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Abstract The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus–host interactions and potential antiviral strategies.

Abstract Carbon dots (CDs) are novel carbon-based nanomaterials that have been used as photosensitizer-mediated photodynamic therapy (PDT) in recent years due to their good photosensitizing activity. Photosensitizers (PSs) are main components of PDT that can produce large amounts of reactive oxygen species (ROS) when stimulated by light source, which have the advantages of low drug resistance and high therapeutic efficiency. CDs can generate ROS efficiently under irradiation and therefore have been extensively studied in disease local phototherapy. In tumor therapy, CDs can be used as PSs or PS carriers to participate in PDT and play an extremely important role. In bacterial infectious diseases, CDs exhibit high bactericidal activity as CDs are effective in disrupting bacterial cell membranes leading to bacterial death upon photoactivation. We focus on recent advances in the therapy of cancer and bacteria with CDs, and also briefly summarize the mechanisms and requirements for PSs in PDT of cancer, bacteria and other diseases. We also discuss the role CDs play in combination therapy and the potential for future applications against other pathogens. Graphical Abstract

Abstract Wood‐decay macrofungi play a vital role in forest ecosystems by promoting nutrient cycling and soil structure, and their evolution is closely related to their host plants. This study investigates the potential evolutionary adaptation of wood‐decay macrofungi to their host plants, focusing on whether these relationships differ between gymnosperms and angiosperms. While previous research has suggested non‐random associations between specific fungi and plant deadwood, direct evidence of evolutionary adaptation has been lacking. Our study, conducted in a subtropical region, utilized metabarcoding techniques to identify deadwood species and associated fungi. We found significant evidence of evolutionary adaptation when considering all sampled species collectively. However, distinct patterns emerged when comparing angiosperms and gymnosperms: a significant evolutionary adaptation was observed of wood‐decay macrofungi to angiosperms, but not to gymnosperms. This variation may be due to the longer evolutionary history and more stable species interactions of gymnosperms, as indicated by a higher modularity coefficient (r = .452), suggesting greater specialization. In contrast, angiosperms, being evolutionarily younger, displayed less stable and more coevolving interactions with fungi, reflected in a lower modularity coefficient (r = .387). Our findings provide the first direct evidence of differential evolutionary adaptation dynamics of these fungi to angiosperms versus gymnosperms, enhancing our understanding of forest ecosystem carbon cycling and resource management.

Nanozymes, nanomaterials exhibiting enzyme‐mimicking activities, have gained considerable interest in biomedicine due to their stability, adjustability, and cost‐efficiency. Among these, metal–organic framework (MOF)‐based nanozymes distinguish themselves by their distinct structure and customizable characteristics. Researchers have explored MOF‐based nanozymes as a platform for developing stimuli‐responsive behaviors. This work first presents the categorization of MOF‐based nanozymes, which are designed to mimic the catalytic functions of oxidases, peroxidases, catalase, superoxide dismutase, hydrolases, and multifunctional enzymes. Crafting MOF‐based nanozymes includes customizing their reactions to particular stimuli, including pH, temperature, light, or biomolecular triggers, ensuring enhanced specificity and potency in catalytic performance amid environmental changes. Moreover, these nanozymes exhibit immense potential in biomedical applications, playing crucial roles in therapeutic interventions like cancer therapy and tissue regeneration. Finally, the article delves into future opportunities and challenges within emerging research frontiers. These stimuli‐responsive MOF‐based nanozymes offer novel avenues for advanced therapeutic strategies, providing prospects for innovative biomedical applications.

