Potential fungicidal and antiaflatoxigenic effects of cinnamon essential oils on Aspergillus flavus inhabiting the stored wheat grains.

Wheat is one of the essential crops for the human and animal nutrition, however, contamination with aflatoxigenic fungi, due to the improper storage conditions and high humidity, was the main global threats. So, preventing the growth of aflatoxigenic fungi in stored wheat grains, by using different essential oils was the main objective of this work. Aspergillus flavus EFBL-MU12 PP087400, EFBL-MU23 PP087401 and EFBL-MU36 PP087403 isolates were the most potent aflatoxins producers inhabiting wheat grains. The effect of storage conditions of wheat grains "humidity, temperature, incubation period, and pH" on growth of A. flavus, was assessed by the response surface methodology using Plackett-Burman design and FCCD. The highest yield of aflatoxins EFBL-MU12 B1 and B2 by A. flavus grown on wheat grains were 145.3 and 7.6 µg/kg, respectively, at incubation temperature 35°C, 16% moisture contents, initial pH 5.0, and incubated for 14 days. The tested oils had a powerful antifungal activity for the growth and aflatoxins production by A. flavus in a concentration-dependent manner. Among these oils, cinnamon oil had the highest fungicidal activity for A. flavus at 0.125%, with about 85-90 % reduction to the aflatoxins B1 and B2, conidial pigmentation and chitin contents on wheat grains. From the SEM analysis, cinnamon oils had the most deleterious effect on A. flavus with morphological aberrations to the conidial heads, vegetative mycelia, alteration in conidiophores identity, hyphae shrank, and winding. To emphasize the effect of the essential oils on the aflatoxins producing potency of A. flavus, the molecular expression of the aflatoxins biosynthetic genes was estimated by RT-qPCR. The molecular expression of nor-1, afLR, pKsA and afLJ genes was suppressed by 94-96%, due to cinnamon oil at 0.062% compared to the control. Conclusively, from the results, cinnamon oils followed by the peppermint oils displayed the most fungicidal activity for the growth and aflatoxins production by A. flavus grown on wheat grains.

In silico evaluation of the potential allergenicity of a fungal biomass from Rhizomucor pusillus for use as a novel food ingredient.

The world's hunger for novel food ingredients drives the development of safe, sustainable, and nutritious novel food products. For foods containing novel proteins, potential allergenicity of the proteins is a key safety consideration. One such product is a fungal biomass obtained from the fermentation of Rhizomucor pusillus. The annotated whole genome sequence of this strain was subjected to sequence homology searches against the AllergenOnline database (sliding 80-amino acid windows and full sequence searches). In a stepwise manner, proteins were designated as potentially allergenic and were further compared to proteins from commonly consumed foods and from humans. From the sliding 80-mer searches, 356 proteins met the conservative >35% Codex Alimentarius threshold, 72 of which shared ≥50% identity over the full sequence. Although matches were identified between R. pusillus proteins and proteins from allergenic food sources, the matches were limited to minor allergens from these sources, and they shared a greater degree of sequence homology with those from commonly consumed foods and human proteins. Based on the in silico analysis and a literature review for the source organism, the risk of allergenic cross-reactivity of R. pusillus is low.

Mitigating drought stress in wheat plants (Triticum Aestivum L.) through grain priming in aqueous extract of spirulina platensis.

