Fluorogenic Rhodamine-Based Chemigenetic Biosensor for Monitoring Cellular NADPH Dynamics.

Ratiometric biosensors employing Förster Resonance Energy Transfer (FRET) enable the real-time tracking of metabolite dynamics. Here, we introduce an approach for generating a FRET-based biosensor in which changes in apparent FRET efficiency rely on the analyte-controlled fluorogenicity of a rhodamine rather than the commonly used distance change between donor-acceptor fluorophores. Our fluorogenic, rhodamine-based, chemigenetic biosensor (FOCS) relies on a synthetic, protein-tethered FRET probe, in which the rhodamine acting as the FRET acceptor switches in an analyte-dependent manner from a dark to a fluorescent state. This allows ratiometric sensing of the analyte concentration. We use this approach to generate a chemigenetic biosensor for nicotinamide adenine dinucleotide phosphate (NADPH). FOCS-NADPH exhibits a rapid and reversible response toward NAPDH with a good dynamic range, selectivity, and pH insensitivity. FOCS-NADPH allows real-time monitoring of cytosolic NADPH fluctuations in live cells during oxidative stress or after drug exposure. We furthermore used FOCS-NADPH to investigate NADPH homeostasis regulation through the pentose phosphate pathway of glucose metabolism. FOCS-NADPH is a powerful tool for studying NADPH metabolism and serves as a blueprint for the development of future fluorescent biosensors.

Maternal mRNA deadenylation is defective in in vitro matured mouse and human oocytes.

Oocyte in vitro maturation is a technique in assisted reproductive technology. Thousands of genes show abnormally high expression in in vitro maturated metaphase II (MII) oocytes compared to those matured in vivo in bovines, mice, and humans. The mechanisms underlying this phenomenon are poorly understood. Here, we use poly(A) inclusive RNA isoform sequencing (PAIso-seq) for profiling the transcriptome-wide poly(A) tails in both in vivo and in vitro matured mouse and human oocytes. Our results demonstrate that the observed increase in maternal mRNA abundance is caused by impaired deadenylation in in vitro MII oocytes. Moreover, the cytoplasmic polyadenylation of dormant Btg4 and Cnot7 mRNAs, which encode key components of deadenylation machinery, is impaired in in vitro MII oocytes, contributing to reduced translation of these deadenylase machinery components and subsequently impaired global maternal mRNA deadenylation. Our findings highlight impaired maternal mRNA deadenylation as a distinct molecular defect in in vitro MII oocytes.

Allelic reprogramming of chromatin states in human early embryos.

The reprogramming of parental epigenomes in human early embryos remains elusive. To what extent the characteristics of parental epigenomes are conserved between humans and mice is currently unknown. Here, we mapped parental haploid epigenomes using human parthenogenetic and androgenetic embryos. Human embryos have a larger portion of genome with parentally specific epigenetic states than mouse embryos. The allelic patterns of epigenetic states for orthologous regions are not conserved between humans and mice. Nevertheless, it is conserved that maternal DNA methylation and paternal H3K27me3 are associated with the repression of two alleles in humans and mice. In addition, for DNA-methylation-dependent imprinting, we report 19 novel imprinted genes and their associated germline differentially methylated regions. Unlike in mice, H3K27me3-dependent imprinting is not observed in human early embryos. Collectively, allele-specific epigenomic reprogramming is different in humans and mice.

Double joystick technique - a modified method facilitates operation of Gartlend type-â ¢ supracondylar humeral fractures in children.

