FecBB mutation enhances follicle stimulating hormone sensitivity of granulosa cells by up-regulating the SMAD1/5-USF1-FSHR signaling pathway.

The FecBB mutation, a single-point mutation (c.A746G; p.Q249R) in bone morphogenetic protein receptor type 1 B (BMPR1B), is associated with increased ovulation quotas and litter size in sheep. However, the regulatory mechanism of the FecBB mutation in increased fecundity remains to be elucidated. Therefore, creating an immortal cell model harboring the FecBB mutation would elucidate the regulatory mechanism of this mutation. Here, we report the creation of a human granulosa cell, COV434, model containing a homozygous FecBB mutation through homology-directed repair (HDR) induced by clustered, regularly-interspaced, short palindromic repeats-CRISPR-associated protein 9 along with a single-stranded oligodeoxynucleotide (ssODN) template. We found that the FecBB mutation enhanced the basal SMAD1/5 signaling activity in COV434 cells, leading to increased expression of FSHR, probably through increased formation of the SMAD1/5-SMAD4 complex to bind to the SBE element, which in turn promotes the binding of USF1 to the regulatory element E-box in the promoter of FSHR. Furthermore, the FecBB mutation substantially enhanced estradiol (E2) synthesis in granulosa cells under follicle stimulating hormone (FSH) stimulation, indicating an enhanced sensitivity to FSH, which may promote the growth of more small follicles into mature follicles, leading to increased fecundity. Our study provides novel insights into the possible regulatory mechanisms of FecBB mutations in increased fecundity.

A Meta-Learning Approach for Classifying Multimodal Retinal Images of Retinal Vein Occlusion With Limited Data.

Purpose: To propose and validate a meta-learning approach for detecting retinal vein occlusion (RVO) from multimodal images with only a few samples. Methods: In this cross-sectional study, we formulate the problem as meta-learning. The meta-training dataset consists of 1254 color fundus (CF) images from 39 different fundus diseases. Two meta-testing datasets include a public domain dataset and an independent dataset from Kandze Prefecture People's Hospital. The proposed meta-learning models comprise two modules: the feature extraction networks and the prototypical networks (PNs). We use two deep learning models (the ResNet and the Contrastive Language-Image Pre-Training networks [CLIP]) for feature extraction. We evaluate the performance of the algorithms using accuracy, area under the receiver operating characteristic curve (AUCROC), F1-score, and recall. Results: CLIP-based PNs outperform across all meta-testing datasets. For the public APTOS dataset, meta-learning algorithms achieve good results with an accuracy of 86.06% and an AUCROC of 0.87 with only 16 training images. In the hospital datasets, meta-learning algorithms show excellent diagnostic capability for detecting RVO with a very low number of shots (AUCROC above 0.99 for n = 4, 8, and 16, respectively). Notably, even though the meta-training dataset does not include fluorescein angiography (FA) images, meta-learning algorithms also have excellent diagnostic capability for detecting RVO from images with a different modality (AUCROC above 0.93 for n = 4, 8, and 16, respectively). Conclusions: The proposed meta-learning models excel in detecting RVO, not only on CF images but also on FA images from a different imaging modality. Translational Relevance: The proposed meta-learning models could be useful in automatically detecting RVO on CF and FA images.

Generating Synthesized Fluorescein Angiography Images From Color Fundus Images by Generative Adversarial Networks for Macular Edema Assessment.

Purpose: To assess the feasibility of generating synthetic fluorescein angiography (FA) images from color fundus (CF) images using pixel-to-pixel generative adversarial network (pix2pixGANs) for clinical applications. Research questions addressed image realism to retinal specialists and utility for assessing macular edema (ME) in Retinal Vein Occlusion (RVO) eyes. Methods: We used a registration-guided pix2pixGANs method trained on the CF-FA dataset from Kham Eye Centre, Kandze Prefecture People's Hospital. A visual Turing test confirmed the realism of synthetic images without novel artifacts. We then assessed the synthetic FA images for assessing ME. Finally, we quantitatively evaluated the synthetic images using Fréchet Inception distance (FID) and structural similarity measures (SSIM). Results: The raw development dataset had 881 image pairs from 349 subjects. Our approach is capable of generating realistic FA images because small vessels are clearly visible and sharp within one optic disc diameter around the macula. Two retinal specialists agreed that more than 85% of synthetic FA images have good or excellent image quality. For ME detection, accuracy was similar for real and synthetic images. FID demonstrated a 38.9% improvement over the previous state-of-the-art (SOTA), and SSIM reached 0.78 compared to the previous SOTA's 0.67. Conclusions: We developed a pix2pixGANs model translating FA images from label-free CF images, yielding reliable synthetic FA images. This suggests potential for noninvasive evaluation of ME in RVO eyes using pix2pix GANs techniques. Translational Relevance: Pix2pixGANs techniques have the potential to assist in the noninvasive clinical assessment of ME in RVO eyes.

