Many studies demonstrated that radio frequency (RF) was an effective pasteurization method for low-moisture foods (LMFs), and our previous study confirmed RF heating stress generated sublethal injured cells (SICs) of Salmonella enterica serovar Typhimurium (S. Typhimurium) in red pepper powder with initial aw >= 0.53. So this study investigated the potential direct protection and cross protection effects of the SICs of S. Typhimurium to multiple stresses, and analyzed fatty acid composition and cell morphology. Results showed that the SICs were repaired after incubating for 5 h, and there were no obvious direct and cross protection effects by exposing to different external stresses (heat, 15% ethanol, pH 3.0 acid buffer solution, 10% salt). According to the fatty acid composition analysis, no significant difference (p > 0.05) between the ratio of unsaturated to saturated fatty acids (UFA/SFA) was observed for SICs of S. Typhimurium and control cells, indicating the same membrane fluidity which can support the experimental results. This study investigated and confirmed there are no direct and cross protection effects for the SICs of S. Typhimurium induced by RF heating stress, and it would be helpful for deeply understand the response of pathogens under RF heating stress.

Antibiotic resistance is a daunting challenge in modern medicine, and novel approaches that minimize the emergence of resistant pathogens are desperately needed. Antimicrobial peptides are newer therapeutics that attempt to do this; however, they fall short because of low to moderate antimicrobial activity, low protease stability, susceptibility to resistance development, and high cost of production. The recently developed random peptide mixtures (RPMs) are promising alternatives. RPMs are synthesized by incorporating a defined proportion of two amino acids at each coupling step rather than just one, making them highly variable but still defined in their overall composition, chain length, and stereochemistry. Because RPMs have extreme diversity, it is unlikely that bacteria would be capable of rapidly evolving resistance. However, their efficacy against pathogens in animal models of human infectious diseases remained uncharacterized. Here, we demonstrated that RPMs have strong safety and pharmacokinetic profiles. RPMs rapidly killed both Pseudomonas aeruginosa and Staphylococcus aureus efficiently and disrupted preformed biofilms by both pathogens. Importantly, RPMs were efficacious against both pathogens in mouse models of bacteremia and acute pneumonia. Our results demonstrate that RPMs are potent broad-spectrum therapeutics against antibiotic-resistant pathogens.

Bacteria show distinct and characteristic fatty acid (FA) patterns which can be modified by environmental conditions. In this study, we cultivated six plant-pathogenic bacteria of agricultural concern and performed a detailed analysis of the fatty acid composition. The study covered four strains of the gram-negative Xanthomonas campestris pathovar (pv) campestris (Xcc), Xanthomonas perforans (Xp), Acidovorax citrulli (Ac) and Pseudomonas syringae pv. tomato (Pst), and two strains of the gram-positive Clavibacter michiganensis subsp. michiganensis (Cmm) and Streptomyces scabies (Ssc). After cultivation, freeze-dried bacteria samples were transesterified and analysed by gas chromatography with mass spectrometry in full scan and selected ion monitoring (SIM) modes. Altogether, 44 different FAs were detected in the six strains with individual contributions of 0.01-43.8% to the total FAs. The variety in the six strains ranged between 12 and 31 individual FAs. The FA composition of Xcc, Xp, Cmm and Ssc were dominated by iso- and anteiso-fatty acids (especially i15:0, a15:0, i16:0), which is typical for most bacteria. In contrast to this, Ac and Pst showed only saturated and monounsaturated FAs. Four of the six bacteria showed similar FA patterns as reported before in the literature. Differences were observed in the case of Cmm where many more FAs were detected in the present study. In addition, to the best of our knowledge, the FA pattern of Xp was presented for the first time.

Electronic noses (e-nose) and optical noses (o-nose) are two emerging approaches for the development of artificial olfactory systems for flavor and smell evaluation. The current work leverages the unique optical properties of semiconducting single-wall carbon nanotubes (SWCNTs) to develop a prototype of a novel paper-based near-infrared optical nose (NIRON). We have drop-dried an array of SWCNTs encapsulated with a wide variety of peptides on a paper substrate and continuously imaged the emitted SWCNTs fluorescence using a CMOS camera. Odors and different volatile molecules were passed above the array in a flow chamber, resulting in unique modulation patterns of the SWCNT photoluminescence (PL). Quartz crystal microbalance (QCM) measurements performed in parallel confirmed the direct binding between the vapor molecules and the peptide-SWCNTs. PL levels measured before and during exposure demonstrate distinct responses to the four tested alcoholic vapors (ethanol, methanol, propanol, and isopropanol). In addition, machine learning tools directly applied to the fluorescence images allow us to distinguish between the aromas of red wine, beer, and vodka. Further, we show that the developed sensor can detect limonene, undecanal, and geraniol vapors, and differentiate between their smells utilizing the PL response pattern. This novel paper-based optical biosensor provides data in real-time, and is recoverable and suitable for working at room temperature and in a wide range of humidity levels. This platform opens new avenues for real-time sensing of volatile chemical compounds, odors, and flavors.