Gartland type-Ⅲ supracondylar humerus fracture (SCHF) is a severe lesion with the feature of difficult reduction. Due to the high failure rate of traditional reduction, a more practical and safer method is needed. This retrospective study aimed to explore the effectiveness of the double joystick technique during the closed reduction of children with type-III fractures. Forty-one children with Gartland type-Ⅲ SCHF underwent closed reduction and percutaneous fixation using the double joystick technique at our hospital between June 2020 and June 2022, and 36 (87.80%) patients were successfully followed up. The affected elbow was evaluated by the joint motion, radiographs, and Flynn's criteria then contrasted with the contralateral elbow at the last follow-up. A group of 29 boys and seven girls with an average age of 6.33 ±â€…2.68 years. The mean time of surgery and hospital stay was 26.61 ±â€…7.51 min and 4.64 ±â€…1.23 days, respectively. After a mean follow-up of 12.85 months, the average Baumann angle was 73.43 ±â€…3.78°, although the average carrying angle (11.33 ±â€…2.17°), flexion angle (143.03 ±â€…5.15°), and extension angle (0.89 ±â€…3.23°) of the affected elbow were less than those of the contralateral elbow ( P  < 0.05), the mean range of motion difference between two sides is only 3.39 ±â€…1.59°, with no complications. Furthermore, 100% of patients recovered satisfactorily, with excellent outcomes (91.67%) and good outcomes (8.33%). The double joystick technique is a safe and effective method that facilitates the closed reduction of Gartland type-Ⅲ SCHF in children without raising the risk of complications.

Population pharmacokinetics of Amisulpride in Chinese patients with schizophrenia with external validation: the impact of renal function.

Introduction: Amisulpride is primarily eliminated via the kidneys. Given the clear influence of renal clearance on plasma concentration, we aimed to explicitly examine the impact of renal function on amisulpride pharmacokinetics (PK) via population PK modelling and Monte Carlo simulations. Method: Plasma concentrations from 921 patients (776 in development and 145 in validation) were utilized. Results: Amisulpride PK could be described by a one-compartment model with linear elimination where estimated glomerular filtration rate, eGFR, had a significant influence on clearance. All PK parameters (estimate, RSE%) were precisely estimated: apparent volume of distribution (645 L, 18%), apparent clearance (60.5 L/h, 2%), absorption rate constant (0.106 h-1, 12%) and coefficient of renal function on clearance (0.817, 10%). No other significant covariate was found. The predictive performance of the model was externally validated. Covariate analysis showed an inverse relationship between eGFR and exposure, where subjects with eGFR= 30 mL/min/1.73 m2 had more than 2-fold increase in AUC, trough and peak concentration. Simulation results further illustrated that, given a dose of 800 mg, plasma concentrations of all patients with renal impairment would exceed 640 ng/mL. Discussion: Our work demonstrated the importance of renal function in amisulpride dose adjustment and provided a quantitative framework to guide individualized dosing for Chinese patients with schizophrenia.

Lung Sound Recognition Method Based on Multi-Resolution Interleaved Net and Time-Frequency Feature Enhancement.

Air pollution and aging population have caused increasing rates of lung diseases and elderly lung diseases year by year. At the same time, the outbreak of COVID-19 has brought challenges to the medical system, which placed higher demands on preventing lung diseases and improving diagnostic efficiency to some extent. Artificial intelligence can alleviate the burden on the medical system by analyzing lung sound signals to help to diagnose lung diseases. The existing models for lung sound recognition have challenges in capturing the correlation between time and frequency information. It is difficult for convolutional neural network to capture multi-scale features across different resolutions, and the fusion of features ignores the difference of influences between time and frequency features. To address these issues, a lung sound recognition model based on multi-resolution interleaved net and time-frequency feature enhancement was proposed, which consisted of a heterogeneous dual-branch time-frequency feature extractor (TFFE), a time-frequency feature enhancement module based on branch attention (FEBA), and a fusion semantic classifier based on semantic mapping (FSC). TFFE independently extracts the time and frequency information of lung sounds through a multi-resolution interleaved net and Transformer, which maintains the correlation between time-frequency features. FEBA focuses on the differences in the influence of time and frequency information on prediction results by branch attention. The proposed model achieved an accuracy of 91.56% on the combined dataset, by an improvement of over 2.13% compared to other models.

Fluorogenic and Cell-Permeable Rhodamine Dyes for High-Contrast Live-Cell Protein Labeling in Bioimaging and Biosensing.

