Characterization of a Chimeric Resilin-Elastin Structural Protein Dedicated to 3D Bioprinting as a Bioink Component.

In this study we propose to use for bioprinting a bioink enriched with a recombinant RE15mR protein with a molecular weight of 26 kDa, containing functional sequences derived from resilin and elastin. The resulting protein also contains RGD sequences in its structure, as well as a metalloproteinase cleavage site, allowing positive interaction with the cells seeded on the construct and remodeling the structure of this protein in situ. The described protein is produced in a prokaryotic expression system using an E. coli bacterial strain and purified by a process using a unique combination of known methods not previously used for recombinant elastin-like proteins. The positive effect of RE15mR on the mechanical, physico-chemical, and biological properties of the print is shown in the attached results. The addition of RE15mR to the bioink resulted in improved mechanical and physicochemical properties and promoted the habitation of the prints by cells of the L-929 line.

Graphene Oxide (GO)-Based Bioink with Enhanced 3D Printability and Mechanical Properties for Tissue Engineering Applications.

Currently, a major challenge in material engineering is to develop a cell-safe biomaterial with significant utility in processing technology such as 3D bioprinting. The main goal of this work was to optimize the composition of a new graphene oxide (GO)-based bioink containing additional extracellular matrix (ECM) with unique properties that may find application in 3D bioprinting of biomimetic scaffolds. The experimental work evaluated functional properties such as viscosity and complex modulus, printability, mechanical strength, elasticity, degradation and absorbability, as well as biological properties such as cytotoxicity and cell response after exposure to a biomaterial. The findings demonstrated that the inclusion of GO had no substantial impact on the rheological properties and printability, but it did enhance the mechanical properties. This enhancement is crucial for the advancement of 3D scaffolds that are resilient to deformation and promote their utilization in tissue engineering investigations. Furthermore, GO-based hydrogels exhibited much greater swelling, absorbability and degradation compared to non-GO-based bioink. Additionally, these biomaterials showed lower cytotoxicity. Due to its properties, it is recommended to use bioink containing GO for bioprinting functional tissue models with the vascular system, e.g., for testing drugs or hard tissue models.

Biodiversity of autotrophic euglenids based on the group specific DNA metabarcoding approach.

This study reports a comprehensive analysis of photoautotrophic euglenids' distribution and biodiversity in 16 small water bodies of various types (including fish ponds, field ponds, rural ponds and park ponds) located in three regions of Poland: Masovia, Masuria and Pomerania during a period of three years. By employing a euglenid specific barcode marker and a curated database of V2 18S rDNA sequences it was possible to identify 97.7 % of euglenid reads at species level. A total of 152 species classified in 13 genera were identified. The number of euglenid species found in one pond varied from 40 to 102. The most common species were Euglena agilis and Euglenaria caudata, found in every analysed waterbody. The highest number of observed species belonged to Trachelomonas and Phacus. Certain species exhibited a tendency to coexist, suggesting the presence of distinct species assemblages. Among them, the most distinctive cluster was associated with water bodies located in the Masuria region, characterized also by the greatest species richness, including many very rare species: Euglenaformis chlorophoenicea, Lepocinclis autumnalis, L. marssonii, Trachelomonas eurystoma, T. manschurica, T. mucosa, T. zuberi, T. zuberi var. nepos.

New Prospects on Neuroimaging in Von Hippel Lindau Disease-A Narrative Review.

(1) Background: Hemangiomas in Von Hippel-Lindau (VHL) syndrome patients are typically benign but pose threats due to their vital locations involving the central nervous system and the retina. An MRI is currently recommended as the gold standard for tumors associated with VHL in the head region. This narrative review aims to comprehensively outline current standards and recent findings related to imaging of retinal and CNS hemangiomas in Von Hippel-Lindau syndrome. (2) Material and Methods: A review in adherence to PRISMA guidelines using the search string "Von Hippel-Lindau hemangioblastoma imaging" was conducted on PUBMED and SCOPUS databases. (3) Results: After reviewing 455 titles and abstracts, 20 publications fulfilling the inclusion criteria were analyzed. The analysis included studies describing MRI, CT, optical coherence tomography, and PET/CT. (4) Conclusion: While MRI remains the gold standard for diagnosing head tumors in Von Hippel-Lindau syndrome, various PET/CT methods show promise as alternative imaging modalities.

Discovery of a new photosynthetic euglenoid in Poland: Euglena mazurica sp. nov. (Euglenales, Euglenaceae).

Herein we describe a new photosynthetic euglenoid species found in Poland - Euglena mazurica. A large population exists in a small, eutrophic body of water located in a pasture near Mikolajki town inside the Masurian Landscape Park (covering a part of the Masurian Lake District in Poland). The unique cell shape (corkscrew-like) discerns it well from other previously described euglenoid species with metabolic cells. The new species possesses two plate-like chloroplasts each with a pyrenoid accompanied by two paramylon caps placed on either side of it (diplopyrenoids). On the phylogenetic tree, the new species is situated within the Euglena clade. Though it is a sister branch to three clades - one representing the similar Euglena agilis, characterized by its fusiform cells and two chloroplasts with diplopyrenoids, the two species are clearly morphologically and molecularly distinct.

Hypogonadism - when does genetic diagnosis help in therapy?

OBJECTIVES: The objective of the study was to describe phenotype-genotype correlation in selected cases with infertility and emphasise the importance of genetic testing as useful tool for proper treatment decision making MATERIAL AND METHODS: Genetic tests were performed in four patients as a part of diagnostic procedure by Sanger sequencing or targeted next generation sequencing (NGS gene panel). RESULTS: We found the genetic causes of hypogonadotropic hypogonadism in 3 males and female with infertility. CONCLUSIONS: Genetic testing is carried out when searching for the genetic causes of clinically identified disorders. Genetic diagnostics may also be extremely helpful in treating hypogonadism but requires the assistance of a clinician endocrinologist or andrologist, as well as a geneticist.

Autoimmune polyendocrine syndromes associated with autoimmune rheumatic diseases.

Autoimmune polyendocrine syndromes (APSs), also called autoimmune polyglandular syndromes, are a group of autoimmune diseases characterized by the co-occurrence of dysfunctions of several (at least two) endocrine glands. They develop under the influence of environmental factors in genetically predisposed people. Autoimmune polyendocrine syndromes may accompany autoimmune rheumatic diseases and worsen their course - APS-2 and APS-3 are the most common. The APS-2 includes the coexistence of, e.g. Hashimoto's disease, celiac disease and rheumatoid arthritis (RA). In APS-3, rheumatic diseases such as RA, systemic lupus erythematosus, and Sjögren's syndrome may coexist with Hashimoto's disease, type 1 diabetes and hypogonadism or other endocrinopathies. Undiagnosed endocrine diseases may be the reason for the intensification of metabolic disorders observed in the course of rheumatic diseases, cause the ineffectiveness of rheumatological treatment and also increase the frequency of bone fractures due to osteoporosis, cardiovascular complications and even miscarriages when coexistent, e.g. Hashimoto's disease with hypothyroiditis, which increases the risk of pregnancy loss. It is important to be able to conduct an extensive interview, paying attention to the symptoms of possible endocrinopathy as well as the features of other autoimmune disorders in the physical examination (e.g. vitiligo or darkening of the skin in Addison's disease). Depending on the history and physical examination, screening for various APSs is advised.

Exome sequencing to explore the possibility of predicting genetic susceptibility to the joint occurrence of polycystic ovary syndrome and Hashimoto's thyroiditis.

A large body of evidence indicates that women with polycystic ovary syndrome (PCOS) have a higher risk of developing Hashimoto's thyroiditis (HT) than healthy individuals. Given the strong genetic impact on both diseases, common predisposing genetic factors are possibly involved but are not fully understood. Here, we performed whole-exome sequencing (WES) for 250 women with sporadic PCOS, HT, combined PCOS and HT (PCOS+HT), and healthy controls to explore the genetic background of the joint occurrence of PCOS and HT. Based on relevant comparative analyses, multivariate logistic regression prediction modeling, and the most informative feature selection using the Monte Carlo feature selection and interdependency discovery algorithm, 77 variants were selected for further validation by TaqMan genotyping in a group of 533 patients. In the allele frequency test, variants in RAB6A, GBP3, and FNDC7 genes were found to significantly (padjusted < 0.05) differentiated the PCOS+HT and PCOS groups, variant in HIF3A differentiated the PCOS+HT and HT groups, whereas variants in CDK20 and CCDC71 differentiated the PCOS+HT and both single disorder groups. TaqMan genotyping data were used to create final prediction models, which differentiated between PCOS+HT and PCOS or HT with a prediction accuracy of AUC = 0.78. Using a 70% cutoff of the prediction score improved the model parameters, increasing the AUC value to 0.87. In summary, we demonstrated the polygenic burden of both PCOS and HT, and many common and intersecting signaling pathways and biological processes whose disorders mutually predispose patients to the development of both diseases.

In silico methods for predicting functional synonymous variants.

Single nucleotide variants (SNVs) contribute to human genomic diversity. Synonymous SNVs are previously considered to be "silent," but mounting evidence has revealed that these variants can cause RNA and protein changes and are implicated in over 85 human diseases and cancers. Recent improvements in computational platforms have led to the development of numerous machine-learning tools, which can be used to advance synonymous SNV research. In this review, we discuss tools that should be used to investigate synonymous variants. We provide supportive examples from seminal studies that demonstrate how these tools have driven new discoveries of functional synonymous SNVs.

Progesterone and Its Metabolites Play a Beneficial Role in Affect Regulation in the Female Brain.

Premenstrual dysphoric disorder is a female affective disorder that is defined by mood symptoms. The condition is linked to unstable progesterone concentrations. Progestin supplementation is given in cases of threatened or recurrent miscarriage and for luteal phase support. Progesterone is essential for implantation, immune tolerance, and modulation of uterine contractility. For a long time, the administration of progestins was associated with an unfavorable impact on mood, leading to negative affect, and, therefore, was contraindicated in existing mood disorders. Establishing the role of the natural progesterone derivative allopregnanolone in advances in the treatment of postpartum depression has shed new light on the general pathophysiology of mood disorders. Allopregnanolone directly interacts with gamma-aminobutyric acid type A (GABA-A) receptors even at nanomolar concentrations and induces significant anti-depressant, anti-stress, sedative, and anxiolytic effects. Postpartum depression is caused by a rapid drop in hormones and can be instantly reversed by the administration of allopregnanolone. Premenstrual dysphoric disorder can also be considered to result from insufficient neuroactive steroid action due to low progesterone derivative concentration, unstable hormone levels, or decreased receptor sensitivity. The decrease in progesterone levels in perimenopause is also associated with affective symptoms and an exacerbation of some psychosomatic syndromes. Bioidentical progesterone supplementation encounters several obstacles, including limited absorption, first-pass effect, and rapid metabolism. Hence, non-bioidentical progestins with better bioavailability were widely applied. The paradoxical, unfavorable effect of progestins on mood can be explained by the fact that progestins suppress ovulation and disturb the endocrine function of the ovary in the luteal phase. Moreover, their distinct chemical structure prevents their metabolism to neuroactive, mood-improving derivatives. A new understanding of progesterone-related mood disorders can translate the study results from case series and observational studies to cohort studies, clinical trials, and novel, effective treatment protocols being developed.

Proposition of a New POLA Index to Assess the Immunomodulatory Properties of the Diet and Its Relationship with the Gut Microbiota, Using the Example of the Incidence of COVID-19 in a Group of People without Comorbidities.

A rise in the incidence of infections with severe acute respiratory syndrome coronavirus 2 has sparked the search for protective strategies against the new pathogen. It is known that individual food components can interact with different immune cells, modulating the immune response of the body. The aim of this study was to develop an index assessing the immunomodulatory potential of diet (POLA index) and to test its utility for the prediction of coronavirus disease 2019 (COVID-19) in a group of healthy young people following a traditional or vegetarian diet. Data on body composition, anthropometric measurements, physical activity, dietary intake, and gut microbiota were obtained from 95 adults (mean age, 34.66 ± 5.76 years). There was a strong correlation between the dietary inflammatory index and the POLA index (r = 0.90; p < 0.0001). Based on Cohen's kappa statistic, there was a good agreement in qualitative interpretation between the two indices (kappa = 0.61; p < 0.0001). People on a diet with beneficial immunomodulatory effects had a lower risk of COVID-19 of approximately 80%, as compared with those on a diet with highly unbeneficial immunomodulatory effects. In daily practice, the POLA index might serve as a useful tool for dietitians to identify individuals whose diet is deficient in ingredients for optimal immune system function and change their dietary behavior to ensure optimal immune function that reduces the risk of infection.

Protocol to identify host-viral protein interactions between coagulation-related proteins and their genetic variants with SARS-CoV-2 proteins.

Here, we describe a bioinformatics pipeline that evaluates the interactions between coagulation-related proteins and genetic variants with SARS-CoV-2 proteins. This pipeline searches for host proteins that may bind to viral protein and identifies and scores the protein genetic variants to predict the disease pathogenesis in specific subpopulations. Additionally, it is able to find structurally similar motifs and identify potential binding sites within the host-viral protein complexes to unveil viral impact on regulated biological processes and/or host-protein impact on viral invasion or reproduction. For complete details on the use and execution of this protocol, please refer to Holcomb et al. (2021).

Laccase immobilization in polyelectrolyte multilayer membranes for 17α-ethynylestradiol removal: Biocatalytic approach for pharmaceuticals degradation.

Enzymatic membrane reactors equipped with multifunctional biocatalytic membranes are promising and sustainable alternatives for removal of micropollutants, including steroid estrogens, under mild conditions. Thus, in this study an effort was made to produce novel multifunctional biocatalytic polyelectrolyte multilayer membranes via polyelectrolyte layer-by-layer assembly with laccase enzyme immobilized between or into polyelectrolyte layers. In this study, multifunctional biocatalytic membranes are considered as systems composed of commercially available filtration membrane modified by polyelectrolytes and immobilized enzymes, which are produced for complex treatment of water pollutants. The multifunctionality of the proposed systems is related to the fact that these membranes are capable of micropollutants removal via simultaneous catalytic conversion, membrane adsorption and membrane rejection making remediation process more complex, however, also more efficient. Briefly, cationic poly-l-lysine and polyethylenimine as well as anionic poly(sodium 4-styrenesulfonate) polyelectrolytes were deposited onto NP010 nanofiltration and UFX5 ultrafiltration membranes to produce systems for removal of 17α-ethynylestradiol. Images from scanning electron microscopy confirm effective enzyme deposition, whereas results of zeta potential measurements indicate introduction of positive charge onto the membranes. Based on preliminary results, four membranes with over 70%, activity retention produced using polyethylenimine in internal and entrapped mode, were selected for degradation tests. Systems based on UFX5 membrane allowed over 60% 17α-ethynylestradiol removal within 100 min, whereas NP010-based systems removed over 75% of estrogen within 150 min. Further, around 80% removal of 17α-ethynylestradiol was possible from the solutions at concentration up to 0.1 mg/L at pH ranging from 4 to 6 and at the pressure up to 3 bar, indicating high activity of the immobilized laccase over wide range of process conditions. Produced systems exhibited also great long-term stability followed by limited enzyme elution from the membrane. Finally, removal of over 70% and 60% of 17α-ethynylestradiol, respectively by NP010 and UFX5 systems after 8 cycles of repeated use indicate high reusability potential of the systems and suggest their practical application in removal of micropollutants, including estrogens.

Synergistic action of laccase treatment and membrane filtration during removal of azo dyes in an enzymatic membrane reactor upgraded with electrospun fibers.

Nowadays, the increasing amounts of dyes present in wastewaters and even water bodies is an emerging global problem. In this work we decided to fabricate new biosystems made of nanofiltration or ultrafiltration membranes combined with laccase entrapped between polystyrene electrospun fibers and apply them for decolorization of aqueous solutions of three azo dyes, C.I. Acid Yellow 23 (AY23), C.I. Direct Blue 71 (DB71) and C.I. Reactive Black 5 (RB5). Besides effective decolorization of the permeate stream, the biosystems also allowed removal of dyes from the retentate stream as a result of enzymatic action. The effect of pH and applied pressure on decolorization efficiencies was investigated, and pH 5 and pressure of 2 bar gave the highest removal efficiencies of 97% for AY23 and 100% for both DB71 and RB5 from permeate solutions while decolorization of retentate for RB5 reached 65% under these conditions. Almost 100% decolorization of all dyes was achieved after three consecutive enzyme membrane cycles. Decolorization was shown to be due to the synergistic action of membrane separation and bioconversion. The biocatalytic action also enabled significant reduction of permeate and retentate toxicity, which is one of the biggest environmental health issues for these types of streams.

Synonymous ADAMTS13 variants impact molecular characteristics and contribute to variability in active protein abundance.

The effects of synonymous single nucleotide variants (sSNVs) are often neglected because they do not alter protein primary structure. Nevertheless, there is growing evidence that synonymous variations may affect messenger RNA (mRNA) expression and protein conformation and activity, which may lead to protein deficiency and disease manifestations. Because there are >21 million possible sSNVs affecting the human genome, it is not feasible to experimentally validate the effect of each sSNV. Here, we report a comprehensive series of in silico analyses assessing sSNV impact on a specific gene. ADAMTS13 was chosen as a model for its large size, many previously reported sSNVs, and associated coagulopathy thrombotic thrombocytopenic purpura. Using various prediction tools of biomolecular characteristics, we evaluated all ADAMTS13 sSNVs registered in the National Center for Biotechnology Information database of single nucleotide polymorphisms, including 357 neutral sSNVs and 19 sSNVs identified in patients with thrombotic thrombocytopenic purpura. We showed that some sSNVs change mRNA-folding energy/stability, disrupt mRNA splicing, disturb microRNA-binding sites, and alter synonymous codon or codon pair usage. Our findings highlight the importance of considering sSNVs when assessing the complex effects of ADAMTS13 alleles, and our approach provides a generalizable framework to characterize sSNV impact in other genes and diseases.

Inhibin-B and FSH Are Good Indicators of Spermatogenesis but Not the Best Indicators of Fertility.

Biochemical markers of spermatogenesis and fertility assessment are important in the practical management of infertile males and the determination of an individual's prognosis. We performed an analysis on 100 males with a male infertility factor. The following study inclusion parameters were analyzed: seminogram, FSH, LH, testosterone, estradiol, prolactin, TSH, and inhibin B concentrations. The patients were subsequently treated by reproductive endocrinologists in accordance with AUA/ASRM and EAU guidelines. The reproductive status was evaluated over a period of 3 years. We found a strong correlation of sperm count with inhibin B (r = 0.74, p < 0.001) and FSH concentration levels (r = −0.46, p < 0.001). Among 95 patients at follow-up, pregnancies occurred for 59 of their partners (48 spontaneous, 5 after IVF−ET, and 6 after IUI). Thirty-six patients remained childless despite the therapy. Sperm count and inhibin B level were the best predictors of natural fertilization (ROC AUC: 0.86 and 0.84; cut-off: 2.7 mln/mL and 45 pg/mL). Although inhibin B and FSH can be used to evaluate spermatogenesis and fertility, the initial sperm concentration appeared to be the best predictor of success. Pregnancy was achieved in a surprisingly large proportion of patients with a very low concentration of inhibin B and a low initial sperm count. It is noteworthy that 81% of the pregnancies were achieved without medically assisted reproduction.

Pharmaceuticals and other contaminants of emerging concern in Admiralty Bay as a result of untreated wastewater discharge: Status and possible environmental consequences.

Considering how the impact of human activity in Antarctica is growing, the aim of this study was to conduct the first assessment of pharmaceuticals and personal care products (PPCPs), other emerging contaminants (ECs), and antibiotic resistance genes present in the western shore of the Admiralty Bay region of King George Island. In total, more than 170 substances were evaluated to assess the potential environmental risks they pose to the study area. The major evaluated source of pollutants in this study is discharged untreated wastewater. The highest PPCP concentrations in wastewater were found for naproxen (2653 ngL-1), diclofenac (747 ngL-1), ketoconazole (760 ngL-1), ibuprofen (477 ngL-1) and acetaminophen (332 ngL-1). Moreover, the concentrations of benzotriazole (6340 ngL-1) and caffeine (3310 ngL-1) were also high. The Risk Quotient values indicate that azole antifungals (ketoconazole), anti-inflammatories (diclofenac, ibuprofen) and stimulants (caffeine) are the main groups responsible for the highest toxic burden. In addition, antibiotic resistance genes integrons (int 1) and sulphonamide resistance genes (sul 1-2) were detected in wastewater and seawater. These results indicate that regular monitoring of PPCPs and other ECs is of great importance in this environment. Additionally, the following mitigation strategies are suggested: (1) to create a centralised record of the medications prescribed and consumed in situ (to improve knowledge of potential contaminants without analysis); (2) to use more environmentally friendly substitutes both for pharmaceuticals and personal care products when possible (limiting consumption at the source); and (3) to apply advanced systems for wastewater treatment before discharge to the recipient (end-of-pipe technologies as a final barrier).

Bioremoval of estrogens by laccase immobilized onto polyacrylonitrile/polyethersulfone material: Effect of inhibitors and mediators, process characterization and catalytic pathways determination.

