The Fe-S cluster biosynthesis in Enterococcus faecium is essential for anaerobic growth and gastrointestinal colonization.

The facultative anaerobic Gram-positive bacterium Enterococcus faecium is a ubiquitous member of the human gut microbiota. However, it has gradually evolved into a pathogenic and multidrug resistant lineage that causes nosocomial infections. The establishment of high-level intestinal colonization by enterococci represents a critical step of infection. The majority of current research on Enterococcus has been conducted under aerobic conditions, while limited attention has been given to its physiological characteristics in anaerobic environments, which reflects its natural colonization niche in the gut. In this study, a high-density transposon mutant library containing 26,620 distinct insertion sites was constructed. Tn-seq analysis identified six genes that significantly contribute to growth under anaerobic conditions. Under anaerobic conditions, deletion of sufB (encoding Fe-S cluster assembly protein B) results in more extensive and significant impairments on carbohydrate metabolism compared to aerobic conditions. Consistently, the pathways involved in this utilization-restricted carbohydrates were mostly expressed at significantly lower levels in mutant compared to wild-type under anaerobic conditions. Moreover, deletion of sufB or pflA (encoding pyruvate formate lyase-activating protein A) led to failure of gastrointestinal colonization in mice. These findings contribute to our understanding of the mechanisms by which E. faecium maintains proliferation under anaerobic conditions and establishes colonization in the gut.

Forest fire susceptibility assessment under small sample scenario: A semi-supervised learning approach using transductive support vector machine.

Forest fires threaten global ecosystems, socio-economic structures, and public safety. Accurately assessing forest fire susceptibility is critical for effective environmental management. Supervised learning methods dominate this assessment, relying on a substantial dataset of forest fire occurrences for model training. However, obtaining precise forest fire location data remains challenging. To address this issue, semi-supervised learning emerges as a viable solution, leveraging both a limited set of collected samples and unlabeled data containing environmental factors for training. Our study employed the transductive support vector machine (TSVM), a key semi-supervised learning method, to assess forest fire susceptibility in scenarios with limited samples. We conducted a comparative analysis, evaluating its performance against widely used supervised learning methods. The assessment area for forest fire susceptibility lies in Dayu County, Jiangxi Province, China, renowned for its vast forest cover and frequent fire incidents. We analyzed and generated maps depicting forest fire susceptibility, evaluating prediction accuracies for both supervised and semi-supervised learning methods across various small sample scenarios (e.g., 4, 8, 12, 16, 20, 24, 28, and 32 samples). Our findings indicate that TSVM exhibits superior prediction accuracy compared to supervised learning with limited samples, yielding more plausible forest fire susceptibility maps. For instance, at sample sizes of 4, 16, and 28, TSVM achieves prediction accuracies of approximately 0.8037, 0.9257, and 0.9583, respectively. In contrast, random forests, the top performers in supervised learning, demonstrate accuracies of approximately 0.7424, 0.8916, and 0.9431, respectively, for the same small sample sizes. Additionally, we discussed three key aspects: TSVM parameter configuration, the impact of unlabeled sample size, and performance within typical sample sizes. Our findings support semi-supervised learning as a promising approach compared to supervised learning for forest fire susceptibility assessment and mapping, particularly in scenarios with small sample sizes.

Embedding of exogenous B cell epitopes on the surface of UreB structure generates a broadly reactive antibody response against Helicobacter pylori.

Since Helicobacter pylori (H. pylori) resistance to antibiotic regimens has increased, vaccination is becoming an increasingly important alternative therapy to control H. pylori infection. UreB, FlaA, AlpB, SabA, and HpaA proteins of H. pylori were previously proved to be used as candidate vaccine antigens. Here, we developed an engineered antigen based on a recombinant chimeric protein containing a structural scaffold from UreB and B cell epitopes from FlaA, AlpB, SabA, and HpaA. The multi-epitope chimeric antigen, named MECU, could generate a broadly reactive antibody response including antigen-specific antibodies and neutralising antibodies against H. pylori urease and adhesins. Moreover, therapeutic immunisation with MECU could reduce H. pylori colonisation in the stomach and protect the stomach in BALB/c mice. This study not only provides promising immunotherapy to control H. pylori infection but also offers a reference for antigen engineering against other pathogens.

A tug-of-war to control plant emission of an airborne alarm signal.