Polystyrene nanoparticles with different particle sizes cause autophagy by ROS/ERS/FOXO1 axis in the Cyprinus carpio kidney affecting immunological function.

Microplastic pollution poses challenges for ecosystems worldwide, and nanoplastics (NPs, 1-1000 nm) have been identified as persistent pollutants. However, although some studies have described the hazards of NPs to aquatic organisms, the toxicological processes of NPs in the common carp kidney and the biotoxicity of differently sized NPs remain unclear. In this study, we used juvenile common carp as an in vivo model that were constantly exposed to freshwater at 1000 µg/L polystyrene nanoparticle (PSNP) concentrations (50, 100, and 400 nm) for 28 days. Simultaneously, we constructed an in vitro model utilizing grass fish kidney cells (CIK) to study the toxicological effects of PSNPs of various sizes. We performed RT-PCR and Western blot assays on the genes involved in FOXO1, HMGB1, HIF-1α, endoplasmic reticulum stress, autophagy, and immunoreaction. According to these results, exposure to PSNPs increased reactive oxygen species (ROS) levels, and the carp kidneys experienced endoplasmic reticulum stress. Additionally, PSNPs promoted renal autophagy by activating the ROS/ERS/FOXO1 (ERS: endoplasmic reticulum stress) pathway, and it affected immunological function by stimulating the ROS/HMGB1/HIF-1α signaling pathway. This study provides new insights into the contamination hazards of NPs in freshwater environments, as well as the harm they pose to the human living environments. The relationship between particle size and the degree of damage caused by PSNPs to organisms is a potential future research direction.

A highly polarizable concentrated dipole glass for ultrahigh energy storage.

Relaxor ferroelectrics are highly desired for pulse-power dielectric capacitors, however it has become a bottleneck that substantial enhancements of energy density generally sacrifice energy efficiency under superhigh fields. Here, we demonstrate a novel concept of highly polarizable concentrated dipole glass in delicately-designed high-entropy (Bi1/3Ba1/3Na1/3)(Fe2/9Ti5/9Nb2/9)O3 ceramic achieved via substitution of multiple heterovalent ferroelectric-active principal cation species on equivalent lattice sites. The atomic-scaled polar heterogeneity of dipoles with different polar vectors between adjacent unit cells enables diffuse reorientation process but disables appreciable growth with electric fields. These unique features cause superior recoverable energy density of ~15.9 J cm-3 and efficiency of ~93.3% in bulk ceramics. We also extend the highly polarizable concentrated dipole glass to the prototype multilayer ceramic capacitor, which exhibits record-breaking recoverable energy density of ~26.3 J cm-3 and efficiency of ~92.4% with excellent temperature and cycle stability. This research presents a distinctive approach for designing high-performance energy-storage dielectric capacitors.

Development and evaluation of a large language model of ophthalmology in Chinese.

