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Soybean (Glycine max L.) is a major global crop. Genetic modification of this crop can contribute to enhance yields, reduce pesticide use, and facilitate sustainability, but there is a need for locally adapted genetically modified varieties. Glyphosate tolerant soybean developed under an agbiogeneric model is an attractive prospect as the technology is now mostly in the public domain. This research evaluates the potential for transformation of three soybean varieties (BR1, BR2, and FNS) and an established variety (SK7). The study investigates the interaction between these genotypes and two A. tumefaciens strains, establishes a selection protocol using glyphosate, compares two explant types and evaluates a bacterial removal protocol. Our findings highlight the importance of plant genotype and bacterial strain specificity in the transformation process, with variety BR1 consistently showing the best performance. While challenges remain, this study advances in the development of glyphosate-tolerant soybean varieties as agbiogenerics. [ABSTRACT FROM AUTHOR]

The product of ozonolysis, glycero-(9,10-trioxolane)-trioleate (ozonide of oleic acid triglyceride, [OTOA]), was incorporated into polylactic acid/polycaprolactone (PLA/PCL) blend films in the amount of 1, 5, 10, 20, 30 and 40% w/w. The morphological, mechanical, thermal and antibacterial properties of the biodegradable PLA/PCL films after the OTOA addition were studied. According to DSC and XRD data, the degree of crystallinity of the PLA/PCL + OTOA films showed a general decreasing trend with an increase in OTOA content. Thus, a significant decrease from 34.0% for the reference PLA/PCL film to 15.7% for the PLA/PCL + 40% OTOA film was established using DSC. Observed results could be explained by the plasticizing effect of OTOA. On the other hand, the PLA/PCL film with 20% OTOA does not follow this trend, showing an increase in crystallinity both via DSC (20.3%) and XRD (34.6%). OTOA molecules, acting as a plasticizer, reduce the entropic barrier for nuclei formation, leading to large number of PLA spherulites in the plasticized PLA/PCL matrix. In addition, OTOA molecules could decrease the local melt viscosity at the vicinity of the growing lamellae, leading to faster crystal growth. Morphological analysis showed that the structure of the films with an OTOA concentration above 20% drastically changed. Specifically, an interface between the PLA/PCL matrix and OTOA was formed, thereby forming a capsule with the embedded antibacterial agent. The moisture permeability of the resulting PLA/PCL + OTOA films decreased due to the formation of uniformly distributed hydrophobic amorphous zones that prevented water penetration. This architecture affects the tensile characteristics of the films: strength decreases to 5.6 MPa, elastic modulus E by 40%. The behavior of film elasticity is associated with the redistribution of amorphous regions in the matrix. Additionally, PLA/PCL + OTOA films with 20, 30 and 40% of OTOA showed good antibacterial properties on Pseudomonas aeruginosa, Raoultella terrigena (Klebsiella terrigena) and Agrobacterium tumefaciens, making the developed films potentially promising materials for wound-dressing applications. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens is a plant pathogen, broadly known as the causal agent of the crown gall disease. The soil bacterium is naturally resistant to beta‐lactam antibiotics by utilizing the inducible beta‐lactamase AmpC. Our picture on the condition‐dependent regulation of ampC expression is incomplete. A known regulator is AmpR controlling the transcription of ampC in response to unrecycled muropeptides as a signal for cell wall stress. In our study, we uncovered the global transcriptional regulator LsrB as a critical player acting upstream of AmpR. Deletion of lsrB led to severe ampicillin and penicillin sensitivity, which could be restored to wild‐type levels by lsrB complementation. By transcriptome profiling via RNA‐Seq and qRT‐PCR and by electrophoretic mobility shift assays, we show that ampD coding for an anhydroamidase involved in peptidoglycan recycling is under direct negative control by LsrB. Controlling AmpD levels by the LysR‐type regulator in turn impacts the cytoplasmic concentration of cell wall degradation products and thereby the AmpR‐mediated regulation of ampC. Our results substantially expand the existing model of inducible beta‐lactam resistance in A. tumefaciens by establishing LsrB as higher‐level transcriptional regulator. [ABSTRACT FROM AUTHOR]

Discovery of new strains of bacteria that inhibit pathogen growth can facilitate improvements in biocontrol and probiotic strategies. Traditional, plate-based co-culture approaches that probe microbial interactions can impede this discovery as these methods are inherently low-throughput, labor-intensive, and qualitative. We report a second-generation, photo-addressable microwell device, developed to iteratively screen interactions between candidate biocontrol agents existing in bacterial strain libraries and pathogens under increasing pathogen pressure. Microwells (0.6 pl volume) provide unique co-culture sites between library strains and pathogens at controlled cellular ratios. During sequential screening iterations, library strains are challenged against increasing numbers of pathogens to quantitatively identify microwells containing strains inhibiting the highest numbers of pathogens. Ring-patterned 365 nm light is then used to ablate a photodegradable hydrogel membrane and sequentially release inhibitory strains from the device for recovery. Pathogen inhibition with each recovered strain is validated, followed by whole genome sequencing. To demonstrate the rapid nature of this approach, the device was used to screen a 293-membered biovar 1 agrobacterial strain library for strains inhibitory to the plant pathogen Agrobacterium tumefaciens sp. 15955. One iterative screen revealed nine new inhibitory strains. For comparison, plate-based methods did not uncover any inhibitory strains from the library (n = 30 plates). The novel pathogen-challenge screening mode developed here enables rapid selection and recovery of strains that effectively suppress pathogen growth from bacterial strain libraries, expanding this microwell technology platform toward rapid, cost-effective, and scalable screening for probiotics, biocontrol agents, and inhibitory molecules that can protect against known or emerging pathogens. [ABSTRACT FROM AUTHOR]

Ridge gourd is cultivated in tropical, subtropical and few varieties like Pusa Nasdar and Satputia temperate regions across the world. It is a rich source of essential vitamins and minerals around the world. However, the production of ridge gourd has been severely affected by diseases among which viral diseases have major impact on its production. The viral disease caused by tomato leaf curl New Delhi virus (ToLCNDV), a member of the genus Begomovirus, family Geminiviridae-an economically important virus affecting the ridge gourd yield. It is transmitted by the whiteflies [Bemisia tabaci (Gennadius)] and causes ridge gourd yellow mosaic disease (RgYMD) in ridge gourds. The present study mainly aimed at the process of development of Agrobacterium coat protein gene construct and in silico analysis of CP. The total DNA from infected leaves of ridge gourd with ToLCNDV was amplified using CP specific primers with amplicon size of 771bp. The purified CP. gene was successfully cloned to the TA cloning vector. The CP gene was further moved into plant expression binary vector pBI121. The gene construct of the plant expression vector was transformed into Agrobacterium tumefaciens. In-silico analysis of the coat protein revealed that the protein contains nuclear export factors BR1 family. BLAST results analysis of coat protein with respect to the other coat proteins of ToLCNDV depicted 80-90 per cent resemblance. Phylogenetic tree revealed the common origin of this protein. [ABSTRACT FROM AUTHOR]

Akkermansia muciniphila is a gram-negative anaerobic bacterium, which represents a part of the commensal human microbiota. Decline in the abundance of A. muciniphila among other microbial species in the gut correlates with severe systemic diseases such as diabetes, obesity, intestinal inflammation and colorectal cancer. Due to its mucin-reducing and immunomodulatory properties, the use of probiotics containing Akkermansia sp. appears as a promising approach to the treatment of metabolic and inflammatory diseases. In particular, a number of studies have focused on the role of A. muciniphila in colorectal cancer. Of note, the results of these studies in mice are contradictory: some reported a protective role of A. muciniphila in colorectal cancer, while others demonstrated that administration of A. muciniphila could aggravate the course of the disease resulting in increased tumor burden. More recent studies suggested the immunomodulatory effect of certain unique surface antigens of A. muciniphila on the intestinal immune system. In this Perspective, we attempt to explain how A. muciniphila contributes to protection against colorectal cancer in some models, while being pathogenic in others. We argue that differences in the experimental protocols of administration of A. muciniphila, as well as viability of bacteria, may significantly affect the results. In addition, we hypothesize that antigens presented by pasteurized bacteria or live A. muciniphila may exert distinct effects on the barrier functions of the gut. Finally, A. muciniphila may reduce the mucin barrier and exerts combined effects with other bacterial species in either promoting or inhibiting cancer development. [ABSTRACT FROM AUTHOR]