The advancement of fluorescence microscopy techniques has opened up new opportunities for visualizing proteins and unraveling their functions in living biological systems. Small-molecule organic dyes, which possess exceptional photophysical properties, small size, and high photostability, serve as powerful fluorescent reporters in protein imaging. However, achieving high-contrast live-cell labeling of target proteins with conventional organic dyes remains a considerable challenge in bioimaging and biosensing due to their inadequate cell permeability and high background signal. Over the past decade, a novel generation of fluorogenic and cell-permeable dyes has been developed, which have substantially improved live-cell protein labeling by fine-tuning the reversible equilibrium between a cell-permeable, nonfluorescent spirocyclic state (unbound) and a fluorescent zwitterion (protein-bound) of rhodamines. In this review, we present the mechanism and design strategies of these fluorogenic and cell-permeable rhodamines, as well as their applications in bioimaging and biosensing.

Limitations of gene editing assessments in human preimplantation embryos.

Range of DNA repair in response to double-strand breaks induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.

Population pharmacokinetics of buprenorphine and naloxone sublingual combination in Chinese healthy volunteers and patients with opioid use disorder: Model-based dose optimization.

The sublingual combination of buprenorphine (BUP) and naloxone (NLX) is a new treatment option for opioid use disorder (OUD) and is effective in preventing drug abuse. This study aimed to explore rational dosing regimen for OUD patients in China via a model-based dose optimization approach. BUP, norbuprenorphine (norBUP), and NLX plasma concentrations of 34 healthy volunteers and 12 OUD subjects after single or repeated dosing were included. A parent-metabolite population pharmacokinetics (popPK) model with transit compartments for absorption was implemented to describe the pharmacokinetic profile of BUP-norBUP. In addition, NLX concentrations were well captured by a one-compartment popPK model. Covariate analysis showed that every additional swallow after the administration within the observed range (0-12) resulted in a 3.5% reduction in BUP bioavailability. This provides a possible reason for the less-than-dose proportionality of BUP. There were no differences in the pharmacokinetic characteristics between BUP or NLX in healthy volunteers and OUD subjects. Ethnic sensitivity analysis demonstrated that the dose-normalized peak concentration and area-under-the-curve of BUP in Chinese were about half of Puerto Ricans, which was consistent with a higher clearance observed in Chinese (166 L / h vs. 270 L / h ). Furthermore, Monte Carlo simulations showed that an 8 mg three-times daily dose was the optimized regimen for Chinese OUD subjects. This regimen ensured that opioid receptor occupancy remained at a maximum (70%) in more than 95% of subjects, at the same time, with NLX plasma concentrations below the withdrawal reaction threshold (4.6  n g / m L ).

Remodeling of maternal mRNA through poly(A) tail orchestrates human oocyte-to-embryo transition.

Poly(A)-tail-mediated post-transcriptional regulation of maternal mRNAs is vital in the oocyte-to-embryo transition (OET). Nothing is known about poly(A) tail dynamics during the human OET. Here, we show that poly(A) tail length and internal non-A residues are highly dynamic during the human OET, using poly(A)-inclusive RNA isoform sequencing (PAIso-seq). Unexpectedly, maternal mRNAs undergo global remodeling: after deadenylation or partial degradation into 3'-UTRs, they are re-polyadenylated to produce polyadenylated degradation intermediates, coinciding with massive incorporation of non-A residues, particularly internal long consecutive U residues, into the newly synthesized poly(A) tails. Moreover, TUT4 and TUT7 contribute to the incorporation of these U residues, BTG4-mediated deadenylation produces substrates for maternal mRNA re-polyadenylation, and TENT4A and TENT4B incorporate internal G residues. The maternal mRNA remodeling is further confirmed using PAIso-seq2. Importantly, maternal mRNA remodeling is essential for the first cleavage of human embryos. Together, these findings broaden our understanding of the post-transcriptional regulation of maternal mRNAs during the human OET.