BACKGROUND: Large language models (LLMs), such as ChatGPT, have considerable implications for various medical applications. However, ChatGPT's training primarily draws from English-centric internet data and is not tailored explicitly to the medical domain. Thus, an ophthalmic LLM in Chinese is clinically essential for both healthcare providers and patients in mainland China. METHODS: We developed an LLM of ophthalmology (MOPH) using Chinese corpora and evaluated its performance in three clinical scenarios: ophthalmic board exams in Chinese, answering evidence-based medicine-oriented ophthalmic questions and diagnostic accuracy for clinical vignettes. Additionally, we compared MOPH's performance to that of human doctors. RESULTS: In the ophthalmic exam, MOPH's average score closely aligned with the mean score of trainees (64.7 (range 62-68) vs 66.2 (range 50-92), p=0.817), but achieving a score above 60 in all seven mock exams. In answering ophthalmic questions, MOPH demonstrated an adherence of 83.3% (25/30) of responses following Chinese guidelines (Likert scale 4-5). Only 6.7% (2/30, Likert scale 1-2) and 10% (3/30, Likert scale 3) of responses were rated as 'poor or very poor' or 'potentially misinterpretable inaccuracies' by reviewers. In diagnostic accuracy, although the rate of correct diagnosis by ophthalmologists was superior to that by MOPH (96.1% vs 81.1%, p>0.05), the difference was not statistically significant. CONCLUSION: This study demonstrated the promising performance of MOPH, a Chinese-specific ophthalmic LLM, in diverse clinical scenarios. MOPH has potential real-world applications in Chinese-language ophthalmology settings.

Generative learning facilitated discovery of high-entropy ceramic dielectrics for capacitive energy storage.

Dielectric capacitors offer great potential for advanced electronics due to their high power densities, but their energy density still needs to be further improved. High-entropy strategy has emerged as an effective method for improving energy storage performance, however, discovering new high-entropy systems within a high-dimensional composition space is a daunting challenge for traditional trial-and-error experiments. Here, based on phase-field simulations and limited experimental data, we propose a generative learning approach to accelerate the discovery of high-entropy dielectrics in a practically infinite exploration space of over 1011 combinations. By encoding-decoding latent space regularities to facilitate data sampling and forward inference, we employ inverse design to screen out the most promising combinations via a ranking strategy. Through only 5 sets of targeted experiments, we successfully obtain a Bi(Mg0.5Ti0.5)O3-based high-entropy dielectric film with a significantly improved energy density of 156 J cm-3 at an electric field of 5104 kV cm-1, surpassing the pristine film by more than eight-fold. This work introduces an effective and innovative avenue for designing high-entropy dielectrics with drastically reduced experimental cycles, which could be also extended to expedite the design of other multicomponent material systems with desired properties.

Bone morphogenetic protein 15 gene disruption affects the in vitro maturation of porcine oocytes by impairing spindle assembly and organelle function.

Bone morphogenetic protein 15 (BMP15) plays a crucial role in the porcine follicular development. However, its exact functions in the in vitro maturation (IVM) of porcine oocytes remain largely unknown. Here, through cytoplasmic injection of a preassembled crRNA-tracrRNA-Cas9 ribonucleoprotein complex, we achieved BMP15 disruption in approximately 54 % of the cultured porcine oocytes. Editing BMP15 impaired the IVM of porcine oocytes, as indicated by the significantly increased abnormal spindle assembly and reduced first polar body (PB1) extrusion. The editing also impaired cytoplasmic maturation of porcine oocytes, as reflected by reduced abundant of Golgi apparatus and impaired functions of mitochondria. The impaired IVM of porcine oocytes by editing BMP15 possibly was associated with the attenuated SMAD1/5 and EGFR-ERK1/2 signaling in the cumulus granulosa cells (CGCs) and the inhibited MOS/ERK1/2 signaling in oocytes. The attenuated MOS/ERK1/2 signaling may contribute to the inactivation of maturation promoting factor (MPF) and the increased abnormal spindle assembly, leading to reduced PB1 extrusion. It also may contribute to reduced Golgi apparatus formation, and impaired functions of mitochondria. These findings expand our understanding of the regulatory role of BMP15 in the IVM of porcine oocytes and provide a basis for manipulation of porcine reproductive performance.

Boosting Thermoelectric Performance via Weakening Carrier-Phonon Coupling in BiCuSeO-Graphene Composites.

