[EMP3 inhibits the formation of heterotypic cell-in-cell structures in hepatocellular carcinoma].

Heterotypic cell-in-cell (heCIC) structures represent a unique intercellular interaction where tumor cells internalize immune cells to enhance the killing efficiency of immune cells. However, the mechanism of heCIC structure formation remains to be fully elucidated. In this study, we explored the role of epithelial membrane protein 3 (EMP3), a PMP-22/EMP/MP20 protein family member highly expressed in the patients with hepatocellular carcinoma and poor prognosis, in the formation of the heCIC structure formed by natural killer cells and hepatocellular carcinoma cells. The analysis of monoclonal hepatocellular carcinoma cell lines revealed that EMP3 presented low expression in the cells with high capability to form heCIC structure and high expression in those with low capability. Knocking down the expression of EMP3 by gene editing promoted the formation of heCIC structures, while overexpression of EMP3 significantly inhibited this process. Additionally, the expression of factors involved in the heCIC structure formation suggested that EMP3 inhibited the formation of heCIC structures by modulating the adhesion ability and cytoskeleton of tumor cells. The findings lay a foundation for enhancing the heCIC-mediated tumor immunotherapy by targeting EMP3.

A study on a fixed-bed for Pb(ii) removal by modified alkaline lignin-sodium alginate composite hydrogel.

In this work, alkaline lignin (AL) co-modified with trimercapto-s-triazine trisodium salt (TMT) and sodium alginate (SA) as a matrix were used to create a composite hydrogel for removing heavy metals, specifically divalent lead (Pb) from water. The obtained hydrogel beads were packed into a fixed bed, and then various operating conditions were explored to assess their impact on the efficiency of Pb(ii) removal. The findings indicated that the optimal removal efficiency for Pb(ii) was attained using an inflow rate of 0.159 L min-1, a hydrogel-II filling height of 40 cm, an initial Pb(ii) concentration of 10 mg L-1, and a bottom inflow direction. In the third adsorption-desorption cycle experiment, the breakthrough curve reached equilibrium after 650 min, in which equilibrium time for the initial breakthrough curve was 855 min, indicating that hydrogel-II exhibit good regeneration capability. This work serves as a foundation for practical applications in removing heavy metals from wastewater.

Haplotype-resolved assembly of a pig genome using single-sperm sequencing.

Single gamete cell sequencing together with long-read sequencing can reliably produce chromosome-level phased genomes. In this study, we employed PacBio HiFi and Hi-C sequencing on a male Landrace pig, coupled with single-sperm sequencing of its 102 sperm cells. A haplotype assembly method was developed based on long-read sequencing and sperm-phased markers. The chromosome-level phased assembly showed higher phasing accuracy than methods that rely only on HiFi reads. The use of single-sperm sequencing data enabled the construction of a genetic map, successfully mapping the sperm motility trait to a specific region on chromosome 1 (105.40-110.70 Mb). Furthermore, with the assistance of Y chromosome-bearing sperm data, 26.16 Mb Y chromosome sequences were assembled. We report a reliable approach for assembling chromosome-level phased genomes and reveal the potential of sperm population in basic biology research and sperm phenotype research.

Unraveling the prognostic significance of RGS gene family in gastric cancer and the potential implication of RGS4 in regulating tumor-infiltrating fibroblast.

Regulator of G-protein signaling (RGS) proteins are regulators of signal transduction mediated by G protein-coupled receptors (GPCRs). Current studies have shown that some molecules in the RGS gene family are related to the occurrence, development and poor prognosis of malignant tumors. However, the RGS gene family has been rarely studied in gastric cancer. In this study, we explored the mutation and expression profile of RGS gene family in gastric cancer, and evaluated the prognostic value of RGS expression. Then we established a prognostic model based on RGS gene family and performed functional analysis. Further studies showed that RGS4, as an independent prognostic predictor, may play an important role in regulating fibroblasts in the immune microenvironment. In conclusion, this study explores the value of RGS gene family in gastric cancer, which is of great significance for predicting the prognosis and guiding the treatment of gastric cancer.

Analysis of the Effect of High-Simulation Teaching on Nursing Students' Learning of Nursing Knowledge on Double J Tubes after Ureteral Soft Scope Lithotomy.

