Infection behavior of Listeria monocytogenes on iceberg lettuce (Lactuca sativa var. capitata).

Iceberg lettuce among leafy vegetables is susceptible to contamination with foodborne pathogens, posing a risk of food microbial safety. Listeria monocytogenes (L. monocytogenes) is a highly lethal pathogen that can survive and proliferate on leafy vegetables. In this paper, the contamination stage, attachment site, internalization pathway, proliferation process, extracellular substance secretion and virulence factors expression of L. monocytogenes on iceberg lettuce were researched. Results showed that the contamination stage of L. monocytogenes on iceberg lettuce was 0-20 min, the proliferation stage was after 20 min. The attachment tissues were stomata and winkles. The internalization distance of L. monocytogenes in the midrib was farther than that in the leaf blade. They enhanced the movement ability of cells by up-regulating the expression of flaA and motA genes, and enhanced the adhesion ability of cells by up-regulating the expression of actA and inla genes, which was beneficial to the proliferation. During proliferation, cells gradually secreted extracellular substances to promote the biofilm formation on iceberg lettuce. The formation of biofilms experienced: individual bacteria, cell aggregation and biofilm maturation. Biofilms were more likely to form on the leaf blade of iceberg lettuce.

Proximity-enabled covalent binding of IL-2 to IL-2Rα selectively activates regulatory T cells and suppresses autoimmunity.

Interleukin-2 (IL-2) is a pleiotropic cytokine that orchestrates bidirectional immune responses via regulatory T cells (Tregs) and effector cells, leading to paradoxical consequences. Here, we report a strategy that exploited genetic code expansion-guided incorporation of the latent bioreactive artificial amino acid fluorosulfate-L-tyrosine (FSY) into IL-2 for proximity-enabled covalent binding to IL-2Rα to selectively promote Treg activation. We found that FSY-bearing IL-2 variants, such as L72-FSY, covalently bound to IL-2Rα via sulfur-fluoride exchange when in proximity, resulting in persistent recycling of IL-2 and selectively promoting the expansion of Tregs but not effector cells. Further assessment of L72-FSY-expanded Tregs demonstrated that L72-FSY maintained Tregs in a central memory phenotype without driving terminal differentiation, as demonstrated by simultaneously attenuated expression of lymphocyte activation gene-3 (LAG-3) and enhanced expression of programmed cell death protein-1 (PD-1). Subcutaneous administration of L72-FSY in murine models of pristane-induced lupus and graft-versus-host disease (GvHD) resulted in enhanced and sustained therapeutic efficacy compared with wild-type IL-2 treatment. The efficacy of L72-FSY was further improved by N-terminal PEGylation, which increased its circulatory retention for preferential and sustained effects. This proximity-enabled covalent binding strategy may accelerate the development of pleiotropic cytokines as a new class of immunomodulatory therapies.

CARs: a new approach for the treatment of autoimmune diseases.

The development of chimeric antigen receptor (CAR)-based therapeutic interventions represented a breakthrough in cancer treatment. Following the success of the CAR-T-cell strategy, this novel therapeutic approach has been applied to other diseases, including autoimmune diseases. Using CAR-T cells to deplete pathological immune cells (i.e., B cells, autoreactive B or T cells, and accessory antigen-presenting cells (APCs)) has resulted in favorable outcomes in diseases characterized by excessive autoantibody levels or hyperactive lymphocyte cell numbers. The importance of immunosuppressive regulatory T cells (Tregs) in restoring immune tolerance has been well established, and CAR-Tregs have shown promising therapeutic potential in treating autoimmune diseases. Moreover, prior experience from the cancer field has provided sufficient paradigms for understanding how to optimize the structure and function of CARs to improve their function, persistence, stability and safety. In this review, we describe the potential application of CAR-T cells and CAR-Tregs in the treatment of autoimmune diseases, and we summarize the currently available strategies of gene editing and synthetic biological tools that have improved the practical application of CAR-based therapies.

The single-cell landscape of cystic echinococcosis in different stages provided insights into endothelial and immune cell heterogeneity.