BiCuSeO is a promising oxygen-containing thermoelectric material due to its intrinsically low lattice thermal conductivity and excellent service stability. However, the low electrical conductivity limits its thermoelectric performance. Aliovalent element doping can significantly improve their carrier concentration, but it may also impact carrier mobility and thermal transport properties. Considering the influence of graphene on carrier-phonon decoupling, Bi0.88 Pb0.06 Ca0.06 CuSeO (BPCCSO)-graphene composites are designed. For further practical application, a rapid preparation method is employed, taking less than 1 h, which combines self-propagating high-temperature synthesis with spark plasma sintering. The incorporation of graphene simultaneously optimizes the electrical properties and thermal conductivity, yielding a high ratio of weighted mobility to lattice thermal conductivity (144 at 300 K and 95 at 923 K). Ultimately, BPCCSO-graphene composites achieve exceptional thermoelectric performance with a ZT value of 1.6 at 923 K, bringing a ≈40% improvement over BPCCSO without graphene. This work further promotes the practical application of BiCuSeO-based materials and this facile and effective strategy can also be extended to other thermoelectric systems.

Bismuth Ferrite-Based Lead-Free High-Entropy Piezoelectric Ceramics.

Piezoelectric ceramics, as essential components of actuators and transducers, have captured significant attention in both industrial and scientific research. The "entropy engineering" approach has been demonstrated to achieve excellent performance in lead-based materials. In this study, the "entropy engineering" approach was employed to introduce the morphotropic phase boundary (MPB) into the bismuth ferrite (BF)-based lead-free system. By employing this strategy, a serial of novel "medium to high entropy" lead-free piezoelectric ceramics were successfully synthesized, namely (1-x)BiFeO3-x(Ba0.2Sr0.2Ca0.2Bi0.2Na0.2)TiO3 (BF-xBSCBNT, x = 0.15-0.5). Our investigation systematically examined the phase structure, domain configuration, and ferroelectric/piezoelectric properties as a function of conformational entropy. Remarkable performances with a largest strain of 0.50% at 100 kV/cm, remanent polarization ∼40.07 µC/cm2, coercive field ∼74.72 kV/cm, piezoelectric coefficient ∼80 pC/N, and d33* ∼500 pm/V were achieved in BF-0.4BSCBNT ceramics. This exceptional performance can be attributed to the presence of MPB, coexisting rhombohedral and cubic phases, along with localized nanodomains. The concept of high-entropy lead-free piezoelectric ceramics in this study provides a promising strategy for the exploration and development of the next generation of lead-free piezoelectric materials.

Eucalyptol ameliorates chlorpyrifos-induced necroptosis in grass carp liver cells by down-regulating ROS/NF-κB pathway.

Chlorpyrifos (Diethoxy-sulfanylidene-(3,5,6-trichloropyridin-2-yl) oxy-λ5-phosphane, CPF) was extensively used organophosphorus pesticide, extensively deteriorating public problem with the enrichment in the water bodies. Eucalyptol (1,3,3-Trimethyl-2-oxabicyclo[2.2.2] octane, EUC), a colorless cyclic monoterpene oxide, has shown anti-inflammatory and anti-oxidation properties. To explore the effect of EUC on CPF-induced necroptosis in the grass carp liver cells (L8824 cells), we treated L8824 cells with 60 mM CPF and 5 µM EUC for 24 h. The results showed that CPF exposed lead to excessive accumulation of reactive oxygen species (ROS) and oxidative stress, activating the NF-κB and RIPK1 pathway, increasing the level of cell necroptosis. However, EUC treatment attenuated the toxic effects of CPF treatment on L8824 cells. In summary, the study demonstrated that CPF induced necroptosis and inflammation, and EUC treatment could decrease CPF-caused cell injury.

2D quasi-layered material with domino structure.

Interlayer coupling strength dichotomizes two-dimensional (2D) materials into layered and non-layered types. Traditionally, they can be regarded as atomic layers intrinsically linked via van der Waals (vdW) forces or covalent bonds, oriented orthogonally to their growth plane. In our work, we report a material system that differentiates from layered and non-layered materials, termed quasi-layered domino-structured (QLDS) materials, effectively bridging the gap between these two typical categories. Considering the skewed structure, the force orthogonal to the 2D QLDS-GaTe growth plane constitutes a synergistic blend of vdW forces and covalent bonds, with neither of them being perpendicular to the 2D growth plane. This unique amalgamation results in a force that surpasses that in layered materials, yet is weaker than that in non-layered materials. Therefore, the lattice constant contraction along this unique orientation can be as much as 7.7%, tantalizingly close to the theoretical prediction of 10.8%. Meanwhile, this feature endows remarkable anisotropy, second harmonic generation enhancement with a staggering susceptibility of 394.3 pm V-1. These findings endow further applications arranged in nonlinear optics, sensors, and catalysis.

