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Abstract Asparagus is a nutritionally dense stem vegetable whose growth and development are correlated with its quality and yield. To investigate the dynamic changes and underlying mechanisms during the elongation and growth process of asparagus stems, we documented the growth pattern of asparagus and selected stem segments from four consecutive elongation stages using physiological and transcriptome analyses. Notably, the growth rate of asparagus accelerated at a length of 25 cm. A significant decrease in the concentration of sucrose, fructose, glucose, and additional sugars was observed in the elongation region of tender stems. Conversely, the levels of auxin and gibberellins(GAs) were elevated along with increased activity of enzymes involved in sucrose degradation. A significant positive correlation existed between auxin, GAs, and enzymes involved in sucrose degradation. The ABA content gradually increased with stem elongation. The tissue section showed that cell elongation is an inherent manifestation of stem elongation. The differential genes screened by transcriptome analysis were enriched in pathways such as starch and sucrose metabolism, phytohormone synthesis metabolism, and signal transduction. The expression levels of genes such as ARF, GA20ox, NCED, PIF4, and otherswere upregulated during stem elongation, while DAO, GA2ox, and other genes were downregulated. The gene expression level was consistent with changes in hormone content and influenced the cell length elongation. Additionally, the expression results of RT-qPCR were consistent with RNA-seq. The observed variations in gene expression levels, endogenous hormones and sugar changes during the elongation and growth of asparagus tender stems offer valuable insights for future investigations into the molecular mechanisms of asparagus stem growth and development and provide a theoretical foundation for cultivation and production practices.

Abstract Sinonasal malignant tumors are a group of uncommon malignancies that account for less than 1% of all tumors. These tumors often involve the maxillary sinus and nasal cavity, with less cumulative incidence in the ethmoidal sinus, sphenoidal sinus, and frontal sinus. The lack of consensus on the management of sinonasal malignancies is due to their rarity, diagnostic challenges, and the heterogeneity of treatments. In this paper, we present a case of endoscopic-assisted medial canthus incision combined with radiotherapy in the treatment of sinonasal malignant tumors, with the aim of providing valuable insights to clinicians on the management of these tumors.

Abstract Several hematologic traits have been suggested to potentially contribute to the formation and rupture of intracranial aneurysms (IA). The purpose of this study is to explore the causal association between hematologic traits and the risk of IA. To explore the causal association between hematologic traits and the risk of IA, we employed two-sample Mendelian randomization (MR) analysis. Two independent summary-level GWAS data were used for preliminary and replicated MR analyses. The inverse variance weighted (IVW) method was employed as the primary method in the MR analyses. The stabilities of the results were further confirmed by a meta-analysis. In the preliminary MR analysis, hematocrit, hemoglobin concentration (p = 0.0047), basophil count (p = 0.0219) had a suggestive inverse causal relationship with the risk of aneurysm-associated subarachnoid hemorrhage (aSAH). The monocyte percentage of white cells (p = 0.00956) was suggestively positively causally correlated with the risk of aSAH. In the replicated MR analysis, only the monocyte percentage of white cells (p = 0.00297) remained consistent with the MR results in the preliminary analysis. The hematocrit, hemoglobin concentration, and basophil count no longer showed significant causal relationship (p > 0.05). Meta-analysis results further confirmed that only the MR result of monocyte percentage of white cells reached significance in the random effect model and fixed effect model. None of the 25 hematologic traits was causally associated with the risk of unruptured intracranial aneurysms (uIA). This study revealed a suggestive positive association between the monocyte percentage of white cells and the risk of aSAH. This finding contributes to a better understanding that monocytes/macrophages could participate in the risk of aSAH.

Abstract Background Triatomines (kissing bugs) are natural vectors of trypanosomes, which are single-celled parasitic protozoans, such as Trypanosoma cruzi, T. conorhini and T. rangeli. The understanding of the transmission cycle of T. conorhini and Triatoma rubrofasciata in China is not fully known. Methods The parasites in the faeces and intestinal contents of the Tr. rubrofasciata were collected, and morphology indices were measured under a microscope to determine the species. DNA was extracted from the samples, and fragments of 18S rRNA, heat shock protein 70 (HSP70) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) were amplified and sequenced. The obtained sequences were then identified using the BLAST search engine, followed by several phylogenetic analyses. Finally, laboratory infections were conducted to test whether Tr. rubrofasciata transmit the parasite to rats (or mice) through bites. Moreover, 135 Tr. rubrofasciata samples were collected from the Guangxi region and were used in assays to investigate the prevalence of trypanosome infection. Results Trypanosoma sp. were found in the faeces and intestinal contents of Tr. rubrofasciata, which were collected in the Guangxi region of southern China and mostly exhibited characteristics typical of epimastigotes, such as the presence of a nucleus, a free flagellum and a kinetoplast. The body length ranged from 6.3 to 33.9 µm, the flagellum length ranged from 8.7 to 29.8 µm, the nucleus index was 0.6 and the kinetoplast length was −4.6. BLAST analysis revealed that the 18S rRNA, HSP70 and gGAPDH sequences of Trypanosoma sp. exhibited the highest degree of similarity with those of T. conorhini (99.7%, 99.0% and 99.0%, respectively) and formed a well-supported clade close to T. conorhini and T. vespertilionis but were distinct from those of T. rangeli and T. cruzi. Laboratory experiments revealed that both rats and mice developed low parasitaemia after inoculation with Trypanosoma sp. and laboratory-fed Tr. rubrofasciata became infected after feeding on trypanosome-positive rats and mice. However, the infected Tr. rubrofasciata did not transmit Trypanosoma sp. to their offspring. Moreover, our investigation revealed a high prevalence of Trypanosoma sp. infection in Tr. rubrofasciata, with up to 36.3% of specimens tested in the field being infected. Conclusions Our study is the first to provide a solid record of T. conorhini from Tr. rubrofasciata in China with morphological and molecular evidence. This Chinese T. conorhini is unlikely to have spread through transovarial transmission in Tr. rubrofasciata, but instead, it is more likely that the parasite is transmitted between Tr. rubrofasciata and mice (or rats). However, there was a high prevalence of T. conorhini in the Tr. rubrofasciata from our collection sites and numerous human cases of Tr. rubrofasciata bites were recorded. Moreover, whether these T. conorhini strains are pathogenic to humans has not been investigated. Graphical Abstract

Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia, an economically important lung disease in pigs. In draft genomes of two Cypriot clinical A. pleuropneumoniae isolates (MIDG3457 and MIDG3459), we previously identified single genomic regions with homology to Mu-like bacteriophage and presented preliminary evidence of active phage. Here, updated Phastest genomic analysis identified two loci in both MIDG3457 and MIDG3459 that were predicted to encode proteins with high homology to, and whose organisation was characteristic of, Mu-like phages. Phylogenetically, the closest matches were with Mannheimia Vb and Glaesserella SuMu phages. Phastest scored the loci as “complete”, indicating they produced active phage. PCR amplification of the Mu-like phage c and tail genes from DNase-treated polyethylene glycol 8000 (PEG)-precipitated supernatants of MIDG3457 and MIDG3459 (grown in either Brain Heart Infusion-NAD or Grace’s Insect Medium-NAD broth) indicated the presence of intact virions. The phages from MIDG3457 and MIDG3459 were named PluMu 3457-1, 3457-2, and PluMu 3459-1 and PluMu 3459-2, respectively. Transmission electron microscopy (TEM) of the PEG-precipitated supernatants of broth-grown MIDG3459 identified virions with icosahedral heads and tails, consistent with other Mu-like phages. We conclude that MIDG3459 produces an active Mu-like phage.

Asparagus, characterized by its high metabolic rate, is susceptible to quality degradation. Proanthocyanidins have antioxidant, antibacterial, antiviral, and other biological functions and can inhibit the production of reactive oxygen species in plants. To enhance the shelf life of asparagus, we investigated the impact of various concentrations of proanthocyanidins on its cold storage and preservation. The findings revealed that proanthocyanidins effectively mitigated water loss, delayed chlorophyll degradation, and prevented firmness decline. Furthermore, they enhanced the activity of antioxidant enzymes (superoxide dismutase, catalase, peroxidase, and polyphenol oxidase), bolstered DPPH free radical scavenging ability, and increased the levels of total phenol, total flavone, rutin, oligomeric procyanidins, proline, and soluble protein. Moreover, proanthocyanidins promoted the accumulation of vitamin C, amino acids, total saponins, and lignin in the later storage stage, contributing to increased mechanical tissue thickness. These results suggest that proanthocyanidins play a crucial role in retarding the deterioration of asparagus quality during storage by affecting the antioxidant capacity and phytochemical (polyphenol,amino acid, total saponin, and lignin) synthesis in asparagus.

Abstract Conjugate vaccines produced either by chemical or biologically conjugation have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccine assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel biotechnological platform that overcomes the limitations of the current conjugate vaccine design method(s). As a model, we focused our design on a leading bioconjugation method using N-oligosaccharyltransferase (OTase), PglB. The installation of MAGIC led to at least twofold increase in glycoconjugate yield via MAGIC when compared to conventional N-OTase based bioconjugation method(s). Then, we improved MAGIC to (a) allow rapid installation of glycoengineering component(s), (b) omit the usage of antibiotics, (c) reduce the dependence on protein induction agents. Furthermore, we show the modularity of the MAGIC platform in performing glycoengineering in bacterial species that are less genetically tractable than the commonly used Escherichia coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against serious bacterial pathogens. We anticipate the utility of the MAGIC platform could enhance vaccines production due to its compatibility with virtually any bioconjugation method, thus expanding vaccine biopreparedness toolbox.

Dwarf apple is one of the most important forms of garden economy, which has become a new engine for rural revitalization. The effective detection of coloring and ripening apples in complex environments is important for the sustainable development of smart agricultural operations. Addressing the issues of low detection efficiency in the greenhouse and the challenges associated with deploying complex target detection algorithms on low-cost equipment, we propose an enhanced lightweight model rooted in YOLOv7. Firstly, we enhance the model training performance by incorporating the Squeeze-and-Excite attention mechanism, which can enhance feature extraction capability. Then, an SCYLLA-IoU (SIoU) loss function is introduced to improve the ability of extracting occluded objects in complex environments. Finally, the model was simplified by introducing depthwise separable convolution and adding a ghost module after up-sampling layers. The improved YOLOv7 model has the highest AP value, which is 10.00%, 5.61%, and 6.00% higher compared to YOLOv5, YOLOv7, and YOLOX, respectively. The improved YOLOv7 model has an MAP value of 95.65%, which provides higher apple detection accuracy compared to other detection models and is suitable for potted dwarf anvil apple identification and detection.

ABSTRACTObjective Endothelial dysfunction is a critical initiating factor in the development of hypertension and related complications. Follistatin-like 1 (FSTL1) can promote endothelial cell function and stimulates revascularization in response to ischemic insult. However, it is unclear whether FSTL1 has an effect on ameliorating endothelial dysfunction in spontaneously hypertensive rats (SHRs).Methods Wistar Kyoto (WKY) and SHRs were treated with a tail vein injection of vehicle (1 mL/day) or recombinant FSTL1 (100 μg/kg body weight/day) for 4 weeks. Blood pressure was measured by tail-cuff plethysmograph, and vascular reactivity in mesenteric arteries was measured using wire myography.Results We found that treatment with FSTL1 reversed impaired endothelium-dependent relaxation (EDR) in mesenteric arteries and lowered blood pressure of SHRs. Decreased AMP-activated protein kinase (AMPK) phosphorylation, elevated endoplasmic reticulum (ER) stress markers, increased reactive oxygen species (ROS), and reduction of nitric oxide (NO) production in mesenteric arteries of SHRs were also reversed by FSTL1 treatment. Ex vivo treatment with FSTL1 improved the impaired EDR in mesenteric arteries from SHRs and reversed tunicamycin (ER stress inducer)-induced ER stress and the impairment of EDR in mesenteric arteries from WKY rats. The effects of FSTL1 were abolished by cotreatment of compound C (AMPK inhibitor).Conclusions These results suggest that FSTL1 prevents endothelial dysfunction in mesenteric arteries of SHRs through inhibiting ER stress and ROS and increasing NO production via activation of AMPK signaling.

