Rapid assessment of enniatins in barley grains using near infrared spectroscopy and chemometric tools.

Barley is an important crop worldwide, and it can be affected by various fungi, among them Fusarium is one of the most relevant due to the economic losses caused by mycotoxin contamination. Enniantins (ENNs) are one of the emergent group of mycotoxins that have been found in grains around the world. Nowadays, the main analytical tools available to evaluate these contaminants are based on chromatographic techniques that are efficient but time-consuming and expensive. In this context, the present study aimed to assess the performance of near infrared (NIR) spectroscopy to detect and/or classify the enniatin (ENN) content on barley grains. Sixty samples of barley grains from three different regions of Brazil were investigated and the ENN content determined by UPLC-MS/MS. The levels found were then used to develop multivariate models based on infrared spectral data. The results indicated high incidence off ENN presence in the samples (>70 %) and the PLS-DA model determined by NIR data showed adequate values of sensitivity and sensibility (100 % and 94.2 %, respectively) distinguishing between contaminated and non-contaminated barley samples, demonstrating NIR as a promising tool to monitoring this emergent mycotoxin.

Novel High-Throughput Strategy for the Aqueous Solubility Assessment of Peptides and Proteins Exhibiting a Propensity for Gelation: Application to the Discovery of Novel Antibacterial Teixobactin Analogues.

A novel high-throughput aqueous solubility assay was developed for peptides and proteins exhibiting a high gelling propensity (in this case, antibacterial teixobactin analogues). By integrating the assessment of gel formation, as indicated by an increase in the solution viscosity, into the peptide equilibrium solubility screening assay, we were able to estimate the "free-flowing solubility", which is defined as the concentration at which the peptide solution not only is fully dissolved but also is a liquid exhibiting ideal flowing characteristics. In this workflow, peptide solutions passing the turbidity assessment were further screened by viscosity measurements based on nanobead-assisted dynamic light scattering analysis in a 96-well plate. The method is able to effectively detect the initiation of peptide gelation and facilitate compound ranking based on their aqueous solubility. The application of such an approach helped confirm that the substitution of Ser3 in teixobactin led to desired physicochemical improvements and provided a focal point for further chemistry structure-activity relationship exploration.

Cyclic depsipeptide mycotoxin exposure may cause human endocrine disruption: Evidence from OECD in vitro stably transfected transcriptional activation assays.

The presence of cyclic depsipeptide mycotoxins in foods and feedstuffs could potentially cause endocrine disrupting effects on humans and wildlife by their inhibition of active steroidogenesis. Therefore, we attempted to assess the human estrogen receptor (ER) and androgen receptor (AR) agonistic/antagonistic effects of representative cyclic depsipeptide mycotoxins, enniatin A1 (ENN A1), and enniatin B1 (ENN B1), by OECD Performand Based Test Guideline (PBTG) No.455, VM7Luc ER transcriptional activation (TA) assay and OECD TG No. 458, 22Rv1/MMTV_GR-KO AR TA assay. No tested cyclic depsipeptide mycotoxins were found to be ER and AR agonists in VM7Luc ER TA and 22Rv1/MMTV_GR-KO AR TA assays. On the other hand, ENN A1, and ENN B1 exhibited the ER and AR antagonistic effects with IC30 and IC50 values in both TA assays. These two cyclic depsipeptide mycotoxins, which were determined as ER and AR antagonists by two in vitro assays, bound to ERα, and AR. Then ENN A1, and ENN B1 inhibited the dimerization of ERα, and AR. These results, for the first time indicated that ENN A1, and ENN B1 could have potential endocrine disrupting effects mediated by interaction of ERα and AR using international standard testing methods to determine the potential endocrine disrupting chemical.

De Novo Resistance to Arg10-Teixobactin Occurs Slowly and Is Costly.