Objectives: Due to the complexity and importance of oral bone structure, oral bone regeneration materials differ from those used in other parts of the body. To study the research trends and hotspots of oral bone regeneration materials, this paper conducts a bibliometric analysis of related papers from 1991 to 2023 (retrieved on 27 September 2023).Materials and methods: Using bibliometric methods, two visualization metric software, Citespace and VOSviewer, were used to analyze 1217 papers in SCIE, including paper analysis, author analysis, country and institution analysis, keyword analysis, and cited literature analysis.Results: ① The number of papers is generally increasing and gradually stabilizing; ② Buser D is the most influential author, while Jung, Ronald E has the highest number of papers and total citations; ③ The United States has the highest number of papers and citation frequency. The University of Bern and the University of Zurich in Switzerland are not only the institutions with the most papers, but also the institutions with the most collaborations with other institutions. ④ Many research directions have persisted for decades since their inception. The field of oral bone; regeneration materials is constantly developing and improving. In recent years, the research direction in this field may mainly focus on the role of blood cells and proteins in bone regeneration. ⑤ In recent years, the types of cited literature mainly include barrier membranes, alveolar ridge augmentation, bone graft materials, histological examination, and in vivo animal experimental models.Conclusion: The United States and Switzerland have a significant influence in the field of oral bone regeneration materials. The research hotspot in recent years is mainly on tissue engineering materials. However, traditional materials still occupy a large proportion in clinical treatment or research. In addition, the combined use of new and old materials has gradually become one of the research hotspots in this field.

Carbon dots (CDs), as a new kind of fluorescent nanomaterials, show great potential for application in several fields due to their unique nano-size effect, easy surface functionalization, controllable photoluminescence, and excellent biocompatibility. Conventional preparation methods for CDs typically involve top-down and bottom-up approaches. Doping is a major step forward in CDs design methodology. Chemical doping includes both non-metal and metal doping, in which non-metal doping is an effective strategy for modulating the fluorescence properties of CDs and improving photocatalytic performance in several areas. In recent years, Metal-doped CDs have aroused the interest of academics as a promising nano-doping technique. This approach has led to improvements in the physicochemical and optical properties of CDs by altering their electron density distribution and bandgap capacity. Additionally, the issues of metal toxicity and utilization have been addressed to a large extent. In this review, we categorize metals into two major groups: transition group metals and rare-earth group metals, and an overview of recent advances in biomedical applications of these two categories, respectively. Meanwhile, the prospects and the challenges of metal-doped CDs for biomedical applications are reviewed and concluded. The aim of this paper is to break through the existing deficiencies of metal-doped CDs and fully exploit their potential. I believe that this review will broaden the insight into the synthesis and biomedical applications of metal-doped CDs.

The distinctive three-dimensional architecture, biological functionality, minimal immunogenicity, and inherent biodegradability of small intestinal submucosa extracellular matrix materials have attracted considerable interest and found wide-ranging applications in the domain of tissue regeneration engineering. This article presents a comprehensive examination of the structure and role of small intestinal submucosa, delving into diverse preparation techniques and classifications. Additionally, it proposes approaches for evaluating and modifying SIS scaffolds. Moreover, the advancements of SIS in the regeneration of skin, bone, heart valves, blood vessels, bladder, uterus, and urethra are thoroughly explored, accompanied by their respective future prospects. Consequently, this review enhances our understanding of the applications of SIS in tissue and organ repair and keeps researchers up-to-date with the latest research advancements in this area.

Abstract Oral and maxillofacial diseases are one of the most prevalent diseases in the world, which not only seriously affect the health of patients’ oral and maxillofacial tissues, but also bring serious economic and psychological burdens to patients. Therefore, oral and maxillofacial diseases require effective treatment. Traditional treatments have limited effects. In recent years, nature exosomes have attracted increasing attention due to their ability to diagnose and treat diseases. However, the application of nature exosomes is limited due to low yield, high impurities, lack of targeting, and high cost. Engineered exosomes can be endowed with better comprehensive therapeutic properties by modifying exosomes of parent cells or directly modifying exosomes, and biomaterial loading exosomes. Compared with natural exosomes, these engineered exosomes can achieve more effective diagnosis and treatment of oral and maxillary system diseases, and provide reference and guidance for clinical application. This paper reviews the engineering modification methods of exosomes and the application of engineered exosomes in oral and maxillofacial diseases and looks forward to future research directions. Graphical Abstract

