RABV induces biphasic actin cytoskeletal rearrangement through Rac1 activity modulation.

Rabies virus (RABV) is highly lethal and triggers severe neurological symptoms. The neuropathogenic mechanism remains poorly understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a Rho-GTPase that is involved in actin remodeling and has been reported to be closely associated with neuronal dysfunction. In this study, by means of a combination of pharmacological inhibitors, small interfering RNA, and specific dominant-negatives, we characterize the crucial roles of dynamic actin and the regulatory function of Rac1 in RABV infection, dominantly in the viral entry phase. The data show that the RABV phosphoprotein interacts with Rac1. RABV phosphoprotein suppress Rac1 activity and impedes downstream Pak1-Limk1-Cofilin1 signaling, leading to the disruption of F-actin-based structure formation. In early viral infection, the EGFR-Rac1-signaling pathway undergoes a biphasic change, which is first upregulated and subsequently downregulated, corresponding to the RABV entry-induced remodeling pattern of F-actin. Taken together, our findings demonstrate for the first time the role played by the Rac1 signaling pathway in RABV infection and may provide a clue for an explanation for the etiology of rabies neurological pathogenesis.IMPORTANCEThough neuronal dysfunction is predominant in fatal rabies, the detailed mechanism by which rabies virus (RABV) infection causes neurological symptoms remains in question. The actin cytoskeleton is involved in numerous viruses infection and plays a crucial role in maintaining neurological function. The cytoskeletal disruption is closely associated with abnormal nervous symptoms and induces neurogenic diseases. In this study, we show that RABV infection led to the rearrangement of the cytoskeleton as well as the biphasic kinetics of the Rac1 signal transduction. These results help elucidate the mechanism that causes the aberrant neuronal processes by RABV infection and may shed light on therapeutic development aimed at ameliorating neurological disorders.

The treatment of oily wastewater by thermo-responsive calcium alginate capsules immobilized Pseudomonas aeruginosa.

A microfluidic strategy of smart calcium alginate (CA) capsules is presented to immobilize Pseudomonas aeruginosa to treat oil slicks effectively. The capsule wall is embedded with poly (N-isopropyl acrylamide) sub-microspheres as thermo-responsive switches. CA capsules, with a diameter of 3.26 mm and a thin wall thickness about 12.8 µm, have satisfying monodispersity, cavity structure, and dense surface structures. The capsules possess excellent encapsulation of bacteria, which are fixed in a restricted space and become more aggregated. It overcomes the disadvantages of a long fermentation production cycle, easy loss of bacteria, and susceptibility to shear effect. The smart CA capsules immobilized with bacteria treat model wastewater containing soybean oil or diesel and display favorable fermentation ability. The capsules can effectively treat oil slicks with high concentration, and it is an economical way for processing oily wastewater. PRACTITIONER POINTS: A thermo-responsive calcium alginate capsule was prepared by microfluidic strategy. Pseudomonas aeruginosa is environmentally friendly in treating oil slicks. The capsules, immobilized bacteria, treat oil slicks effectively. This study provides an economical way for processing different oily water.

Endogenous Melanin and Hydrogen-Based Specific Activated Theranostics Nanoagents: A Novel Multi-Treatment Paradigm for Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by excessive proliferation of rheumatoid arthritis synovial fibroblasts (RASFs) and accumulation of inflammatory cytokines. Exploring the suppression of RASFs and modulation of the RA microenvironment is considered a comprehensive strategy for RA. In this work, specifically activated nanoagents (MAHI NGs) based on the hypoxic and weakly acidic RA microenvironment are developed to achieve a second near-infrared fluorescence (NIR-II FL)/photoacoustic (PA) dual-model imaging-guided multi-treatment. Due to optimal size, the MAHI NGs passively accumulate in the diseased joint region and undergo rapid responsive degradation, precisely releasing functionalized components: endogenous melanin-nanoparticles (MNPs), hydrogen gas (H2), and indocyanine green (ICG). The released MNPs play a crucial role in ablating RASFs within the RA microenvironment through photothermal therapy (PTT) guided by accurate PA imaging. However, the regional hyperthermia generated by PTT may exacerbate reactive oxygen species (ROS) production and inflammatory response following cell lysis. Remarkably, under the acidic microenvironment, the controlled release of H2 exhibits precise synergistic antioxidant and anti-inflammatory effects with MNPs. Moreover, the ICG, the second near-infrared dye currently approved for clinical use, possesses excellent NIR-II FL imaging properties that facilitate the diagnosis of deep tissue diseases and provide the right time-point for PTT.

