Small-cell lung cancer (SCLC) is a highly aggressive tumor with high proliferation and metastatic potential. Due to its nature, it constantly remains one of the leading causes of cancer-related deaths around the world. Moreover, therapeutic options for SCLC patients are still limited. Even though our knowledge of SCLC has increased in recent years, it remains insufficient to identify reliable biomarkers and precisely guide therapy. High intra- and inter-patient heterogeneity appears to be one of the leading issues. Moreover, SCLC has the ability to reprogram during the course of the disease and under the pressure of treatment. This may result in the development of resistance to initially effective therapy. Although identification of molecular subtypes was a major breakthrough in studies on SCLC, defining the subtype alone seems to be insufficient to personalize patients’ management. Therefore, there is ongoing research to identify additional biomarkers. Copy number alterations (CNAs) are known to affect genes expression in various oncological cohorts, and SCLC is reported to exhibit a high CNA burden. Analysis of numerical alterations may be one of the keys to uncovering its drivers and diversity. In this review, we discuss nomenclature and methodological issues concerning CNAs assessment. Subsequently, we summarize current knowledge regarding the most frequent CNAs in the SCLC cohort, as well as the latest studies that point directions worth further investigation. We put emphasis both on tumor tissue- and liquid biopsy-derived CNAs as each of these approaches seems to be highly valuable for uncovering SCLC heterogeneity.

Ginger (Zingiber officinale), due to its diverse therapeutic properties, continues to attract researchers’ attention. One of these properties is its anticonvulsant activity, which, as our previous studies have shown, may result from the presence of 6-gingerol, a compound that reduced pentylenetetrazole (PTZ)-induced hyperlocomotion in zebrafish larvae. In the present study, we further investigated the anticonvulsant effects of 6-gingerol using additional seizure and epilepsy models in both zebrafish larvae and mice. First, we observed that 6-gingerol reduced the overexpression of the neuronal PAS domain protein (npas4) gene in PTZ-exposed zebrafish larvae, but it did not affect the expression of brain-derived neurotrophic factor (bdnf). The anticonvulsant effect of 6-gingerol was evident in the ethyl 2-ketopent-4-enoate seizure test in zebrafish larvae but was not observed in the pilocarpine model or in scn1lab (Dravet syndrome model) and cacna1aa (absence seizure model) zebrafish morphants. In mice, 6-gingerol penetrated the blood-brain barrier and inhibited psychomotor seizures induced by 6 Hz electrical stimulation at an intensity of 32 mA, but it was ineffective at 44 mA. We did not observe any anticonvulsant effects of 6-gingerol in the maximal electroshock test or timed intravenous PTZ infusion seizure test in mice. In summary, our findings provide further evidence for the anticonvulsant effects of 6-gingerol; however, additional investigations are required to clarify its mechanisms of action.

Background: Currently, increasing attention is being paid to the role of genes other than CFTR and their variants as factors modifying the course of cystic fibrosis (CF). One such gene is SLC26A9, which encodes a protein involved in chloride and bicarbonate transport across the epithelial cell membrane. Variants of SLC26A9, such as c.229G>A (p.Gly77Ser) and c.1885C>T (p.Pro629Ser), have been described in patients with severe and rapidly progressive CF. The aim of this study was to identify SLC26A9 variants in a group of 20 patients with CF. Methods: DNA was isolated from blood samples and collected from all patients. Fragments of exons 3 and 17 of the SLC26A9 gene were amplified by PCR and sequenced using the Sanger method. Results: An SLC26A9 variant was identified in two siblings. These patients were diagnosed with CF in adulthood and presented with moderate pulmonary symptoms without exocrine pancreatic insufficiency. In both siblings carrying the CFTR variants p.Phe508del and c.3140-26A>G, the SLC26A9 variant c.1847C>T (p.Pro616Leu) was detected. This variant has not been widely described in the literature and has not previously been associated with CF. Conclusions: The c.1847C>T (p.Pro616Leu) variant is located near a domain that may affect the transport function of the SLC26A9 protein. However, patients in whom the variant was identified did not present a severe disease phenotype. Further studies on larger patient cohorts are required, and at present this variant should be considered of uncertain significance in CF.