Human zygotic genome activation is initiated from paternal genome.

Although parental genomes undergo extensive epigenetic reprogramming to be equalized after fertilization, whether they play different roles in human zygotic genome activation (ZGA) remains unknown. Here, we mapped parental transcriptomes by using human parthenogenetic (PG) and androgenetic (AG) embryos during ZGA. Our data show that human ZGA is launched at the 8-cell stage in AG and bi-parental embryos, but at the morula stage in PG embryos. In contrast, mouse ZGA occurs at the same stage in PG and AG embryos. Mechanistically, primate-specific ZNF675 with AG-specific expression plays a role in human ZGA initiated from paternal genome at the 8-cell stage. AG-specifically expressed LSM1 is also critical for human maternal RNA degradation (MRD) and ZGA. The allelic expressions of ZNF675 and LSM1 are associated with their allelically epigenetic states. Notably, the paternally specific expressions of ZNF675 and LSM1 are also observed in diploid embryos. Collectively, human ZGA is initiated from paternal genome.

Lung Sound Recognition Method Based on Wavelet Feature Enhancement and Time-Frequency Synchronous Modeling.

Lung diseases are serious threats to human health and life, therefore, an accurate diagnosis of lung diseases is significant. The use of artificial intelligence to analyze lung sounds can aid in diagnosing lung diseases. Most of the existing lung sound recognition methods ignore the correlation between the time-domain and frequency-domain information of the lung sounds. Additionally, the spectrograms used in these models do not adequately capture the detailed features of the lung sounds. This paper proposes a model based on wavelet feature enhancement and time-frequency synchronous modeling, comprising a dual wavelet analysis module (DWAM), a cubic network, and an attention module. DWAM in the model performed a dual wavelet transformation on the spectrograms to extract the detailed features of the lung sounds. The cubic network comprised multiple cubic gated recursive units to capture the correlation of the time-frequency of the lung sounds using the time-frequency synchronous modeling. The attention module, which includes temporal and channel attention, was used to enhance the time-domain and channel dimension features. In the combined dataset and the International Conference on Biomedical and Health Informatics 2017 dataset, the suggested framework outperforms existing models by more than 1.36% and 4.28%, respectively.

Functional substitution of zona pellucida with modified sodium hyaluronate gel in human embryos.

PURPOSE: Zona pellucida-free (ZP-free) embryos often fail to achieve good developmental outcomes and are routinely discarded in assisted reproductive laboratories. Existing attempts to rescue ZP-free embryos are not widely used due to operational complexity and high technical requirements. To handle cases with missing ZP, we applied modified sodium hyaluronate gel (MSHG) to embryo culture to determine if it can function as a substitute for human zona pellucida. METHODS: The developmental process and the blastocyst formation rate of embryos were analyzed in both mouse and human. The first clinical application of MSHG was reported, and the pregnancy outcome was continuously followed up. RESULTS: Human and mouse ZP-free embryos cultured with MSHG showed a blastocyst formation rate similar to ZP-intact embryos. MSHG improves blastocysts formation rate by maintaining blastomere spatial arrangement at early stages. Compared to ZP-free embryos, the proportion of tetrahedrally arranged blastomeres at the 4-cell stage increased significantly in embryos cultured with MSHG in humans. A ZP-free blastocyst cultured in MSHG with the highest score was successfully implanted after day 5 transplantation and developed normally. CONCLUSION: These data demonstrate that MSHG can substitute the function of zona pellucida and rescue human ZP-free embryos during assisted reproductive technology.

Analyzing Prognostic Hub Genes in the Microenvironment of Cutaneous Melanoma by Computer Integrated Bioinformatics.