Nisin, derived from Lactococcus lactis, is a well-known natural food preservative. In the present study, the gene of nisin was transformed to carrot by Agrobacterium tumefaciens strain LBA4404 harboring the recombinant binary vector pBI121 containing neomycin phosphotransferase II (nptII) gene, peptide signal KDEL, and Kozak sequence. The integration of nisin and nptII transgenes into the plant genome was confirmed by polymerase chain reaction (PCR) and dot blot analysis. The gene expression was also performed by RT-PCR and Enzyme-Linked Immunosorbent Assay. The level of nisin expressed in one gram of transgenic plant ranged from 0.05 to 0.08 μg/ml. The stability of nisin varied in orange and peach juices depending on the temperature on the 70th day. The leaf protein extracted from the transgenic plant showed a significant preservative effect of nisin in peach and orange juice. A complete inhibition activity against Staphylococcus aureus and Escherichia coli in orange juice was observed within 24 h. After 24 h, log 1 and log 2 were obtained in a peach juice containing Staphylococcus aureus and Escherichia coli, respectively. Results of HPLC indicated that Chlorogenic and Chicoric acid compounds were increased in transgenic plants, but this increase was not significant. The study of determining the genetic stability of transgenic plants in comparison with non-transgenic plants showed high genetic stability between non-transgenic plants and transgenic plants. This study confirmed the significant inhibitory effect of nisin protein on gram-positive and gram-negative bacteria. [ABSTRACT FROM AUTHOR]

BACKGROUND: The production of oral vaccines by transgenic plants has great advantages and broad application prospects. At present, there are few reports on the study of the tomato transient expression system. It is necessary to optimize tomato transient expression conditions, which can greatly improve the genetic transformation efficiency of plants. OBJECTIVE: The transient expression system of eggplant cotyledon was established by Agrobacterium tumefaciens-mediated injection to detect the transcription level of exogenous genes in plant expression vector pCAMBIA-E8-APB-DOCK8. METHODS: Through the cultivation of tomato bacterial-free seedlings, significant differences in the number of sprouts, seedlings and agronomic traits of different tomato varieties were observed, and the tomato varieties suitable for this experiment were selected. The plant expression vector pCAMBIA-E8-APBDOCK8 constructed earlier was transformed into Agrobacterium tumefaciens EHA105, and then the tomato cotyledons, the concentration of Agrobacterium tumefaciens injected, the amount of Agrobacterium tumefaciens injected, the age of seedlings and the days of coculture were screened. The transient expression system of eggplant cotyledon mediated by Agrobacterium tumefaciens was established to observe the transcriptional level of the reporter gene. RESULTS AND CONCLUSION: The transient expression system of eggplant cotyledon was preliminarily established. The suitable tomato variety was selected as European Dahong (apple type), and the selected material was tomato cotyledon. The concentration of Agrobacterium for injection was A600:0.2-0.7, and the amount of Agrobacterium for injection was 50 μL. The seedling age was 7-10 days, and the total culture time was 24-48 hours. It provides a convenient, fast and effective method for the analysis of exogenous gene protein expression in the later stage. [ABSTRACT FROM AUTHOR]

A reliable and simple Agrobacterium-mediated transformation system for the unicellular green algae model organism Chlamydomonas reinhardtii has been developed. The protocol has been successfully employed with both neomycin phosphotransferase II (nptII) and the phleomycin resistance (bleI) genes coupled with the selective agents paromomycin and zeocin, respectively. A set of binary vectors were assembled that carry the selectable marker cassettes under control either of the Rbcs2 alone or fused to the HSP270A leader sequence, PsaD, or ß-tubulin2 promoters. The corresponding T-DNA elements also harbored a cassette with a codon-optimized version of yellow fluorescence protein (YFP) under control of the Rbcs2 promoter in which the YFP open reading frame was interrupted with the first intron of Rbcs2 to prevent expression in Agrobacterium tumefaciens. The resultant binary vectors were introduced into A. tumefaciens strain C58C1/pMP90, and the derived transconjugants were used for transformation studies with the walled C. reinhardtii strain CC124. Estimated transformation frequencies ranged from 0.09 to 2.86 colonies per 106 cells inoculated. Molecular characterizations on a subset of the transgenic lineages revealed that most of the transgenic events harbored single locus insertions. Moreover, sequencing of captured junction fragments about the T-DNA insertion site showed that minimal disruption of the C. reinhardtii genome occurred. However, the transgenic lineages often harbored truncated T-DNA regions within the non-selectable marker gene cassettes. [ABSTRACT FROM AUTHOR]

Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and Agrobacterium tumefaciens cells was performed at 24–36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes' integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in P. inflatum. [ABSTRACT FROM AUTHOR]

The worldwide occurrence of diabetic kidney disease (DKD) is swiftly rising, primarily attributed to the growing population of individuals affected by type 2 diabetes. This surge has been transformed into a substantial global concern, placing additional strain on healthcare systems already grappling with significant demands. The pathogenesis of DKD is intricate, originating with hyperglycemia, which triggers various mechanisms and pathways: metabolic, hemodynamic, inflammatory, and fibrotic which ultimately lead to renal damage. Within each pathway, several mediators contribute to the development of renal structural and functional changes. Some of these mediators, such as inflammatory cytokines, reactive oxygen species, and transforming growth factor β are shared among the different pathways, leading to significant overlap and interaction between them. While current treatment options for DKD have shown advancement over previous strategies, their effectiveness remains somewhat constrained as patients still experience residual risk of disease progression. Therefore, a comprehensive grasp of the molecular mechanisms underlying the onset and progression of DKD is imperative for the continued creation of novel and groundbreaking therapies for this condition. In this review, we discuss the current achievements in fundamental research, with a particular emphasis on individual factors and recent developments in DKD treatment. [ABSTRACT FROM AUTHOR]

Crown gall is a globally recognized bacterial disease caused by Agrobacterium tumefaciens. This bacterium is characterized by its potential to infect a wide range of plants, specifically fruit trees. In Morocco, almond trees (Prunus amygdalus) are among the most heavily affected species by this disease. In our study, all tested bacteria were isolated in the laboratory from infected almond samples exhibiting tumors at the crown and root of the sampled trees collected from northwestern of Morocco. Biochemical, pathogenicity and molecular identification were performed to identify the causal agent of almond crown gall. Molecular identification was done using a duplex PCR (A/C9 and A/E9) targeting virD2gene located in the pTi plasmid, which is the oncogenic element of the bacterium. The results showed that 12 from 20 isolates were identified as tumorigenic A. tumefaciens with the presence of pTi. Moreover, these isolates were found to exhibit tumorigenic properties both in vitro and in vivo when tested on indicator plants, specifically tomato and carrot discs. The efficacy of agrocin synthesized by Agrobacterium radiobacter strain K1026, a biopesticide known as NoGall, was tested in vitro and in planta against tumorigenic strains of A. tumefaciens. Our results revealed a significant inhibitory effect in vitro, with inhibition percentages ranging from 29.5% to 38.9%. Additionally, we observed a complete reduction in tumor growth at the inoculation sites on indicator plants, with a 100% reduction percentage.

Subviral agents are nucleic acids which lack the features for classification as a virus. Tombusvirus-like associated RNAs (tlaRNAs) are subviral positive-sense, single-stranded RNAs that replicate autonomously, yet depend on a coinfecting virus for encapsidation and transmission. TlaRNAs produce abundant subgenomic RNA (sgRNA) upon infection. Here, we investigate how the well-studied tlaRNA, ST9, produces sgRNA and its function. We found ST9 is a noncoding RNA, due to its lack of protein coding capacity. We used resistance assays with eukaryotic Exoribonuclease-1 (XRN1) to investigate sgRNA production via incomplete degradation of genomic RNA. The ST9 3' untranslated region stalled XRN1 very near the 5' sgRNA end. Thus, the XRN family of enzymes drives sgRNA accumulation in ST9-infected tissue by incomplete degradation of ST9 RNA. This work suggests tlaRNAs are not just parasites of viruses with compatible capsids, but also mutually beneficial partners that influence host cell RNA biology.