OBJECTIVE: To study the effect of high-simulation teaching on nursing students' learning knowledge related to stoma tube care after ureteral flexible mirror lithotripsy. METHODS: A total of 80 nursing students who were admitted to our hospital from January 2020 to December 2022 were selected as the study objects. They were divided into the control group (traditional teaching) and observation group (high-simulation teaching based on traditional teaching) in accordance with teaching method. General demographic information and specialty theory, Objective Structured Clinical Examination, Chinese Critical Thinking Disposition Inventory, Teaching Quality Evaluation Scale and System for Evaluation of Teaching Qualities scores were collected from both groups of nursing students. Data were analysed with t- and chi-square tests. RESULTS: The general demographics of the two groups were not statistically significantly different (p > 0.05). No significant differences in examination scores, clinical skills, thinking skills, teaching quality and nursing students' satisfaction were found between the two groups before teaching (p > 0.05). Examination scores, clinical skills, thinking skills, teaching quality and nursing students' satisfaction were higher in the observation group than in the control group after teaching (p < 0.05). CONCLUSIONS: High-simulation teaching can effectively improve theoretical and clinical skill examination results, strengthen critical thinking, and improve teaching quality and nursing students' overall satisfaction with teaching. Therefore, it has application value.

Multi-site isomerization of synergistically regulated stimuli-responsive AIE materials toward multi-level decryption.

Stimuli-responsive aggregation-induced emission (AIE) materials are highly sensitive and rapidly responsive to external signals, making them ideal solid materials for anti-counterfeiting encryption. However, the limited conformational and packing variations resulting from regio-isomerization with a single substituent restricts the stimuli-responsive behavior of these materials. In this work, several AIE-active regio-structural isomers based on the salicylaldehyde Schiff base scaffold have been straightforwardly obtained through multiple substitutions with bromide and triphenylamine moieties. Solvent-effect experiments demonstrate their different orders of charge-transfer and excited-state intramolecular proton transfer upon photoexcitation, indicating the regulation of excited-state processes via multi-site isomerization. These isomers also demonstrate mechanochromism and acidichromism, allowing for adjustable stimuli-responsive effects. As a demonstration, p-Br-TPA with both mechanochromism and acidichromism can be synergistically utilized for multi-level decryption. This study successfully regulates the evolution of excited states through multi-site isomerization, offering a general approach for achieving tunable stimuli-responsive properties in AIE-active salicylaldehyde Schiff bases toward multi-level decryption.

Transcription Factor SATB2 Regulates Skeletal Muscle Cell Proliferation and Migration via HDAC4 in Pigs.

Skeletal muscle development remarkably affects meat production and growth rate, regulated by complex regulatory mechanisms in pigs. Specific AT sequence-binding protein 2 (SATB2) is a classic transcription factor and chromatin organizer, which holds a profound effect in the regulation of chromatin remodeling. However, the regulation role of SATB2 concerning skeletal muscle cell fate through chromatin remodeling in pigs remains largely unknown. Here, we observed that SATB2 was expressed higher in the lean-type compared to the obese-type pigs, which also enriched the pathways of skeletal muscle development, chromatin organization, and histone modification. Functionally, knockdown SATB2 led to decreases in the proliferation and migration markers at the mRNA and protein expression levels, respectively, while overexpression SATB2 had the opposite effects. Further, we found histone deacetylase 4 (HDAC4) was a key downstream target gene of SATB2 related to chromatin remodeling. The binding relationship between SATB2 and HDAC4 was confirmed by a dual-luciferase reporter system and ChIP-qPCR analysis. Besides, we revealed that HDAC4 promoted the skeletal muscle cell proliferation and migration at the mRNA and protein expression levels, respectively. In conclusion, our study indicates that transcription factor SATB2 binding to HDAC4 positively contributes to skeletal muscle cell proliferation and migration, which might mediate the chromatin remodeling to influence myogenesis in pigs. This study develops a novel insight into understanding the molecular regulatory mechanism of myogenesis, and provides a promising gene for genetic breeding in pigs.

Natural Acceptor of Coumarin-Isomerized Red-Emissive BioAIEgen for Monitoring Cu2+ Concentration in Live Cells via FLIM.

Artificial aggregation-induced emission luminogens (AIEgens) have flourished in bio-applications with the development of synthetic chemistry, which however are plagued by issues like singularity in structures and non-renewability. The unique structures and renewability of biomass moieties can compensate for these drawbacks, but their properties are hard to design and regulate due to their confined structures. Therefore, it appears to be a reasonable approach to derive AIEgens from abundant biomass (BioAIEgens), integrating the bilateral advantages of both synthetic and natural AIEgens. In this work, the blue-violet emissive coumarin with its lactone structure serving as a rare natural acceptor, is utilized to construct donor-π-acceptor typed BioAIE isomers incorporating the propeller-like and electron-donating triphenylamine (TPA) unit. The results show that Cm-p-TPA undergoes charge transfer with its keto form, emitting red light at 600 nm, which can be applied to monitor Cu2+ concentration during mitophagy using fluorescence lifetime imaging microscopy because of the excellent biocompatibility, photostability, and specific recognition to Cu2+ . This work not only demonstrates the feasibility of utilizing positional isomerization to modulate excited-state evolutions and resultant optical properties, but also provides evidence for the rationality of constructing biologically-active BioAIEgens via a biomass-derivatization concept.

