Red Kale (Brassica oleracea L. ssp. acephala L. var. sabellica) Induces Apoptosis in Human Colorectal Cancer Cells In Vitro.

This article presents the results of studies investigating the effect of red kale (Brassica oleracea L. ssp. acephala L. var. sabellica) extract on cancer cells (HT-29). The cytotoxicity of the red kale extract was assessed using MTT and LDH assays, while qRT-PCR was employed to analyze the expression of genes associated with the p53 signaling pathway to elucidate the effect of the extract on cancer cells. Furthermore, HPLC-ESI-QTOF-MS/MS was applied to identify bioactive compounds present in red kale. The obtained results indicated that red kale extract reduced the viability and suppressed the proliferation of HT-29 cells (the IC50 value of 60.8 µg/mL). Additionally, mRNA expression analysis revealed significant upregulation of several genes, i.e., casp9, mapk10, mapk11, fas, kat2 b, and ubd, suggesting the induction of cell apoptosis through the caspase-dependent pathway. Interestingly, the study revealed a decrease in the expression of genes including cdk2 and cdk4 encoding cell cycle-related proteins, which may lead to cell cycle arrest. Furthermore, the study identified certain bioactive compounds, such as sinigrin, spirostanol, hesperetin and usambarensine, which could potentially contribute to the apoptotic effect of red kale extracts. However, further investigations are necessary to elucidate the specific role of these individual compounds in the anti-cancer process.

Comparison of the Effect of Cold Plasma with Conventional Preservation Methods on Red Wine Quality Using Chemometrics Analysis.

In this study, the effect of cold plasma (CP) on the physicochemical and biological properties of red wine was investigated in comparison with the effects of the conventional preservation method and the combined method. In addition, the effect of storage time after the application of each of the analyzed methods was evaluated. The study examined the effects of the different preservation methods on the pH, color, phenolic content, antioxidant activity and microbiological purity of the red wine. Chemometric analysis was used to discover the relationship between the preservation method used and wine quality. In the wine samples tested, a reduction in phenolic compounds and a decrease in antioxidant activity were noted after storage. This effect was mildest for preservation methods with the addition of potassium metabisulphite and those in which a mixture of helium and nitrogen was used as the working gas. On a positive note, the CP treatment did not affect the color of the wine in a way perceptible to the consumer: ∆E*-1.12 (He/N2; 5 min). In addition, the lowest growth of microorganisms was detected in the CP-treated samples. This indicates the potential of cold plasma as an alternative method to the use of potassium metabisulfite in wine production, which may contribute to its wider use in the alcohol industry in the future.

Biological Activity of an Epilobium angustifolium L. (Fireweed) Infusion after In Vitro Digestion.

The biological activity of an in vitro digested infusion of Epilobium angustifolium (fireweed) was examined in a model system of intestinal epithelial and colon cancer tissues. The content of selected phenolic compounds in the digested aqueous extract of fireweed was determined using HPLC-ESI-QTOF-MS/MS. Biological activity was examined using the human colon adenocarcinoma cell lines HT-29 and CaCo-2 and the human colon epithelial cell line CCD 841 CoTr. Cytotoxicity was assessed by an MTT assay, a Neutral Red uptake assay, May-Grünwald-Giemsa staining, and a label-free Electric Cell-Substrate Impedance Sensing cytotoxicity assay. The effect of the infusion on the growth of selected intestinal bacteria was also examined. The extract inhibited the growth of intestinal cancer cells HT-29. This effect can be attributed to the activity of quercetin and kaempferol, which were the most abundant phenolic compounds found in the extract after in vitro digestion. The cytotoxicity of the fireweed infusion was dose-dependent. The highest decrease in proliferation (by almost 80%) compared to the control was observed in HT-29 line treated with the extract at a concentration of 250 µg/mL. The fireweed infusion did not affect the growth of beneficial intestinal bacteria, but it did significantly inhibit E. coli. The cytotoxic effect of the fireweed extract indicates that it does not lose its biological activity after in vitro digestion. It can be concluded that the fireweed infusion has the potential to be used as a supporting agent in colon cancer therapy.

