Determination of 465 psychoactive substances, drugs and their metabolites in urine by LC-MS/MS.

Due to the emergence of novel psychoactive substances on the drug market, there is a growing demand for analytical methods allowing identification and determination of as many psychoactive substances as possible in the shortest possible time, which can be easily expanded to include additional analytes appearing in street trade. Immunochemical methods are not sufficient to meet constantly growing requirements. Therefore, the aim of the study was to develop an analytical method enabling quick analysis of urine samples for psychoactive substances, drugs and their metabolites. Liquid-liquid extraction (LLE) and liquid chromatography coupled with mass spectrometry (LC-MS/MS) were used for this purpose. Using available analytical standards, the operating parameters of the mass spectrometer were selected for each of the 477 analytes, and MRM (multiple reaction monitoring) acquisition was selected for each of them. The use of analytical standards allowed for the identification of analytes separated on the chromatography column. The exceptions are methylmethcathinone isomers (3-MMC and 4-MMC), which we were unable to separate using the gradient elution method used. Extraction using acetonitrile with the addition of ammonium formate and formic acid allowed us to obtain high recoveries without the use of ß-glucuronidase. Recovery values ranged from 18.43 to 119.94%. The matrix effect was eliminated by obtaining a calibration curve in the matrix. The developed analytical method was validated in accordance with SWGTOX guidelines. Only 12 substances did not meet the validation criteria (CV: ±20% and bias: ±20%). Thus the validated method makes it possible to determine 465 psychoactive substances in just 30 minutes. In the validation process, values such as the limit of detection (LOD) and the limit of quantification (LOQ) were also determined. The LODs are in the range of 3-30 ng mL-1, and the LOQs are in the range of 10-100 ng mL-1. The method was successfully applied to toxicological analyses of urine samples, which was an opportunity to develop it further to meet the needs of toxicology.

From KIT-mutated into wild-type: dedifferentiation of gastrointestinal stromal tumor in adolescent patient-a case report.

Background: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract, usually found in elderly adults. It is infrequent among pediatric patients and usually differs from adult-type disease in terms of histopathology and molecular features. Case Description: We describe the management of the disease in a 10-year-old female patient diagnosed with a GIST of the stomach. In total, she has undergone successively total tumor resection, unsuccessful imatinib treatment and subtotal gastric resection at relapse. The first genetic test from primary tumor confirmed KIT mutations in exons 13 and 17, while the repeated genetic screening using tumor sample from subtotal gastric resection revealed no KIT or platelet-derived growth factor receptor α (PDGFRA) genes mutations. Such dedifferentiation from adult type (thus KIT-mutated) into wild-type (without KIT or PDGFRA mutation) has not been reported so far to the best of our knowledge. Currently, the patient is observed, and no further pharmacological nor surgical treatment has been administered. Conclusions: The case underlines the importance of genetic profiling combined with non-standard diagnostics (both histopathological and radiological) due to the treatment efficacy prediction. We moreover emphasize the necessity to create worldwide standards on the diagnostics and treatment of GIST in pediatric patients that would include options of targeted therapies.

The Characteristics, Uses, and Biases of Studies Related to Malignancies Using Google Trends: Systematic Review.