Homology-Activated Ultrasensitive Nanomedicines for Precise NIR-II FL/MRI Imaging-Guided "Knock-On" Dynamic Therapy in Rheumatoid Arthritis.

Stimuli-responsive nanomedicines represent a pivotal technology for in situ on-demand drug release and offer multiple advantages over conventional drug delivery systems to combat rheumatoid arthritis(RA). However, the lack of sensitivity to a single-stimuli source or the inability to synchronize multi-stimuli responses can easily lead to challenges in achieving precise-theranostics of RA. Herein, a homology-activated ultrasensitive nanomedicines MnO2-CQ4T-GOx(MCG NMs) is designed for NIR-II fluorescence(NIR-II FL)/magnetic resonance imaging(MRI)-guided effective "knock-on" dynamic anti-RA therapy. Building upon the characteristics of the RA-microenvironment, the MCG innovatively construct a MnO2-Mn2+ system, which can normalized activation sites. The ultrasensitive-responsive degradation is achieved using the multi-stimuli processes in the RA-microenvironment, triggering release of functional small molecules. The produced Mn2+ can exert Fenton-like activity to generate •OH from H2O2, thus providing the effective chemodynamic therapy(CDT). Moreover, the up-regulation of H2O2 by GOx-catalysis not only sensitizes the MnO2-Mn2+ system but also achieves self-enhancing CDT efficacy. The NIR-II FL quenching of CQ4T-BSA in the aggregated state occurs in MCG NMs, which can be rapidly and precisely "turn-on" via the MnO2-Mn2+ system. Meanwhile, the integration of activated Mn2+-based MRI imaging has successfully developed an activatable dual-modal imaging. Feedback imaging-guided precise photodynamic therapy of CQ4T-BSA can achieve efficient "knock-on" dynamic therapy for RA.

A Single NIR-II Laser-Triggered Self-Enhancing Photo/Enzyme-coupled Three-in-One Nanosystems for Breast Cancer Phototheranostics.

Multifunctional phototheranostics, employing precise and non-invasive techniques, is widely developed to enhance theranostic efficiency of breast cancer (BC), reduce side-effects, and improve quality of life. Integrating all phototheranostic modalities into a single photosensitizer for highly effective BC treatment is particularly challenging due to the potential inefficiency and time consumption associated with repeated switching of multiple-wavelength lasers. Herein, a novel single NIR-II laser-triggered three-in-one nanosystem(PdCu NY) is rationally designed, which enables dual-modal (NIR-II FL/NIR-II PA) imaging-guided self-enhancing photothermal-photodynamic therapy (PTT-PDT) in NIR-II window. The PdCu NY based on optimal Pd/Cu molar-ratio(1:11) can be easily fabricated and large-scale production for simultaneous PTT-PDT against BC under a single 1064nm laser irradiation. Significantly, the PdCu NY acted as a promising photocatalyst for decomposition of H2O into O2 upon the same laser irradiation. In addition, the inherent catalase (CAT)-like activity of PdCu NYs enables photo-enzyme dual-catalytic O2 supply to effectively alleviate hypoxia, achieving self-enhanced PDT efficiency. These PTT-PDT self-enhanced nanosystems demonstrate precise lesion localization and complete tumor ablation using a single 1064nm laser source by "one-laser, multi-functions" strategy. More importantly, this study not only reports a three-in-one PdCu-based phototheranostic agent, but also sheds light on the exploration of versatile biosafety nanosystems for clinical applications.

Fluorocarbonylation via palladium/phosphine synergistic catalysis.