Single-cell long-read sequencing in human cerebral organoids uncovers cell-type-specific and autism-associated exons.

Dysregulation of alternative splicing has been repeatedly associated with neurodevelopmental disorders, but the extent of cell-type-specific splicing in human neural development remains largely uncharted. Here, single-cell long-read sequencing in induced pluripotent stem cell (iPSC)-derived cerebral organoids identifies over 31,000 uncatalogued isoforms and 4,531 cell-type-specific splicing events. Long reads uncover coordinated splicing and cell-type-specific intron retention events, which are challenging to study with short reads. Retained neuronal introns are enriched in RNA splicing regulators, showing shorter lengths, higher GC contents, and weaker 5' splice sites. We use this dataset to explore the biological processes underlying neurological disorders, focusing on autism. In comparison with prior transcriptomic data, we find that the splicing program in autistic brains is closer to the progenitor state than differentiated neurons. Furthermore, cell-type-specific exons harbor significantly more de novo mutations in autism probands than in siblings. Overall, these results highlight the importance of cell-type-specific splicing in autism and neuronal gene regulation.

Infernape uncovers cell type-specific and spatially resolved alternative polyadenylation in the brain.

Differential polyadenylation sites (PAs) critically regulate gene expression, but their cell type-specific usage and spatial distribution in the brain have not been systematically characterized. Here, we present Infernape, which infers and quantifies PA usage from single-cell and spatial transcriptomic data and show its application in the mouse brain. Infernape uncovers alternative intronic PAs and 3'-UTR lengthening during cortical neurogenesis. Progenitor-neuron comparisons in the excitatory and inhibitory neuron lineages show overlapping PA changes in embryonic brains, suggesting that the neural proliferation-differentiation axis plays a prominent role. In the adult mouse brain, we uncover cell type-specific PAs and visualize such events using spatial transcriptomic data. Over two dozen neurodevelopmental disorder-associated genes such as Csnk2a1 and Mecp2 show differential PAs during brain development. This study presents Infernape to identify PAs from scRNA-seq and spatial data, and highlights the role of alternative PAs in neuronal gene regulation.

Protocol for high-sensitivity western blot on murine hematopoietic stem cells.

Hematopoietic stem cells (HSCs) sustain hematopoiesis during homeostasis and regeneration. However, their limited availability poses a challenge for protein analysis. Here, we present a protocol for performing high-sensitivity western blot on HSCs using two techniques that enhance HSC isolation from mice and boost sensitivity for low cell numbers. We describe steps for isolating murine bone marrow cells, antibody staining, and cell sorting and post-sort analysis. We then detail a western blot procedure suitable for low numbers of HSCs. For complete details on the use and execution of this protocol, please refer to Li et al (2022).1,2.

Circular RNA transcriptome across multiple tissues reveal skeletal muscle-specific circPSME4 regulating myogenesis.

There are significant differences in meat production, growth rate and other traits between Western commercial pigs and Chinese local pigs. Comparative transcriptome approaches have identified many coding and non-coding candidate genes associated various traits. However, the expression and function of circular RNAs (circRNAs) in different pig tissues are largely unknown. In this study, we conducted a comprehensive analysis of the genome-wide circRNA expression profile across ten tissues in Luchuan (a Chinese local breed) and Duroc (a Western commercial breed) pigs. We identified a total of 56,254 circRNAs, of which 42.9 % were not previously annotated. We found that 33.7 % of these circRNAs were differentially expressed. Enrichment analysis revealed that differentially expressed circRNAs might contribute to the phenotypic differentiation between Luchuan and Duroc pigs. We identified 538 tissue-specific circRNAs, most of which were specifically expressed in the brain and skeletal muscle. Competitive endogenous RNA network analysis suggested that skeletal muscle-specific circPSME4 was co-expressed with MYOD1 and targeted by ssc-miR-181d-3p. Functional analysis revealed that circPSME4 knockdown could promote the proliferation and differentiation of myoblasts. Together, our findings provide valuable resources of circRNAs for animal breeding and biomedical research. We demonstrated that circPSME4 is a novel regulator of skeletal muscle development.

