The Existing Methods and Novel Approaches in Mycotoxins’ Detection

Mycotoxins represent a wide range of secondary, naturally occurring and practically unavoidable fungal metabolites. They contaminate various agricultural commodities like cereals, maize, peanuts, fruits, and feed at any stage in pre- or post-harvest conditions. Consumption of mycotoxin-contaminated food and feed can cause acute or chronic toxicity in human and animals. The risk that is posed to public health have prompted the need to develop methods of analysis and detection of mycotoxins in food products. Mycotoxins wide range of structural diversity, high chemical stability, and low concentrations in tested samples require robust, effective, and comprehensible detection methods. This review summarizes current methods, such as chromatographic and immunochemical techniques, as well as novel, alternative approaches like biosensors, electronic noses, or molecularly imprinted polymers that have been successfully applied in detection and identification of various mycotoxins in food commodities. In order to highlight the significance of sampling and sample treatment in the analytical process, these steps have been comprehensively described.     

An Integrative Approach of an In Vitro Measurement of the Digestibility of Triacylglycerols of Human Milk

Several studies have been published regarding the effect of different factors on the digestion of milk lipids, considering their natural structural arrangement as milk fat globules and the efficiency of the digestive enzymes in the lipolysis of such complex structures. During digestion, the lipolytic products are dispersed in vesicles and micelles, which are the source for absorption of digested lipids. Therefore, it is necessary to consider the isolation of the micellar phase from the digesta to appropriately determine the amounts and classes of lipids which are bioaccessible. This study presents an integrative approach that included an isolation procedure to separate the micellar fraction from undigested and non-micellar parts, and the distribution of digested milk lipids in micelles determined directly through chromatographic techniques. Four groups of five full term mothers donated colostrum or mature milk. Two sets of samples were analyzed directly (raw), and two sets were pasteurized and then analyzed. Our data revealed that the profile of digested milk lipids is different depending on the lactation period and processing stage, while the carbon atom number distribution of the digested triacylglycerols in the micellar fraction provides a substantial information regarding the acylglycerols species that are less available for absorption.     

Garcinielliptone G from Garcinia subelliptica Induces Apoptosis in Acute Leukemia Cells

Cytotoxicity and apoptosis-inducing properties of compounds isolated from Garcinia subelliptica leaves were investigated. The hexane-soluble portion of MeOH extracts of G. subelliptica leaves that showed cytotoxic activity was separated to yield seven compounds 1–7. Chemical structure analysis using NMR spectroscopy and mass spectrometry confirmed that compound 1 was canophyllol, and compounds 2–7 were garcinielliptones N, O, J, G, F, and garcinielliptin oxide, respectively. Among them, garcinielliptone G (5) showed growth inhibition by causing apoptosis in THP-1 and Jurkat cells derived from human acute monocytic leukemia and T lymphocyte cells, respectively. Apoptosis induced by garcinielliptone G (5) was demonstrated by the detection of early apoptotic cells with fluorescein-labeled Annexin V and increases in cleaved caspase-3 and cleaved PARP protein levels. However, the addition of caspase inhibitor Z-VAD-FMK did not affect growth arrest or apoptosis induction. These results suggest that garcinielliptone G (5) can induce both caspase-3 activation and caspase-independent apoptosis. Therefore, garcinielliptone G (5) may be a potential candidate for acute leukemia treatment.     

Film-Forming Spray of Water-Soluble Chitosan Containing Liposome-Coated Human Epidermal Growth Factor for Wound Healing

Human epidermal growth factor (hEGF) has been known to have excellent wound-healing activity. However, direct application to the wound area can lead to low hEGF bioavailability due to protease enzymes or endocytosis. The use of liposomes as coatings and carriers can protect hEGF from degradation by enzymes, chemical reactions, and immune reactions. Sustained release using a matrix polymer can also keep the levels of hEGF in line with the treatment. Therefore, this study aimed to develop a film-forming spray of water-soluble chitosan (FFSWSC) containing hEGF-liposomes as a potential wound dressing. The hEGF-liposomes were prepared using the hydration film method, and the preparation of the FFSWSC was achieved by the ionic gelation method. The hydration film method produced hEGF-liposomes that were round and spread with a Z-average of 219.3 nm and encapsulation efficiency of 99.87%, whereas the film-forming solution, which provided good sprayability, had a formula containing 2% WSC and 3% propylene glycol with a viscosity, spray angle, droplet size, spray weight, and occlusion factor of 21.94 ± 0.05 mPa.s, 73.03 ± 1.28°, 54.25 ± 13.33 µm, 0.14 ± 0.00 g, and 14.57 ± 3.41%, respectively. The pH, viscosity, and particle size of the FFSWSC containing hEGF-liposomes were stable during storage for a month in a climatic chamber (40 ± 2 °C, RH 75 ± 5%). A wound healing activity test on mice revealed that hEGF-liposomes in FFSWSC accelerated wound closure significantly, with a complete wound closure on day 6. Based on the findings, we concluded that FFSWSC containing hEGF-liposomes has the potential to be used as a wound dressing.     

