A novel machine learning-based imputation strategy for missing data in step-stress accelerated degradation test.

The presence of missing data is a significant data quality issue that negatively impacts the accuracy and reliability of data analysis. This issue is especially relevant in the context of accelerated tests, particularly for step-stress accelerated degradation tests. While missing data can occur due to objective factors or human error, high missing rate is an inevitable pattern of missing data that will occur during the conversion process of accelerated test data. This type of missing data manifests as a degradation dataset with unequal measuring intervals. Therefore, developing a more appropriate imputation method for accelerated test data is essential. In this study, we propose a novel hybrid imputation method that combines the LSSVM and RBF models to address missing data problems. A comparison is conducted between the proposed model and various traditional and machine learning imputation methods using simulation data, to justify the advantages of the proposed model over the existing methods. Finally, the proposed model is implemented on real degradation datasets of the super-luminescent diode (SLD) to validate its performance and effectiveness in dealing with missing data in step-stress accelerated degradation test. Additionally, due to the generalizability of the proposed method, it is expected to be applicable in other scenarios with high missing data rates.

[FMECA and Failure Data Analysis of Domestic Wheeled Walking-Aid].

The gradual acceleration of the aging population in China has led to an increased demand for mobility aids, and the reliability of domestic wheeled walking-aid is one of their important attributes, but there is little research on the reliability of mobility aids. This paper conducts the failure mode, effects and criticality analysis on domestic wheeled walking-aid. By collecting, collating and analyzing the 26 failure modes, the key modules are the chassis and the lifting system. The key modules are obtained, and the failure data is analyzed to find out that the lifetime distribution model is log-normal and the 0.95 lower confidence limit for reliability life of product t 0.9 is 2489.4 hours. The study aims to provide ideas for the reliability analysis of other active medical devices and calls for the formation of a reliability study review point for the industry that meets the requirements of Chinese medical device regulations.

Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells.

Base editors show promise for treating human genetic diseases, but most current systems use deaminases, which cause off-target effects and are limited in editing type. In this study, we constructed deaminase-free base editors for cytosine (DAF-CBE) and thymine (DAF-TBE), which contain only a cytosine-DNA or a thymine-DNA glycosylase (CDG/TDG) variant, respectively, tethered to a Cas9 nickase. Multiple rounds of mutagenesis by directed evolution in Escherichia coli generated two variants with enhanced base-converting activity-CDG-nCas9 and TDG-nCas9-with efficiencies of up to 58.7% for C-to-A and 54.3% for T-to-A. DAF-BEs achieve C-to-G/T-to-G editing in mammalian cells with minimal Cas9-dependent and Cas9-independent off-target effects as well as minimal RNA off-target effects. Additional engineering resulted in DAF-CBE2/DAF-TBE2, which exhibit altered editing windows from the 5' end to the middle of the protospacer and increased C-to-G/T-to-G editing efficiency of 3.5-fold and 1.2-fold, respectively. Compared to prime editing or CGBEs, DAF-BEs expand conversion types of base editors with similar efficiencies, smaller sizes and lower off-target effects.

BPA interferes with granulosa cell development and oocyte meiosis in mouse preantral follicles.

Bisphenol A (BPA) is an established environmental endocrine disruptor and can interfere with the development of female germ cells. However, the underlying mechanisms are still unclear. We investigated the effects of BPA on granulosa cell development and meiosis of oocytes using in vitro culture system of mouse preantral follicles. Preantral follicles from D14 mouse ovary were treated with 10 µg/mL BPA in vitro for 11 days. The adherent area of follicles was measured. On D11, cumulus cell expansion was observed. The meiosis recovery rate was calculated. Western blot detected P53, proliferating cell nuclear antigen (PCNA), estrogen receptor α (ERα), and cyclin B1. ELISA measured estrogen and progesterone levels. Immunofluorescence detected Cx37 on oocyte membrane. Gap junction communication was assessed. We found that BPA significantly promoted the expressions of PCNA and ERα in granulosa cells and the secretion of estrogen and progesterone by granulosa cells on D10 and significantly increased the attachment area of the follicles on D8 and D10. However, it reduced the expansion of cumulus cells, Cx37 expression, and the gap junction communication between cumulus cells and oocytes on D11. BPA promoted the recovery of oocytes from meiosis, interrupted the expression of cyclin B1 protein in arrested germinal vesicle breakdown (GVBD) oocytes, and reduced the in vitro maturation rate of oocytes. These GVBD oocytes were live without apoptosis or death. Conclusively, BPA disturbs the development of granulosa cells and the meiosis progression of oocytes by decreasing gap junction communication between oocytes and the granulosa cells as well as regulating cyclin B1 expression in GVBD oocytes.

