Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome.

Lactylation was initially discovered on human histones. Given its nascence, its occurrence on nonhistone proteins and downstream functional consequences remain elusive. Here we report a cyclic immonium ion of lactyllysine formed during tandem mass spectrometry that enables confident protein lactylation assignment. We validated the sensitivity and specificity of this ion for lactylation through affinity-enriched lactylproteome analysis and large-scale informatic assessment of nonlactylated spectral libraries. With this diagnostic ion-based strategy, we confidently determined new lactylation, unveiling a wide landscape beyond histones from not only the enriched lactylproteome but also existing unenriched human proteome resources. Specifically, by mining the public human Meltome Atlas, we found that lactylation is common on glycolytic enzymes and conserved on ALDOA. We also discovered prevalent lactylation on DHRS7 in the draft of the human tissue proteome. We partially demonstrated the functional importance of lactylation: site-specific engineering of lactylation into ALDOA caused enzyme inhibition, suggesting a lactylation-dependent feedback loop in glycolysis.

Lactyllysine Esterification Enables Efficient Lactylprotein Expression via Genetic Code Expansion and Supports Functional Proteomics Studies.

Lysine lactylation has recently been discovered and demonstrated to be an essential player in immunity, cancer and neurodegenerative diseases. Genetic code expansion (GCE) technique is powerful in uncovering lactylation functions, since it allows site-specific incorporation of lactyllysine (Klac) into proteins of interest (POIs) in living cells. However, the inefficient uptake of Klac into cells, due to its high hydrophilicity, results in limited expression of lactylated POIs. To address this challenge, here we designed esterified Klac derivatives, exemplified by ethylated Klac (KlacOEt), to enhance Klac's lipophilicity and improve its cellular uptake. The expression level of site-specifically lactylated POIs was doubled using KlacOEt in both Escherichia coli and HEK293T cells. Immunoprecipitation mass spectrometry analysis verified the significantly increased yield of the precisely lactylated fructose-bisphosphate aldolase A using KlacOEt. Furthermore, in conjunction with the Target Responsive Accessibility Profiling approach, we found that lactylation at ALDOA-K147 altered the protein's conformation, which may explain the lactylation-induced reduction in enzyme activity. Together, we demonstrate that, through enhancing the yield of lactylated proteins with Klac esters via GCE, we are able to site-specifically reveal the effects of lactylation on POIs' interactions, conformations and activities using a suite of functional proteomics and biochemical tools.

Carbene Footprinting Directs Design of Genetically Encoded Proximity-Reactive Protein Binders.

Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI-IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRASG12D/RAF1, which lacks crystal structure, and generated a covalent binder to KRASG12D. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.

Augmented Physics-Based Models for High-Order Markov Filtering.

Hybrid physics-based data-driven models, namely, augmented physics-based models (APBMs), are capable of learning complex state dynamics while maintaining some level of model interpretability that can be controlled through appropriate regularizations of the data-driven component. In this article, we extend the APBM formulation for high-order Markov models, where the state space is further augmented with past states (AG-APBM). Typically, state augmentation is a powerful method for state estimation for a high-order Markov model, but it requires the exact knowledge of the system dynamics. The proposed approach, however, does not require full knowledge of dynamics, especially the Markovity order. To mitigate the extra computational burden of such augmentation we propose an approximated-state APBM (AP-APBM) implementation leveraging summaries from past time steps. We demonstrate the performance of AG- and AP-APBMs in an autoregressive model and a target-tracking scenario based on the trajectory of a controlled aircraft with delay-feedback control. The experiments showed that both proposed strategies outperformed the standard APBM approach in terms of estimation error and that the AP-APBM only degraded slightly when compared to AG-APBM. For example, the autoregressive (AR) model simulation in our settings showed that AG-APBM and AP-APBM reduced the estimate error by 31.1% and 26.7%. The time cost and memory usage were reduced by 37.5% and 20% by AP-APBM compared to AG-APBM.