Bacterial pathogens are rapidly evolving resistance to all clinically available antibiotics. One part of the solution to this complex issue is to better understand the resistance mechanisms to new and existing antibiotics. Here, we focus on two antibiotics. Teixobactin is a recently discovered promising antibiotic that is claimed to "kill pathogens without detectable resistance" (L. L. Ling, T. Schneider, A. J. Peoples, A. L. Spoering, et al., Nature 517:455-459, 2015, https://doi.org/10.1038/nature14098). Moenomycin A has been extensively used in animal husbandry for over 50 years with no meaningful antibiotic resistance arising. However, the nature, mechanisms, and consequences of the evolution of resistance to these "resistance-proof" compounds have not been investigated. Through a fusion of experimental evolution, whole-genome sequencing, and structural biology, we show that Staphylococcus aureus can develop significant resistance to both antibiotics in clinically meaningful timescales. The magnitude of evolved resistance to Arg10-teixobactin is 300-fold less than to moenomycin A over 45 days, and these are 2,500-fold and 8-fold less than evolved resistance to rifampicin (control), respectively. We have identified a core suite of key mutations, which correlate with the evolution of resistance, that are in genes involved in cell wall modulation, lipid synthesis, and energy metabolism. We show the evolution of resistance to these antimicrobials translates into significant cross-resistance against other clinically relevant antibiotics for moenomycin A but not Arg10-teixobactin. Lastly, we show that resistance is rapidly lost in the absence of antibiotic selection, especially for Arg10-teixobactin. These findings indicate that teixobactin is worth pursuing for clinical applications and provide evidence to inform strategies for future compound development and clinical management.

Decision making in the pharmaceutical industry - A tale of three antibiotics.

There is a mounting crisis in treatment of bacterial diseases. The appearance of nosocomial infections produced by multi-drug resistant bacteria is rapidly increasing and at the same time the pharmaceutical industry has been abandoning new antibiotic discovery. To help understand why, we investigated the decision-making processes behind three novel antibiotics that were initially discovered in the late 1980's and early 1990's: daptomycin, linezolid, and lysobactin. Each antibiotic was investigated by two highly qualified scientific organizations that came to opposing opinions regarding the clinical utility and commercial potential of the drug. After reviewing the literature and interviewing key scientific staff members working on each of these molecules, we have identified factors needed to generate positive development decisions. Organizational factors included decision timing, therapeutic area focus, organizational support for risk taking and the presence of a project champion. Technical factors included investment in the optimization of dosing for improved drug exposure, toxicological evaluation of the purified eutomer from a diastereomer and the failure to develop an effective research formulation.

Integrated Assessment of Viral Transcription, Antigen Presentation, and CD8+ T Cell Function Reveals Multiple Limitations of Class I-Selective Histone Deacetylase Inhibitors during HIV-1 Latency Reversal.

Clinical trials investigating histone deacetylase inhibitors (HDACi) to reverse HIV-1 latency aim to expose reservoirs in antiretroviral (ARV)-treated individuals to clearance by immune effectors, yet have not driven measurable reductions in the frequencies of infected cells. We therefore investigated the effects of the class I-selective HDACi nanatinostat and romidepsin on various blocks to latency reversal and elimination, including viral splicing, antigen presentation, and CD8+ T cell function. In ex vivo CD4+ T cells from ARV-suppressed individuals, both HDACi significantly induced viral transcription, but not splicing nor supernatant HIV-1 RNA. In an HIV-1 latency model using autologous CD8+ T cell clones as biosensors of antigen presentation, neither HDACi-treated CD4+ T cell condition induced clone degranulation. Both HDACi also impaired the function of primary CD8+ T cells in viral inhibition assays, with nanatinostat causing less impairment. These findings suggest that spliced or cell-free HIV-1 RNAs are more indicative of antigen expression than unspliced HIV-RNAs and may help to explain the limited abilities of HDACi to generate CD8+ T cell targets in vivoIMPORTANCE Antiretroviral (ARV) drug regimens suppress HIV-1 replication but are unable to cure infection. This leaves people living with HIV-1 burdened by a lifelong commitment to expensive daily medication. Furthermore, it has become clear that ARV therapy does not fully restore health, leaving individuals at elevated risk for cardiovascular disease, certain types of cancers, and neurocognitive disorders, as well as leaving them exposed to stigma. Efforts are therefore under way to develop therapies capable of curing infection. A key focus of these efforts has been on a class of drugs called histone deacetylase inhibitors (HDACi), which have the potential of exposing hidden reservoirs of HIV-1 to elimination by the immune system. Unfortunately, clinical trial results with HDACi have thus far been disappointing. In the current study, we integrate a number of experimental approaches to build a model that provides insights into the limited activity of HDACi in clinical trials and offers direction for future approaches.

