Spatially organized cellular communities form the developing human heart.

The heart, which is the first organ to develop, is highly dependent on its form to function1,2. However, how diverse cardiac cell types spatially coordinate to create the complex morphological structures that are crucial for heart function remains unclear. Here we integrated single-cell RNA-sequencing with high-resolution multiplexed error-robust fluorescence in situ hybridization to resolve the identity of the cardiac cell types that develop the human heart. This approach also provided a spatial mapping of individual cells that enables illumination of their organization into cellular communities that form distinct cardiac structures. We discovered that many of these cardiac cell types further specified into subpopulations exclusive to specific communities, which support their specialization according to the cellular ecosystem and anatomical region. In particular, ventricular cardiomyocyte subpopulations displayed an unexpected complex laminar organization across the ventricular wall and formed, with other cell subpopulations, several cellular communities. Interrogating cell-cell interactions within these communities using in vivo conditional genetic mouse models and in vitro human pluripotent stem cell systems revealed multicellular signalling pathways that orchestrate the spatial organization of cardiac cell subpopulations during ventricular wall morphogenesis. These detailed findings into the cellular social interactions and specialization of cardiac cell types constructing and remodelling the human heart offer new insights into structural heart diseases and the engineering of complex multicellular tissues for human heart repair.

Amplification-Free Digital Immunoassay down to the Attomolar Level by Synergistic Sedimentation of Brownian Motion Suppression and Dehydration Transfer.

Amplification-free digital immunoassays (DIAs) typically utilize optical nanoparticles to enhance single immunocomplex molecule detection. The efficiency and uniformity of transferring the nanoparticles from a bulk solution to a solid surface determine the limit of detection (LOD) and the accuracy of DIAs. Previous methods suffer from issues like low efficiency, nonuniform distribution, and particle aggregation. Here, we present a novel technique named synergistic sedimentation of Brownian motion suppression and dehydration transfer (SynSed) for nanoparticles using water-soluble polymers. The efficiency of transferring quantum dots (QDs) was increased from 10.7 to 91.4%, and the variation in QD distribution was restricted to 8.8%. By incorporating SynSed into DIAs, we achieved a remarkable reduction in the LOD (down to 3.9 aM) for carcinoembryonic antigen and expanded the dynamic range to cover 3 orders of magnitude in concentration, ranging from 0.01 to 10 fM. DIAs enhanced with SynSed possess ultrahigh sensitivity, advanced accuracy, and specificity, offering a great premise in early disease diagnostics, risk stratification, and treatment response monitoring.

LncCMRR Plays an Important Role in Cardiac Differentiation by Regulating the Purb/Flk1 Axis.

As crucial epigenetic regulators, long noncoding RNAs (lncRNAs) play critical functions in development processes and various diseases. However, the regulatory mechanism of lncRNAs in early heart development is still limited. In this study, we identified cardiac mesoderm-related lncRNA (LncCMRR). Knockout (KO) of LncCMRR decreased the formation potential of cardiac mesoderm and cardiomyocytes during embryoid body differentiation of mouse embryonic stem (ES) cells. Mechanistic analyses showed that LncCMRR functionally interacted with the transcription suppressor PURB and inhibited its binding potential at the promoter region of Flk1, which safeguarded the transcription of Flk1 during cardiac mesoderm formation. We also carried out gene ontology term and signaling pathway enrichment analyses for the differentially expressed genes after KO of LncCMRR, and found significant correlation of LncCMRR with cardiac muscle contraction, dilated cardiomyopathy, and hypertrophic cardiomyopathy. Consistently, the expression level of Flk1 at E7.75 and the thickness of myocardium at E17.5 were significantly decreased after KO of LncCMRR, and the survival rate and heart function index of LncCMRR-KO mice were also significantly decreased as compared with the wild-type group. These findings indicated that the defects in early heart development led to functional abnormalities in adulthood heart of LncCMRR-KO mice. Conclusively, our findings elucidate the main function and regulatory mechanism of LncCMRR in cardiac mesoderm formation, and provide new insights into lncRNA-mediated regulatory network of mouse ES cell differentiation.