A New Denoising Method for Belt Conveyor Roller Fault Signals.

In the process of the intelligent inspection of belt conveyor systems, due to problems such as its long duration, the large number of rollers, and the complex working environment, fault diagnosis by acoustic signals is easily affected by signal coupling interference, which poses a great challenge to selecting denoising methods of signal preprocessing. This paper proposes a novel wavelet threshold denoising algorithm by integrating a new biparameter and trisegment threshold function. Firstly, we elaborate on the mutual influence and optimization process of two adjustment parameters and three wavelet coefficient processing intervals in the BT-WTD (the biparameter and trisegment of wavelet threshold denoising, BT-WTD) denoising model. Subsequently, the advantages of the proposed threshold function are theoretically demonstrated. Finally, the BT-WTD algorithm is applied to denoise the simulation signals and the vibration and acoustic signals collected from the belt conveyor experimental platform. The experimental results indicate that this method's denoising effectiveness surpasses that of traditional threshold function denoising algorithms, effectively addressing the denoising preprocessing of idler roller fault signals under strong noise backgrounds while preserving useful signal features and avoiding signal distortion problems. This research lays the theoretical foundation for the non-contact intelligent fault diagnosis of future inspection robots based on acoustic signals.

Super-Selective Intra-arterial Thrombolytic Therapy for Peripheral Facial Paralysis After Postauricular Hyaluronic Acid Injection for the Esthetic Correction of Lying Ears.

BACKGROUND: Hyaluronic acid (HA) injection in the auricular base is one of the most popular and non-surgical cosmetic procedures for correcting lying ears and optimizing the facial profile because of its minimal invasiveness, immediate effect and safety (Li et al. in Aesthet Surg J 44: 746-75, 2024). But we have recently discovered that this treatment may lead to a new and rare complication called peripheral facial paralysis that has never been reported before. Until now, the etiology, clinical traits, treatment strategies, outcomes and possible reversibility have not been characterized. METHODS: In the present study, we enrolled 4 patients with peripheral facial paralysis after subcutaneous postauricular HA filler injection. Preoperative digital subtraction angiography revealed a vascular embolism. Then, the patients underwent super-selective facial arterial thrombolytic therapy via hyaluronidase and papaverine injections. Simultaneously, general symptomatic treatment and nutritional therapy were performed. RESULTS: The patients were relieved of their clinical symptoms and the significant improvement was observed in terms of motor function in her left facial areas after treatment. The auricular skin necrosis of all patients was restored to near normal appearance. CONCLUSION: Our results indicate that super-selective facial arterial thrombolytic therapy is feasible for patients with peripheral facial paralysis induced by HA embolism. It was also beneficial in the recovery from skin necrosis. The therapy was shown to be worthy of clinical application. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The rice transcription factor Nhd1 regulates root growth and nitrogen uptake by activating nitrogen transporters.

Plants adjust root architecture and nitrogen (N) transporter activity to meet the variable N demand, but their integrated regulatory mechanism remains unclear. We have previously reported that a floral factor in rice (Oryza sativa), N-mediated heading date-1 (Nhd1), regulates flowering time. Here, we show that Nhd1 can directly activate the transcription of the high-affinity ammonium (NH4+) transporter 1;3 (OsAMT1;3) and the dual affinity nitrate (NO3-) transporter 2.4 (OsNRT2.4). Knockout of Nhd1 inhibited root growth in the presence of NO3- or a low concentration of NH4+. Compared to the wild-type (WT), nhd1 and osamt1;3 mutants showed a similar decrease in root growth and N uptake under low NH4+ supply, while nhd1 and osnrt2.4 mutants showed comparable root inhibition and altered NO3- translocation in shoots. The defects of nhd1 mutants in NH4+ uptake and root growth response to various N supplies were restored by overexpression of OsAMT1;3 or OsNRT2.4. However, when grown in a paddy field with low N availability, nhd1 mutants accumulated more N and achieved a higher N uptake efficiency (NUpE) due to the delayed flowering time and prolonged growth period. Our findings reveal a molecular mechanism underlying the growth duration-dependent NUpE.

Rice circadian clock regulator Nhd1 controls the expression of the sucrose transporter gene OsSUT1 and impacts carbon-nitrogen balance.

Interdependent metabolic and transport processes of carbon (C) and nitrogen (N) regulate plant growth and development, while the regulatory pathways remain poorly defined. We previously reported that rice circadian clock N-mediated heading date-1 (Nhd1) regulates growth duration-dependent N use efficiency. Here, we report that knockout of Nhd1 in rice reduced the rate of photosynthesis and the sucrose ratio of sheaths to blades, but increased the total C to N ratio and free amino acids. Leaf RNA-seq analysis indicated that mutation of Nhd1 dramatically altered expression of the genes linked to starch and sucrose metabolism, circadian rhythm, and amino acid metabolic pathways. We identified that Nhd1 can directly activate the transcriptional expression of sucrose transporter-1 (OsSUT1). Knockout of Nhd1 suppressed OsSUT1 expression, and both nhd1 and ossut1 mutants showed similar shorter height, and lower shoot biomass and sucrose concentration in comparison with the wild type, while overexpression of OsSUT1 can restore the defective sucrose transport and partially ameliorate the reduced growth of nhd1 mutants. The Nhd1-binding site of the OsSUT1 promoter is conserved in all known rice genomes. The positively related variation of Nhd1 and OsSUT1 expression among randomly selected indica and japonica varieties suggests a common regulatory module of Nhd1-OsSUT1-mediated C and N balance in rice.

In-situ construction of fluorescent probes for hydrogen peroxide detection in mitochondria and lysosomes with on-demand modular assembling and double turn-on features.

Due to the diverse H2O2 distribution in organelles, fluorescent probes were usually required to be prepared separately, which limited the convenience and practicability. Herein, we reported a flexible strategy to in-situ construct H2O2 fluorescent probes in different organelles. A tetrazine fused probe TP was developed with rapid click reaction capacity and sensitive H2O2 response. When treated with H2O2, the turn-on fluorescence was effectively quenched by the tetrazine part. Only after click reaction with dienophiles, the fluorescence resumed. In application, cells were firstly treated with triphenylphosphorus tagged norbornene (TPP-NB) to label mitochondria, which was followed by the introduction of probe TP to trigger click reaction. The in-situ constructed probe P1 served as a local H2O2 sensor. In a similar way, probe P2 was in-situ constructed in lysosomes via probe TP and morpholine tagged norbornene (MP-NB). With this on-demand modular assembling and double turn-on features, our strategy to construct fluorescent probes presented high flexibility and anti-interference performance, which was expected to inspired more applications in biological studies.

Cavity-dumped burst-mode Nd:YAG laser master-oscillator power-amplifier system with a flat-top beam output realized by gain profile-controlled side pumping.

We report a compact cavity-dumped burst-mode Nd:YAG laser master-oscillator power-amplifier system with a flat-top intensity distribution across the output-beam section. Custom-designed gain profile-controlled diode side pumping modules providing flat-top and concave gain profiles were utilized to generate a uniform beam profile and suppress thermal lensing during amplification, respectively. Bursts with an energy of 2.0 J and duration of 1.6 ms were operated at 10 Hz. Within the bursts, single pulses with an energy of 12.7 mJ and pulse width of 3.3 ns were achieved at 100 kHz.

Visible-Light-Induced 1,6-Enynes Triggered C-Br Bond Homolysis of Bromomalonates: Solvent-Controlled Divergent Synthesis of Carbonylated and Hydroxylated Benzofurans.