Aphids represent a major threat to crops. Hundreds of different viruses are aphid-borne. Upon aphid attack, plants release volatile organic compounds (VOCs) as airborne alarm signals to turn on the airborne defense (AD) of neighboring plants, thereby repelling aphids as well as reducing aphid fitness and virus transmission. This phenomenon provides a critical community-wide plant protection to fend off aphids, but the underlying molecular basis remains undetermined for a long time. In a recent article, Gong et al. established the NAC2-SAMT1 module as the core component regulating the emission of methyl-salicylate (MeSA), a major component of VOCs in aphid-attacked plants. Furthermore, they showed that SABP2 protein is critical for the perception of volatile MeSA signal by converting MeSA to Salicylic Acid (SA), which is the cue to elicit AD against aphids at the community level. Moreover, they showed that multiple viruses use a conserved glycine residue in the ATP-dependent helicase domain in viral proteins to shuttle NAC2 from the nucleus to the cytoplasm for degradation, leading to the attenuation of MeSA emission and AD. These findings illuminate the functional roles of key regulators in the complex MeSA-mediated airborne defense process and a counter-defense mechanism used by viruses, which has profound significance in advancing the knowledge of plant-pathogen interactions as well as providing potential targets for gene editing-based crop breeding.

Cellular roadmaps of viroid infection.

Viroids are single-stranded circular noncoding RNAs that infect plants. According to the International Committee on Taxonomy of Viruses, there are 44 viroids known to date. Notably, more than 20 000 distinct viroid-like RNA sequences have recently been identified in existing sequencing datasets, suggesting an unprecedented complexity in biological roles of viroids and viroid-like RNAs. Interestingly, a human pathogen, hepatitis delta virus (HDV), also replicates via a rolling circle mechanism like viroids. Therefore, knowledge of viroid infection is informative for research on HDV and other viroid-like RNAs reported from various organisms. Here, we summarize recent advancements in understanding viroid shuttling among subcellular compartments for completing replication cycles, emphasizing regulatory roles of RNA motifs and structural dynamics in diverse biological processes. We also compare the knowledge of viroid intracellular trafficking with known pathways governing cellular RNA movement in cells. Future investigations on regulatory RNA structures and cognate factors in regulating viroid subcellular trafficking and replication will likely provide new insights into RNA structure-function relationships and facilitate the development of strategies controlling RNA localization and function in cells.

A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription.

Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.

A nuclear import pathway exploited by pathogenic noncoding RNAs.

The prevailing view of intracellular RNA trafficking in eukaryotic cells is that RNAs transcribed in the nucleus either stay in the nucleus or cross the nuclear envelope, entering the cytoplasm for function. However, emerging evidence illustrates that numerous functional RNAs move in the reverse direction, from the cytoplasm to the nucleus. The mechanism underlying RNA nuclear import has not been well elucidated. Viroids are single-stranded circular noncoding RNAs that infect plants. Using Nicotiana benthamiana, tomato (Solanum lycopersicum), and nuclear-replicating viroids as a model, we showed that cellular IMPORTIN ALPHA-4 (IMPa-4) is likely involved in viroid RNA nuclear import, empirically supporting the involvement of Importin-based cellular pathway in RNA nuclear import. We also confirmed the involvement of a cellular protein (viroid RNA-binding protein 1 [VIRP1]) that binds both IMPa-4 and viroids. Moreover, a conserved C-loop in nuclear-replicating viroids serves as a key signal for nuclear import. Disrupting C-loop impairs VIRP1 binding, viroid nuclear accumulation, and infectivity. Further, C-loop exists in a subviral satellite noncoding RNA that relies on VIRP1 for nuclear import. These results advance our understanding of subviral RNA infection and the regulation of RNA nuclear import.

Studies on Viroid Shed Light on the Role of RNA Three-Dimensional Structural Motifs in RNA Trafficking in Plants.

RNAs play essential roles in various biological processes. Mounting evidence has demonstrated that RNA subcellular localization and intercellular/systemic trafficking govern their functions in coordinating plant growth at the organismal level. While numerous types of RNAs (i.e., mRNAs, small RNAs, rRNAs, tRNAs, and long noncoding RNAs) have been found to traffic in a non-cell-autonomous fashion within plants, the underlying regulatory mechanism remains unclear. Viroids are single-stranded circular noncoding RNAs, which entirely rely on their RNA motifs to exploit cellular machinery for organelle entry and exit, cell-to-cell movement through plasmodesmata, and systemic trafficking. Viroids represent an excellent model to dissect the role of RNA three-dimensional (3D) structural motifs in regulating RNA movement. Nearly two decades of studies have found multiple RNA 3D motifs responsible for viroid nuclear import as well as trafficking across diverse cellular boundaries in plants. These RNA 3D motifs function as "keys" to unlock cellular and subcellular barriers and guide RNA movement within a cell or between cells. Here, we summarize the key findings along this line of research with implications for future studies on RNA trafficking in plants.