BACKGROUND: The internet is a primary source of health information for patients, supplementing physician care. Google Trends (GT), a popular tool, allows the exploration of public interest in health-related phenomena. Despite the growing volume of GT studies, none have focused explicitly on oncology, creating a need for a systematic review to bridge this gap. OBJECTIVE: We aimed to systematically characterize studies related to oncology using GT to describe its utilities and biases. METHODS: We included all studies that used GT to analyze Google searches related to malignancies. We excluded studies written in languages other than English. The search was performed using the PubMed engine on August 1, 2022. We used the following search input: "Google trends" AND ("oncology" OR "cancer" or "malignancy" OR "tumor" OR "lymphoma" OR "multiple myeloma" OR "leukemia"). We analyzed sources of bias that included using search terms instead of topics, lack of confrontation of GT statistics with real-world data, and absence of sensitivity analysis. We performed descriptive statistics. RESULTS: A total of 85 articles were included. The first study using GT for oncology research was published in 2013, and since then, the number of publications has increased annually. The studies were categorized as follows: 22% (19/85) were related to prophylaxis, 20% (17/85) pertained to awareness events, 11% (9/85) were celebrity-related, 13% (11/85) were related to COVID-19, and 47% (40/85) fell into other categories. The most frequently analyzed cancers were breast (n=28), prostate (n=26), lung (n=18), and colorectal cancers (n=18). We discovered that of the 85 studies, 17 (20%) acknowledged using GT topics instead of search terms, 79 (93%) disclosed all search input details necessary for replicating their results, and 34 (40%) compared GT statistics with real-world data. The most prevalent methods for analyzing the GT data were correlation analysis (55/85, 65%) and peak analysis (43/85, 51%). The authors of only 11% (9/85) of the studies performed a sensitivity analysis. CONCLUSIONS: The number of studies related to oncology using GT data has increased annually. The studies included in this systematic review demonstrate a variety of concerning topics, search strategies, and statistical methodologies. The most frequently analyzed cancers were breast, prostate, lung, colorectal, skin, and cervical cancers, potentially reflecting their prevalence in the population or public interest. Although most researchers provided reproducible search inputs, only one-fifth used GT topics instead of search terms, and many studies lacked a sensitivity analysis. Scientists using GT for medical research should ensure the quality of studies by providing a transparent search strategy to reproduce results, preferring to use topics over search terms, and performing robust statistical calculations coupled with sensitivity analysis.

Temperature, Salinity and Garlic Additive Shape the Microbial Community during Traditional Beetroot Fermentation Process.

Plant-based traditional fermented products are attracting a lot of interest in global markets. An example of them is beetroot leaven, which is valued for its high bioactive compound content. The variety of production recipes and the spontaneous nature of red beet fermentation favor its high diversity. This study aimed to analyze the impact of external factors-temperature, brine salinity, and garlic dose-on the beetroot fermentation and bacterial metapopulation responsible for this process. The research results confirmed the significant influence of the selected and analyzed factors in shaping the leaven physicochemical profile including organic acid profile and betalain content. Analysis of bacterial populations proved the crucial importance of the first 48 h of the fermentation process in establishing a stable metapopulation structure and confirmed that this is a targeted process driven by the effect of the analyzed factors. Lactobacillaceae, Enterobacteriaceae, and Leuconostocaceae were observed to be the core microbiome families of the fermented red beet. Regardless of the impact of the tested factors, the leaven maintained the status of a promising source of probiotic bacteria. The results of this research may be helpful in the development of the regional food sector and in improving the quality and safety of traditionally fermented products such as beetroot leaven.

2,4-D versus 2,4-D based ionic liquids: Effect of cation on herbicide biodegradation, tfdA genes abundance and microbiome changes during soil bioaugmentation.

The commercial formulations of herbicides rely on surfactants which increase the efficiency of active substance. Herbicidal ionic liquids (ILs), in which cationic surfactants are combined with herbicidal anions, allow for additives' reduction and ensure very good herbicide performance with lower doses. We aimed to test the impact of synthetic and natural cations on biological degradation of 2,4-dichlorophenoxyacetic acid (2,4-D). Although primary biodegradation was high, the mineralization in agricultural soil indicated incomplete conversion of ILs to CO2. Even the introduction of naturally-derived cations resulted in an increase in the herbicide's half-lives - from 32 days for [Na][2,4-D] to 120 days for [Chol][2,4-D] and 300 days for the synthetic tetramethylammonium derivative [TMA][2,4-D]. Bioaugmentation with 2,4-D-degrading strains improves the herbicides' degradation, which was reflected by higher abundance of tfdA genes. Microbial community analysis confirmed that hydrophobic cationic surfactants, even those based on natural compounds, played a negative role on microbial biodiversity. Our study provides a valuable indication for further research related to the production of a new generation of environmentally friendly compounds. Moreover, the results shed a new light on the ionic liquids as independent mixtures of ions in the environment, as opposed to treating them as new type of environmental pollutants.

Successful Treatment of Large B-Cell Lymphoma in a Child with Compound Heterozygous Mutation in the ATM Gene.