In the present study, two C-type lectins (designated as VpClec-3 and VpClec-4) were identified and characterized from the manila clam Venerupis philippinarum. Multiple alignment and phylogenetic relationship analysis strongly suggested that VpClec-3 and VpClec-4 belong to the C-type lectin family. In nonstimulated clams, the VpClec-3 transcript was dominantly expressed in the hepatopancreas, while the VpClec-4 transcript was mainly expressed in gill tissues. Both VpClec-3 and VpClec-4 mRNA expression was significantly upregulated following Vibrio anguillarum challenge. Recombinant VpClec-4 (rVpClec-4) was shown to bind lipopolysaccharide (LPS) and glucan in vitro, whereas recombinant VpClec-3 (rVpClec-3) only bound to glucan. In addition, rVpClec-3 and rVpClec-4 displayed broad agglutination activities towards Vibrio harveyi, Vibrio splendidus and V. anguillarum, while no agglutination activities towards Enterobacter cloacae or Aeromonas hydrophila were observed in rVpClec-3. Moreover, hemocyte phagocytosis was significantly enhanced by rVpClec-3 and rVpClec-4. All the results showed that VpClecs function as pattern recognition receptors (PRRs) with distinct recognition spectra and are potentially involved in the innate immune responses of V. philippinarum.

Meat products contain valuable nutrients that are important for human health and development but are also highly susceptible to colonization by microorganisms. This can lead to spoilage and serious foodborne illnesses. Natural antimicrobial peptides, produced by many organisms as part of their innate immune system to fight microbial infections, have great potential as food preservatives. In this study, we explored the effect of ternary antimicrobial random peptide mixtures (RPMs) on food spoilage bacteria in minced turkey meat. Amendment of RPMs to meat led to significant reductions in bacterial abundance in experimental tests, and RPMs worked synergistically with nitrite to reduce bacterial loads. Using high-throughput 16S ribosomal RNA gene amplicon sequencing, we characterized the effect of RPMs and nitrite on meat microbial community structure before and during incubation under refrigerated conditions. Our findings reveal strong antimicrobial activity for RPMs against spoilage bacteria in meat, including Listeria monocytogenes and Pseudomonas putida. These results demonstrate the potential of RPMs as a safer preservative for reducing spoilage in meat and other food products.

In the current study, we evaluated the antimicrobial activity of randomly-sequenced peptide mixtures (RPMs) bearing hydrophobic and cationic residues thatwere immobilized on beads. We showed that these beads exhibit high and broad bactericidal activity against various pathogenic bacteria while possessing minimal hemolytic activity.

Random peptide mixtures (RPMs) have been recently proposed as powerful antimicrobial compounds. These are unique mixtures of peptides synthesized by random combination of a cationic and a hydrophobic amino acid. Here, we introduce a new type of antimicrobial compounds, short lipo-RPMs, which result fromN-palmitoylation of RPMs. We report the characterization of 5-mer lipo-RPMs containingl-phenylalanine andd-lysine, named p-FdK5. p-FdK5 had high antibacterial activity against several bacterial strains and was able to reduce disease severity caused by a plant pathogen. We further synthesized and studied all 32 (2(5)) possible lipopeptides that compose the p-FdK5 mixture. We showed that the antibacterial activity of specific lipopeptides depends on the peptide hydrophobicity and on the location of the hydrophobic amino acids relative to the palmitic acid. Interestingly, synergism assays revealed positive interactions between different sequence-specific lipopeptides in terms of antimicrobial activity.

A protease is an enzyme that catalyzes proteolysis of proteins into smaller polypeptides or single amino acids. As crucial elements in many biological processes, proteases have been shown to be informative biomarkers for several pathological conditions in humans, animals, and plants. Therefore, fast, reliable, and cost-effective protease biosensors suitable for point-of-care (POC) sensing may aid in diagnostics, treatment, and drug discovery for various diseases. This work presents an affordable and simple paper-based dipstick biosensor that utilizes peptide-encapsulated single-wall carbon nanotubes (SWCNTs) for protease detection. Upon enzymatic digestion of the peptide, a significant drop in the photoluminescence (PL) of the SWCNTs was detected. As the emitted PL is in the near-infrared region, the developed biosensor has a good signal to noise ratio in biological fluids. One of the diseases associated with abnormal protease activity is pancreatitis. In acute pancreatitis, trypsin concentration could reach up to 84 mu g/mL in the urine. For proof of concept, we demonstrate the feasibility of the proposed biosensor for the detection of the abnormal levels of trypsin activity in urine samples.