Bactrian camels inhabiting desert and semi-desert regions of China are valuable animal models for studying adaptation to desert environments and heat stress. In this study, 16S rRNA technology was employed to investigate the distribution characteristics and differences of mucosal microorganisms in the anterior gland area, posterior gland area, third gland area, cardia gland area, gastric fundic gland area and pyloric gland area of 5-peak adult healthy Bactrian camels. We aimed to explore the possible reasons for the observed microbial distribution from the aspects of histological structure and mucosal immunity. Bacteroides and Fibrobacteria accounted for 59.54% and 3.22% in the gland area, respectively, and 52.37% and 1.49% in the wrinkled stomach gland area, respectively. The gland area showed higher abundance of Bacteroides and Fibrobacteria than the wrinkled stomach gland area. Additionally, the anterior gland area, posterior gland area, third gland area, and cardia gland area of Bactrian camels mainly secreted acidic mucus, while the gastric fundic gland area mainly secreted neutral mucus and the pyloric region mainly secreted a mixture of acidic and neutral mucus. The results of immunohistochemistry techniques demonstrated that the number of IgA+ cells in the anterior glandular area, posterior glandular area, third glandular area, and cardia gland area was significantly higher than that in the fundic and pyloric gland area (p < 0.05), and the difference in IgA+ between the fundic and pyloric gland area was not significant (p > 0.05). The study revealed a large number of bacteria that can digest and degrade cellulose on the mucosa of the gastric gland area of Bactrian camels. The distribution of IgA+ cells, the structure of the mucosal tissue in the glandular region, and the composition of the mucus secreted on its surface may have a crucial influence on microbial fixation and differential distribution.

Background/purpose: Parathyroid hormone-related protein (PTHrP) is an important regulatory factor in the growth, development and remodeling of bone or cartilage, and acts through its sole receptor, parathyroid hormone receptor-1 (PTH1R). The present study aimed to research the expression changes of PTHrP, PTH1R and other relevant factors in condylar cartilage during the progress of temporomandibular joint osteoarthritis (TMJOA). Materials and methods: The animal model of TMJOA was constructed by the “resin-modified method”, and Sprague Dawley (SD) rats were euthanized at 2 weeks, 4 weeks, 6 weeks and 8 weeks after occlusal elevation. The histological changes of condylar cartilage were observed by X-ray, hematoxylin-eosin (HE) and safranine O-fast green (SO-FG) staining. The expressions of PTHrP, PTH1R, Ki67, Collagen II (Col II), Collagen X (Col X) and Caspase 3 in each group were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). Results: TMJOA progression was time-dependent. In the experimental group, PTHrP expression was unimodal with a peak at 4 weeks, but PTH1R expression showed a decreasing trend. From 2 weeks to 8 weeks in the experimental group, Col X expression rather than Caspase 3 expression was negatively related to PTHrP's, which has no positive relation to Ki67 or Col II. These results demonstrated abnormal occlusal load may be an important pathogenic factor of TMJOA. Conclusion: It may be one of the reasons of TMJOA progression that PTHrP can't play an effective role due to the low expression of PTH1R. PTHrP may be a direct factor regulating the hypertrophic differentiation of chondrocytes, but it does not directly regulate the proliferation and apoptosis of chondrocytes, and the realization of both regulatory effects may depend on the inhibition of hypertrophic differentiation.

Abstract Background Kushenol A is natural flavonoid extract discovered in recent years, with potential anti‐tumor activity. Its role in breast cancer is poorly understood. Methods To investigate biological function of Kushenol A in breast cancer (BC), Cell Counting Kit‐8 assay, colony formation assay, flow cytometry, western blotting, qPCR analysis, and xenograft mouse model were performed. Results We found that Kushenol A treatment reduced proliferative capability and induced G0/G1 phase cell cycle arrest and apoptosis of BC cells in a concentration‐dependent manner. Besides, Kushenol A treatment contributed to the upregulation of apoptosis‐related and cell cycle‐associated genes. In nude mice, Kushenol A administration repressed BC xenograft tumor growth. Mechanistically, phosphorylation levels of AKT and mTOR were markedly attenuated in Kushenol A‐treated BC cells; however, there were no significant differences in total AKT and mTOR expressions. Moreover, PI3K inhibitor combined with Kushenol A exhibited synergistic inhibitory activity on cell proliferation. Conclusions Taken together, our findings suggested that Kushenol A suppressed BC cell proliferation by modulating PI3K/AKT/mTOR signaling pathway. Kushenol A may be a promising therapeutic drug for treating BC.