Ataxia-telangiectasia (AT) is a multisystemic neurodegenerative inborn error of immunity (IEI) characterized by DNA repair defect, chromosomal instability, and hypersensitivity to ionizing radiation. Impaired DNA double-strand break repair determines a high risk of developing hematological malignancies, especially lymphoproliferative diseases. Poor response to treatment, excessive chemotherapy toxicities, and the need for avoiding exposure to ionizing radiation make the successful clinical management of patients with AT challenging for oncologists. We describe the favorable outcome of the LBCL with IRF4 rearrangement at stage III in a 7-year-old female patient diagnosed with AT. The patient was treated according to the B-HR arm of the INTER-B-NHL-COP 2010 protocol, including the administration of rituximab, cyclophosphamide, methotrexate, prednisone, etc. She presented excessive treatment toxicities despite individually reduced doses of methotrexate and cyclophosphamide. However, in the MRI there was no significant reduction in pathologic lymph nodes after three immunochemotherapy courses. Therefore, a lymph node biopsy was taken. Its subsequent histopathological examination revealed tuberculosis-like changes, though tuberculosis suspicion was excluded. After two following immunochemotherapy courses, PET-CT confirmed complete remission. From March 2022 onwards, the patient has remained in remission under the care of the outpatient children's oncology clinic.

Development and Validation of the LC-MS/MS Method for Determination of 130 Natural and Synthetic Cannabinoids in Cannabis Oil.

Dietary supplements are widely available products used by millions of people around the world. Unfortunately, the procedure of adding pharmaceutical and psychoactive substances has recently been observed, in order to increase the effectiveness of supplements in the form of hemp oils. For this reason, it is extremely important to develop analytical methods for the detection of substances prohibited in dietary supplements and food products. In the present study, using the LC-MS/MS technique, an innovative method for the detection and quantification of 117 synthetic cannabinoids and 13 natural cannabinoids in dietary supplements and food products in the form of oils during one 13-min chromatographic run was developed. Each method was fully validated by characterization of the following parameters: The limit of detection was set to 0.1 ng/mL (100 µg/g, 0.01%). The limit of quantification ranged from 0.05 ng/mL to 50 ng/mL. The criteria assumed for systematic error caused by methodological bias (±20%) resulting from the recovery of analytes after the extraction process, as well as the coefficient of variation (CV) (≤20%), were met for all 130 tested compounds. The positive results of the validation confirmed that the developed methods met the requirements related to the adequacy of their application in a given scope. Additionally, methods developed using the LC-MS/MS technique were verified via proficiency tests. The developed analytical procedure was successfully used in the analysis of hemp oils and capsules containing them in the studied dietary supplements.

Latest Advances in the Management of Pediatric Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumor is the most common mesenchymal neoplasm of the gastrointestinal tract, usually found in elderly adults. It is infrequent among pediatric patients and usually differs biologically from adult-type diseases presenting mutations of KIT and PDGFR genes. In this population, more frequent is the wild-type GIST possessing SDH, TRK, RAS, NF1 mutations, among others. Both tumor types require individualized treatment with kinase inhibitors that are still being tested in the pediatric population due to the different neoplasm biology. We review the latest updates to the management of pediatric gastrointestinal tumors with a particular focus on the advances in molecular biology of the disease that enables the definition of possible resistance. Emerging treatment with kinase inhibitors that could serve as targeted therapy is discussed, especially with multikinase inhibitors of higher generation, the effectiveness of which has already been confirmed in the adult population.

Targeting CD38 in Neoplasms and Non-Cancer Diseases.

