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Various industrial waste binders (IWBs) are being recycled in soil stabilization to save cement consumption. However, the coupled effects brought out by combined IWBs on stabilized soils are still unclear. IWBs are categorized into two typical categories (IWB-A and IWB-B) referring to their chemical role in this study. The alkali-source effect, pore-filling effect and cementation damage effect by IWBs in soil stabilization are explored. A series of mechanical and microscopic tests is performed on stabilized clay with different proportions of IWB-A and IWB-B. Moreover, initial water contents and cement contents of cement-stabilized clay are varied to examine the evolution of coupled effect with void ratio and cementation level. The results indicate that the alkali-source effect strengthens the cementation bonds and increases the early strength by 0.5–1.3 times, whereas the pore-filling effect improves the microfabric especially for the specimen with a large void ratio. The alkali-source effect increases soil cohesion cu at the pre-yield stage, and the pore-filling effect increases frictional angle φu at the post-yield stage. The cementation damage effect is remarkable at a low void ratio, which may result in many extruded pores among soil aggregates. The strength evolution with IWB proportions can be well stimulated by considering the coupled alkali-source effect, pore-filling effect and cementation damage effect. The optimal proportion of IWBs corresponds to an optimal combination of coupled effect.

Abstract Background Laccases can oxidize a broad spectrum of substrates, offering promising applications in various sectors, such as bioremediation, biomass fractionation in future biorefineries, and synthesis of biochemicals and biopolymers. However, laccase discovery and optimization with a desirable pH optimum remains a challenge due to the labor-intensive and time-consuming nature of the traditional laboratory methods. Results This study presents a machine learning (ML)-integrated approach for predicting pH optima of basidiomycete fungal laccases, utilizing a small, curated dataset against a vast metagenomic data. Comparative computational analyses unveiled the structural and pH-dependent solubility differences between acidic and neutral-alkaline laccases, helping us understand the molecular bases of enzyme pH optimum. The pH profiling of the two ML-predicted alkaline laccase candidates from the basidiomycete fungus Lepista nuda further validated our computational approach, showing the accuracy of this comprehensive method. Conclusions This study uncovers the efficacy of ML in the prediction of enzyme pH optimum from minimal datasets, marking a significant step towards harnessing computational tools for systematic screening of enzymes for biotechnology applications. Graphical Abstract

Abstract Acidic microenvironments is a cancer progression driver, unclear core mechanism hinders the discovery of new diagnostic or therapeutic targets. ASIC3 is an extracellular proton sensor and acid-sensitive, but its role in acidic tumor microenvironment of colorectal cancer is not reported. Functional analysis data show that colorectal cancer cells respond to specific concentration of lactate to accelerate invasion and metastasis, and ASIC3 is the main actor in this process. Mechanism reveal de novo lipid synthesis is a regulatory process of ASIC3, down-regulated ASIC3 increases and interacts with ACC1 and SCD1, which are key enzymes in de novo lipid synthesis pathway, this interaction results in increased unsaturated fatty acids, which in turn induce EMT to promote metastasis, and overexpression of ASIC3 reduces acidic TME-enhanced colorectal cancer metastasis. Clinical samples of colorectal cancer also exhibit decreased ASIC3 expression, and low ASIC3 expression is associated with metastasis and stage of colorectal cancer. This study is the first to identify the role of the ASIC3-ACC1/SCD1 axis in acid-enhanced colorectal cancer metastasis. The expression pattern of ASIC3 in colorectal cancer differs significantly from that in other types of cancers, ASIC3 may serve as a novel and reliable marker for acidic microenvironmental in colorectal cancer, and potentially a therapeutic target.

In order to improve the safety and preservability of fermented vegetables, strain Leuconostoc mesenteroides AP7, with potent inhibitory effects on foodborne pathogens and bacteria causing paocai over-acidification, was selected from 8 Leuconostoc strains derived from traditional fermented vegetables in Yunnan, China. The main antimicrobial substance of AP7 was determined by eliminating the effects of organic acid and H2O2 and through protease sensitivity. The acid stability and thermal stability were analyzed and potential bacteriocin gene clusters were mined from the whole genome sequence of AP7. The results showed that the culture supernatant still had obvious antibacterial activity after excluding the influence of acid and H2O2, and after protease treatment, the antibacterial effect decreased significantly. It was speculated that the antibacterial substance in the concentrated culture supernatant was bacteriocin. The bacteriocin was sensitive to pH changes, had high thermal stability, and had a molecular weight of 6.51~14.4 kDa. Whole genome sequencing showed that the whole genome of AP7 contained 1 chromosome (1948310 bp) and 2 plasmids (37366 and 20698 bp), with a GC content of 37.7%. There was a gene cluster with Enterocin_X_chain_beta bacteriocin as the core. Its encoded product was predicted to be a positively charged hydrophilic stable protein, the secondary structure was dominated by α-helix, and the tertiary structure was mainly composed of loose peptide chains at both ends and an α-helix in the middle. In summary, the bacteriocin-producing Leuconostoc mesenteroides AP7 has excellent antibacterial properties and has the potential to be applied to the fermentation and preservation of acidic foods.

Abstract High-altitude hypoxia acclimatization requires whole-body physiological regulation in highland immigrants, but the underlying genetic mechanism has not been clarified. Here we use sheep as an animal model for low-to-high altitude translocation. We generate multi-omics data including whole-genome sequences, time-resolved bulk RNA-Seq, ATAC-Seq and single-cell RNA-Seq from multiple tissues as well as phenotypic data from 20 bio-indicators. We characterize transcriptional changes of all genes in each tissue, and examine multi-tissue temporal dynamics and transcriptional interactions among genes. Particularly, we identify critical functional genes regulating the short response to hypoxia in each tissue (e.g., PARG in the cerebellum and HMOX1 in the colon). We further identify TAD-constrained cis-regulatory elements, which suppress the transcriptional activity of most genes under hypoxia. Phenotypic and transcriptional evidence indicate that antenatal hypoxia could improve hypoxia tolerance in offspring. Furthermore, we provide time-series expression data of candidate genes associated with human mountain sickness (e.g., BMPR2) and high-altitude adaptation (e.g., HIF1A). Our study provides valuable resources and insights for future hypoxia-related studies in mammals.

Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease and is closely associated with metabolic diseases such as obesity, type 2 diabetes mellitus (T2DM), and metabolic syndrome. However, effective treatment strategies for NAFLD are still lacking. In recent years, progress has been made in understanding the pathogenesis of NAFLD, identifying multiple therapeutic targets and providing new directions for drug development. This review summarizes the recent advances in the treatment of NAFLD, focusing on the mechanisms of action of natural products, small-synthetic-molecule drugs, and combination therapy strategies. This review aims to provide new insights and strategies in treating NAFLD.

IntroductionBreast cancer is a heterogeneous disease comprising various molecular subtypes, including Luminal A, Luminal B, human epidermal growth factor receptor-2 (HER2) positive, and triple negative types, each with distinct biological characteristics and behaviors. Triple negative breast cancer (TNBC) remains a particularly challenging subtype worldwide. Our study aims to evaluate whether Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (18F-FDG PET/CT) parameters, clinical pathological features, and biochemical indicators serve as prognostic risk factors for TNBC. Additionally, we explore correlations between biochemical indicators and 18F-FDG PET/CT parameters.MethodsWe conducted a retrospective analysis of 95 TNBC patients who underwent preoperative 18F-FDG PET/CT examinations at Tianjin Medical University Cancer Institute and Hospital from 2013 to 2018. Collected data included 18F-FDG PET/CT parameters, clinical and pathological features, and biochemical indicators. We used Kaplan-Meier survival analysis and multivariate Cox regression analysis to evaluate associations between 18F-FDG PET/CT parameters/biochemical indicators and disease free survival (DFS)/overall survival (OS). The log-rank test determined significant differences in survival curves, and the Spearman correlation coefficient analyzed correlations between quantitative variables. Visualization and analysis were performed using R packages.ResultsAmong 95 TNBC patients, mean standardized uptake value (SUVmean) was significantly correlated with DFS. Fasting blood glucose (FBG), α- L-fucosylase (AFU) and Creatine kinase (CK) were independent predictors of DFS, while Precursor albumin (PALB) and CK were independent predictors of OS. FBG showed correlations with SUVpeak and SUVmean, and CK was correlated with peak standardized uptake value (SUVpeak). Our results indicated that 18F-FDG PET/CT parameters and biochemical indicators may constitute a new prognostic model for TNBC patients post-surgery.DiscussionWe found that SUVmean, FBG, AFU and CK are predictive factors for DFS in TNBC patients post-surgery, while PALB and CK are predictive factors for OS, which prompts us to pay more attention to these indicators in clinical practice. Also 18F-FDG PET/CT parameters and biochemical indicators have potential utility in constituting a new prognostic model for TNBC patients post-surgery.

BackgroundThe effectiveness of Tuina therapy has been confirmed in treating pain of patients with cervical spondylosis (CS), however, its therapeutic mechanism is still unclear. This study aimed to observe the changes of regional brain activity following Tuina therapy in patients with painful CS based on resting-state functional magnetic resonance imaging (rs-fMRI) data.MethodsA total of 27 patients with CS and 27 healthy subjects (HCs) were enrolled in this study. All patients received Tuina therapy every 2 days for 2 weeks. The clinical manifestations of patients were evaluated by the Visual Analog Scale (VAS) and Neck Disability Index (NDI) before and after treatment. In addition, rs-fMRI data were collected and preprocessed in all patients before and after treatment, as well as HCs. HCs underwent a 1-time rs-fMRI scan, whereas CS patients underwent 2-times of rs-fMRI scan. The measure of regional homogeneity (ReHo) was calculated and compared between groups. Finally, relationships between altered brain regions and clinical characteristics were evaluated by Pearson’s correlation analysis.ResultsAfter Tuina therapy, VAS and NDI scores of patients decreased. Before treatment, CS patients showed higher ReHo values in the left middle temporal gyrus, left thalamus, right anterior and posterior cingulate gyrus, left inferior parietal gyrus and lower ReHo values in the right gyrus rectus when compared with HCs. After treatment, CS patients exhibited higher ReHo values in the left inferior temporal gyrus, right anterior and posterior cingulate gyrus, left inferior parietal gyrus and lower ReHo values in the right rectus gyrus when compared with HCs. CS patients after treatment demonstrated higher ReHo values in the left inferior occipital gyrus when compared with those before treatment. Positive correlations were found between ReHo values of the right rectus gyrus and VAS, NDI scores in CS patients before treatment. Differences of VAS scores between before and after treatment were negatively correlated with ReHo values of the left inferior temporal gyrus in CS patients after treatment.ConclusionThis study demonstrated the presence of asynchronous activity in certain brain regions in CS patients, which might be associated with pain and cervical spine dysfunction. Tuina therapy might modulate asynchronous activity of abnormal brain regions, which might contribute to the effectiveness of Tuina therapy in alleviating pain and cervical spine dysfunction in CS patients.