Cuproptosis, a copper-induced mechanism of mitochondrial-related cell death, has been implicated as a breakthrough in the treatment of cancer and has become a new treatment strategy. Furthermore, long non-coding RNA (lncRNA) can change the biological activities of tumor cells. Worldwide, lung squamous cell carcinoma (LUSC) is among the most common annoying tumors. LncRNAs related to cuproptosis are not researched at LUSC. Our research intends to develop a signature on the basis of cuproptosis-associated lncRNAs, which can predict LUSC prognosis and investigate LUSC immunological features. The TCGA database was used to retrieve LUSC transcriptome, clinical, and gene mutation data. For statistical analysis, we utilized the R program. We created a signature consisting of three cuproptosis-related lncRNAs in this investigation (including AC002467.1, LINC01740, and LINC02345). Survival analyses and Receiver Operating Characteristic curves demonstrated that this signature possessed powerful predictive capability. The signature’s ability to predict was confirmed by a Receiver Operating Characteristic curve and principal component analysis. Notably, the patient with a high-risk score and a high tumor mutation burden level had a lower survival time. Furthermore, the tumor immune dysfunction and exclusion analysis showed that these individuals with low-risk scores may benefit from immunotherapy. The signature constructed by three cuproptosis-associated lncRNAs may be prognostic markers of LUSC. It contributes to immunotherapy and offers LUSC’s therapy a new treatment direction.

In recent years, mechanoluminescent (ML) materials have shown great potential in stress sensing, mechanical energy collection and conversion, so they have attracted wide attention in the field of stomatology. In the early stage of this study, BaSi2O2N2:Eu2+ ML phosphors were synthesized by two-step high temperature solid state method, and then mixed with Polydimethylsiloxane (PDMS) in different proportions to obtain BaSi2O2N2:Eu2+/PDMS ML composites with different mass fractions (10%,20%,30%,40%,50%). Then its biosafety was evaluated by Cell Counting Kit-8 (CCK-8), Calcein-AM/PI fluorescence staining, hemolysis, oral mucosal irritation, acute and subacute systemic toxicity tests. The experimental results show that the biosafety of BaSi2O2N2:Eu2+/PDMS ML composite elastomers with different mass fraction is in line with the existing standards, and other related properties can be further studied.

Lithium disilicate (Li2Si2O5, referred as LD) glass ceramics with unique aesthetic properties are ideal dental restorative materials. However, their applications are limited due to the lower flexural strength than polycrystalline ceramics. Herein, micro-nano-LD whiskers were utilized to facilitate the formation of crystallization sites and further growth of crystals in SiO2-Li2O glasses during the heat treatment process. Meanwhile, an orthogonal experiment with four-factor three-level was designed, and the optimum heat treatment conditions for preparing LD glass-ceramics with excellent flexural strength were found. The order of influencing extent of heat treatment conditions is crystallization temperature > crystallization time > nucleation temperature > nucleation time. In addition, the crystallization behavior, phase formation, microstructure and flexural strength of glass were measured at different heat treatment temperatures. Remarkably, the optimal LD glass-ceramics sample has a strong flexural strength at 342 MPa, in which plentiful crystal grains with uniform and dense distribution are observed. These results indicate that micro-nano-LD whisker-reinforced glass-ceramics obtained by optimized heat treatment program offer a potential candidate for dental applications.