Introduction: Hydatid cysts and angiogenesis are the key characteristics of cystic echinococcosis, with immune cells and endothelial cells mediating essential roles in disease progression. Recent single-cell analysis studies demonstrated immune cell infiltration after Echinococcus granulosus infection, highlighting the diagnostic and therapeutic potential of targeting certain cell types in the lesion microenvironment. However, more detailed immune mechanisms during different periods of E. granulosus infection were not elucidated. Methods: Herein, we characterized immune and endothelial cells from the liver samples of mice in different stages by single-cell RNA sequencing. Results: We profiled the transcriptomes of 45,199 cells from the liver samples of mice at 1, 3, and 6 months after infection (two replicates) and uninfected wild-type mice. The cells were categorized into 26 clusters with four distinct cell types: natural killer (NK)/T cells, B cells, myeloid cells, and endothelial cells. An SPP1+ macrophage subset with immunosuppressive and pro-angiogenic functions was identified in the late infection stage. Single-cell regulatory network inference and clustering (SCENIC) analysis suggested that Cebpe, Runx3, and Rora were the key regulators of the SPP1+ macrophages. Cell communication analysis revealed that the SPP1+ macrophages interacted with endothelial cells and had pro-angiogenic functions. There was an obvious communicative relationship between SPP1+ macrophages and endothelial cells via Vegfa-Vegfr1/Vegfr2, and SPP1+ macrophages interacted with other immune cells via specific ligand-receptor pairs, which might have contributed to their immunosuppressive function. Discussion: Our comprehensive exploration of the cystic echinococcosis ecosystem and the first discovery of SPP1+ macrophages with infection period specificity provide deeper insights into angiogenesis and the immune evasion mechanisms associated with later stages of infection.

Research advances on the contamination of vegetables by Enterohemorrhagic Escherichia coli: pathways, processes and interaction.

Enterohemorrhagic Escherichia coli is considered one of the primary bacterial pathogens that cause foodborne diseases because it can survive in meat, vegetables and so on. Understanding of the effect of vegetable characteristics on the adhesion and proliferation process of EHEC is necessary to develop control measures. In this review, the amount and methods of adhesion, the internalization pathway and proliferation process of EHEC have been described during the vegetable contamination. Types, cultivars, tissue characteristics, leaf age, and damage degree can affect EHEC adhesion on vegetables. EHEC cells contaminate the root surface of vegetables through soil and further internalize. It can also contaminate the stem scar tissue of vegetables by rain or irrigation water and internalize the vertical axis, as well as the stomata, necrotic lesions and damaged tissues of vegetable leaves. After EHEC adhered to the vegetables, they may further proliferate and form biofilms. Leaf and fruit tissues were more sensitive to biofilm formation, and shedding rate of biofilms on epidermis tissue was faster. Insights into the mechanisms of vegetable contamination by EHEC, including the role of exopolysaccharides and proteins responsible for movement, adhesion and oxidative stress response could reveal the molecular mechanism by which EHEC contaminates vegetables.

Genotyping and subtyping of Cryptosporidium spp. and Giardia duodenalis isolates from two wild rodent species in Gansu Province, China.

Cryptosporidium spp. and Giardia duodenalis are commonly detected intestinal protozoa species in humans and animals, contributing to global gastroenteritis spread. The present study examined the prevalence and zoonotic potential of Cryptosporidium spp. and G. duodenalis in Himalayan marmots and Alashan ground squirrels in China's Qinghai-Tibetan Plateau area (QTPA) for the first time. Four hundred ninety-eight intestinal content samples were collected from five counties of QTPA of Gansu province, China. All samples were examined for Cryptosporidium spp. and G. duodenalis by PCR amplification. The resultant data were statistically analyzed by chi-square, Fisher's test and Bonferroni correction using SPSS software 25. 0. Cryptosporidium positive samples were further subtyped through analysis of the 60-kDa glycoprotein (gp60) gene sequence. A total of 11 and 8 samples were positive for Cryptosporidium spp. and G. duodenalis, respectively. Prevalence of Cryptosporidium spp. and G. duodenalis were 2.5% (10/399) and 1.5% (6/399) in Himalayan marmots, 1.0% (1/99) and 2.0% (2/99) in Alashan ground squirrels, respectively. Sequence analysis confirmed the presence of C. rubeyi (n = 2), ground squirrel genotype II (n = 7), chipmunk genotype V (n = 1) and horse genotype (n = 1). The horse genotype was further subtyped as novel subtype VIbA10. G. duodenalis zoonotic assemblages A (n = 1), B (n = 6), E (n = 1) were identified in the present study. This is the first study to identify Cryptosporidium spp. and G. duodenalis in Himalayan marmots and Alashan ground squirrels, suggesting the potential zoonotic transmission of the two pathogens in QTPA.

Investigation on the Microbial Diversity of Fresh-Cut Lettuce during Processing and Storage Using High Throughput Sequencing and Their Relationship with Quality.