Text CAPTCHAs are crucial security measures deployed on global websites to deter unauthorized intrusions. The presence of anti-attack features incorporated into text CAPTCHAs limits the effectiveness of evaluating them, despite CAPTCHA recognition being an effective method for assessing their security. This study introduces a novel color augmentation technique called Variational Color Shift (VCS) to boost the recognition accuracy of different networks. VCS generates a color shift of every input image and then resamples the image within that range to generate a new image, thus expanding the number of samples of the original dataset to improve training effectiveness. In contrast to Random Color Shift (RCS), which treats the color offsets as hyperparameters, VCS estimates color shifts by reparametrizing the points sampled from the uniform distribution using predicted offsets according to every image, which makes the color shifts learnable. To better balance the computation and performance, we also propose two variants of VCS: Sim-VCS and Dilated-VCS. In addition, to solve the overfitting problem caused by disturbances in text CAPTCHAs, we propose an Auto-Encoder (AE) based on Large Separable Kernel Attention (AE-LSKA) to replace the convolutional module with large kernels in the text CAPTCHA recognizer. This new module employs an AE to compress the interference while expanding the receptive field using Large Separable Kernel Attention (LSKA), reducing the impact of local interference on the model training and improving the overall perception of characters. The experimental results show that the recognition accuracy of the model after integrating the AE-LSKA module is improved by at least 15 percentage points on both M-CAPTCHA and P-CAPTCHA datasets. In addition, experimental results demonstrate that color augmentation using VCS is more effective in enhancing recognition, which has higher accuracy compared to RCS and PCA Color Shift (PCA-CS).

Abstract Background A lumbar puncture procedure’s success depends on a competent physician minimizing the risk of failing to get a sample and avoiding complications such as post-dural headache. A new virtual-reality simulator might be helpful in deciding when a physician is competent to perform lumbar puncture. We aimed to investigate validity evidence for a simulator-based test in lumbar puncture and establish a pass/fail standard to allow a mastery learning training program. Methods Validity evidence was investigated using Messick’s framework by including participants who were novices, intermediates, or experienced in lumbar puncture. Each participant performed two lumbar puncture procedures on the simulator, and fifty-nine predefined simulator metrics were automatically recorded. Cronbach’s alpha was used to explore internal consistency reliability. Intergroup comparisons were made using independent sample t-tests with Tukey’s correction for multiple comparisons. The learning effect was explored using paired sample t-test analysis, and a pass/fail standard was established using the contrasting groups’ method. Results 73 novices, 18 intermediates, and 19 physicians performed the test resulting in a total of 220 procedures. 25 metrics (42.4%) had good discriminatory ability, and the reliability of these metrics was good, Cronbach’s α = 0.81. The experienced physicians were significantly better than the novices (18.3 vs. 13.3, p

Abstract Background Colorectal cancer (CRC) ranks among the most prevalent malignancies worldwide, characterized by its complex etiology and slow research progress. Diabetes, as an independent risk factor for CRC, has been widely certified. Consequently, this study centers on elucidating the intricacies of CRC cells initiation and progression within a high‐glucose environment. Methods A battery of assays was employed to assess the proliferation and metastasis of CRC cells cultured under varying glucose concentrations. Optimal glucose levels conducive to cells' proliferation and migration were identified. Western blot analyses were conducted to evaluate alterations in apoptosis, autophagy, and EMT‐related proteins in CRC cells under high‐glucose conditions. The expression of PI3K/AKT/mTOR pathway‐associated proteins was assessed using western blot. The effect of high glucose on xenograft growth was investigated in vivo by MC38 cells, and changes in inflammatory factors (IL‐4, IL‐13, TNF‐α, IL‐5, and IL‐12) were measured via serum ELISA. Results Our experiments demonstrated that elevated glucose concentrations promoted both the proliferation and migration of CRC cells; the most favorable glucose dose is 20 mM. Western blot analyses revealed a decrease in apoptotic proteins, such as Bim, Bax, and caspase‐3 with increasing glucose levels. Concurrently, the expression of EMT‐related proteins, including N‐cadherin, vimentin, ZEB1, and MMP9, increased. High‐glucose cultured cells exhibited elevated levels of PI3K/AKT/mTOR pathway proteins. In the xenograft model, tumor cells stimulated by high glucose exhibited accelerated growth, larger tumor volumes, and heightened KI67 expression of immunohistochemistry. ELISA experiments revealed higher expression of IL‐4 and IL‐13 and lower expression of TNF‐α and IL‐5 in the serum of high‐glucose‐stimulated mice. Conclusion The most favorable dose and time for tumor cells proliferation and migration is 20 mM, 48 h. High glucose fosters CRC cell proliferation and migration while suppressing autophagy through the activation of the PI3K/AKT/mTOR pathway.

Abstract The patterns of communication among different chondrocyte subtypes in human cartilage degeneration and regeneration help us understand the microenvironment of osteoarthritis and optimize cell-targeted therapies. Here, a single-cell transcriptome dataset of chondrocytes is used to explore the synergistic and communicative patterns of different chondrocyte subtypes. We collected 1600 chondrocytes from 10 patients with osteoarthritis and analyzed the active communication patterns for the first time based on network analysis and pattern recognition at the single-cell level. Manifold learning and quantitative contrasts were performed to analyze conserved and specific communication pathways. We found that ProCs (Proliferative chondrocytes), ECs (Effector chondrocytes), preHTCs (Prehypertrophic chondrocytes), HTCs (Hypertrophic chondrocytes), and FCs (Fibrocartilage chondrocytes) are more active in incoming and outgoing signaling patterns, which is consistent with studies on their close functional cooperation. Among them, preHTCs play multiple roles in chondrocyte communication, and ProCs and preHTCs have many overlapping pathways. These two subtypes are the most active among all chondrocyte subtypes. Interestingly, ECs and FCs are a pair of “mutually exclusive” subtypes, of which ECs are predominant in incoming patterns and FCs in outgoing patterns. The active signaling pathways of ECs and FCs largely do not overlap. COLLAGEN and LAMININ are the main pivotal pathways, which means they are very important in the repair and expansion of joint homeostasis. Notably, only preHTCs assume multiple roles (including sender, receiver, mediator, and influencer) and are involved in multiple communication pathways. We have examined their communication patterns from the perspective of cellular interactions, revealed the relationships among different chondrocyte subtypes, and, in particular, identified a number of active subtypes and pathways that are important for targeted therapy in the osteoarthritic microenvironment. Our findings provide a new research paradigm and new insights into understanding chondrocyte activity patterns in the osteoarthritic microenvironment.