Chemodiversity of Cyanobacterial Toxins Driven by Future Scenarios of Climate Warming and Eutrophication.

Climate change and eutrophication are two environmental threats that can alter the structure of freshwater ecosystems and their service functions, but we know little about how ecosystem structure and function will evolve in future scenarios of climate warming. Therefore, we created different experimental climate scenarios, including present-day conditions, a 3.0 °C increase in mean temperature, and a "heatwaves" scenario (i.e., an increase in temperature variability) to assess the effects of climate change on phytoplankton communities under simultaneous stress from eutrophication and herbicides. We show that the effects of climate warming, particularly heatwaves, are associated with elevated cyanobacterial abundances and toxin production, driven by a change from mainly nontoxic to toxic Microcystis spp. The reason for higher cyanobacterial toxin concentrations is likely an increase in abundances because under the dual pressures of climate warming and eutrophication individual Microcystis toxin-producing ability decreased. Eutrophication and higher temperatures significantly increased the biomass of Microcystis, leading to an increase in the cyanobacterial toxin concentrations. In contrast, warming alone did not produce higher cyanobacterial abundances or cyanobacterial toxin concentrations likely due to the depletion of the available nutrient pool. Similarly, the herbicide glyphosate alone did not affect abundances of any phytoplankton taxa. In the case of nutrient enrichment, cyanobacterial toxin concentrations were much higher than under warming alone due to a strong boost in biomass of potential cyanobacterial toxin producers. From a broader perspective our study shows that in a future warmer climate, nutrient loading has to be reduced if toxic cyanobacterial dominance is to be controlled.

Establishing a novel and sensitive assay for bioactivity determination of anti-CD25 antibodies.

Anti-CD25 antibodies have been approved for renal transplantation and has been used prior to and during transplantation by the Food and Drug Administration (FDA). However, no reported bioassays have been reflected the mechanism of action (MOA) of anti-CD25 antibodies. Here, we describe the development and validation of a reporter gene assay (RGA) based on the engineered C8166-STAT5RE-Luc cells expressing endogenous IL-2 receptors and a STAT5-inducible element-driven firefly luciferase in C8166 cell lines. The RGA was fully validated according to the International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for the Human Use-Q2 (ICH-Q2). After optimization, the assay showed excellent specificity, linearity, accuracy, precision, and robustness. Due to the MOA relatedness and the excellent assay performance, the RGA is suitable for exploring the critical quality attributes (CQAs), release inspection, comparability and stability of anti-CD25 mAbs.

Reactivation of mutant p53 in esophageal squamous cell carcinoma by isothiocyanate inhibits tumor growth.

p53 mutations are prevalent in human cancers; approximately half of patients with esophageal cancer present these mutations. Mutant p53 (mutp53) exerts oncogenic functions that promote malignant tumor progression, invasion, metastasis, and drug resistance, resulting in poor prognosis. Some small molecules have been shown to mitigate the oncogenic function of mutp53 by restoring its wild-type activity. Although these molecules have been evaluated in clinical trials, none have been successfully used in the clinic. Here, we investigated the antitumor effects of phenethyl isothiocyanate (PEITC) in p53-mutant esophageal squamous cell carcinoma (ESCC) and elucidated its mechanism to identify new therapeutic strategies. We observed that p53R248Q is a DNA contact mutation and a structural mutation and that PEITC can restore the activity of p53R248Q in vitro and in vivo, further clarifying the antitumor activity of PEITC in cancers with different types of p53 mutations. PEITC can inhibit ESCC growth, induce apoptosis, and arrest cell cycle progression and has a preferential selectivity for ESCC with p53 mutations. Mechanistic studies showed that PEITC induced apoptosis and arrested cells at G2/M transition in cells expressing the p53R248Q mutant by restoring the wild-type conformation and transactivation function of p53; these effects were concentration dependent. Furthermore, PEITC inhibited the growth of subcutaneous xenografts in vivo and restored p53 mutant activity in xenografts. According to these findings, PEITC has antitumor effects, with its ability to restore p53R248Q activity being a key molecular event responsible for these effects.

A Novel Predictor of Pathologic Complete Response for Neoadjuvant Immunochemotherapy in Resectable Locally Advanced Esophageal Squamous Cell Carcinoma.