Purslane (Portulaca oleracea L.) is highly valued for its nutritional, medicinal, and ecological significance. Genetic transformation in plants provides a powerful tool for gene manipulation, allowing for the investigation of important phenotypes and agronomic traits at the genetic level. To develop an effective genetic transformation method for purslane, various organ tissues were used as explants for callus induction and shoot regeneration. Leaf tissue exhibited the highest dedifferentiation and regeneration ability, making it the optimal explant for tissue culture. By culturing on Murashige and Skoog (MS) medium supplemented with varying concentrations of 6-benzyleaminopurine (6-BA) and 1-naphthaleneacetic acid (NAA), somatic cells from leaf explants could be developed into calli, shoots, and roots. The shoot induction results of 27 different purslane accessions elucidated the impact of genotype on somatic-cell regeneration capacity and further confirmed the effectiveness of the culture medium in promoting shoot regeneration. On this basis, a total of 17 transgenic plants were obtained utilizing the genetic transformation method mediated by Agrobacterium. The assessment of GUS staining, hygromycin selection, and polymerase chain reaction (PCR) amplification of the transgenic plants as well as their progeny lines indicated that the method established could effectively introduce foreign DNA into the purslane nucleus genome, and that integration was found to be stably inherited by offspring plants. Overall, the present study demonstrates the feasibility and reliability of the Agrobacterium-mediated genetic transformation method for introducing and integrating foreign DNA into the purslane genome, paving the way for further research and applications in purslane genetic modification.

Crown gall is a soil-borne bacterial disease caused by Agrobacterium tumefaciens, leading to significant economic losses in many plant species. For the assessment of the biological and chemical products on crown gall, each plant’s crown region and roots were wounded, and then were dipped into their respective treatments. After the treatments, the plants were inoculated with a suspension of pathogenic A. tumefaciens isolate FBG1034 and maintained in a greenhouse for six months to assess them for gall formation. A quantitative real-time PCR assay was performed to quantify the A. tumefaciens using the chvE gene. Biological products such as the Agrobacterium radiobacter strain K1026, and strains 1 and 2, resulted in the lowest average root gall diameter and significantly reduced the crown gall diameter to stem diameter ratio, and the chemical product copper octanoate reduced the number of crown and root galls as well as the crown and root gall diameter compared to the inoculated, non-treated control. Moreover, both the A. radiobacter strain K1026 and strain 1 treatments resulted in an approximately 85% and 65% reduction in crown and root gall incidence, respectively, in both of the trials compared to the inoculated, non-treated plants. The findings of this study indicate that the use of biological and chemical products could help to suppress crown and root gall disease in rose plants.

Abstract With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficient Agrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence of uidA and β-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO3 and NH4NO3, and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches.

The telomere resolvase, TelA, forms the hairpin telomeres of the linear chromosome of Agrobacterium tumefaciens in a process referred to as telomere resolution. Telomere resolution is a unique DNA cleavage and rejoining reaction that resolves replicated telomere junctions into a pair of hairpin telomeres. Telomere resolvases utilize a reaction mechanism with similarities to that of topoisomerase-IB enzymes and tyrosine recombinases. The reaction proceeds without the need for high-energy cofactors due to the use of a covalent, enzyme-cleaved DNA intermediate that stores the bond energy of the cleaved bonds in 3'-phosphotyrosyl linkages. The cleaved DNA strands are then refolded into a hairpin conformation and the 5'-OH ends of the refolded strands attack the 3'-phosphotyrosine linkages in order to rejoin the DNA strands into hairpin telomeres. Because this kind of reaction mechanism is, in principle, reversible it is unclear how TelA controls the direction of the reaction and propels the reaction to completion. We present evidence that TelA forms and/or stabilizes a pre-cleavage intermediate that features breakage of the four central basepairs between the scissile phosphates prior to DNA cleavage to help propel the reaction forwards, thus preventing abortive cleavage and rejoining cycles that regenerate the substrate DNA. We identify eight TelA sidechains, located in the hairpin-binding module and catalytic domains of TelA, implicated in this process. These mutants were deficient for telomere resolution on parental replicated telomere junctions but were rescued by introduction of substrate modifications that mimic unwinding of the DNA between the scissile phosphates. [ABSTRACT FROM AUTHOR]

Interspecific and intraspecific competition and facilitation have been a focus of study in plant-plant interactions, but their influence on plant recruitment of soil microbes is unknown. In this greenhouse microcosm experiment, three cover crops (alfalfa, brassica, and fescue) were grown alone, in paired mixtures, and all together under different densities. For all monoculture trials, total pot biomass increased as density increased. Monoculture plantings of brassica were associated with the bacteria Azospirillum spp., fescue with Ensifer adhaerens, and alfalfa with both bacterial taxa. In the polycultures of cover crops, for all plant mixtures, total above-ground alfalfa biomass increased with density, and total above ground brassica biomass remained unchanged. For each plant mixture, differential abundances highlighted bacterial taxa which had not been previously identified in monocultures. For instance, mixtures of all three plants showed an increase in abundance of Planctomyces sp. SH-PL14 and Sandaracinus amylolyticus which were not represented in the monocultures. Facilitation was best supported for the alfalfa-fescue interaction as the total above ground biomass was the highest of any mixture. Additionally, the bulk soil microbiome that correlated with increasing plant densities showed increases in plant growth-promoting rhizobacteria such as Achromobacter xylosoxidans, Stentotrophomonas spp., and Azospirillum sp. In contrast, Agrobacterium tumefaciens, a previously known generalist phytopathogen, also increased with alfalfa-fescue plant densities. This could suggest a strategy by which, after facilitation, a plant neighbor could culture a pathogen that could be more detrimental to the other. [ABSTRACT FROM AUTHOR]

Neoadjuvant chemotherapy (NAC) is a well-established treatment modality for locally advanced breast cancer (BC). However, it can also result in severe toxicities while controlling tumors. Therefore, reliable predictive biomarkers are urgently needed to objectively and accurately predict NAC response. In this study, we integrated single-cell and bulk RNA-seq data to identify nine genes associated with the prognostic response to NAC: NDRG1, CXCL14, HOXB2, NAT1, EVL, FBP1, MAGED2, AR and CIRBP. Furthermore, we constructed a prognostic risk model specifically linked to NAC. The clinical independence and generalizability of this model were effectively demonstrated. Additionally, we explore the underlying cancer hallmarks and microenvironment features of this NAC response-related risk score, and further assess the potential impact of risk score on drug response. In summary, our study constructed and validated a nine-gene signature associated with NAC prognosis, which was accomplished through the integration of single-cell and bulk RNA data. The results of our study are of crucial significance in the prediction of the efficacy of NAC in BC, and may have implications for the clinical management of this disease. [ABSTRACT FROM AUTHOR]

The clinical application of chimeric antigen receptor (CAR) T‐cell therapy has rapidly changed the treatment options for terminally ill patients with defined blood‐borne cancer types. However, CAR T‐cell therapy can lead to severe therapy‐associated toxicities including CAR‐related hematotoxicity, ON‐target OFF‐tumor toxicity, cytokine release syndrome (CRS) or immune effector cell‐associated neurotoxicity syndrome (ICANS). Just as CAR T‐cell therapy has evolved regarding receptor design, gene transfer systems and production protocols, the management of side effects has also improved. However, because of measures taken to abrogate adverse events, CAR T‐cell viability and persistence might be impaired before complete remission can be achieved. This has fueled efforts for the development of extrinsic and intrinsic strategies for better control of CAR T‐cell activity. These approaches can mediate a reversible resting state or irreversible T‐cell elimination, depending on the route chosen. Control can be passive or active. By combination of CAR T‐cells with T‐cell inhibiting compounds, pharmacologic control, mostly independent of the CAR construct design used, can be achieved. Other strategies involve the genetic modification of T‐cells or further development of the CAR construct by integration of molecular ON/OFF switches such as suicide genes. Alternatively, CAR T‐cell activity can be regulated intracellularly through a self‐regulation function or extracellularly through titration of a CAR adaptor or of a priming small molecule. In this work, we review the current strategies and mechanisms to control activity of CAR T‐cells reversibly or irreversibly for preventing and for managing therapy‐associated toxicities. [ABSTRACT FROM AUTHOR]