Visible-light-induced 1,6-enyne-triggered C-Br bond homolysis of bromomalonates has been developed. This transition-metal-free, photocatalyst-free, and oxidant- and additive-free protocol affords an efficient approach for divergent synthesis of carbonylated and hydroxylated benzofurans from 1,6-enynes and bromomalonates under mild conditions. Significantly, mechanistic studies reveal that the homolysis of C-Br bonds appears to experience an energy-transfer pathway, and the atom-transfer radical addition products are the key intermediates to generate carbonylated and hydroxylated benzofurans.

The effectiveness of emergency knowledge training of pediatric medical workers based on the knowledge, skills, simulation model: a quasi-experimental study.

BACKGROUND: Basic life support and advanced life support are essential emergency management skills for medical workers, and pediatricians' first aid skills can be improved through emergency knowledge training. METHODS: A controlled pre-post-intervention quasi-experimental study design was used. The study setting was a tertiary children's hospital in China. In November 2019, a KSS model of emergency knowledge learning was developed and tested, and pediatric medical workers (N = 1448) were trained with it. The outcome measures were based on an emergency knowledge questionnaire devised by the authors that measured the effectiveness of training by comparing the pre-and post-training scores of the particpants. RESULTS: Pediatric medical workers scored significantly higher in total emergency knowledge after the training course than before [75.00 (62.50, 85.00) versus 100.00 (95.00, 100.00); P = 0.00]. Basic life support and advanced life support knowledge score significantly improved after training. Teamwork scores were significantly higher after the training than before [5.00 (5.00, 10.00) versus 10.00 (10.00, 10.00); P = 0.00]. Scores were significantly higher after the training (P < 0.001), especially for case analysis questions (P = 0.00). The attitudes of the medical workers towards the training were all positive and affirmative. CONCLUSION: The KSS model was shown to be effective in improving the emergency knowledge of pediatric medical workers. Future research will be to explore the effectiveness of the model with different participants and at other hospitals or other institutions such as schools, encouraging more people to participate in and evaluate the model to promote its optimization. TRIAL REGISTRATION: Hunan Children's Hospital, HCHLL-2018-03.

New Mechanism of Gemcitabine and Its Phosphates: DNA Polymerization Disruption via 3'-5' Exonuclease Inhibition.

Gemcitabine (dFdC), a modified deoxycytidine (dC) widely used in tumor treatment, is a prodrug that is phosphorylated to generate mono-, di-, and triphosphates. The triphosphate (dFdCTP) is incorporated into DNA to terminate DNA synthesis in cancer. Some incorporated dFdC nucleotides can be partially removed by the 3'-5' exonuclease activity, namely its editing function, and the others escape the editing. However, whether there is an active mechanism for dFdC to escape the editing remains unclear. We have first discovered that unlike dFdC, its mono-, di-, and triphosphates can inhibit the 3'-5' exonuclease of DNA polymerase I, suppress editing, and allow the active escaping mechanism, whereas its polymerase activity is not remarkably affected. As such, these phosphates can prevent the removal of the incorporated dFdC residue, thereby actively blocking DNA extension and synthesis. The inhibition efficiency of these phosphates follows the increased order of the mono-, di-, and triphosphates of gemcitabine (dFdC < dFdCMP < dFdCDP < dFdCTP). In addition, after the deletion of the 3'-5' exonuclease of cellular DNA polymerase I, the Escherichia coli mutant is more sensitive to dFdCTP than is wild-type E. coli. Our new discovery of the ability of these dFdC phosphates to inhibit exonuclease activity suggests a novel anticancer mechanism of gemcitabine and its phosphate derivatives.

Applied stretch initiates directional invasion through the action of Rap1 GTPase as a tension sensor.