Emerging value of the viroid model in molecular biology and beyond.

Viroids are single-stranded circular noncoding RNAs that infect plants. Research in the past five decades has deciphered the viroid genome structures, viroid replication cycles, numerous host factors for viroid infection, viroid motifs for intracellular and intercellular trafficking, interactions with host defense machinery, etc. In this review, we mainly focus on some significant questions that remain to be tackled, centered around (1) how the RNA polymerase II machinery performs transcription on RNA templates of nuclear-replicating viroids, (2) how viroid RNAs coordinate multiple structural elements for diverse functions, and (3) how viroid RNAs activate plant immunity. Research on viroids has led to seminal discoveries in biology, and we expect the research directions outlined in this review to continue providing key knowledge inspiring other areas of biology.

Hot Deformation Behavior, Processing Maps and Microstructural Evolution of the Mg-2.5Nd-0.5Zn-0.5Zr Alloy.

Isothermal hot compression experiments were conducted on Mg-2.5Nd-0.5Zn-0.5Zr alloy to investigate hot deformation behavior at the temperature range of 573-773 K and the strain rate range of 0.001 s-1-10 s-1 using a Gleeble-3500D thermomechanical simulator. The results showed that the rheological curve showed a typical work hardening stage, and there were three different stages: work hardening, transition and steady state. A strain compensation constitutive model was established to predict the flow stress of the Mg-2.5Nd-0.5Zn-0.5Zr alloy, and the results proved that it had high predictability. The main deformation mechanism of the Mg-2.5Nd-0.5Zn-0.5Zr alloy was dislocation climbing. The processing maps were established to distinguish the unstable region from the working region. The maps showed that the instability generally occurred at high strain rates and low temperatures, and the common forms of instability were cracking and flow localization. The optimum machining range of the alloy was determined to be 592-773 K and 0.001-0.217 s-1. With the increase in deformation temperature, the grain size of the alloy grew slowly at the 573-673 K temperature range and rapidly at the 673-773 K temperature range.

Facile nanostructured zinc oxide coating technique for antibacterial and antifouling air filters with low pressure drop.

Air filters effectively filtrate external contaminants including pathogenic bioaerosols; however, they also act as culture sites for the pathogenic bacteria captured in nutrient organic pollutants. Although many researchers have applied various antibacterial coatings to filters, the coating application inevitably increased the pressure drop, leading to the low efficiency and high energy consumption of the purification system. Herein, we report a simple nanostructured zinc oxide (ZnO) coating technique to confer a polypropylene nonwoven filter with superior antibacterial, antifouling and anti-biofilm properties without an additional pressure drop. For aerodynamic coating designs, filters were directly immersed into low concentration precursor solutions to enable the sedimentation of the ZnO sol-gel particles on the filter fibers according to fluid dynamic. The precursor concentration affected the surface topology and so properties of the as-fabricated coating. 0.07 M precursor solution produced a rose-like nanostructured coating exhibiting no pressure-drop increase. The large specific surface area and hydrophobic surface killed and then repelled the attached bacteria effectively. As a result, the bare filter promoted the growth and consequent biofilm formation of the surface bacteria in a favorable environment for the growth of microorganisms, while the coated filter successfully suppressed biofilm development.

Analysis on RNA Motif-Based RNA Trafficking in Plants.

Systemic RNA trafficking widely exists in plants and is critical for integrating the healthy development and responses to environmental cues. Viroids, single-stranded circular noncoding RNAs that infect plants, have been used as a model to delineate the mechanism underlying systemic RNA trafficking. Recent work on viroids has shown that structural motifs are critical to direct RNA trafficking through distinct cellular boundaries. Here, we describe the methods for generating mutational variants using site-directed mutagenesis and infection assays to unravel the function of RNA motifs. This approach can be modified to study other RNA motif-based biological processes.

Molten salt synthesis of KCl-preintercalated C3N4 nanosheets with abundant pyridinic-N as a superior anode with 10 K cycles in lithium ion battery.