CD38 is a myeloid antigen present both on the cell membrane and in the intracellular compartment of the cell. Its occurrence is often enhanced in cancer cells, thus making it a potential target in anticancer therapy. Daratumumab and isatuximab already received FDA approval, and novel agents such as MOR202, TAK079 and TNB-738 undergo clinical trials. Also, novel therapeutics such as SAR442085 aim to outrank the older antibodies against CD38. Multiple myeloma and immunoglobulin light-chain amyloidosis may be effectively treated with anti-CD38 immunotherapy. Its role in other hematological malignancies is also important concerning both diagnostic process and potential treatment in the future. Aside from the hematological malignancies, CD38 remains a potential target in gastrointestinal, neurological and pulmonary system disorders. Due to the strong interaction of CD38 with TCR and CD16 on T cells, it may also serve as the biomarker in transplant rejection in renal transplant patients. Besides, CD38 finds its role outside oncology in systemic lupus erythematosus and collagen-induced arthritis. CD38 plays an important role in viral infections, including AIDS and COVID-19. Most of the undergoing clinical trials focus on the use of anti-CD38 antibodies in the therapy of multiple myeloma, CD19- B-cell malignancies, and NK cell lymphomas. This review focuses on targeting CD38 in cancer and non-cancerous diseases using antibodies, cell-based therapies and CD38 inhibitors. We also provide a summary of current clinical trials targeting CD38.

One-step extraction and determination of 513 psychoactive substances, drugs, and their metabolites from hair by LC-MS/MS.

The emergence of new psychoactive substances on the market is a significant problem on a global scale. This type of substance in society is associated with many negative consequences, such as traffic accidents, accidents at work, rape, homicide, poisoning, or overdose deaths. The analysis of these substances in biological samples is very important for further legal action and saving lives. Therefore, laboratories face a tremendous challenge in tackling the evolving drug market. The paper describes the optimization of the analytical LC-MS/MS method to identify and determine 513 psychoactive substances in hair samples. A method of chromatographic separation was developed, and the working parameters of the mass spectrometer were selected for each analyte. The method has been validated, and the results are as follows: the limit of quantification of the developed method ranges from 0.025 to 1.25 ng/mg hair. The mean recovery of the tested analytes ranges from 80 to 120%. The achieved coefficient of variation in within-run precision ranged from 1.05 to 19.99%. The results achieved for BIAS are in the range of ± 20%.

Overview of analytical methods for determining novel psychoactive substances, drugs and their metabolites in biological samples.

Recent years have witnessed a growing in interest in psychoactive substances, particularly those available in e-commerce. These have led to an increase in the number of drug-related poisonings, deaths, and road accidents. Illegal drugs are available on an unprecedented scale and cause previously unknown adverse effects, which creates a challenge for analysts to find rapid methods for identifying these substances and taking appropriate action in the shortest possible time. New psychoactive substances (NPSs) can be lethal at very low concentrations, which give particularly serious cause for concern. These drugs are easily accessible and often regarded (or claimed) to be safe, which encourages many people, in particular young people, to try them. The widespread use of these substances is compounded by the awareness that they are difficult to detect with the existing rapid screening tests. Simple, fast, sensitive, and specific methods for determining the largest possible number of black-market psychoactive substances and their metabolites are therefore essential. Such methods will facilitate treatment and increase the effectiveness of measures aiming to reduce drug addiction. The objective of this review article was to critically compare the most commonly used analytical methods for determining NPS and their metabolites in biological material, with special emphasis on the sample preparation process, and to highlight the possibilities offered by the existing analytical methods.

Effect of Processing Treatment and Modified Atmosphere Packing on Carrot's Microbial Community Structure by Illumina MiSeq Sequencing.

The aim of this study was to analyze the microbiome of carrot (Daucus carota subsp. sativus) subjected to minimal pre-treatment (rinsing in organic acid solution) and packaging in a high-oxygen modified atmosphere, and then stored for 17 days under refrigeration conditions (4 °C). The highest levels of bacteria in the carrot microbiome were characterized, at almost 78%, by bacteria belonging to the Enterobacteriaceae and Pseudomonadaceae families. Rinsing in a solution of ascorbic and citric acids resulted in the improvement of microbiological quality in the first day of storage. However, the use of a high-oxygen modified atmosphere extended the shelf life of the minimally processed product. Compared to carrots stored in air, those stored in high oxygen concentration were characterized by a greater ratio of bacteria belonging to the Serratia and Enterobacter genera, and a lower ratio belonging to the Pseudomonas and Pantoea genera. Moreover, the ß-biodiversity analysis confirmed that the oxygen concentration was the main factor influencing the differentiation of the metabiomes of the stored carrots. The bacterial strains isolated from carrots identified by molecular methods were mostly pathogenic or potentially pathogenic microorganisms. Neither the minimal pre-treatment nor packaging in high-oxygen atmosphere was able to eliminate the threat of pathogenic bacteria emerging in the product.