Deep-trap luminescent materials have attracted great attention for optical information storage applications. However, the flexible luminescence films based on red luminescence materials with temperature and infrared response are scarce. In this study, we have successfully developed various novel flexible red emitting films based on Eu2+/Dy3+ co-doped Sr2Si5N8 phosphors through screen-printing and spin-coating technologies, respectively. Interestingly, the fabricated flexible luminescence films exhibit unique temperature and infrared responsive properties for optical information storage by releasing photons in response to thermal or infrared stimulation. Notably, deep-trap red emitting Eu2+/Dy3+ co-doped Sr2Si5N8 phosphors are crucial for the optical information storage properties of the films. Two emission peaks of Sr2Si5N8:Dy3+ phosphors at 476 nm and 577 nm are observed under excitation at 345 nm, corresponding to the radiative transition occurs from the 4F9/2 level to the 6H13/2 and 6H15/2 levels of Dy3+. When Dy3+ and Eu2+ ions are co-doped in Sr2Si5N8, the energy transfer from Dy3+ to Eu2+ in Sr2Si5N8 matrix is found and the decay time confirms that Dy3+ ions can be acted as deep trap centers to storage photons. For Sr2Si5N8:Eu2+,Dy3+ phosphors and the corresponding flexible luminescence films, the specific patterns (for example apple and note patterns) are firstly recorded under NUV or blue light excitation and then reappear through thermal stimulation or near-infrared photo-stimulation (980 nm laser). This work not only validates the feasibility of Sr2Si5N8:Eu2+,Dy3+ phosphors as deep-trap red emitting luminescence materials, but also suggests the applications of flexible luminescence films for optical information storage.

Bacterial cellulose (BC) has been widely used in tissue engineering due to its unique spatial structure and suitable biological properties. In this study, a small biologically active Arginine-Glycine-Aspartic acid-Serine (RGDS) tetrapeptide was incorporated on the porous BC surface followed by a low-energy CO2 laser etching operation. As a result, different micropatterns were established on the BC surface with RGDS only anchored on the raised platform surface of the micropatterned BC (MPBC). Material characterization showed that all micropatterned structures exhibited platforms with a width of ~150 μm and grooves with a width of ~100 μm and a depth of ~300 μm, which displayed distinct hydrophilic and hydrophobic properties. The resulting RGDS-MPBC could hold the material integrity, as well as the microstructure morphology under a humid environment. In-vitro and in-vivo assays on cell migration, collagen deposition, and histological analysis revealed that micropatterns led to significant impacts on wound healing progress compared to the BC without surface-engineered micropatterns. Specifically, the basket-woven micropattern etched on the BC surface exhibited the optimal wound healing outcome with the presence of fewer macrophages and the least scar formation. This study further addresses the potential of adopting surface micropatterning strategies to promote skin wounds towards scar-free outcomes.

Abstract In this paper, we propose an iteration algorithm for finding a split common fixed point of an asymptotically nonexpansive mapping in the frameworks of two real Banach spaces. Under some suitable conditions imposed on the sequences of parameters, some strong convergence theorems are proved, which also solve some variational inequalities that are closely related to optimization problems. The results here generalize and improve the main results of other authors.

Lithium disilicate (Li2Si2O5) glass-ceramics are an ideal material for dental restoration; however, their intrinsic brittleness and low defect tolerance limit the scope of their clinical applications. In this study, Li2Si2O5 whiskers were creatively synthesized via a mild-condition hydrothermal reaction. Self-reinforced Li2Si2O5 glass-ceramics were sintered by introducing the Li2Si2O5 whiskers, and their effects on phase, microstructure, and mechanical properties were systematically studied. The crystal-growth and toughening mechanisms were also discussed. The results showed that the Li2Si2O5 whiskers played an important role in inducing crystallization, and improving the microstructure and properties of the glass-ceramics. With increasing amounts of Li2Si2O5 whiskers, the crystallinities increased slightly, and the average crystal size also increased. The microstructure was composed of crystals of bimodal size distributions, in which some large, rod-like Li2Si2O5 crystals epitaxially grew along with the whiskers, and small crystals directly crystallized from the parent glass-ceramic powders. The Li2Si2O5 glass-ceramics exhibited high flexural strength (389.5 ± 11.77 MPa, LDW3), and fracture toughness (3.46 ± 0.10 MPa·m1/2, LDW5). The improved properties were attributed mainly to crack deflection and bridge-toughening mechanisms.