Despite the growing importance of fluorinated organic compounds in pharmaceuticals, agrochemicals, and materials science, the introduction of fluorine into organic molecules is still a challenge, and no catalytic fluorocarbonylation of aryl/alkyl boron compounds has been reported to date. Herein, we present the development of palladium and phosphine synergistic redox catalysis of fluorocarbonylation of potassium aryl/alkyl trifluoroborate. Trifluoromethyl arylsulfonate (TFMS), which was used as a trifluoromethoxylation reagent, an easily handled and bench-scale reagent, has been employed as an efficient source of COF2. The reaction operates under mild conditions with good to excellent yields and tolerates diverse complex scaffolds, which allows efficient late-stage fluorocarbonylation of marked small-molecule drugs. Mechanistically, the key intermediates of labile Brettphos-Pd(II)-OCF3 complex and difluoro-Brettphos were synthesized and spectroscopically characterized, including X-ray crystallography. A detailed reaction mechanism involving the synergistic redox catalytic cycles Pd(II)/(0) and P(III)/(V) was proposed, and multifunction of phosphine ligand was identified based on 19F NMR, isotope tracing, synthetic, and computational studies.

Cold Exposure-induced Alterations in the Brain Peptidome and Gut Microbiome Are Linked to Energy Homeostasis in Mice.

Energy homeostasis of mammals during cold exposure involves complicated neural regulation and is affected by gut microbiota. However, the regulatory mechanism remains unclear partially due to a lack of comprehensive knowledge of the signaling molecules involved. Herein, we performed region-resolvable quantitative profiling of the brain peptidome using cold-exposed mouse models and interrogated the interaction between gut microbes and brain peptides in response to cold. Region-specific alterations in the brain peptidome were observed during chronic cold exposure and were correlated with gut microbiome composition. Several proSAAS-derived peptides exhibited a positive correlation with Lactobacillus. The hypothalamus-pituitary axis exhibited a sensitive response to cold exposure. We obtained a candidate pool of bioactive peptides that potentially participate in the regulation of cold-induced energy homeostasis. Intervention with cold-adapted microbiota in mice decreased the abundance of hypothalamic neurokinin B and subsequently contributed to shifting the fuel source for energy consumption from lipids to glucose. Collectively, this study demonstrated that gut microbes modulate brain peptides contributing to energy metabolism, providing a data resource for understanding the regulatory mechanism of energy homeostasis upon cold exposure.

[Finite element analysis of the effect of knee movable unicompartmental prosthesis insertion shape and mounting position on stress distribution in the knee joint after replacement].

In unicompartmental replacement surgery, there are a wide variety of commercially available unicompartmental prostheses, and the consistency of the contact surface between the common liner and the femoral prosthesis could impact the stress distribution in the knee after replacement in different ways. Medial tibial plateau fracture and liner dislocation are two common forms of failure after unicompartmental replacement. One of the reasons is the mismatch in the mounting position of the unicompartmental prosthesis in the knee joint, which may lead to failure. Therefore, this paper focuses on the influence of the shape of the contact surface between the liner and the femoral prosthesis and the mounting position of the unicompartmental prosthesis on the stress distribution in the knee joint after replacement. Firstly, a finite element model of the normal human knee joint was established, and the validity of the model was verified by both stress and displacement. Secondly, two different shapes of padded knee prosthesis models (type A and type B) were developed to simulate and analyze the stress distribution in the knee joint under single-leg stance with five internal or external rotation mounting positions of the two pads. The results showed that under a 1 kN axial load, the peak contact pressure of the liner, the peak ACL equivalent force, and the peak contact pressure of the lateral meniscus were smaller for type A than for type B. The liner displacement, peak contact pressure of the liner, peak tibial equivalent force, and peak ACL equivalent force were the smallest for type A at 3° of internal rotation in all five internal or external rotation mounting positions. For unicompartmental replacement, it is recommended that the choice of type A or type B liner for prosthetic internal rotation up to 6° should be combined with other factors of the patient for comprehensive analysis. In conclusion, the results of this paper may reduce the risk of liner dislocation and medial tibial plateau fracture after unicompartmental replacement, providing a biomechanical reference for unicompartmental prosthesis design.

Quantitative Analysis of the Protein Methylome Reveals PARP1 Methylation is involved in DNA Damage Response.