The Influence of Bacteria Causing Subclinical Mastitis on the Structure of the Cow's Milk Microbiome.

Mastitis is the most expensive disease of dairy cattle across the world and is the main reason for the use of antibiotics in animal husbandry. The aim of this study was to analyze the microbiome of raw milk obtained from a semi-subsistence farm located in the Kuyavian-Pomeranian Voivodeship in Poland. Milk from healthy cows and from cows with subclinical mastitis was analyzed. The following pathogenic bacteria were found in milk from individuals with subclinical mastitis: Escherichia coli or Streptococcus agalactiae. The composition of drinking milk was assessed on the basis of 16S rRNA gene sequencing using the Ion Torrent platform. Based on the conducted research, significant changes in the composition of the milk microbiome were found depending on the physiological state of the cows. The microbiome of milk from healthy cows differed significantly from the milk from cows with subclinical mastitis. Two phyla dominated in the milk from healthy cows: Firmicutes and Proteobacteria, in equal amounts. On the contrary, in the milk from cows with diagnosed subclinical mastitis, one of the types dominated: either Firmicutes or Proteobacteria, and was largely predominant. Moreover, the milk microflora from the ill animals were characterized by lower values of the determined biodiversity indicators than the milk from healthy cows. The presence of pathogenic bacteria in the milk resulted in a significant reduction in the share of lactic acid bacteria in the structure of the population of microorganisms, which are of great importance in the production technology of regional products.

The Raw Milk Microbiota from Semi-Subsistence Farms Characteristics by NGS Analysis Method.

The aim of this study was to analyze the microbiome of raw milk obtained from three semi-subsistence farms (A, B, and C) located in the Kuyavian-Pomeranian Voivodeship in Poland. The composition of drinking milk was assessed on the basis of 16S rRNA gene sequencing using the Ion Torrent platform. Based on the conducted research, significant changes in the composition of the milk microbiome were found depending on its place of origin. Bacteria belonging to the Bacillus (17.0%), Corynebacterium (12.0%) and Escherichia-Shigella (11.0%) genera were dominant in the milk collected from farm A. In the case of the milk from farm B, the dominant bacteria belonged to the Acinetobacter genus (21.0%), whereas in the sample from farm C, Escherichia-Shigella (24.8%) and Bacillus (10.3%) dominated the microbiome. An analysis was performed using the PICRUSt tool (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) in order to generate a profile of genes responsible for bacterial metabolism. The conducted analysis confirmed the diversity of the profile of genes responsible for bacterial metabolism in all the tested samples. On the other hand, simultaneous analysis of six KEGG Orthologs (KO), which participated in beta-lactam resistance responsible for antibiotic resistance of bacteria, demonstrated that there is no significant relationship between the predicted occurrence of these orthologs and the place of existence of microorganisms. Therefore, it can be supposed that bacterial resistance to beta-lactam antibiotics occurs regardless of the environmental niche, and that the antibiotic resistance maintained in the population is a factor that shapes the functional structure of the microbial consortia.

Heavy Metals as a Factor Increasing the Functional Genetic Potential of Bacterial Community for Polycyclic Aromatic Hydrocarbon Biodegradation.

The bioremediation of areas contaminated with hydrocarbon compounds and heavy metals is challenging due to the synergistic toxic effects of these contaminants. On the other hand, the phenomenon of the induction of microbial secretion of exopolysaccharides (EPS) under the influence of heavy metals may contribute to affect the interaction between hydrophobic hydrocarbons and microbial cells, thus increasing the bioavailability of hydrophobic organic pollutants. The purpose of this study was to analyze the impact of heavy metals on the changes in the metapopulation structure of an environmental consortium, with particular emphasis on the number of copies of orthologous genes involved in exopolysaccharide synthesis pathways and the biodegradation of hydrocarbons. The results of the experiment confirmed that the presence of heavy metals at concentrations of 50 mg·L-1 and 150 mg·L-1 resulted in a decrease in the metabolic activity of the microbial consortium and its biodiversity. Despite this, an increase in the biological degradation rate of polycyclic aromatic hydrocarbons was noted of 17.9% and 16.9%, respectively. An assessment of the estimated number of genes crucial for EPS synthesis and biodegradation of polycyclic aromatic hydrocarbons confirmed the relationship between the activation of EPS synthesis pathways and polyaromatic hydrocarbon biodegradation pathways. It was established that microorganisms that belong to the Burkholderiales order are characterized by a high representation of the analyzed orthologs and high application potential in areas contaminated with heavy metals and hydrocarbons.