Although it is known that a stiffening of the stroma and the rearrangement of collagen fibers within the extracellular matrix facilitate the movement of tumor cells away from the primary lesion, the underlying mechanisms responsible are not fully understood. We now show that this invasion, which can be initiated by applying tensional loads to a three-dimensional collagen gel matrix in culture, is dependent on the Rap1 GTPases (Rap1a and Rap1b, referred to collectively as Rap1). Under these conditions Rap1 activity stimulates the formation of focal adhesion structures that align with the tensional axis as single tumor cells move into the matrix. These effects are mediated by the ability of Rap1 to induce the polarized polymerization and retrograde flow of actin, which stabilizes integrins and recruits vinculin to preformed adhesions, particularly those near the leading edge of invasive cells. Rap1 activity also contributes to the tension-induced collective invasive elongation of tumor cell clusters and it enhances tumor cell growth in vivo Thus, Rap1 mediates the effects of increased extracellular tension in multiple ways that are capable of contributing to tumor progression when dysregulated.

Bioinert Control of Zwitterionic Poly(ethylene terephtalate) Fibrous Membranes.

Poly(ethylene terephtalate) (PET)-based materials face general biofouling issues that we addressed by grafting a copolymer of glycidyl methacrylate and sulfobetaine methacrylate, poly(GMA- r-SBMA). The grafting procedure involved a dip-coating step followed by UV-exposure and led to successful grafting of the copolymer as evidenced by X-ray photoelectron spectroscopy and zeta potential measurements. It did not modify the pore size nor the porosity of the PET membranes. In addition, their surface hydrophilicity was considerably improved, with a water contact angle falling to 30° in less than 20 s and 0° in less than 1 min. The effect of copolymer concentration in the coating bath (dip-coating procedure) and UV exposure time (UV step) were scrutinized during biofouling studies involving several bacteria such as Escherichia coli and Stenotrophomonas maltophilia, but also whole blood and HT1080 fibroblasts cells. The results indicate that if all conditions led to improved biofouling mitigation, due to the efficiency of the zwitterionic copolymer and grafting procedure, a higher concentration (15 mg/mL) and longer UV exposure time (at least 10 min) enhanced the grafting density which reflected on the biofouling results and permitted a better general biofouling control regardless of the nature of the biofoulant (bacteria, blood cells, fibroblasts).

Curcumin Attenuates Asthmatic Airway Inflammation and Mucus Hypersecretion Involving a PPARγ-Dependent NF-κB Signaling Pathway In Vivo and In Vitro.

Asthma is characterized by airway inflammation and mucus hypersecretion. Curcumin possessed a potent anti-inflammatory property involved in the PPARγ-dependent NF-κB signaling pathway. Then, the aim of the current study was to explore the value of curcumin in asthmatic airway inflammation and mucus secretion and its underlying mechanism. In vivo, mice were sensitized and challenged by ovalbumin (OVA) to induce chronic asthma. Airway inflammation and mucus secretion were analyzed. In vitro, BEAS-2B cells were obtained. MCP-1, MUC5AC, and PPARγ expression and the phosphorylation of NF-κB p65 and NF-κB p65 DNA-binding activity were measured in both the lungs and BEAS-2B cells. shRNA-PPARγ was used to knock down PPARγ expression. We found that OVA-induced airway inflammation and mucus hypersecretion in mice, OVA and IL-4-induced upregulation of MCP-1 and MUC5AC, suppression of PPARγ, and activation and translocation of NF-κB p65 were notably improved by curcumin both in vivo and in vitro. Our data also showed that these effects of curcumin were significantly abrogated by shRNA-PPARγ. Taken together, our results indicate that curcumin attenuated OVA-induced airway inflammation and mucus hypersecretion in mice and suppressed OVA- and IL-4-induced upregulation of MCP-1 and MUC5AC both in vivo and in vitro, most likely through a PPARγ-dependent NF-κB signaling pathway.

Modelling Thermally Induced Non-Equilibrium Gas Flows by Coupling Kinetic and Extended Thermodynamic Methods.