Novel Primer Sets for Rapid Detection of Zymoseptoria tritici in Wheat.

Zymoseptoria tritici is a fungal pathogen causing losses in wheat yields. Here, we present new primer sets for species-specific identification of this microorganism in wheat leaf samples using conventional PCR. Primer sets were validated in silico using tools available in genetic databases. Furthermore, in vitro tests were also carried out on 190 common wheat samples with visual symptoms of Septoria tritici blotch (STB) collected in Poland in three growing seasons (2015, 2016, 2017). The designed primer sets showed full hybridization to the available genetic resources deposited in the NCBI GenBank database, and their high specificity and sensitivity were demonstrated on wheat leaf samples and selected fungal strains.

Genome and Pangenome Analysis of Lactobacillus hilgardii FLUB-A New Strain Isolated from Mead.

The production of mead holds great value for the Polish liquor industry, which is why the bacterium that spoils mead has become an object of concern and scientific interest. This article describes, for the first time, Lactobacillus hilgardii FLUB newly isolated from mead, as a mead spoilage bacteria. Whole genome sequencing of L. hilgardii FLUB revealed a 3 Mbp chromosome and five plasmids, which is the largest reported genome of this species. An extensive phylogenetic analysis and digital DNA-DNA hybridization confirmed the membership of the strain in the L. hilgardii species. The genome of L. hilgardii FLUB encodes 3043 genes, 2871 of which are protein coding sequences, 79 code for RNA, and 93 are pseudogenes. L. hilgardii FLUB possesses three clustered regularly interspaced short palindromic repeats (CRISPR), eight genomic islands (44,155 bp to 6345 bp), and three (two intact and one incomplete) prophage regions. For the first time, the characteristics of the genome of this species were described and a pangenomic analysis was performed. The concept of the pangenome was used not only to establish the genetic repertoire of this species, but primarily to highlight the unique characteristics of L. hilgardii FLUB. The core of the genome of L. hilgardii is centered around genes related to the storage and processing of genetic information, as well as to carbohydrate and amino acid metabolism. Strains with such a genetic constitution can effectively adapt to environmental changes. L. hilgardii FLUB is distinguished by an extensive cluster of metabolic genes, arsenic detoxification genes, and unique surface layer proteins. Variants of MRS broth with ethanol (10-20%), glucose (2-25%), and fructose (2-24%) were prepared to test the strain's growth preferences using Bioscreen C and the PYTHON script. L. hilgardii FLUB was found to be more resistant than a reference strain to high concentrations of alcohol (18%) and sugars (25%). It exhibited greater preference for fructose than glucose, which suggests it has a fructophilic nature. Comparative genomic analysis supported by experimental research imitating the conditions of alcoholic beverages confirmed the niche specialization of L. hilgardii FLUB to the mead environment.

Prebiotic Potential of Oligosaccharides Obtained by Acid Hydrolysis of α-(1→3)-Glucan from Laetiporus sulphureus: A Pilot Study.

Increasing knowledge of the role of the intestinal microbiome in human health and well-being has resulted in increased interest in prebiotics, mainly oligosaccharides of various origins. To date, there are no reports in the literature on the prebiotic properties of oligosaccharides produced by the hydrolysis of pure fungal α-(1→3)-glucan. The aim of this study was to prepare α-(1→3)-glucooligosaccharides (α-(1→3)-GOS) and to perform initial evaluation of their prebiotic potential. The oligosaccharides were obtained by acid hydrolysis of α-(1→3)-glucan isolated from the fruiting bodies of Laetiporus sulphureus and then, characterized by HPLC. Fermentation of α-(1→3)-GOS and reference prebiotics was compared in in vitro pure cultures of Lactobacillus, Bifidobacterium, and enteric bacterial strains. A mixture of α-(1→3)-GOS, notably with a degree of polymerization of 2 to 9, was obtained. The hydrolysate was utilized for growth by most of the Lactobacillus strains tested and showed a strong bifidogenic effect, but did not promote the growth of Escherichia coli and Enterococcus faecalis. α-(1→3)-GOS proved to be effective in the selective stimulation of beneficial bacteria and can be further tested to determine their prebiotic functionality.