Beauvericin, A Fusarium Mycotoxin: Anticancer Activity, Mechanisms, and Human Exposure Risk Assessment.

Beauvericin (BEA) is a cyclic hexadepsipeptide, which derives from Cordyceps cicadae. It is also produced by Fusarium species, which are parasitic to maize, wheat, rice and other important commodities. BEA increases ion permeability in biological membranes by forming a complex with essential cations, which may affect ionic homeostasis. Its ion-complexing capability allows BEA to transport alkaline earth metal and alkali metal ions across cell membranes. Importantly, increasing lines of evidence show that BEA has an anticancer effect and can be potentially used in cancer therapeutics. Normally, BEA performs the anticancer effect due to the induced cancer cell apoptosis via a reactive oxygen species-dependent pathway. Moreover, BEA increases the intracellular Ca2+ levels and subsequently regulates the activity of a series of signalling pathways including MAPK, JAK/STAT, and NF-κB, and finally causes cancer cell apoptosis. In vivo studies further show that BEA reduces tumour volumes and weights. BEA especially targets differentiated and invasive cancer types. Currently, the anticancer activity of BEA is a hot topic; however, there is no review article to discuss the anticancer activity of BEA. Therefore, in this review, we have mainly summarized the anticancer activity of BEA and thoroughly discussed its underlying mechanisms. In addition, the human exposure risk assessment of BEA is also discussed. We hope that this review will provide further information for understanding the anticancer mechanisms of BEA.

Assessment of cytotoxic and genotoxic effects of enniatin-A in vitro.

Enniatin A (EN-A) is a Fusarium mycotoxin which is a common contaminant in grains and especially in maize and it causes serious loss of product. The aim of this study was to investigate the cytotoxic effects using 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) assay in human cervix carcinoma (HeLa) cell line, and genotoxic effects of EN-A using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (MN) and comet assays in human lymphocytes. The cells were treated with 0.07, 0.14, 0.29, 0.57, 1.15, 2.29, 4.59 and 9.17 µM concentrations of EN-A. It exhibited cytotoxic effects in HeLa cell lines especially when the concentrations were increased. The half-inhibitory value (IC50) was determined as 1.15 µM concentration for 24 h and 0.57 µM concentration for 48 h. However, EN-A failed to affect the frequency of CAs, SCEs and MN in human lymphocytes. Only a slight increase was observed in the frequency of SCEs at 0.57 µM concentration over 48 h. The replication (RI) and nuclear division (NDI) indices were not affected. On the contrary, EN-A decreased the mitotic index (MI) significantly at all concentrations compared to the negative control and solvent control (except at 0.29 µM for 24 h, and except at 0.14, 0.29 and 0.57 µM for 48 h). Treatments over 2.29 µM showed toxic effects in human lymphocytes. EN-A significantly increased comet tail intensity (except at 0.07 and 0.57 µM) in isolated human lymphocytes. The results of this study demonstrate that EN-A has an obvious cytotoxic effect especially when the EN-A concentration was increased. In addition, EN-A could exhibit a mild genotoxic effect.

Preclinical Assessment of a 68Ga-DOTA-Functionalized Depsipeptide as a Radiodiagnostic Infection Imaging Agent.