Attack behaviour detection of the pig is a valid method to protect the health of pig. Due to the farm conditions and the illumination changes of the piggery, the images of the pig in the videos are often being overlapped, which lead to difficulties in recognizing pig attack behaviour. We propose an improved YOLOX target detection model to overcome these difficulties. The improvements of the proposed model are: (1) the normalization attention mechanism is adopted to gain global information in the last block of the neck network and (2) the loss function IoU in YOLOX is replaced by DIoU to improve the detection accuracy. The pig attack behaviour considered in this paper includes the ear biting, the tail biting, the head to head collision and the head to body collision. The dataset is builded from the artificially observed attack video segments by using the inter-frame difference method. In the pig attack behaviour detection experiments, the improved YOLOX model achieves 93.21% precision which is 5.30% higher than the YOLOX model. The experiment results show that the improved YOLOX can realize pig attack behaviour detection with high precision.

Abstract Background Mood swings have been observed in patients with intracranial aneurysm (IA), but it is still unknown whether mood swings can affect IA. Aim To explore the causal association between mood swings or experiencing mood swings and IA through a two‐sample Mendelian randomization (MR) study. Methods Summary‐level statistics of mood swings, experiencing mood swings, IA, aneurysm‐associated subarachnoid hemorrhage (aSAH), and non‐ruptured IA (uIA) were collected from the genome‐wide association study. Two‐sample MR and various sensitivity analyses were employed to explore the causal association between mood swings or experiencing mood swings and IA, or aSAH, or uIA. The inverse‐variance weighted method was used as the primary method. Results Genetically determined mood swings (odds ratio [OR] = 5.23, 95% confidence interval (95%CI): 1.65–16.64, p = .005) and experiencing mood swings (OR = 2.50, 95%CI: 1.37–4.57, p = .003) were causally associated with an increased risk of IA. Mood swings (OR = 5.67, 95%CI: 1.40–23.04, p = .015) and experiencing mood swings were causally associated with the risk of aSAH (OR = 2.91, 95%CI: 1.47–5.75, p = .002). Neither mood swings (OR = 1.95, 95%CI: .31–12.29, p = .478) nor experiencing mood swings (OR = 1.20, 95%CI: .48–3.03, p = .693) were associated with uIA. Conclusions Mood swings and experiencing mood swings increased the risk of IA and aSAH incidence. These results suggest that alleviating mood swings may reduce IA rupture incidence and aSAH incidence.

Extracellular vesicle (EV) production by bacteria is an important mechanism for microbial communication and host-pathogen interaction. EVs of some bacterial species have been reported to contain nucleic acids. However, the role of small RNAs (sRNAs) packaged in EVs is poorly understood. Here, we report on the RNA cargo of EVs produced by the pig pathogen Actinobacillus pleuropneumoniae, the causal agent of porcine pleuropneumonia, a disease which causes substantial economic losses to the swine industry worldwide. The EVs produced by aerobically and anaerobically grown bacteria were only slightly different in size and distribution. Total cell and outer membrane protein profiles and lipid composition of A. pleuropneumoniae whole cell extracts and EVs were similar, although EVs contained rough lipopolysaccharide compared to the smooth form in whole cells. Approximately 50% of Galleria mellonella larvae died after the injection of EVs. RNAseq, RT-PCR, protection from nuclease degradation, and database searching identified previously described and 13 novel A. pleuropneumoniae sRNAs in EVs, some of which were enriched compared to whole cell content. We conclude that A. pleuropneumoniae EVs contain sRNAs, including those known to be involved in virulence, and some with homologs in other Pasteurellaceae and/or non-Pasteurellaceae. Further work will establish whether the novel sRNAs in A. pleuropneumoniae EVs play any role in pathogenesis.

Abstract Magnetic fluids are the suspensions composed of magnetic nanoparticles, surfactants, and non-magnetic carrier liquids. Magnetic fluids are widely used in various fields, especially in sealing, because of their excellent features, including rapid magnetic response, flexible flow ability, tunable magneto-viscous effect, and reliable self-repairing capability. Here, we provide an in-depth, comprehensive insight into the theoretical analyses and diverse applications of magnetic fluids in sealing from three categories: static sealing, rotary sealing, and reciprocating sealing. We summarize the magnetic fluid sealing mechanisms and the development of magnetic fluid seals from 1960s to the present, particularly focusing on the recent progress of magnetic fluid seals. Although magnetic fluid sealing technology has been commercialized and industrialized, many difficulties still exist in its applications. At the end of the review, the present challenges and future prospects in the progress of magnetic fluid seals are also outlined.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a leading cause of infectious disease mortality. Animal infection models have contributed substantially to our understanding of TB, yet their biological and non-biological limitations are a research bottleneck. There is a need for more ethically acceptable, economical, and reproducible TB infection models capable of mimicking key aspects of disease. Here, we demonstrate and present a basic description of how Galleria mellonella (the greater wax moth, Gm) larvae can be used as a low cost, rapid, and ethically more acceptable model for TB research. This is the first study to infect Gm with the fully virulent MTB H37Rv, the most widely used strain in research. Infection of Gm with MTB resulted in a symptomatic lethal infection, the virulence of which differed from both attenuated Mycobacterium bovis BCG and auxotrophic MTB strains. The Gm-MTB model can also be used for anti-TB drug screening, although CFU enumeration from Gm is necessary for confirmation of mycobacterial load reducing activity of the tested compound. Furthermore, comparative virulence of MTB isogenic mutants can be determined in Gm. However, comparison of mutant phenotypes in Gm against conventional models must consider the limitations of innate immunity. Our findings indicate that Gm will be a practical, valuable, and advantageous additional model to be used alongside existing models to advance tuberculosis research.

Context Among the Tujia people, the root or rhizome of Trillium tschonoskii Maxim.in Bull.Acad (TTM) is considered a miraculous herb for headaches. Previous studies have shown ethyl acetate extract (TTM1) can protect SH-SY5Y cells against glutamate injury. Objective This study clarified TTM1’s mechanism against glutamate-induced cell damage, focusing on the regulation of apoptosis. The compounds were separated, identified, and performed molecular docking with pro-apoptotic proteins. Materials and Methods SH-SY5Y cells were treated with glutamate (2 mM) for 12 hour, and the effect of TTM1 (2.5, 5, 10, and 20 μg/mL) was evaluated with MTT and LDH release assays, taking EGb761(40 μg/mL) as a control. Cell apoptosis was detected with Hoechst 33258 and Annexin V-FITC and measurements of intracellular calcium and caspase-3. The major components were separated and identified by LCMS-IT-TOF and NMR, then the proapoptotic activity of TTM1 was confirmed by molecular docking method. Results TTM1 protected SH-SY5Y cells by resisting apoptosis, TTM1 (10 and 20 μg/mL) decreased apoptotic bodies and nuclear fragments, increased the proportion of normal cells to 68.38 ± 5.63% and 92.80 ± .88%, decreased VA cells to 4.30 ± .76% and 3.58 ± .45% and caspase-3 to .365 ± .034 and .344 ± .047 ng/mL.TTM1 (10 μg/mL) decreased intracellular free calcium to 2.77 ± .40. Polyphyllin VI and pennogenin 3-O-β-chacotrioside were identified in TTM1 at 15.04% and 2.84%, and had potential anti-apoptosis activities. Discussion and Conclusions Folk records of TTM for headache may be related to its anti-apoptosis of nerve cells. Identification and content determination of index components based on effective extract provides research paradigms for rare and endangered ethnic plants.