The Toxic Effect of Herbicidal Ionic Liquids on Biogas-Producing Microbial Community.

The aim of the study was to evaluate the effect of herbicidal ionic liquids on the population changes of microorganisms used in a batch anaerobic digester. The influence of the following ionic liquids: benzalkonium (2,4-dichlorophenoxy)acetate (BA)(2,4-D), benzalkonium (4-chloro-2-methylphenoxy)acetate (BA)(MCPA), didecyldimethylammonium (2,4-dichlorophenoxy)acetate (DDA)(2,4-D), didecyldimethylammonium (4-chloro-2-methylphenoxy)acetate (DDA)(MCPA), as well as reference herbicides (4-chloro-2-methylphenoxy)acetic acid (MCPA) and (2,4-dichlorophenoxy)acetic acid (2,4-D) in the form of sodium salts on biogas production efficiency was investigated. The effective concentration (EC50) values were determined for all tested compounds. (MCPA)- was the most toxic, with an EC50 value of 38.6⁻41.2 mg/L. The EC50 for 2,4-D was 55.7⁻59.8 mg/L. The addition of the test substances resulted in changes of the population structure of the microbiota which formed the fermentation pulp. The research was based on 16S rDNA analysis with the use of the Next Generation Sequencing method and the MiSeq platform (Illumina, San Diego, CA, USA). There was a significant decrease in bacteria belonging to Firmicutes and Archaea belonging to Euryarchaeota. A significant decrease of the biodiversity of the methane fermentation microbiota was also established, which was expressed by the decrease of the operational taxonomic units (OTUs) and the value of Shannon's entropy. In order to determine the functional potential of bacterial metapopulations based on the 16SrDNAprofile, the PICRUSt(Phylogenetic Investigation of Communities by Reconstruction of Unobserved States)tool was used, which allowed to determine the gene potency of microorganisms and their ability to biodegrade the herbicides. In the framework of the conducted analysis, no key genes related to the biodegradation of MCPA or 2,4-D were found, and the observed decrease of their content in the supernatant liquid was caused by their sorption on bacterial biomass.

Characterization of specific spoilage organisms (SSOs) in vacuum-packed ham by culture-plating techniques and MiSeq next-generation sequencing technologies.

BACKGROUND: Knowledge regarding microaerophilic and anaerobic specific spoilage organisms (SSOs) is crucial for an appropriate evaluation of vacuum-packed ham. The objective of this study was to characterize the SSO community in vacuum-packed ham by a culture-dependent technique and MiSeq next-generation sequencing (NGS) platform. The relation between changes among the SSO group in the ham and changes in sensory characteristics of the product was also assessed. RESULTS: In the study, conventional microbiological analyses were employed in order to establish the participation of several groups of microorganisms in the deterioration of vacuum-packed ham. The diversity of the SSO group in the product was further assessed with the use of MiSeq NGS technology. The bacteria identified in sliced cooked ham belonged mostly to four phyla, namely Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes. A temperature of 4 °C favoured the development of mesophilic and psychrophilic/psychrotrophic flora, mainly Lactobacillaceae, Enterobacteriaceae and Micrococcaceae families. A high ratio of Brochothrix thermosphacta species and new, cold-tolerant Clostridium spp. was also observed. The growth of these microorganisms facilitated changes in the pH value and organoleptic characteristics of the product. CONCLUSION: This study confirms that the combination of culturing and MiSeq NGS technology improves the microbial evaluation of food. © 2016 Society of Chemical Industry.