The study assessed a radiolabeled depsipeptide conjugate (68Ga-DOTA-TBIA101) for its potential as an imaging agent targeting infection or infection-associated inflammation. 68Ga-labeled DOTA-TBIA101 imaging was performed in (NZR1) healthy rabbits; (NZR2) rabbits bearing muscular sterile inflammation and Staphylococcus aureus (SA) infection; and (NZR3) rabbits infected with Mycobacterium tuberculosis (MTB) combined with a subcutaneous scruff infection of SA in the same animal. All animals were imaged using a PET/CT scanner at 5 and 60 min post injection. Images showed elevated accumulation of 68Ga-DOTA-TBIA101 in the sterile muscular inflammation site (T/NT ratio = 2.6 ± 0.37 (5 min) and 2.8 ± 2.3 (60 min)) and muscles infected with MTB (T/NT ratio = 2.6 ± 0.35 (5 min) and 2.8 ± 0.16 (60 min)). The findings suggest that 68Ga-DOTA-TBIA101-PET/CT may detect MTB-associated inflammation, although more foundational studies need to be performed to rationalize the diagnostic value of this technique.

Conformation-Based Design and Synthesis of Apratoxin A Mimetics Modified at the α,ß-Unsaturated Thiazoline Moiety.

We have demonstrated design, synthesis, and biological evaluation of apratoxin A mimetics. In the first generation, the moCys moiety was replaced with seven simple amino acids as their 3D structures can be similar to that of apratoxin A. Apratoxins M1-M7 were synthesized using solid-phase peptide synthesis and solution-phase macrolactamization. Apratoxin M7, which contains a piperidinecarboxylic acid moiety, exhibited potent cytotoxicity against HCT-116 cells. In the second generation, substitution of each amino acid residue in the tripeptide Tyr(Me)-MeAla-MeIle moiety in apratoxin M7 led to the development of the highly potent apratoxin M16 possessing biphenylalanine (Bph) instead of Tyr(Me), which exhibited an IC50 value of 1.1 nM against HCT-116 cells. Moreover, compared to apratoxin A, apratoxin M16 exhibited a similarly high level of growth inhibitory activity against various cancer cell lines. The results indicate that apratoxin M16 could be a potential candidate as an anticancer agent.

Mycotoxin risk assessment for consumers of groundnut in domestic markets in Nigeria.

The fungal and multi-mycotoxin profiles of groundnuts sold in domestic markets in Nigeria as well as the associated risk to consumers were assessed in the present study. Four hundred fungal isolates representing mainly Aspergillus [58.6%: Aspergillus section Flavi (37.1%) and A. niger-clade (21.5%)], Penicillium (40.9%) and Fusarium (0.5%) were isolated from 82 (97.6%, n=84) groundnut samples collected from four agro-ecological zones (AEZs) of Nigeria. The incidence of aflatoxin-producing A. flavus isolates (71%) was significantly (p<0.05) higher in the groundnuts than that of the non-aflatoxigenic isolates (29%). Fifty-four fungal metabolites [including aflatoxins (AFB1, AFB2, AFG1, AFG2 and AFM1), beauvericin (BEAU), cyclopiazonic acid (CPA), moniliformin, nivalenol and ochratoxin A] and four bacterial metabolites were detected in the groundnuts by liquid chromatography tandem mass spectrometry. Aflatoxins (39%; max: 2076µg/kg; mean: 216µg/kg) were detected in more samples than any other mycotoxin. About 25, 23 and 14% of the samples respectively were above the 2µg/kg AFB1, 4 and 20µg/kg total aflatoxin limits of the European Union and US FDA respectively. The mean margins of exposure of AFB1 and total aflatoxins for adult consumers were 1665 and 908, respectively, while mean estimated daily intake values for infants, children and adults were <0.1% for BEAU and 4% for CPA. Consumers of mycotoxin contaminated groundnuts in Nigeria may therefore be at a risk of liver cancer in addition to other combinatory effects of mycotoxin/metabolite cocktails. There is need for increased targeted interventions in the groundnut value chain in Nigeria for public health benefits.

