Rapid and Sensitive Detection of Shiga Toxin-Producing Escherichia coli (STEC) from Food Matrices Using the CANARY Biosensor Assay.

Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Previously, we developed a rapid, sensitive, and potentially portable assay that identified STEC by detecting Shiga toxin (Stx) using a B-cell based biosensor platform. We applied this assay to detect Stx2 present in food samples that have been implicated in previous STEC foodborne outbreaks (milk, lettuce, and beef). The STEC enrichment medium, modified Tryptone Soy Broth (mTSB), inhibited the biosensor assay, but dilution with the assay buffer relieved this effect. Results with Stx2a toxoid-spiked food samples indicated an estimated limit of detection (LOD) of ≈4 ng/mL. When this assay was applied to food samples inoculated with STEC, it was able to detect 0.4 CFU/g or 0.4 CFU/mL of STEC at 16 h post incubation (hpi) in an enrichment medium containing mitomycin C. Importantly, this assay was even able to detect STEC strains that were high expressors of Stx2 at 8 hpi. These results indicate that the STEC CANARY biosensor assay is a rapid and sensitive assay applicable for detection of STEC contamination in food with minimal sample processing that can complement the current Food Safety Inspection Service (US) methodologies for STEC.

Chemical Compositions of Lianqiao (Forsythia suspensa) Extracts and Their Potential Health Benefits.

This study evaluated the fruits of Forsythia suspensa (Lianqiao), an important economic crop, for the chemical components of its water and ethanol extracts, inhibitory effects on SARS-CoV-2 virus spike protein binding to ACE2, inhibition of ACE2 activity, and capacity to scavenge free radicals. A total of 42 compounds were tentatively identified in the extracts via HPLC-MS/MS analysis. The water extract showed a greater ACE2 inhibition but a weaker inhibition on SARS-CoV-2 spike protein binding to ACE2 than the ethanol extract on a per-botanical-weight-concentration basis. The phenolic content was found to be greater in the water extract at 45.19 mg GAE/g dry botanical weight than in the ethanol extract (6.89 mg GAE/g dry botanical). Furthermore, the water extract had greater scavenging capacities against HO●, DPPH●, and ABTS●+ at 448.48, 66.36, and 121.29 µmol TE/g dry botanical, respectively, as compared to that of the ethanol extract (154.04, 3.55, and 33.83 µmol TE/g dry botanical, respectively). These results warrant further research into, and the development of, the potential COVID-19-preventive applications of Lianqiao and its extracts.

Biomimetic Capsid-like Nanoshells Self-assembled from Homopolypeptides.

The preparation of capsid-like nanoshells and the elucidation of their formation pathways are crucial for the application potential of capsid-like nanomaterials. In this study, we have prepared biomimetic capsid-like nanoshells (CLNs) through the solution self-assembly of poly (ß-phenethyl-L-aspartate) homopolypeptide (PPLA). The formation of CLNs is governed by an aggregation-fusion mechanism. Initially, PPLA molecules self-assemble into small spherical assemblies as subunits and the initial nuclei are formed through fusing some subunits. Subsequently, additional subunits rapidly fuse onto these nuclei, leading to the growth of full or partial CLNs during the growth phase. Moreover, the suitable condition benefiting CLNs formation is clarified by a morphological phase diagram based on the initial PPLA concentration against water content. Molecular-level measurements suggest that the molecular flexibility of PPLA is a key factor in the arrangement and fusion of subunits for the formation of CLNs. These findings offer new perspectives for a deeper understanding of the formation pathways of capsid-like nanoshells derived from synthetic polymers.

Camrelizumab-induced anaphylactic reaction: a case report and literature review.