Abstract Background Nonalcoholic fatty liver disease (NAFLD) is now the major contributor to chronic liver disease. Disorders of lipid metabolism are a major element in the emergence of NAFLD. This research intended to explore lipid metabolism-related clusters in NAFLD and establish a prediction biomarker. Methods The expression mode of lipid metabolism-related genes (LMRGs) and immune characteristics in NAFLD were examined. The “ConsensusClusterPlus” package was utilized to investigate the lipid metabolism-related subgroup. The WGCNA was utilized to determine hub genes and perform functional enrichment analysis. After that, a model was constructed by machine learning techniques. To validate the predictive effectiveness, receiver operating characteristic curves, nomograms, decision curve analysis (DCA), and test sets were used. Lastly, gene set variation analysis (GSVA) was utilized to investigate the biological role of biomarkers in NAFLD. Results Dysregulated LMRGs and immunological responses were identified between NAFLD and normal samples. Two LMRG-related clusters were identified in NAFLD. Immune infiltration analysis revealed that C2 had much more immune infiltration. GSVA also showed that these two subtypes have distinctly different biological features. Thirty cluster-specific genes were identified by two WGCNAs. Functional enrichment analysis indicated that cluster-specific genes are primarily engaged in adipogenesis, signalling by interleukins, and the JAK-STAT signalling pathway. Comparing several models, the random forest model exhibited good discrimination performance. Importantly, the final five-gene random forest model showed excellent predictive power in two test sets. In addition, the nomogram and DCA confirmed the precision of the model for NAFLD prediction. GSVA revealed that model genes were down-regulated in several immune and inflammatory-related routes. This suggests that these genes may inhibit the progression of NAFLD by inhibiting these pathways. Conclusions This research thoroughly emphasized the complex relationship between LMRGs and NAFLD and established a five-gene biomarker to evaluate the risk of the lipid metabolism phenotype and the pathologic results of NAFLD.

Diffusion models have attracted considerable scholarly interest for their outstanding performance in generative tasks. However, current style transfer techniques based on diffusion models still rely on fine-tuning during the inference phase to optimize the generated results. This approach is not merely laborious and resource-demanding but also fails to fully harness the creative potential of expansive diffusion models. To overcome this limitation, this paper introduces an innovative solution that utilizes a pretrained diffusion model, thereby obviating the necessity for additional training steps. The scheme proposes a Feature Normalization Mapping Module with Cross-Attention Mechanism (INN-FMM) based on the dual-path diffusion model. This module employs soft attention to extract style features and integrate them with content features. Additionally, a parameter-free Similarity Attention Mechanism (SimAM) is employed within the image feature space to facilitate the transfer of style image textures and colors, while simultaneously minimizing the loss of structural content information. The fusion of these dual attention mechanisms enables us to achieve style transfer in texture and color without sacrificing content integrity. The experimental results indicate that our approach exceeds existing methods in several evaluation metrics.

(1) Background: Atopic dermatitis (AD) is characterized as a chronic inflammatory skin disease with a significant incidence rate. The pathophysiological mechanisms underlying AD remain incompletely understood. However, extensive research demonstrates that a complex interplay among genetic, immune, and environmental factors contributes to the disruption of skin barrier function. Inflammation is identified as one of the pathological mechanisms in AD. Recombined mussel adhesive protein exhibits anti-inflammatory properties. However, recombinant mussel adhesive protein has been used less frequently for AD, so we explored the therapeutic effect of recombinant mussel adhesive protein for AD and the potential mechanism. (2) Methods: We established a mice model of AD in vivo and an LPS-induced inflammation model in HaCaT cells in vitro. Through assessment of skin lesion scores, itch frequency, transepidermal water loss, skin microcirculation, HE staining, Elisa assays for IL-6, IL-12, IL-13, IL-4, IL-5, IFN-γ, IgE, and TNF-α, immunohistochemical staining for filaggrin and CK14, Masson staining, and Western blot analysis of NF-κB p65, P-P65, Keap1, and Nrf2, the effects of recombined mussel adhesive protein on AD symptoms, pathology, inflammation, and its mechanisms are investigated. (3) Results: The recombined mussel adhesive protein significantly improved the compromised skin barrier, reduced scratching frequency in mice, decreased transepidermal water loss, and lowered the expression of inflammatory factors, thus ameliorating skin inflammation damage. Mechanistically, recombined mussel adhesive protein downregulated the expression of P-p65/p65 and Keap1 while upregulating the level of Nrf2. (4) Conclusions: Overall, our results demonstrate the effectiveness of recombined mussel adhesive protein in attenuating DNFB-induced AD by inhibiting NF-κB and activating the Keap1/Nrf2 signaling pathway. Thus, recombined mussel adhesive protein is a promising therapeutic candidate for the treatment of AD.

Determining the compressibility of slurry with high water contents via self-weight consolidation test usually requires excessive testing time. To overcome such shortcoming, double drainage self-weight sedimentation test (DDSST) is used in this study. The stress equilibrium at the steady state under double drainage condition is analyzed, based on which the relation between effective stress σ′and the distribution of water contents is investigated. Accordingly, the procedure for calculating σ′- e relation with obtained distribution of water contents is presented. To verify the feasibility of determining σ′- e relation of slurry with DDSST, DDSSTs were conducted on two types of slurry. Calculated results according to the given procedure show that, the σ′- e relation obtained via DDSST is basically the same with the one obtained via single drainage self-weight sedimentation test (SDSST), which demonstrate the effectiveness of DDSST in determining the compressibility of slurry. Furthermore, using DDSST rather than SDSST could significantly shorten the testing time and reduce experimental material usage.