Combined exposure of polystyrene microplastics and benzo[a]pyrene in rat: Study of the oxidative stress effects in the liver.

Microplastics (MPs) and benzo[a]pyrene (B[a]P) are prevalent environmental pollutants. Numerous studies have extensively reported their individual adverse effects on organisms. However, the combined effects and mechanisms of exposure in mammals remain unknown. Thus, this study aims to investigate the potential effects of oral administration of 0.5µm polystyrene (PS) MPs (1 mg/mL or 5 mg/mL), B[a]P (1 mg/mL or 5 mg/mL) and combined (1 mg/mL or 5 mg/mL) on 64 male SD rats by gavage method over 6-weeks. The results demonstrate that the liver histopathological examination showed that the liver lobules in the combined (5 mg/kg) group had blurred and loose boundaries, liver cord morphological disorders, and significant steatosis. The levels of AST, ALT, TC, and TG in the combined dose groups were significantly higher than those in the other groups, the combined (5 mg/kg) group had the lowest levels of antioxidant enzymes and the highest levels of oxidants. The expression of Nrf2 was lowest and the expression of P38, NF-κB, and TNF-α was highest in the combined (5 mg/kg) group. In conclusion, these findings indicate that the combination of PSMPs and B[a]P can cause the highest levels of oxidative stress and elicit markedly enhanced toxic effects, which cause severe liver damage.

Unveiling Complexity and Multipotentiality of Early Heart Fields.

[Figure: see text].

Homogeneous immunoassay utilizing fluorescence resonance energy transfer from quantum dots to tyramide dyes deposited on full immunocomplexes.

There is an urgent need for homogeneous immunoassays that offer sufficient sensitivity for routine clinical practice. In this study, we have developed a highly sensitive, fluorescence resonance energy transfer (FRET)-based homogeneous immunoassay. Unlike previous FRET-based homogeneous immunoassays, where acceptors were attached to antibody molecules located far from the donor, we employed acceptors to label the entire sandwich-structured immunocomplex, including two antibodies and one antigen. As a result, the FRET signal was amplified by a factor of 10, owing to the reduced distance between the donor and acceptors. We validated our method by quantifying carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) in PBS buffer and blank plasma. The limits of detection (LOD) for CEA and AFP in both PBS buffer and blank plasma were comparable, reaching sub-femtomolar levels. Furthermore, we successfully quantified CEA and AFP in three human plasma samples, thereby confirming the reliability of our method for clinical applications.

The value of serum Sema4D level in predicting the prognosis of patients with acute ST-segment elevation myocardial infarction and with high thrombus burden.

BACKGROUND: Acute ST-segment elevation myocardial infarction (STEMI) is a serious cardiovascular disease. High thrombus burden is an independent risk factor for poor prognosis of acute myocardial infarction. However, there is no study on the correlation between soluble semaphorin 4D (sSema4D) level and high thrombus burden in patients with STEMI. PURPOSE: This study aimed to investigate the relationship between sSema4D level and the thrombus burden of STEMI and further explore its effect on the main predictive value of the occurrence of major adverse cardiovascular events (MACE). METHODS: From October 2020 to June 2021, 100 patients with STEMI diagnosed in our hospital's cardiology department were selected. According to the thrombolysis in myocardial infarction(TIMI)score, STEMI patients were divided into high thrombus burden groups (55 cases) and non-high thrombus burden groups (45 cases) 0.74 patients with stable coronary heart disease (CHD) were selected as stable CHD group, and 75 patients with negative coronary angiography (CAG) were selected as control group. Serum sSema4D levels were measured in 4 groups. The correlation between serum sSema4D and high-sensitivity C-reactive protein (hs-CRP) in patients with STEMI was analyzed. The relationship of serum sSema4D levels between the high and non-high thrombus burden group was evaluated. The effect of sSema4D levels on the occurrence of MACE was explored in one year after percutaneous coronary intervention. RESULTS: Serum sSema4D level was positively correlated with hs-CRP level in STEMI patients (P < 0.05) with a correlation coefficient of 0.493. The sSema4D level was significantly higher in the high versus non-high thrombus burden group (22.54(20.82,24.17), P < 0.05). Moreover, MACE occurred in 19 cases in high thrombus burden group and 3 cases in non-high thrombus burden group. The results of Cox regression analysis showed that sSema4D was an independent predictor of MACE (OR = 1.497,95% CI: 1.213-1.847, P < 0.001). CONCLUSION: The sSema4D level is associated with coronary thrombus burden and is an independent risk factor for MACE.