Microbial community distribution in vegetables can affect their quality. This study analyzed the distribution of the microbial community at various stages during processing and storage with the microbial diversity analysis, and evaluated the correlation between the dominant bacteria and sensory quality of lettuce using correspondence analysis with multiple regression analysis. Results showed that the process of washing, cutting, then disinfection and dewatering could change the community distribution and dominant bacteria in lettuce, and maintain better texture, morphology, aroma, color qualities of lettuce. The total number of colonies and relative abundance of Xanthomonas in fresh-cut lettuce decreased, while Afipia and Ralstonia increased during processing and pre-storage (storage for 6 h, 12 h and 1 d). After storage for 3 d, the total number of colonies in lettuce increased (more than 5 log CFU/g), especially the relative abundance of Pseudomonas, which led to the obvious deterioration of the sensory quality of lettuce. Throughout the process, the number of Bacillus cereus, Staphylococcus aureus, and E. coli was less than 100 CFU/g and 3 MPN/g. The number of typical pathogenic bacteria, Salmonella, Listeria monocytogenes and E. coli O157:H7, was below the detection limit. Overall, the prevention and control of psychrotrophic Pseudomonas in lettuce was still necessary. These results will provide useful information for the fresh-cut lettuce industry.

Adhesion mechanism and biofilm formation of Escherichia coli O157:H7 in infected cucumber (Cucumis sativus L.).

Cucumber is usually eaten as a raw vegetable and easily contaminated by pathogenic microorganisms; the contamination process includes colonization, proliferation, and biofilm formation. In this study, plate counting was used to determine the stage of E. coli O157:H7 colonization/proliferation in cucumber epidermis and fruit. Expression of E. coli genes associated with adhesion, movement and oxidative stress response during colonization and proliferation in cucumber was evaluated with fluorescence real-time quantitative PCR. Scanning electron microscopy imaging was used to observe biofilm formation over time in different cucumber tissues at 4 °C and 25 °C. During colonization (at 0-45 and 0-30 min in epidermis and fruit, respectively), escV, fliC, espA, escN, espF, espG, espZ, nleA, tir, and ycbR genes were upregulated. The escC was downregulated, while map and espH expression did not vary. During proliferation (after 45 and 30 min in epidermis and fruit, respectively), fliC was downregulated, whereas the outer membrane protein intimin gene and oxidative stress genes rpoS and sodB were upregulated. During storage, 25 °C was more favorable for biofilm formation than 4 °C. The ability of biofilm formation on the vascular system was the strongest, and the biofilm on epidermis sloughed off earlier than that on other tissues. Clarifying the process of E. coli O157:H7 contaminating cucumbers provided useful information for the development of prevention and control methods of fresh-cut cucumber.

Use of aqueous ozone rinsing to improve the disinfection efficacy and shorten the processing time of ultrasound-assisted washing of fresh produce.

In minimal processing industry, chlorine is widely used in the disinfection process and ultrasound (US) increases the disinfection efficacy of chlorine and reduces the cross-contamination incidence during washing. Tap water (TW), which has no disinfection effect, is generally used to rinse off sanitizer residues on the surface of disinfected fresh-cut vegetables. In this study, aqueous ozone (AO), a low-cost and residue-free sanitizer, was used to replace TW rinsing in combination with US (28 kHz)-chlorine (free chlorine [FC] at 10 ppm, a concentration recommended for industrial use) for the disinfection of fresh-cut lettuce as a model. US-chlorine (40 s) + 2.0 ppm AO (60 s) treatment resulted in browning spots on lettuce surface at the end of storage. In contrast, US-chlorine (40 s) + 1.0 ppm AO (60 s) did not lead to deterioration of the sensory quality (sensory crispness, color, and flavor) and a change in total color difference, and the activities of browning-related enzymes were significantly lower. Moreover, US-chlorine (40 s) + 1.0 ppm of AO (60 s) treatment led to significantly lower counts of Escherichia coli O157:H7, Salmonella Typhimurium, aerobic mesophilic (AMC), and molds and yeasts (M&Y) on days 0-7 than US-chlorine (60 s) + TW (60 s) and single 1.0 ppm AO (120 s) treatments, suggesting that AO provided an additional disinfection effect over TW, while reducing the overall processing time by 20 s. Cell membrane permeability analysis (alkaline phosphatase, protein, nucleotide, and adenosine triphosphate leakage) showed that the combination with 1.0 ppm AO caused more severe cell membrane damage in E. coli O157:H7 and S. Typhimurium, explaining the higher disinfection efficacy. 16S rRNA sequencing revealed that following US-chlorine (40 s) + 1.0 ppm of AO (60 s) treatment, Massilia and Acinetobacter had higher relative abundances (RAs) on day 7 than after US-chlorine (60 s) + TW (60 s) treatment, whereas the RAs of Escherichia-Shigella was significantly lower, indicating that the former treatment has a superior capacity in maintaining a stable microbial composition. This explains from an ecological point of view why US-chlorine (40 s) + 1.0 ppm of AO (60 s) led to the lowest AMC and M&Y counts during storage. The study results provide evidence that AO has potential as an alternative to TW rinsing to increase the disinfection efficacy of US-chlorine.

Effect of NUDT15 polymorphisms on early hematological safety of low-dose azathioprine in Chinese patients with pemphigus vulgaris: A prospective cohort study.

Azathioprine (AZA) is the preferred immunosuppressant for treating pemphigus vulgaris (PV), with discontinuation mainly attributed to hematological adverse events (AE). Reportedly, nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) polymorphisms have been strongly associated with thiopurine-induced leukopenia. To investigate hematological AE of low-dose AZA based on NUDT15 genotypes among patients with PV, a prospective cohort study was conducted in patients with PV, followed-up for the first 8 weeks after AZA administration. All patients were divided into wild homozygous and heterozygous NUDT15 groups. Both groups initiated AZA at low dose (50 mg/day) and continued with different dose-escalating approaches. Bone marrow suppression was considered the principal outcome. Overall, 62 patients with PV were enrolled (48 in the wild homozygous NUDT15 group vs. 14 in the heterozygous NUDT15 group). Except for median maintenance doses of AZA, no statistically significant differences were observed between the two groups in terms of age, sex, white blood cells, neutrophil count, platelet count, hemoglobin level, median final doses of corticosteroids (mg prednisone equivalent), pemphigus disease area index, and anti-desmoglein 1/3 autoantibodies. In both groups, patients presented similar hematological AE and treatment responses after administration of different low-dose AZA treatment strategies. Low-dose AZA based on NUDT15 genotypes can reduce the risk of early hematological AE among patients with PV.

Inhibitory effect of modified atmosphere packaging on Escherichia coli O157:H7 in fresh-cut cucumbers (Cucumis sativus L.) and effectively maintain quality during storage.

Modified atmosphere packaging (MAP) can inhibit microbial growth and prolong shelf life of fresh-cut cucumbers. This study compared the effects of different packaging gases on the growth of E. coli O157:H7 and sensory characteristics of fresh-cut cucumbers. Changes in key movement, adhesion, and oxidative stress genes expression of strain under optimal MAP and air were determined. Cell population density, the extracellular carbohydrate complex content and expression of curli fimbriae were evaluated. Results revealed that the growth of E. coli O157:H7 in fresh-cut cucumbers could be effectively inhibited under MAP (atmosphere = 2% O2, 7% CO2, 91% N2), and better maintained the sensory characteristics. Furthermore, the inhibition mechanism was revealed by inhibiting the expression of movement (fliC), adhesion (eaeA) and oxidative stress (rpoS and sodB) genes in E. coli O157:H7, reducing biofilm formation, extracellular carbohydrate production and curli fimbriae expression. Proper MAP can maintain the quality and safety of fresh-cut cucumbers.

Site-specific PEGylation of interleukin-2 enhances immunosuppression via the sustained activation of regulatory T cells.

The preferential activation of regulatory T (Treg) cells by interleukin-2 (IL-2), which selectively binds to the trimeric IL-2 receptor (IL-2R) on Treg cells, makes this cytokine a promising therapeutic for the treatment of autoimmune diseases. However, IL-2 has a narrow therapeutic window and a short half-life. Here, we show that the pharmacokinetics and half-life of IL-2 can be substantially improved by orthogonally conjugating the cytokine to poly(ethylene glycol) (PEG) moieties via a copper-free click reaction through the incorporation of azide-bearing amino acids at defined sites. Subcutaneous injection of a PEGylated IL-2 that optimally induced sustained Treg-cell activation and expansion over a wide range of doses through highly selective binding to trimeric IL-2R led to enhanced therapeutic efficacy in mouse models of lupus, collagen-induced arthritis and graft-versus-host disease without compromising the immune defences of the host against viral infection. Site-specific PEGylation could be used more generally to engineer cytokines with improved therapeutic performance for the treatment of autoimmune diseases.

Monitoring of transfer and internalization of Escherichia coli from inoculated knives to fresh cut cucumbers (Cucumis sativus L.) using bioluminescence imaging.