A large number of mixed soils exist in nature, including sand–clay mixtures, silt–clay mixtures and clay–clay mixtures. The physical parameters of soils are the basis for investigating their mechanical properties (compression, permeability, strength). More importantly, the liquid limits play an important role in establishing the relationships between physical and mechanical properties. The liquid limit tests of sand–clay mixtures were performed on different kinds of original clays with different sand contents. These data together with those compiled from the literature were used to investigate the change in liquid limits of mixed soils. The results show that the liquid limits of mixed soils are influenced by the types of original soils and the contents of each constituent. In sand–clay mixtures, the liquid limits increase with the increasing clay contents. The empirical equation is proposed to predict the liquid limits of sand–clay mixtures based on the liquid limits of original clays and clay contents. By analogy, the method for predicting liquid limits of mixed soils including two types of original soils with known liquid limits is acquired, such as silt–clay mixtures and clay–clay​ mixtures. Such a method is also extrapolated to the mixed soils with more types of original soils.

Renal tubular epithelial cells (TECs) are vulnerable to mitochondrial dysregulation, which is an integral part of diabetic kidney disease (DKD). We found that CD36 knockout ameliorated mitochondrial dysfunction and diabetic kidney injury in mice, improved renal function, glomerular hypertrophy, tubular injury, tubulointerstitial fibrosis, and kidney cell apoptosis. Furthermore, CD36 knockout conferred protection against diabetes-induced mitochondrial dysfunction and restored renal tubular cells and mitochondrial morphology. CD36 knockout also restored mitochondrial fatty acid oxidation (FAO) and enhanced FAO-associated respiration in diabetic TECs. CD36 was found to alter cellular metabolic pathways in diabetic kidneys partly via PDK4 the –AMPK axis inactivation. Because CD36 protects against DKD by improving mitochondrial function and restoring FAO, it can serve as a potential therapeutic target.

Text-based CAPTCHAs remain the most widely adopted security scheme, which is the first barrier to securing websites. Deep learning methods, especially Convolutional Neural Networks (CNNs), are the mainstream approach for text CAPTCHA recognition and are widely used in CAPTCHA vulnerability assessment and data collection. However, verification code recognizers are mostly deployed on the CPU platform as part of a web crawler and security assessment; they are required to have both low complexity and high recognition accuracy. Due to the specifically designed anti-attack mechanisms like noise, interference, geometric deformation, twisting, rotation, and character adhesion in text CAPTCHAs, some characters are difficult to efficiently identify with high accuracy in these complex CAPTCHA images. This paper proposed a recognition model named Adaptive CAPTCHA with a CNN combined with an RNN (CRNN) module and trainable Adaptive Fusion Filtering Networks (AFFN), which effectively handle the interference and learn the correlation between characters in CAPTCHAs to enhance recognition accuracy. Experimental results on two datasets of different complexities show that, compared with the baseline model Deep CAPTCHA, the number of parameters of our proposed model is reduced by about 70%, and the recognition accuracy is improved by more than 10 percentage points in the two datasets. In addition, the proposed model has a faster training convergence speed. Compared with several of the latest models, the model proposed by the study also has better comprehensive performance.

IntroductionLactococcus is a genus of lactic acid bacteria used in the dairy industry as a starter. Lactococci have been found to produce altogether more than 40 different bacteriocins, ribosomally synthesized antimicrobial proteins. All known Lactococcus spp. bacteriocins belong to classes I and II, which are mainly heat-resistant peptides. No class III bacteriocins, bigger heat-sensitive proteins, including phage tail-like bacteriocins, have been found from the Lactococcus spp. Unlike phage tail-like bacteriocins, prophage lysins have not been regarded as bacteriocins, possibly because phage lysins contribute to autolysis, degrading the host's own cell wall.MethodsWild-type Lactococcus lactis strain LAC460, isolated from spontaneously fermented idli batter, was examined for its antimicrobial activity. We sequenced the genome, searched phage lysins from the culture supernatant, and created knock-out mutants to find out the source of the antimicrobial activity.Results and discussionThe strain LAC460 was shown to kill other Lactococcus strains with protease- and heat-sensitive lytic activity. Three phage lysins were identified in the culture supernatant. The genes encoding the three lysins were localized in different prophage regions in the chromosome. By knock-out mutants, two of the lysins, namely LysL and LysP, were demonstrated to be responsible for the antimicrobial activity. The strain LAC460 was found to be resistant to the lytic action of its own culture supernatant, and as a consequence, the phage lysins could behave like bacteriocins targeting and killing other closely related bacteria. Hence, similar to phage tail-like bacteriocins, phage lysin-like bacteriocins could be regarded as a novel type of class III bacteriocins.

Goats with diverse economic phenotypic traits play an important role in animal husbandry. However, the genetic mechanisms underlying complex phenotypic traits are unclear in goats. Genomic studies of variations provided a lens to identify functional genes. In this study, we focused on the worldwide goat breeds with outstanding traits and used whole-genome resequencing data in 361 samples from 68 breeds to detect genomic selection sweep regions. We identified 210–531 genomic regions with six phenotypic traits, respectively. Further gene annotation analysis revealed 332, 203, 164, 300, 205, and 145 candidate genes corresponding with dairy, wool, high prolificacy, poll, big ear, and white coat color traits. Some of these genes have been reported previously (e.g., KIT, KITLG, NBEA, RELL1, AHCY, and EDNRA), while we also discovered novel genes, such as STIM1, NRXN1, LEP, that may be associated with agronomic traits like poll and big ear morphology. Our study found a set of new genetic markers for genetic improvement in goats and provided novel insights into the genetic mechanisms of complex traits.