High-fat diet (HFD) leads to enhancement in various parameters of mice like weight, fasting glucose levels, adipose tissue, and also the liver weight in male C57 BL/6 J mice. Additionally, high-fat diet causes severe liver damage with significant increase in the level of aspartate amino transferase (AST) and alanine transaminase (ALT). The variations in microbiota induced by different diet were analyzed by Illumina MiSeq platform with sequencing of 16S ribosomal RNA (rRNA) gene, and QIIME pipeline was used. The population of Proteobacteria was found to be higher in HFD cecum sample as compared to other treatments. Microbiota analysis suggests that phylum Proteobacteria and Firmicutes were found to be higher in high-fat diet groups as compared to mice fed with normal diet (ND). At the genus level,Bacteroidesshowed higher population in HFD diet. Bacterial strains belonging toEnterobacteriaceaelikeEscherichia, Klebsiella, andShigellawere also dominant in HFD treatments. Furthermore, we explored the effects of ethanol productionin vitrowith supplementation of dietary fibers following inoculation of ND and HFD microbiotas. HFD microbiota of cecum and feces showed high level (P< 0.05) of ethanol production with 2% fructooligosaccharide (FOS) as compared to 2% galactomannan. Microbial fermentation also generated short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate. High levels (P< 0.05) of propionate were found after fermentation of FOS with HFD cecum and feces microbiota. The present study highlights the HFD-induced population of phylum Proteobacteria and genusBacteroidesfor ethanol production using FOS as a dietary supplement, and these findings may imply on the harmful effect of HFD even at the microbiota level.

The quorum sensing (QS) system of , an opportunistic human pathogen, utilizes the autoinducing PapR peptide signal that mediates the activation of the pleiotropic virulence regulator PlcR. A set of synthetic 7-mer PapR-derived peptides (PapR; ADLPFEF) have been shown to inhibit efficiently the PlcR regulon activity and the production of virulence factors, reflected by a loss in hemolytic activity without affecting bacterial growth. Interestingly, these first potent synthetic inhibitors involved D-amino acid or alanine replacements of three amino acids; proline, glutamic acid, and phenylalanine of the heptapeptide PapR. To better understand the role of these three positions in PlcR activity, we report herein the second generation design, synthesis, and characterization of PapR-derived combinations, alternate double and triple alanine and D-amino acids replacement at these positions. Our findings generate a new set of non-native PapR-derived peptides that inhibit the PlcR regulon activity and the production of virulence factors. Using the amino acids substitution strategy, we revealed the role of proline and glutamic acid on PlcR regulon activation. Moreover, we demonstrated that the D-Glutamic acid substitution was crucial for the design of stronger PlcR antagonists. These peptides represent potent synthetic inhibitors of QS and constitute new and readily accessible chemical tools for the study of the PlcR system. Our method might be applied to other quorum sensing systems to design new anti-virulence agents.

Antibiotic resistance in bacteria has become a serious threat to public health, and therefore there is an urgent need to develop new classes of antimicrobial agents. Nowadays, natural antimicrobial peptides (AMPs) and their synthetic derivatives are considered as promising alternatives to traditional antibiotics. The broad molecular diversity of AMPs, in terms of sequences and structures, suggests that their activity does not depend on specific features of amino acid sequence or peptide conformation. We therefore selected two common properties of AMPs, (high percentage of hydrophobic and cationic amino acids), to develop a novel approach to synthesize random antimicrobial peptide mixtures (RPMs). Instead of incorporating a single amino acid at each coupling step, a mixture of hydrophobic and cationic amino acids in a defined proportion is coupled. This results in a mixture that contains up to 2n sequences, where n is the number of the coupling step, of random peptides with a defined composition, stereochemistry, and controlled chain length. We have discovered that RPMs of hydrophobic and cationic α-amino acids, such as phenylalanine and lysine, display strong and broad antimicrobial activity towards Gram-negative, Gram-positive, clinically isolated antibiotic resistant "superbugs", and several plant pathogenic bacteria. This review summarizes our efforts to explore the mode of action of RPMs and their potential as bioactive agents for multiple applications, including the prevention of biofilm formation and degradation of mature biofilm (related to human health), reduction of disease severity in plant bacterial disease models (related to crop protection), and inhibition of bacterial growth in milk (related to food preservation). All our findings illustrate the effectiveness of RPMs and their great potential for various applications.

