear factor erythroid 2 like 1 (NFE2L1 or NRF1) regulates constitutive and inducible expression of proteasome subunits and assembly chaperones. The precursor of NRF1 is integrated into the endoplasmic reticulum (ER) and can be retrotranslocated from the ER to the cytosol where it is processed by ubiquitin-directed endoprotease DDI2. DDI2 cleaves and activates NRF1 only when NRF1 is highly polyubiquitinated. It remains unclear how retrotranslocated NRF1 is primed with large amount of ubiquitin and/or very long polyubiquitin chain for subsequent processing. Here, we report that E3 ligase UBE4A catalyzes ubiquitination of retrotranslocated NRF1 and promotes its cleavage. Depletion of UBE4A reduces the amount of ubiquitin modified on NRF1, shortens the average length of polyubiquitin chain, decreases NRF1 cleavage efficiency and causes accumulation of non-cleaved, inactivated NRF1. Expression of a UBE4A mutant lacking ligase activity impairs the cleavage, likely due to a dominant negative effect. UBE4A interacts with NRF1 and the recombinant UBE4A can promote ubiquitination of retrotranslocated NRF1 in vitro. In addition, knocking out UBE4A reduces transcription of proteasomal subunits in cells. Our results indicate that UBE4A primes NRF1 for DDI2-mediated activation to facilitate expression of proteasomal genes.

The pregnancy outcomes of day-5 poor-quality and day-6 high-quality blastocysts in single blastocyst transfer cycles.

OBJECTIVE: This study compared the outcomes of single blastocyst transfer cycles, using day- 5 poor-quality blastocysts and day-6 high-quality blastocysts. METHODS: We analyzed 462 frozen-thawed embryo transfer (FET) cycles performed at our center from January 2014 to December 2019. The cycles were divided into two groups: a day-5 poor-quality blastocyst transfer group (group A) and a day-6 high-quality blastocyst transfer group (group B). The clinical outcomes were tested. RESULTS: In groups A and B, respectively, the clinical pregnancy rate (CPR; 61.65% vs. 67.17%, p=0.258), implantation rate (IR; 61.65% vs. 67.17%, p=0.258), and live birth rate (LBR; 69.51% vs. 77.83%, p=0.134) showed no significant differences. Moreover, when day-3 embryo quality was considered, the CPR, IR, and LBR were also similar in group A and group B (p>0.05). CONCLUSION: The clinical outcomes of day-5 poor-quality blastocysts and day-6 high-quality blastocysts were similar, suggesting that the developmental speed of the embryo might be more important than embryo quality for the clinical outcomes of single blastocyst transfer in FET cycles.

Automation in vitrification and thawing of mouse oocytes and embryos.

Vitrification is a common technique for cryopreserving oocytes or embryos. However, manual vitrification is tedious and labor-intensive, and can be subject to variations caused by human factors. To address these challenges, we developed an automated vitrification-thawing system (AVTS) based on a cryo-handle. Our study firstly assessed the efficiency of cryoprotectant exchange through comparing the osmolalities of fresh and collected solutions during automated vitrification and thawing, and evaluated the cooling and warming rates of the cryo-handle. We also compared mouse oocyte survival, fertilization and embryo development after thawing and ICSI, and the development of re-frozen cleavage embryos between manual operation and automated system. The results showed that the osmolalities of collected samples were within normal range and comparable to fresh solutions. Furthermore, the automated system could obtain the reliable cooling and warming rates. Particularly, there were no significant differences in oocyte survival rates, fertilization rates, and subsequent embryo development and its quality between two procedures. Our findings suggest that AVTS has no impact on osmolalities of vitrification and thawing solutions, ensuring the proper exchange of cryoprotectants. The cryo-handle also shows the ability to achieve reliable cooling and warming rates, which benefits for the cryopreservation and thawing process. Moreover, the results from mouse oocytes and embryos indicate that automated system has effectively maintained the survival and fertilization of frozen oocytes and supported subsequent embryo development. Therefore, the automated vitrification and thawing system will inevitably represent a superior alternative to manual operation.