HMGN1 enhances CRISPR-directed dual-function A-to-G and C-to-G base editing.

C-to-G base editors have been successfully constructed recently, but limited work has been done on concurrent C-to-G and A-to-G base editing. In addition, there is also limited data on how chromatin-associated factors affect the base editing. Here, we test a series of chromatin-associated factors, and chromosomal protein HMGN1 was found to enhance the efficiency of both C-to-G and A-to-G base editing. By fusing HMGN1, GBE and ABE to Cas9, we develop a CRISPR-based dual-function A-to-G and C-to-G base editor (GGBE) which is capable of converting simultaneous A and C to G conversion with substantial editing efficiency. Accordingly, the HMGN1 role shown in this work and the resulting GGBE tool further broaden the genome manipulation capacity of CRISPR-directed base editors.

Obtaining the best igRNAs for bystander-less correction of all ABE-reversible pathogenic SNVs using high-throughput screening.

Imperfect -gRNA (igRNA) provides a simple strategy for single-base editing of a base editor. However, a significant number of igRNAs need to be generated and tested for each target locus to achieve efficient single-base reversion of pathogenic single nucleotide variations (SNVs), which hinders the direct application of this technology. To provide ready-to-use igRNAs for single-base and bystander-less correction of all the adenine base editor (ABE)-reversible pathogenic SNVs, we employed a high-throughput method to edit all 5,253 known ABE-reversible pathogenic SNVs, each with multiple systematically designed igRNAs, and two libraries of 96,000 igRNAs were tested. A total of 1,988 SNV loci could be single-base reversed by igRNA with a >30% efficiency. Among these 1,988 loci, 378 SNV loci exhibited an efficiency of more than 90%. At the same time, the bystander editing efficiency of 76.62% of the SNV loci was reduced to 0%, while remaining below 1% for another 18.93% of the loci. These ready-to-use igRNAs provided the best solutions for a substantial portion of the 4,657 pathogenic/likely pathogenic SNVs. In this work, we overcame one of the most significant obstacles of base editors and provide a ready-to-use platform for the genetic treatment of diseases caused by ABE-reversible SNVs.

Automated high-throughput genome editing platform with an AI learning in situ prediction model.

A great number of cell disease models with pathogenic SNVs are needed for the development of genome editing based therapeutics or broadly basic scientific research. However, the generation of traditional cell disease models is heavily dependent on large-scale manual operations, which is not only time-consuming, but also costly and error-prone. In this study, we devise an automated high-throughput platform, through which thousands of samples are automatically edited within a week, providing edited cells with high efficiency. Based on the large in situ genome editing data obtained by the automatic high-throughput platform, we develop a Chromatin Accessibility Enabled Learning Model (CAELM) to predict the performance of cytosine base editors (CBEs), both chromatin accessibility and the context-sequence are utilized to build the model, which accurately predicts the result of in situ base editing. This work is expected to accelerate the development of BE-based genetic therapies.

[Research on the current situations of reliability and evaluation in Chinese active medical device industry].