Characterization and Exposure Assessment of Emetic Bacillus cereus and Cereulide Production in Food Products on the Dutch Market.

The emetic toxin cereulide, which can be produced by Bacillus cereus, can be the cause of food poisoning upon ingestion by the consumer. The toxin causes vomiting and is mainly produced in farinaceous food products. This article includes the prevalence of B. cereus and of cereulide in food products in The Netherlands, a characterization of B. cereus isolates obtained, cereulide production conditions, and a comparison of consumer exposure estimates with those of a previous exposure assessment. Food samples (n = 1,489) were tested for the presence of B. cereus; 5.4% of the samples contained detectable levels (>10(2) CFU/g), and 0.7% contained levels above 10(5) CFU/g. Samples (n = 3,008) also were tested for the presence of cereulide. Two samples (0.067%) contained detectable levels of cereulide at 3.2 and 5.4 µg/kg of food product. Of the 481 tested isolates, 81 produced cereulide and/or contained the ces gene. None of the starch-positive and hbl-containing isolates possessed the ces gene, whereas all strains contained the nhe genes. Culture of emetic B. cereus under nonoptimal conditions revealed a delay in onset of cereulide production compared with culture under optimal conditions, and cereulide was produced in all cases when B. cereus cells had been in the stationary phase for some time. The prevalence of cereulide-contaminated food approached the prevalence of contaminated products estimated in an exposure assessment. The main food safety focus associated with this pathogen should be to prevent germination and growth of any B. cereus present in food products and thus prevent cereulide production in foods.

Enniatin-containing solutions for oromucosal use: Quality-by-design ex-vivo transmucosal risk assessment of composition variability.

Fusafungine, a mixture of the cyclic hexadepsipeptides enniatins, is currently on the market for the treatment of upper respiratory tract diseases because of its bacteriostatic and anti-inflammatory effects. In this study, a quality-by-design risk assessment was performed with two objectives: (i) investigate whether enniatins are able to permeate the mucosa and reach blood circulation, as the summary of product characteristics indicates this is not the case, and if so, to quantify their transmucosal kinetics and (ii) study the influence of excipient concentration variability on mucosal permeation. First, the concentration of the two main excipients isopropyl myristate and ethanol, known penetration enhancers, in several marketed samples was determined using GC-FID. Then, the transmucosal kinetics of the enniatins were quantitatively evaluated for different dose solutions, using porcine buccal mucosa in an ex-vivo in-vitro Franz diffusion cell set-up, with UHPLC-MS/MS bioanalytics. This study demonstrated that enniatins are capable of permeating the mucosa. However, no risk of a significant different transmucosal permeability with varying excipient concentrations was detected.

An efficient and cost-effective approach to kahalalide F N-terminal modifications using a nuisance algal bloom of Bryopsis pennata.

BACKGROUND: Kahalalide F (KF) and its isomer iso-kahalalide F (isoKF), both of which can be isolated from the mollusk Elysia rufescens and its diet alga Bryopsis pennata, are potent cytotoxic agents that have advanced through five clinical trials. Due to a short half-life, narrow spectrum of activity, and a modest response in patients, further efforts to modify the molecule are required to address its limitations. In addition, due to the high cost in producing KF analogues using solid phase peptide synthesis (SPPS), a degradation and reconstruction approach was employed using natural KF from a seasonal algal bloom to generate KF analogues. METHODS: N-protected KF was carefully hydrolyzed at the amide linkage between l-Thr12 and d-Val13 using dilute HCl. The synthesis of the C-terminal fragment began with the formation of hexanoic succinimide ester, followed by a reaction with dipeptides. The final coupling reaction was performed between the semisynthesized Fmoc-KF hydrolysis product and the C-terminal fragment, followed by the deprotection of the Fmoc group. RESULTS: Six KF analogues with an addition of an amino acid residue on the N-terminal chain, d-Val14-isoKF (2), Val13-Val14-isoKF (3), d-Leu14-isoKF (4), d-Pro14-isoKF (5), d-Phe14-isoKF (6), and 3,4-2F-d-Phe14-isoKF (7) were prepared using semisynthesis at the exposed N-terminal chain. CONCLUSIONS: The overall yield of the medication was 45%. This approach is economical, efficient and amendable to large-scale production while eliminated a nuisance algal bloom. GENERAL SIGNIFICANCE: B. pennata blooms are capable of producing KF in good yields. The semisynthesis from the natural product produced N-terminal modifications for the construction of inexpensive semisynthetic KF libraries.

