Immunotherapy-induced hepatitis in metastatic colorectal cancer: a systematic review and meta-analysis.

Recent advances in immunotherapy using immune checkpoint inhibitors (ICIs) for various cancers have also highlighted a rise in immune-related adverse events, including hepatitis, potentially leading to the discontinuation of treatment. This study aimed to evaluate the prevalence of hepatitis in metastatic colorectal cancer (mCRC) patients undergoing different ICI therapies. An extensive search of PubMed, PubMed Central, and Google Scholar up to November 2023 identified relevant studies. After excluding non-English articles, case reports, reviews, ongoing trials, and studies combining other therapies, five studies qualified for inclusion. Data extraction and statistical analyses were performed using Excel and Comprehensive Meta-Analysis software, respectively. Results from a subgroup analysis indicated that the incidence of hepatitis was comparable among patients treated with PD-1 monotherapy, PDL-1 monotherapy, and combination PD-1 and CTLA-4 therapy, with rates of 2.6%, 2.2%, and 1.7% for any grade and 2.1%, 2.2%, and 1.7% for grade ≥3 hepatitis, respectively. Naive-treated mCRC patients exhibited higher hepatitis rates than those previously treated (3.2% vs 1.6% and 2.6% vs 1.6% for any grade and grade ≥3, respectively). This study underscores the similar risk of hepatitis across different ICI therapies, with an increased incidence in naive-treated mCRC patients.

Benzalkonium chloride induced acute toxicity and its multifaceted implications on growth, hematological metrics, biochemical profiles, and stress-responsive biomarkers in tilapia (Oreochromis mossambicus).

This study aimed to investigate the toxic effects of benzalkonium chloride (BAC) on Oreochromis mossambicus, a freshwater fish species. Probit analysis was used to determine the lethal concentration (LC50) of BAC for different exposure periods (24, 48, 72, and 96 h). The viability of fish exposed to BAC was assessed using the general threshold survival models (GUTS) and confirmed with relevant datasets to evaluate model accuracy. Experimental groups of fish were exposed to BAC concentrations equivalent to 10% and 20% of the 96-h LC50 for 45 days. The study revealed significant alterations in various parameters during sublethal BAC exposure. These effects included decreased specific growth rate (SGR), red blood cell count (RBC), hemoglobin (Hb) concentration, hematocrit (Ht) value, plasma protein, and albumin levels, as well as acetylcholinesterase (AChE) activities in both gills and liver. Additionally, an increase in gastrosomatic index (GSI), feed conversion ratio (FCR), plasma glucose and creatinine concentrations, alanine aminotransferase (ALT), aspartate aminotransferase (AST) enzymatic activities, catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) levels were observed in the exposed fish's gills and liver. Furthermore, the study found that glutathione S-transferase (GST) and glutathione peroxidase (GPx) levels initially increased and then decreased in both gills and liver after exposure to BAC. Correlation matrix analysis, multivariate multiple regression (MMR), canonical correspondence analysis (CCA), integrated biomarker response (IBR), and biomarker response index (BRI) were utilized to assess the impact of BAC on fish, highlighting significant effects on multiple biomarkers in O. mossambicus following surfactant exposure. Thus, the study provides valuable insights into the toxic effects of BAC on this fish species, emphasizing the importance of monitoring such pollutants in aquatic environments.

Biodegradation of chemical contamination by lactic acid bacteria: A biological tool for food safety.

Chemical pollutants such as mycotoxins and pesticides exert harmful effects on human health such as inflammation, oxidative stress, and cancer. Several strategies were applied for food decontamination, including physicochemical and biological strategies. The present review comprehensively discussed the recent efforts related to the biodegradation of eight food chemical contaminants, including mycotoxins, acrylamide, biogenic amines, N-nitrosamines, polycyclic aromatic hydrocarbons, bisphenol A, pesticides, and heavy metals by lactic acid bacteria (LAB). Biological detoxification by LAB such as Lactobacillus is a promising approach to remove the risks related to the presence of chemical and environmental pollutants in foodstuffs. It is a safe, efficient, environmentally friendly, and low-cost strategy to remove hazardous compounds. LAB can directly decrease these chemical pollutants by degradation or adsorption. Also, it can indirectly reduce the content of these pollutants by reducing their precursors. Hence, LAB can contribute to reducing chemical pollutants in contaminated foods and enhance food safety.