Thermally induced non-equilibrium gas flows have been simulated in the present study by coupling kinetic and extended thermodynamic methods. Three different types of thermally induced gas flows, including temperature-discontinuity- and temperature-gradient-induced flows and radiometric flow, have been explored in the transition regime. The temperature-discontinuity-induced flow case has shown that as the Knudsen number increases, the regularised 26 (R26) moment equation system will gradually loss its accuracy and validation. A coupling macro- and microscopic approach is employed to overcome these problems. The R26 moment equations are used at the macroscopic level for the bulk flow region, while the kinetic equation associated with the discrete velocity method (DVM) is applied to describe the gas close to the wall at the microscopic level, which yields a hybrid DVM/R26 approach. The numerical results have shown that the hybrid DVM/R26 method can be faithfully used for the thermally induced non-equilibrium flows. The proposed scheme not only improves the accuracy of the results in comparison with the R26 equations, but also extends their capability with a wider range of Knudsen numbers. In addition, the hybrid scheme is able to reduce the computational memory and time cost compared to the DVM.

GLP-1 Analogue Liraglutide Enhances SP-A Expression in LPS-Induced Acute Lung Injury through the TTF-1 Signaling Pathway.

The reduction of pulmonary surfactant (PS) is essential for decreased pulmonary compliance and edema in acute lung injury (ALI). Thyroid transcription factor-1 (TTF-1) plays a major role in the regulation of surfactant protein-A (SP-A), the most abundant protein component of PS. Simultaneously, the glucagon-like peptide-1 (GLP-1) analogue can enhance SP-A expression in the lung. However, the underlying mechanism is still unknown. The purpose of this study was to explore whether liraglutide, a GLP-1 analogue, upregulates SP-A expression through the TTF-1 signaling pathway in ALI. In vivo, a murine model of ALI was induced by lipopolysaccharide (LPS). Pulmonary inflammation, edema, insulin level, ultrastructural changes in type II alveolar epithelial (ATII) cells, and SP-A and TTF-1 expression were analyzed. In vitro, rat ATII cells were obtained. SP-A and TTF-1 expression in cells was measured. ShRNA-TTF-1 transfection was performed to knock down TTF-1 expression. Our data showed that LPS-induced lung injury and increase in insulin level, and LPS-induced reduction of SP-A and TTF-1 expression in both the lung and cells, were significantly compromised by liraglutide. Furthermore, we also found that these effects of liraglutide were markedly blunted by shRNA-TTF-1. Taken together, our findings suggest that liraglutide enhances SP-A expression in ATII cells and attenuates pulmonary inflammation in LPS-induced ALI, most likely through the TTF-1 signaling pathway.

Design of high energy laser pulse delivery in a multimode fiber for photoacoustic tomography.

In photoacoustic tomography (PAT), delivering high energy pulses through optical fiber is critical for achieving high quality imaging. A fiber coupling scheme with a beam homogenizer is demonstrated for coupling high energy pulses in a single multimode fiber. This scheme can benefit PAT applications that require miniaturized illumination or internal illumination with a small fiber. The beam homogenizer is achieved by using a cross cylindrical lens array, which provides a periodic spatial modulation on the phase of the input light. Thus the lens array acts as a phase grating which diffracts the beam into a 2D diffraction pattern. Both theoretical analysis and experiments demonstrate that the focused beam can be split into a 2D spot array that can reduce the peak power on the fiber tip surface and thus enhance the coupling performance. The theoretical analysis of the intensity distribution of the focused beam is carried out by Fourier optics. In experiments, coupled energy at 48 mJ/pulse and 60 mJ/pulse have been achieved and the corresponding coupling efficiency is 70% and 90% in a 1000-µm and a 1500-µm-core-diameter fiber, respectively. The high energy pulses delivered by the multimode fiber are further tested for PAT imaging in phantoms. PAT imaging of a printed dot array shows a large illumination area of 7 cm2 under 5 mm thick chicken breast tissue. In vivo imaging is also demonstrated on the human forearm. The large improvement in coupling energy can potentially benefit PAT with single fiber delivery to achieve large area imaging and deep penetration detection.