Potentilla alba Extracts Affect the Viability and Proliferation of Non-Cancerous and Cancerous Colon Human Epithelial Cells.

The aim of this study was to determine the anti-tumor activity of extracts isolated from Potentilla alba L. on human colon cancer cells of the HT-29 line and on non-cancer colon epithelial cells of the CCD 841 CoTr line. The research methods we used to determine the cytotoxic and proliferative properties were 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR) assays, the ability to produce nitric oxide, the Griess method, and the biochemical properties like 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods indicating reduction activity of tested samples. Finally, the effects of the extracts on the morphology and cell counts were assessed by May-Grünwald-Giemsa staining. After a comprehensive analysis of all the experiments, the extracts were found to demonstrate cytotoxic properties, they stimulated the division of non-cancer cells, and they were able to scavenge free radicals. In the NR method, the cell viability dropped to approximately 80% compared to the control. In the MTT assay, tumor cell proliferation decreased to 9.5% compared to the control. Therefore, we concluded that this plant has medical potential.

A Report on Fungal (1→3)-α-d-glucans: Properties, Functions and Application.

The cell walls of fungi are composed of glycoproteins, chitin, and α- and ß-glucans. Although there are many reports on ß-glucans, α-glucan polysaccharides are not yet fully understood. This review characterizes the physicochemical properties and functions of (1→3)-α-d-glucans. Particular attention has been paid to practical application and the effect of glucans in various respects, taking into account unfavourable effects and potential use. The role of α-glucans in plant infection has been proven, and collected facts have confirmed the characteristics of Aspergillus fumigatus infection associated with the presence of glucan in fungal cell wall. Like ß-glucans, there are now evidence that α-glucans can also stimulate the immune system. Moreover, α-d-glucans have the ability to induce mutanases and can thus decompose plaque.

Steinernema poinari (Nematoda: Steinernematidae): a new symbiotic host of entomopathogenic bacteria Xenorhabdus bovienii.

Three strains of symbiotic bacteria were isolated from an entomopathogenic nematode Steinernema poinari retrieved from soil in eastern Poland. Using 16S rDNA, recA, gltX, gyrB, and dnaN gene sequences for phylogenetic analysis, these strains were shown to belong to the species Xenorhabdus bovienii. The nucleotide identity between the studied S. poinari microsymbionts and other X. bovienii strains calculated for 16S rDNA and concatenated sequences of four protein-coding genes was 98.7-100% and 97.9-99.5%, respectively. The phenotypic properties of the isolates also supported their close phylogenetic relationship with X. bovienii. All three tested X. bovienii strains of different Steinernema clade origin supported the recovery of infective juveniles and subsequent development of the nematode population. However, the colonization degree of new infective juvenile generations was significantly affected by the bacterial host donor/recipient. The colonization degree of infective juveniles reared on bacterial symbionts deriving from a non-cognate clade of nematodes was extremely low, but proved the possible host-switching between non-related Steinernema species.

Distribution of Cell Envelope Proteinases Genes among Polish Strains of Lactobacillus helveticus.

Most of the lactic acid bacteria (LAB) are able to grow in milk mainly due to the activity of a complex and well-developed proteolytic system. Cell envelope-associated proteinases (CEPs) begin casein hydrolysis and allow for releasing the peptides, enclosed in the structure of native milk proteins that are essential for growth of Lactobacillus helveticus. The biodiversity of genes encoding CEPs among L. helveticus strains can have an effect on some technological parameters such as acid production, bacterial growth rate in milk as well as liberation of biologically active peptides. The study reveals significant differences in the presence of various variants of CEPs encoding genes among ten novel Polish strains and indicates the intraspecific diversity exhibited by L. helveticus. In terms of distribution of CEPs genes, four different genetic profiles were found among the microorganisms analyzed. Furthermore, the strains exhibited also various levels of proteolytic activity. Molecular analysis revealed that prtH3 is the most abundant CEPs-encoding gene among the strains investigated. The results indicate also that ecological niche and environmental conditions might affect proteolytic properties of L. helveticus strains. The greatest variety in terms of quantity of the detected CEP encoding genes was noticed in L. helveticus 141, T105 and T104 strains. In these strains, the combination of three nucleotide gene sequences (prtH/prtH2/prtH3) was identified. Interestingly, T104 and T105 exhibited the highest proteolytic activity and also the fastest dynamic of milk acidification among the tested strains of L. helveticus.