BACKGROUND: The study focuses on the global challenge of drought stress, which significantly impedes wheat production, a cornerstone of global food security. Drought stress disrupts cellular and physiological processes in wheat, leading to substantial yield losses, especially in arid and semi-arid regions. The research investigates the use of Spirulina platensis aqueous extract (SPAE) as a biostimulant to enhance the drought resistance of two Egyptian wheat cultivars, Sakha 95 (drought-tolerant) and Shandawel 1 (drought-sensitive). Each cultivar's grains were divided into four treatments: Cont, DS, SPAE-Cont, and SPAE + DS. Cont and DS grains were presoaked in distilled water for 18 h while SPAE-Cont and SPAE + DS were presoaked in 10% SPAE, and then all treatments were cultivated for 96 days in a semi-field experiment. During the heading stage (45 days: 66 days), two drought treatments, DS and SPAE + DS, were not irrigated. In contrast, the Cont and SPAE-Cont treatments were irrigated during the entire experiment period. At the end of the heading stage, agronomy, pigment fractions, gas exchange, and carbohydrate content parameters of the flag leaf were assessed. Also, at the harvest stage, yield attributes and biochemical aspects of yielded grains (total carbohydrates and proteins) were evaluated. RESULTS: The study demonstrated that SPAE treatments significantly enhanced the growth vigor, photosynthetic rate, and yield components of both wheat cultivars under standard and drought conditions. Specifically, SPAE treatments increased photosynthetic rate by up to 53.4%, number of spikes by 76.5%, and economic yield by 190% for the control and 153% for the drought-stressed cultivars pre-soaked in SPAE. Leaf agronomy, pigment fractions, gas exchange parameters, and carbohydrate content were positively influenced by SPAE treatments, suggesting their effectiveness in mitigating drought adverse effects, and improving wheat crop performance. CONCLUSION: The application of S. platensis aqueous extract appears to ameliorate the adverse effects of drought stress on wheat, enhancing the growth vigor, metabolism, and productivity of the cultivars studied. This indicates the potential of SPAE as an eco-friendly biostimulant for improving crop resilience, nutrition, and yield under various environmental challenges, thus contributing to global food security.

Phage vB_Ec_ZCEC14 to treat antibiotic-resistant Escherichia coli isolated from urinary tract infections.

Escherichia coli is a commensal bacterial species in the human gastrointestinal tract; however, it could be pathogenic and cause severe infections in intra and extra-intestinal sites. Uropathogenic E. coli accounts for 80-90% of urinary tract infections that can result in urosepsis and septic shock. Consequently, multidrug-resistant uropathogenic E. coli poses a considerable risk to the healthcare system worldwide. Phage therapy is demonstrated as an optimistic solution to over-the-counter antibiotics that contribute to the global issue of multidrug-resistant bacteria. This study aims to isolate a novel phage that could be implemented to cure urinary tract infections mediated by multidrug-resistant E. coli. Twenty-seven E. coli isolates were collected from patients with urinary tract infections to assess the antibacterial efficacy of phage vB_Ec_ZCEC14. Phage kinetics were encountered against the E. coli strain (EC/4), in addition to evaluating phage stability under various temperatures, pH values, and UV exposure periods. Full genome sequencing and morphological analysis were conducted for further phage characterization, which revealed that phage vB_Ec_ZCEC14 belongs to the family Straboviridae. Phage vB_Ec_ZCEC14 showed thermal tolerance at 80 ℃, pH stability between pH 3 and pH 12, and endurance to UV exposure for 45 min. The phage-host interaction results revealed that phage vB_Ec_ZCEC14 has strong and steady antibacterial action at lower concentrations (MOI 0.1). The study findings strongly indicate that phage vB_Ec_ZCEC14 holds significant promise as a potential therapeutic alternative for treatment of antibiotic-resistant uropathogenic E. coli.

The role of identified and characterized bacteriophage ZCEC13 in controlling pathogenic and multidrug-resistant Escherichia coli in wastewater: in vitro study.

The spread and development of Multi-Drug Resistant (MDR) bacteria in wastewater became beyond control and a global public health concern. The conventional disinfectants used in wastewater treatment methods have been becoming increasingly ineffective against a range of pathogenic and MDR bacteria. Bacteriophages are considered a novel approach to microbial control. Therefore, this study aims to explore the possibility of using phages against pathogenic and MDR Escherichia coli strains isolated from wastewater treatment plants. The wastewater samples were collected from two different treatment plants for E. coli isolation. The antibiotic sensitivity profile and occurrence of virulence and resistant genes were tested in 28 E. coli isolates. Phage ZCEC13 was selected based on its promising activity and host range to undergo identification and characterization. ZCEC13 was evaluated by transmission electron microscopy, genomic sequencing, in vitro lytic activity and tested for its stability under different conditions such as pH, Ultraviolet light exposure, and temperature. The results reported that ZCEC13 belongs to the Caudoviricetes class, with a high antibacterial dynamic. Phage ZCEC13 displayed high stability at different pH values ranging from 2 to 12, good tolerance to temperatures from -4 to 65°C, and high stability at UV exposure for 120 min. Respectively, the findings showed stability of the phage under several conditions and high efficiency in killing MDR bacteria isolated from the treatment plants. Further studies are encouraged to analyse the efficacy of phages as a microbial control agent in wastewater treatment plants.

Morphological, biological, and genomic characterization of Klebsiella pneumoniae phage vB_Kpn_ZC2.