When UDG and hAPE1 Meet Cyclopurines. How (5′R) and (5′S) 5′,8-Cyclo-2′-deoxyadenosine and 5′,8-Cyclo-2′-deoxyguanosine Affect UDG and hAPE1 Activity?

Ionizing radiation is a factor that seriously damages cellular mechanisms/macromolecules, e.g., by inducing damage in the human genome, such as 5′,8-cyclo-2′-deoxypurines (cdPus). CdPus may become a component of clustered DNA lesions (CDL), which are notably unfavorable for the base excision repair system (BER). In this study, the influence of 5′S and 5′R diastereomers of 5′,8-cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) on the uracil-DNA glycosylase (UDG) and human AP site endonuclease 1 (hAPE1) activity has been taken under consideration. Synthetic oligonucleotides containing 2′-deoxyuridine (dU) and cdPu were used as a model of single-stranded CDL. The activity of the UDG and hAPE1 enzymes decreased in the presence of RcdG compared to ScdG. Contrary to the above, ScdA reduced enzyme activity more than RcdA. The presented results show the influence of cdPus lesions located within CDL on the activity of the initial stages of BER dependently on their position toward dU. Numerous studies have shown the biological importance of cdPus (e.g., as a risk of carcinogenesis). Due to that, it is important to understand how to recognize and eliminate this type of DNA damage from the genome.     

Tissue-Specific Regulation of HNK-1 Biosynthesis by Bisecting GlcNAc

Human natural killer—1 (HNK-1) is a sulfated glyco-epitope regulating cell adhesion and synaptic functions. HNK-1 and its non-sulfated forms, which are specifically expressed in the brain and the kidney, respectively, are distinctly biosynthesized by two homologous glycosyltransferases: GlcAT-P in the brain and GlcAT-S in the kidney. However, it is largely unclear how the activity of these isozymes is regulated in vivo. We recently found that bisecting GlcNAc, a branching sugar in N-glycan, suppresses both GlcAT-P activity and HNK-1 expression in the brain. Here, we observed that the expression of non-sulfated HNK-1 in the kidney is unexpectedly unaltered in mutant mice lacking bisecting GlcNAc. This suggests that the biosynthesis of HNK-1 in the brain and the kidney are differentially regulated by bisecting GlcNAc. Mechanistically, in vitro activity assays demonstrated that bisecting GlcNAc inhibits the activity of GlcAT-P but not that of GlcAT-S. Furthermore, molecular dynamics simulation showed that GlcAT-P binds poorly to bisected N-glycan substrates, whereas GlcAT-S binds similarly to bisected and non-bisected N-glycans. These findings revealed the difference of the highly homologous isozymes for HNK-1 synthesis, highlighting the novel mechanism of the tissue-specific regulation of HNK-1 synthesis by bisecting GlcNAc.     

Tetrandrine Suppresses Human Brain Glioblastoma GBM 8401/luc2 Cell-Xenografted Subcutaneous Tumors in Nude Mice In Vivo

Tetrandrine (TET), a bisbenzylisoquinoline (BBI) alkaloid, is isolated from the plant Stephania tetrandra S. Moore and has a wide range of biological activity, including anticancer properties in vitro and in vivo. At first, we established a luciferase-expressing stable clone that was named GBM 8401/luc2 cells. Herein, the primary results indicated that TET reduced the total cell viability and induced cell apoptosis in GBM 8401/luc2 human glioblastoma cells. However, there is no available information showing that TET suppresses glioblastoma cells in vivo. Thus, we investigated the effects and mechanisms of TET on a GBM 8401/luc2 cell-generated tumor in vivo. After the tumor volume reached 100–120 mm³ in subcutaneously xenografted nude mice, all of the mice were randomly divided into three groups: Group I was treated with phosphate-buffered solution (PBS) containing 0.1% dimethyl sulfoxide, Group II with 25 mg/kg of TET, and Group III with 50 mg/kg of TET. All mice were given the oral treatment of PBS or TET by gavage for 21 days, and the body weight and tumor volumes were recorded every 5 days. After treatment, individual tumors, kidneys, livers, and spleens were isolated from each group. The results showed that TET did not affect the body weights, but it significantly decreased the tumor volumes. The TET treatment at 50 mg/kg had a two-fold decrease in tumor volumes than that at 25 mg/kg when compared to the control. TET decreased the total photon flux, and treatment with TET at 50 mg/kg had a lower total photon flux than that at 25 mg/kg, as measured by a Xenogen IVIS imaging system. Moreover, the higher TET treatment had lower tumor volumes and weights than those of the lower dose. The apoptosis-associated protein expression in the tumor section was examined by immunohistochemical analysis, and the results showed that TET treatment reduced the levels of c-FLIP, MCL-1, and XIAP but increased the signals of cleaved-caspase-3, -8, and -9. Furthermore, the hematoxylin and eosin (H & E) staining of kidney, liver, and spleen tissues showed no significant difference between the TET-treated and control groups. Overall, these observations demonstrated that TET suppressed subcutaneous tumor growth in a nude-mice model via the induction of cell apoptosis.     