The presence of micropollutants in water, wastewater and soil are a global problem due to their persistent effect on ecosystems and human health. Although there are many methods of removal of environmental pollutants, they are often ineffective for degradation of pharmaceuticals, including estrogens. In presented work we proposed fabrication of electrospun material from polyacrylonitrile/polyethersulfone (PAN/PES) as a support for laccase immobilization by covalent binding. Oxidoreductase was attached to the electrospun fibers using polydopamine as a linker and produced system was used for degradation of two estrogens: 17ß-estradiol (E2) and 17α-ethynylestradiol (EE2). It was shown that 92% of E2 and 100% of EE2 were degraded after 24 h of the process. Moreover, the effect of surfactants, metal ions and mediators on conversion efficiencies of estrogens was investigated and it was confirmed that immobilized enzyme possessed higher resistance to inhibitory agents as well as thermal and storage stability, compared to its native form. Finally, estrogenic activities of E2 and EE2 solutions decreased around 99% and 87%, respectively, after enzymatic conversion, that corresponds to significant reduction of the total organic carbon and formation of low-toxic final products of estrogens degradation.

Chitosan-based formulation of hemagglutinin antigens for oculo-nasal booster vaccination of chickens against influenza viruses.

Avian influenza viruses (AIVs) and especially highly pathogenic (HP) AIVs of the H5 and H7 subtypes are of both veterinary and public health concern worldwide. In response to the demand for effective vaccines against H5N1 HPAIVs, we produced recombinant protein based on hemagglutinin (HA), a protective viral antigen. A fragment of the HA ectodomain, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. Finally, the protein was formulated in Tris-HCl buffer of pH 8.0 or PBS of pH 7.4 to obtain antigens denoted rH5-1 and rH5-2, respectively. The systemic prime and boost immunizations proved that rH5-1 adsorbed to aluminum hydroxide induces anti-H5 HA neutralizing antibodies and protective immune responses against H5N1 HPAIVs in chickens. The present studies were aimed at stimulating immune responses via the mucosal routes using the systemic prime-mucosal boost strategy. Efficacy trials were performed in commercial layer chickens. For systemic and mucosal immunizations, H5 HA antigens were adjuvanted with aluminum hydroxide and chitosan glutamate, respectively. The first dose of rH5-2 was administered subcutaneously, while its second dose was administered subcutaneously, intraocularly, oculo-nasally, or intranasally. rH5-1 was delivered to the subcutaneously primed chickens by the intranasal route. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition tests. Intraocularly and oculo-nasally delivered rH5-2 mixed with chitosan glutamate was capable of stimulating anti-H5 HA IgY antibody responses in the subcutaneously primed chickens; however, it was ineffective when administered by the intranasal route. Efficient intranasal boosting was achieved using rH5-1. The enhanced production of antigen-specific antibodies was reflected in the development of H5-subtype specific and hemagglutination inhibiting antibodies. Conclusively, the subcutaneous prime and oculo-nasal boost vaccination is proposed as the target strategy for future optimization.

Insights into the microbial community of treated wastewater, its year-round variability and impact on the receiver, using cultivation, microscopy and amplicon-based methods.

Apart from chemical constituents, wastewater treatment plant (WWTP) effluents also release microorganisms that can be important to the receiving water bodies either from a sanitary point of view, or taking to the account the biogeochemical potential of the recipients. However, little is known about the treated wastewater microbial community, its composition, seasonal changes, functions and fate in the waters of the receiver. Thus, this study presents a synergistic approach coupling new and traditional methods: analytical chemistry, classical microbiology (cultivation- and microscopy-based methods), as well as Next Generation Sequencing and a quantitative real-time polymerase chain reaction (qPCR). The results show that in terms of bacterial community composition, treated wastewater differed from the environmental samples, irrespectively if they were related or unrelated to the WWTP effluent discharge. The canonical correspondence analysis (CCA) taking into account chemical parameters and taxonomical biodiversity indirectly confirmed the seasonal deterioration of the treated wastewater quality as a result of temperature-driven change of activated sludge community structure and biomass washout (observed also by DAPI staining). Despite seasonal fluctuations of total suspended solids and inter-related parameters (such as COD, BOD, TN, TP), the treated wastewater quality remained within current discharge limits. It was due to treatment processes intensively adjusted by WWTP operators, particularly those necessary to maintain an appropriate rate of autotrophic processes of nitrification and to support biological phosphorus removal. This can explain the observed microbiome composition similarity among WWTP effluents at high taxonomic levels. Obtained data also suggest that besides wastewater treatment efficiency, WWTP effluents are still sources of both human-related microorganisms as well as bacteria equipped in genes involved in N-cycling. Their potential of participation in nutrients cycling in the receivers is widely unknown and require critical attention and better understanding.