BACKGROUND: Bacteriophages (phages) are one of the most promising alternatives to traditional antibiotic therapies, especially against multidrug-resistant bacteria. Klebsiella pneumoniae is considered to be an opportunistic pathogen that can cause life-threatening infections. Thus, this study aims at the characterization of a novel isolated phage vB_Kpn_ZC2 (ZCKP2, for short). METHODS: The phage ZCKP2 was isolated from sewage water by using the clinical isolate KP/08 as a host strain. The isolated bacteriophage was purified and amplified, followed by testing of its molecular weight using Pulse-Field Gel Electrophoresis (PFGE), transmission electron microscopy, antibacterial activity against a panel of other Klebsiella pneumoniae hosts, stability studies, and whole genome sequencing. RESULTS: Phage ZCKP2 belongs morphologically to siphoviruses as indicated from the Transmission Electron Microscopy microgram. The Pulsed Field Gel Electrophoresis and the phage sequencing estimated the phage genome size of 48.2 kbp. Moreover, the absence of lysogeny-related genes, antibiotic resistance genes, and virulence genes in the annotated genome suggests that phage ZCKP2 is safe for therapeutic use. Genome-based taxonomic analysis indicates that phage ZCKP2 represents a new family that has not been formally rated yet. In addition, phage ZCKP2 preserved high stability at different temperatures and pH values (-20 - 70 °C and pH 4 - 9). For the antibacterial activity, phage ZCKP2 maintained consistent clear zones on KP/08 bacteria along with other hosts, in addition to effective bacterial killing over time at different MOIs (0.1, 1, and 10). Also, the genome annotation predicted antibacterial lytic enzymes. Furthermore, the topology of class II holins was predicted in some putative proteins with dual transmembrane domains that contribute significantly to antibacterial activity. Phage ZCKP2 characterization demonstrates safety and efficiency against multidrug-resistant K. pneumoniae, hence ZCKP2 is a good candidate for further in vivo and phage therapy clinical applications.

Bacteriophage as a potential therapy to control antibiotic-resistant Pseudomonas aeruginosa infection through topical application onto a full-thickness wound in a rat model.

BACKGROUND: Antibiotic-resistant Pseudomonas aeruginosa (P. aeruginosa) is one of the most critical pathogens in wound infections, causing high mortality and morbidity in severe cases. However, bacteriophage therapy is a potential alternative to antibiotics against P. aeruginosa. Therefore, this study aimed to isolate a novel phage targeting P. aeruginosa and examine its efficacy in vitro and in vivo. RESULTS: The morphometric and genomic analyses revealed that ZCPA1 belongs to the Siphoviridae family and could infect 58% of the tested antibiotic-resistant P. aeruginosa clinical isolates. The phage ZCPA1 exhibited thermal stability at 37 °C, and then, it decreased gradually at 50 °C and 60 °C. At the same time, it dropped significantly at 70 °C, and the phage was undetectable at 80 °C. Moreover, the phage ZCPA1 exhibited no significant titer reduction at a wide range of pH values (4-10) with maximum activity at pH 7. In addition, it was stable for 45 min under UV light with one log reduction after 1 h. Also, it displayed significant lytic activity and biofilm elimination against P. aeruginosa by inhibiting bacterial growth in vitro in a dose-dependent pattern with a complete reduction of the bacterial growth at a multiplicity of infection (MOI) of 100. In addition, P. aeruginosa-infected wounds treated with phages displayed 100% wound closure with a high quality of regenerated skin compared to the untreated and gentamicin-treated groups due to the complete elimination of bacterial infection. CONCLUSION: The phage ZCPA1 exhibited high lytic activity against MDR P. aeruginosa planktonic and biofilms. In addition, phage ZCPA1 showed complete wound healing in the rat model. Hence, this research demonstrates the potential of phage therapy as a promising alternative in treating MDR P. aeruginosa.

Safety evaluation of Neurospora crassa mycoprotein for use as a novel meat alternative and enhancer.