Weissella strains are often detected in spontaneously fermented foods. Because of their abilities to produce lactic acid and functional exopolysaccharides as well as their probiotic traits, Weissella spp. improve not only the sensorial properties but also nutritional values of the fermented food products. However, some Weissella species have been associated with human and animal diseases. In the era of vast genomic sequencing, new genomic/genome data are becoming available to the public on daily pace. Detailed genomic analyses are due to provide a full understanding of individual Weissella species. In this study, the genomes of six Weissella paramesenteroides strains were de novo sequenced. The genomes of 42 W. paramesenteroides strains were compared to discover their metabolic and functional potentials in food fermentation. Comparative genomics and metabolic pathway reconstructions revealed that W. paramesenteroides is a compact group of heterofermentative bacteria with good capacity of producing secondary metabolites and vitamin Bs. Since the strains rarely harbored plasmid DNA, they did not commonly possess the genes associated with bacteriocin production. All 42 strains were shown to bear vanT gene from the glycopeptide resistance gene cluster vanG. Yet none of the strains carried virulence genes.

Cell senescence is closely related to autophagy. In this article, we identified a natural nucleoside analogue, cordycepin, that has the ability to significantly improve lysosomal function, enhance the activity of the lysosomal representative protease cathepsin B (CTSB), and promote the expression of the functional protein lysosomal‐associated membrane protein 2 (LAMP2) on the lysosomal membrane. Cordycepin then restores the damaged autophagy level of aging cells by activating the classic AMPK and mTOR–p70S6K signaling pathways, thus inhibiting cell senescence in an H2O2‐induced stress‐induced premature senescence (SIPS) cell model. This study provides new theoretical support for the further development of cordycepin and clinical antiaging drugs to inhibit cell senescence and suggests that the regulatory mechanisms of lysosomes in senescent cells should be considered when treating age‐related diseases.

In this paper, the properties of superfine cement with different mesh sizes are observed from both macro and micro perspectives through laboratory tests, and the fluidity, stability, and mechanical properties of superfine cement are studied in depth. The results show that increasing the mesh size of superfine cement can reduce the influence of the water-cement ratio on slurry, reduce the fluidity of superfine cement, and enhance its stability. The strength of the superfine cement stone specimen will increase with the increase in cement mesh number, and the higher the mesh number, the shorter the time required for the superfine cement stone specimen to reach its maximum strength. Considering the performance, cost, and storage difficulty, the superfine cement with the mesh size of K700 is more cost-effective. Using K700 superfine cement grout can effectively reduce the impact of geostress on the surrounding rock of the roadway and inhibit the deformation and damage of the surrounding rock.

Summary Fungal laccases are attracting enzymes for sustainable valorization of biorefinery lignins. To improve the lignin oxidation capacity of two previously characterized laccase isoenzymes from the white‐rot fungus Obba rivulosa, we mutated their substrate‐binding site at T1. As a result, the pH optimum of the recombinantly produced laccase variant rOrLcc2‐D206N shifted by three units towards neutral pH. O. rivulosa laccase variants with redox mediators oxidized both the dimeric lignin model compound and biorefinery poplar lignin. Significant structural changes, such as selective benzylic α‐oxidation, were detected by nuclear magnetic resonance analysis, although no polymerization of lignin was observed by gel permeation chromatography. This suggests that especially rOrLcc2‐D206N is a promising candidate for lignin‐related applications.

In the escalating battle against antimicrobial resistance, there is an urgent need to discover and investigate new antibiotic strategies. Bacteriophages are untapped reservoirs of such potential antimicrobials. This study focused on Hypothetical Proteins of Unknown Function (HPUFs) from a Staphylococcus phage Stab21. We examined its HPUFs for bactericidal activity against E. coli using a Next Generation Sequencing (NGS)-based approach. Among the 96 HPUFs examined, 5 demonstrated cross-species toxicity towards E. coli, suggesting the presence of shared molecular targets between E. coli and S. aureus. One toxic antibacterial HPUF (toxHPUF) was found to share homology with a homing endonuclease. The implications of these findings are profound, particularly given the potential broad applicability of these bactericidal agents. This study confirms the efficacy of NGS in streamlining the screening process of toxHPUFs, contributes significantly to the ongoing exploration of phage biology, and offers promises in the search for potent antimicrobial agents.

In order to study the coupling vibration between a bridge and a train under the action of crosswind loads, a dynamic interaction model of the wind–train–bridge system is established considering the geometric nonlinear factors of a long-span suspension bridge. A calculation frame is composed, and a corresponding computer program is written. A long-span highway–railway suspension bridge scheme is studied as an example. The linear and nonlinear vibration responses of the bridge under the simultaneous action of both train loads and wind loads are compared using the self-written program, and the influence of wind velocity and train speed on the dynamic responses of the bridge is studied. The results show that the large displacement nonlinearity of the structure does not influence the changing tendency of bridge displacement and acceleration time histories, but reduces the maximum values of the responses. The geometric nonlinear influence on the bridge accelerations is more obvious than that on the displacements. The natural frequencies of this long-span suspension bridge are very low and it is sensitive to wind action. The changes in train speed and average wind velocity have a great influence on the maximum value of bridge displacement, especially when the lateral deformation and acceleration increase sharply with the wind velocity, and the coupling vibration between wind, train, and the bridge can easily occur. The research results can provide references for the safe operation and maintenance of long-span bridges.