Purpose: Neoadjuvant immunochemotherapy (nICT) for resectable locally advanced esophageal squamous cell carcinoma (LA-ESCC) has attracted widespread attention recently, whose safety and clinical benefit was observed in clinical researches. This study aimed to develop and validate a novel predictor systemic inflammation-tumor markers index (SITI) to predict the pathological complete response (pCR) for resectable LA-ESCC patients receiving nICT. Patients and Methods: A total of 147 LA-ESCC patients who underwent nICT followed by surgery from February 2020 to April 2022 were included in the study. The dynamic change of inflammatory indexes was compared at baseline, after two cycles of nICT and postoperative one month. Least absolute shrinkage and selection operator (LASSO) regression was performed to avoid collinearity and identify key indexes, with SITI constructed. After univariate and multivariate stepwise forward logistic analyses, a nomogram for pCR prediction was developed. Results: 41(27.9%) patients achieved pCR among 147 resectable LA-ESCC patients received nICT. Compared with baseline, most inflammatory indexes were significantly decreased at postoperative one month. 5 key indexes were identified and then a predictive index named SITI was constructed. The result showed that lower SITI and earlier clinical tumor node metastasis (cTNM) stage were more likely to achieve pCR. The nomogram for pCR prediction had excellent discrimination performance (C-index = 0.791). Conclusion: The SITI is an independent predictor for pCR in resectable LA-ESCC patients received nICT. To our knowledge, our nomogram is the first model using systemic inflammation-tumor markers for pCR prediction and may be a promising predictor to effectively differentiate pCR for nICT in LA-ESCC patients.

Method validation of a bridging immunoassay in combination with acid-dissociation and bead treatment for detection of anti-drug antibody.

Anti-drug antibody (ADA) positivity is correlated with disease relapse risk when treated with monoclonal antibody (mAb) therapeutics. ADA evaluation can assist with interpreting pharmacokinetic, pharmacological, and toxicology results. Here, we established an ADA assay based on two steps of acid dissociation combined with a bridging immunoassay to provide a comprehensive validation strategy. The three-tiered sample analysis process included screening, confirmation, and titration assays using therapeutic HLX26 (targeting lymphocyte activation gene-3 [LAG-3]) as an example. The cut points were determined by testing 50 individual normal human serum samples, including screening cut point (SCP) (SNR: 1.08), confirmatory cut point (CCP) (% inhibition: 12.65), and titration cut point (TCP) (sample-to-noise ratio [SNR]: 1.17). The assay sensitivity, low positive control (LPC), and high positive control (HPC) titer acceptable range were also set up as 33.0 ng/mL, 41.0 ng/mL, and 320-1280, respectively. After full validation, both the intra-assay and inter-assay precision testing passed with coefficient of variations (CVs) < 20%. The assay enabled excellent drug tolerance up to 768.0 µg/mL at the HPC level and 291.0 µg/mL at the LPC level, while the tolerance of target interference was up to 74.0 ng/mL of soluble LAG3. Moreover, no false-positive results were observed in the presence of 5% hemolyzed serum samples and 150 mg/dL of triglyceride in the serum samples, no hook effect was observed, and the stability performed normally under room temperature for 24 h, 2-8 °C for 7 d, and six freeze/thaw cycles. In summary, this ADA assay is feasible and could be used for evaluating the immunogenicity of HLX26 in clinical trials.

MicroRNA-200c-5p Regulates Migration and Differentiation of Myoblasts via Targeting Adamts5 in Skeletal Muscle Regeneration and Myogenesis.

Skeletal muscle, as a regenerative organization, plays a vital role in physiological characteristics and homeostasis. However, the regulation mechanism of skeletal muscle regeneration is not entirely clear. miRNAs, as one of the regulatory factors, exert profound effects on regulating skeletal muscle regeneration and myogenesis. This study aimed to discover the regulatory function of important miRNA miR-200c-5p in skeletal muscle regeneration. In our study, miR-200c-5p increased at the early stage and peaked at first day during mouse skeletal muscle regeneration, which was also highly expressed in skeletal muscle of mouse tissue profile. Further, overexpression of miR-200c-5p promoted migration and inhibited differentiation of C2C12 myoblast, whereas inhibition of miR-200c-5p had the opposite effect. Bioinformatic analysis predicted that Adamts5 has potential binding sites for miR-200c-5p at 3'UTR region. Dual-luciferase and RIP assays further proved that Adamts5 is a target gene of miR-200c-5p. The expression patterns of miR-200c-5p and Adamts5 were opposite during the skeletal muscle regeneration. Moreover, miR-200c-5p can rescue the effects of Adamts5 in the C2C12 myoblast. In conclusion, miR-200c-5p might play a considerable function during skeletal muscle regeneration and myogenesis. These findings will provide a promising gene for promoting muscle health and candidate therapeutic target for skeletal muscle repair.