The emergence of global antibiotic resistance necessitates the urgent need to develop new and effective antimicrobial agents. Combination of two antimicrobial agents can potentially improve antimicrobial potency and mitigate the development of resistance. Therefore, we have utilized metal molecular doping methodology whereby antimicrobial random peptides mixture (RPMs) are entrapped in a bactericidal copper metal matrix. The copper/RPM composite exhibits greater antimicrobial activity toward methicillin-resistant Staphylococcus aureus (MRSA) than either copper or RPMs alone. Our findings indicate that this bactericidal antimicrobial biomaterial could be utilized to efficiently eradicate antibiotic-resistant pathogenic bacteria for health, agricultural and environmental applications.

Light emitting diodes of wavelength 410 nm were used to inactivate Listeria monocytogenes stains on paperboard, an increasingly popular food packaging material. The integrity of the cell membranes was examined using differential fluorescent staining. Scanning electron microscopy (SEM) was used to obtain a deeper understanding of L. monocytogenes stain formation on paperboard and the damage caused to the cells by the LEDs. While the planktonic L. monocytogenes population could be completely inactivated following a brief lag phase that lasted about 20 min, the illumination of the sessile population left some persisters despite immediate commencement of the inactivation. Planktonic populations of inocula sized 3, 5 and 7 log CFU/mL were reduced below the detection limit in 54, 80 and 84 min respectively, whereas it took 120 and 390 min to reach constancy in the sessile populations of inocula sized 5 and 7 log CFU/cm2. The number of membrane-damaged cells was seen to increase with the illumination time. SEM images provided evidence of the protection conferred by the stain on the underlying cells. This study demonstrates that blue LEDs have the potential to reduce the risk of L. monocytogenes contamination from paperboard cartons under refrigeration.

ABSTRACT Recombinant chicken prolactin (chPRL), expressed in Escherichia coli and purified as a monomer, was successfully PEGylated and purified to homogeneity as a mono-PEGylated protein (PEG-chPRL). Its biological activity was estimated by its ability to interact with human prolactin receptor extracellular domain (hPRLR-ECD) and stimulate PRLR-mediated proliferation in Nb2-11C cells. PEG-chPRL activity in a cell bioassay was 10-fold lower than that of non-PEGylated chPRL, but only 2-fold lower in a binding assay to hPRLR-ECD. The CD spectra of non-PEGylated and PEGylated chPRL were almost identical and similar to that of hPRL, indicating proper refolding. Although the PEGylation of chPRL resulted in lower activity in vitro, PEG-chPRL was absorbed more slowly than chPRL, remained in the circulation 16 h longer. Furthermore the effects of PEG-chPRL injections in chickens on subsequent corticosteroid levels in blood were significantly profound compared to chPRL. These favorable PEGylation-induced pharmacokinetic alterations should improve efficacy of PEG-chPRL in in vivo experiments, as dosing frequency can be reduced due to its prolonged persistence in the circulation, and thus reduce the frequency of dosing. Furthermore, hydrophobic interaction chromatography was successfully adopted to isolate PEG-chPRL as a better alternative for separation of PEGylated PRL, and is likely to be successfully applicable to other proteins.

To provide new tools for in vitro and in vivo prolactin (PRL) research, novel protocols for large-scale preparation of untagged human PRL (hPRL), a hPRL antagonist (del 1-9-G129R hPRL) that acts as a pure antagonist of hPRL in binding to hPRL receptor extracellular domain (hPRLR-ECD), and hPRLR-ECD are demonstrated. The interaction of del 1-9-G129R hPRL with hPRLR-ECD was demonstrated by competitive non-radioactive binding assay using biotinylated hPRL as the ligand and hPRLR-ECD as the receptor, by formation of stable 1:1 complexes with hPRLR-ECD under non-denaturing conditions using size-exclusion chromatography, and by surface plasmon resonance methodology. In all three types of experiments, the interaction of del 1-9-G129R hPRL was equal to that of unmodified hPRL. Del 1-9-G129R hPRL inhibited the hPRL-induced proliferation of Baf/LP cells stably expressing hPRLR. Overall, the biological properties of del 1-9-G129R hPRL prepared by the protocol described herein were similar to those of the antagonist prepared using the protocol reported in the original study; however, the newly described protocol improved yields by >6-fold. To provide long-lasting hPRL as a new reagent needed for in vivo experiments, we prepared its mono-pegylated analogue and found that pegylation lowers its biological activity in a homologous in vitro assay. As its future use will require the development of a PRL antagonist with highly elevated affinity, del 1-9-G129R hPRL was expressed on the surface of yeast cells. It retained its binding capacity for hPRLR-ECD, and this methodology was shown to be suitable for future development of high-affinity hPRL antagonists using a library of randomly mutated open reading frame of del 1-9-G129R hPRL and selecting high-affinity mutants by yeast surface display methodology.