Eucalyptol alleviates avermectin exposure-induced apoptosis and necroptosis of grass carp hepatocytes by regulating ROS/NLRP3 axis.

The wide application of Avermectin (AVM) has caused pollution of surface water and damage to non-target organisms. A growing body of evidence supports the most prominent role of Eucalyptol (EUC) is antioxidation. To the purpose of explore the injury mechanism of Avermectin on grass carp hepatocytes and the antagonistic effect of Eucalyptol, 5.7 µM AVM and/or 20 µM EUC were used to treat grass carp hepatocytes for 24 h to establish hepatocyte exposure model. The results showed that Avermectin exposure significantly increased the contents of reactive oxygen species (ROS) and malondialdehyde (MDA) in cells, reduced the activities of superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC). Also, the expressions of NLRP3 inflammasome-related genes including NLRP3, ASC, and Caspase-1, the necroptosis-related genes including RIPK1, RIPK3, and MLKL and apoptotic genes including Bax, Caspase-3, and Caspase-9 were all up-regulated. Meanwhile, the expressions of Caspase-8 and Bcl-2 were significantly decreased upon exposure to Avermectin. However, the toxicity was significantly alleviated with the treatment of EUC or N-acetyl-l-cysteine (NAC). The above results indicated that eucalyptol alleviated AVM exposure-induced apoptosis and necroptosis of grass carp hepatocytes by regulating the ROS/NLRP3 signaling pathway.

2D MXenes Embedded Perovskite Hydrogels for Efficient and Stable Solar Evaporation.

Solar evaporation is a facile and promising technology to efficiently utilize renewable energy for freshwater production and seawater desalination. Here, the fabrication of self-regenerating hydrogel composed of 2D-MXenes nanosheets embedded in perovskite La 0.6Sr 0.4Co 0.2Fe 0.8O3- δ (LSCF)/polyvinyl alcohol hydrogels for efficient solar-driven evaporation and seawater desalination is reported. The mixed dimensional LSCF/Ti3C2 composite features a localized surface plasmonic resonance effect in the polymeric network of polyvinyl alcohol endows excellent evaporation rates (1.98 kg m-2 h-1) under 1 k Wm-2 or one sun solar irradiation ascribed by hydrophilicity and broadband solar absorption (96%). Furthermore, the long-term performance reveals smooth mass change (13.33 kg m-2) during 8 h under one sun. The composite hydrogel prompts the dilution of concentrated brines and redissolves it back to water (1.2 g NaCl/270 min) without impeding the evaporation rate without any salt-accumulation. The present research offers a substantial opportunity for solar-driven evaporation without any salt accumulation in real-life applications.

Molecular characterization of the follicular development of BMP15-edited pigs.

In brief: The regulatory role of BMP15 on porcine ovarian follicular development still remains unclear. This study reveals that biallelic editing of BMP15 impairs SMAD signaling and inhibits granulosa cell proliferation, resulting in porcine follicular development arrest and ovarian hypoplasia. Abstract: Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor beta (TGF-ß) superfamily, which is critical for facilitating ovarian folliculogenesis in mono-ovulatory mammalian species but is not essential in polyovulatory mice. Our previously established BMP15-edited pigs presented varied female reproductive phenotypes, suggesting the important role of BMP15 in ovarian folliculogenesis in polyovulatory pigs. To understand the regulatory mechanism underlying the effect of BMP15 on porcine ovarian follicular development, we molecularly characterized infertile biallelic-BMP15-edited gilts with ovarian hypoplasia. We found that an absence of BMP15 proteins in biallelic-BMP15-edited gilts can lead to premature activation of primordial follicles, possibly through the upregulation of KITLG-KIT-PI3K-AKT signaling pathways. However, this absence severely impaired SMAD (Sma and Mad proteins from Caenorhabditis elegans and Drosophila, respectively) signaling, causing severely reduced granulosa cell proliferation, leading to the arrest of follicular development during the preantral stage and ovarian hypoplasia, resulting in complete infertility. Our study expands the understanding of the molecular functions of BMP15 in nonrodent polyovulatory mammals.