Isolation and characterization of a new fructophilic Lactobacillus plantarum FPL strain from honeydew.

In the present study, a Lactobacillus plantarum FPL strain exhibiting fructophilic behavior has been isolated for the first time from honeydew. It is a probably syntrophic bacterium inhabiting the gastrointestinal tract of Coccus hesperidum L. and taking part in sugar metabolism. The promising growth characteristics and biochemical properties of Lb. plantarum FPL indicate that this may be a facultatively fructophilic species, whose properties are not associated with the loss of the alcohol/acetaldehyde dehydrogenase gene. The article attempts to classify the peculiar behavior of this strain by means of tests that are characteristic for FLAB as well as through a classic identification approach. In this study, we used a reference strain Lb. plantarum NRRL B-4496, which showed no fructophilic properties. With the FLAB group, the new strain shares the habit, such as a fructose-rich environment, the preference of this sugar for growth, and similar growth curves. However, it exceeds FLAB in terms of osmotolerance to high sugar content. The fructophilic Lb. plantarum FPL strain can proliferate and grow on a medium wherein the sugar concentration is 45 and 50% (w/v). Our findings indicate that honeydew can be a promising source of new fructophilic lactic acid bacteria.

Strains of Photorhabdus spp. associated with polish Heterorhabditis isolates: their molecular and phenotypic characterization and symbiont exchange.

The relationships between six bacterial symbionts of the entomopathogenic nematodes Heterorhabditis bacteriophora and Heterorhabditis megidis from Poland to species and subspecies of the genus Photorhabdus were evaluated. This study was based on phylogenetic analysis of sequence data of five genes: 16S rRNA, gyrB, recA, gltX, and dnaN. The bacteria were also characterized phenotypically by biochemical and physiological tests. Our results have revealed that the Photorhabdus strains isolated from H. megidis belong to P. temperata, subsp. temperata and subsp. cinerea. Isolates from H. bacteriophora represent P. luminescens subs. kayaii and P. temperata subs. cinerea. This study for the first time provides evidence for H. bacteriophora and P. temperata subsp. cinerea symbiotic association. In addition, we tested whether the microsymbionts of the Polish H. bacteriophora and H. megidis isolates support the development of non-native nematode host population and colonization of their infective juveniles. It has been shown that the studied Photorhabdus strains can readily swap their nematode host, both at intra- and interspecies level. It supports the hypothesis of different symbiotic associations in the Heterorhabditis-Photorhabdus lineage.

Studies on the removal of Cd ions by gastrointestinal lactobacilli.

Accumulation of toxic metal ions in food and water is nowadays a growing health-related problem. One detoxification method involves the use of microorganisms naturally inhabiting the gastrointestinal tract (GIT). The purpose of this study was to prove that lactic acid bacteria derived from the GIT are able to effectively remove Cd2+ from water solution. Seven strains of lactobacilli, out of 11 examined, showed tolerance to high concentrations of cadmium ions. The metal-removal efficiencies of these seven lactobacilli ranged from 6 to 138.4 µg/h mg. Among these bacteria, Lactobacillus gallinarum and Lactobacillus crispatus belonged to the highest (85%) Cd-removal efficiency class. An analysis of the zeta potential (ζ) indicated that the bacterial cell surface had a negative charge at the pH ranging from 3 to 10. The presence of carboxyl, amide, and phosphate groups was favorable for Cd2+ binding to the cell surface, which found confirmation in FTIR-ATR spectra. Elemental SEM/EDS analysis and TEM imaging not only confirmed the adsorption of Cd2+ on the cell envelope but also gave us a reason to suppose that Lb. crispatus accumulates metal ions inside the cell. Our findings open perspectives for further research on the new biological function of GIT lactobacilli as natural biosorbents.

