Murine trophoblast organoids as a model for trophoblast development and CRISPR-Cas9 screening.

The placenta becomes one of the most diversified organs during placental mammal radiation. The main in vitro model for studying mouse trophoblast development is the 2D differentiation model of trophoblast stem cells, which is highly skewed to certain lineages and thus hampers systematic screens. Here, we established culture conditions for the establishment, maintenance, and differentiation of murine trophoblast organoids. Murine trophoblast organoids under the maintenance condition contain stem cell-like populations, whereas differentiated organoids possess various trophoblasts resembling placental ones in vivo. Ablation of Nubpl or Gcm1 in trophoblast organoids recapitulated their deficiency phenotypes in vivo, suggesting that those organoids are valid in vitro models for trophoblast development. Importantly, we performed an efficient CRISPR-Cas9 screening in mouse trophoblast organoids using a focused sgRNA (single guide RNA) library targeting G protein-coupled receptors. Together, our results establish an organoid model to investigate mouse trophoblast development and a practicable approach to performing forward screening in trophoblast lineages.

Genetic lineage tracing identifies cardiac mesenchymal-to-adipose transition in an arrhythmogenic cardiomyopathy model.

Arrhythmogenic cardiomyopathy (ACM) is one of the most common inherited cardiomyopathies, characterized by progressive fibrofatty replacement in the myocardium. However, the cellular origin of cardiac adipocytes in ACM remains largely unknown. Unraveling the cellular source of cardiac adipocytes in ACM would elucidate the underlying pathological process and provide a potential target for therapy. Herein, we generated an ACM mouse model by inactivating desmosomal gene desmoplakin in cardiomyocytes; and examined the adipogenic fates of several cell types in the disease model. The results showed that SOX9+, PDGFRa+, and PDGFRb+ mesenchymal cells, but not cardiomyocytes or smooth muscle cells, contribute to the intramyocardial adipocytes in the ACM model. Mechanistically, Bmp4 was highly expressed in the ACM mouse heart and functionally promoted cardiac mesenchymal-to-adipose transition in vitro.

15N Natural Abundance of C3 and C4 Herbaceous Plants and Its Response to Climatic Factors along an Agro-Pastoral Zone of Northern China.

The nitrogen isotope composition of plants (δ15N) can comprehensively reflect information on climate change and ecosystems' nitrogen cycle. By collecting common herbs and soil samples along the 400 mm isoline of mean annual precipitation (MAP) in the agro-pastoral zone of North China (APZNC) and measuring their δ15N values, the statistical characteristics of foliar δ15N of herbs and the responses of foliar δ15N to the MAP and mean annual temperature (MAT) were analyzed. The results showed that: (1) the δ15N values of all herbs investigated varied from -5.5% to 15.25%. Among them, the δ15N value range of C3 herbs (-5.5~15.00%) was wider than that of C4 herbs (-2.17~15.25%), but the average value (3.27%) of C3 herbs was significantly lower than that of C4 herbaceous plants (5.55%). This difference provides an important method for identifying plants of different photosynthetic types by nitrogen isotope technology. (2) Along the transect from northeast to southwest, the δ15N of both C3 and C4 herbs decreased with the increase in the MAP, but not significantly for C3 herbs. The inverse relationship between the nitrogen isotopic signatures of herbs and MAP is consistent with previous studies. However, the MAP in the APZNC is found to only explain a small amount of the observed variance in the δ15N herbs (C3 herbs: 10.40%; C4 herbs: 25.03%). (3) A strong negative relationship was found between δ15N of herbs and MAT across the transect (C3 herbs: -0.368%/°C; C4 herbs: -0.381%/°C), which was contrary to the global pattern and some regional patterns. There was no significant difference in the δ15N responses of two different photosynthetic herbs to temperature, but the effect of temperature on the variances of δ15N of C3 and C4 herbs was significantly greater than that of precipitation. This suggests that temperature is a key factor affecting foliar δ15N of herbs in this transect. The above findings may be of value to global change researchers studying the processes of the nitrogen cycle and gaining an insight into climate dynamics of the past.

Chronic Kidney Disease and Cognitive Impairment: The Kidney-Brain Axis.

Background: Cognitive impairment, increasingly recognized as a major social burden, is commonly found in chronic kidney disease (CKD) patients. Summary: Vascular damage, uremic toxicity, oxidative stress, and peripheral/central inflammation induced by CKD might be involved in brain lesions and ultimately result in cognitive decline. Uncovering the pathophysiology of CKD-associated cognitive impairment is important for early diagnosis and prevention, which undoubtedly prompts innovative pharmacological treatments. Key Messages: Here, we sequentially review the current understanding and advances in the epidemiology, risk factors, and pathological mechanisms of cognitive impairment in CKD. Furthermore, we summarize the currently available therapeutic strategies for cognitive impairment in CKD.