Unfavorable epidemiological forecasts indicating a significant increase in cancer incidence and mortality, as well as limitations of traditional cancer treatment methods, prompt the search for new, more effective therapeutic strategies. In response to the difficulties in treating cancer resulting from the significant heterogeneity of the tumor microenvironment and the presence of hypoxic and necrotic zones, anaerobic bacteria from the Clostridiaceae family, particularly those of the Clostridium genus, are attracting increasing interest. These bacteria can selectively grow in hypoxic areas of tumors while showing no affinity for healthy tissues. An additional advantage of these bacteria is their ability to produce toxins and enzymes that enable the lysis of cancer cells and activate the immune response. One of the most promising and distinctive strains is Clostridium novyi-NT, which lacks virulence factors and, in studies, has been shown to induce a strong cytolytic response. Modern genetic engineering has enabled the modification of Clostridiaceae bacteria to express enzymes that activate prodrugs, thereby significantly reducing systemic toxicity while targeting tumor tissue. Combining Clostridiaceae spore therapy with conventional treatments, such as chemotherapy, radiotherapy, or immunotherapy, significantly enhances their effectiveness, resulting in a highly beneficial synergistic therapeutic effect. The purpose of this review is to discuss the role and potential of Clostridiaceae bacteria in innovative anticancer therapies.

Biologically active peptides can be obtained with various research methods, depending on the starting material, biological activity, and intended use. To use the most efficient method, it is worth combining in silico and in vitro experiments. Among the tools that can support an in silico analysis are databases such as the Antimicrobial Peptide Database (AMPD) or BIOPEP-UWM. The aim of this study was to make an in silico hydrolysis of peptides with anticancer properties selected from the AMP database, using pepsin, trypsin, and chymotrypsin. Most peptides obtained had properties inhibiting ACE and dipeptidyl peptidase IV activity. Among the resulting peptides, those with the sequence AR, CF, ER, TF, IY, ER, AW, GF, TW, SK and IM are potentially resistant to peptidase from microbial action. An analysis of the peptides’ characteristics showed that peptides with the sequence AR, EK, ER and SK are well-soluble in water and have high affinity for protein and ligand binding. Peptides with the sequence TF, IL and PF are unstable. Thermostable peptides are PGL, IL, GL, IY, VF, PL, IM and QL. The results of the study may be used to design in vitro experiments.

The possibilities of small-cell lung cancer (SCLC) therapy were strictly limited for years, leading to high patient mortality rates. New approaches to SCLC treatment are being proposed, including chemoimmunotherapy. However, biomarkers enabling appropriate personalization of therapy in SCLC patients have not been identified yet. Even though molecular subtyping (ASCL1, NEUROD1, POU2F3, and YAP1) seems pivotal in the management of SCLC, expression of other genes might be potentially valuable during patients’ stratification. Due to their crucial role in tumorigenesis and SCLC invasiveness, benefits arising from MET and SLFN11 gene evaluation are suggested. Our study was designed to evaluate the relationship between the mRNA expression of these genes and chemoimmunotherapy efficacy in SCLC patients. A total of 35 patients with extensive-stage SCLC (ES-SCLC) treated with first-line chemoimmunotherapy were involved in the study. mRNA expression of MET and SLFN11 genes was evaluated using the RT-qPCR technique in FFPE tissue collected from all patients. Molecular results were correlated with clinicopathological features and outcome of disease (OS, PFS). We detected SLFN11 expression in 60% (21 of 35) of the samples. SLFN11 expression was higher in patients with longer PFS (p = 0.05) and with the T4 feature in the TNM scale (p = 0.08). MET mRNA was expressed in all FFPE tissues. We observed that risk of progression and death was higher in patients with higher expression of MET mRNA (p = 0.06 and p = 0.04, respectively). Our study showed that MET and SLFN11 expression might serve as additional biomarkers for prediction of chemoimmunotherapy efficacy in ES-SCLC patients.

The composition of the gut microbiome of patients with advanced non-small cell lung cancer is currently considered a factor influencing the effectiveness of treatment with immune checkpoint inhibitors. We aimed to evaluate the baseline gut microbiome composition in patients before receiving first-line immunotherapy alone or combined with chemoimmunotherapy. We performed 16S rRNA sequencing based on hypervariable regions. Stool samples were collected from 52 patients with advanced NSCLC treated with immunotherapy or chemoimmunotherapy before treatment. We found that the Ruminococcaceae family, species Alistipes sp. genus Eubacterium ventriosum group and genus Marvinbryantia may be intestinal, microbiological predictors of response to treatment. Genus Akkermansia and species from the [Clostridum] leptum group predicted the length of PFS (progression-free survival). Longer OS (overall survival) is associated with bacteria from the Ruminococcaceae family genera [Eubacterium] ventriosum group, Marvinbryantia, Colidextribacter and species [Clostridum] leptum. Bacteria that have an adverse effect (shortening of PFS or OS) on the response to treatment using immune checkpoint inhibitors are Rothia genus, Streptococus salivarius, Streptococus, Family XIII AD3011 group and Family XIII AD3011 group, s. uncultured bacterium. The composition of intestinal flora can be a predictive factor for immunotherapy in NSCLC patients. Specific bacteria can be positively or negatively associated with response to treatment, progression-free survival, and overall survival. They can be potentially used as predictive markers in NSCLC patients treated with immunotherapy.

Small-cell lung cancer (SCLC) remains one of the deadliest cancers worldwide. Patients' survival remains poor due to its rapid growth, high metastatic rate and limited possibilities of treatment. For many years, SCLC management has been based mostly on chemo and radiotherapy. However, new therapeutic approaches have been proposed in the past few years, including immunotherapy, which is currently implemented in clinical practice. Unfortunately, in many cases, response to therapy, especially chemotherapy, remains poor, or the patient becomes resistant to initially effective treatment. One of the crucial problems during SCLC patient care is a lack of appropriate predictive biomarkers for various therapeutic approaches. Another critical issue is the scarcity of collected tissue during biopsy, which may be insufficient or of too poor quality for analysis. A liquid biopsy might be the key to solving both of those problems as it is collected in a non-invasive way and enables the measurement of various biomarkers, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs). In this review, we discuss various approaches to potentially incorporating liquid biopsy into clinical application - as a companion to imaging during SCLC diagnostics, a new approach to molecular subtyping, and a material enabling predictive or prognostic biomarkers assessment. We also summarize ongoing clinical trials encompassing SCLC patients in which liquid biopsy is collected and examined.

The use of botulinum toxins in various oncological conditions brings long-term results in the treatment of chronic pain without burdening internal organs, as is the case with the use of opioids. Numerous reports indicate a possible apoptotic effect achieved in vitro and in vivo. Most literature data suggest that using botulinum toxins to support oncological treatment gives positive results, and in most cases, it is free of side effects. The article reviews the literature on the use of botulinum toxins in experiments carried out on cancer cell lines and their impact on cancer at the level of reactions occurring at the cellular level. This literature review presents a collection of the most important reports on using various types of botulinum toxins in supporting oncological therapies, considering their analgesic and apoptotic effects. The work presents comprehensive research on large groups of patients, the most important case reports, and clinical trials. The collected information seems to confirm that using botulinum toxins creates an opportunity to support oncological therapies with comprehensive benefits for the patient, improving the quality of life and anticancer effect.

Introduction. Immunotherapy can be effective in some patients with small-cell lung cancer (SCLC) with good performance status. However, the factors contributing to sustained treatment responses remain unclear. The intestinal microbiome profile has emerged as a potential biomarker for the effectiveness of chemoimmunotherapy in SCLC. Evidence suggests that microbiome diversity, both in richness and abundance, may influence immunotherapy outcomes. The presence or absence of specific bacterial populations may also be linked to treatment success or failure. This pilot study compared the gut microbiome profile in two SCLC patients with partial responses to chemoimmunotherapy but had distinctly different outcomes. Material and methods. Metagenomic analysis of the gut microbiome was performed using stool samples from two patients collected prior to treatment initiation. Gut microbiome composition was determined based on next-generation sequencing of the hypervariable regions of the 16S rRNA gene. Both patients received a treatment regimen consisting of atezolizumab, carboplatin and etoposide. Results. The progression-free survival (PFS) and overall survival (OS) were 5.7 and 10.2 months for the first patient and 19.9 and 34.9 months for the second patient, respectively. The patient with early progression exhibited reduced species-level diversity compared to the long-term responder. Additionally, bacteria from the families Lachnospiraceae, Akkermansiaceae were found to be more prevalent in the patient with greater and Desulfovibrionaceae were more abundant in the patient with short survival than in patients with prolonged response. Conclusions. Bacteria residing in the gut may affect the efficacy of chemoimmunotherapy in SCLC patients and represents a promising candidate for predictive biomarkers of treatment response and efficacy.

IntroductionExpression of PD-L1 on cancer cells is the only validated predictive factor for immunotherapy in NSCLC (Non-Small Cell Lung Cancer) patients. However, on this basis, it is difficult to predict the occurrence of resistance to immune checkpoint inhibitors (ICIs). MicroRNAs are widely studied as biomarkers of cancers. Our study was designed to determine whether microRNAs can be sensitive predictive factors in the qualification of NSCLC patients to first-line immunotherapy or chemoimmunotherapy.Material and methodsThe two-stage research on validation group (n=20) and study group (n=35) of patients with advanced NSCLC was conducted. Analysis of microRNAs expression by qPCR in plasma collected prior to the start of immunotherapy (pembrolizumab) or chemoimmunotherapy (combination of pembrolizumab with chemotherapy) was made. Broad-spectrum analysis of microRNAs expression was used in the studied group. Three microRNAs selected in that group as important for the effectiveness of ICIs were then examined in the validation group.ResultsIn the studied group, significantly higher expression of miRNA-126-3p, miR-144-3p and miR-146-5p was observed in patients with long PFS compared to those with short PFS. In the validation group, low miRNA-126 expression indicated lower median progression-free survival and overall survival (2.3 vs. 5.0 months and 5.2 vs 11.2, respectively). These patients had a significantly higher risk of progression (HR= 2.92, 95% CI: 1.01 to 8.40, p=0.04) and death (HR=3.64, 95% CI: 1.22 to 10.84, p=0.02).ConclusionOur study showed that the expression of miR-126 in blood plasma may be a predictive factor for the effectiveness of first-line immunotherapy or chemoimmunotherapy in advanced NSCLC patients.

Neuropeptide Y is a neurotransmitter in the nervous system and belongs to the orexigenic system that increases appetite. Its excessive secretion leads to obesity. Leptin is a pro-inflammatory adipokine (produced in adipose tissue) induced in obesity and may mediate increased antitumor immunity in obesity (including the promotion of M1 macrophages). Leptin and neuropeptide Y gene polymorphisms, causing increased leptin levels and the occurrence of obesity, and lipid profile disorders, may increase the effectiveness of immunotherapy. In 121 patients with advanced NSCLC without mutations in the EGFR gene and rearrangements of the ALK and ROS1 genes, undergoing immunotherapy (1st and 2nd line of treatment) or chemoimmunotherapy (1st line of treatment), we assessed BMI, lipid profile, PD-L1 expression on cancer cells using the immunohistochemical method (clone SP263 antibody), leptin concentration in blood serum by ELISA, polymorphisms in the promoter region of the genes for leptin (LEP) and neuropeptide Y (NPY) by real-time PCR. Leptin concentration was significantly higher in obese patients than in patients with normal or low weight (p = 0.00003) and in patients with disease stabilization compared to patients with progression observed during immunotherapy (p = 0.012). Disease control occurred significantly more often in patients with the GA or AA genotype than patients with the GG genotype in the rs779039 polymorphism of the LEP gene. The median PFS in the entire study group was five months (95

ObjectivePreclinical models of seizures and epilepsy in rodents contributed substantially to the discovery of currently available antiseizure medications. These were also broadly used for investigation of processes of epileptogenesis. Nevertheless, rodent models pose some limitations, thus, new models using alternative species are in high demand. The aim of this study was to describe a new model of seizures/epilepsy induced by the cholinomimetic agent, pilocarpine (PILO), in larval zebrafish.MethodsLocal field potential (LFP) recordings were conducted to analyze electroencephalographic discharges and correlate it with larval behavior. Hematoxylin and eosin (H&E) staining, as well as TUNEL staining were performed to analyze morphology and apoptosis, respectively. Real-time quantitative polymerase chain reaction (qRT-PCR) was undertaken for gene expression analysis.ResultsAcute exposure to PILO, in a concentration-dependent manner, induces electroencephalographic discharges in larval zebrafish, which behaviorally manifest as decreased locomotion and moving time, but enhanced movement velocity. The PILO-induced seizure-like activity is behaviorally distinct from this induced by the application of chemoconvulsant pentylenetetrazole (PTZ). Zebrafish larvae previously exposed to PILO (2 h), after a washing out period, exhibit spontaneous, unprovoked discharges and apoptotic changes in their brains.SignificanceHere, we comprehensively investigated a new model of PILO-induced seizures/epilepsy in larval zebrafish. We propose that this model may be used to study epileptogenesis and for antiseizure drug screening purposes.

BackgroundThe newest method of treatment for patients with NSCLC (non-small cell lung cancer) is immunotherapy directed at the immune checkpoints PD-1 (Programmed Cell Death 1) and PD-L1 (Programmed Cell Death Ligand 1). PD-L1 is the only validated predictor factor for immunotherapy efficacy, but it is imperfect. Some patients do not benefit from immunotherapy and may develop primary or secondary resistance. This study aimed to assess the intestinal resistome composition of non-small cell lung cancer (NSCLC) patients treated with immune checkpoint inhibitors in the context of clinical features and potentially new prediction factors for assessing immunotherapy efficacy.MethodsThe study included 30 advanced NSCLC patients, 19 (57%) men and 11 (33%) women treated with first- or second-line immunotherapy (nivolumab, pembrolizumab or atezolizumab). We evaluated the patient’s gut resistome composition using the high sensitivity of targeted metagenomics.ResultsStudies have shown that resistome richness is associated with clinical and demographic factors of NSCLC patients treated with immunotherapy. Smoking seems to be associated with an increased abundance of macrolides, lincosamides, streptogramins and vancomycin core resistome. The resistome of patients with progression disease appears to be more abundant and diverse, with significantly higher levels of genomic markers of resistance to lincosamides (lnuC). The resistance genes lnuC, msrD, ermG, aph(6), fosA were correlated with progression-free survival or/and overall survival, thus may be considered as factors potentially impacting the disease.ConclusionThe results indicate that the intestinal resistome of NSCLC patients with immune checkpoint inhibitors treatment differs depending on the response to immunotherapy, with several distinguished markers. Since it might impact treatment efficacy, it must be examined more deeply.

Background:The use of immunotherapy in treatment of non-small cell lung cancer (NSCLC) patients with the BRAF gene mutations is an area of active research and is an item of clinical trials. While BRAF mutations are relatively infrequent in NSCLC patients, comprising approximately 1-3% of cases, the V600E substitution stands out as the most prevalent subtype of BRAF mutations. The presence of this mutation in cancer cells qualifies the patients for first-line therapy with BRAF and MEK inhibitors. This study aims to evaluate the efficacy of immunotherapy in NSCLC patients with BRAF mutations. We presented a series of seven NSCLC cases with BRAF mutations, four of whom received immunotherapy or chemoimmunotherapy. Methods:We observed benefit from immunotherapy in all patients, but its duration depended on comorbidities and the presence of brain metastases. Utilization of the next generation sequencing (NGS) technique causes high detection frequency of BRAF mutations (4.7% of patients), although mutations other than V600E may predominate (4 out of 7 patients). Results:In patients receiving immune checkpoint inhibitors (ICIs)-based therapy, the median progression-free survival (PFS) was 17 months from the start of immunotherapy, the overall objective response rate (ORR) was 50%, and disease control was achieved in all patients. Conclusions:Immunotherapy can benefit NSCLC patients with BRAF mutations, though its efficacy is affected by comorbidities and brain metastases. The use of NGS enhances mutation detection, highlighting the need for personalized treatment approaches in NSCLC management. The varying responses to treatments among the patients emphasize the complexity of NSCLC management and the necessity for a personalized approach.