The graphitic carbon nitride is considered as the promising anode of lithium ion battery due to its high theoretical capacity (>1000 mAh g-1) and easy synthesis method. But the electrochemical inactivity and the structural collapse during cycles lead to its poor electrochemical performance in practice. Here, an interesting molten salt method is used to obtain the KCl-preintercalated carbon nitride nanosheets with abundant N vacancies and pyridinic-N. The KCl as a prop enhances the interlayer distance and the structural stability. And the N vacancy and the pyridinic-N increase the conductivity, the active sites and the reversibility of Li+ storage. Thus, the optimized electrode shows a higher specific discharge capacity (389 mAh g-1 at 0.1 A g-1) and a longer cyclic life (66% capacity retention after 10 K cycles at 3.0 A g-1) compared to those of bulk g-C3N4.

Turning commercial MnO2 (≥85 wt%) into high-crystallized K+-doped LiMn2O4 cathode with superior structural stability by a low-temperature molten salt method.

The obtainment of low-cost, easily prepared and high-powered LiMn2O4 is the key to achieve its wide application in various electronic devices. In this work, a mild and easily scaled molten salt method (KCl@LiCl) is utilized to convert commercial MnO2 to the high-performance LiMn2O4. At the same reaction temperature, the molten salt method leads to the formation of K+-doped LiMn2O4 with higher crystallinity compared to the conventional solid state method, which contributes to the improved inner charge transfer. The Li3PO4 protective layer is coated on the surface of K+-doped LiMn2O4 to elevate the interfacial stability and the Li+ transfer on interface. Thus, the optimized electrode shows a higher specific discharge capacity (103/60 mAh g-1 at 0.02/2 A g-1) and a longer cyclic life (80 mAh g-1 after 500 cycles at 0.5 A g-1) compared to those of LiMn2O4 by solid state method (49/2 mAh g-1 at 0.02/2 A g-1 and 20 mAh g-1 after 500 cycles at 0.5 A g-1).

Evaluation of an attenuated Listeria monocytogenes as a vaccine vector to control Helicobacter pylori infection.

The increasing resistance of Helicobacter pylori (H. pylori) to antibiotics has limited the efficacy of antibiotic therapy in the treatment of H. pylori-associated gastric diseases. The vaccine as an alternative method is becoming a safe and effective way to address this problem. In previous studies, live vector vaccines have proved to be effective in controlling H. pylori infection. Attenuated Listeria monocytogenes (L. monocytogenes) is a potential candidate vector applied in clinical trials, which can deliver foreign antigens and induce a broad immune response. To further explore the effectiveness of L. monocytogenes as a vaccine vector against H. pylori, attenuated L. monocytogenes-based vaccine EGDeΔactA/inlB(EGDeAB)-MECU was constructed to secrete a multi-epitope chimeric antigen (MECU) containing multiple B cell epitopes from H. pylori antigens. EGDeAB-MECU could secrete MECU stably. After immunized by gavage and intravenous injection, both EGDeAB and EGDeAB-MECU could significantly decrease gastric H. pylori colonization and induce a high level of specific antibodies against H. pylori. In conclusion, attenuated L. monocytogenes had an immunotherapeutic effect on H. pylori-infected mice, indicating its further development as a promising candidate vaccine vector for the H. pylori vaccine.

Systematic identification of a panel of strong promoter regions from Listeria monocytogenes for fine-tuning gene expression.

BACKGROUND: Attenuated Listeria monocytogenes (Lm) has been widely used as a vaccine vector in the prevention and treatment of pathogen infection and tumor diseases. In addition, previous studies have proved that the attenuated Lm can protect zebrafish from Vibrio infections, indicating that the attenuated Lm has a good application prospect in the field of aquatic vaccines. However, the limitation mainly lies in the lack of a set of well-characterized natural promoters for the expression of target antigens in attenuated Lm. RESULTS: In our study, candidate strong promoters were identified through RNA-seq analysis, and characterized in Lm through enhanced green fluorescent protein (EGFP). Nine native promoters that showed stronger activities than that of the known strong promoter P36 under two tested temperatures (28 and 37 °C) were selected from the set, and P29 with the highest activity was 24-fold greater than P36. Furthermore, we demonstrated that P29 could initiate EGFP expression in ZF4 cells and zebrafish embryos. CONCLUSIONS: This well-characterized promoter library can be used to fine-tune the expression of different proteins in Lm. The availability of a well-characterized promoter toolbox of Lm is essential for the analysis of yield increase for biotechnology applications.