Protein methylation plays important roles in DNA damage response. To date, proteome-wide profiling of protein methylation upon DNA damage has been not reported yet. In this study, using HILIC affinity enrichment combined with MS analysis, we conducted a quantitative analysis of the methylated proteins in HEK293T cells in response to IR treatment. In total, 235 distinct methylation sites responding to IR treatment were identified, and 38% of them were previously unknown. Multiple RNA-binding proteins were differentially methylated upon DNA damage stress. Furthermore, we identified 14 novel methylation sites in DNA damage response-related proteins. Moreover, we validated the function of PARP1 K23 methylation in repairing IR-induced DNA lesions. K23 methylation deficiency sensitizes cancer cells to radiation and HU-induced replication stress. In addition, PARP1 K23 methylation participates in the resolution of stalled replication forks by regulating PARP1 binding to damaged forks. Taken together, this study generates a data resource for global protein methylation in response to IR-induced DNA damage and reveals a critical role of PARP1 K23 methylation in DNA repair.

SiamMixer: A Lightweight and Hardware-Friendly Visual Object-Tracking Network.

Siamese networks have been extensively studied in recent years. Most of the previous research focuses on improving accuracy, while merely a few recognize the necessity of reducing parameter redundancy and computation load. Even less work has been done to optimize the runtime memory cost when designing networks, making the Siamese-network-based tracker difficult to deploy on edge devices. In this paper, we present SiamMixer, a lightweight and hardware-friendly visual object-tracking network. It uses patch-by-patch inference to reduce memory use in shallow layers, where each small image region is processed individually. It merges and globally encodes feature maps in deep layers to enhance accuracy. Benefiting from these techniques, SiamMixer demonstrates a comparable accuracy to other large trackers with only 286 kB parameters and 196 kB extra memory use for feature maps. Additionally, we verify the impact of various activation functions and replace all activation functions with ReLU in SiamMixer. This reduces the cost when deploying on mobile devices.

A Compact High-Quality Image Demosaicking Neural Network for Edge-Computing Devices.

Image demosaicking has been an essential and challenging problem among the most crucial steps of image processing behind image sensors. Due to the rapid development of intelligent processors based on deep learning, several demosaicking methods based on a convolutional neural network (CNN) have been proposed. However, it is difficult for their networks to run in real-time on edge computing devices with a large number of model parameters. This paper presents a compact demosaicking neural network based on the UNet++ structure. The network inserts densely connected layer blocks and adopts Gaussian smoothing layers instead of down-sampling operations before the backbone network. The densely connected blocks can extract mosaic image features efficiently by utilizing the correlation between feature maps. Furthermore, the block adopts depthwise separable convolutions to reduce the model parameters; the Gaussian smoothing layer can expand the receptive fields without down-sampling image size and discarding image information. The size constraints on the input and output images can also be relaxed, and the quality of demosaicked images is improved. Experiment results show that the proposed network can improve the running speed by 42% compared with the fastest CNN-based method and achieve comparable reconstruction quality as it on four mainstream datasets. Besides, when we carry out the inference processing on the demosaicked images on typical deep CNN networks, Mobilenet v1 and SSD, the accuracy can also achieve 85.83% (top 5) and 75.44% (mAP), which performs comparably to the existing methods. The proposed network has the highest computing efficiency and lowest parameter number through all methods, demonstrating that it is well suitable for applications on modern edge computing devices.

Identification of PIK3CA multigene mutation patterns associated with superior prognosis in stomach cancer.