Metagenomic Analysis of Soil Bacterial Community and Level of Genes Responsible for Biodegradation of Aromatic Hydrocarbons.

The aim of the studies was to compare the composition of soil bacterial metabiomes originating from urbanized areas and areas con¬taminated with hydrocarbons with those from agricultural soil and forest soil obtained from a protected wild-life park area. It should be noted that hydrocarbons are everywhere therefore bacteria capable of their utilization are present in every soil type. In the hydrocarbon-contaminated soil and in the soil of anthropogenic origin, the bacteria belonging to Gammaproteobacteria were dominant (28.4-36.6%), whereas in the case of agricultural soil and protected wild-life park soil their ratios decreased (22.8-23.0%) and were similar to that of Alphaproteobacteria. No statistically significant changes were observed in terms of the Operational Taxonomic Unit identified in the studies soils, however, based on the determined alpha-diversity it can be established that contaminated soils were characterized by lower biodiversity indices compared to agricultural and forest soils. Furthermore, the dioxygenase level was also evaluated in the studied soils, which are genes encoding crucial enzymes for the decomposition of mono- and polycyclic aromatic hydrocarbons during the biodegradation of diesel oil (PAHRHDαGN, PAHRHDαGP, xylE, Cat 2,3, ndoB). It was concluded that both the population structure of the soil metabiome and the number of genes crucial for biodegradation processes differed significantly between the soils. The level of analysed genes showed a similar trend, as their highest number in relations to genes encoding 16S RNA was determined in urban and hydrocarbon-contaminated soil.

Influence of soil contamination with PAH on microbial community dynamics and expression level of genes responsible for biodegradation of PAH and production of rhamnolipids.

The aim of this study was to evaluate the effect of bioaugmentation and addition of rhamnolipids on the biodegradation of PAHs in artificially contaminated soil, expression of genes crucial for the biodegradation process (PAHRHDαGN, PAHRHDαGP), and the synthesis of rhamnolipids as well as population changes in the soil bacterial metabiome. The positive effect of bioaugmentation and addition of rhamnolipids on the bioremediation of the majority of PAHs was confirmed during the early stages of treatment, especially in case of the most structurally complicated compounds. The results of metagenomic analysis indicated that the initial changes in the soil metabiome caused by bioaugmentation diminished after 3 months and that the community structure in treated soil was similar to control. The survival period of bacteria introduced into the soil via bioaugmentation reached a maximum of 3 months. The increased expression of genes observed after addition of PAH into the soil also returned to the initial conditions after 3 months.

High Voltage Electrochemiluminescence (ECL) as a New Method for Detection of PAH During Screening for PAH-Degrading Microbial Consortia.

The search for new bacterial consortia capable of removing PAH from the environment is associated with the need to employ novel, simple, and economically efficient detection methods. A fluorimetric method (FL) as well as high voltage electrochemiluminescence (ECL) on a modified surface of an aluminum electrode were used in order to determine the changes in the concentrations of PAH in the studied aqueous solutions. The ECL signal (the spectrum and emission intensity for a given wavelength) was determined with the use of an apparatus operating in single photon counting mode. The dependency of ECL and FL intensity on the concentration of naphthalene, phenanthrene, and pyrene was linear in the studied concentration range. The biodegradation kinetics of the particular PAH compounds was determined on the basis of the obtained spectroscopic determinations. It has been established that the half-life of naphthalene, phenanthrene, and pyrene at initial concentrations of 50 mg/l (beyond the solubility limit) reached 41, 75, and 130 h, accordingly. Additionally, the possibility of using ECL for rapid determination of the soluble fraction of PAH directly in the aqueous medium has been confirmed. Metagenomic analysis of the gene encoding 16S rRNA was conducted on the basis of V4 hypervariable region of the 16S rRNA gene and allowed to identify 198 species of bacteria that create the S4consortium. The consortium was dominated by Gammaproteobacteria (78.82 %), Flavobacteria (9.25 %), Betaproteobacteria (7.68 %), Sphingobacteria (3.76 %), Alphaproteobacteria (0.42 %), Clostridia (0.04 %), and Bacilli (0.03 %).