[Research progress in the rapid preparation of monoclonal antibodies of mouse hybridoma].

Mouse hybridoma monoclonal antibody is the most commonly used antibody in immunology because of its stable source, easy preparation in later stage and high yield. The traditional time-consuming and laborious hybridoma preparation technology could not meet the growing market demand. In this paper, we describe the rapid preparation techniques involved in antigen design and screening, B cell enrichment and screening, transgenic myeloma cells, fusion technology improvement, positive hybridoma cell screening and rapid detection of monoclonal antibody performance, to provide a reference for rapid preparation of mouse hybridoma monoclonal antibody.

Identification and evaluation of a panel of strong constitutive promoters in Listeria monocytogenes for improving the expression of foreign antigens.

Attenuated Listeria monocytogenes could be a potential vaccine vector for the immunotherapy of tumors or pathogens. However, the lack of reliable promoters has limited its ability to express foreign antigens. In the present study, 21 promoters were identified from Listeria monocytogenes through RNA-seq analysis under two pH conditions of pH 7.4 and pH 5.5. Based on the constructed fluorescence report system, 7 constitutive promoters exhibited higher strength than Phelp (1.8-fold to 5.4-fold), a previously reported strong promoter. Furthermore, the selected 5 constitutive promoters exhibited higher UreB production activity than Phelp (1.1-fold to 8.3-fold). Notably, a well-characterized constitutive promoter P18 was found with the highest activity of fluorescence intensity and UreB production. In summary, the study provides a panel of strong constitutive promoters for Listeria monocytogenes and offers a theoretical basis for mining constitutive promoters in other organisms. KEY POINTS: • Twenty-one promoters were identified from L. monocytogenes through RNA-seq. • Fluorescent tracer of L. monocytogenes (P18) was performed in vitro and in vivo. • A well-characterized constitutive promoter P18 could improve the expression level of a foreign antigen UreB in L. monocytogenes.

Enhanced Polymerization and Surface Hardness of Colloidal Siloxane Films via Electron Beam Irradiation.

Electron beam (EB) curing is a foldable hard coating process and has attracted significant research attention in the field of flexible electronic devices. In this study, we report a method for enhancing material surface hardness with low-energy EB curing in a short time. The low-energy EB improved the coating hardness of films by inducing cross-linking polymerization of the silicon-containing monomer. The hardness of the cured coating layer was measured as 3 H using a pencil hardness tester, and the transparency of the coating was higher than 90%. Owing to a series of cross-linking reactions between Si-O-C and Si-OH groups under EB curing and the formation of Si-Si bonds, the cured layer exhibited remarkable durability in the 100000-flexible cycle test. Additionally, the natural oxidation of the C-O groups on the surface of the coating formed carboxyl groups that improved the hydrophilic properties of the coating layer. To the best of our knowledge, this is the first study to propose that the hardness of polyethylene terephthalate films can be improved using low-energy EBs to rapidly cure silicon-containing coatings. Our results provide a novel and commercially viable approach for improving the hardness of touch screens and foldable displays.

A Novel Design of Multi-epitope Vaccine Against Helicobacter pylori by Immunoinformatics Approach.

Helicobacter pylori (H. pylori) is a gram-negative spiral bacterium that caused infections in half of the world's population and had been identified as type I carcinogen by the World Health Organization. Compared with antibiotic treatment which could result in drug resistance, the vaccine therapy is becoming a promising immunotherapy option against H. pylori. Further, the multi-epitope vaccine could provoke a wider immune protection to control H. pylori infection. In this study, the in-silico immunogenicity calculations on 381 protein sequences of H. pylori were performed, and the immunogenicity of selected proteins with top-ranked score were tested. The B cell epitopes and T cell epitopes from three well performed proteins UreB, PLA1, and Omp6 were assembled into six constructs of multi-epitope vaccines with random orders. In order to select the optimal constructs, the stability of the vaccine structure and the exposure of B cell epitopes on the vaccine surface were evaluated based on structure prediction and solvent accessible surface area analysis. Finally Construct S1 was selected and molecular docking showed that it had the potential of binding TLR2, TLR4, and TLR9 to stimulate strong immune response. In particular, this study provides good suggestions for epitope assembly in the construction of multi-epitope vaccines and it may be helpful to control H. pylori infection in the future. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s10989-020-10148-x) contains supplementary material, which is available to authorized users.