A Bibliometric Analysis of Diagnosis Related Groups from 2013 to 2022.

Purpose: As an important management method of the global healthcare system, diagnosis related groups (DRGs) classify patients into different cost groups and pay more attention to the equitable distribution of medical resources and the quality of medical services. At present, most countries have used DRGs to help medical institutions and doctors to treat patients more accurately, avoid the waste of medical resources, and improve treatment efficiency. Methods: The Web of Science database was searched to collect all relevant literature on DRGs from 2013 to 2022. The literature information was imported into CiteSpace, Vosviewer, and Histcite for data analysis and visualization of the results. Analyze the cooperative relationship among the countries, institutions, journals, and authors. The usage trend of keywords; Highlight the content of the cited articles. Results: The number of articles published in this decade was stable, and the number of citations in 2014 was the highest. The United States and Germany, as the first countries to use the DRGs system, are ahead of other countries in terms of the number and quality of articles. We have carried out content research on the articles with high citations, and summarized the application range of DRGs; classification method; advantages and disadvantages of the application. In general, the development trend of DRGs in foreign countries is to continuously optimize the classification method, expand the scope of application, and improve the application effect. These provide support and reference for the improvement of medical services and the perfection of the medical insurance system. Conclusion: The application of DRGs can improve the quality and efficiency of medical services, and reduce the waste of medical expenses. It can also promote the rational allocation of medical resources and the equity of medical services. In the future, DRGs will pay more attention to the personalized diagnosis and treatment and fine management of patients, and the sharing and standardization of medical data, to promote the development of medical informatization.

Albicanol antagonizes PFF-induced mitochondrial damage and reduces inflammatory factors by regulating innate immunity.

As an environmental pollutant, profenofos (PFF) can seriously endanger human health through the food chain. Albicanol is a sesquiterpene compound with antioxidant, anti-inflammatory, and anti-aging properties. Previous studies have shown that Albicanol can antagonize apoptosis and genotoxicity caused by PFF exposure. However, the toxicity mechanism of PFF regulating hepatocyte immune function, apoptosis, and programmed necrosis and the role of Albicanol in this process have not been reported yet. In this study, grass carp hepatocytes (L8824) were treated with PFF (200 µM) or combined with Albicanol (5 ×10-5 µg mL-1) for 24 h to establish an experimental model. The results of JC-1 probe staining and Fluo-3 AM probe staining showed increased free calcium ions and decreased mitochondrial membrane potential in L8824 cells after PFF exposure, suggesting that PFF exposure may lead to mitochondrial damage. Real-time quantitative PCR and Western blot results showed that PFF exposure could increase the transcription of innate immunity-related factors (C3, Pardaxin 1, Hepcidin, INF-γ, IL-8, and IL-1ß) in L8824 cells. PFF up-regulated the TNF/NF-κB signaling pathway and the expression of caspase-3, caspase-9, Bax, MLKL, RIPK1, and RIPK3 and down-regulated the expression of Caspase-8 and Bcl-2. Albicanol can antagonize the above-mentioned effects caused by PFF exposure. In conclusion, Albicanol antagonized the mitochondrial damage, apoptosis, and necroptosis of grass carp hepatocytes caused by PFF exposure by inhibiting the TNF/NF-κB pathway in innate immunity.

First paleoproteome study of fossil fish otoliths and the pristine preservation of the biomineral crystal host.

Otoliths are calcium carbonate components of the stato-acoustical organ responsible for hearing and maintenance of the body balance in teleost fish. During their formation, control over, e.g., morphology and carbonate polymorph is influenced by complex insoluble collagen-like protein and soluble non-collagenous protein assemblages; many of these proteins are incorporated into their aragonite crystal structure. However, in the fossil record these proteins are considered lost through diagenetic processes, hampering studies of past biomineralization mechanisms. Here we report the presence of 11 fish-specific proteins (and several isoforms) in Miocene (ca. 14.8-14.6 Ma) phycid hake otoliths. These fossil otoliths were preserved in water-impermeable clays and exhibit microscopic and crystallographic features indistinguishable from modern representatives, consistent with an exceptionally pristine state of preservation. Indeed, these fossil otoliths retain ca. 10% of the proteins sequenced from modern counterparts, including proteins specific to inner ear development, such as otolin-1-like proteins involved in the arrangement of the otoliths into the sensory epithelium and otogelin/otogelin-like proteins that are located in the acellular membranes of the inner ear in modern fish. The specificity of these proteins excludes the possibility of external contamination. Identification of a fraction of identical proteins in modern and fossil phycid hake otoliths implies a highly conserved inner ear biomineralization process through time.