BACKGROUND: PIK3CA is the second most frequently mutated gene in cancers and is extensively studied for its role in promoting cancer cell resistance to chemotherapy or targeted therapy. However, PIK3CA functions have mostly been investigated at a lower-order genetic level, and therapeutic strategies targeting PIK3CA mutations have limited effects. Here, we explore crucial factors interacting with PIK3CA mutations to facilitate a significant marginal survival effect at the higher-order level and identify therapeutic strategies based on these marginal factors. METHODS: Mutations in stomach adenocarcinoma (STAD), breast adenocarcinoma (BRCA), and colon adenocarcinoma (COAD) samples from The Cancer Genome Atlas (TCGA) database were top-selected and combined for Cox proportional-hazards model analysis to calculate hazard ratios of mutation combinations according to overall survival data and define criteria to acquire mutation combinations with considerable marginal effects. We next analyzed the PIK3CA + HMCN1 + LRP1B mutation combination with marginal effects in STAD patients by Kaplan-Meier, transcriptomic differential, and KEGG integrated pathway enrichment analyses. Lastly, we adopted a connectivity map (CMap) to find potentially useful drugs specifically targeting LRP1B mutation in STAD patients. RESULTS: Factors interacting with PIK3CA mutations in a higher-order manner significantly influenced patient cohort survival curves (hazard ratio (HR) = 2.93, p-value = 2.63 × 10- 6). Moreover, PIK3CA mutations interacting with higher-order combination elements distinctly differentiated survival curves, with or without a marginal factor (HR = 0.26, p-value = 6.18 × 10- 8). Approximately 3238 PIK3CA-specific higher-order mutational combinations producing marginal survival effects were obtained. In STAD patients, PIK3CA + HMCN1 mutation yielded a substantial beneficial survival effect by interacting with LRP1B (HR = 3.78 × 10- 8, p-value = 0.0361) and AHNAK2 (HR = 3.86 × 10- 8, p-value = 0.0493) mutations. We next identified 208 differentially expressed genes (DEGs) induced by PIK3CA + HMCN1 compared with LRP1B mutation and mapped them to specific KEGG modules. Finally, small-molecule drugs such as geldanamycin (connectivity score = - 0.4011) and vemurafenib (connectivity score = - 0.4488) were selected as optimal therapeutic agents for targeting the STAD subtype with LRP1B mutation. CONCLUSIONS: Overall, PIK3CA-induced marginal survival effects need to be analyzed. We established a framework to systematically identify crucial factors responsible for marginal survival effects, analyzed mechanisms underlying marginal effects, and identified related drugs.

Selective C-H trifluoromethoxylation of (hetero)arenes as limiting reagent.

Methods for direct C-H trifluoromethoxylation of arenes and heteroarenes are rare, despite the importance of trifluoromethoxylated compounds for pharmaceuticals, agrochemicals, and material sciences. Especially selective C-H trifluoromethoxylation of pyridines remains a formidable challenge. Here we show a general late-stage C-H trifluoromethoxylation of arenes and heteroarenes as limiting reagent with trifluoromethoxide anion. The reaction is mediated by silver salts under mild reaction conditions, exhibiting broad substrate scope and wide functional-group compatibility. In addition, ortho-position selective C-H trifluoromethoxylation of pyridines is observed. The method is not only applicable to the gram-scale synthesis of trifluoromethoxylated products but also allows efficient late-stage C-H trifluoromethoxylation of marketed small-molecule drugs, common pharmacophores and natural products.

Entry of Challenge Virus Standard (CVS) -11 into N2a cells via a clathrin-mediated, cholesterol-, dynamin-, pH-dependent endocytic pathway.

BACKGROUND: Rabies virus (RABV), a member of Lyssavirus of Rhabdoviridae family, is a kind of negative-strand RNA virus. The zoonosis caused by RABV leads to high mortality in animals and humans. Though with the extensive investigation, the mechanisms of RABV entry into cells have not been well characterized. METHODS: Chemical inhibitors and RNA interference (RNAi) were used to analysis RABV internalization pathway. The expression level of viral N protein was examined by quantitative PCR and western blot, and the virus infection in the cells was visualized by fluorescence microscopy. RESULTS: We firstly examined the endocytosis pathway of the challenge virus standard (CVS) -11 strain in N2a cells. Chlorpromazine treatment and knockdown of clathrin heavy chain (CHC) significantly reduced viral entry, which proved clathrin was required. Meanwhile neither nystatin nor knocking down caveolin-1 (Cav1) in N2a cells had an effect on CVS-11 infection, suggesting that caveolae was independent for CVS-11 internalization. And when cholesterol of cell membrane was extracted by MßCD, viral infection was strongly impacted. Additionally by using the specific inhibitor dynasore and ammonium chloride, we verified that dynamin and a low-pH environment were crucial for RABV infection, which was confirmed by confocal microscopy. CONCLUSIONS: Our results demonstrated that CVS-11 entered N2a cells through a clathrin-mediated, cholesterol-, pH-, dynamin-required, and caveolae-independent endocytic pathway.