κ-Casein as a source of short-chain bioactive peptides generated by Lactobacillus helveticus.

This paper explores the ability of Lactobacillus helveticus strains to release sequences of short biologically active peptides (containing 2-10 amino acid residues) from casein. The proteolytic enzymes of the tested strains exhibit different patterns of cleavage of CN fractions. The modification of κ-casein (κ-CN) with pyrrolidone carboxylic acid inhibits the proteolytic activity of strains L. helveticus 141 and the reference strain (DSMZ 20075), while the modification with phosphothreonine inhibits enzymes of all the tested bacteria. The peptide sequencing analysis indicated that the examined strains produced functional peptides very efficiently. κ-CN proved to be the main source of short peptides released by bacterial enzymes, and the hydrolysis of κ-CN yielded eighty-two bioactive peptides. The hydrolysis of αS2-casein, αS1-casein, and ß-casein yielded six, two, and one short-chain bioactive peptides, respectively. The isolated bioactive peptides exhibited antioxidative, opioid, stimulating, hypotensive, immunomodulating, antibacterial, and antithrombotic activities. A vast majority of the isolated bioactive peptides caused inhibition of the angiotensin-converting enzyme and dipeptidyl peptidase IV. The role of hydrolysis products as neuropeptides is also pointed out. The highest number of cleavage sites in κ-casein and functional activities of short-chain peptides were obtained in hydrolyzates produced by L. helveticus strain T105.

[The use of oligonucleotide aptamers in cancer therapy]. / Wykorzystanie aptamerów oligonukleotydowych w terapii nowotworów.

Aptamers are a new class of molecules which originated in the 1990s. They are usually RNA or DNA oligonucleotides, the length of which ranges between 20 and 80 nt. They are produced using the SELEX method that allows one to obtain aptamers that bind to virtually any molecule of interest, providing a high specificity. Aptamers are an alternative to antibodies because on the one hand, their sensitivity is at a similar or sometimes even higher level, while on the other hand they do not show immunogenicity, and may be synthesized in vitro. To date, a broad range of different applications of aptamers has been described: as components of biosensors, or use in various laboratory techniques, such as microarrays or chromatography. One of the most important is the use of aptamers in medicine, especially in the fight against cancer. They can be used both for diagnosis and for the eradication of cancers - particularly through the delivery of drugs. Currently, most transport-related research is devoted to the delivery of chemotherapeutic drugs, such as doxorubicin. This was used in research on liver cancer cells, prostate, and acute lymphoblastic leukemia blast cells. Another possibility is to use aptamers to deliver siRNAs. In this way inhibition of the quality control process of the mRNA in tumor cells is possible. An aptamer complex with the drug allows for direct delivery of the active substance in a particular cell type, substantially eliminating the non-specific effect of the drug.

The effect of cell surface components on adhesion ability of Lactobacillus rhamnosus.

The aim of this study was to analyze the cell envelope components and surface properties of two phenotypes of Lactobacillus rhamnosus isolated from the human gastrointestinal tract. The ability of the bacteria to adhere to human intestinal cells and to aggregate with other bacteria was determined. L. rhamnosus strains E/N and PEN differed with regard to the presence of exopolysaccharides (EPS) and specific surface proteins. Transmission electron microscopy showed differences in the structure of the outer cell surface of the strains tested. Bacterial surface properties were analyzed by Fourier transform infrared spectroscopy, fatty acid methyl esters and hydrophobicity assays. Aggregation capacity and adhesion of the tested strains to the human colon adenocarcinoma cell line HT29 was determined. The results indicated a high adhesion and aggregation ability of L. rhamnosus PEN, which possessed specific surface proteins, had a unique fatty acid content, and did not synthesize EPS. Adherence of L. rhamnosus was dependent on specific interactions and was promoted by surface proteins (42-114 kDa) and specific fatty acids. Polysaccharides likely hindered bacterial adhesion and aggregation by masking protein receptors. This study provides information on the cell envelope constituents of lactobacilli that influence bacterial aggregation and adhesion to intestinal cells. This knowledge will help to understand better their specific contribution in commensal-host interactions and adaptation to this ecological niche.