Caspase-8 contributes to an immuno-hot microenvironment by promoting phagocytosis via an ecto-calreticulin-dependent mechanism.

BACKGROUND: Caspase-8 (Casp8) acts as an initiator in cell apoptosis signaling. However, the role of Casp8 in tuning the tumor immune microenvironment remains controversial due to the complicated crosstalk between immune-tolerogenic apoptotic cell death and immunogenic cell death cascades. METHODS: The Cancer Genome Atlas (TCGA) and publicly accessible immune checkpoint blockade (ICB)-treated cohorts were used to investigate the clinical relevance of Casp8. A tumor-bearing mouse model was used to characterize changes in the tumor microenvironment and to explore the efficacy of ICB treatment under Casp8 knockout conditions. RESULTS: By exploring TCGA datasets, we showed that the expression level of Casp8 was associated with an immuno-hot microenvironment across various solid tumor types. Casp8 deficiency leads to decreased CD8+ T cell infiltration and resistance to anti-PD-L1 therapy in a mouse model. Mechanistically, Casp8 deficiency or pharmacological disruption results in impaired ecto-calreticulin transition in tumor cells, which in turn hampers antigen presentation in draining lymph nodes. Furthermore, radiotherapy restored sensitivity to anti-PD-L1 treatment via elevated calreticulin surface expression. CONCLUSIONS: Our data revealed a causative role of Casp8 in modulating the immunogenicity of tumor cells and responsiveness to ICB immunotherapies and proposed radiotherapy as a salvage approach to overcome Casp8 deficiency-mediated ICB resistance.

Efficient editing BMP15 in porcine oocytes through microinjection of CRISPR ctRNP.

Bone morphogenetic protein 15 (BMP15) is an X-linked gene encoding an oocyte secreted factor, which plays varied functions in the female fertility between mono-ovulatory and poly-ovulatory mammalian species. We previously found that knockout of BMP15 completely blocked porcine follicular development at preantral stages. However, the specific function of BMP15 on porcine oocytes in vitro maturation remains largely unknown. Here, we injected the pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complex into the cytoplasm of germinal vesicle stage porcine oocytes to disrupt BMP15. The ctRNP composed of Cas9 nuclease and crRNA-tracrRNA complex at 1.2/1 content ratio. The tested crRNA-tracrRNA complex concentration ranging from 50 to 200 ng/µL, all presented effective editing of BMP15 in porcine oocytes, and the 125 ng/µL crRNA-tracrRNA complex presented the highest editing efficiency (39.23 ± 3.33%). Surprisingly, we found approximately 95% edited oocytes presented monoallelic mutations, and only 5% edited oocytes harbored biallelic mutations. Interestingly, the coinjected two crRNAs guided the ctRNP complex to concurrently cut within a 10 bp window of the PAM (protospacer adjacent motif), resulting in a precise deletion within BMP15 in 85.9% edited oocytes, and additional deletion happened in 14.1% edited oocytes, which resulted in large fragment deletions in BMP15. Most deletions caused frameshift and introduced premature stop codon in BMP15, resulting in the disruption of BMP15 protein expression, which was confirmed by the Western blot analysis showing the reduced BMP15 protein expression in ctRNP injected oocytes. The disruption of BMP15 attenuated the activation of SMAD1/5/8 signaling, and impaired cumulus expansion of porcine cumulus cell-oocyte complexes (COCs). Our study proved that delivering CRISPR ctRNP into porcine oocytes by microinjection was able to edit BMP15 efficiently, providing a new strategy to investigate the functions of oocyte-specific secreted factors in oocyte in vitro maturation.