A Clinical Observation Study for Transnasal Endoscopic Marsupialization in the Treatment of Maxillary Cyst.

The aim of this study was to investigate the feasibility and evaluate the clinical effects of transnasal marsupialization of maxillary cyst under nasal endoscope. Twenty patients with maxillary bone cysts were treated with endoscopic marsupialization. According to the location of the maxillary bone cyst, the opening window was selected in the nasal base or the inferior or middle nasal meatus. The cyst wall of the window was removed, and the fluid was drained out. The diameter of the opening window was more than 1 cm, so that the cyst and the maxillary sinus can be fused into a cavity. This ensured nasal drainage through the cyst cavity and nasal cavity or maxillary sinus. The patients were followed up for 6 to 18 months. No serious complications occurred. The cyst wall epithelialized 2 or 3 months after the operation, and the cyst cavity drained well with no recurrence. Intranasal marsupialization under a nasal endoscope is a feasible alternative for the treatment of maxillary cysts. It makes the procedure simple, safe, less traumatic, has definite clinical effects, and low recurrence rate. Especially suitable for maxillary cysts protruding into the nasal floor or maxillary sinus.

Low-Numerical Aperture Microscope Objective Boosted by Liquid-Immersed Dielectric Microspheres for Quantum Dot-Based Digital Immunoassays.

Quantum dot (QD)-based digital immunoassays play an important role in ultrasensitive biomarker detection. However, the requirement of an objective with a high numerical aperture (NA) limits the application of this immunoassay. Here, high-quality imaging of massive single-QDs was achieved by the combination of an air objective (20×/0.4 NA) and liquid-immersed microspheres (150 µm, n = 2.2). The signal-to-noise ratio was comparable to that of a 100×/1.4 NA oil objective. Digital analysis of prostate-specific antigen (PSA) was performed within the dynamic range of 0-50 ng/mL and a limit of detection of 0.17 ng/mL. The measured serum data from the PSA were close to the values provided by a hospital. Using a low-magnification and low-NA objective may reduce the barrier of microscopy miniaturization and is beneficial to popularize biomolecular digital analysis.

In-situ immobilization of cadmium-polluted upland soil: A ten-year field study.

In-situ immobilization is an effective and economically viable strategy for remediation of soil extensively polluted with heavy metals. The long-term sustainability is critical for the remediation practice. In the present study, a ten-year experiment was performed in a Cd-polluted agricultural field to evaluate the long-term stability of lime, silicon fertilizer (SF), fused calcium magnesium phosphate fertilizer (FCMP), bone charcoal, steel slag, and blast furnace slag with one-off application. All amendments had no significant effect on biomass but significantly reduced Cd uptake by Artemisia selengensis at higher dose. Among them, SF and FCMP applied at 1% could reduce Cd uptake by more than 40% to meet the Chinese maximum permissible limit for Cd content in food products (50 µg kg-1). These amendments stimulated high Cd immobilization by increasing the soil pH and decreasing the soil acid-extractable Cd content, which were closely associated with Cd uptake. In addition, the two amendments altered the soil microbial structure and stimulated metabolism pathways, including amino acid, carbohydrate, and lipid metabolism, which are beneficial for soil function and quality. The results proved that SF and FCMP at 1% are stable and ecologically safe amendments, suitable for long-term Cd immobilization, and provide a strategy to mitigate the risk of food product contamination in heavy-metal-polluted soil.

Nonstochastic Protein Counting Analysis for Precision Biomarker Detection: Suppressing Poisson Noise at Ultralow Concentration.