Active medical device is a kind of medical device which is widely used. In order to realize the goal of high-quality development, product with high reliability is a necessary requirement for the domestic active medical device industry. By means of literature research, data collection, field research, materials comprehensive combing and analysis, this paper systematically analyzes and studies the current situations and the existing problems of reliability and evaluation from the dimensions of Chinese active medical device industry policy, enterprise situation and evaluation method. In addition, by considering the technical characteristics of reliability work, concrete suggestions for solving the problems are given from the directions of standard and guiding principle, so as to provide reference for active medical device industry to develop scientific and objective reliability technical standard system and guiding principle, which are in accord with the current characteristics of Chinese active medical device industry and supervision.

Astaxanthin improves the development of the follicles and oocytes through alleviating oxidative stress induced by BPA in cultured follicles.

This study is to investigate whether astaxanthin could alleviate the oxidative stress damages of follicles induced by BPA and improve the development of the cultured follicles and oocytes. Compared with BPA group, the survival rate, antrum formation rate, oocyte maturation rate and adherence area of the D8 and D10 follicles of the BPA+Asta group were significantly higher. The estrogen and progesterone in the culture medium of BPA+Asta group were significantly higher. PCNA in D8 and D10 granulosa cells and ERα in D10 granulosa cells of follicles in BPA+Asta group were significantly higher. The levels of malondialdehyde in the follicle culture medium, levels of ROS in the oocytes, the expression levels of caspase 3 and cathepsin B in the oocytes of the BPA+Asta group were significantly lower. However, the mitochondrial membrane potential, and the expression levels of antioxidant genes (CAT, SOD1 and SOD2) and anti-apoptotic gene Bcl-2 in the oocytes in the BPA+Asta group were significantly higher. Astaxanthin improves the development of follicles and oocytes through increasing the antioxidant capacity of follicles and oocytes, and relieving the BPA-induced oxidative stress during follicular development and oocyte maturation.

Molecular Mechanism of the Cytosine CRISPR Base Editing Process and the Roles of Translesion DNA Polymerases.

CRISPR-mediated base editing causes damage to DNA, mainly uracil, apurinic/apyrimidinic (AP) sites, and nicks, which require various DNA repair mechanisms to complete the base conversion process. Currently, there are only hypotheses explaining the base editing process, but the molecular mechanism and roles of the repair systems in the process are relatively unknown. To explore the mechanism of base editing repair, a base editor, nCas9-PmCDA1, was applied in the model eukaryote, Saccharomyces cerevisiae, either with the wild type or its derivatives with genes encoding translesion DNA synthesis (TLS) polymerases knocked out. We found that C-to-G and C-to-A conversions resulted mainly from the repair of AP sites created by Ung and required Polζ as an extender. Rev1 is the main TLS polymerase for specifically incorporating Cs on the opposite position of AP sites to cause the dominant C-to-G conversion, while Polδ incorporates Ts or As on the opposite of AP sites, resulting in C-to-A and C-to-T conversions. Polη is not involved in the repair of AP sites caused by the base editor. Furthermore, our data suggested that the indels of base editing are mainly caused by the breakage of AP sites. Different from the current hypothesis model of the base editing mechanism, this work first elucidates the key roles of TLS polymerases in the cytosine base editing process. This work also suggests a new direction for the development of genomic and base editing techniques by employing, manipulating, and engineering TLS polymerases.

Endothelial nitric oxide synthase activation is required for heparin receptor effects on vascular smooth muscle cells.

Published studies indicate that TMEM184A is a heparin receptor that interacts with and transduces stimulation from heparin in vascular cells. Previous studies have indicated that heparin increases endothelial nitric oxide synthase (eNOS) activity in bovine endothelial cells. However, the precise mechanism remains unknown. In this study, we investigated the impact of heparin treatment and TMEM184A on eNOS's activation and the role of eNOS in heparin signaling in the cloned A7r5 rat vascular smooth muscle cell line and confirmed results in endothelial cells. We employed a combination of TMEM184A knockdown A7r5 cells along with transient eNOS knockdown and enzyme inhibitor strategies. The results indicate that heparin induces phosphorylation of eNOS. eNOS can be immunoprecipitated with TMEM184A and is internalized to the perinuclear region in a TMEM184A-dependent manner in response to heparin. We also examined how heparin treatment leads to phosphorylation of eNOS and confirmed that TMEM184A and Ca2+ were required to mediate heparin-elicited eNOS phosphorylation. Evidence supporting the involvement of transient receptor potential cation channel subfamily V member 4 with TMEM184A in this eNOS activation process is also presented.