The thermal performance of a novel exterior coating material for commonly used grain and food-grain oil structures was investigated. Grain structures included a concrete squat silo and a concrete warehouse while the edible oil structure was a concrete sided tank. The exterior coating provided excellent moisture runoff and solar reflectance properties and is best described as a superamphiphobic self-cleaning passive subambient daytime radiative cooling (SSC-PSDRC) coating. The coating exhibited a remarkable subambient daytime cooling effect in various structures in different climatic regions. Compared with the roof surface temperatures of a cool white-coated concrete grain silo and a gray carbon iron-based edible oil storage tank, those of the PSDRC coated top surfaces could be reduced by 37 °C and 33 °C, respectively. The roof surface temperature of a warehouse painted with a cool-white coating—with a solar reflectance of 0.9 and an emissivity of 0.85—and that of a warehouse with the roof installed with aluminised polymer waterproof membranes were 19 °C and 18 °C higher than that of the PSDRC warehouse, respectively. Consequently, the interior temperature of the wheat pile in the PSDRC grain silo was 10 °C lower than that in the control squat silo. With the inner loop flow temperature control system operating, the interior air temperatures of the PSDRC west-facing separate space were 6 °C and 3 °C higher than those of the cool-white coated and control west-facing separate spaces, respectively. Even after the application of PSDRC coating for only a few days, the interior air temperature of the PSDRC oil storage tank was reduced by 38 °C, and the interior temperature of the oil storage tank was reduced by 4 °C. Furthermore, in practical applications, the coating showed impressive superamphiphobic self-cleaning capabilities and super aging resistance. The wide applications of the coating would have far-reaching, global implications for maintaining grain and edible oil products, particularly in the sub-tropical climates.

Abstract Current research on spherical parallel mechanisms (SPMs) mainly focus on surgical robots, exoskeleton robots, entertainment equipment, and other fields. However, compared with the SPM, the structure types and research contents of the SPM are not abundant enough. In this paper, a novel two-degree-of-freedom (2DOF) SPM with symmetrical structure is proposed and analyzed. First, the models of forward kinematics and inverse kinematics are established based on D-H parameters, and the Jacobian matrix of the mechanism is obtained and verified. Second, the workspace of the mechanism is obtained according to inverse kinematics and link interference conditions. Next, rotational characteristics analysis shows that the end effector can achieve continuous rotation about an axis located in the mid-plane and passing through the rotation center of the mechanism. Moreover, the rotational characteristics of the mechanism are proved, and motion planning is carried out. A numerical example is given to verify the kinematics analysis and motion planning. Finally, some variant mechanisms can be synthesized. This work lays the foundation for the motion control and practical application of this 2DOF SPM.

ABSTRACT 12-Bis-THA Cl2 [12,12′-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications. IMPORTANCE While we face an acute threat from antibiotic resistant bacteria and a lack of new classes of antibiotic, there are many effective antimicrobials which have limited application due to concerns regarding their toxicity and which could be more useful if such risks are reduced or eliminated. We modified a bolalipid antiseptic used in throat lozenges to see if it could be made more effective against some of the highest-priority bacteria and less toxic. We found that structural modifications that rendered the lipid more toxic against human cells made it less toxic in infection models and we could effectively treat caterpillars infected with either Mycobacterium tuberculosis, methicillin resistant Staphylococcus aureus, or Acinetobacter baumannii. The study provides a rationale for further adaptation toward diversifying the range of indications in which this class of antimicrobial may be used.

Glioma is the most common primary malignant tumor of the central nervous system in clinical practice. Most adult diffuse gliomas have poor efficacy after standard treatment, especially glioblastoma. With the in-depth understanding of brain immune microenvironment, immunotherapy as a new treatment has attracted much attention. In this study, through analyzing a large number of glioma cohorts, we reported that TSPAN7, a member of the tetraspanin family, decreased in high-grade gliomas and low expression was associated with poor prognosis in glioma patients. Meanwhile, the expression pattern of TSPAN7 was verified in glioma clinical samples and glioma cell lines by qPCR, Western Blotting and immunofluorescence. In addition, functional enrichment analysis showed that cell proliferation, EMT, angiogenesis, DNA repair and MAPK signaling pathways were activated in the TSPAN7 lower expression subgroup. Lentiviral plasmids were used to overexpress TSPAN7 in U87 and LN229 glioma cell lines to explore the anti-tumor role of TSPAN7 in glioma. Moreover, by analyzing the relationship between TSPAN7 expression and immune cell infiltration in multiple datasets, we found that TSPAN7 was significantly negatively correlated with the immune infiltration of tumor-related macrophages, especially M2-type macrophages. Further analysis of immune checkpoints showed that, the expression level of TSPAN7 was negatively correlated with the expression of PD-1, PD-L1 and CTLA-4. Using an independent anti-PD-1 immunotherapy cohorts of GBM, we demonstrated that TSPAN7 expression may had a synergistic effect with PD-L1 on the response to immunotherapy. Based on the above findings, we speculate that TSPAN7 can serve as a biomarker for prognosis and a potential immunotherapy target in glioma patients.