The pregnancy outcomes of day-5 poor-quality and day-6 high-quality blastocysts in single blastocyst transfer cycles / 대한생식의학회지

Objective@#This study compared the outcomes of single blastocyst transfer cycles, using day- 5 poor-quality blastocysts and day-6 high-quality blastocysts. @*Methods@#We analyzed 462 frozen-thawed embryo transfer (FET) cycles performed at our center from January 2014 to December 2019. The cycles were divided into two groups: a day-5 poor-quality blastocyst transfer group (group A) and a day-6 high-quality blastocyst transfer group (group B). The clinical outcomes were tested. @*Results@#In groups A and B, respectively, the clinical pregnancy rate (CPR; 61.65% vs. 67.17%, p=0.258), implantation rate (IR; 61.65% vs. 67.17%, p=0.258), and live birth rate (LBR; 69.51% vs. 77.83%, p=0.134) showed no significant differences. Moreover, when day-3 embryo quality was considered, the CPR, IR, and LBR were also similar in group A and group B (p>0.05). @*Conclusion@#The clinical outcomes of day-5 poor-quality blastocysts and day-6 high-quality blastocysts were similar, suggesting that the developmental speed of the embryo might be more important than embryo quality for the clinical outcomes of single blastocyst transfer in FET cycles.

Erratum: Lentinan-functionalized Selenium Nanoparticles target Tumor Cell Mitochondria via TLR4/TRAF3/MFN1 pathway: Erratum.

[This corrects the article DOI: 10.7150/thno.46467.].

The effect of ion environment changes on retention protein behavior during whey ultrafiltration process.

The factors affecting membrane fouling are very complex. In this study, the membrane fouling process was revealed from the perspective of ion environment changes, which affected the whey protein structure during ultrafiltration. It was found that the concentrations of Ca2+ and Na+ were overall increased and the concentrations of K+, Mg2+ and Zn2+ were decreased at an ultrafiltration time of 11 min, which made more hydrophilic groups buried inside and increased the content of α-helix, leading to more protein aggregation. The relatively higher K+ ratio in retention could lead to an antiparallel ß-sheet configuration, aspartic acid, glutamic acid and tryptophan increased, which resulted in more protein aggregation and deposition on the membrane surface at 17 min. When the ion concentration and ratio restored the balance and were close to the initial state in retention, the protein surface tension decreased, and the hydrophilic ability increased at 21-24 min.

An Improved Logistic Regression Method for Assessing the Performance of Track and Field Sports.

Track and field is an important part of sports. Track and field athletes are an important reserve force for the development of national sports. An accurate assessment of track and field athletes' performance can help them develop more appropriate training programs and improve their performance. In order to assess the performance of track and field athletes better, this paper proposes an improved logistic regression method. Firstly, this method uses factor analysis to reduce the data dimensions of the factors that affect the performance of track and field athletes, and uses the principal component analysis to select common factors and their corresponding values. Then, according to the common factors, a binary logistic regression model is established to evaluate the performance of track and field athletes. Experiments show that the method can effectively evaluate the performance of track and field athletes and is suitable for athletes of different track and field sports. It has high accuracy, fast evaluation efficiency, and good universality of performance evaluation. For different numbers of athletes, the proposed method has a lower error evaluation index, higher evaluation accuracy, and better evaluation quality. Compared with the other two methods, the proposed method has the shortest evaluation time and is more effective for the performance evaluation of track and field athletes.

TMUB1 is an endoplasmic reticulum-resident escortase that promotes the p97-mediated extraction of membrane proteins for degradation.