Radio frequency (RF) dielectric heating has been investigated to inactivate pathogens in some low-moisture foods. This study was aimed to evaluate RF inactivation effects on Salmonella typhimurium in red pepper powders, by focusing on the influence of sample initial water activity (aw) and applying Weibull model to describe the inactivation curves. The experimental results showed that RF heating rate increased when aw was in the range of 0.57–0.71, but decreased after aw reached to 0.71. During the come-up time of RF heating, 2–3 log reduction of the pathogen was achieved depending on aw levels. Increasing initial aw could first increased log reductions and then decreased the log reductions, optimum aw level was 0.71 for RF inactivation of Salmonella in red pepper powders. For red pepper powders with aw of 0.71, RF heating to 70 °C (come-up time was 110 s) with holding time over 60 s could achieve >5 log reduction of S. typhimurium. Weibull model well fitted the survival curves of the pathogen with goodness of fit (R2 > 0.93, RMSE<0.29). Scale factor (b) of the model increased with treatment temperature increasing, while the shape factor (n) was independent on temperature. This study provided basic guideline for using RF heating to inactivate Salmonella in red pepper powders.

Many types of crops are severely affected by at least one important bacterial disease. Chemical control of bacterial plant diseases in the field vastly relies on copper-based bactericides, yet with limited efficacy. In this study, we explored the potential of two random peptide mixture (RPM) models as novel crop protection agents. These unique peptide mixtures consist of random combination of l-phenylalanine and l- or d-lysine (FK-20 and FdK-20, respectively) along the 20 mer chain length of the peptides. Both RPMs displayed powerful bacteriostatic and bactericidal activities towards strains belonging to several plant pathogenic bacterial genera, for example, Xanthomonas, Clavibacter and Pseudomonas. In planta studies in the glasshouse revealed that RPMs significantly reduced disease severity of tomato and kohlrabi plants infected with Xanthomonas perforans and Xanthomonas campestris pv. campestris respectively. Moreover, RPM effects on reduction in disease severity were similar to those exerted by the commercial copper-based bactericide Kocide 2000 that was applied at a 12-fold higher concentration of the active compound relative to the RPM treatments. Importantly, the two tested RPM compounds had no toxic effect on survival of bees and Caco-2 mammalian cells. This study demonstrates the potential of these innovative RPMs to serve as crop protection agents against crop diseases caused by phytopathogenic bacteria.

The shelf life of pasteurized bovine milk is limited by microorganism activity as surviving bacteria continue to grow in the bovine milk, eventually causing milk spoilage. In the current study, we used matrix-assisted laser desorption ionization time of flight mass spectrometry to identify pasteurized bovine milk-associated mesophilic and psychrotrophic bacteria. We have recently designed random cationic peptide mixtures that possess strong antimicrobial and antibiofilm properties. These compounds are cheap and easy to synthesize and represent a new class of antimicrobial agents. Here, we show that the random peptide mixtures are able to efficiently eradicate the bacteria identified as associated with pasteurized bovine milk, and reduced significantly the growth of Bacillus subtilis in milk. We propose these antimicrobial peptides as potential candidates for integration in bioactive milk and food packaging to prevent bacterial growth and extend the shelf life of food.

Fish growth hormones (GHs) play an important role in regulating growth, metabolism, reproduction, osmoregulation, and immunity and have thus garnered attention for their application in aquaculture. Zebrafish GH (zGH) cDNA or rainbow trout GH (rtGH) cDNA was cloned into the pMon3401 vector, expressed in MON105-competent Escherichia coli and purified to homogeneity. Their biological activity was evidenced by their ability to interact with ovine GH receptor extracellular domain and stimulate GH receptor-mediated proliferation in FDC-P1-3B9 cells stably transfected with rabbit GH receptor. The relative affinity of zGH and rtGH, estimated by IC50, was about 38-fold and 512-fold lower, respectively, than ovine GH. This is likely the reason for the low biological activity in cells with rabbit GH receptor, ~ 36-fold lower for zGH and ~ 107-fold lower for rtGH than for human GH. This was not due to improper refolding, as evidenced by circular dichroism analysis. Predicting the activity of fish GHs is problematic as there is no one single optimal in vitro bioassay; heterologous assays may be ambiguous, and only homologous assays are suitable for measuring activity.