Trichothecenes in Food and Feed,Relevance to Human and Animal Health and Methods of Detection: A Systematic Review

Trichothecene mycotoxins are sesquiterpenoid compounds primarily produced by fungi in taxonomical genera such as Fusarium, Myrothecium, Stachybotrys, Trichothecium, and others, under specific climatic conditions on a worldwide basis. Fusarium mold is a major plant pathogen and produces a number of trichothecene mycotoxins including deoxynivalenol (or vomitoxin), nivalenol, diacetoxyscirpenol, and T-2 toxin, HT-2 toxin. Monogastrics are sensitive to vomitoxin, while poultry and ruminants appear to be less sensitive to some trichothecenes through microbial metabolism of trichothecenes in the gastrointestinal tract. Trichothecene mycotoxins occur worldwide however both total concentrations and the particular mix of toxins present vary with environmental conditions. Proper agricultural practices such as avoiding late harvests, removing overwintered stubble from fields, and avoiding a corn/wheat rotation that favors Fusarium growth in residue can reduce trichothecene contamination of grains. Due to the vague nature of toxic effects attributed to low concentrations of trichothecenes, a solid link between low level exposure and a specific trichothecene is difficult to establish. Multiple factors, such as nutrition, management, and environmental conditions impact animal health and need to be evaluated with the knowledge of the mycotoxin and concentrations known to cause adverse health effects. Future research evaluating the impact of low-level exposure on livestock may clarify the potential impact on immunity. Trichothecenes are rapidly excreted from animals, and residues in edible tissues, milk, or eggs are likely negligible. In chronic exposures to trichothecenes, once the contaminated feed is removed and exposure stopped, animals generally have an excellent prognosis for recovery. This review shows the occurrence of trichothecenes in food and feed in 2011–2020 and their toxic effects and provides a summary of the discussions on the potential public health concerns specifically related to trichothecenes residues in foods associated with the exposure of farm animals to mycotoxin-contaminated feeds and impact to human health. Moreover, the article discusses the methods of their detection.     

Atomistic De-novo Inhibitor Generation-Guided Drug Repurposing for SARS-CoV-2 Spike Protein with Free-Energy Validation by Well-Tempered Metadynamics

Computational drug design is increasingly becoming important with new and unforeseen diseases like COVID-19. In this study, we present a new computational de novo drug design and repurposing method and applied it to find plausible drug candidates for the receptor binding domain (RBD) of SARS-CoV-2 (COVID-19). Our study comprises three steps: atom-by-atom generation of new molecules around a receptor, structural similarity mapping to existing approved and investigational drugs, and validation of their binding strengths to the viral spike proteins based on rigorous all-atom, explicit-water well-tempered metadynamics free energy calculations. By choosing the receptor binding domain of the viral spike protein, we showed that some of our new molecules and some of the repurposable drugs have stronger binding to RBD than hACE2. To validate our approach, we also calculated the free energy of hACE2 and RBD, and found it to be in an excellent agreement with experiments. These pool of drugs will allow strategic repurposing against COVID-19 for a particular prevailing conditions.     

Sensory Perception of Non-Deuterated and Deuterated Organic Compounds

The chemical background of olfactory perception has been subject of intensive research, but no available model can fully explain the sense of smell. There are also inconsistent results on the role of the isotopology of molecules. In experiments with human subjects it was found that the isotope effect is weak with acetone and D₆-acetone. In contrast, clear differences were observed in the perception of octanoic acid and D₁₅-octanoic acid. Furthermore, a trained sniffer dog was initially able to distinguish between these isotopologues of octanoic acid. In chromatographic measurements, the respective deuterated molecule showed weaker interaction with a non-polar liquid phase. Quantum chemical calculations give evidence that deuterated octanoic acid binds more strongly to a model receptor than non-deuterated. In contrast, the binding of the non-deuterated molecule is stronger with acetone. The isotope effect is calculated in the framework of statistical mechanics. It results from a complicated interplay between various thermostatistical contributions to the non-covalent free binding energies and it turns out to be very molecule-specific. The vibrational terms including non-classical zero-point energies play about the same role as rotational/translational contributions and are larger than bond length effects for the differential isotope perception of odor for which general rules cannot be derived.