Membrane protein clients of endoplasmic reticulum (ER)-associated degradation must be retrotranslocated from the ER membrane by the AAA-ATPase p97 for proteasomal degradation. Before direct engagement with p97, client transmembrane domains (TMDs) that have partially or fully crossed the membrane must be constantly shielded to avoid non-native interactions. How client TMDs are seamlessly escorted from the membrane to p97 is unknown. Here, we identified ER-anchored TMUB1 as a TMD-specific escortase. TMUB1 interacts with the TMD of clients within the membrane and holds ∼10-14 residues of a hydrophobic sequence that is exposed out of membrane, using its transmembrane and cytosolic regions, respectively. The ubiquitin-like domain of TMUB1 recruits p97, which can pull client TMDs from bound TMUB1 into the cytosol. The disruption of TMUB1 escortase activity impairs retrotranslocation and stabilizes retrotranslocating intermediates of client proteins within the ER membrane. Thus, TMUB1 promotes TMD segregation by safeguarding the TMD movement from the membrane to p97.

Reduction of mtDNA heteroplasmy in mitochondrial replacement therapy by inducing forced mitophagy.

Mitochondrial replacement therapy (MRT) has been used to prevent maternal transmission of disease-causing mutations in mitochondrial DNA (mtDNA). However, because MRT requires nuclear transfer, it carries the risk of mtDNA carryover and hence of the reversion of mtDNA to pathogenic levels owing to selective replication and genetic drift. Here we show in HeLa cells, mouse embryos and human embryos that mtDNA heteroplasmy can be reduced by pre-labelling the mitochondrial outer membrane of a donor zygote via microinjection with an mRNA coding for a transmembrane peptide fused to an autophagy receptor, to induce the degradation of the labelled mitochondria via forced mitophagy. Forced mitophagy reduced mtDNA carryover in newly reconstructed embryos after MRT, and had negligible effects on the growth curve, reproduction, exercise capacity and other behavioural characteristics of the offspring mice. The induction of forced mitophagy to degrade undesired donor mtDNA may increase the clinical feasibility of MRT and could be extended to other nuclear transfer techniques.

Nickel-Catalyzed Reductive Csp2-Csp3 Cross Coupling Using Phosphonium Salts.

A nickel-catalyzed reductive cross coupling with phosphonium salts and allylic C(sp3)-O bond electrophiles, which granted direct construction of the C(sp2)-C(sp3) bond, is successfully developed. The protocol features broad substrate scope, high-functional-group tolerance, and heterocycle compatibility. Notably, the much more challenging reductive cross coupling with heterocyclic thiazolylphosphonium salts has also been accomplished for the first time.

Household poverty in people with severe mental illness in rural China: 1994-2015.

BACKGROUND: Little is known about poverty trends in people with severe mental illness (SMI) over a long time span, especially under conditions of fast socioeconomic development. AIMS: This study aims to unravel changes in household poverty levels among people with SMI in a fast-changing rural community in China. METHOD: Two mental health surveys, using ICD-10, were conducted in the same six townships of Xinjin county, Chengdu, China. A total of 711 and 1042 people with SMI identified in 1994 and 2015, respectively, participated in the study. The Foster-Greer-Thorbecke poverty index was adopted to measure the changes in household poverty. These changes were decomposed into effects of growth and equity using a static decomposition method. Factors associated with household poverty in 1994 and 2015 were examined and compared by regression analyses. RESULTS: The proportion of poor households, as measured by the headcount ratio, increased significantly from 29.8% in 1994 to 39.5% in 2015. Decomposition showed that poverty in households containing people with SMI had worsened because of a redistribution effect. Factors associated with household poverty had also changed during the study period. The patient's age, ability to work and family size were of paramount significance in 2015. CONCLUSIONS: This study shows that the levels of poverty faced by households containing people with SMI has become more pressing with China's fast socioeconomic development. It calls for further integration of mental health recovery and targeted antipoverty interventions for people with SMI as a development priority.

Lentinan-functionalized Selenium Nanoparticles target Tumor Cell Mitochondria via TLR4/TRAF3/MFN1 pathway.