Development of quantitative structure-activity relationship models to predict potential nephrotoxic ingredients in traditional Chinese medicines.

The broad use of traditional Chinese medicines (TCMs) and the accompanied incidences of kidney injury have attracted considerable interest in investigating the responsible toxic ingredients. It is challenging to evaluate toxicity of TCMs since they contain complex mixtures of phytochemicals. Quantitative structure-activity relationship (QSAR) is an efficient tool to predict toxicity and QSAR study on TCMs-induced nephrotoxicity remains lacked. We developed QSAR models using three datasets of 609 compounds: natural products, drugs, and mixed (contained both kinds of data) datasets. Each dataset was used for modelling by utilizing artificial neural networks (ANN) and support vector machines (SVM) algorithms separately. Both internal and external validations were performed on each model. Six QSAR models were developed and yielded reliable performance in the internal validation. For external validation, 30 ingredients in the TCMs were predicted well by the natural product models (accuracy: ANN 96.7%, SVM 93.3%). The mixed models (accuracy: ANN 76.7%, SVM 66.7%) showed a better performance than the drug models (accuracy: ANN 50%, SVM 53.3%). Particularly, natural product models produced the most reliable results. It has the application not only on screening the nephrotoxic ingredients in TCMs, but it is also helpful at prioritizing the subsequent toxicity testing of natural products.

A Life Prediction Model of Multilayered PTH Based on Fatigue Mechanism.

Plated through hole (PTH) plays a critical role in printed circuit board (PCB) reliability. Thermal fatigue deformation of the PTH material is regarded as the primary factor affecting the lifetime of electrical devices. Numerous research efforts have focused on the failure mechanism model of PTH. However, most of the existing models were based on the one-dimensional structure hypothesis without taking the multilayered structure and external pad into consideration. In this paper, the constitutive relation of multilayered PTH is developed to establish the stress equation, and finite element analysis (FEA) is performed to locate the maximum stress and simulate the influence of the material properties. Finally, thermal cycle tests are conducted to verify the accuracy of the life prediction results. This model could be used in fatigue failure portable diagnosis and for life prediction of multilayered PCB.

Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity.

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies against specific peptides within the predicted sequence allow some localization experiments. However, other possible interactions with the antibodies often cannot be excluded. This situation provided an opportunity to develop a set of assays dependent on the protein sequence. Specifically, a construct containing the gene sequence coupled to the GFP coding sequence at the C-terminal end of the protein was obtained and employed for these purposes. Experiments to characterize localization, ligand affinity, and gain of function were originally designed and carried out to confirm the identification of TMEM184A as a heparin receptor1. In addition, the construct can be employed for studies addressing membrane topology questions and detailed protein-ligand interactions. The present report presents a range of experimental protocols based on the GFP-TMEM184A construct expressed in vascular cells that could easily be adapted for other novel proteins.

[In vivo study of the renoprotective effects of EGCG in diabetic db/db mice].