Laccases are multi-copper oxidases that use molecular oxygen as the electron acceptor to oxidize phenolic and indirectly also non-phenolic substrates by mechanisms involving radicals. Due to their eco-friendliness and broad substrate specificity, laccases span a wide range of biotechnological applications. We have heterologously expressed a laccase from the coprophilic basidiomycete Coprinopsis cinerea (CcLcc9) in the methylotrophic yeast Pichia pastoris. The recombinant CcLcc9 (rCcLcc9) oxidized 2,6-dimethoxyphenol in the neutral pH range, and showed thermostability up to 70°C. The rCcLcc9 efficiently oxidized veratryl alcohol to veratraldehyde in the presence of low molecular weight mediators syringyl nitrile, methyl syringate and violuric acid, which are syringyl-type plant phenolics that have shown potential as natural co-oxidants for lignocellulosic materials. In addition, rCcLcc9 is able to depolymerize biorefinery hardwood lignin in the presence of methyl syringate and syringyl nitrile as indicated by gel permeation chromatography, and infrared spectral and nucleic magnetic resonance analyses. Furthermore, we showed that several added-value aromatic compounds, such as vanillin, vanillic acid, syringaldehyde, syringic acid and p-hydroxybenzoic acid, were formed during sequential biocatalytic chemical degradation of biorefinery lignin, indicating that rCcLcc9 harbors a great potential for sustainable processes of circular economy and modern biorefineries.

Abstract Background The incidence and mortality rates of gastric cancer (GC) rank in top five among all malignant tumors. Chemokines and their receptor-signaling pathways reportedly play key roles in the metastasis of malignant tumor cells. Receptor activator of nuclear factor κB ligand (RANKL) is a member of the tumor necrosis factor family, with strong chemokine-like effects. Some studies have pointed out that the RANKL/RANK pathway is vital for the metastasis of cancer cells, but the specific mechanisms in GC remain poorly understood. Results This study reports original findings in cell culture models and in patients with GC. Flow cytometry and western blotting analyses showed that RANK was expressed in BGC-823 and SGC-7901 cells in particular. Chemotaxis experiments and wound healing assay suggested that RANKL spurred the migration of GC cells. This effect was offset by osteoprotegerin (OPG), a decoy receptor for RANKL. RANKL contributed to the activation of human epidermal growth factor receptor (HER) family pathways. The lipid raft core protein, caveolin 1 (Cav-1), interacted with both RANK and human epidermal growth factor receptor-1(EGFR). Knockdown of Cav-1 blocked the activation of EGFR and cell migration induced by RANKL. Moreover, RANK-positive GC patients who displayed higher levels of EGFR expression had poor overall survival. Conclusions In summary, we confirmed that with the promotion of RANKL, RANK and EGFR can form complexes with the lipid raft core protein Cav-1, which together promote GC cell migration. The formation of the RANK-Cav-1-EGFR complex provides a novel mechanism for the metastasis of GC. These observations warrant confirmation in independent studies, in vitro and in vivo. They also inform future drug target discovery research and innovation in the treatment of GC progression.

ABSTRACT The dynamics of quasispecies afford RNA viruses a great fitness on cell tropism and host range. To study the quasispecies features and the intra-host evolution of SARS-CoV-2, we collected nine confirmed patients and sequenced the haplotypes of spike gene using a single-molecule real-time platform. Fourteen samples were extracted from sputum, nasopharyngeal swabs, or stool, which in total produced 283,655 high-quality circular consensus sequences. We observed a stable quasispecies structure that one master mutant (mean abundance ∼0.70), followed by numerous minor mutants (mean abundance ∼1.21 × 10−3). Under high selective pressure, minor mutants may obtain a fitness advantage and become the master ones. The later predominant substitution D614G existed in the minor mutants of more than one early patient. An epidemic variant had a possibility to be independently originated from multiple hosts. The mutant spectrums covered ∼85% amino acid variations of public genomes (GISAID; frequency ≥ 0.1) and likely provided an advantage mutation pool for the current/future epidemic variants. Notably, 32 of 35 collected antibody escape substitutions were preexistent in the early quasispecies. Virus populations in different tissues/organs revealed potentially independent replications. The quasispecies complexity of sputum samples was significantly lower than that of nasopharyngeal swabs (P = 0.02). Evolution analysis revealed that three continuous S2 domains (HR1, CH, and CD) had undergone a positive selection. Cell fusion-related domains may play a crucial role in adapting to the intrahost immune system. Our findings suggested that future epidemiologic investigations and clinical interventions should consider the quasispecies information that has missed by routine single consensus genome. IMPORTANCE RNA virus population in a host does not consist of a consensus single haplotype but rather an ensemble of related sequences termed quasispecies. The dynamics of quasispecies afford SARS-CoV-2 a great ability on genetic fitness during intrahost evolution. The process is likely achieved by changing the genetic characteristics of key functional genes, such as the spike glycoprotein. Previous studies have applied the next-generation sequencing (NGS) technology to evaluate the quasispecies of SARS-CoV-2, and results indicated a low genetic diversity of the spike gene. However, the NGS platform cannot directly obtain the full haplotypes without assembling, and it is also difficult to predict the extremely low-frequency variations. Therefore, we introduced a single-molecule real-time technology to directly obtain the haplotypes of the RNA population and further study the quasispecies features and intrahost evolution of the spike gene.

Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS–PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology.

The double row layout problem is to arrange a number of machines on both sides of a straight aisle so as to minimize the total material handling cost. Aiming at the random distribution of product demands, we study a stochastic robust double row layout problem (SR-DRLP). A mixed integer programming (MIP) model is established for SR-DRLP. A surrogate model is used to linearize the nonlinear term in the MIP to achieve a mixed integer linear programming model, which can be readily solved by an exact method to yield high-quality solutions (layouts) for small-scale SR-DRLPs. Furthermore, we propose a hybrid approach combining a local search and an exact approach (LS-EA) to solve large-scale SR-DRLPs. Firstly, a local search is designed to optimize the machine sequences on two rows and the clearance from the most left machine on row 1 to the left boundary. Then, the exact location of each machine is further optimized by an exact approach. The LS-EA is applied to six problem instances ranging from 8 to 50 machines. Experimental results show that the surrogate model is effective and LS-EA outperforms the comparison approaches.

Leber's hereditary optic neuropathy (LHON) is a disease that leads to blindness. Gene therapy has been investigated with some success, and could lead to important advancements in treating LHON. This was a prospective, open-label trial involving 9 LHON patients at Tongji Hospital, Wuhan, China, from August 2011 to December 2015. The purpose of this study was to evaluate the long-term outcomes of gene therapy for LHON. Nine LHON patients voluntarily received an intravitreal injection of rAAV2-ND4. Systemic examinations and visual function tests were performed during the 36-month follow-up period to determine the safety and efficacy of this gene therapy. Based on successful experiments in an animal model of LHON, 1 subject also received an rAAV2-ND4 injection in the second eye 12 months after gene therapy was administered in the first eye. Recovery of visual acuity was defined as the primary outcome of this study. Changes in the visual field, visual evoked potential (VEP), optical coherence tomography findings, liver and kidney function, and antibodies against AAV2 were defined as secondary endpoints. Eight patients (Patients 2–9) received unilateral gene therapy and visual function improvement was observed in both treated eyes (Patients 4, 6, 7, and 8) and untreated eyes (Patients 2, 3, 4, 6 and 8). Visual regression fluctuations, defined as changes in visual acuity greater than or equal to 0.3 logMAR, were observed in Patients 2 and 9. Age at disease onset, disease duration, and the amount of remaining optic nerve fibers did not have a significant effect on the visual function improvement. The visual field and pattern reversal VEP also improved. The patient (Patient 1) who received gene therapy in both eyes had improved visual acuity in the injected eye after the first treatment. Unfortunately, visual acuity in this eye decreased 3 months after he received gene therapy in the second eye. Animal experiments suggested that ND4 expression remains stable in the contralateral eye after intravitreal injections. No serious safety problem was observed in the 3-year follow-up of the 9 participants enrolled in this virus-based gene therapy. Meanwhile, our results support the use of intravitreal rAAV2-ND4 as an aggressive maneuver in our clinical trial. Further study in additional patients and in these 9 subjects is needed to better understand the effects of rAAV2-ND4 gene therapy on LHON and to increase the applications of this technique.

The lytic phage, fHe-Kpn01 was isolated from sewage water using an extended-spectrum beta-lactamase-producing strain of Klebsiella pneumoniae as a host. The genome is 43,329 bp in size and contains direct terminal repeats of 222 bp. The genome contains 56 predicted genes, of which proteomics analysis detected 29 different proteins in purified phage particles. Comparison of fHe-Kpn01 to other phages, both morphologically and genetically, indicated that the phage belongs to the family Podoviridae and genus Drulisvirus. Because fHe-Kpn01 is strictly lytic and does not carry any known resistance or virulence genes, it is suitable for phage therapy. It has, however, a narrow host range since it infected only three of the 72 tested K. pneumoniae strains, two of which were of capsule type KL62. After annotation of the predicted genes based on the similarity to genes of known function and proteomics results on the virion-associated proteins, 22 gene products remained annotated as hypothetical proteins of unknown function (HPUF). These fHe-Kpn01 HPUFs were screened for their toxicity in Escherichia coli. Three of the HPUFs, encoded by the genes g10, g22, and g38, were confirmed to be toxic.

Orexins are hypothalamic neuropeptides that regulate feeding, reward, wakefulness and energy homeostasis. The present study sought to characterize the involvement of orexin A in glucose metabolism in HepG2 human hepatocellular carcinoma cells, and investigated the role of hypoxia-inducible factor-1α (HIF-1α) in the response. HepG2 cells were exposed to different concentrations of orexin A (10-9 to 10-7 M) in vitro, without or with the orexin receptor 1 (OX1R) inhibitor (SB334867), HIF-1α inhibitor (YC-1) or a combination of both inhibitors. Subsequently, OX1R, HIF-1α expression and localization, glucose uptake, glucose transporter 1 (GLUT1) expression and ATP content were measured. We further investigated the intracellular fate of glucose by measuring the gene expression of pyruvate dehydrogenase kinase 1 (PDK1), lactate dehydrogenase (LDHA) and pyruvate dehydrogenase B (PDHB), as well as metabolite levels including lactate generation and mitochondrial pyruvate dehydrogenase (PDH) activity. The activity of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was also assessed. Our results showed that the expression of OX1R was predominantly located in the nucleus in HepG2 cells. Orexin A oxygen-independently promoted the mRNA and protein expression of HIF-1α as well as its nuclear accumulation in HepG2 cells and the elevated HIF-1α protein was associated, at least partly, with the activation of the PI3K/Akt/mTOR pathway. Orexin A stimulated GLUT1 expression, glucose uptake as well as ATP generation in HepG2 cells via OX1R acting through the HIF-1α pathway. Moreover, orexin A inhibited LDHA, PDK1 expression and lactate production, stimulated PDHB expression and PDH enzyme activity independent of HIF-1α. Our results indicated that orexin signaling facilitated the glucose flux into mitochondrial oxidative metabolism rather than glycolysis in HepG2 cells. These findings provide new insight into the regulation of glucose metabolism by orexin A in hepatocellular carcinoma cells.

The asymmetric unit of the title compound, C6H5Cl2NO2S, contains two molecules with similar conformations (r.m.s. overlay fit for the non-H atoms = 0.067 Å). Atoms attached to the pendent Csp3—S bond are arranged in a staggered conformation with one of the Cl atoms anti to the C atom in the aromatic ring [C—S—C—Cl torsion angles = 178.41 (11) and −176.70 (13)°]. In the crystal, molecules are linked by C—H...N and C—H...O hydrogen bonds, generating a three-dimensional network, and weak aromatic π–π stacking is also observed [centroid–centroid separation = 3.8902 (17) Å].