Cultivation of filamentous fungi to produce sustainable, nutrient rich meat replacements has recently attracted significant commercial and research interest. Here, we report evidence for the safety and nutritional value of Neurospora crassa mycoprotein, a whole mycelium food ingredient produced by fermentation and minimal downstream processing. N. crassa has a long history of human use in fermented foods and in molecular biology research. A survey of studies that used N. crassa in animal feed revealed no adverse effects to the health of the animals. Furthermore, a review of the literature found no reports of confirmed allergenicity or toxicity in humans involving N. crassa. Genomic toxigenicity analysis and in vitro testing did not identify any toxins in N. crassa mycoprotein. Two independent genomic allergenicity studies did not identify proteins that would be considered a particular risk for allergenic potential. Furthermore, nutritional analysis demonstrated that N. crassa mycoprotein is a good source of complete protein and is rich in fiber, potassium, and iron. Taken together, the presented data and the history of human use without evidence of human or animal harm indicate that foods containing N. crassa can generally be regarded as safe.

Development of a Sequence Searchable Database of Celiac Disease-Associated Peptides and Proteins for Risk Assessment of Novel Food Proteins.

Celiac disease (CeD) is an autoimmune enteropathy induced by prolamin and glutelin proteins in wheat, barley, rye, and triticale recognized by genetically restricted major histocompatibility (MHC) receptors. Patients with CeD must avoid consuming these proteins. Regulators in Europe and the United States expect an evaluation of CeD risks from proteins in genetically modified (GM) crops or novel foods for wheat-related proteins. Our database includes evidence-based causative peptides and proteins and two amino acid sequence comparison tools for CeD risk assessment. Sequence entries are based on the review of published studies of specific gluten-reactive T cell activation or intestinal epithelial toxicity. The initial database in 2012 was updated in 2018 and 2022. The current database holds 1,041 causative peptides and 76 representative proteins. The FASTA sequence comparison of 76 representative CeD proteins provides an insurance for possible unreported epitopes. Validation was conducted using protein homologs from Pooideae and non-Pooideae monocots, dicots, and non-plant proteins. Criteria for minimum percent identity and maximum E-scores are guidelines. Exact matches to any of the 1,041 peptides suggest risks, while FASTA alignment to the 76 CeD proteins suggests possible risks. Matched proteins should be tested further by CeD-specific CD4/8+ T cell assays or in vivo challenges before their use in foods.

Isolation, Characterization, and Genomic Analysis of Three Novel E. coli Bacteriophages That Effectively Infect E. coli O18.

Escherichia coli (E. coli) is one of the most common pathogenic bacteria worldwide. Avian pathogenic E. coli (APEC) causes severe systemic disease in poultry (Colibacillosis), and accordingly, has an extreme risk to the poultry industry and public health worldwide. Due to the increased rate of multi-drug resistance among these bacteria, it is necessary to find an alternative therapy to antibiotics to treat such infections. Bacteriophages are considered one of the best solutions. This study aimed to isolate, characterize, and evaluate the potential use of isolated bacteriophages to control E. coli infections in poultry. Three novel phages against E. coli O18 were isolated from sewage water and characterized in vitro. The genome size of the three phages was estimated to be 44,776 bp, and the electron microscopic analysis showed that they belonged to the Siphoviridae family, in the order Caudovirales. Phages showed good tolerance to a broad range of pH and temperature. The complete genomes of three phages were sequenced and deposited into the GenBank database. The closely related published genomes of Escherichia phages were identified using BLASTn alignment and phylogenetic trees. The prediction of the open reading frames (ORFs) identified protein-coding genes that are responsible for functions that have been assigned such as cell lysis proteins, DNA packaging proteins, structural proteins, and DNA replication/transcription/repair proteins.

Topically Applied Bacteriophage to Control Multi-Drug Resistant Klebsiella pneumoniae Infected Wound in a Rat Model.

(Background): Multi-drug-resistant Klebsiella pneumoniae (MDR-KP) has steadily grown beyond antibiotic control. Wound infection kills many patients each year, due to the entry of multi-drug resistant (MDR) bacterial pathogens into the skin gaps. However, a bacteriophage (phage) is considered to be a potential antibiotic alternative for treating bacterial infections. This research aims at isolating and characterizing a specific phage and evaluate its topical activity against MDR-KP isolated from infected wounds. (Methods): A lytic phage ZCKP8 was isolated by using a clinical isolate KP/15 as a host strain then characterized. Additionally, phage was assessed for its in vitro host range, temperature, ultraviolet (UV), and pH sensitivity. The therapeutic efficiency of phage suspension and a phage-impeded gel vehicle were assessed in vivo against a K. pneumoniae infected wound on a rat model. (Result): The phage produced a clear plaque and was classified as Siphoviridae. The phage inhibited KP/15 growth in vitro in a dose-dependent pattern and it was found to resist high temperature (˂70 °C) and was primarily active at pH 5; moreover, it showed UV stability for 45 min. Phage-treated K. pneumoniae inoculated wounds showed the highest healing efficiency by lowering the infection. The quality of the regenerated skin was evidenced via histological examination compared to the untreated control group. (Conclusions): This research represents the evidence of effective phage therapy against MDR-KP.