Progestin-Primed Ovarian Stimulation Protocol for Patients With Endometrioma.

Objective: To evaluate the pregnancy outcomes of progestin-primed ovarian stimulation (PPOS) protocol for patients with endometrioma underwent in vitro fertilization/intra-cytoplasmic sperm injection embryo transfer (IVF/ICSI-ET). Design: Observational retrospective cohort study. Setting: University affiliated reproductive center. Study Participants: 605 infertile patients with endometrioma underwent IVF/ICSI-ET from January 2016 to March 2021 were included in this study. Methods: Multivariable logistic regression analyses were conducted to determine the independent effect of controlled ovarian stimulation (COS) protocols on reproductive outcomes of first embryo transfer (ET) cycles. The live birth was primary outcome, the implantation rate, biochemical pregnancy, clinical pregnancy and ongoing pregnancy were secondary outcomes. Results: Compared to PPOS protocol, the probability of implantation showed no significant difference with ultra-long gonadotrophin-releasing hormone agonist (GnRHa) protocol and gonadotrophin-releasing hormone antagonist (GnRHant) protocol (OR 1.7, 95% CI 0.9-3.1, OR 1.2, 95% CI 0.7-2.1, respectively). The PPOS protocol was correlated with a significantly lower biochemical pregnancy and clinical pregnancy than ultra-long GnRHa protocol in the multivariable logistic regression analysis (OR 2.3, 95% CI 1.1-4.9, OR 2.4, 95% CI 1.1-5.3, respectively). However, there was no significant difference in terms of biochemical pregnancy, clinical pregnancy and ongoing pregnancy between PPOS and GnRHant protocol (OR 1.4, 95% CI 0.7-2.7, OR 1.3, 95% CI 0.7-2.4, OR 1.1, 95% CI 0.6-2.3, respectively). In addition, compared to PPOS protocol, ultra-long GnRHa protocol and GnRHant protocol demonstrated no statistical difference in ongoing pregnancy (OR 2.0, 95% CI 0.9-4.5, OR 2.1, 95% CI 0.6-2.3, respectively). Notably, the ultra-long GnRHa protocol was associated with a significant higher probability of live birth than PPOS protocol both in crude analysis and multivariable logistic regression analysis (OR 2.6, 95% CI 1.3-5.1, OR 2.5, 95% CI 1.1-5.7, respectively). Nevertheless, no statistical difference was found in live birth between PPOS and GnRHant protocol either in crude analysis and multivariable logistic regression analysis (OR1.2, 95% CI 0.6-2.3, OR 1.2, 95% CI 0.6-2.5, respectively). Conclusions: Based on the reproductive outcomes of the first ET cycles in patients with endometrioma, PPOS protocol may associated with inferior reproductive outcomes in terms of biochemical pregnancy, clinical pregnancy and live birth than ultra-long GnRHa protocol. However, there was no significant difference in implantation rate, clinical pregnancy, ongoing pregnancy and live birth between PPOS and GnRHant protocol.

A Bi-Layer Hydrogel Cardiac Patch Made of Recombinant Functional Proteins.

The development of minimally invasive cardiac patches, either as hemostatic dressing or treating myocardial infarction, is of clinical significance but remains a major challenge. Designing such patches often requires simultaneous consideration of several material attributes, including bioabsorption, non-toxicity, matching the mechanic properties of heart tissues, and working efficiently in wet and dynamic environments. Using genetically engineered multi-domain proteins, a printed bi-layer proteinaceous hydrogel patch for heart failure treatments is reported. The intrinsic self-healing nature of hydrogel materials physically enables seamless interfacial integration of two disparate hydrogel layers and functionally endows the cardiac patches with the combinatorial advantages of each layer. Leveraging the biocompatibility, structural stability, and tunable drug release properties of the bi-layer hydrogel, promising effects of hemostasis, fibrosis reduction, and heart function recovery on mice is demonstrated with two myocardium damage models. Moreover, this proteinaceous patch is proved biodegradable in vivo without any additive inflammations. In conclusion, this work introduces a promising new type of minimally invasive patch based on genetically modified double-layer protein gel for treating heart-related injuries or diseases.