Background: ZBTB42 is a transcription factor that belongs to the ZBTB transcript factor family and plays an important role in skeletal muscle development. Dysregulation of ZBTB42 expression can lead to a variety of diseases. However, the function of ZBTB42 in glioma development has not been studied by now.Methods: We analyzed the expression of ZBTB42 in LGG and GBM via the The Cancer Genome Atlas CGA and Chinese Glioma Genome Atlas database. Gene Ontology, KEGG, and GSVA analyses were performed to illustrate ZBTB42-related pathways. ESTIMATE and CIBERSORT were applied to calculate the immune score and immune cell proportion in glioma. One-class logistic regression OCLR algorithm was used to study the stemness of glioma. Multivariate Cox analysis was employed to detect the prognostic value of five ZBTB42-related genes.Results: Our results show that ZBTB42 is highly expressed in glioma and may be a promising prognostic factor for Low Grade Glioma and GBM. In addition, ZBTB42 is related to immune cell infiltration and may play a role in the immune suppression microenvironment. What’s more, ZBTB42 is correlated with stem cell markers and positively associated with glioma stemness. Finally, a five genes nomogram based on ZBTB42 was constructed and has an effective prognosis prediction ability.Conclusion: We identify that ZBTB42 is a prognostic biomarker for Low Grade Glioma and GBM and its function is related to the suppressive tumor microenvironment and stemness of glioma.

BackgroundSupratentorial hemangioblastoma is an extremely rare neoplasm. The aim of this study is to delineate the clinical features among cystic and solid supratentorial hemangioblastoma patients and evaluate the risk factors for progression-free survival (PFS).MethodsWe conducted a literature search in PubMed for histopathologically identified supratentorial hemangioblastoma between 1947 and 2021 and extracted and collected the clinical features of patients treated at our own institute. The rate of PFS was determined using Kaplan–Meier analysis. Differences in categorical factors, such as the location of tumor and diagnosis of von Hippel–Lindau disease, were analyzed using the Pearson χ2 test. A Cox regression analysis was performed to evaluate the association between various variates and survival outcomes.ResultsA total of 237 cases of supratentorial hemangioblastoma were identified from 169 studies. A survival analysis found that patients with cystic tumors had a significantly better prognosis than those with solid tumors (log-rank, p = 0.0122). Cox regression analysis suggested that cystic hemangioblastoma (hazard ratio (HR): 0.186, 95% CI: 0.043–0.803, p

Abstract Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines.

Abstract Computing the grain boundary (GB) counterparts to bulk phase diagrams represents an emerging research direction. Using a classical embrittlement model system Ga-doped Al alloy, this study demonstrates the feasibility of computing temperature- and composition-dependent GB diagrams to represent not only equilibrium thermodynamic and structural characters, but also mechanical properties. Specifically, hybrid Monte Carlo and molecular dynamics (MC/MD) simulations are used to obtain the equilibrium GB structure as a function of temperature and composition. Simulated GB structures are validated by aberration-corrected scanning transmission electron microscopy. Subsequently, MD tensile tests are performed on the simulated equilibrium GB structures. GB diagrams are computed for not only GB adsorption and structural disorder, but also interfacial structural and chemical widths, MD ultimate tensile strength, and MD tensile toughness. This study suggests a research direction to investigate GB composition–structure–property relationships via computing GB diagrams of thermodynamic, structural, and mechanical (or potentially other) properties.

As a stem variety of lettuce, the fleshy stem is the main product organ of stem lettuce. The molecular mechanism of fleshy stem expansion in stem lettuce is a complex biological process. In the study, the material accumulation, gene expression, and morphogenesis during fleshy stem expansion process were analyzed by the comparative analysis of metabolome, transcriptome and the anatomical studies. The anatomical studies showed that the occurrence and activity of vascular cambium mainly led to the development of fleshy stems; and the volume of pith cells gradually increased and arranged tightly during the expansion process. A total of 822 differential metabolites and 9,383 differentially expressed genes (DEGs) were identified by the metabolomics and transcriptomics analyses, respectively. These changes significantly enriched in sugar synthesis, glycolysis, and plant hormone anabolism. The expression profiles of genes in the sugar metabolic pathway gradually increased in fleshy stem expansion process. But the sucrose content was the highest in the early stage of fleshy stem expansion, other sugars such as fructose and glucose content increased during fleshy stem expansion process. Plant hormones, including IAA, GA, CTK, and JA, depicted important roles at different stem expansion stages. A total of 1,805 DEGs were identified as transcription factors, such as MYB, bHLH, and bZIP, indicating that these transcription factor families might regulate the fleshy stems expansion in lettuce. In addition, the expression patterns identified by qRT-PCR were consistent with the expression abundance identified by the transcriptome data. The important genes and metabolites identified in the lettuce fleshy stem expansion process will provide important information for the further molecular mechanism study of lettuce fleshy stem growth and development.

With the rapid development of aerospace technology, the vibration problem of the spacecraft flexible structure urgently needs to be solved. Magnetic fluids are a type of multi-functional smart materials, which can be employed in shock absorbers to eliminate these vibrations. Referring to the calculation methods of stiffness coefficients of other passive dampers, the stiffness coefficient formula of magnetic fluid shock absorbers (MFSAs) was derived and refined. Meanwhile, a series of varying stiffness magnetic fluid shock absorbers (VS-MFSAs) were proposed and fabricated based on the second-order buoyancy principle. The range of stiffness coefficients covered by these VS-MFSAs contains the optimal stiffness coefficient estimated by formulas. The repulsive force measurement and vibration attenuation experiments were conducted on these VS-MFSAs. In the case of small amplitude, the relationship between the repulsive force and the offset distance was linear, which means the stiffness was linear. The simulation and experiment curves of the stiffness were in good agreement. The results of vibration attenuation experiments demonstrated that the rod length and the magnetic fluid mass influence the damping efficiency of VS-MFSAs. In addition, these results verified that the VS-MFSA with the optimal stiffness coefficient performed best. Therefore, the stiffness coefficient formula can guide the design of MFSAs.

The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.

The aim of the study was to analyze acoustic emission (AE) signals generated in different magnetic fluid sealing processes to estimate the status of the seal. Based on a magnetic fluid rotary sealing experimental setup, the AE sensor is respectively attached to the shell at the corresponding position of the seal, and the external pressure is applied to the magnetic fluid seal by a gas supply system. As the pressure gradually increases, the magnetic fluid sealing rings under different pole teeth are gradually broken, and processes such as pressure transfer between the seal stages occur. These processes generate different AE signals, which are collected by the sensors. The results show that the root-mean-square values of AE signals generated at the moment of sealing failure are different from those generated before and after. At the same time, by analyzing the characteristic values of AE signals generated in the whole process, we can infer the time when the rupture begins under different pole pieces and further estimate the status of the magnetic fluid seal. For the first time, this research demonstrates the experimental and data analysis procedures of AE technology for magnetic fluid seals.