Quantitative Assessment of Destruxins from Strawberry and Maize in the Lower Parts per Billion Range: Combination of a QuEChERS-Based Extraction Protocol with a Fast and Selective UHPLC-QTOF-MS Assay.

The entomopathogenic fungus Metarhizium brunneum is widely applied as a biological pest control agent. Consequently, its use has to be accompanied by a risk management approach, which includes the need to monitor the fate of its bioactive metabolites in the environment, for example, in treated crops. A fast and selective UHPLC-QTOF-MS method was developed to monitor the presence of secreted destruxins in two model food plants for the application of this fungal biocontrol agent, namely, strawberry and maize. The liquid chromatography-mass spectrometric assay for destruxin trace analysis is combined with a novel QuEChERS-based extraction protocol. The whole assay was optimized for the application in these crops, and it allows quantitative analysis of the major M. brunneum metabolites destruxin A, 1, destruxin B, 2, and destruxin E, 3, down to the parts per billion range. In strawberry, limits of quantitation (LOQs) were found to be <2.0 ppb for all analytes; in maize LOQs were found to be <3.2 ppb for destruxin A and destruxin B. Destruxin E showed a distinctive loss of recovery in maize and was excluded from further quantitative analysis in this crop. For both crops assay linearities ranged from the LOQs to 100 ppb, interassay repeatabilities (RSD) were found to be better than 16.4%, and accuracies ranged from 83.5 to 105.3% (assessed at four spiking levels between 5 and 75 ppb).

Synthesis, 68Ga-radiolabeling, and preliminary in vivo assessment of a depsipeptide-derived compound as a potential PET/CT infection imaging agent.

Noninvasive imaging is a powerful tool for early diagnosis and monitoring of various disease processes, such as infections. An alarming shortage of infection-selective radiopharmaceuticals exists for overcoming the diagnostic limitations with unspecific tracers such as (67/68)Ga-citrate or (18)F-FDG. We report here TBIA101, an antimicrobial peptide derivative that was conjugated to DOTA and radiolabeled with (68)Ga for a subsequent in vitro assessment and in vivo infection imaging using Escherichia coli-bearing mice by targeting bacterial lipopolysaccharides with PET/CT. Following DOTA-conjugation, the compound was verified for its cytotoxic and bacterial binding behaviour and compound stability, followed by (68)Gallium-radiolabeling. µPET/CT using (68)Ga-DOTA-TBIA101 was employed to detect muscular E. coli-infection in BALB/c mice, as warranted by the in vitro results. (68)Ga-DOTA-TBIA101-PET detected E. coli-infected muscle tissue (SUV = 1.3-2.4) > noninfected thighs (P = 0.322) > forearm muscles (P = 0.092) > background (P = 0.021) in the same animal. Normalization of the infected thigh muscle to reference tissue showed a ratio of 3.0 ± 0.8 and a ratio of 2.3 ± 0.6 compared to the identical healthy tissue. The majority of the activity was cleared by renal excretion. The latter findings warrant further preclinical imaging studies of greater depth, as the DOTA-conjugation did not compromise the TBIA101's capacity as targeting vector.