Camrelizumab is an immune checkpoint inhibitor clinically used to treat various types of tumours. In this study, the authors provided the first report of a case of an anaphylactic reaction induced by camrelizumab in the treatment of a patient with squamous cell carcinoma of the floor of the mouth. The patient, a 58-year-old man, was diagnosed with advanced squamous cell carcinoma of the floor of the mouth, with cancer infiltration and multiple metastases. He underwent treatment for nine cycles, in which cycles 1-5 he received camrelizumab, albumin-bound paclitaxel, and cisplatin (200 mg of camrelizumab each time, every 3 weeks), with no adverse reactions; in cycle 6, he received albumin-bound paclitaxel and cisplatin, with no adverse reactions; and in cycles 7-9, he received camrelizumab and albumin-bound paclitaxel. However, 30 min after 8th administration of camrelizumab (cycle 9), he suddenly developed sweating, a pale complexion, clamminess and cyanosis of the limbs (percutaneous arterial oxygen saturation [SpO2] = 82%, blood pressure [BP] = 79/49 mmHg, heart rate [HR] = 83 beats/min [bpm] and respiratory rate [RR) = 12 bpm). The patient underwent intravenous infusion of methylprednisolone (80 mg) combined with dopamine to boost the BP; he regained consciousness 20 min later, and many parts of his skin appeared smooth, with no desquamation and accompanied by itching erythema, especially on the upper limbs. Approximately 2 h after treatment, the patient's skin erythema subsided (vital sign monitoring results: SpO2 = 100%, BP = 122/84 mmHg, HR = 91 bpm and RR = 17 bpm); the patient did not complain about his obvious discomfort. Despite the rarity of acute anaphylactic reactions among immune-related adverse reactions, great importance should be given to anaphylactic reactions of camrelizumab due to its extensive clinical application.

Nomogram to predict ventilator-associated pneumonia in large vessel occlusion stroke after endovascular treatment: a retrospective study.

Background: Ventilator-Associated Pneumonia (VAP) severely impacts stroke patients' prognosis after endovascular treatment. Hence, this study created a nomogram to predict the occurrence of VAP after endovascular treatment. Methods: The individuals with acute ischemic stroke and large vessel occlusion (AIS-LVO) who received mechanical ventilation and endovascular therapy between July 2020 and August 2023 were included in this retrospective study. The predictive model and nomogram were generated by performing feature selection optimization using the LASSO regression model and multifactor logistic regression analysis and assessed the evaluation, verification and clinical application. Results: A total of 184 individuals (average age 61.85 ± 13.25 years, 73.37% male) were enrolled, and the rate of VAP occurrence was found to be 57.07%. Factors such as the Glasgow Coma Scale (GCS) score, duration of stay in the Intensive Care Unit (ICU), dysphagia, Fazekas scale 2 and admission diastolic blood pressure were found to be associated with the occurrence of VAP in the nomogram that demonstrating a strong discriminatory power with AUC of 0.862 (95% CI, 0.810-0.914), and a favorable clinical net benefit. Conclusion: This nomogram, comprising GCS score, ICU duration, dysphagia, Fazekas scale 2 and admission diastolic blood pressure, can aid clinicians in predicting the identification of high-risk patients for VAP following endovascular treatment in large vessel occlusion stroke.

Chemical Composition of Turmeric (Curcuma longa L.) Ethanol Extract and Its Antimicrobial Activities and Free Radical Scavenging Capacities.

Turmeric (Curcuma longa L.) is a perennial tuberous plant from the genus Curcuma (Zingiberaceae) and has been widely used in foods for thousands of years. The present study examined the ethanol extract of turmeric for its chemical composition, antimicrobial activity, and free radical scavenging properties. UHPLC-MS/MS analysis tentatively identified eight compounds in the turmeric extract. Potential antimicrobial effects of 0.1, 1.0, and 10 mg turmeric equivalents (TE)/mL were evaluated in vitro against a variety of Gram-negative bacteria (i.e., Escherichia coli, Klebsiella pneumoniae, and Pseudomonas sp.) and Gram-positive bacteria (i.e., Enterococcus faecalis, Listeria innocua, and Staphylococcus aureus). Concentrations of 0.1 and 1.0 mg TE/mL inhibited the growth of S. aureus and significantly suppressed that of Pseudomonas sp., E. faecalis, and L. innocua. The growth of all strains, including E. coli, was inhibited by 10 mg TE/mL. Moreover, free radical scavenging capacities were determined using HO●, ABTS●+, and DPPH● (HOSC, ABTS, and RDSC, respectively) radicals. The turmeric ethanol extract had a TPC value of 27.12 mg GAE/g, together with HOSC, RDSC, and ABTS values of 1524.59, 56.38, and 1.70 µmol TE/g, respectively. Our results suggest that turmeric extract has potential applications for use in functional foods to reduce microbial burdens and oxidative stress-related health problems.

Streptococcal pyrogenic exotoxin B is a superantigen that induces murine splenocyte proliferation and secretion of IL-2 and IFN-γ ex vivo.

Streptococcus pyogenes is a significant human pathogen, producing a range of virulence factors, including streptococcal pyrogenic exotoxin B (SpeB) that is associated with foodborne outbreaks. It was only known that this cysteine protease mediates cleavage of transmembrane proteins to permit bacterial penetration and is found in 25% of clinical isolates from streptococcal toxic shock syndrome patients with extreme inflammation. Its interaction with host and streptococcal proteins has been well characterized, but doubt remains about whether it constitutes a superantigen. In this study, for the first time it is shown that SpeB acts as a superantigen, similarly to other known superantigens such as staphylococcal enterotoxin A or streptococcal pyrogenic exotoxin type C, by inducing proliferation of murine splenocytes and cytokine secretion, primarily of interleukin-2 (IL-2), as shown by cytometric bead array analysis. IL-2 secretion was confirmed by enzyme-linked immunosorbent assay (ELISA) as well as secretion of interferon-γ. ELISA showed a dose-dependent relationship between SpeB concentration in splenocyte cells and IL-2 secretion levels, and it was shown that SpeB retains activity in milk pasteurized for 30 min at 63°C.

Genetic association between smoking and DLCO in idiopathic pulmonary fibrosis patients.

BACKGROUND: Observational studies have shown that smoking is related to the diffusing capacity of the lungs for carbon monoxide (DLCO) in individuals with idiopathic pulmonary fibrosis (IPF). Nevertheless, further investigation is needed to determine the causal effect between these two variables. Therefore, we conducted a study to investigate the causal relationship between smoking and DLCO in IPF patients using two-sample Mendelian randomization (MR) analysis. METHODS: Large-scale genome-wide association study (GWAS) datasets from individuals of European descent were analysed. These datasets included published lifetime smoking index (LSI) data for 462,690 participants and DLCO data for 975 IPF patients. The inverse-variance weighting (IVW) method was the main method used in our analysis. Sensitivity analyses were performed by MR‒Egger regression, Cochran's Q test, the leave-one-out test and the MR-PRESSO global test. RESULTS: A genetically predicted increase in LSI was associated with a decrease in DLCO in IPF patients [ORIVW = 0.54; 95% CI 0.32-0.93; P = 0.02]. CONCLUSIONS: Our study suggested that smoking is associated with a decrease in DLCO. Patients diagnosed with IPF should adopt an active and healthy lifestyle, especially by quitting smoking, which may be effective at slowing the progression of IPF.

Immature persimmon residue as a novel biosorbent for efficient removal of Pb(II) and Cr(VI) from wastewater: Performance and mechanisms.

Owing to the powerful affinity of tannin toward heavy metal ions, it is frequently immobilized on adsorbents to enhance their adsorption properties. However, natural adsorbents containing tannin have been overlooked owing to its water solubility. Herein, a novel natural adsorbent based on the immature persimmon residue (IPR) with soluble tannin removed was fabricated to eliminate Pb(II) and Cr(VI) in aquatic environments. The insoluble tannin in IPR endowed it with prosperous properties for eliminating Pb(II) and Cr(VI), and the IPR achieved maximum Pb(II) and Cr(VI) adsorption quantities of 68.79 mg/g and 139.40 mg/g, respectively. Kinetics and isothermal adsorption analysis demonstrated that the removal behavior was controlled by monolayer chemical adsorption. Moreover, the IPR exhibited satisfactory Pb(II) and Cr(VI) removal efficiencies even in the presence of multiple coexisting ions and showed promising regeneration potential after undergoing five consecutive cycles. Additionally, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) analysis unveiled that the elimination mechanisms were primarily electrostatic attraction, chelation and reduction. Overall, the IPR, as a tannin-containing biosorbent, was verified to possess substantial potential for heavy metal removal, which can provide new insights into the development of novel natural adsorbents from the perspective of waste resource utilization.

Development of a Rapid and Sensitive CANARY Biosensor Assay for the Detection of Shiga Toxin 2 from Escherichia coli.

Shiga-toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). The current Food Safety Inspection Service (FSIS) testing methods for STEC use the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) protocol, which includes enrichment, cell plating, and genomic sequencing and takes time to complete, thus delaying diagnosis and treatment. We wanted to develop a rapid, sensitive, and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology. Five potential biosensor cell lines were evaluated for their ability to detect Stx2. The results using the best biosensor cell line (T5) indicated that this biosensor was stable after reconstitution with assay buffer covered in foil at 4 °C for up to 10 days with an estimated limit of detection (LOD) of ≈0.1-0.2 ng/mL for days up to day 5 and ≈0.4 ng/mL on day 10. The assay detected a broad range of Stx2 subtypes, including Stx2a, Stx2b, Stx2c, Stx2d, and Stx2g but did not cross-react with closely related Stx1, abrin, or ricin. Additionally, this assay was able to detect Stx2 in culture supernatants of STEC grown in media with mitomycin C at 8 and 24 h post-inoculation. These results indicate that the STEC CANARY biosensor developed in this study is sensitive, reproducible, specific, rapid (≈3 min), and may be applicable for surveillance of the environment and food to protect public health.

Chemical Compositions of Scutellaria baicalensis Georgi. (Huangqin) Extracts and Their Effects on ACE2 Binding of SARS-CoV-2 Spike Protein, ACE2 Activity, and Free Radicals.

The water and ethanol extracts of huangqin, the roots of Scutellaria baicalensis Georgi. with potential antiviral properties and antioxidant activities, were investigated for their chemical profiles and their abilities to interfere with the interaction between SARS-CoV-2 spike protein and ACE2, inhibiting ACE2 activity and scavenging free radicals. A total of 76 compounds were tentatively identified from the extracts. The water extract showed a greater inhibition on the interaction between SARS-CoV-2 spike protein and ACE2, but less inhibition on ACE2 activity than that of the ethanol extract on a per botanical weight concentration basis. The total phenolic content was 65.27 mg gallic acid equivalent (GAE)/g dry botanical and the scavenging capacities against HO●, DPPH●, and ABTS●+ were 1369.39, 334.37, and 533.66 µmol trolox equivalent (TE)/g dry botanical for the water extract, respectively. These values were greater than those of the ethanol extract, with a TPC of 20.34 mg GAE/g, and 217.17, 10.93, and 50.21 µmol TE/g against HO●, DPPH●, and ABTS●+, respectively. The results suggested the potential use of huangqin as a functional food ingredient in preventing COVID-19.

Alterations in mitochondrial structure and function in response to environmental temperature changes in Apostichopus japonicus.

Global temperatures have risen as a result of climate change, and the resulting warmer seawater will exert physiological stresses on many aquatic animals, including Apostichopus japonicus. It has been suggested that the sensitivity of aquatic poikilothermal animals to climate change is closely related to mitochondrial function. Therefore, understanding the interaction between elevated temperature and mitochondrial functioning is key to characterizing organisms' responses to heat stress. However, little is known about the mitochondrial response to heat stress in A. japonicus. In this work, we investigated the morphological and functional changes of A. japonicus mitochondria under three representative temperatures, control temperature (18 °C), aestivation temperature (25 °C) and heat stress temperature (32 °C) temperatures using transmission electron microscopy (TEM) observation of mitochondrial morphology combined with proteomics and metabolomics techniques. The results showed that the mitochondrial morphology of A. japonicus was altered, with decreases in the number of mitochondrial cristae at 25 °C and mitochondrial lysis, fracture, and vacuolization at 32 °C. Proteomic and metabolomic analyses revealed 103 differentially expressed proteins and 161 differential metabolites at 25 °C. At 32 °C, the levels of 214 proteins and 172 metabolites were significantly altered. These proteins and metabolites were involved in the tricarboxylic acid (TCA) cycle, substance transport, membrane potential homeostasis, anti-stress processes, mitochondrial autophagy, and apoptosis. Furthermore, a hypothetical network of proteins and metabolites in A. japonicus mitochondria in response to temperature changes was constructed based on proteomic and metabolomic data. These results suggest that the dynamic regulation of mitochondrial energy metabolism, resistance to oxidative stress, autophagy, apoptosis, and mitochondrial morphology in A. japonicus may play important roles in the response to elevated temperatures. In summary, this study describes the response of A. japonicus mitochondria to temperature changes from the perspectives of morphology, proteins, and metabolites, which provided a better understanding the mechanisms of mitochondrial regulation under environment stress in marine echinoderms.

Inhibiting the NLRP3 Inflammasome with MCC950 Alleviates Neurological Impairment in the Brain of EAE Mice.

Multiple sclerosis (MS) is a chronic disease that is characterized by demyelination and neuronal damage. Experimental autoimmune encephalomyelitis (EAE) mice are used to model the disease progression of MS and mirror MS-like pathology. Previous researches have confirmed that inhibition of NLRP3 inflammasome significantly alleviated the severity of EAE mice and the demyelination of spinal cord, but its effect on neuronal damage and oligodendrocyte loss in the brain remains unclear. In this study, female C57BL/6 mice were immunized with MOG35-55 and PTX to establish experimental autoimmune encephalomyelitis (EAE) model. MCC950, a selective NLRP3 inflammasome inhibitor, was used to investigate the effect of NLRP3 inflammasome on the pathological changes and glial cell activation in the brain of EAE mice by immunohistochemistry. Our results demonstrated that MCC950 ameliorated the neuronal damage, demyelination, and oligodendrocyte loss in the brain of EAE mice. This protective effect of MCC950 may be attributed to its ability to suppress the activation of glial cells and prevents microglia polarization to M1 phenotype. Our work indicates that inhibition of NLRP3 inflammasome has the therapeutic effects of neuroprotection through immunomodulation and is a promising therapeutic strategy for MS.

A novel Shiga toxin 2a neutralizing antibody therapeutic with low immunogenicity and high efficacy.

Shiga toxin-producing Escherichia coli infections are difficult to treat due to the risk of antibiotic-induced stress upregulating the production of toxins, medical treatment is consequently limited to supportive care to prevent the development of hemolytic uremic syndrome (HUS). Here, we introduce a potentially therapeutic humanized mouse monoclonal antibody (Hu-mAb 2-5) targeting Stx2a, the most common Shiga toxin subtype identified from outbreaks. We demonstrate that Hu-mAb 2-5 has low immunogenicity in healthy adults ex vivo and high neutralizing efficacy in vivo, protecting mice from mortality and HUS-related tissue damage.

Symbionts in Hodgkinia-free cicadas and their implications for co-evolution between endosymbionts and host insects.

IMPORTANCE: Obligate symbionts in sap-sucking hemipterans are harbored in either the same or different organs, which provide a unique perspective for uncovering complicated insect-microbe symbiosis. Here, we investigated the distribution of symbionts in adults of 10 Hodgkinia-free cicada species of 2 tribes (Sonatini and Polyneurini) and the co-phylogeny between 65 cicada species and related symbionts (Sulcia and YLSs). We revealed that YLSs commonly colonize the bacteriome sheath besides the fat bodies in these two tribes, which is different with that in most other Hodgkinia-free cicadas. Co-phylogeny analyses between cicadas and symbionts suggest that genetic variation of Sulcia occurred in Sonatini and some other cicada lineages and more independent replacement events in the loss of Hodgkinia/acquisition of YLS in Cicadidae. Our results provide new information on the complex relationships between auchenorrhynchans and related symbionts.

Detection of Cleaved Stx2a in the Blood of STEC-Infected Patients.

Typical hemolytic uremic syndrome (HUS) is mainly caused by Shiga toxin-producing Escherichia coli (STEC) releasing Shiga toxin 2 (Stx2). Two different structures of this AB5 toxin have been described: uncleaved, with intact B and A chains, and cleaved, with intact B and a nicked A chain consisting of two fragments, A1 and A2, connected by a disulfide bond. Despite having the same toxic effect on sensitive cells, the two forms differ in their binding properties for circulating cells, serum components and complement factors, thus contributing to the pathogenesis of HUS differently. The outcome of STEC infections and the development of HUS could be influenced by the relative amounts of uncleaved or cleaved Stx2 circulating in patients' blood. Cleaved Stx2 was identified and quantified for the first time in four out of eight STEC-infected patients' sera by a method based on the inhibition of cell-free translation. Cleaved Stx2 was present in the sera of patients with toxins bound to neutrophils and in two out of three patients developing HUS, suggesting its involvement in HUS pathogenesis, although in association with other bacterial or host factors.

A Sensitive, Cell-Based Assay for Measuring Low-Level Biological Activity of α-Amanitin.

α-Amanitin is one of the primary toxins produced by the poisonous mushroom genus, Amanita. Because it is odorless and tasteless, it is an important cause of death from the consumption of misidentified mushrooms. To study the thermal stability of α-amanitin, novel cell-based assays were developed to measure the toxin's activity, based on the inhibition of RNA polymerase II by α-amanitin. First, an MTT-formazan cell viability assay was used to measure the biological activity of α-amanitin through the inhibition of cellular activity. This method can detect 10 µg/mL of α-amanitin in a time-dependent manner. Second, a more sensitive quantitative PCR approach was developed to examine its inhibition of viral replication. The new RT-qPCR assay enabled the detection of 100 ng/mL. At this level, α-amanitin still significantly reduced adenovirus transcription. Third, a simpler GFP expression-based assay was developed with an equal sensitivity to the RT-qPCR assay. With this assay, aqueous α-amanitin heated at 90 °C for 16 h or treated in the microwave for 3 min retained its biological activity when tested in HEK293 cells, but a slight reduction was observed when tested in Vero cells. Beyond detecting the activity of α-amanitin, the new method has a potential application for detecting the activity of other toxins that are RNA polymerase inhibitors.

Genomic and Phenotypic Characterization of Shiga Toxin-Producing Escherichia albertii Strains Isolated from Wild Birds in a Major Agricultural Region in California.

Escherichia albertii is an emerging foodborne pathogen. To better understand the pathogenesis and health risk of this pathogen, comparative genomics and phenotypic characterization were applied to assess the pathogenicity potential of E. albertii strains isolated from wild birds in a major agricultural region in California. Shiga toxin genes stx2f were present in all avian strains. Pangenome analyses of 20 complete genomes revealed a total of 11,249 genes, of which nearly 80% were accessory genes. Both core gene-based phylogenetic and accessory gene-based relatedness analyses consistently grouped the three stx2f-positive clinical strains with the five avian strains carrying ST7971. Among the three Stx2f-converting prophage integration sites identified, ssrA was the most common one. Besides the locus of enterocyte effacement and type three secretion system, the high pathogenicity island, OI-122, and type six secretion systems were identified. Substantial strain variation in virulence gene repertoire, Shiga toxin production, and cytotoxicity were revealed. Six avian strains exhibited significantly higher cytotoxicity than that of stx2f-positive E. coli, and three of them exhibited a comparable level of cytotoxicity with that of enterohemorrhagic E. coli outbreak strains, suggesting that wild birds could serve as a reservoir of E. albertii strains with great potential to cause severe diseases in humans.

Chemical Composition of Rosemary (Rosmarinus officinalis L.) Extract and Its Inhibitory Effects on SARS-CoV-2 Spike Protein-ACE2 Interaction and ACE2 Activity and Free Radical Scavenging Capacities.

This study evaluated the chemical composition of rosemary water extract (RWE) and its influence on mechanisms by which the SARS-CoV-2 virus enters into cells as a potential route for reducing the risk of COVID-19 disease. Compounds in RWE were identified using UHPLC-MS/MS. The inhibitory effect of RWE was then evaluated on binding between the SARS-CoV-2 spike protein (S-protein) and ACE2 and separately on ACE2 activity/availability. Additionally, total phenolic content (TPC) and free radical scavenging capacities of RWE against HO•, ABTS•+, and DPPH• were assessed. Twenty-one compounds were tentatively identified in RWE, of which tuberonic acid hexoside was identified for the first time in rosemary. RWE dose of 33.3 mg of rosemary equivalents (RE)/mL suppressed the interaction between S-protein and ACE2 by 72.9%, while rosmarinic and caffeic acids at 3.3 µmol/mL suppressed the interaction by 36 and 55%, respectively. RWE at 5.0, 2.5, and 0.5 mg of RE/mL inhibited ACE2 activity by 99.5, 94.5, and 68.6%, respectively, while rosmarinic acid at 0.05 and 0.01 µmol/mL reduced ACE2 activity by 31 and 8%, respectively. RWE had a TPC value of 72.5 mg GAE/g. The results provide a mechanistic basis on which rosemary may reduce the risk of SARS-CoV-2 infection and the development of COVID-19.

Enzymatic Cleavage of Stx2a in the Gut and Identification of Pancreatic Elastase and Trypsin as Possible Main Cleavers.

Shiga toxins (Stxs), especially the Stx2a subtype, are the major virulence factors involved in enterohemorrhagic Escherichia coli (EHEC)-associated hemolytic uremic syndrome (eHUS), a life-threatening disease causing acute kidney injury, especially in children. After oral transmission and colonization in the gut, EHEC release Stx. Intracellular cleavage of the Stx A subunit, when followed by reduction, boosts the enzymatic activity that causes damage to targeted cells. This cleavage was assumed to be mostly mediated by furin during Stx intracellular trafficking. To investigate whether this cleavage could occur in the intestine, even prior to entering target cells, Stx2a A subunit structure (intact or cleaved) was characterized after its exposure to specific host factors present in human stool. The molecular weight of Stx2a A subunit/fragments was determined by immunoblotting after electrophoretic separation under reducing conditions. In this study, it was demonstrated that Stx2a is cleaved by certain human stool components. Trypsin and chymotrypsin-like elastase 3B (CELA3B), two serine proteases, were identified as potential candidates that can trigger the extracellular cleavage of Stx2a A subunit directly after its secretion by EHEC in the gut. Whether the observed cleavage indeed translates to natural infections and plays a role in eHUS pathogenesis has yet to be determined. If so, it seems likely that a host's protease profile could affect disease development by changing the toxin's biological features.