Protein counting analysis obtains the quantitative results of specific protein through counting the number of target signals and displays a great value in disease diagnosis. Current protein counting techniques just stochastically count a small portion of the target signal, which causes a considerable information loss and limits the accuracy and precision of the protein assay at ultralow concentration. Here, we present a nonstochastic and ultrasensitive protein counting method through combining multiround evaporation-induced particle sedimentation, grid-assisted multiframe imaging, and microsphere-enhanced high-resolution signals. Using carcinoembryonic antigen (CEA) as the model, the dynamic range was from 5 × 10-18 M (aM) to 5 × 10-16 M, and the limit of detection was 4.9 aM. For CEA-spiked plasma detection, the relative standard deviation and the relative error of CEA concentrations were both lower than 8.0%, and the recoveries reached 92.5% and 98.8% for 20.0 aM and 40.0 aM CEA respectively. Two clinical plasma samples were measured by the standard addition method, and the results showed little deviation with the values provided by the hospital. The established approach suppresses Poisson noise of the stochastic counting, offers ultrahigh sensitivity, and features a remarkable potential in early disease screening.

USP44 suppresses proliferation and enhances apoptosis in colorectal cancer cells by inactivating the Wnt/ß-catenin pathway via Axin1 deubiquitination.

Colorectal cancer (CRC) is the leading cause of cancer death, and its 5-year survival rate remains unsatisfactory. Recent studies have revealed that ubiquitin-specific protease 44 (USP44) is a cancer suppressor or oncogene depending on the type of neoplasm. However, its role in CRC remains unclear. Here, we found that the USP44 expression level was markedly decreased in CRC, and USP44 overexpression inhibited proliferation while enhancing apoptosis in CRC cells, suggesting that USP44 is a cancer suppressor in CRC. We then investigated if USP44 functioned through regulating the Wnt/ß-catenin pathway. We found that USP44 overexpression increased the Axin1 protein while decreasing ß-catenin, c-myc, and cyclin D1 proteins, suggesting that USP44 inhibited the activation of the Wnt/ß-catenin pathway. Moreover, we found that two Wnt/ß-catenin activators, LiCl and SKL2001, both attenuated oeUSP44-mediated proliferation and apoptosis in CRC cells. Collectively, these data points indicated that USP44 inhibited proliferation while promoting apoptosis in CRC cells by inhibiting the Wnt/ß-catenin pathway. Interestingly, we observed that USP44 overexpression did not affect the Axin1 mRNA level. Further study uncovered that USP44 interacted with Axin1 and reduced the ubiquitination of Axin1. Furthermore, Axin1 knock-down abolished the effects of oeUSP44 on proliferation, apoptosis, and Wnt/ß-catenin activity in CRC cells. Taken together, this study demonstrates that USP44 inhibits proliferation while enhancing apoptosis in CRC cells by inactivating the Wnt/ß-catenin pathway via Axin1 deubiquitination. USP44 is a cancer suppressor in CRC and a potential target for CRC therapy.

Sin3a-Tet1 interaction activates gene transcription and is required for embryonic stem cell pluripotency.

Sin3a is a core component of histone-deacetylation-activity-associated transcriptional repressor complex, playing important roles in early embryo development. Here, we reported that down-regulation of Sin3a led to the loss of embryonic stem cell (ESC) self-renewal and skewed differentiation into mesendoderm lineage. We found that Sin3a functioned as a transcriptional coactivator of the critical Nodal antagonist Lefty1 through interacting with Tet1 to de-methylate the Lefty1 promoter. Further studies showed that two amino acid residues (Phe147, Phe182) in the PAH1 domain of Sin3a are essential for Sin3a-Tet1 interaction and its activity in regulating pluripotency. Furthermore, genome-wide analyses of Sin3a, Tet1 and Pol II ChIP-seq and of 5mC MeDIP-seq revealed that Sin3a acted with Tet1 to facilitate the transcription of a set of their co-target genes. These results link Sin3a to epigenetic DNA modifications in transcriptional activation and have implications for understanding mechanisms underlying versatile functions of Sin3a in mouse ESCs.

Expression of ILK in renal stroma is essential for multiple aspects of renal development.