Melt-blowing technology is an important method for directly preparing micro-nanofiber materials by drawing polymer melts with high temperature and high velocity air flow. During the drawing process, the melt-blowing fiber not only undergoes a phase change, but also has an extremely complex coupling effect with the drawing airflow. Therefore, in the numerical calculation of the flow field, the existence of melt-blowing fibers is often ignored. In this paper, based on the volume of fluid method, a numerical study of the flexible fiber/air-coupling flow field of an annular melt-blowing die is carried out with the aid of computational fluid dynamics software. The results show that the pressure distribution in the different central symmetry planes of the ring die at the same time was basically the same. However, the velocity distribution may have been different; the velocity on the spinning line varied with time; the pressure changes on the spinning line were small; and velocity fluctuations around the spinning line could cause whiplash of the fibers.

Background Bronchus-associated lymphoid tissue (BALT), distributed in the bronchial mucosa, plays a critical role in maintaining the mucosal immune homeostasis of the lower respiratory tract. The bronchial tree is a functional structure for gas exchange with the outside environment and maintains basic lung morphology. Methods To explore the structural and distributive characteristics of BALT in Bactrian camels, twelve healthy adult Bactrian camels were divided into two groups (six in each group). The lungs, bronchial tree and BALT were observed and analysed systematically through anatomical and histological methods. Results The results showed that Bactrian camel lungs were constituted by the left cranial lobe, left caudal lobe, right cranial lobe, right caudal lobe and accessory lobe, but lacked the middle lobe. The cranial lobe was narrow and small, the caudal lobe was extremely developed (almost four times the cranial lobe in size), and the accessory lobe was smaller than the cranial lobe; the bronchial tree, an unequal dichotomy with a tracheobronchial branch, was composed of dorsal, ventral, lateral and medial bronchiole systems. Isolated lymphoid follicles (the chief type) and aggregates of lymphoid follicles revealed two types of BALT, and germinal centres, follicle-associated epithelium and high endothelial venules could be observed in some well-developed BALT. Additionally, BALT was scattered along the bronchial tree in the entire lung, and the density increased from the trachea to the lower graded branches (densest in the bronchioles) and then decreased, with the occasional location around respiratory bronchioles or among the pulmonary mesenchyme. In the conducting portion, BALT was primarily located in the mucosa lamina propria but was also found in the submucosa, under the muscular layer, and around the submucosal glands and cartilage. Conclusion The results demonstrated that the lung morphology of Bactrian camels was similar to that of horses, but the bronchial branches were more closely related to those of ruminants. These characteristics were in accordance with the morphological and structural variation regularity of lungs with species evolution. BALT was mainly scattered in the conducting portion, and bronchioles, as the final “checkpoint” in the surveillance, capture and recognition of antigens before pulmonary exchange, were the pivotal locational position of BALT. However, BALT at different depths of the bronchial wall of the conducting portion might be at different developmental stages. Our study provided evidence for further insight into the mucosal immunomodulatory mechanism of BALT in the respiratory system of Bactrian camels.