De Novo Synthesis of Orthogonally-Protected C2-Fluoro Digitoxoses and Cymaroses: Development and Application for the Synthesis of Fluorinated Digoxin.

Inspired by Roush's pioneering work on rare sugars, we have developed a scalable, stereoselective, de novo synthesis of orthogonally protected C2-fluoro digitoxose and cymarose, utilizing Sharpless kinetic resolution and organocatalytic fluorination as key steps. The utility of this strategy is demonstrated by the synthesis of a fluorinated analogue of digoxin, which indicates the fluorine on the sugar ring may have a significant impact on biological activity.

Functional amyloid-chitin hybrid ink coupled with flexible fabrication approaches for diverse macro and micro-structures.

The precise fabrication of artificially designed molecular complexes into ordered structures resembling their natural counterparts would find broad applications but remains a major challenge in the field. Here we genetically design chitin-binding domain (CBD)-containing amyloid proteins, and rationally fabricate well-ordered CBD-containing functional amyloid-chitin complex structures by coupling a top-down manufacturing process with a bottom-up self-assembly. Our fabrication approach starts with the dissolution of both CBD-containing functional amyloid and chitin in hexafluoroisopropanol (HFIP) to make a hybrid ink. This hybrid ink platform, coupled with multiple fabrication methods including airbrushing, electrospinning and soft-lithography, produces a series of unique freestanding structures. The structural features of the products, such as the ability to direct the light path and mimicking of the extracellular matrix enable applications in functional light gratings and cell culture, respectively. Further genetic engineering of the protein component allowed tunable functionalization of these materials, including nanoparticle immobilization and protein conjugation, resulting in broad applications in electronic devices and enzyme immobilization. Our technological platform can drive new advances in biocatalysis, tissue engineering, biomedicine, photonics and electronics.

A single point mutation in the hyaC gene affects Pasteurella multocida serovar A capsule production and virulence.

Pasteurella multocida (P. multocida) is a Gram-negative bacterium which causes diseases in poultry, livestock, and humans, resulting in huge economic losses. P. multocida serovar A CQ6 (PmCQ6) is a naturally occurring attenuated strain with a thin capsule. Thus, we aimed to explore why this strain is less virulent and produces less capsule compared with P. multocida serovar A strain CQ2 (PmCQ2). Analysis of capsular polysaccharide synthesis genes in PmCQ6 revealed that, compared with PmCQ2, there was only a single point mutation in the initiation codon sequence of the hyaC gene. To test whether this point mutation caused capsular deficiency and reduced virulence, we rescued this hyaC mutation and observed a restoration of capsule production and higher virulence. Transcriptome analysis showed that the hyaC point mutation led to a downregulation of capsule synthesis and/or iron utilization related-genes. Taken together, the results indicate that the start codon mutation of hyaC is an important factor affecting the capsule synthesis and virulence of PmCQ6.

PDGFRb+ mesenchymal cells, but not NG2+ mural cells, contribute to cardiac fat.

Understanding cellular origins of cardiac adipocytes (CAs) can offer important implications for the treatment of fat-associated cardiovascular diseases. Here, we perform lineage tracing studies by using various genetic models and find that cardiac mesenchymal cells (MCs) contribute to CAs in postnatal development and adult homeostasis. Although PDGFRa+ and PDGFRb+ MCs both give rise to intramyocardial adipocytes, PDGFRb+ MCs are demonstrated to be the major source of intramyocardial adipocytes. Moreover, we find that PDGFRb+ cells are heterogenous, as PDGFRb is expressed not only in pericytes and smooth muscle cells (SMCs) but also in some subendocardial, pericapillary, or adventitial PDGFRa+ fibroblasts. Dual-recombinase-mediated intersectional genetic lineage tracing reveals that PDGFRa+PDGFRb+ double-positive periendothelial fibroblasts contribute to intramyocardial adipocytes. In contrast, SMCs and NG2+ pericytes do not contribute to CAs. These in vivo findings demonstrate that PDGFRb+ MCs, but not NG2+ coronary vascular mural cells, are the major source of intramyocardial adipocytes.

Rational Design of Near-Infrared-Absorbing Pt(II)-Chelated Azadipyrromethene Dyes as a New Generation of Photosensitizers for Synergistic Phototherapy.