Kidney development involves reciprocal and inductive interactions between the ureteric bud (UB) and surrounding metanephric mesenchyme. Signals from renal stromal lineages are essential for differentiation and patterning of renal epithelial and mesenchymal cell types and renal vasculogenesis; however, underlying mechanisms remain not fully understood. Integrin-linked kinase (ILK), a key component of integrin signaling pathway, plays an important role in kidney development. However, the role of ILK in renal stroma remains unknown. Here, we ablated ILK in renal stromal lineages using a platelet-derived growth factor receptor B ( Pdgfrb) -Cre mouse line, and the resulting Ilk mutant mice presented postnatal growth retardation and died within 3 wk of age with severe renal developmental defects. Pdgfrb-Cre;Ilk mutant kidneys exhibited a significant decrease in UB branching and disrupted collecting duct formation. From E16.5 onward, renal interstitium was disorganized, forming medullary interstitial pseudocysts. Pdgfrb-Cre;Ilk mutants exhibited renal vasculature mispatterning and impaired glomerular vascular differentiation. Impaired glial cell-derived neurotrophic factor/Ret and bone morphogenetic protein 7 signaling pathways were observed in Pdgfrb-Cre;Ilk mutant kidneys. Furthermore, phosphoproteomic and Western blot analyses revealed a significant dysregulation of a number of key signaling pathways required for kidney morphogenesis, including PI3K/AKT and MAPK/ERK in Pdgfrb-Cre;Ilk mutants. Our results revealed a critical requirement for ILK in renal-stromal and vascular development, as well as a noncell autonomous role of ILK in UB branching morphogenesis.

Comparative proteomics illustrates the molecular mechanism of potato (Solanum tuberosum L.) tuberization inhibited by exogenous gibberellins in vitro.

Among the multiple environmental signals and hormonal factors regulating potato tuberization, gibberellins (GAs) are important components of the signaling pathways in these processes. To understand the GAs-signaling response mechanism of potato tuberization, a comparative proteomics approach was applied to analyze proteome change of potato tuberization in vitro subjected to a range of exogenous GA3 treatments (0, 0.01, 0.1 and 1.0 µM) using two-dimensional gel electrophoresis. Quantitative image analyses showed that a total of 37 protein spots have their abundance significantly altered more than 2-fold. Among these proteins, 13 proteins were up-regulated, 13 proteins were down-regulated, one protein was absent and 10 proteins were induced after treatment by exogenous GA3 . The MALDI-TOF/TOF MS analyses led to the identification of differentially abundant proteins that are mainly involved in bioenergy and metabolism, storage, signaling, cell defense and rescue, transcription, chaperones, transport. Furthermore, the comparative analysis of GA3 -responsive proteome allowed for general elucidation of underlying molecular mechanisms of potato tuberization inhibited by exogenous GA3 . Most of these cellular processes were not conducive to the transition from stolon elongation to tuber formation, including a blockage of starch and storage protein accumulation, the accelerated carbohydrate catabolism, a blockage of JA biosynthesis but an elevated endogenous GAs level, the amplification of GA3 signal transduction by other signaling pathways, and the regulation of cellular RNA metabolism for controlling tuberization. Our results firstly integrated physiology and proteome data to provide new insights into GA3 -signaling response mechanisms of potato tuberization in vitro.

Visual and colorimetric determination of H2O2 and glucose based on citrate-promoted H2O2 sculpturing of silver nanoparticles.

Isotropic silver nanoparticles (iAg NPs) can be easily prepared at low costs, have a low electrochemical potential and high extinction coefficient. An effective colorimetric assay for H2O2 is reported here based on the finding that H2O2 can induce the shape transformation of citrate-capped iAg NPs with the help of citrate. The substantial shape variation affords an apparent surface plasmon resonance (SPR) shift, accompanied by a vivid color change from light yellow to mauve. The color change can be observed visually if the concentration of H2O2 is 2 µM or higher. A good linear relationship was obtained over the concentration range of 0.2-32 µM with a limit of detection of 90 nM. By making use of glucose oxidase, the method is further extended to glucose detection. Glucose at a concentration as low as 10 µM can be well determined with bare eyes. Benefitting from the high selectivity, the detection of glucose in human serum is realized, and the results are in good agreement with those provided by a clinical analyzer. Graphical abstract Schematic presentation of a colorimetric assay for H2O2 and glucose based on citrate-promoted H2O2-mediated shape transformation of the isotropic silver nanoparticles (Ag NPs). The shape variation of isotropic Ag NPs induces a color change from light yellow to mauve.