Slicing may cause the risk of cross-contamination in cucumber. In this study, knife inoculated with Escherichia coli (E. coli) was used to cut cucumbers, bioluminescence imaging (BLI) was used to visualize the possible distribution and internalization of E. coli during cutting and storage. Results showed that the initial two slices resulted in greater bacterial transfer. The bacterial transfer exhibited a fluctuating decay trend, E. coli was most distributed at the initial cutting site. The contaminated area on the surface of cucumber slices decreased during the storage period, which can be attributed to the death and internalization of E. coli. The maximum internalization distance of E. coli was about 2-3 mm, and did not further spread after 30 min from inoculation. Hence, our results provide useful information for risk management in both home and industrial environment.

Elucidating Escherichia Coli O157:H7 Colonization and Internalization in Cucumbers Using an Inverted Fluorescence Microscope and Hyperspectral Microscopy.

Contamination of fresh cucumbers (Cucumis sativus L.) with Escherichia coli O157:H7 can impact the health of consumers. Despite this, the pertinent mechanisms underlying E. coli O157:H7 colonization and internalization remain poorly documented. Herein we aimed to elucidate these mechanisms in cucumbers using an inverted fluorescence microscope and hyperspectral microscopy. We observed that E. coli O157:H7 primarily colonized around the stomata on cucumber epidermis without invading the internal tissues of intact cucumbers. Once the bacterial cells had infiltrated into the internal tissues, they colonized the cucumber placenta and vascular bundles (xylem vessels, in particular), and also migrated along the xylem vessels. Moreover, the movement rate of E. coli O157:H7 from the stalk to the flower bud was faster than that from the flower bud to the stalk. We then used hyperspectral microscope imaging to categorize the infiltrated and uninfiltrated areas with high accuracy using the spectral angle mapper (SAM) classification method, which confirmed the results obtained upon using the inverted fluorescence microscope. We believe that our results are pivotal for developing science-based food safety practices, interventions for controlling E. coli O157:H7 internalization, and new methods for detecting E. coli O157:H7-plant interactions.

Reduction of Escherichia coli O157:H7, Listeria monocytogenes, and Naturally Present Microbe Counts on Lettuce using an Acid Mixture of Acetic and Lactic Acid.

Lactic acid (LA) and acetic acid (AA) are independently used to disinfect fresh leaf vegetables. LA has a higher efficacy but costs more than AA. Herein, we compared the disinfection efficacy of LA, AA, and their mixture on lettuce to determine whether the cheaper acid mixture shows similar or more efficacy than LA. Quality analysis indicated that the acid mixture and individual acids did not cause additional loss of instrument color and polyphenolic content compared with that of the control; however, visible defects were observed at AA concentrations exceeding 0.8%. Analysis of Escherichia coli O157:H7, Listeria monocytogenes, and naturally present microbes (aerobic mesophilic and psychrotrophic bacteria, coliforms, molds, and yeasts) showed that the acid mixture led to the highest reduction in microbial count during storage. 16S rRNA sequencing was further employed to understand the effects of the acid mixture and individual acids on lettuce microbial ecology. During storage, the acid mixture and individual acids significantly decreased the abundance of Massilia spp. and Alkanindiges spp. but there was a marked increase in Escherichia-Shigella abundance (LA: 0.003-58.82%; AA: 0.01-55.34%; acid mixture: undetected to 50.71%; control: 0.007-33.09%), indicating that acid disinfection altered the microbial ecology to stimulate Escherichia-Shigella growth. These results enhance our understanding of the relationship between lettuce disinfection and ecological changes.

Reduction of Escherichia coli O157:H7 and naturally present microbes on fresh-cut lettuce using lactic acid and aqueous ozone.

Lactic acid (LA) is an effective sanitizer for disinfection of fresh produce. Tap water is generally used to wash disinfected fresh produce because sanitizer residues negatively affect the quality and organoleptic properties of the produce. However, tap water is ineffective for secondary disinfection compared with sanitizers. Thus, we propose a disinfection method using LA plus aqueous ozone (AO), an oxidizing sanitizer that does not lead to secondary residue. We compared the proposed method of 1% LA (90 s) plus 1 mg L-1 AO (30 s) or 2 mg L-1 AO (30 s) with the traditional method of 100 ppm chlorine (120 s) or 1% LA (120 s) plus tap water (30 s) and 2 mg L-1 AO (150 s). Microbial analysis showed that LA plus AO led to the greatest reductions in microbes (Escherichia coli O157:H7, aerobic mesophilic counts, aerobic psychrophilic counts, moulds, and yeasts) during storage (0-5 days at 5 °C). Quality analysis (colour, sensory qualities, electrolyte leakage, polyphenolic content, and weight loss) showed that LA + AO did not cause additional quality loss compared with tap water treatment. These results indicate that the hurdle technology proposed (LA plus AO) has a good potential for use in fresh produce disinfection.