The biological mechanisms underlying meat quality remain unclear. Currently, many studies report that the gastrointestinal microbiota is essential for animal growth and performance. However, it is uncertain which bacterial species are specifically associated with the meat quality traits. In this study, 16S rDNA and metagenomic sequencing were performed to explore the composition and function of microbes in various gastrointestinal segments of Tan sheep and Dorper sheep, as well as the relationship between microbiota and meat quality (specifically, the fatty acid content of the muscle). In the ruminal, duodenal, and colonic microbiome, several bacteria were uniquely identified in respective breeds, including Agrobacterium tumefaciens, Bacteroidales bacterium CF, and several members of the family Oscillospiraceae. The annotation of GO, KEGG, and CAZYme revealed that these different bacterial species were linked to the metabolism of glucose, lipids, and amino acids. Additionally, our findings suggested that 16 microbial species may be essential to the content of fatty acids in the muscle, especially C12:0 (lauric acid). 4 bacterial species, including Achromobacter xylosoxidans, Mageeibacillus indolicus, and Mycobacterium dioxanotrophicus, were positively correlated with C12:0, while 13 bacteria, including Methanobrevibacter millerae, Bacteroidales bacterium CF, and Bacteroides coprosuis were negatively correlated with C12:0. In a word, this study provides a basic data for better understanding the interaction between ruminant gastrointestinal microorganisms and the meat quality traits of hosts. [ABSTRACT FROM AUTHOR]

Biological degradation of natural product glycosides involves, alongside hydrolysis, β-elimination for glycosidic bond cleavage. Here, we discover an O-glycoside β-eliminase (OGE) from Agrobacterium tumefaciens that converts the C3-oxidized O-β-d-glucoside of phloretin (a plant-derived flavonoid) into the aglycone and the 2-hydroxy-3-keto-glycal elimination product. While unrelated in sequence, OGE is structurally homologous to, and shows effectively the same Mn2+ active site as, the C-glycoside deglycosylating enzyme (CGE) from a human intestinal bacterium implicated in β-elimination of 3-keto C-β-d-glucosides. We show that CGE catalyzes β-elimination of 3-keto O- and C-β-d-glucosides while OGE is specific for the O-glycoside substrate. Substrate comparisons and mutagenesis for CGE uncover positioning of aglycone for protonic assistance by the enzyme as critically important for C-glycoside cleavage. Collectively, our study suggests convergent evolution of active site for β-elimination of 3-keto O-β-d-glucosides. C-Glycoside cleavage is a specialized feature of this active site which is elicited by substrate through finely tuned enzyme-aglycone interactions. Biological degradation of glycosides involves, alongside hydrolysis, β-elimination for glycosidic bond cleavage. Here, the authors report an O-glycoside β-eliminase from Agrobacterium tumefaciens that converts the C3-oxidized O-β-d-glucoside of phloretin into the aglycone and the 2-hydroxy-3-keto-d-glycal elimination product, and suggest convergent evolution of β-eliminase active sites for the cleavage of natural product 3-keto-O-glycosides. [ABSTRACT FROM AUTHOR]

Objective: To detect the HPV genotype and integration sites in patients with high-risk HPV infection at different stages of photodynamic therapy using nanopore technology and to evaluate the treatment effect. Methods: Four patients with HPV infection were selected and subjected to photodynamic therapy, and cervical exfoliated cell was sampled at before treatment, after three courses of treatment and six courses of treatment, their viral abundance and insertion sites were analyzed by nanopore technology, and pathological examinations were performed before and after treatment. In this study, we developed a novel assay that combined viral sequence enrichment and Nanopore sequencing for identification of HPV genotype and integration sites at once. The assay has obvious advantages over qPCR or NGS-based methods, as it has better sensitivity after viral sequences enrichment and can generate long-reads (kb to Mb) for better detection rate of structure variations, moreover, fast turn-around time for real-time viral sequencing and analysis. Results: The pathological grade was reduced in all four patients after photodynamic therapy. Virus has been cleared in two cases after treatment, the virus amount reduced after treatment but not completely cleared in one case, and two type viruses were cleared and one type virus persisted after treatment in the last patient with multiple infection. Viral abundance and the number of integration sites were positively correlated. Gene enrichment analysis showed complete viral clearance in 1 patient and 3 patients required follow-up. Conclusion: Nanopore sequencing can effectively monitor the abundance of HPV viruses and integration sites to show the presence status of viruses, and combined with the results of gene enrichment analysis, the treatment effect can be dynamically assessed. [ABSTRACT FROM AUTHOR]

NieR is a TetR family transcriptional repressor previously shown to regulate the NaOCl-inducible efflux pump NieAB in Agrobacterium tumefaciens. NieR is an ortholog of Escherichia coli NemR that specifically senses hypochlorite through the redox switch of a reversible sulfenamide bond between C106 and K175. The amino acid sequence of NieR contains only one cysteine. NieR has C104 and R166, which correspond to C106 and K175 of NemR, respectively. The aim of this study was to investigate the redox-sensing mechanism of NieR under NaOCl stress. C104 and R166 were subjected to mutagenesis to determine their roles. Although the substitution of R166 by alanine slightly reduced its DNA-binding activity, NieR retained its repressor function. By contrast, the DNA-binding and repression activities of NieR were completely lost when C104 was replaced by alanine. C104 substitution with serine only partially impaired the repressor function. Mass spectrometry analysis revealed an intermolecular disulfide bond between the C104 residues of NieR monomers. This study demonstrates the engagement of C104 in the mechanism of NaOCl sensing. C104 oxidation induced the formation of a disulfide-linked dimer that was likely to alter conformation, thus abolishing the DNA-binding ability of NieR and derepressing the target genes. [Display omitted] • Agrobacterium tumefaciens NieR is a thiol-based oxidation sensor for hypochlorite stress. • C104 oxidation induced the formation of a disulfide-linked dimer. • Ala and Ser mutations of C104 differentially affect DNA-binding and repressor activities of NieR. [ABSTRACT FROM AUTHOR]

For almost 50 years, tobacco (Nicotiana tabacum) BY-2 cells have been widely recognized as an important cell line for plant biology. The cell line grows rapidly, can be synchronized to a high degree, and is excellent for imaging; over the years, these features have led to many high-impact discoveries. However, certain other uses of this cell line are virtually unknown. In the early days, I was involved in distributing the cells to laboratories around the world. Many of these scientists wanted to study the cell cycle; however, I also distributed the cells to scientists who were elucidating the mechanism of plant transformation by Agrobacterium tumefaciens. In fact, BY-2 cells played an essential role in the identification and analysis of Vir genes on the Ti plasmid; likewise, the cells were important for discovering the factor that induces the expression of Vir genes. Thus, BY-2 cells were crucial for the development of modern plant biotechnology. Here, I recount the story of how this came to pass and explain why the use of BY-2 cells in this work was never recognized. [ABSTRACT FROM AUTHOR]

Cancer immunotherapy has emerged as a groundbreaking method of treating malignancies. However, cancer immunotherapy can only benefit a small percentage of patients, and the numerous side effects that might develop during treatment reduce its effectiveness or even put patients' lives in jeopardy. Surprisingly, the gut microbiome Akkermansia muciniphila (A. muciniphila) can significantly inhibit carcinogenesis and improve anti-tumor effects, thus increasing the effectiveness of cancer immunotherapy and decreasing the likelihood of side effects. In this review, we focus on the effects of A. muciniphila on the human immune system and the positive impacts of A. muciniphila on cancer immunotherapy, which can build on strengths and improve weaknesses of cancer immunotherapy. The potential clinical applications of A. muciniphila on cancer immunotherapy are also proposed, which have great prospects for anti-tumor therapy. [ABSTRACT FROM AUTHOR]