Comparative Analysis of Tentacle Extract and Nematocyst Venom: Toxicity, Mechanism, and Potential Intervention in the Giant Jellyfish Nemopilema nomurai.

The giant jellyfish Nemopilema nomurai sting can cause local and systemic reactions; however, comparative analysis of the tentacle extract (TE) and nematocyst venom extract (NV), and its toxicity, mechanism, and potential intervention are still limited. This study compared venom from TE and NV for their composition, toxicity, and efficacy in vitro and in vivo used RAW264.7 cells and ICR mice. A total of 239 and 225 toxin proteins were identified in TE and NV by proteomics, respectively. Pathological analysis revealed that TE and NV caused heart and liver damage through apoptosis, necrosis, and inflammation, while TE exhibited higher toxicity ex vivo and in vivo. Biochemical markers indicated TE and NV elevated creatine kinase, lactatedehydrogenase, and aspartate aminotransferase, with the TE group showing a more significant increase. Transcriptomics and Western blotting indicated both venoms increased cytokines expression and MAPK signaling pathways. Additionally, 1 mg/kg PACOCF3 (the phospholipase A2 inhibitor) improved survival from 16.7% to 75% in mice. Our results indicate that different extraction methods impact venom activities, tentacle autolysis preserves toxin proteins and their toxicity, and PACOCF3 is a potential antidote, which establishes a good extraction method of jellyfish venom, expands our understanding of jellyfish toxicity, mechanism, and provides a promising intervention.

Multi-omics integration analysis: Tools and applications in environmental toxicology.

Nowadays, traditional single-omics study is not enough to explain the causality between molecular alterations and toxicity endpoints for environmental pollutants. With the development of high-throughput sequencing technology and high-resolution mass spectrometry technology, the integrative analysis of multi-omics has become an efficient strategy to understand holistic biological mechanisms and to uncover the regulation network in specific biological processes. This review summarized sample preparation methods, integration analysis tools and the application of multi-omics integration analyses in environmental toxicology field. Currently, omics methods have been widely applied being as the sensitivity of early biological response, especially for low-dose and long-term exposure to environmental pollutants. Integrative omics can reveal the overall changes of genes, proteins, and/or metabolites in the cells, tissues or organisms, which provide new insights into revealing the overall toxicity effects, screening the toxic targets, and exploring the underlying molecular mechanism of pollutants.

Norfloxacin affects inorganic nitrogen compound transformation in tailwater containing Corbicula fluminea.

The Asian clam, Corbicula fluminea, commonly used in engineered wetlands receiving tailwater, affects nitrogen compound transformation in water. This study investigates how a commonly observed antibiotic in tailwater, norfloxacin, impact nitrogen compound transformation in tailwater containing C. fluminea. The clam was exposed to artificial tailwater with norfloxacin (0, 0.2, 20, and 2000 µg/L) for 15 days. Water properties, C. fluminea ecotoxicity responses, microorganism composition and nitrification- or denitrification-related enzyme activities were measured. Results revealed norfloxacin-induced increases and reductions in tailwater NH4+ and NO2- concentrations, respectively, along with antioxidant system inhibition, organ histopathological damage and disruption of water filtering and digestion system. Microorganism composition, especially biodiversity indices, varied with medium (clam organs and exposure water) and norfloxacin concentrations. Norfloxacin reduced NO2- content by lowering the ratio between microbial nitrifying enzyme (decreased hydroxylamine oxidoreductase and nitrite oxidoreductase activity) and denitrifying enzyme (increased nitrate reductase and nitrite reductase activity) in tailwater. Elevated NH4+ content resulted from upregulated ammonification and inhibited nitrification of microorganisms in tailwater, as well as increased ammonia emission from C. fluminea due to organ damage and metabolic disruption of the digestion system. Overall, this study offers insights into using benthic organisms to treat tailwater with antibiotic residues, especially regarding nitrogen treatment.