Magnetic fluid seals have long been thought to be a successful sealing form while sealing liquids are always a challenge. The instability of the liquid–liquid interface under the washout has become the key technical problem that hinders the realization of sealing liquid. This work mainly presents an experimental study about the influence of high viscosity and magnetoviscous effects on washout resistance. Three engine oil-based magnetic fluids of different viscosities were prepared with two kinds of surfactants. The magnetoviscous effects of the prepared magnetic fluids under different working conditions were found through rheological experiments. The viscosity of the three samples decreased at most by about 100 times with the shear rate increasing. An experimental platform was designed and built for the washout tests. The entire process of magnetic fluids being washed away was obtained experimentally. The magnetic fluid of higher viscosity can remain stationary with lower magnetic force. The quantitative results show that the viscosity of the magnetic fluid has a significant influence on washout resistance under a magnetic field.

The accurate segmentation of apple leaf disease spots is the key to identifying the classification of apple leaf diseases and disease severity. Therefore, a DeepLabV3+ semantic segmentation network model with an actors spatial pyramid pool module (ASPP) was proposed to achieve effective extraction of apple leaf lesion features and to improve the apple leaf disease recognition and disease severity diagnosis compared with the classical semantic segmentation network models PSPNet and GCNet. In addition, the effects of the learning rate, optimizer, and backbone network on the performance of the DeepLabV3+ network model with the best performance were analyzed. The experimental results show that the mean pixel accuracy (MPA) and mean intersection over union (MIoU) of the model reached 97.26% and 83.85%, respectively. After being deployed into the smartphone platform, the detection time of the detection system was 9s per image for the portable and intelligent diagnostics of apple leaf diseases. The transfer learning method provided the possibility of quickly acquiring a high-performance model under the condition of small datasets. The research results can provide a precise guide for the prevention and precise control of apple diseases in fields.

Asparagus belongs to the Liliaceae family and has important economic and pharmacological value. Lignin plays a crucial role in cell wall structural integrity, stem strength, water transport, mechanical support and plant resistance to pathogens. In this study, various biological methods were used to study the mechanism of shading on the asparagus lignin accumulation pathway. The physiological results showed that shading significantly reduced stem diameter and cell wall lignin content. Microstructure observation showed that shading reduced the number of vascular bundles and xylem area, resulting in decreased lignin content, and thus reducing the lignification of asparagus. Cinnamic acid, caffeic acid, ferulic acid and sinapyl alcohol are crucial intermediate metabolites in the process of lignin synthesis. Metabolomic profiling showed that shading significantly reduced the contents of cinnamic acid, caffeic acid, ferulic acid and sinapyl alcohol. Transcriptome profiling identified 37 differentially expressed genes related to lignin, including PAL, C4H, 4CL, CAD, CCR, POD, CCoAOMT, and F5H related enzyme activity regulation genes. The expression levels of POD, CCoAOMT, and CCR genes were significantly decreased under shading treatment, while the expression levels of CAD and F5H genes exhibited no significant difference with increased shading. The downregulation of POD, CCoAOMT genes and the decrease in CCR gene expression levels inhibited the activities of the corresponding enzymes under shading treatment, resulting in decreased downstream content of caffeic acid, ferulic acid, sinaperol, chlorogenic acid and coniferin. A significant decrease in upstream cinnamic acid content was observed with shading, which also led to decreased downstream metabolites and reduced asparagus lignin content. In this study, transcriptomic and metabolomic analysis revealed the key regulatory genes and metabolites of asparagus lignin under shading treatment. This study provides a reference for further understanding the mechanism of lignin biosynthesis and the interaction of related genes.

Mammalian infection models have contributed significantly to our understanding of the host-mycobacterial interaction, revealing potential mechanisms and targets for novel antimycobacterial therapeutics. However, the use of conventional mammalian models such as mice, are typically expensive, high maintenance, require specialized animal housing, and are ethically regulated. Furthermore, research using Mycobacterium tuberculosis (MTB), is inherently difficult as work needs to be carried out at biosafety level 3 (BSL3). The insect larvae of Galleria mellonella (greater wax moth), have become increasingly popular as an infection model, and we previously demonstrated its potential as a mycobacterial infection model using Mycobacterium bovis BCG. Here we present a novel BSL2 complaint MTB infection model using G. mellonella in combination with a bioluminescent ΔleuDΔpanCD double auxotrophic mutant of MTB H37Rv (SAMTB lux) which offers safety and practical advantages over working with wild type MTB. Our results show a SAMTB lux dose dependent survival of G. mellonella larvae and demonstrate proliferation and persistence of SAMTB lux bioluminescence over a 1 week infection time course. Histopathological analysis of G. mellonella, highlight the formation of early granuloma-like structures which matured over time. We additionally demonstrate the drug efficacy of first (isoniazid, rifampicin, and ethambutol) and second line (moxifloxacin) antimycobacterial drugs. Our findings demonstrate the broad potential of this insect model to study MTB infection under BSL2 conditions. We anticipate that the successful adaptation and implementation of this model will remove the inherent limitations of MTB research at BSL3 and increase tuberculosis research output.