Antibacterial activity of the lipopetides produced by Bacillus amyloliquefaciens M1 against multidrug-resistant Vibrio spp. isolated from diseased marine animals.

In this work, the antibacterial activity of the lipopeptides produced by Bacillus amyloliquefaciens M1 was examined against multidrug-resistant Vibrio spp. and Shewanella aquimarina isolated from diseased marine animals. A new and cheap medium which contained 1.0 % soybean powder, 1.5 % wheat flour, pH 7.0 was developed. A crude surfactant concentration of 0.28 mg/ml was obtained after 18 h of 10-l fermentation and diameter of the clear zone on the plate seeded with Vibrio anguillarum was 34 mm. A preliminary characterization suggested that the lipopeptide N3 produced by B. amyloliquefaciens M1 was the main product and contained the surfactin isoforms with amino acids (GLLVDLL) and hydroxy fatty acids (of 12-15 carbons in length). The evaluation of the antibacterial activity of the lipopeptide N3 was carried out against S. aquimarina and nine species of Vibrio spp.. It was found that all the Vibrio spp. and S. aquimarina showed resistance to several different antibiotics, suggesting that they were the multidrug resistance. It was also indicated that all the Vibrio spp. strains and S. aquimarina were sensitive to the surfactin N3, in particular V. anguillarum. The results demonstrated that the lipopeptides produced by B. amyloliquefaciens M1 had a broad spectrum of action, including antibacterial activity against the pathogenic Vibrio spp. with multidrug-resistant profiles. After the treatment with the lipopeptide N3, the cell membrane of V. anguillarum was damaged, and the whole cells of the bacterium were disrupted.

Antifungal activity of the lipopeptides produced by Bacillus amyloliquefaciens anti-CA against Candida albicans isolated from clinic.

The bacterium Bacillus amyloliquefaciens anti-CA isolated from mangrove system was found to be able to actively kill Candida albicans isolated from clinic. The bacterial strain anti-CA could produce high level of bioactive substance, amylase and protease in the cheap medium containing 2.0 % soybean meal, 2.0 % wheat flour, pH 6.5 within 26 h. After purification, the main bioactive substance was confirmed to be a cyclic lipopeptide containing a heptapeptide, L-Asp→L-Leu→L-Leu→L-Val→L-Val→L-Glu→L-Leu and a 3-OH fatty acid (15 carbons). In addition to C. albicans, the purified lipopeptide can also kill many yeast strains including Metschnikowia bicuspidata, Candida tropicalis, Yarrowia lipolytica and Saccharomyces cerevisiae. After treated by the purified lipopeptide, both the whole cells and protoplasts of C. albicans were destroyed.

Trehalose accumulation from cassava starch and release by a highly thermosensitive and permeable mutant of Saccharomycopsis fibuligera.

Highly thermosensitive and permeable mutants are the mutants from which intracellular contents can be released when they are incubated both in low osmolarity water and at non-permissive temperature (usually 37°C). After mutagenesis by using nitrosoguanidine, a highly thermosensitive and permeable mutant named A11-b was obtained from Saccharomycopsis fibuligera A11-12, a trehalose overproducer in which the acid protease gene has been disrupted. Of the total trehalose, 73.8% was released from the mutant cells suspended in distilled water after they had been treated at 37°C overnight. However, only 10.0% of the total trehalose was released from the cells of S. fibuligera A11-12 treated under the same conditions. The cell volume of the mutant cells suspended in distilled water and treated at 37°C overnight was much bigger than that of S. fibuligera A11-12 treated under the same conditions. The cell growth and trehalose accumulation of the mutant were almost the same as those of S. fibuligera A11-12 during the cultivation at the flask level and in a 5-l fermentor. Both could accumulate around 28.0% (w/w) trehalose from cassava starch. After purification, the trehalose crystal from the aqueous extract of the mutant was obtained.