Cutaneous melanoma (CM) is attracting increasing attention due to high mortality. In response to this, we synthetically analyze the CM dataset from the TCGA database and explore microenvironment-related genes that effectively predict patient prognosis. Immune/stromal scores of cases are calculated using the ESTIMATE algorithm and are significantly associated with overall patient survival. Then, differentially expressed genes are identified by comparing the immune score and stromal score, also prognostic genes are subsequently screened. Functional analysis shows that these genes are enriched in different activities of immune system. Moreover, 19 prognosis-related hub genes are extracted from the protein-protein interaction network, of which four unreported genes (IL7R, FLT3, C1QC, and HLA-DRB5) are chosen for validation. A significant negative relationship is found between the expression levels of the 4 genes and pathological stages, notably T grade. Furthermore, the K-M plots and TIMER results show that these genes have favorable value for CM prognosis. In conclusion, these results give a novel insight into CM and identify IL7R, FLT3, C1QC, and HLA-DRB5 as crucial roles for the diagnosis and treatment of CM.

Low-Temperature Gas Plasma Combined with Antibiotics for the Reduction of Methicillin-Resistant Staphylococcus aureus Biofilm Both In Vitro and In Vivo.

Biofilm infections in wounds seriously delay the healing process, and methicillin-resistant Staphylococcus aureus is a major cause of wound infections. In addition to inactivating micro-organisms, low-temperature gas plasma can restore the sensitivity of pathogenic microbes to antibiotics. However, the combined treatment has not been applied to infectious diseases. In this study, low-temperature gas plasma treatment promoted the effects of different antibiotics on the reduction of S. aureus biofilms in vitro. Low-temperature gas plasma combined with rifampicin also effectively reduced the S. aureus cells in biofilms in the murine wound infection model. The blood and histochemical analysis demonstrated the biosafety of the combined treatment. Our findings demonstrated that low-temperature gas plasma combined with antibiotics is a promising therapeutic strategy for wound infections.

A Color-Shifting Near-Infrared Fluorescent Aptamer-Fluorophore Module for Live-Cell RNA Imaging.

Fluorescent light-up RNA aptamers (FLAPs) have become promising tools for visualizing RNAs in living cells. Specific binding of FLAPs to their non-fluorescent cognate ligands results in a dramatic fluorescence increase, thereby allowing RNA imaging. Here, we present a color-shifting aptamer-fluorophore system, where the free dye is cyan fluorescent and the aptamer-dye complex is near-infrared (NIR) fluorescent. Unlike other reported FLAPs, this system enables ratiometric RNA imaging. To design the color-shifting system, we synthesized a series of environmentally sensitive benzopyrylium-coumarin hybrid fluorophores which exist in equilibrium between a cyan fluorescent spirocyclic form and a NIR fluorescent zwitterionic form. As an RNA tag, we evolved a 38-nucleotide aptamer that selectively binds the zwitterionic forms with nanomolar affinity. We used this system as a light-up RNA marker to image mRNAs in the NIR region and demonstrated its utility in ratiometric analysis of target RNAs expressed at different levels in single cells.

The Mutation of the Ap3b1 Gene Causes Uterine Hypoplasia in Pearl Mice.

The pearl (pe) mouse mutant has been identified as a model for Hermansky-Pudlak syndrome and bears a mutation in the beta3A subunit of the AP-3 complex, which has a core function in the biogenesis and function of various lysosomal-related organelles. Through large-scale mating, we found that female pearl mice also displayed reduced fertility with a smaller litter size. Abnormal uteri in both 1-month-old and 3-month-old mice were observed as having short and thin uterine horns, indicating abnormal development. Histological studies revealed that the endometrial epithelium and endometrial stoma of the uterus were both thinner than those in the normal controls. We examined some key factors in uterine development, including the Hoxa10, Hoxa11, and Wnt5a genes, and found that they all presented lower mRNA and protein levels. The pearl mouse could serve as a model for uterine hypoplasia, a common problem in female infertility.

Correction to: The Mutation of the Ap3b1 Gene Causes Uterine Hypoplasia in Pearl Mice.

The authors are deeply sorry that, due to an unintentional mistake, the proof-editing procedure was skipped. A major mistake must be corrected: Fig. 2C contains pictures from a mislabeled folder and should be replaced as shown in the updated Fig. 2 below.