Evaluating potential risks of food allergy of novel food sources based on comparison of proteins predicted from genomes and compared to www.AllergenOnline.org.

Potential proteins from three novel food sources (Chlorella variabilis, Galdieria sulphuraria, and Fusarium strain flavolapis) were predicted from genomic sequences and were evaluated for potential risks of allergic cross-reactivity by comparing the predicted amino acid sequences against the allergens in the www.AllergenOnline.org (AOL) database. The preliminary analysis used CODEX Alimentarius limits of >35% identity over 80 amino acids to evaluate the predicted proteins which include many evolutionarily conserved proteins. Regulators might expect clinical serum IgE tests based on identity matches above the criteria if the proteins were introduced in genetically engineered crops. Some regulators have the same expectations for proteins in novel foods. To address the inequality of extensively conserved sequences, we compared the predicted proteins from curated genomes of 23 highly diverse allergenic species from animals, plants and arthropods as well as humans to AOL sequences and compiled identities. Identity matches greater than CODEX limits (>35% ID over 80 AA) are common for many proteins that are conserved through extensive evolution but are not predictive of published allergy risks based on observed taxonomic cross-reactivity. Therefore, we recommend changes in the allergen databases or methods of identifying matches for risk evaluation of new food sources. Our results provide critical data for redefining allergens in AOL or for providing guidance on more predictive sequence identity matches for risk assessment of possible risks of food allergy.

Synthesis of novel naphthalene-heterocycle hybrids with potent antitumor, anti-inflammatory and antituberculosis activities.

Multitarget-directed drugs (hybrid drugs) constitute an efficient avenue for the treatment of multifactorial diseases. In this work, novel naphthalene hybrids with different heterocyclic scaffolds such as nicotinonitrile, pyran, pyranopyrazole, pyrazole, pyrazolopyridine, and azepine were efficiently synthesized via tandem reactions of 3-formyl-4H-benzo[h]chromen-4-one 1 with different nucleophilic reagents. Analysis of these hybrids using PASS online software indicated different predicted biological activities such as anticancer, antimicrobial, antiviral, antiprotozoal, anti-inflammatory, etc. By focusing on antitumor, anti-inflammatory, and antituberculosis activities, many compounds revealed remarkable activities. While 3c, 3e, and 3h were more potent than doxorubicin in the case of HepG-2 cell lines, 3a-e, 3i, 6, 8, 10, 11, and 12b were more potent in the case of MCF-7. Moreover, compounds 3c, 3h, 8, 10, 3d, and 12b manifested superior activity and COX-2 selectivity to the reference anti-inflammatory Celecoxib. Regarding antituberculosis activity, 3c, 3d, and 3i were found to be the most promising with MIC less than 1 µg mL-1. The molecular docking studies showed strong polar and hydrophobic interactions with the novel naphthalene-heterocycle hybrids that were compatible with experimental evaluations to a great extent.

Unorthodox synthesis, biological activity and DFT studies of novel and multifunctionalized naphthoxocine derivatives.

A new promising protocol has been developed for the synthesis of scarce oxocine derivatives 3a-e and 6 through addition of amine-based nucleophiles such as hydroxylamine hydrochloride, primary amine and hydrazide to chromonylidene benzothiazol-2-ylacetonitrile 2 in refluxing dioxane under metal free reaction conditions in moderate to good yields. Other nitrogen nucleophiles such as piperidine, hydrazine and thiosemicarbazide failed to afford the corresponding oxocinols, and instead pyridine derivatives 7, 8 and 10 were obtained exclusively. Predictive study for the biological activities using PASS (prediction of activity spectra for biologically active substances) online software showed optimistic activities for oxocinols 3a-e in the treatment of cancer, influenza A and microbial infections. Additionally, DFT studies of oxocine derivatives 3a-e and 6 indicated the presence of required thermodynamics parameters for the application in dye-sensitized solar cells (DSSCs).