Actinobacillus pleuropneumoniae (APP) is the causative agent of porcine pleuropneumonia, resulting in high economic impact worldwide. There are currently 19 known serovars of APP, with different ones being predominant in specific geographic regions. Outbreaks of pleuropneumonia, characterized by sudden respiratory difficulties and high mortality, can occur when infected pigs are brought into naïve herds, or by those carrying different serovars. Good biosecurity measures include regular diagnostic testing for surveillance purposes. Current gold standard diagnostic techniques lack sensitivity (bacterial culture), require expensive thermocycling machinery (PCR) and are time consuming (culture and PCR). Here we describe the development of an isothermal point-of-care diagnostic test - utilizing recombinase polymerase amplification (RPA) for the detection of APP, targeting the species-specific apxIVA gene. Our APP-RPA diagnostic test achieved a sensitivity of 10 copies/μL using a strain of APP serovar 8, which is the most prevalent serovar in the UK. Additionally, our APP-RPA assay achieved a clinical sensitivity and specificity of 84.3 and 100%, respectively, across 61 extracted clinical samples obtained from farms located in England and Portugal. Using a small subset (n = 14) of the lung tissue samples, we achieved a clinical sensitivity and specificity of 76.9 and 100%, respectively) using lung imprints made on FTA cards tested directly in the APP-RPA reaction. Our results demonstrate that our APP-RPA assay enables a suitable rapid and sensitive screening tool for this important veterinary pathogen.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a disease of major impact on pig health, welfare, and productivity globally. Serovar 8 (APP) is the predominant clinical serovar in Norway and the United Kingdom (UK), and has been isolated from clinical cases in Denmark. The primary objective of this study was to characterize the genetic variability of isolates of A. pleuropneumoniae APP8 in the Norwegian population. The secondary objectives were to determine the within-host variability of APP8; to compare the APP8 bacterial populations in Norway, Denmark, and the UK, including antimicrobial resistance (AMR) gene profiles and to assess the effect of national differences in antimicrobial drug use and restricted animal movement on the occurrence of resistance. Isolates of APP8 from the UK (n=67), Denmark (n=22), and Norway (n=123) collected between 1983 and 2020 were compared using whole genome sequencing. To investigate genetic variability within individual hosts, an additional 104 APP8 isolates from the lungs of six Norwegian pigs were compared. Very low within-host variation was observed (≤ 2 single nucleotide polymorphisms). The phylogeny of 123 Norwegian APP8 isolates from 76 herds revealed some within-herd genetic variation, but substantial geographical clustering. When inferring the relatedness of the three international APP8 collections, the topology highlighted the existence of two distinct monophyletic branches characterized by the Norwegian and UK isolates, respectively. Three Danish isolates were scattered across the UK branch, whereas the remaining 19 Danish isolates clustered in two monophyletic groups nested in the Norwegian branch. Coalescence analysis, performed to estimate the divergences from a common ancestor, indicated a last common ancestor several centuries ago. The phylogenetic analyses also revealed striking differences in occurrence of AMR genes, as these were 23-times more prevalent among the UK isolates than among the Norwegian isolates. An increased understanding of the effects of population strategies is helpful in surveillance and control of infectious diseases.

Actinobacillus pleuropneumoniae (APP), the causative agent of porcine pleuropneumonia, is highly contagious and responsible for high morbidity, mortality, and economic losses in the swine industry worldwide, but quick serotyping and diagnosis are still not widely available. In this study, we sought to validate the use of Whatman FTA® cards for collection and processing of A. pleuropneumoniae isolates, or porcine lung tissue samples, for direct use in diagnostic multiplex PCRs. We have optimized the processing of 3-mm discs punched from FTA® cards loaded with cultured A. pleuropneumoniae, or imprinted on lesioned regions of lung tissue, with only three distilled water washes before addition into our APP-multiplex PCR (mPCR) assay for rapid, low-cost identification and serotyping. DNA captured on FTA® cards generated the same diagnostic PCR results as DNA extracted using commercial kits for 85 A. pleuropneumoniae clinical isolate cultures and 22 lung samples. Additionally, bacterial DNA bound to FTA® cards was detectable by PCR after 6 months of storage at 37°C. This study provides simple, efficient, rapid, and practical sample processing for detection and molecular serotyping of A. pleuropneumoniae.

Animal models have long been used in tuberculosis research to understand disease pathogenesis and to evaluate novel vaccine candidates and anti-mycobacterial drugs. However, all have limitations and there is no single animal model which mimics all the aspects of mycobacterial pathogenesis seen in humans. Importantly mice, the most commonly used model, do not normally form granulomas, the hallmark of tuberculosis infection. Thus there is an urgent need for the development of new alternative in vivo models. The insect larvae, Galleria mellonella has been increasingly used as a successful, simple, widely available and cost-effective model to study microbial infections. Here we report for the first time that G. mellonella can be used as an infection model for members of the Mycobacterium tuberculosis complex. We demonstrate a dose-response for G. mellonella survival infected with different inocula of bioluminescent Mycobacterium bovis BCG lux, and demonstrate suppression of mycobacterial luminesence over 14 days. Histopathology staining and transmission electron microscopy of infected G. mellonella phagocytic haemocytes show internalization and aggregation of M. bovis BCG lux in granuloma-like structures, and increasing accumulation of lipid bodies within M. bovis BCG lux over time, characteristic of latent tuberculosis infection. Our results demonstrate that G. mellonella can act as a surrogate host to study the pathogenesis of mycobacterial infection and shed light on host-mycobacteria interactions, including latent tuberculosis infection.

The larvae of the insect Galleria mellonella, have recently been established as a non-mammalian infection model for the Mycobacterium tuberculosis complex (MTBC). To gain further insight into the potential of this model, we applied proteomic (label-free quantification) and transcriptomic (gene expression) approaches to characterise the innate immune response of G. mellonella to infection with Mycobacterium bovis BCG lux over a 168 h time course. Proteomic analysis of the haemolymph from infected larvae revealed distinct changes in the proteome at all time points (4, 48, 168 h). Reverse transcriptase quantitative PCR confirmed induction of five genes (gloverin, cecropin, IMPI, hemolin, and Hdd11), which encoded proteins found to be differentially abundant from the proteomic analysis. However, the trend between gene expression and protein abundance were largely inconsistent (20%). Overall, the data are in agreement with previous phenotypic observations such as haemocyte internalization of mycobacterial bacilli (hemolin/β-actin), formation of granuloma-like structures (Hdd11), and melanization (phenoloxidase activating enzyme 3 and serpins). Furthermore, similarities in immune expression in G. mellonella, mouse, zebrafish and in vitro cell-line models of tuberculosis infection were also identified for the mechanism of phagocytosis (β-actin). Cecropins (antimicrobial peptides), which share the same α-helical motif as a highly potent peptide expressed in humans (h-CAP-18), were induced in G. mellonella in response to infection, giving insight into a potential starting point for novel antimycobacterial agents. We believe that these novel insights into the innate immune response further contribute to the validation of this cost-effective and ethically acceptable insect model to study members of the MTBC.