Rationale: Malignant ascites caused by cancer cells results in poor prognosis and short average survival time. No effective treatment is currently available for malignant ascites. In this study, the effects of lentinan (LNT)-functionalized selenium nanoparticles (Selene) on malignant ascites were evaluated. Furthermore, the mechanism of Selene targeting mitochondria of tumor cells were also investigated. Methods: Selene were synthesized and characterized by TEM, AFM and particle size analysis. The OVCAR-3 and EAC cells induced ascites models were used to evaluate the effects of Selene on malignant ascites. Proteomic analysis, immunofluorescence, TEM and ICP-MS were used to determine the location of Selene in tumor cells. Mitochondrial membrane potential, ROS, ATP content, and caspase-1/3 activity were detected to evaluate the effect of Selene on mitochondrial function and cell apoptosis. Immunofluorescence, Co-IP, pull-down, duolink, Western blot, and FPLC were used to investigate the pathway of Selene targeting mitochondria. Results: Selene could effectively inhibit ascites induced by OVCAR-3 and EAC cells. Selene was mainly located in the mitochondria of tumor cells and induced apoptosis of tumor cells. The LNT in Selene was involved in caveolae-mediated endocytosis through the interaction between toll-like receptor-4 (TLR4) and caveolin 1 (CAV1). Furthermore, the Selene in the endocytic vesicles could enter the mitochondria via the mitochondrial membrane fusion pathway, which was mediated by TLR4/TNF receptor associated factor 3 (TRAF3)/mitofusin-1 (MFN1) protein complex. Conclusion: Selene is a candidate anticancer drug for the treatment of malignant ascites. And TLR4/TRAF3/MFN1 may be a specific nano-drug delivery pathway that could target the mitochondria.

Isolation, X-ray crystal structure of the new diterpene and identification of others lignans and flavonoids from the fresh needles of Pinus massoniana.

Three new compounds, namely massonside C (1), massonianoside F (2), and 3, 8-dimethyl- herbacetin-7-O-ß-D-glucopyranoside (3), together with five known compounds (4-8), were isolated from the fresh needles of Pinus massoniana. Their structures were established by 1D, 2D NMR, HRMS and comparison with the literature data. The absolute configuration of 1 was confirmed by a combination of X-ray single crystal analysis. All isolated compounds were evaluated for the protective effect of human umbilical vein endothelial cells against oxidative damage.

A single-nucleotide polymorphism induced alternative splicing in Tacr3 involves in hypoxic-ischemic brain damage.

Single-nucleotide polymorphism (SNP) and Alternative splicing (AS) were found to be implicated in certain diseases, nevertheless, the contributions of mRNA SNPs and AS to pathogenesis in developing rat brains with hypoxic-ischemic encephalopathy (HIE) remained largely vague. Additionally, the disease associated with Tacr3 was normosmic congenital hypogonadotropic hypogonadism, while the relationship between HIE and Tacr3 remained largely elusive. The current study was designed to investigate the differentially expressed mRNAs and related SNPs as well as AS in neonatal rats subjected to HIE to identify if the exhibition of AS was associated with SNPs under pathological condition. Firstly, we used postnatal day 7 Sprague-Dawley rats to construct neonatal HIE model, and analyzed the expression profiles of SNP mRNA in hypoxic-ischemic (HI) and sham brains by using RNA sequencing. Then four genes, including Mdfic, Lpp, Bag3 and Tacr3, connecting with HIE and exhibiting SNPs and AS were identified by bioinformatics analysis. Moreover, combined with exonic splicing enhancer (ESE) and alternative splice site predictor (ASSP) analysis, we found that Tacr3 is associated specifically with HIE through 258547789 G > A SNP in inside the Alt First Exon and 258548573 G > A SNP in outside the Alt First Exon. Taken together, our study provides new evidence to understand the role of Tacr3 in HIE and it is possibly a potential target for the treatment of HIE in future clinic trial.