Background: To identify novel gene combinations and to develop an early diagnostic model for Polycystic Ovary Syndrome (PCOS) through the integration of artificial neural networks (ANN) and random forest (RF) methods.Methods: We retrieved and processed gene expression datasets for PCOS from the Gene Expression Omnibus (GEO) database. Differential expression analysis of genes (DEGs) within the training set was performed using the "limma" R package. Enrichment analyses on DEGs using gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), and immune cell infiltration. The identification of critical genes from DEGs was then performed using random forests, followed by the developing of new diagnostic models for PCOS using artificial neural networks.Results: We identified 130 up-regulated genes and 132 down-regulated genes in PCOS compared to normal samples. Gene Ontology analysis revealed significant enrichment in myofibrils and highlighted crucial biological functions related to myofilament sliding, myofibril, and actin-binding. Compared with normal tissues, the types of immune cells expressed in PCOS samples are different. A random forest algorithm identified 10 significant genes proposed as potential PCOS-specific biomarkers. Using these genes, an artificial neural network diagnostic model accurately distinguished PCOS from normal samples. The diagnostic model underwent validation using the independent validation set, and the resulting area under the receiver operating characteristic curve (AUC) values was consistent with the anticipated outcomes.Conclusion: Utilizing unique gene combinations, this research created a diagnostic model by merging random forest techniques with artificial neural networks. The AUC indicated a notably superior performance of the diagnostic model. [ABSTRACT FROM AUTHOR]

Background: Our previous study explored the molecular signatures of generalized aggressive periodontitis (GAgP) using gingival tissues through omics‐based‐whole‐genome transcriptomic analysis. This continuation study aimed to investigate the whole protein profiling of these gingival samples through liquid chromatography‐mass spectroscopy/mass spectroscopy (LC‐MS/MS) analysis and to validate the identified proteins through immunohistochemistry to provide further evidence for the quality of the results. Methods: In previous study, gene expression patterns were identified in gingival tissues from 23 GAgP and 25 control individuals. In the current study, comparative proteomic analysis was performed on isolated proteins from the same study groups using LC‐MS/MS analysis. The data from the transcriptomics study published before and the proteomics data were integrated to reveal any common genes and proteins. Additionally, immunohistochemical analysis was conducted to further investigate the findings. Results: The most upregulated proteins in patients compared to controls were ITGAM, AZU1, MMP9, BPI, UGGG1, MZB1, TRFL, PDIA6, PRDX4, and PLG. The top six pathways associated with these proteins were involved in innate immune system, post‐translational protein phosphorylation, interleukin‐4 and ‐13 signaling, toll‐like receptors cascades, and extracellular matrix organization. Based on the integration and validation analysis of transcriptomics and proteomics data, as well as immunohistochemical analysis, MZB1 was identified as a shared gene and protein that were upregulated in the patients. Conclusions: MZB1 is a protein that is involved in the development of B cells and the production of antibodies. Its upregulation in periodontitis suggests that there may be a dysregulation of the immune response in this condition, and MZB1 may be a potent biomarker for periodontitis. [ABSTRACT FROM AUTHOR]

Microplastics (MPs) with slow degradation rates carry toxins and pathogens from surroundings, accumulate and pollute the environment. They bio‐accumulate on humans and other lifeforms leading to health concerns, including inflammatory lesions, oxidative stress and increased cancer risk, thus requiring immediate remediation actions. This review summarizes, categorizes and analyses recent findings on MP source, transit and environmental toxicity and explores their microbe‐mediated breakdown. Various micro‐organisms such as fungi, bacteria, algae and protists interact with and build biofilm on MP surfaces and alter their surface morphology for their degradation. Pure strains and microbial consortia have been successfully able to degrade MPs. Extracellular enzymes are produced by these micro‐organisms that convert the complex recalcitrant polymeric structure of MP to simpler forms. Further, knowledge of factors associated with MP degradation along with the development of genetic tools enhances the rate of microbial degradation with consortium having an advantage over single bacterium‐mediated MP transformation, which has been discussed. [ABSTRACT FROM AUTHOR]

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Introduction: Chimeric antigen receptor-engineered T cells (CAR-Ts) are investigated in various clinical trials for the treatment of cancer entities beyond hematologic malignancies. A major hurdle is the identification of a target antigen with high expression on the tumor but no expression on healthy cells, since "ontarget/ off-tumor" cytotoxicity is usually intolerable. Approximately 90% of carcinomas and leukemias are positive for the Thomsen-Friedenreich carbohydrate antigen CD176, which is associated with tumor progression, metastasis and therapy resistance. In contrast, CD176 is not accessible for ligand binding on healthy cells due to prolongation by carbohydrate chains or sialylation. Thus, no "on-target/off-tumor" cytotoxicity and low probability of antigen escape is expected for corresponding CD176-CAR-Ts. Methods: Using the anti-CD176 monoclonal antibody (mAb) Nemod-TF2, the presence of CD176 was evaluated on multiple healthy or cancerous tissues and cells. To target CD176, we generated two different 2nd generation CD176-CAR constructs differing in spacer length. Their specificity for CD176 was tested in reporter cells as well as primary CD8+ T cells upon co-cultivation with CD176+ tumor cell lines as models for CD176+ blood and solid cancer entities, as well as after unmasking CD176 on healthy cells by vibrio cholerae neuraminidase (VCN) treatment. Following that, both CD176-CARs were thoroughly examined for their ability to initiate target-specific T-cell signaling and activation, cytokine release, as well as cytotoxicity. Results: Specific expression of CD176 was detected on primary tumor tissues as well as on cell lines from corresponding blood and solid cancer entities. CD176-CARs mediated T-cell signaling (NF-kB activation) and T-cell activation (CD69, CD137 expression) upon recognition of CD176+ cancer cell lines and unmasked CD176, whereby a short spacer enabled superior target recognition. Importantly, they also released effector molecules (e.g. interferon-g, granzyme B and perforin), mediated cytotoxicity against CD176+ cancer cells, and maintained functionality upon repetitive antigen stimulation. Here, CD176L-CAR-Ts exhibited slightly higher proliferation and mediator-release capacities. Since both CD176-CAR-Ts did not react towards CD176-control cells, their response proved to be target-specific. Discussion: Genetically engineered CD176-CAR-Ts specifically recognize CD176 which is widely expressed on cancer cells. Since CD176 is masked on most healthy cells, this antigen and the corresponding CAR-Ts represent a promising approach for the treatment of various blood and solid cancers while avoiding "on-target/off-tumor" cytotoxicity. [ABSTRACT FROM AUTHOR]

Background: The unfolding protein response is a critical biological process implicated in a variety of physiological functions and disease states across eukaryotes. Despite its significance, the role and underlying mechanisms of the response in the context of ischemic stroke remain elusive. Hence, this study endeavors to shed light on the mechanisms and role of the unfolding protein response in the context of ischemic stroke. Methods: In this study, mRNA expression patterns were extracted from the GSE58294 and GSE16561 datasets in the GEO database. The screening and validation of protein response-related biomarkers in stroke patients, as well as the analysis of the immune effects of the pathway, were carried out. To identify the key genes in the unfolded protein response, we constructed diagnostic models using both random forest and support vector machine-recursive feature elimination methods. The internal validation was performed using a bootstrapping approach based on a random sample of 1,000 iterations. Lastly, the target gene was validated by RT-PCR using clinical samples. We utilized two algorithms, CIBERSORT and MCPcounter, to investigate the relationship between the model genes and immune cells. Additionally, we performed uniform clustering of ischemic stroke samples based on expression of genes related to the UPR pathway and analyzed the relationship between different clusters and clinical traits. The weighted gene co-expression network analysis was conducted to identify the core genes in various clusters, followed by enrichment analysis and protein profiling for the hub genes from different clusters. Results: Our differential analysis revealed 44 genes related to the UPR pathway to be statistically significant. The integration of both machine learning algorithms resulted in the identification of 7 key genes, namely ATF6, EXOSC5, EEF2, LSM4, NOLC1, BANF1, and DNAJC3. These genes served as the foundation for a diagnostic model, with an area under the curve of 0.972. Following 1000 rounds of internal validation via randomized sampling, the model was confirmed to exhibit high levels of both specificity and sensitivity. Furthermore, the expression of these genes was found to be linked with the infiltration of immune cells such as neutrophils and CD8 T cells. The cluster analysis of ischemic stroke samples revealed three distinct groups, each with differential expression of most genes related to the UPR pathway, immune cell infiltration, and inflammatory factor secretion. The weighted gene co-expression network analysis showed that all three clusters were associated with the unfolded protein response, as evidenced by gene enrichment analysis and the protein landscape of each cluster. The results showed that the expression of the target gene in blood was consistent with the previous analysis. Conclusion: The study of the relationship between UPR and ischemic stroke can help to better understand the underlying mechanisms of the disease and provide new targets for therapeutic intervention. For example, targeting the UPR pathway by blocking excessive autophagy or inducing moderate UPR could potentially reduce tissue injury and promote cell survival after ischemic stroke. In addition, the results of this study suggest that the use of UPR gene expression levels as biomarkers could improve the accuracy of early diagnosis and prognosis of ischemic stroke, leading to more personalized treatment strategies. Overall, this study highlights the importance of the UPR pathway in the pathology of ischemic stroke and provides a foundation for future studies in this field. [ABSTRACT FROM AUTHOR]