Drug screening models have a vital role in the development of novel antimycobacterial agents which are urgently needed to tackle drug-resistant tuberculosis (TB). We recently established the larvae of the insect Galleria mellonella (greater wax moth) as a novel infection model for the Mycobacterium tuberculosis complex. Here we demonstrate its use as a rapid and reproducible screen to evaluate antimycobacterial drug efficacy using larvae infected with bioluminescent Mycobacterium bovis BCG lux. Treatment improved larval survival outcome and, with the exception of pyrazinamide, was associated with a significant reduction in in vivo mycobacterial bioluminescence over a 96 h period compared to the untreated controls. Isoniazid and rifampicin displayed the greatest in vivo efficacy and survival outcome. Thus G. mellonella, infected with bioluminescent mycobacteria, can rapidly determine in vivo drug efficacy, and has the potential to significantly reduce and/or replace the number of animals used in TB research.

The electro-hydraulic servo pump control system (EHSPCS) is a volume control system that uses a permanent magnet synchronous motor (PMSM) with a fixed displacement pump to directly drive and control the hydraulic cylinder. The energy transmission law of the system is very complicated due to the transformation of electrical, mechanical and hydraulic energy as well as other energy fields, and qualitative analysis of the energy transfer efficiency is difficult. Energy transfer analysis of the EHSPCS under different working conditions and loads is proposed in this paper. First, the energy flow transfer mechanism was analyzed, and the mathematical and energy transfer models of the key components of the system were established to explore the energy characteristic state transition rule. Second, a power bond diagram model was built, its state equation and state matrix were deduced, and a system simulation model was built. Finally, combined with the EHSPCS experimental platform, simulation experiments were carried out on the dynamic position following and steady-state position holding conditions of the system, and the variation rules of the power of each energy characteristic state and the system energy transfer efficiency under different loads were obtained. The research results provide a foundation for the study of power matching and energy-saving mechanism of the EHSPCS.

Evidence of plasmids carrying the tetracycline resistance gene, tet(B), was found in the previously reported whole genome sequences of 14 United Kingdom, and 4 Brazilian, isolates of Actinobacillus pleuropneumoniae. Isolation and sequencing of selected plasmids, combined with comparative sequence analysis, indicated that the four Brazilian isolates all harbor plasmids that are nearly identical to pB1001, a plasmid previously found in Pasteurella multocida isolates from Spain. Of the United Kingdom isolates, 13/14 harbor plasmids that are (almost) identical to pTetHS016 from Haemophilus parasuis. The remaining United Kingdom isolate, MIDG3362, harbors a 12666 bp plasmid that shares extensive regions of similarity with pOV from P. multocida (which carries blaROB-1, sul2, and strAB genes), as well as with pTetHS016. The newly identified multi-resistance plasmid, pM3362MDR, appears to have arisen through illegitimate recombination of pTetHS016 into the stop codon of the truncated strB gene in a pOV-like plasmid. All of the tet(B)-carrying plasmids studied were capable of replicating in Escherichia coli, and predicted origins of replication were identified. A putative origin of transfer (oriT) sequence with similar secondary structure and a nic-site almost identical to that of RP4 was also identified in these plasmids, however, attempts to mobilize them from an RP4-encoding E. coli donor strain were not successful, indicating that specific conjugation machinery may be required.

OBJECTIVE:The prognostic significance of CD147 expression in esophageal cancer patients remains controversial. Using a meta-analysis, we investigated the prognostic and clinicopathologic characteristics of CD147 in esophageal cancer. METHODS:A comprehensive literature search of the PubMed (1966-2016), EMBASE (1980-2016), Cochrane Library (1996-2016), Web of Science (1945-2016), China National Knowledge Infrastructure (1982-2016), and Wanfang databases (1988-2016) was performed to identify studies of all esophageal cancer subtypes. Correlations between CD147 expression and survival outcomes and clinicopathological features were analyzed using meta-analysis methods. RESULTS:Seventeen studies were included. High CD147 expression reduced the 3-year survival rate (OR = 3.26, 95% CI = (1.53, 6.93), p = 0.02) and 5-year survival rate(OR = 4.35, 95% CI = (2.13, 8.90), p < 0.0001). High CD147 expression reduced overall survival in esophageal cancer (HR = 1.60, 95% CI = (1.19, 2.15), p = 0.02). Additionally, higher CD147 expression was detected in esophageal cancer tissues than noncancerous tissues (OR = 9.45, 95% CI = (5.39, 16.59), p < 0.00001), normal tissues (OR = 12.73, 95% CI = (3.49, 46.46), p = 0.0001), para-carcinoma tissues (OR = 12.80, 95% CI = (6.57, 24.92), p < 0.00001), and hyperplastic tissues (OR = 3.27, 95% CI = (1.47, 7.29), p = 0.004). CD147 expression was associated with TNM stage (OR = 3.66, 95% CI = (2.20, 6.09), p < 0.00001), tumor depth (OR = 7.97, 95% CI = (4.13, 15.38), p < 0.00001), and lymph node status (OR = 5.14, 95% CI = (2.03,13.01), p = 0.0005), but not with tumor differentiation, age, or sex. CONCLUSION:Our meta-analysis suggests that CD147 is an efficient prognostic factor in esophageal cancer. High CD147 expression in patients with esophageal cancer was associated with worse survival outcomes and common clinicopathological indicators of poor prognosis.

Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N-linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO, ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering.

ICEApl1 was identified in the whole genome sequence of MIDG2331, a tetracycline-resistant (MIC = 8 mg/L) serovar 8 clinical isolate of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. PCR amplification of virB4, one of the core genes involved in conjugation, was used to identify other A. pleuropneumoniae isolates potentially carrying ICEApl1. MICs for tetracycline were determined for virB4 positive isolates, and shotgun whole genome sequence analysis was used to confirm presence of the complete ICEApl1. The sequence of ICEApl1 is 56083 bp long and contains 67 genes including a Tn10 element encoding tetracycline resistance. Comparative sequence analysis was performed with similar integrative conjugative elements (ICEs) found in other members of the Pasteurellaceae. ICEApl1 is most similar to the 59393 bp ICEHin1056, from Haemophilus influenzae strain 1056. Although initially identified only in serovar 8 isolates of A. pleuropneumoniae (31 from the UK and 1 from Cyprus), conjugal transfer of ICEApl1 to representative isolates of other serovars was confirmed. All isolates carrying ICEApl1 had a MIC for tetracycline of 8 mg/L. This is, to our knowledge, the first description of an ICE in A. pleuropneumoniae, and the first report of a member of the ICEHin1056 subfamily in a non-human pathogen. ICEApl1 confers resistance to tetracycline, currently one of the more commonly used antibiotics for treatment and control of porcine pleuropneumonia.

We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains.