Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.

The receptor-interacting serine-threonine kinase 3 (RIP3) is a key signaling molecule in the programmed necrosis (necroptosis) pathway. This pathway plays important roles in a variety of physiological and pathological conditions, including development, tissue damage response, and antiviral immunity. Here, we report the identification of a small molecule called (E)-N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide--hereafter referred to as necrosulfonamide--that specifically blocks necrosis downstream of RIP3 activation. An affinity probe derived from necrosulfonamide and coimmunoprecipitation using anti-RIP3 antibodies both identified the mixed lineage kinase domain-like protein (MLKL) as the interacting target. MLKL was phosphorylated by RIP3 at the threonine 357 and serine 358 residues, and these phosphorylation events were critical for necrosis. Treating cells with necrosulfonamide or knocking down MLKL expression arrested necrosis at a specific step at which RIP3 formed discrete punctae in cells. These findings implicate MLKL as a key mediator of necrosis signaling downstream of the kinase RIP3.

The structure of a minimum amyloid fibril core formed by necroptosis-mediating RHIM of human RIPK3.

Receptor-interacting protein kinases 3 (RIPK3), a central node in necroptosis, polymerizes in response to the upstream signals and then activates its downstream mediator to induce cell death. The active polymeric form of RIPK3 has been indicated as the form of amyloid fibrils assembled via its RIP homotypic interaction motif (RHIM). In this study, we combine cryogenic electron microscopy and solid-state NMR to determine the amyloid fibril structure of RIPK3 RHIM-containing C-terminal domain (CTD). The structure reveals a single protofilament composed of the RHIM domain. RHIM forms three ß-strands (referred to as strands 1 through 3) folding into an S shape, a distinct fold from that in complex with RIPK1. The consensus tetrapeptide VQVG of RHIM forms strand 2, which zips up strands 1 and 3 via heterozipper-like interfaces. Notably, the RIPK3-CTD fibril, as a physiological fibril, exhibits distinctive assembly compared with pathological fibrils. It has an exceptionally small fibril core and twists in both handedness with the smallest pitch known so far. These traits may contribute to a favorable spatial arrangement of RIPK3 kinase domain for efficient phosphorylation.

FBANet: Transfer Learning for Depression Recognition Using a Feature-Enhanced Bi-Level Attention Network.

The House-Tree-Person (HTP) sketch test is a psychological analysis technique designed to assess the mental health status of test subjects. Nowadays, there are mature methods for the recognition of depression using the HTP sketch test. However, existing works primarily rely on manual analysis of drawing features, which has the drawbacks of strong subjectivity and low automation. Only a small number of works automatically recognize depression using machine learning and deep learning methods, but their complex data preprocessing pipelines and multi-stage computational processes indicate a relatively low level of automation. To overcome the above issues, we present a novel deep learning-based one-stage approach for depression recognition in HTP sketches, which has a simple data preprocessing pipeline and calculation process with a high accuracy rate. In terms of data, we use a hand-drawn HTP sketch dataset, which contains drawings of normal people and patients with depression. In the model aspect, we design a novel network called Feature-Enhanced Bi-Level Attention Network (FBANet), which contains feature enhancement and bi-level attention modules. Due to the limited size of the collected data, transfer learning is employed, where the model is pre-trained on a large-scale sketch dataset and fine-tuned on the HTP sketch dataset. On the HTP sketch dataset, utilizing cross-validation, FBANet achieves a maximum accuracy of 99.07% on the validation dataset, with an average accuracy of 97.71%, outperforming traditional classification models and previous works. In summary, the proposed FBANet, after pre-training, demonstrates superior performance on the HTP sketch dataset and is expected to be a method for the auxiliary diagnosis of depression.

Image-free multi-character recognition.

The recently developed image-free sensing technique decouples semantic information directly from compressed measurements without image reconstruction, which maintains the advantages of both the light hardware and software. However, the existing attempts have failed to classify multi-semantic information with multiple targets in the practical fieldof-view. In this Letter, we report a novel image-free sensing technique to tackle the multi-target recognition challenge for the first time, to the best of our knowledge. Different from the convolutional layer stack of image-free single-pixel networks, the reported convolutional recurrent neural network (CRNN) uses the bidirectional LSTM architecture to predict the distribution of multiple characters simultaneously. The framework enables capture of the long-range dependencies, providing a high recognition accuracy of multiple characters. We demonstrate the technique's effectiveness in license plate detection, which achieves a recognition accuracy of 87.60% at a sampling rate of 5% with a refresh rate higher than 100 FPS.

Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3.

Programmed necrotic cell death induced by the tumor necrosis factor alpha (TNF-α) family of cytokines is dependent on a kinase cascade consisting of receptor-interacting kinases RIP1 and RIP3. How these kinase activities cause cells to die by necrosis is not known. The mixed lineage kinase domain-like protein MLKL is a functional RIP3 substrate that binds to RIP3 through its kinase-like domain but lacks kinase activity of its own. RIP3 phosphorylates MLKL at the T357 and S358 sites. Reported here is the development of a monoclonal antibody that specifically recognizes phosphorylated MLKL in cells dying of this pathway and in human liver biopsy samples from patients suffering from drug-induced liver injury. The phosphorylated MLKL forms an oligomer that binds to phosphatidylinositol lipids and cardiolipin. This property allows MLKL to move from the cytosol to the plasma and intracellular membranes, where it directly disrupts membrane integrity, resulting in necrotic death.

Depth acquisition in single-pixel imaging with multiplexed illumination.

Single-pixel imaging (SPI) has drawn wide attentions due to its high signal-to-noise ratio and wide working spectrum, providing a feasible solution when array sensors are expensive or not available. In the conventional SPI, the target's depth information is lost in the acquisition process due to the 3D-to-1D projection. In this work, we report an efficient depth acquisition method that enables the existing SPI systems to obtain reflectance and depth information without any additional hardware. The technique employs a multiplexed illumination strategy that contains both random and sinusoidal codes, which simultaneously encode the target's spatial and depth information into the single measurement sequence. In the reconstruction phase, we build a convolutional neural network to decode both spatial and depth information from the 1D measurements. Compared to the conventional scene acquisition method, the end-to-end deep-learning reconstruction reduces both sampling ratio (30%) and computational complexity (two orders of magnitude). Both simulations and experiments validate the method's effectiveness and high efficiency for additional depth acquisition in single-pixel imaging without additional hardware.

Photophysical properties of a boron analogue of coumarin.

In this report, we present a study into the structure and electronic properties of difluoroboronsalicylaldoxime (DFBS), a boron-based structural analog of coumarin. The modification of the heterocyclic ring of coumarin with boron results in a compound with similar structural parameters and molecular orbitals to coumarin. DFT and TDDFT calculations reveal a significant stabilization of the LUMO in DFBS; this is supported by a ∼40 nm red shift of the lowest electronic transition in the absorption spectrum. Interestingly, DFBS is emissive, while unmodified coumarin is effectively non-radiative. Comparisons between DFBS, emissive coumarin variants, and unmodified coumarin suggest that the charge transfer character of the transition contributes to the fluorescence.

Diagnosis of Invasive Nonfunctional Pituitary Adenomas by Serum Extracellular Vesicles.

The invasiveness evaluation of nonfunctional pituitary adenoma (NFPAs) is crucial for the prediction of the malignant potential and for making surgical plans of NFPAs. Current invasiveness evaluation of NFPAs is based on neuroimaging, which can hardly predict the invasive potential and dynamically monitor disease progress. Here we used microbead-assisted flow cytometry to detect and analyze the serum extracellular vesicles (EVs) from 30 NFPAs patients (15 invasive and 15 noninvasive). Lower expressions of folate receptor 1 (FOLR1) and epithelial cell adhesion molecule (EpCAM) were found in serum EVs from the invasive NFPAs patients compared to the noninvasive ones [area under the curve (AUC) of 0.94 for FOLR1 and 0.88 for EpCAM]. Meanwhile, increased mRNA expression of vimentin and N-cadherin, two mesenchymal markers, was found in serum EVs from the invasive NFPAs patients compared to the noninvasive ones. Consistent results were observed in the tumor tissue that invasive NFPAs have lower expression of the epithelial markers while higher expression of the mesenchymal markers. These results suggested the possible role of epithelial-mesenchymal transition (EMT) in the invasiveness of NFPAs. Pituitary tumor transforming gene 1 (PTTG1) mRNA in serum EVs was also found to be an indicator for invasive NFPAs and is related with EMT. These results provide a method for the blood-based diagnosis and invasiveness evaluation of NFPAs and would be beneficial to the diagnosis, prognosis prediction, and surgical risk evaluation of NFPAs.

A cytosolic heat shock protein 90 and cochaperone CDC37 complex is required for RIP3 activation during necroptosis.

Receptor-interacting protein kinase 3, RIP3, and a pseudokinase mixed lineage kinase-domain like protein, MLKL, constitute the core components of the necroptosis pathway, which causes programmed necrotic death in mammalian cells. Latent RIP3 in the cytosol is activated by several upstream signals including the related kinase RIP1, which transduces signals from the tumor necrosis factor (TNF) family of cytokines. We report here that RIP3 activation following the induction of necroptosis requires the activity of an HSP90 and CDC37 cochaperone complex. This complex physically associates with RIP3. Chemical inhibitors of HSP90 efficiently block necroptosis by preventing RIP3 activation. Cells with knocked down CDC37 were unable to respond to necroptosis stimuli. Moreover, an HSP90 inhibitor that is currently under clinical development as a cancer therapy was able to prevent systemic inflammatory response syndrome in rats treated with TNF-α. HSP90 and CDC37 cochaperone complex-mediated protein folding is thus an important part of the RIP3 activation process during necroptosis.

In Vitro and In Vivo Anti-Candida albicans Activity of a Scorpion-Derived Peptide.

The increasing infection and drug resistance frequency has encouraged the exploration of new and effective anti-Candida albicans agents. In this study, CT-K3K7, a scorpion antimicrobial peptide derivative, effectively inhibit the growth of C. albicans. CT-K3K7 killed C. albicans cells in a dose-dependent manner, mainly by damaging the plasma membrane. CT-K3K7 could also disrupt the nucleus and interact with nucleic acid. Moreover, CT-K3K7 induced C. albicans cells necrosis via a reactive oxygen species (ROS)-related pathway. Furthermore, CT-K3K7 inhibited the hyphal and biofilm formation of C. albicans. In the mouse skin subcutaneous infection model, CT-K3K7 significantly prevented skin abscess formation and reduced the number of C. albicans cells recovered from the infection area. Taken together, CT-K3K7 has the potential to be a therapeutic for C. albicans skin infections.

Antifungal efficacy of scorpion derived peptide K1K8 against Candida albicans in vitro and in vivo.

As an alternative class of antimicrobial agents, antimicrobial peptides (AMPs) have gained significant attention. In this study, K1K8, a scorpion AMP derivative, showed effective activity against Candida albicans including clinically resistant strains. K1K8 killed C. albicans cells mainly by damaging the cell membrane and inducing necrosis via an ROS-related pathway. K1K8 could also interact with DNA after damaging the nuclear envelope. Moreover, K1K8 inhibited hyphal development and biofilm formation of C. albicans in a dose-dependent manner. In the mouse skin infection model, K1K8 significantly decreased the counts of C. albicans cells in the infection area. Overall, K1K8 is a potential anti-infective agent against skin infections caused by C. albicans.

Focusing on the value of cooperative learning in physical education: a bibliometric analysis.

The shift toward cooperative learning has highlighted the growing advantages of individual learning modes during the transition. Nevertheless, a systematic compilation of the precise classification and developmental dynamics of cooperative learning in PE has been absent. This study aimed to organize the existing progress and significance of collaborative learning. The study entailed a meticulous systematic review process, examining 169 articles in this domain with the aid of visualization software. The results of the study indicate that the overall use of cooperative learning in physical education is on the rise and will reach its highest level in 2021; Second, the keywords, major core scholars, journals, countries, and major research topics; the visual knowledge map reveals the major research topics of intrinsic motivation, cooperative learning, motor skills, self-learning, written expression, and pedagogical models. The research primarily centers on primary and secondary education, followed by teacher training and higher education. At the primary and secondary school levels, there is a specific focus on aspects such as motivation, teacher-student relationships, and the group atmosphere. This research also explores sustainable development and training for PE teachers, model integration, and its influence on students' intrinsic motivation; and finally, the future directions of cooperative learning in PEare summarized. This study provides meaningful and valuable information on how cooperative learning models can be used and developed in various teaching and learning environments, physical education teacher education, and overall student development.

Nanostructures and Nanotechnologies for the Detection of Extracellular Vesicle.

Liquid biopsy has been taken as a minimally invasive examination and a promising surrogate to the clinically applied tissue-based test for the diagnosis and molecular analysis of cancer. Extracellular vesicles (EVs) carry complex molecular information from the tumor, allowing for the multicomponent analysis of cancer and would be beneficial to personalized medicine. In this review, the advanced nanomaterials and nanotechniques for the detection and molecular profiling of EVs, highlight the advantages of nanotechnology in the high-purity isolation and the high-sensitive and high-specific identification of EVs, are summarized. An outlook on the clinical application of nanotechnology-based liquid biopsy in the diagnosis, prognostication, and surveillance of cancer is also provided. It provides information for developing liquid biopsy based on EVs by discussing the advantages and challenges of functionalized nanomaterials and various nanotechnologies.

Enhanced photodynamic therapy through multienzyme-like MOF for cancer treatment.

Overcoming resistance to apoptosis is a major challenge in cancer therapy. Recent research has shown that manipulating mitochondria, the organelles critical for energy metabolism in tumor cells, can increase the effectiveness of photodynamic therapy and trigger apoptosis in tumor cells. However, there is currently insufficient research and effective methods to exploit mitochondrial damage to induce apoptosis in tumor cells and improve the effectiveness of photodynamic therapy. In this study, we present a novel nanomedicine delivery and therapeutic system called PyroFPSH, which utilizes a nanozymes-modified metal-organic framework as a carrier. PyroFPSH exhibits remarkable multienzyme-like activities, including glutathione peroxidase (GPx) and catalase (CAT) mimicry, allowing it to overcome apoptosis resistance, reduce endogenous glutathione levels, and continuously generate reactive oxygen species (ROS). In addition, PyroFPSH can serve as a carrier for the targeted delivery of sulfasalazine, a drug that can induce mitochondrial depolarization in tumor cells, thereby reducing oxygen consumption and energy supply in the mitochondria of tumor cells and weakening resistance to other synergistic treatment approaches. Our experimental results highlight the potential of PyroFPSH as a versatile nanoplatform in cancer treatment. This study expands the biomedical applications of nanomaterials as platforms and enables the integration of various novel therapeutic strategies to synergistically improve tumor therapy. It deepens our understanding of multienzyme-mimicking active nanocarriers and mitochondrial damage through photodynamic therapy. Future research can further explore the potential of PyroFPSH in clinical cancer treatment and improve its drug loading capacity, biocompatibility and targeting specificity. In summary, PyroFPSH represents a promising therapeutic approach that can provide new insights and possibilities for cancer treatment.

m6 A Reader YTHDF1-Targeting Engineered Small Extracellular Vesicles for Gastric Cancer Therapy via Epigenetic and Immune Regulation.

N6 -methyladenosine (m6 A) modulators decide the fate of m6 A-modified transcripts and drive cancer development. RNA interference targeting m6 A modulators promise to be an emerging cancer therapy but is challenging due to its poor tumor targeting and high systematic toxicity. Here engineered small extracellular vesicles (sEVs) with high CD47 expression and cyclic arginine-glycine-aspartic (c(RGDyC)) modification are developed for effective delivery of short interfering RNA against m6 A reader YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) to treat gastric cancer via epigenetic and immune regulation. This nanosystem efficiently depletes YTHDF1 expression and suppresses gastric cancer progression and metastasis through hampering frizzled7 translation and inactivating Wnt/ß-catenin pathway in an m6 A dependent manner. Loss of YTHDF1 mediates overexpression of interferon (IFN)-γ receptor 1 and enhances IFN-γ response, promoting expression of major histocompatibility complex class I on tumor cells to achieve self-presentation of the immunogenic tumor cells to stimulate strong cytotoxic T lymphocytes responses. CD47 expression on the engineered sEVs can competitively bind with signal regulatory protein α to enhance phagocytosis of the tumor cells by tumor-associated macrophages. This versatile nanoplatform provides an efficient and low toxic strategy to inhibit epigenetic regulators and holds great potential in promoting immunotherapy.

HSPA8 acts as an amyloidase to suppress necroptosis by inhibiting and reversing functional amyloid formation.

Ultra-stable fibrous structure is a hallmark of amyloids. In contrast to canonical disease-related amyloids, emerging research indicates that a significant number of cellular amyloids, termed 'functional amyloids', contribute to signal transduction as temporal signaling hubs in humans. However, it is unclear how these functional amyloids are effectively disassembled to terminate signal transduction. RHIM motif-containing amyloids, the largest functional amyloid family discovered thus far, play an important role in mediating necroptosis signal transduction in mammalian cells. Here, we identify heat shock protein family A member 8 (HSPA8) as a new type of enzyme - which we name as 'amyloidase' - that directly disassembles RHIM-amyloids to inhibit necroptosis signaling in cells and mice. Different from its role in chaperone-mediated autophagy where it selects substrates containing a KFERQ-like motif, HSPA8 specifically recognizes RHIM-containing proteins through a hydrophobic hexapeptide motif N(X1)φ(X3). The SBD domain of HSPA8 interacts with RHIM-containing proteins, preventing proximate RHIM monomers from stacking into functional fibrils; furthermore, with the NBD domain supplying energy via ATP hydrolysis, HSPA8 breaks down pre-formed RHIM-amyloids into non-functional monomers. Notably, HSPA8's amyloidase activity in disassembling functional RHIM-amyloids does not require its co-chaperone system. Using this amyloidase activity, HSPA8 reverses the initiator RHIM-amyloids (formed by RIP1, ZBP1, and TRIF) to prevent necroptosis initiation, and reverses RIP3-amyloid to prevent necroptosis execution, thus eliminating multi-level RHIM-amyloids to effectively prevent spontaneous necroptosis activation. The discovery that HSPA8 acts as an amyloidase dismantling functional amyloids provides a fundamental understanding of the reversibility nature of functional amyloids, a property distinguishing them from disease-related amyloids that are unbreakable in vivo.

Recent advances in lipid nanovesicles for targeted treatment of spinal cord injury.

The effective regeneration and functional restoration of damaged spinal cord tissue have been a long-standing concern in regenerative medicine. Treatment of spinal cord injury (SCI) is challenging due to the obstruction of the blood-spinal cord barrier (BSCB), the lack of targeting of drugs, and the complex pathophysiology of injury sites. Lipid nanovesicles, including cell-derived nanovesicles and synthetic lipid nanovesicles, are highly biocompatible and can penetrate BSCB, and are therefore effective delivery systems for targeted treatment of SCI. We summarize the progress of lipid nanovesicles for the targeted treatment of SCI, discuss their advantages and challenges, and provide a perspective on the application of lipid nanovesicles for SCI treatment. Although most of the lipid nanovesicle-based therapy of SCI is still in preclinical studies, this low immunogenicity, low toxicity, and highly engineerable nanovesicles will hold great promise for future spinal cord injury treatments.

Guanylyl cyclase-D in the olfactory CO2 neurons is activated by bicarbonate.

Atmospheric CO(2) is an important environmental cue that regulates several types of animal behavior. In mice, CO(2) responses of the olfactory sensory neurons (OSNs) require the activity of carbonic anhydrase to catalyze the conversion of CO(2) to bicarbonate and the opening of cGMP-sensitive ion channels. However, it remains unknown how the enhancement of bicarbonate levels results in cGMP production. Here, we show that bicarbonate activates cGMP-producing ability of guanylyl cyclase-D (GC-D), a membrane GC exclusively expressed in the CO(2)-responsive OSNs, by directly acting on the intracellular cyclase domain of GC-D. Also, the molecular mechanism for GC-D activation is distinct from the commonly believed model of "release from repression" for other membrane GCs. Our results contribute to our understanding of the molecular mechanisms of CO(2) sensing and suggest diverse mechanisms of molecular activation among membrane GCs.

Clinical effect of day case arthroscopic surgery in tibial-eminence fracture in adults using button plates.

Background: The tibial-eminence fracture (TEF) is an anterior cruciate-ligament avulsion fracture with a low incidence. Many surgical techniques have been described, but none of them allow early functional exercise, and there are many postoperative complications. Purposes: This study aimed to evaluate the early clinical efficacy and complications of day case arthroscopic-surgery treatment of adult TEF with button plates. Methods: We retrospectively analyzed patients with TEF treated with arthroscopic surgery. Clinical subjective evaluation included International Knee Documentation Committee (IKDC) subjective score, Lysholm Knee Score, and Visual Analog Scale (VAS) score. Knee joint scores were evaluated by Lysholm score. Clinical objective assessment included the Lachman test, anterior-drawer test (ADT), IKDC, and range of motion. We assessed patient quality of life using a life summary table. Assessment of fracture healing and internal fixation was based on lateral x-rays of the knee joint. We measured and evaluated patient satisfaction at the last follow-up in accordance with Marsh criteria. Results: At final follow-up (average follow-up time, 28.23 ± 3.14 months), we evaluated results from 22 patients (22 knees). Average patient age during surgery was 33.64 ± 6.96 years. Average time from injury to surgery was 6.59 ± 1.47 h. Postoperative function was better than pre-operative function in all patients. IKDC subjective score, Lysholm score, and VAS score were better at final follow-up than before surgery. Differences in Lachman test and ADT scores before and after surgery were statistically significant. According to Intra-articular button position classification, 6 patients (6 knees) showed ideal position (A), 16 patients (16 knees) showed nearly ideal position (B), and none of the patients had nonideal position (C). The fractures of 22 patients healed completely; 2 patients had a 5°-10° knee joint dysfunction, and 1 had an abnormal knee sound. According to intra-articular button position classification, the rate of ideal position was 100%. Patient satisfaction rate was 81.8%. Conclusion: Day surgery using double-button plates to treat TEF could achieve anatomical reduction, power and stability, as well as good clinical efficacy.

Early Education Application Software Based on Artificial Intelligence VR Technology.

In order to solve the problems of restricted classroom and lack of repeated training in good curriculum, the design and development of a school life adaptation curriculum based on VR is proposed. This study identified six life adaptation themes for design and development (recognizing facial expressions, crossing the road, how to get lost, shopping, taking public transport, and job interview) and set the objectives, design principles, specific content, teaching evaluation, and other aspects of virtual reality life adaptation course. Combined with the characteristics of virtual reality, the implementation suggestions and matters needing attention are put forward. The results showed that, in the recognition of human facial expressions, 51 experts thought it was appropriate, 3 experts thought it needed to be modified, and no expert thought it was inappropriate. On the topic of crossing the road, 46 experts thought it was appropriate, eight thought it needed to be modified, and no expert thought it was inappropriate. On the theme of taking public transportation, 48 experts thought it was appropriate, 6 experts thought it needed to be modified, and no expert thought it was inappropriate. For the three major topics of how to get lost, shopping, and job interview, 2-3 experts put forward different opinions and think that the topic is not appropriate mainly because the grade arrangement is not appropriate. Conclusion. The VR-based curriculum for school life adaptation has been reviewed by experts and revised by researchers. It is scientific, interesting, and operable, solves the teaching problems of life adaptation teachers, and provides a safe, reliable, and effective practice channel for students.