Litchi (Litchi chinensis Sonn.) is a type of commercially prevalent subtropical and tropical fruit. Since litchi has a highly heterozygous genetic background and a long reproductive cycle, conventional breeding methods (such as hybridization) have limited ability to nurture new litchi cultivars. Here, an efficient and stable Agrobacterium tumefaciens-mediated genetic transformation of embryogenic callus was established in 'Feizixiao' litchi. Transgenic materials were verified using polymerase chain reaction (PCR) analysis, b-glucuronidase (GUS) assay, and green fluorescent protein (GFP) assay. To implement the technology of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology in 'Feizixiao' litchi and verify the validity of these transformation systems, the litchi polyphenol oxidase gene (LcPPO, JF926153) was knocked out. Various categories of mutations, covering base insertions, deletions, and substitutions, were found in transgenic materials via sequence analysis. The transformation system achieved high feasibility and efficiency, and the system of CRISPR/Cas9 was successfully employed to edit genes in 'Feizixiao' litchi. This work provides an essential foundation for investigating the functions of genes and accelerating litchi genetic improvement. [ABSTRACT FROM AUTHOR]

This work proposes the design of β-keto esters as antibacterial compounds. The design was based on the structure of the autoinducer of bacterial quorum sensing, N-(3-oxo-hexanoyl)-l-homoserine lactone (3-oxo-C6-HSL). Eight β-keto ester analogues were synthesised with good yields and were spectroscopically characterised, showing that the compounds were only present in their β-keto ester tautomer form. We carried out a computational analysis of the reactivity and ADME (absorption, distribution, metabolism, and excretion) properties of the compounds as well as molecular docking and molecular dynamics calculations with the LasR and LuxS quorum-sensing (QS) proteins, which are involved in bacterial resistance to antibiotics. The results show that all the compounds exhibit reliable ADME properties and that only compound 7 can present electrophile toxicity. The theoretical reactivity study shows that compounds 6 and 8 present a differential local reactivity regarding the rest of the series. Compound 8 presents the most promising potential in terms of its ability to interact with the LasR and LuxS QS proteins efficiently according to its molecular docking and molecular dynamics calculations. An initial in vitro antimicrobial screening was performed against the human pathogenic bacteria Pseudomonas aeruginosa and Staphylococcus aureus as well as the phytopathogenic bacteria Pseudomonas syringae and Agrobacterium tumefaciens. Compounds 6 and 8 exhibit the most promising results in the in vitro antimicrobial screening against the panel of bacteria studied. [ABSTRACT FROM AUTHOR]

Crown gall, caused by Agrobacterium tumefaciens (syn. Rhizobium radiobacter), is characterized by gall formation on crowns, stems or roots on many plant species. We describe the occurrence of this disease for the first time on cannabis (Cannabis sativa L. marijuana) plants in two licenced production facilities in British Columbia. Galls were observed on crowns, stems and roots of several genotypes, including 'White Rhino', 'Pink Kush' and 'Sour Kush'. The affected plants displayed no other visible symptoms. The incidence of crown gall was extremely low, estimated at 0.01% of total plants. Isolations made from root and stem galls on MacConkey medium (selective for Gram -ve bacteria) and on D1 medium (selective for Agrobacterium spp.) yielded a range of bacterial species, which did not include Agrobacterium sp. However, the presence of Agrobacterium in gall tissues was confirmed following PCR amplification with primers for the indole-acetic acid (iaa) H gene on the T-DNA region of Agrobacterium spp. and showed > 99% similarity to A. tumefaciens. Artificial inoculations were conducted using an A. tumefaciens strain from ATCC (Rhizobium radiobacter, strain designation TT134 [6-1-2, CIP 104 336], cat. 15955) which resulted in gall formation on five cannabis genotypes tested. These galls contained the iaaH gene as determined by PCR and the pathogen was successfully re-isolated on MacConkey agar medium at 4 and 10 weeks post-inoculation. Our findings confirm the occurrence of A. tumefaciens on naturally infected and artificially infected cannabis plants grown under greenhouse conditions and demonstrate reproducible gall symptoms on several cannabis genotypes following A. tumefaciens inoculation. [ABSTRACT FROM AUTHOR]

The pupillary light reflex (PLR) is an important biomarker for the detection and management of traumatic brain injury (TBI). We investigated the performance of PupilScreen, a smartphone-based pupillometry app, in classifying healthy control subjects and subjects with severe TBI in comparison to the current gold standard NeurOptics pupillometer (NPi-200 model with proprietary Neurological Pupil Index [NPi] TBI severity score). A total of 230 PLR video recordings taken using both the PupilScreen smartphone pupillometer and NeurOptics handheld device (NPi-200) pupillometer were collected from 33 subjects with severe TBI (sTBI) and 132 subjects who were healthy without self-reported neurological disease. Severe TBI status was determined by Glasgow Coma Scale (GCS) at the time of recording. The proprietary NPi score was collected from the NPi-200 pupillometer for each subject. Seven PLR curve morphological parameters were collected from the PupilScreen app for each subject. A comparison via t-test and via binary classification algorithm performance using NPi scores from the NPi-200 and PLR parameter data from the PupilScreen app was completed. This was used to determine how the frequently used NPi-200 proprietary NPi TBI severity score compares to the PupilScreen app in ability to distinguish between healthy and sTBI subjects. Binary classification models for this task were trained for the diagnosis of healthy or severe TBI using logistic regression, k-nearest neighbors, support vector machine, and random forest machine learning classification models. Overall classification accuracy, sensitivity, specificity, area under the curve, and F1 score values were calculated. Median GCS was 15 for the healthy cohort and 6 (interquartile range 2) for the severe TBI cohort. Smartphone app PLR parameters as well as NPi from the digital infrared pupillometer were significantly different between healthy and severe TBI cohorts; 33% of the study cohort had dark eye colors defined as brown eyes of varying shades. Across all classification models, the top performing PLR parameter combination for classifying subjects as healthy or sTBI for PupilScreen was maximum diameter, constriction velocity, maximum constriction velocity, and dilation velocity with accuracy, sensitivity, specificity, area under the curve (AUC), and F1 score of 87%, 85.9%, 88%, 0.869, and 0.85, respectively, in a random forest model. The proprietary NPi TBI severity score demonstrated greatest AUC value, F1 score, and sensitivity of 0.648, 0.567, and 50.9% respectively using a random forest classifier and greatest overall accuracy and specificity of 67.4% and 92.4% using a logistic regression model in the same classification task on the same dataset. The PupilScreen smartphone pupillometry app demonstrated binary healthy versus severe TBI classification ability greater than that of the NPi-200 proprietary NPi TBI severity score. These results may indicate the potential benefit of future study of this PupilScreen smartphone pupillometry application in comparison to the NPi-200 digital infrared pupillometer across the broader TBI spectrum, as well as in other neurological diseases. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to analyze the hub genes of heart failure with reduced ejection fraction (HFrEF) treated with Empagliflozin using RNA sequencing (RNA-seq) and bioinformatics methods, including machine learning.Methods: From February 2021 to February 2023, nine patients with HFrEF were enrolled from our hospital's cardiovascular department. In addition to routine drug treatment, these patients received 10 mg of Empagliflozin once daily for two months. Efficacy was assessed and RNA-seq was performed on peripheral blood before and after treatment with empagliflozin. HFrEF-related hub genes were identified through bioinformatics analyses including differential gene expression analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, immune infiltration analysis, machine learning, immune cell correlation analysis and clinical indicator correlation analysis.Results: The nine patients included in this study completed a two-month treatment regimen, with an average age of 62.11 ± 6.36 years. By performing bioinformatics analysis on the transcriptome from the treatment groups, 42 differentially expressed genes were identified, with six being up-regulated and 36 being down-regulated (|log2FC|> 1 and adj.pvalue< 0.05). Immune infiltration analysis of these genes demonstrated a significant difference in the proportion of plasma between the pre-treatment and post-treatment groups (p< 0.05). Two hub genes, GTF2IP14 and MTLN, were finally identified through machine learning. Further analysis of the correlation between the hub genes and immune cells suggested a negative correlation between GTF2IP14 and naive B cells, and a positive correlation between MTLN and regulatory T cells and resting memory CD4+ T cells (p< 0.05).Conclusion: Through RNA-seq and bioinformatics analysis, this study identified GTF2IP14 and MTLN as the hub genes of HFrEF, and their mechanisms may be related to immune inflammatory responses and various immune cells. [ABSTRACT FROM AUTHOR]