Viscosity based droplet size controlling in negative pressure driven droplets generator for large-scale particle synthesis.

The poor control and regulation of droplets limit the applications of negative pressure driven droplet generator (NPDDG). Here, we present a simple method to control droplet size in NPDDG via varying the oil viscosity. Depending on the infinite intersolubility of high viscous mineral oil and low viscous hexadecane, we obtain a series of oils with kinematic viscosities linearly varied from 4.2 to 194.6 mm2 /s. By using these oils as continuous phases, monodisperse droplets are fabricated with controllable size in NPDDG. This viscosity-based droplet regulation method is effective, reliable, and compatible with scale-up processes. Compared with droplet generator driven by positive pressure, the presented method can fabricate hydrogel particles massively, without complicated multilayer chip structure and complex fluid controlling, which may extend the potential of NPDDG in droplets based high-throughput assay or large-scale materials synthesis.

Single Gold Nanoparticle-Based Colorimetric Detection of Picomolar Mercury Ion with Dark-Field Microscopy.

Mercury severely damages the environment and human health, particularly when it accumulates in the food chain. Methods for the colorimetric detection of Hg(2+) have increasingly been developed over the past decade because of the progress in nanotechnology. However, the limits of detection (LODs) of these methods are mostly either comparable to or higher than the allowable maximum level (10 nM) in drinking water set by the US Environmental Protection Agency. In this study, we report a single Au nanoparticle (AuNP)-based colorimetric assay for Hg(2+) detection in solution. AuNPs modified with oligonucleotides were fixed on the slide. The fixed AuNPs bound to free AuNPs in the solution in the presence of Hg(2+) because of oligonucleotide hybridization. This process was accompanied by a color change from green to yellow as observed under an optical microscope. The ratio of changed color spots corresponded with Hg(2+) concentration. The LOD was determined as 1.4 pM, which may help guard against mercury accumulation. The proposed approach was applied to environmental samples with recoveries of 98.3 ± 7.7% and 110.0 ± 8.8% for Yuquan River and industrial wastewater, respectively.

Requirement for integrin-linked kinase in neural crest migration and differentiation and outflow tract morphogenesis.

BACKGROUND: Neural crest defects lead to congenital heart disease involving outflow tract malformation. Integrin-linked-kinase (ILK) plays important roles in multiple cellular processes and embryogenesis. ILK is expressed in the neural crest, but its role in neural crest and outflow tract morphogenesis remains unknown. RESULTS: We ablated ILK specifically in the neural crest using the Wnt1-Cre transgene. ILK ablation resulted in abnormal migration and overpopulation of neural crest cells in the pharyngeal arches and outflow tract and a significant reduction in the expression of neural cell adhesion molecule (NCAM) and extracellular matrix components. ILK mutant embryos exhibited an enlarged common arterial trunk and ventricular septal defect. Reduced smooth muscle differentiation, but increased ossification and neurogenesis/innervation were observed in ILK mutant outflow tract that may partly be due to reduced transforming growth factor ß2 (TGFß2) but increased bone morphogenetic protein (BMP) signaling. Consistent with these observations, microarray analysis of fluorescence-activated cell sorting (FACS)-sorted neural crest cells revealed reduced expression of genes associated with muscle differentiation, but increased expression of genes of neurogenesis and osteogenesis. CONCLUSIONS: Our results demonstrate that ILK plays essential roles in neural crest and outflow tract development by mediating complex crosstalk between cell matrix and multiple signaling pathways. Changes in these pathways may collectively result in the unique neural crest and outflow tract phenotypes observed in ILK mutants.