Distribution, transformation, and toxic effects of the flame retardant tetrabromobisphenol S and its derivatives in the environment: A review.

As widely used alternative brominated flame retardants, tetrabromobisphenol S (TBBPS) and its derivatives have attracted increasing amounts of attention in the field of environmental science. Previous studies have shown that TBBPS and its derivatives easily accumulate in environmental media and may cause risks to environmental safety and human health. Therefore, to explore the environmental behaviours of TBBPS and its derivatives, in this paper, we summarized relevant research on the distribution of these compounds in water, the atmosphere, soil and food/biota, as well as their transformation mechanisms (biological and nonbiological) and toxic effects. The summary results show that TBBPS and its derivatives have been detected in water, the atmosphere, soil, and food/biota globally, making them a ubiquitous pollutant. These compounds may be subject to adsorption, photolysis or biological degradation after being released into the environment, which in turn increases their ecological risk. TBBPS and its derivatives can cause a series of toxic effects, such as neurotoxicity, hepatotoxicity, cytotoxicity, thyrotoxicity, genotoxicity and phytotoxicity, to cells or living organisms in in vitro and in vivo exposure. Toxicological studies suggest that TBBPS as an alternative to TBBPA is not entirely environmentally friendly. Finally, we propose future directions for research on TBBPS and its derivatives, including the application of new technologies in studies on the migration, transformation, toxicology and human exposure risk assessment of TBBPS and its derivatives in the environment. This review provides useful information for obtaining a better understanding of the behaviour and potential toxic effects of TBBPS and its derivatives in the environment.

First Record of Malformation in Seahorses Attributed to the Oil Spill off the Brazilian Coast in 2019.

In 2019, there was an environmental catastrophe in Brazil, when more than 5000 tons of unknown origin crude oil invaded beaches and mangroves. Two years later, two monitoring areas were selected to study seahorses' offspring: Massangana River estuary (apparently healthy area) and Cocaia Island (affected area). Thirty-six reproductive events of Hippocampus reidi (Syngnathidae) couples from these two areas were monitored to analyze the offspring. At the apparently healthy area, no newborns with malformations were found. However, the offspring from Cocaia Island showed a mean of 19.73% (±5.23) malformations in newborns. It is argued that the toxic/teratogenic effects of polycyclic aromatic hydrocarbons have affected the population in two ways: directly through the induction of mutations in the germ cells of the species and through a drastic reduction of the population (bottleneck effect) whose density observed today recovered through consanguineous couplings, potentiating deleterious genotypes in the offspring. Environ Toxicol Chem 2024;43:1996-2004. © 2024 SETAC.

Current research on ecotoxicity of metal-based nanoparticles: from exposure pathways, ecotoxicological effects to toxicity mechanisms.

Metal-based nanoparticles have garnered significant usage across industries, spanning catalysis, optoelectronics, and drug delivery, owing to their diverse applications. However, their potential ecological toxicity remains a crucial area of research interest. This paper offers a comprehensive review of recent advancements in studying the ecotoxicity of these nanoparticles, encompassing exposure pathways, toxic effects, and toxicity mechanisms. Furthermore, it delves into the challenges and future prospects in this research domain. While some progress has been made in addressing this issue, there is still a need for more comprehensive assessments to fully understand the implications of metal-based nanoparticles on the environment and human well-being.

Research advance of occupational exposure risks and toxic effects of semiconductor nanomaterials.

In recent years, semiconductor nanomaterials, as one of the most promising and applied classes of engineered nanomaterials, have been widely used in industries such as photovoltaics, electronic devices, and biomedicine. However, occupational exposure is unavoidable during the production, use, and disposal stages of products containing these materials, thus posing potential health risks to workers. The intricacies of the work environment present challenges in obtaining comprehensive data on such exposure. Consequently, there remains a significant gap in understanding the exposure risks and toxic effects associated with semiconductor nanomaterials. This paper provides an overview of the current classification and applications of typical semiconductor nanomaterials. It also delves into the existing state of occupational exposure, methodologies for exposure assessment, and prevailing occupational exposure limits. Furthermore, relevant epidemiological studies are examined. Subsequently, the review scrutinizes the toxicity of semiconductor nanomaterials concerning target organ toxicity, toxicity mechanisms, and influencing factors. The aim of this review is to lay the groundwork for enhancing the assessment of occupational exposure to semiconductor nanomaterials, optimizing occupational exposure limits, and promoting environmentally sustainable development practices in this domain.

Cardiovascular toxic effects of nanoparticles and corresponding molecular mechanisms.

Rapid advancements in nanotechnology have been integrated into various disciplines, leading to an increased prevalence of nanoparticle exposure. The widespread utilization of nanomaterials and heightened levels of particulate pollution have prompted government departments to intensify their focus on assessing the safety of nanoparticles (NPs). The cardiovascular system, crucial for maintaining human health, has emerged as vulnerable to damage from nanoparticle exposure. A mounting body of evidence indicates that interactions can occur when NPs come into contact with components of the cardiovascular system, contributing to adverse cardiovascular disease (CVD). However, the underlying molecular mechanisms driving these events remain elusive. This work provides a comprehensive review of recent advance on nanoparticle-induced adverse cardiovascular events and offers insight into the associated molecular mechanisms. Finally, the influencing factors of NPs-induced cardiovascular toxicity are discussed.

An insight on the plausible biological and non-biological detoxification of heavy metals in tannery waste: A comprehensive review.

A key challenge for the tannery industries is the volume of tannery waste water (TWW) generated during the processing of leather, releasing various forms of toxic heavy metals resulting in uncontrolled discharge of tannery waste (TW) into the environment leading to pollution. The pollutants in TW includes heavy metals such as chromium (Cr), cadmium (Cd), lead (Pb) etc, when discharged above the permissible limit causes ill effects on humans. Therefore, several researchers have reported the application of biological and non-biological methods for the removal of pollutants in TW. This review provides insights on the global scenario of tannery industries and the harmful effects of heavy metal generated by tannery industry on micro and macroorganisms of the various ecological niches. It also provides information on the process, advantages and disadvantages of non-biological methods such as electrochemical oxidation, advanced oxidation processes, photon assisted catalytic remediation, adsorption and membrane technology. The various biological methods emphasised includes strategies such as constructed wetland, vermitechnology, phytoremediation, bioaugmentation, quorum sensing and biofilm in the remediation of heavy metals from tannery wastewater (TWW) with special emphasize on chromium.

Tire and road wear particles in the aquatic organisms - A review of source, properties, exposure routes, and biological effects.

With the continuous development of the modern social economy, rubber has been widely used in our daily life. Tire and road wear particles (TRWPs) are generated by friction between tires and the road surface during the processes of driving, acceleration, and braking. TRWPs can be divided into three main components according to their source: tire tread, brake wear, and road wear. Due to urban runoff, TRWPs flow with rainwater into the aquatic environment and influence the surrounding aquatic organisms. As an emerging contaminant, TRWPs with the characteristics of small particles and strong toxicity have been given more attention recently. Here, we summarized the existing knowledge of the physical and chemical properties of TRWPs, the pathways of TRWPs into the water body, and the exposure routes of TRWPs. Furthermore, we introduced the biological effects of TRWPs involved in size, concentration, and shape, as well as key toxic compounds involved in heavy metals, polycyclic aromatic hydrocarbons (PAHs), N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), and benzothiazole on aquatic organisms, and attempted to find the relevant factors influencing the toxic effects of TRWPs. In the context of existing policies that ignore pollution from TRWPs emissions in the aquatic environment, we also proposed measures to mitigate the impact of TRWPs in the future, as well as an outlook for TRWPs research.

Pollution-related changes in nest microbiota: Implications for growth and fledging in three passerine birds.