Pt(II) photosensitizers are emerging as novel Pt anticancer agents for cancer photodynamic therapy (PDT) to avoid uncontrollable toxicity of cisplatin. However, the application of Pt(II) photosensitizers is limited by tumor hypoxia and the poor penetration depth of excitation light. To overcome these drawbacks, exploiting the next generation of Pt anticancer agents is of urgent need. According to theoretical calculations, novel near-infrared (NIR)-absorbing Pt(II)-chelated azadipyrromethene dyes (PtDP-X, where X = N, C, and S) were designed. Importantly, spin-orbit coupling of the Pt atom could promote the intersystem crossing of a singlet-to-triplet transition for converting oxygen to singlet oxygen (1O2), and the azadipyrromethene skeleton could provide a strong photothermal effect. As expected, PtDP-X exhibited intense NIR absorption and synergistic PDT and photothermal effects with low dark cytotoxicity. Furthermore, water-soluble and biocompatible PtDP-N nanoparticles (PtDP-N NPs) were prepared that achieved effective tumor cell elimination with low side effects under 730 nm light irradiation in vitro and in vivo. This pioneering work could push the exploitation of NIR-absorbing metal-chelated azadipyrromethene dyes, so as to promote the positive evolution of phototherapy agents.

Flame Retardancy and Thermal Behavior of an Unsaturated Polyester Modified with Kaolinite-Urea Intercalation Complexes.

Organic modified kaolinite-urea intercalation complex (KUIC) was prepared using dimethyl sulfoxide (DMSO) as the precursor of kaolinite intercalation. Its structure was characterized by Fourier transform infrared (FTIR) and X-ray diffraction (XRD). Subsequently, as a synergistic agent, KUIC was combined with flame retardant ammonium polyphosphate (APP) to improve the flame retardant and smoke suppression performance of unsaturated polyester (UP) resin. A cone calorimeter (CONE) was used to study its flame retardancy and smoke suppression, and a scanning electron microscope (SEM) and thermogravimetry (TG) were used to study the micro morphology of the char and flame retardant mechanism. The results show that 12 phr of APP and 3 phr of KUIC were doped into UP to obtain a 28.0% limiting oxygen index (LOI) value. Compared with UP, the heat release rate and smoke production of UP/APP/KUIC composites were greatly decreased. Meanwhile, KUIC indeed enhanced the mechanical properties of UP.

No Evidence for Erythro-Myeloid Progenitor-Derived Vascular Endothelial Cells in Multiple Organs.

RATIONALE: Endothelial cells are thought to emerge de novo from the mesoderm to form the entire circulatory system. Recently, erythro-myeloid progenitors (EMPs) have been proposed to be another remarkable developmental origin for blood vessels in multiple organs, including the hindbrain, liver, lung, and heart, as demonstrated by lineage tracing studies using different genetic tools. These observations challenge the current consensus that intraembryonic vessels are thought to expand solely by the proliferation of preexisting endothelial cells. Resolution of this controversy over the developmental origin of endothelial cells is crucial for developing future therapeutics for vessel-dependent organ repair and regeneration. OBJECTIVE: To examine the contribution of EMPs to intraembryonic endothelial cells. METHODS AND RESULTS: We first used a transgenic mouse expressing a tamoxifen-inducible Mer-iCre fusion protein driven by the Csf1r (colony stimulating factor 1 receptor) promoter. Genetic lineage tracing based on Csf1r-Mer-iCre-Mer showed no contribution of EMPs to brain endothelial cells identified by several markers. We also generated a knock-in mouse line by inserting an internal ribosome entry site-iCre cassette into the 3' untranslated region of Csf1r gene to further investigate the cellular fates of EMPs. Similarly, we did not find any Csf1r-ires-iCre traced endothelial cells in brain, liver, lung, or heart in development either. Additionally, we found that Kit (KIT proto-oncogene receptor tyrosine kinase) was expressed not only in EMPs but also in embryonic hindbrain endothelial cells. Therefore, Kit promoter-driven recombinase, such as Kit-CreER, is a flawed tool for lineage tracing when examining the contribution of EMPs to hindbrain endothelial cells. We also traced CD45 (protein tyrosine phosphatase receptor type C; Ptprc)+ circulating EMPs and did not find any CD45 lineage-derived endothelial cells during development. CONCLUSIONS: Our study suggested that EMPs are not the origin of intraembryonic endothelial cells.

Improved method for damping coefficient measurement based on spectral analysis of a self-mixing signal.

In this paper, the self-mixing interference subject to weak optical feedback has been used to measure the damping vibration. By analyzing the spectrum of the signal, the damping coefficient can be extracted precisely from the nth-order Bessel functions, which are determined by the dominant harmonic order of the frequency spectrum. Theoretical derivation and signal processing are presented. Four kinds of vibrating targets with different damping coefficients are measured. Experimental results show that standard deviation and root mean square error of data are less than 0.2 and 0.1, respectively, which means fitted values are stable as well as having a very high fitting precision.