OBJECTIVE: To explore the renoprotective effects of (-)-epigallocatechin-3-gallate (EGCG) and its potential mechanism in type 2 diabetic db/db mice. METHODS: 8-week-old db/db mice (6 h fasting plasma glucose >16.7 mmol/L) were allocated randomly into Control group (non-intervention group, n=8), EGCG A group (50 mg·kg(-1)·d(-1,)n=8), EGCG B group (100 mg·kg(-1)·d(-1,)n=8). Before the study and after the intervention (in the 4(th)and 8(th)week), the body weight, the level of fasting plasma glucose, oral glucose tolerance test (OGTT) were measured and 24 h urine samples were collected. 24 h proteinuria was measured by routine chemical method. The levels of angiotensin Ⅱ(AngⅡ), fasting plasma insulin and urinary 8-OHdG were measured with enzyme-linked immunosorbent assay (ELISA). The protein expression levels of angiotensin Ⅱ type 1 receptor (AT-1R), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit P22-phox, NADPH oxidase subunit P47-phox, phospho-extracellular regulated protein kinases (p-Erk1/2), phospho-P38 mitogen-activated protein kinase (p-P38MAPK), phospho -phosphatidylinositol 3-hydroxy kinase (p-PI3K) and phospho-protein kinase B (p-AKT) were determined by Western blot. The renal pathological changes were examined by the method of PAS (periodic acid-Schiff stain). RESULTS: After 8 weeks of treatment with EGCG, the level of fasting plasma glucose decreased[(14.4±1.0) mmol/L, (14.2±0.7) mmol/L vs. (17.2±0.8) mmol/L]; the level of fasting plasma insulin increased[(13.2±1.2)mU/L, (13.4±1.3) mU/L vs. (9.9±1.0) mU/L]; the area under the curve (AUC) of OGTT decreased[(49.3±1.8) mmol·L(-1)·h(-1,)(44.8±0.7) mmol·L(-1)·h(-1)vs. (60.0±0.8) mmol·L(-1)·h(-1)]; the level of 24 h proteinuria[(8.8±1.0) mg, (8.6±1.1) mg vs. (11.7±1.3) mg]and urinary 8-OHdG[(90±5) ng/d, (78±5) ng/d vs. (118±10) ng/d]decreased; the level of serum Ang-Ⅱ[(498±23) ng/L, (511±19) ng/L vs. (688±17) ng/L]and renal cortex AngⅡ[(367±5) ng/L, (384±10) ng/L vs. (406±7) ng/L]decreased; the expression levels of AT-1R, P22-phox, P47-phox, p-Erk1/2, p-P38MAPK downregulated obviously and the expression levels of p-PI3K, p-AKT increased significantly (P<0.05), and renal pathology improved as compared with the control group. After 8 weeks of treatment with EGCG, the level of urinary 8-OHdG decreased (P=0.007) and the AUC of OGTT also decreased (P=0.01) in EGCG B group when compared with the EGCG A group. CONCLUSION: EGCG protects the kidney in diabetic db/db mice via anti-oxidative stress pathway, as well as inhibiting Erk1/2-P38MAPK pathway and improving PI3K-AKT signaling transduction pathway.

Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin.

Vascular cell responses to exogenous heparin have been documented to include decreased vascular smooth muscle cell proliferation following decreased ERK pathway signaling. However, the molecular mechanism(s) by which heparin interacts with cells to induce those responses has remained unclear. Previously characterized monoclonal antibodies that block heparin binding to vascular cells have been found to mimic heparin effects. In this study, those antibodies were employed to isolate a heparin binding protein. MALDI mass spectrometry data provide evidence that the protein isolated is transmembrane protein 184A (TMEM184A). Commercial antibodies against three separate regions of the TMEM184A human protein were used to identify the TMEM184A protein in vascular smooth muscle cells and endothelial cells. A GFP-TMEM184A construct was employed to determine colocalization with heparin after endocytosis. Knockdown of TMEM184A eliminated the physiological responses to heparin, including effects on ERK pathway activity and BrdU incorporation. Isolated GFP-TMEM184A binds heparin, and overexpression results in additional heparin uptake. Together, these data support the identification of TMEM184A as a heparin receptor in vascular cells.

A Model of BGA Thermal Fatigue Life Prediction Considering Load Sequence Effects.

Accurate testing history data is necessary for all fatigue life prediction approaches, but such data is always deficient especially for the microelectronic devices. Additionally, the sequence of the individual load cycle plays an important role in physical fatigue damage. However, most of the existing models based on the linear damage accumulation rule ignore the sequence effects. This paper proposes a thermal fatigue life prediction model for ball grid array (BGA) packages to take into consideration the load sequence effects. For the purpose of improving the availability and accessibility of testing data, a new failure criterion is discussed and verified by simulation and experimentation. The consequences for the fatigue underlying sequence load conditions are shown.