Mutations in the Kirsten rat sarcoma virus (KRAS) gene are the most common mutations in NSCLC, and they occur in 25-40% of patients with lung adenocarcinoma. Sotorasib, a selective KRAS inhibitor, is an anticancer drug used in NSCLC patients with a G12C mutation in the KRAS gene. In previously treated patients, this therapy was safer and more effective than docetaxel chemotherapy. Heterogeneity refers to differences between tumor cells within a single tumor as well as in primary and metastatic lesions. It may influence the response to targeted therapies and the development of acquired resistance to these therapies. It is assumed that sotorasib efficacy is lower in patients with known tumor molecular heterogeneity, which may be common in patients exposed to tobacco smoke. This case report presents a 63-year-old woman with advanced NSCLC and a confirmed G12C mutation in the KRAS gene detected with the real-time PCR technique. A later next-generation sequencing (NGS) examination did not show the presence of this mutation. However, the NGS study was performed on material from a different metastatic lesion. The negative NGS result from this material was confirmed by the real-time PCR technique. The patient had a short-term benefit from first-line chemotherapy and second-line nivolumab immunotherapy (disease stabilization). Due to progression (progression of measurable lesions and new metastases to the CNS), the patient received brain radiotherapy and then sotorasib in the third line of treatment. However, the effectiveness of KRAS inhibition was limited. Regression of the lesion with a detected mutation in the KRAS gene and progression of lesions without this mutation were observed. Sotorasib therapy was terminated. The woman died two years after diagnosis, not benefiting from subsequent lines of therapy. NSCLC heterogeneity (presence of mutations in only some clones of cancer cells) may be responsible for primary and acquired resistance to molecularly targeted therapies, including KRAS inhibitors.

Endometrial cancer (EC) accounts for 90% of uterine cancer cases. It is considered not only one of the most common gynecological malignancies but also one of the most frequent cancers among women overall. Nowadays, the differentiation of EC subtypes is based on immunohistochemistry and molecular techniques. It is considered that patients’ prognosis and the implementation of the appropriate treatment depend on the cancer subtype. Patients with pathogenic variants in POLE have the most favorable outcome, while those with abnormal p53 protein have the poorest. Therefore, in patients with POLE mutation, the de-escalation of postoperative treatment may be considered, and patients with abnormal p53 protein should be subjected to intensive adjuvant therapy. Patients with a DNA mismatch repair (dMMR) deficiency are classified in the intermediate prognosis group as EC patients without a specific molecular profile. Immunotherapy has been recognized as an effective treatment method in patients with advanced or recurrent EC with a mismatch deficiency. Thus, different adjuvant therapy approaches, including targeted therapy and immunotherapy, are being proposed depending on the EC subtype, and international guidelines, such as those published by ESMO and ESGO/ESTRO/ESP, include recommendations for performing the molecular classification of all EC cases. The decision about adjuvant therapy selection has to be based not only on clinical data and histological type and stage of cancer, but, following international recommendations, has to include EC molecular subtyping. This review describes how molecular classification could support more optimal therapeutic management in endometrial cancer patients.

The cluster of genes determining the production of botulinum toxins is an attribute of not only the Clostridium botulinum species. This cluster is also found in other members of the Clostridium genus, such as C. baratii, C. butyricum, and C. sporogenes. The occurrence of a botulinum-like cluster has also been recorded in strains of other genera, i.e., Enterococcus faecium, as well as in a Gram-negative species isolated from freshwater sediments; however, the biological activity of bont-related genes has not been noted. It can be said that the mentioned species have a dual nature. Another species with a dual nature is C. butyricum. This bacterium is a common human and animal gut commensal bacterium and is also frequently found in the environment. Although non-toxigenic strains are currently used as probiotics in Asia, other strains have been implicated in pathological conditions, such as botulism in infants or necrotizing enterocolitis in preterm neonates. Additionally, C. baratii strains are rare opportunistic pathogens associated with botulism intoxication. They have been isolated from food and soil and can be carried asymptomatically or cause botulism outbreaks in animals and humans. In addition to the mentioned clostridia, the other microorganisms considered as non-toxigenic have also been suspected of carrying botulinum cluster Gram-negative bacteria, such as Chryseobacterium piperi isolated from freshwater sediments; however, the biological activity of bont-related genes has not been noted. Additionally, Enterococcus faecium strains have been discovered carrying BoNT-related clusters (BoNT/En). Literature data regarding the heterogeneity of BoNT-producing strains indicate the requirement to reclassify C. botulinum species and other microorganisms able to produce BoNTs or possess botulinum-like gene clusters. This article aims to show the dual nature of Clostridium strains not belonging to the C. botulinum species that are sporadically able to carry bont clusters, which are usually considered saprophytic and even probiotic, and bont-like clusters in microorganisms from other genera. The aim was also to consider the genetic mechanisms of botulinum cluster expression in strains that are considered opportunistic and the microbiological safety aspects associated with their occurrence in the environment.