Differential analysis of urinary albumin for membranous nephropathy patients by online capillary isoelectric focusing - Mass spectrometry.

Membranous nephropathy (MN) is one of the most common causes of primary glomerular diseases worldwide. The M-type phospholipase A2 receptor (PLA2R), an antigen expressed in more than 70% of cases of idiopathic membranous nephropathy (IMN), is a biomarker which is now used by physicians for clinical diagnosis. Despite the prevalence of PLA2R in the cases of MN, it is not always effective to use PLA2R for differentiating primary or secondary MNs. On the other hand, urinary albumin assay is one of the de facto tests for kidney function testing for several decades. In this work, urinary albumin species between primary and secondary MN patients are compared using a newly developed capillary isoelectric focusing - mass spectrometry (CIEF-MS) technology. The distinct patterns of cationic and acidic urinary albumin species, as revealed by this novel CIEF-MS technology, suggest potential applications of this differential analysis for subtyping of membranous nephropathy. Further investigation of these cationic human albumin species in urine may provide clues to the disease onset and development of MN, thus facilitating treatment. In addition, this novel workflow of using CIEF-MS for urinary protein analysis may be beneficial to the research, pathology, prognosis, and diagnosis of many other types of kidney diseases, such as chronic kidney disease, diabetic nephrology, etc.

CARF, As An Oncogene, Promotes Colorectal Cancer Stemness By Activating ERBB Signaling Pathway.

INTRODUCTION: The role of CARF, a calcium-responsive transcription factor, in colorectal cancer initiation and development is still unknown. Here, we report that CARF promotes colorectal cancer stemness through ERBB signaling pathway. MATERIALS AND METHODS: Both colorectal cancer cell lines and primary cells were used in this study. The levels of target mRNA and protein in the cells were examined by qRT-PCR and Western blot. Gene manipulation was achieved by the lentivirus delivery system. Luciferase reporter gene assay was employed to analyze the transcriptional activity of the promoter. ChIP assay was performed for the examination of the binding between CARF and the promoters of MAPK8 and JUN. Kaplan-Meier survival curve was generated by the R2 program. Correlation analysis was performed using Spearman correlation analysis. RESULTS: Aberrant upregulation of CARF has been found in tumor tissues of colorectal cancer patients and associated with poor prognosis. Ectopic expression of CARF promoted the sphere-formation activities, as well as the expression of stem cell markers in colorectal cancer cells and knockdown of CARF, inhibited these activities. The mechanistic analysis showed that CARF directly binds to the promoter of MAPK8 and JUN, promotes the expression of MAPK8 and JUN, activates the ERBB signaling pathway, and thereby promotes the maintenance of the stemness in colorectal cancer cells. CONCLUSION: CARF, as an oncogene, promotes colorectal cancer stemness by activating ERBB signaling pathway. The ERBB signaling pathway that serves as the main downstream effector of CARF could be an efficient drug target for colorectal cancer caused by aberrant expression of CARF.

Sodium oligomannate therapeutically remodels gut microbiota and suppresses gut bacterial amino acids-shaped neuroinflammation to inhibit Alzheimer's disease progression.

Recently, increasing evidence has suggested the association between gut dysbiosis and Alzheimer's disease (AD) progression, yet the role of gut microbiota in AD pathogenesis remains obscure. Herein, we provide a potential mechanistic link between gut microbiota dysbiosis and neuroinflammation in AD progression. Using AD mouse models, we discovered that, during AD progression, the alteration of gut microbiota composition leads to the peripheral accumulation of phenylalanine and isoleucine, which stimulates the differentiation and proliferation of pro-inflammatory T helper 1 (Th1) cells. The brain-infiltrated peripheral Th1 immune cells are associated with the M1 microglia activation, contributing to AD-associated neuroinflammation. Importantly, the elevation of phenylalanine and isoleucine concentrations and the increase of Th1 cell frequency in the blood were also observed in two small independent cohorts of patients with mild cognitive impairment (MCI) due to AD. Furthermore, GV-971, a sodium oligomannate that has demonstrated solid and consistent cognition improvement in a phase 3 clinical trial in China, suppresses gut dysbiosis and the associated phenylalanine/isoleucine accumulation, harnesses neuroinflammation and reverses the cognition impairment. Together, our findings highlight the role of gut dysbiosis-promoted neuroinflammation in AD progression and suggest a novel strategy for AD therapy by remodelling the gut microbiota.