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Abstract Analysis of amatoxins is of great importance as these cyclic peptides contribute to a high number of fatalities each year. Development of analytical approaches needs to focus on rapid, sensitive, and reliable methods. By establishing an affinity column chromatography-based assay using the monoclonal amanitin antibody AMA9G3 and liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) for the trace detection of α-, β-, and γ-amanitin in human urine samples to confirm ingestion, we report the first approach that extents the current status of amatoxin analysis. The presented procedure allows detection of amatoxins in human urine down to 1 ng/mL. The method was successfully validated qualitatively for α- and γ-amanitin according to international recommendations. A proof of concept was performed by analyzing 37 urine samples after suspected amatoxin consumption submitted for regular clinical toxicological analysis. Using this antibody-based enrichment strategy, acute amatoxin intoxications can be determined within 90 min and due to the high sensitivity and selectivity, a comparable approach using target specific antibodies may also be used for other toxicological relevant peptides.

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.

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.

The United States is a principal producer of tree nuts (almonds, pistachios, and walnuts), resulting in the generation of excess of tree-nutshell by-products each year, with few market outlets. A nutshell is an essential, lignocellulosic layer that protects a kernel (seed) from the environment during cultivation. The objective of this study was to develop nutshell by-products as herbicide delivery systems, which would not only enable sustainable weed control in fields but also increases nutshell value and reduce the cost of waste disposal. We recently identified a natural salicylaldehyde (SA) that emits volatiles with both herbicidal and antifungal properties. In this study, walnut shell particles saturated with 0.8 to 1.6 M SA were developed as delivery vehicles for SA to soil, which allowed for the controlled release of an SA fumigant for weed control. The pre- and post-emergent herbicidal efficacy of SA was investigated using model monocot (Lolium arundinaceum (Schreb.) Darbysh; turfgrass) and dicot (Brassica rapa var. pekinensis; Chinese cabbage) plants. We compared (1) the effects of different types of solvents for dissolving SA (dimethyl sulfoxide (DMSO) and ethanol (60%, v/v)), and (2) the effect of covering soil with plastic layers (i.e., soil pasteurization) or not covering soil during SA fumigation using nutshells. Results: In the pre-emergent herbicidal testing with the soil covered, the dicot plants exhibited levels of higher susceptibility to SA in DMSO emitted from nutshells when compared to the monocot plants. The seed germination frequencies in the dicots were 15% and 1% with 0.8 and 1.6 M SA, respectively, while those in the monocots were 32% and 18%, respectively, under the same test conditions. In the post-emergent herbicidal testing with the soil covered, the growth of both the monocot and dicot plants was completely prevented after 5 to 7 days of SA fumigation, resulting in the deaths of entire plants. It was noteworthy that in the post-emergent herbicidal testing, SA dissolved in ethanol (60%, v/v) completely disrupted the growth of the monocot and dicot plants as early as 3 days after SA emission from the nutshells, even without the soil being covered. Tree-nutshell particles could serve as effective SA delivery vehicles with controlled release capabilities for SA. The SA exhibited pre- and post-emergent herbicidal activities against the monocot and dicot plants at most growth stages. SA (0.8 and 1.6 M) dissolved in ethanol (60%, v/v) might exert a synergism for higher herbicidal activity after emission from nutshells. Since tree nuts capture/store a substantial amount of carbon over their life-cycles, the new and sustainable utility of using nutshells not only reduces carbon emissions but also valorizes tree-nut by-products, thus benefitting the tree-nut industry.

Abstract Background The clustering of Big Four contributors to morbidity and mortality—alcohol misuse, smoking, poor diet, and physical inactivity—may further elevate chronic health risk, but there is limited information about their specific combinations and associated health risks for racial/ethnic minority groups. We aimed to examine patterns of clustering in risk behaviors for White, Black, Hispanic, and Asian American adults and their associations with diabetes and hypertension. As these behaviors may be socioeconomically-patterned, we also examined associations between clustering and socioeconomic status (SES). Methods Latent class analyses and multinomial and logistic regressions were conducted using a nationally-representative sample of United States (US) adults ages 40–70 (N = 35,322) from Waves 2 (2004–2005) and 3 (2012–2013) of the National Epidemiologic Survey on Alcohol and Related Conditions. Obesity was used as a proxy for unhealthy diet. The outcomes were diabetes and hypertension. Results A relatively-healthy-lifestyle class was found only among White adults. Common patterns of unhealthy clustering were found across groups with some variations: the obese-inactive class among White, Black, and Hispanic adults (and the inactive class among Asian adults); the obese-inactive-smoking class among White, Black, and Hispanic adults; the smoking-risky-drinking class among White and Hispanic adults; and the smoking-risky-drinking-inactive class among Black and Asian adults. Positive associations of unhealthier clustering (having a greater number of risk behaviors) with lower SES (i.e., family income and education) and with health conditions were more consistent for Whites than for other groups. For racial minority groups, lower education than income was more consistently associated with unhealthy clusters. The associations between unhealthier clustering and diabetes and hypertension were less clear for Blacks and Asians than for Whites, with no significant association observed for Hispanics. Conclusion Concerted efforts to address clustered risk behaviors in most US adults, particularly in racial/ethnic minority groups given the high prevalence of unhealthy clustering, are warranted.

Abstract Background We previously reported that the tomato glycoalkaloid tomatine inhibited the growth of Trichomonas vaginalis strain G3, Tritrichomonas foetus strain D1, and Tritrichomonas foetus-like strain C1 that cause disease in humans and farm and domesticated animals. The increasing prevalence of antibiotic resistance requires development of new tools to enhance or replace medicinal antibiotics. Methods Wild tomato plants were harvested and divided into leaves, stems, and fruit of different colors: green, yellow, and red. Samples were freeze dried and ground with a handheld mill. The resulting powders were evaluated for their potential anti-microbial effects on protozoan parasites, bacteria, and fungi. A concentration of 0.02% (w/v) was used for the inhibition of protozoan parasites. A high concentration of 10% (w/v) solution was tested for bacteria and fungi as an initial screen to evaluate potential anti-microbial activity and results using this high concentration limits its clinical relevance. Results Natural powders derived from various parts of tomato plants were all effective in inhibiting the growth of the three trichomonads to varying degrees. Test samples from leaves, stems, and immature ‘green’ tomato peels and fruit, all containing tomatine, were more effective as an inhibitor of the D1 strain than those prepared from yellow and red tomato peels which lack tomatine. Chlorogenic acid and quercetin glycosides were present in all parts of the plant and fruit, while caffeic acid was only found in the fruit peels. Any correlation between plant components and inhibition of the G3 and C1 strains was not apparent, although all the powders were variably effective. Tomato leaf was the most effective powder in all strains, and was also the highest in tomatine. S. enterica showed a minor susceptibility while B. cereus and C. albicans fungi both showed a significant growth inhibition with some of the test powders. The powders inhibited growth of the pathogens without affecting beneficial lactobacilli found in the normal flora of the vagina. Conclusions The results suggest that powders prepared from tomato leaves, stems, and green tomato peels and to a lesser extent from peels from yellow and red tomatoes offer potential multiple health benefits against infections caused by pathogenic protozoa, bacteria, and fungi, without affecting beneficial lactobacilli that also reside in the normal flora of the vagina.

Equine-derived antitoxin (BAT®) is the only treatment for botulism from botulinum neurotoxin serotype G (BoNT/G). BAT® is a foreign protein with potentially severe adverse effects and is not renewable. To develop a safe, more potent, and renewable antitoxin, humanized monoclonal antibodies (mAbs) were generated. Yeast displayed single chain Fv (scFv) libraries were prepared from mice immunized with BoNT/G and BoNT/G domains and screened with BoNT/G using fluorescence-activated cell sorting (FACS). Fourteen scFv-binding BoNT/G were isolated with KD values ranging from 3.86 nM to 103 nM (median KD 20.9 nM). Five mAb-binding non-overlapping epitopes were humanized and affinity matured to create antibodies hu6G6.2, hu6G7.2, hu6G9.1, hu6G10, and hu6G11.2, with IgG KD values ranging from 51 pM to 8 pM. Three IgG combinations completely protected mice challenged with 10,000 LD50s of BoNT/G at a total mAb dose of 6.25 μg per mouse. The mAb combinations have the potential for use in the diagnosis and treatment of botulism due to serotype G and, along with antibody combinations to BoNT/A, B, C, D, E, and F, provide the basis for a fully recombinant heptavalent botulinum antitoxin to replace the legacy equine product.

Abstract Human trichomoniasis, caused by the pathogenic parasitic protozoan Trichomonas vaginalis, is the most common non-viral sexually transmitted disease that contributes to reproductive morbidity in affected women and possibly to prostate cancer in men. Tritrichomonas foetus strains cause the disease trichomoniasis in farm animals (cattle, bulls, pigs) and diarrhea in domestic animals (cats and dogs). Because some T. vaginalis strains have become resistant to the widely used drug metronidazole, there is a need to develop alternative treatments, based on safe natural products that have the potential to replace and/or enhance the activity of lower doses of metronidazole. To help meet this need, this overview collates and interprets worldwide reported studies on the efficacy of structurally different classes of food, marine, and medicinal plant extracts and some of their bioactive pure compounds against T. vaginalis and T. foetus in vitro and in infected mice and women. Active food extracts include potato peels and their glycoalkaloids α-chaconine and α-solanine, caffeic and chlorogenic acids, and quercetin; the tomato glycoalkaloid α-tomatine; theaflavin-rich black tea extracts and bioactive theaflavins; plant essential oils and their compounds (+)-α-bisabolol and eugenol; the grape skin compound resveratrol; the kidney bean lectin, marine extracts from algae, seaweeds, and fungi and compounds that are derived from fungi; medicinal extracts and about 30 isolated pure compounds. Also covered are the inactivation of drug-resistant T. vaginalis and T. foetus strains by sensitized light; anti-trichomonad effects in mice and women; beneficial effects of probiotics in women; and mechanisms that govern cell death. The summarized findings will hopefully stimulate additional research, including molecular-mechanism-guided inactivations and human clinical studies, that will help ameliorate adverse effects of pathogenic protozoa.

Abstract Background Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus. Previous studies have identified neutralizing antibodies from Zika patients that bind to quaternary epitopes across neighboring envelope (E) proteins, called E dimer epitopes (EDE). An asparagine-linked glycan on the “glycan loop” (GL) of the ZIKV envelope protein protects the functionally important “fusion loop” on the opposite E subunit in the dimer, and EDE antibodies have been shown to bind to both of these loops. Human EDE antibodies have been divided into two subclasses based on how they bind to the glycan loop region: EDE1 antibodies do not require glycosylation for binding, while EDE2 antibodies strongly rely on the glycan for binding. Methods ZIKV GL was expressed on tobacco mosaic virus nanoparticles. Mice were immunized with GL or full-length monomeric E and the immune response was analyzed by testing the ability of sera and monoclonal antibodies to bind to GL and to neutralize ZIKV in in vitro cellular assay. Results We report here the existence of ZIKV moderately neutralizing antibodies that bind to E monomers through epitopes that include the glycan loop. We show that sera from human Zika patients contain antibodies capable of binding to the unglycosylated glycan loop in the absence of the rest of the envelope protein. Furthermore, mice were inoculated with recombinant E monomers and produced neutralizing antibodies that either recognize unglycosylated glycan loop or require glycan for their binding to monomeric E. We demonstrate that both types of antibodies neutralize ZIKV to some extent in a cellular virus neutralization assay. Conclusions Analogous to the existing EDE antibody nomenclature, we propose a new classification for antibodies that bind to E monomer epitopes (EME): EME1 and EME2 for those that do not require and those that do require glycan for binding to E, respectively.

Background: The COVID-19 pandemic caused numerous stressors that may have been linked to substance use among college students. Methods: We analyzed data from the Fall 2020 Healthy Minds Study (N = 15,995), a non-probability sample of students attending one of 28 universities, who completed an online survey during the COVID-19 pandemic (September – December 2020). Using multivariable logistic regression, we examined the associations between COVID-19 stressors (concern, racial/ethnic discrimination, financial distress, infection, illness of loved one, death of loved one, caregiving) and substance use (alcohol, cigarette, marijuana), adjusting for age, gender, race/ethnicity, and international student status. All COVID-19 stressors were included in the same weighted logistic regression models. Results: About 46.89% of the sample reported drinking any alcohol (in the past 2 weeks), 7.38% used any cigarettes, and 16.87% used any marijuana over the past month. Multivariable logistic regression models showed that infection and caregiving were significantly associated with alcohol use; racial/ethnic discrimination and financial distress were associated with smoking cigarettes; and concern and infection were associated with marijuana use. Conclusions: COVID-19 stressors were related to substance use, though the strength and significance of the associations varied depending on the stressors and the type of substance.

Antimicrobial efficacy of the water or methanolic extracts of three medicinal mushrooms Taiwanofungus camphoratus, Agaricus blazei Murrill, and Ganoderma lucidum (Curtis) P. Karst were investigated against yeast and filamentous fungal pathogens as well as against commensal and pathogenic bacteria. The methanolic extract of T. camphoratus (TcM) exhibited both potent antifungal and antibacterial activity, while the water extract of T. camphoratus (TcW) showed limited antibacterial activity against Listeria monocytogenes. Neither the methanolic nor water extracts of A. blazei and G. lucidum exhibited antimicrobial activity. In the risk assessment testing monitoring the development of fungal tolerance to mushroom extracts in food matrices, two P. expansum mitogen-activated protein kinase (MAPK) mutants exhibited a tolerance to TcM. In a proof-of-concept bioassay using the natural benzoic salicylaldehyde (SA), P. expansum and A. fumigatus MAPK antioxidant mutants showed similar tolerance to SA, suggesting that natural ingredients in TcM such as benzoic derivatives could negatively affect the efficacy of TcM when antioxidant mutants are targeted. Conclusion: TcM could be developed as a food ingredient having antimicrobial potential. The antimicrobial activity of TcM operates via the intact MAPK antioxidant signaling system in microbes, however, mutants lacking genes in the MAPK system escape the toxicity triggered by TcM. Therefore, caution should be exercised in the use of TcM so as to not adversely affect food safety and quality by triggering the resistance of antioxidant mutants in contaminated food.

We would like to comment on a recent study where the redox-modulatory anti-protozoal drug chloroquine has shown to trigger a crosstalk between the antioxidant and cell wall integrity systems in the yeast Saccharomyces cerevisiae. This note discusses the redox-active potential of the natural compound 2-hydroxy-4-methoxybenzaldehyde (2H4M) which could also serve as a potent redox-cycler in fungal (yeast, molds) pathogens. Using S. cerevisiae as a molecular tool, we determined how 2H4M negatively affected both the antioxidant and cell wall integrity systems of fungi, thus indicating a similar crosstalk between the two systems under 2H4M-induced toxicity. The crosstalk functions as a fungal defense against redox-active drugs/compounds or environmental cues, and therefore, could be an effective target for antifungal treatment.

The fungal antioxidant system is one of the targets of the redox-active polyene antifungal drugs, including amphotericin B (AMB), nystatin (NYS), and natamycin (NAT). Besides medical applications, NAT has been used in industry for preserving foods and crops. In this study, we investigated two parameters (pH and food ingredients) affecting NAT efficacy. In the human pathogen, Aspergillus fumigatus, NAT (2 to 16 μg mL−1) exerted higher activity at pH 5.6 than at pH 3.5 on a defined medium. In contrast, NAT exhibited higher activity at pH 3.5 than at pH 5.6 against foodborne fungal contaminants, Aspergillus flavus, Aspergillus parasiticus, and Penicillium expansum, with P. expansum being the most sensitive. In commercial food matrices (10 organic fruit juices), food ingredients differentially affected NAT antifungal efficacy. Noteworthily, NAT overcame tolerance of the A. fumigatus signaling mutants to the fungicide fludioxonil and exerted antifungal synergism with the secondary metabolite, kojic acid (KA). Altogether, NAT exhibited better antifungal activity at acidic pH against foodborne fungi; however, the ingredients from commercial food matrices presented greater impact on NAT efficacy compared to pH values. Comprehensive determination of parameters affecting NAT efficacy and improved food formulation will promote sustainable food/crop production, food safety, and public health.

Quercetin is a flavonoid that has been shown to have health-promoting capacities due to its potent antioxidant activity. However, the effect of chronic intake of quercetin on the gut microbiome and diabetes-related biomarkers remains unclear. Male C57BL/6J mice were fed HF or HF supplemented with 0.05% quercetin (HFQ) for 6 weeks. Diabetes-related biomarkers in blood were determined in mice fed high-fat (HF) diets supplemented with quercetin. Mice fed the HFQ diet gained less body, liver, and adipose weight, while liver lipid and blood glucose levels were also lowered. Diabetes-related plasma biomarkers insulin, leptin, resistin, and glucagon were significantly reduced by quercetin supplementation. In feces, quercetin supplementation significantly increased the relative abundance of Akkermansia and decreased the Firmicutes/Bacteroidetes ratio. The expression of genes Srebf1, Ppara, Cyp51, Scd1, and Fasn was downregulated by quercetin supplementation. These results indicated that diabetes biomarkers are associated with early metabolic changes accompanying obesity, and quercetin may ameliorate insulin resistance.

Colored common beans are associated with health promoting and chronic disease prevention effects. Male C57BL/6J mice were fed high-fat (HF) diets supplemented with cooked black turtle beans (HFB) to prevent obesity related insulin resistance. Mice on both HF and HFB were obese compared to mice fed a low-fat (LF) diet. Plasma low density lipoprotein (LDL) and triglyceride concentrations of mice fed HFB diet were 28% and 36.6% lower than those on HF diet. Homeostatic model assessment of insulin resistance (HOMA-IR) index of mice fed HFB diet was 87% lower than that of mice fed HF diet. Diabetes related biomarkers, gastric inhibitory polypeptide (GIP), leptin, glucagon, and inflammatory cytokines interleukin 4 (IL-4) and IL-5, 10 and 12, IFN-g and TNF-α were significantly affected by HFB diet. Pparα, Cyp7a1 and Fasn were down-regulated by HFB diet while LDL-R, Srebp-2, Adipoq and Slc2a4 were up-regulated by HFB diet. The ratio of Firmicutes/Bacteroidetes (F/B) was also decreased 64.1% by HFB diet compared to HF diet. The results indicated that cooked black turtle bean consumption could ameliorate insulin resistance and lower plasma LDL in mice fed HF diet through glucose signaling pathway and JNK/c-Jun pathway. Meanwhile, cooked black turtle bean consumption restored the gut microbiome.

Trichomoniasis in humans, caused by the protozoal parasite Trichomonas vaginalis, is the most common non-viral sexually transmitted disease, while Tritrichomonas foetus causes trichomonosis, an infection of the gastrointestinal tract and diarrhea in farm animals and domesticated cats. As part of an effort to determine the inhibitory effects of plant-based extracts and pure compounds, seven commercially available cherry tomato varieties were hand-peeled, freeze-dried, and pounded into powders. The anti-trichomonad inhibitory activities of these peel powders at 0.02% concentration determined using an in vitro cell assay varied widely from 0.0% to 66.7% against T. vaginalis G3 (human); from 0.9% to 66.8% for T. foetus C1 (feline); and from 0.0% to 81.3% for T. foetus D1 (bovine). The organic Solanum lycopersicum var. cerasiforme (D) peels were the most active against all three trichomonads, inhibiting 52.2% (G3), 66.8% (C1), and 81.3% (D1). Additional assays showed that none of the powders inhibited the growth of foodborne pathogenic bacteria, pathogenic fungi, or non-pathogenic lactobacilli. Tomato peel and pomace powders with high content of described biologically active compounds could serve as functional food and feed additives that might help overcome adverse effects of wide-ranging diseases and complement the treatment of parasites with the anti-trichomonad drug metronidazole.

Control of fungal pathogens is increasingly problematic due to the limited number of effective drugs available for antifungal therapy. Conventional antifungal drugs could also trigger human cytotoxicity associated with the kidneys and liver, including the generation of reactive oxygen species. Moreover, increased incidences of fungal resistance to the classes of azoles, such as fluconazole, itraconazole, voriconazole, or posaconazole, or echinocandins, including caspofungin, anidulafungin, or micafungin, have been documented. Of note, certain azole fungicides such as propiconazole or tebuconazole that are applied to agricultural fields have the same mechanism of antifungal action as clinical azole drugs. Such long-term application of azole fungicides to crop fields provides environmental selection pressure for the emergence of pan-azole-resistant fungal strains such as Aspergillus fumigatus having TR34/L98H mutations, specifically, a 34 bp insertion into the cytochrome P450 51A (CYP51A) gene promoter region and a leucine-to-histidine substitution at codon 98 of CYP51A. Altogether, the emerging resistance of pathogens to currently available antifungal drugs and insufficiency in the discovery of new therapeutics engender the urgent need for the development of new antifungals and/or alternative therapies for effective control of fungal pathogens. We discuss the current needs for the discovery of new clinical antifungal drugs and the recent drug repurposing endeavors as alternative methods for fungal pathogen control.

Plant-derived anthraquinones were evaluated in cell assays for their inhibitory activities against the parasitic protozoa Trichomonas vaginalis human strain G3 that causes the sexually transmitted disease trichomoniasis in women, Tritrichomonas foetus bovine strain D1 that causes sexually transmitted diseases in farm animals (bulls, cows, and pigs), Tritrichomonas foetus-like strain C1 that causes diarrhea in domestic animals (cats and dogs), and bacteria and fungi. The anthraquinones assessed for their inhibitory activity were anthraquinone, aloe-emodin (1,8-dihydroxy-3-hydroxymethylanthraquinone), anthrarufin (1,5-dihydroxyanthraquinone), chrysazin (1,8-dihydroxyanthraquinone), emodin (1,3,8-trihydroxy-6-methylanthraquinone), purpurin (1,2,4-trihydroxyanthraquinone), and rhein (1,8-dihydroxy-3-carboxyanthraquinone). Their activities were determined in terms of IC50 values, defined as the concentration that inhibits 50% of the cells under the test conditions and calculated from linear dose response plots for the parasitic protozoa, and zone of inhibition for bacteria and fungi, respectively. The results show that the different substituents on the anthraquinone ring seem to influence the relative potency. Analysis of the structure–activity relationships in protozoa indicates that the aloe-emodin and chrysazin with the highest biological activities merit further study for their potential to help treat the diseases in women and domestic and farm animals. Emodin also exhibited antifungal activity against Candida albicans. The suggested mechanism of action and the additional reported beneficial biological properties of anthraquinones suggest that they have the potential to ameliorate a broad spectrum of human diseases.

Abrin, a highly toxic plant toxin, is a potential bioterror weapon. Work from our laboratory and others have shown that abrin is highly resistant to both thermal and pH inactivation methods. We sought to evaluate the effectiveness of selected food processing thermal inactivation conditions against abrin in economically important food matrices (whole milk, non-fat milk, liquid egg, and ground beef). The effectiveness of toxin inactivation was measured via three different assays: (1) In vitro cell free translation (CFT) assay, (2) Vero cell culture cytotoxicity; and the in vivo mouse intraperitoneal (ip) bioassay. For both whole and non-fat milk, complete inactivation was achieved at temperatures of ≥ 80 °C for 3 min or 134 °C for 60 s, which were higher than the normal vat/batch pasteurization or the high temperature short time pasteurization (HTST). Toxin inactivation in liquid egg required temperatures of ≥ 74 °C for 3 min higher than suggested temperatures for scrambled eggs (22% solids) and plain whole egg. Additionally, the ground beef (80:20%) matrix was found to be inhibitory for full toxin activity in the mouse bioassay while retaining some activity in both the cell free translation assay and Vero cell culture cytotoxicity assay.

Botulinum neurotoxin (BoNT) intoxication can lead to the disease botulism, characterized by flaccid muscle paralysis that can cause respiratory failure and death. Due to the significant morbidity and mortality costs associated with BoNTs high toxicity, developing highly sensitive, rapid, and field-deployable assays are critically important to protect the nation’s food supply against either accidental or intentional contamination. We report here that the B-cell based biosensor assay CANARY® (Cellular Analysis and Notification of Antigen Risks and Yields) Zephyr detects BoNT/A holotoxin at limits of detection (LOD) of 10.0 ± 2.5 ng/mL in assay buffer. Milk matrices (whole milk, 2% milk and non-fat milk) with BoNT/A holotoxin were detected at similar levels (7.4⁻7.9 ng/mL). BoNT/A complex was positive in carrot, orange, and apple juices at LODs of 32.5⁻75.0 ng/mL. The detection of BoNT/A complex in solid complex foods (ground beef, smoked salmon, green bean baby puree) ranged from 14.8 ng/mL to 62.5 ng/mL. Detection of BoNT/A complex in the viscous liquid egg matrix required dilution in assay buffer and gave a LOD of 171.9 ± 64.7 ng/mL. These results show that the CANARY® Zephyr assay can be a highly useful qualitative tool in environmental and food safety surveillance programs.

Due to the increased interest in their application in the treatment of infectious diseases, boron-containing compounds have received a significant coverage in the literature. Herein, a small set of novel cinnamoly-oxaborole amides were synthesized and screened against nagana Trypanosoma brucei brucei for antitrypanosomal activity. Compound 5g emerged as a new hit with an in vitro IC50 value of 0.086 μM against T. b. brucei without obvious inhibitory activity against HeLa cell lines. The same series was also screened against other human pathogens, including Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), for which moderate to weak activity (10 to >125 μM) was observed. Similarly, these compounds exhibited moderate activity against the human protozoal pathogen Trichomonas vaginalis with no observed effect on common microbiome bacterial species. The cross-species inhibitory activity presents the possibility of these compounds serving as broad-spectrum antibiotics for these prevalent three human pathogens.

Abrin, one of most potent toxins known to man, is derived from the rosary pea (jequirity pea), Abrus precatorius and is a potential bioterror weapon. The temperature and pH stability of abrin was evaluated with an in vitro cell free translation (CFT) assay, a Vero cell culture cytotoxicity assay, and an in vivo mouse bioassay. pH treatment of abrin had no detrimental effect on its stability and toxicity as seen either in vitro or in vivo. Abrin exposure to increasing temperatures did not completely abrogate protein translation. In both the cell culture cytotoxicity model and the mouse bioassay, abrin’s toxic effects were completely abrogated if the toxin was exposed to temperatures of 74 °C or higher. In the cell culture model, 63 °C-treated abrin had a 30% reduction in cytotoxicity which was validated in the in vivo mouse bioassay with all mice dying but with a slight time-to-death delay as compared to the non-treated abrin control. Since temperature inactivation did not affect abrin’s ability to inhibit protein synthesis (A-chain), we hypothesize that high temperature treatment affected abrin’s ability to bind to cellular receptors (affecting B-chain). Our results confirm the absolute need to validate in vitro cytotoxicity assays with in vivo mouse bioassays.

Abrin, one of the most highly potent toxins in the world, is derived from the plant, Abrus precatorius. Because of its high toxicity, it poses potential bioterror risks. Therefore, a need exists for new reagents and technologies that would be able to rapidly detect abrin contamination as well as lead to new therapeutics. We report here a group of abrin-specific monoclonal antibodies (mAbs) that recognize abrin A-chain, intact A–B chain toxin, and agglutinin by Western blot. Additionally, these mAbs were evaluated for their ability to serve as capture antibodies for a sandwich (capture) ELISA. All possible capture–detector pairs were evaluated and the best antibody pair identified and optimized for a capture ELISA. The capture ELISA based on this capture–detector mAb pair had a limit of detection (L.O.D) of ≈1 ng/mL measured using three independent experiments. The assay did not reveal any false positives with extracts containing other potential ribosome-inactivating proteins (RIPs). Thus, this new capture ELISA uses mAbs for both capture and detection; has no cross-reactivity against other plant RIPs; and has a sensitivity comparable to other reported capture ELISAs using polyclonal antibodies as either capture or detector.

Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins known to man and are threats to public health and safety. Previous work from our laboratory showed that both BoNT serotype A complex and holotoxin can bind and transit through the intestinal epithelia to disseminate in the blood. The timing of BoNT/A toxin internalization was shown to be comparable in both the Caco-2 in vitro cell culture and in the oral mouse intoxication models. Probiotic microorganisms have been extensively studied for their beneficial effects in not only maintaining the normal gut mucosa but also protection from allergens, pathogens, and toxins. In this study, we evaluate whether probiotic microorganisms will block BoNT/A uptake in the in vitro cell culture system using Caco-2 cells. Several probiotics tested (Saccharomyces boulardii, Lactobacillus acidophilus, Lactobacillus rhamnosus LGG, and Lactobacillus reuteri) blocked BoNT/A uptake in a dose-dependent manner whereas a non-probiotic strain of Escherichia coli did not. We also showed that inhibition of BoNT/A uptake was not due to the degradation of BoNT/A nor by sequestration of toxin via binding to probiotics. These results show for the first time that probiotic treatment can inhibit BoNT/A binding and internalization in vitro and may lead to the development of new therapies.