Drug‐induced liver injury is one of the main challenges that leads to the withdrawal of several drugs in the clinical setting. Cyclosporine is one of the drugs that its long‐term administration exerts devastating effects on the hepatocytes. In the present study, we aimed to evaluate the effect of ferulic acid, a natural compound found in plants, on cyclosporine‐mediated hepatotoxicity. Forty‐eight male Wistar rats were treated with cyclosporine and/or ferulic acid to evaluate the function as well as the morphology of liver cells. We found that ferulic acid dose‐dependently recovered the functional as well as histopathological parameters of liver cells, as revealed by the improvement of hepatocellular vacuolation, portal fibroplasia, and necrosis. Moreover, this phenolic compound was able to restore the balance of the redox system in cyclosporine‐treated rats by activating the nuclear factor (NF) erythroid 2‐related factor 2 (Nrf2)/hemeoxygenase‐1 (HO‐1) signaling axis. Of note, the protective effects of ferulic acid against cyclosporine‐mediated liver toxicity were not restricted only to induction of the potential antioxidant property, as in the presence of this agent, the expression of pro‐inflammatory cytokines such as NF‐κB, tumor necrosis factor (TNF)‐α, and interleukin‐1β was also diminished. Ferulic acid also shifted the equilibrium between the expression levels of proapoptotic to antiapoptotic proteins and thereby prevented the development of cyclosporine‐induced liver injury. Overall, these findings highlighted that ferulic acid can reduce cyclosporine‐induced liver injury due to its antioxidant properties. [ABSTRACT FROM AUTHOR]

Norovirus (NoV) is the most common cause of diarrheal episodes globally. Issues with in vitro cultivation systems, genetic variation, and animal models have hindered vaccine development. Plant-derived virus-like particles (VLPs) may address some of these concerns because they are highly immunogenic, can be administered by different routes, and can be rapidly produced to accommodate emerging viral strains. NoV VLPs (NoVLP) composed of the surface viral protein (VP) 1 of the GI and GII genogroups were produced in Nicotiana benthamiana using an Agrobacterium tumefaciens -based recombinant transient expression system. Leaves from infiltrated plants were harvested and NoVLPs were extracted and purified. The safety and immunogenicity of the GII.4 NoVLP, the genotype currently causing most human disease, were subsequently examined in rabbits and mice. Fifteen GI and GII NoVLPs were successfully expressed in N. benthamiana and were structurally similar to NoV virions, as determined by cryogenic transmission electron microscopy. The NoVLP was well-tolerated, with no local or systemic signs of toxicity in rabbits. Three intramuscular doses of the GII.4 NoVLP adjuvanted with aluminum hydroxide induced robust IgG titers, IgG-secreting cells, histo-blood group antigen blocking titers, and IFNγ-secreting T cells in mice. In addition to circulating antibodies, oral administration of the NoVLP in mice induced significant IgA levels in feces, indicative of a mucosal response. The plant-made NoVLP vaccine was safe and immunogenic in mice and rabbits. Multi-modal vaccination, combining oral and intramuscular administration could be considered for future clinical development to maximize systemic and mucosal immune responses. [ABSTRACT FROM AUTHOR]

Climate change results in exceptional environmental conditions and drives the migration of pathogens to which local plants are not adapted. Biotic stress disrupts plants' metabolism, fitness, and performance, ultimately impacting their productivity. It is therefore necessary to develop strategies for improving plant resistance by promoting stress responsiveness and resilience in an environmentally friendly and sustainable way. The aim of this study was to investigate whether priming tobacco plants with a formulation containing silicon-stabilised hybrid lipid nanoparticles functionalised with quercetin (referred to as GS3 phyto-courier) can protect against biotic stress triggered by Agrobacterium tumefaciens leaf infiltration. Tobacco leaves were primed via infiltration or spraying with the GS3 phyto-courier, as well as with a buffer (B) and free quercetin (Q) solution serving as controls prior to the biotic stress. Leaves were then sampled four days after bacterial infiltration for gene expression analysis and microscopy. The investigated genes increased in expression after stress, both in leaves treated with the phyto-courier and control solutions. A trend towards lower values was observed in the presence of the GS3 phyto-courier for genes encoding chitinases and pathogenesis-related proteins. Agroinfiltrated leaves sprayed with GS3 confirmed the significant lower expression of the pathogenesis-related gene PR-1a and showed higher expression of peroxidase and serine threonine kinase. Microscopy revealed swelling of the chloroplasts in the parenchyma of stressed leaves treated with B; however, GS3 preserved the chloroplasts' mean area under stress. Furthermore, the UV spectrum of free Q solution and of quercetin freshly extracted from GS3 revealed a different spectral signature with higher values of maximum absorbance (Amax) of the flavonoid in the latter, suggesting that the silicon-stabilised hybrid lipid nanoparticles protect quercetin against oxidative degradation. [ABSTRACT FROM AUTHOR]

• This is the first study that successfully expressed genes encoding chitinase 42 kDa from Trichoderma asperellum SH16 in peanuts (Arachis hypogaea) cultivar L14 through Agrobacterium tumefaciens -mediated transformation. • Two synthetic genes (syncodChi42–1 and syncodChi42–2) optimized for codon usage for plant expression proved to be more efficient than their original gene (Chi42) from T. asperellum SH16 in transgenic peanuts. • The high expression level of two synthetic genes in peanut roots as well as their strong resistance to Sclerotium rolfsii promise potential applications in the field of transgenic crops against phytopathogenic fungi. This study aims to extracellularly express the 42 kDa chitinase from Trichoderma asperellum SH16 in peanut (Arachis hypogaea) roots under the regulation of a tissue-specific Asy promoter and the driving of the signal peptide of the rice amylase 3D gene. The 42 kDa chitinase genes were employed in this study, which included one wild-type sequence (Chi42) and two synthetic sequences (syncodChi42–1 and syncodChi42–2) optimized for codon usage for plant expression. From 102 42 kDa chitinase transgenic peanut lines, seven elite lines were discovered, among them two for syncodChi42–1, three for syncodChi42–2, and two for Chi42. The study also revealed chitinase from two synthetic genes had higher expression levels than chitinase from the wild-type gene, with the highest specific activities of approximately 901 U/mg and 1124 U/mg, respectively, compared to 739 U/mg. All seven elite transgenic peanut lines exhibited strong extracellular chitinase activity, and some of them were able to fight the soil-borne pathogenic fungus Sclerotium rolfsii in both in vitro and in vivo assays. The significant chitinase activity of seven elite transgenic peanut lines, particularly five lines carrying one of two synthetic genes, suggests that transgenic crops might be used to prevent phytopathogenic fungi. [ABSTRACT FROM AUTHOR]