The effect of moonlighting proteins on the adhesion and aggregation ability of Lactobacillus helveticus.

The goal of this study was to identify moonlighting proteins in Lactobacillus helveticus that play an important role in adhesion and aggregation. The label-free method was used for identification and analysis of expression of cellular proteins. The analysis revealed the presence of eight moonlighting proteins in the cell envelope of Lb. helveticus. The tested strains mainly differed with respect to the presence of S-layer proteins and the level of expression of moonlighting proteins in Lb. helveticus strain T159. These surface proteins give the cell a hydrophobic character and play a role in specific interactions with intestinal epithelium cells and with other bacteria. In Lb. helveticus T159, the S-layer associated with moonlighting proteins could act as adherence factors, which was evidenced by the high capability of adhesion, auto- and coaggregation. The hydrophobicity, adhesion and aggregation abilities provide biological activities in food products and they are regarded as an important criterion for probiotic selection.

Variability of S-layer proteins in Lactobacillus helveticus strains.

The presence of S-layer proteins in the cell envelope of Lactobacillus helveticus may be technologically important. S-layer proteins are the adhesion site for cell envelope proteinase, which forms the proteolytic pathway in bacteria. Eleven strains of L. helveticus were examined for the presence of S-layer proteins and slpH genes. S-layer proteins from six strains were identified and sequenced. Multiple alignments of the deduced amino acid sequences demonstrated a strong sequence conservation of all Slp studied. Transmission Electron Microscopy analysis of the cells revealed the typical cell wall architecture of the S-layer. This is the first report on characterisation of glycosylated S-layer proteins from different strains of L. helveticus. The amino acid composition, the secondary structure, and the physical properties of these proteins were found to be quite similar to those of S-layer proteins from other lactobacilli. However, PCR analysis revealed that five of the examined strains of L. helveticus did not have slpH genes. This finding suggests that S-layer protein genes cannot be considered as housekeeping genes and cannot be used as molecular markers for L. helveticus.

Optimization of culture conditions for exopolysaccharide production by a probiotic strain of Lactobacillus rhamnosus E/N.

The effects of culture conditions on exopolysaccharides (EPS) production by a probiotic Lb. rhamnosus E/N have been studied using the Plackett-Burman design. Process optimization was performed in stationary cultures to maximize the production of EPS. In order to verify the optimal conditions, an analysis was performed of EPS production in fermentation culture. Batch fermentation was carried out at working volume of 2.51. The optimal temperature, pH, carbon source, and nitrogen source conditions were 37 degrees C, pH 5.0, galactose, and yeast extract, respectively. EPS production was improved by 210.28 mg/l in stationary cultures compared to 134.2 mg/l in a control grown on commercial MRS medium. The fermentor experiment showed the possibility of increasing EPS biosynthesis by 175.8%. Our results clearly demonstrate that in the case of Lb. rhamnosus E/N specific culture conditions can enhance EPS production for possible application in the industry.

Bifidogenic and antioxidant activity of exopolysaccharides produced by Lactobacillus rhamnosus E/N cultivated on different carbon sources.

Lactobacillus rhamnosus E/N is a probiotic bacterium, which synthesizes exopolysaccharides (EPS) with significant bifidogenic and antioxidant activities. The sugar composition of the EPSs produced depended on carbohydrates used as a carbon source in the growth media. Five Bifidobacterium strains were tested in vitro for their ability to utilize all the EPSs studied. The highest bifidogenic activity was revealed by EPSs obtained from Lactobacillus cultures supplemented with Gal, Lac, and Mal as the only carbon source, while significant antioxidant effects were observed in EPSs isolated from growth media enriched with galactose, lactose, and sucrose.