Abstract Background The intestinal phase is the early invasion stage of Trichinella spiralis (T. spiralis), in which muscle larvae invade intestine epithelial cells and then develop into adult worms to breed newborn larvae. Thus, intestinal infective larvae are first exposed to the immune system of the host, and antigens from the worms may be the earliest marker in the diagnosis of trichinellosis and may contribute to vaccine development to prevent Trichinella infections in pigs. Methods A cDNA library of intestinal infective larvae of T. spiralis at 6 hours post infection (p.i.) was constructed and immunoscreened using serum collected from pigs that were infected with T. spiralis at 26 days p.i. T. spiralis cystatin-like protein (Ts-CLP) gene encoding a 45.9 kDa protein was cloned and expressed in Escherichia coli. The rabbit antisera were generated and used to determine the location of Ts-CLP in the parasite. Transcription levels of Ts-CLP in different developmental stages of T. spiralis were observed by RT-PCR. The potential application of recombinant Ts-CLP in diagnosis against T. spiralis infection was tested by ELISA. The immune protection of recombinant Ts-CLP protein against T. spiralis infection was evaluated in mice. Results Thirty-three positive clones were selected from cDNA library, among which 20 clones encoded the same novel cystatin-like protein (Ts-CLP). Immunolocalisation and real-time quantitative PCR revealed that native Ts-CLP was localised primarily to β-stichocytes and that the Ts-clp gene was transcribed and expressed in all developmental stages of T. spiralis. The recombinant protein rTs-CLP was recognised by pig antiserum as early as 15 days p.i., and could induce protective immunity in mice, with a 61.21% reduction in the number of muscle larvae. Conclusions These data preliminarily suggested that Ts-CLP may play an important role in the early infection of T. spiralis and that recombinant Ts-CLP protein is a candidate antigen for diagnosis and vaccine development in Trichinella infections.

Sodium houttuyfonate (SH), an addition compound of sodium bisulfite and houttuynin, showed in vitro antibacterial activity against 21 Staphylococcus aureus (S. aureus) strains grown in planktonic cultures. Microarray results showed decreased levels of autolysin atl, sle1, cidA and lytN transcripts in the SH-treated strain as compared to the control strain, consistent with the induction of the autolytic repressors lrgAB and sarA and with the downregulation of the positive regulators agrA and RNAIII. Triton X-100-induced autolysis was significantly decreased by SH in S. aureus ATCC 25923, and quantitative bacteriolytic assays and zymographic analysis demonstrated SH-mediated reduction of extracellular murein hydrolase activity in these cells. Anti-biofilm assay showed that SH is poorly active against S. aureus grown in biofilm cultures, whereas SH diminished the amounts of extracellular DNA (eDNA) of S. aureus in a dose-dependent manner, which suggested that SH may impede biofilm formation by reducing the expression of cidA to inhibit autolysis and eDNA release in the early phase. Some of the microarray results were confirmed by real-time RT-PCR.

Deoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the Trichinella spiralis (T. spiralis) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. XM_003370715.1) contains the HKD motif in its C-terminus domain.In this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from T. spiralis were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (Ts-Pt, 885 bp) and has been deposited in GenBank under accession number KF984291. The recombinant protein (rTs-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that rTs-Pt was recognised by serum from T. spiralis-infected mice; the anti-rTs-Pt serum recognised crude antigens but not ES antigens. The Ts-Pt gene was examined at all T. spiralis developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that Ts-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. rTs-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C.This study indicated that Ts-Pt was classified as a somatic protein in different T. spiralis developmental stages, and demonstrated for the first time that an expressed DNase II protein from T. spiralis had nuclease activity.

The formation of extracellular traps (ETs) has recently been recognized as a novel defense mechanism in several types of innate immune cells. It has been suggested that these structures are toxic to microbes and contribute significantly to killing several pathogens. However, the role of ETs formed by macrophages (METs) in defense against microbes remains little known. In this study, we demonstrated that a subset of murine J774A.1 macrophage cell line (8% to 17%) and peritoneal macrophages (8.5% to 15%) form METs-like structures (METs-LS) in response to Escherichia coli and Candida albicans challenge. We found only a portion of murine METs-LS, which are released by dying macrophages, showed detectable killing effects on trapped E. coli but not C. albicans. Fluorescence and scanning electron microscopy analyses revealed that, in vitro, both microorganisms were entrapped in J774A.1 METs-LS composed of DNA and microbicidal proteins such as histone, myeloperoxidase and lysozyme. DNA components of both nucleus and mitochondrion origins were detectable in these structures. Additionally, METs-LS formation occurred independently of ROS produced by NADPH oxidase, and this process did not result in cell lysis. In summary, our results emphasized that microbes induced METs-LS in murine macrophage cells and that the microbicidal activity of these METs-LS differs greatly. We propose the function of METs-LS is to contain invading microbes at the infection site, thereby preventing the systemic diffusion of them, rather than significantly killing them.