Societal Impact Statement: Lateral gene transfer (LGT) refers to the transmission of genetic material without sexual reproduction. LGT is widespread in a number of plant species, including grasses. But how these genes of foreign origin got there is presently unknown. In this review, we show that transformation techniques used to genetically modify organisms could occur in the wild and be responsible for the frequently observed grass‐to‐grass LGTs. The distinction between natural evolutionary processes and genetic engineering might be arbitrary, and its validity will be further debated as agricultural biotechnology becomes more widely used and examples of "natural genetic engineering" through LGT increase. Summary: Lateral gene transfer (LGT) is the transmission of genetic material among species without sexual reproduction. LGT was initially thought to be restricted to prokaryotes, but it has since been reported in a wide range of eukaryotes, including plants. Grasses seem to be particularly prone to LGT and frequently exchange genes among species. However, the mechanism(s) facilitating these transfers in this economically and ecologically important group of plants are debated. Here, we review vector‐mediated, direct tissue‐to‐tissue contact, wide‐crossing and reproductive contamination LGT mechanisms and discuss the likelihood of each in light of recent studies. Of particular relevance are transformation approaches that require minimal human intervention to transfer DNA among grasses in the lab that could mimic the mechanisms facilitating grass‐to‐grass LGT in the wild. These approaches include relatively simple techniques, such as pollen tube pathway‐mediated transformation, that take advantage of the permeability of the reproductive process to introduce alien genetic material from a third individual into an embryo. This process could be easily mirrored in the wild where pollen from one species lands on the stigma of another, acting as a source of alien DNA that can ultimately contaminate the reproductive process. This contamination is likely to be prevalent in wind pollinated species such as grasses, where the rates of illegitimate pollination will be high. In conclusion, plant transformation methods requiring minimal intervention are likely paralleled in the wild where they act as the mechanism underpinning LGT between distantly related grass species. [ABSTRACT FROM AUTHOR]

Cinnamomum camphora is an excellent evergreen broad-leaved tree species with strong stress tolerance, but its molecular character revelation as well as ecological and economic value improvement were limited due to the lack of a genetic transformation system. To establish a simple and efficient transient transformation system for uncovering the molecular mechanism of plant tolerating stresses and promoting the selective breeding of good varieties, the infection method, co-cultivation time, infection solution concentration, and growth density of Agrobacterium tumefaciens containing green fluorescent protein (GFP)-based calmodulin protein 3 gene (GCaMP3) were identified by monitoring the fluorescence emitted from GCaMP3 bound to Ca2+. Meanwhile, the transient transformation effects were evaluated via cytoplasmic Ca2+ concentration variations at high temperatures of 35 °C and 40 °C. When C. camphora leaves were infected with A. tumefaciens containing GCaMP3 via injection and soaking, no significant difference was detected in the fluorescence intensity over 48 h, indicating that the two infection methods had the same transient transformation efficiency. By prolonging the co-cultivation time, the fluorescence intensity gradually increased, reached its strongest at the 48th h, and then gradually declined. For the infection solution concentration, an OD600 of 0.7 led to the strongest fluorescence intensity, with an increase of 42.2%, 13.7%, 4.2%, and 14.2%, respectively, compared to that at OD600 of 0.5, 0.6, 0.8, and 0.9. When A. tumefaciens growth density OD600 was 0.5–0.7, the strongest fluorescence intensity was detected after transient transformation. Combining these optimum conditions, GCaMP3 was transferred into C. camphora, which indicated the variations in cytoplasmic Ca2+ concentration at high temperatures, with the fluorescence intensity at 35 °C and 40 °C increasing by 12.6% and 30.6%, respectively, in contrast to that at 28 °C. Therefore, it should be an efficient transient transformation system for C. camphora, with A. tumefaciens growth density OD600 of 0.5–0.7, infection solution concentration OD600 of 0.7, and co-cultivation time of 48 h by using both injection and soak infection methods, which is beneficial for uncovering the Ca2+ signal transduction in the plant tolerating stresses and promoting its molecular biology development and selective breeding of good varieties. [ABSTRACT FROM AUTHOR]

N-acyl homoserine lactones (AHLs) are small, diffusible chemical signal molecules that serve as social interaction tools for bacteria, enabling them to synchronize their collective actions in a density-dependent manner through quorum sensing (QS). The QS activity from epiphytic bacteria of the red macroalgae Porphyra haitanensis, along with its involvement in biofilm formation and regulation, remains unexplored in prior scientific inquiries. Therefore, this study explores the AHL signal molecules produced by epiphytic bacteria. The bacterium isolated from the surface of P. haitanensis was identified as Pseudoalteromonas galatheae by 16s rRNA gene sequencing and screened for AHLs using two AHL reporter strains, Agrobacterium tumefaciens A136 and Chromobacterium violaceum CV026. The crystal violet assay was used for the biofilm-forming phenotype. The inferences revealed that P. galatheae produces four different types of AHL molecules, i.e., C4-HSL, C8-HSL, C18-HSL, and 3-oxo-C16-HSL, and it was observed that its biofilm formation phenotype is regulated by QS molecules. This is the first study providing insights into the QS activity, diverse AHL profile, and regulatory mechanisms that govern the biofilm formation phenotype of P. galatheae. These findings offer valuable insights for future investigations exploring the role of AHL producing epiphytes and biofilms in the life cycle of P. haitanensis. [ABSTRACT FROM AUTHOR]

The Agrobacterium growth pole ring (GPR) protein forms a hexameric ring at the growth pole (GP) that is essential for polar growth. GPR is large (2,115 amino acids) and contains 1,700 amino acids of continuous α-helices. To dissect potential GPR functional domains, we created deletions of regions with similarity to human apolipoprotein A-IV (396 amino acids), itself composed of α-helical domains. We also tested deletions of the GPR C terminus. Deletions were inducibly expressed as green fluorescent protein (GFP) fusion proteins and tested for merodiploid interference with wild-type (WT) GPR function, for partial function in cells lacking GPR, and for formation of paired fluorescent foci (indicative of hexameric rings) at the GP. Deletion of domains similar to human apolipoprotein A-IV in GPR caused defects in cell morphology when expressed in trans to WT GPR and provided only partial complementation to cells lacking GPR. Agrobacterium-specific domains A-IV-1 and A-IV-4 contain predicted coiled coil (CC) regions of 21 amino acids; deletion of CC regions produced severe defects in cell morphology in the interference assay. Mutants that produced the most severe effects on cell shape also failed to form paired polar foci. Modeling of A-IV-1 and A-IV-4 reveals significant similarity to the solved structure of human apolipoprotein A-IV. GPR C-terminal deletions profoundly blocked complementation. Finally, peptidoglycan (PG) synthesis is abnormally localized circumferentially in cells lacking GPR. The results support the hypothesis that GPR plays essential roles as an organizing center for membrane and PG synthesis during polar growth.IMPORTANCE Bacterial growth and division are extensively studied in model systems (Escherichia coli, Bacillus subtilis, and Caulobacter crescentus) that grow by dispersed insertion of new cell wall material along the length of the cell. An alternative growth mode-polar growth-is used by some Actinomycetales and Proteobacteria species. The latter phylum includes the family Rhizobiaceae, in which many species, including Agrobacterium tumefaciens, exhibit polar growth. Current research aims to identify growth pole (GP) factors. The Agrobacterium growth pole ring (GPR) protein is essential for polar growth and forms a striking hexameric ring structure at the GP. GPR is long (2,115 amino acids), and little is known about regions essential for structure or function. Genetic analyses demonstrate that the C terminus of GPR, and two internal regions with homology to human apolipoproteins (that sequester lipids), are essential for GPR function and localization to the GP. We hypothesize that GPR is an organizing center for membrane and cell wall synthesis during polar growth.

Plants rely on innate immune systems to defend against a wide variety of biotic attackers. Key components of innate immunity include cell-surface pattern-recognition receptors (PRRs), which recognize pest- and pathogen-associated molecular patterns (PAMPs). Unlike other classes of receptors that often have visible cell-death immune outputs upon activation, PRRs generally lack rapid methods for assessing function. Here, we describe a genetically encoded bioluminescent reporter of immune activation by heterologously expressed PRRs in the model organism Nicotiana benthamiana. We characterized N. benthamiana transcriptome changes in response to Agrobacterium tumefaciens and subsequent PAMP treatment to identify pattern-triggered immunity (PTI)-associated marker genes, which were then used to generate promoter-luciferase fusion fungal bioluminescence pathway (FBP) constructs. A reporter construct termed pFBP_2xNbLYS1::LUZ allows for robust detection of PTI activation by heterologously expressed PRRs. Consistent with known PTI signaling pathways, reporter activation by receptor-like protein (RLP) PRRs is dependent on the known adaptor of RLP PRRs, i.e., SOBIR1. The FBP reporter minimizes the amount of labor, reagents, and time needed to assay function of PRRs and displays robust sensitivity at biologically relevant PAMP concentrations, making it ideal for high throughput screens. The tools described in this paper will be powerful for investigations of PRR function and characterization of the structure-function of plant cell-surface receptors. [Graphic: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.