Non-ferrous smelters emit toxic metals into the environment, posing a threat to wildlife health. Despite the acknowledged role of microbes in host health, the impact of such emissions on host-associated microbiota, especially in wild birds, remains largely unexplored. This study investigates the associations of metal pollution, fitness, and nest microbiota (serving as a proxy for early-life microbial environment) which may influence the nestling health and development. Our study focuses on three passerine birds, the great tit (Parus major), blue tit (Cyanistes caeruleus), and pied flycatcher (Ficedula hypoleuca), within control and metal-polluted sites around a Finnish copper-nickel smelter. The polluted sites had been contaminated with arsenic (As), cadmium (Cd), copper (Cu), nickel (Ni), and zinc (Zn). We performed bacterial 16S rRNA sequencing and metal analyses on 90 nests and monitored nestling body mass, fledging success, and various biotic and abiotic factors. Our findings revealed species-specific responses to metal exposure in terms of both fitness and nest microbiota. P. major and C. caeruleus showed sensitivity to pollution, with decreased nestling growth and fledging in the polluted zone. This was accompanied by a shift in the bacterial community composition, which was characterized by an increase in some pathogenic bacteria (in P. major and C. caeruleus nests) and by a decrease in plant-associated bacteria (within C. caeruleus nests). Conversely, F. hypoleuca and their nest microbiota showed limited responses to pollution, indicating greater tolerance to pollution-induced environmental changes. Although pollution did not correlate with nest alpha diversity or the most abundant bacterial taxa across all species, certain potential pathogens within the nests were enriched in polluted environments and negatively correlated with nestling fitness parameters. Our results suggest that metal pollution may alter the nest bacterial composition in some bird species, either directly or indirectly through environmental changes, promoting pathogenic bacteria and potentially impacting bird survival.

Assessment of toxicity of pyriproxyfen, Bacillus thuringiensis, and malathion and their mixtures used for mosquito control on embryo-larval development and behavior of zebrafish.

Pyriproxyfen (PPF), Bacillus thuringiensis israelensis (BTI), and malathion (MLT) are widely used worldwide to control the population of mosquitos that transmit arboviruses. The current work aimed to evaluate the toxicity of these single pesticides and their binary mixtures of PPF + BTI, PPF + MLT, and MLT + BTI on the embryo-larval stage of zebrafish (Danio rerio) as an animal model. Epiboly, mortality, apical endpoints, affected animals, heart rate, morphometric, thigmotaxis, touch sensitivity, and optomotor response tests were evaluated. PPF and MLT and all mixtures reduced the epiboly percentage. Mortality increased significantly in all exposed groups, except BTI, with MLT being the most toxic. The observed apical endpoints were pericardial and yolk sac edemas, and tail and spine deformation. Exposure to MLT showed a higher percentage of affected animals. A reduction in heart rate was also observed in MLT- and PPF + MLT-exposed groups. The PPF + MLT mixture decreased head measurements. Behavioral alterations were observed, with a decrease in thigmotaxis and touch sensitivity responses in PPF + MLT and MLT + BTI groups. Finally, optomotor responses were affected in all groups. The above data obtained suggest that the MLT + PFF mixture has the greatest toxicity effects. This mixture affected embryo-larval development and behavior and is close to the reality in several cities that use both pesticides for mosquito control rather than single pesticides, leading to a reevaluation of the strategy for mosquito control.

Arsenic inorganic exposure, metabolism, genetic biomarkers and its impact on human health: A mini-review.

Inorganic arsenic species exist in the environment as a result of both natural sources, such as volcanic and geothermal activities, and geological formations, as well as anthropogenic activities, including smelting, exploration of fossil fuels, coal burning, mining, and the use of pesticides. These species deposit in water, rocks, soil, sediments, and the atmosphere. Arsenic-contaminated drinking water is a global public health issue because of its natural prevalence and toxicity. Therefore, chronic exposure to arsenic can have deleterious effect on humans, including cancer and other diseases. This work describes the mechanisms of environmental exposure to arsenic, molecular regulatory factors involved in its metabolism, genetic polymorphisms affecting individual susceptibility and the toxic effects of arsenic on human health (oxidative stress, DNA damage and cancer). We conclude that the role of single nucleotide variants affecting urinary excretion of arsenic metabolites are highly relevant and can be used as biomarkers of the intracellular retention rates of arsenic, showing new avenues of research in this field.

Immunotherapy-induced colitis in metastatic colorectal cancer: a systematic review and meta-analysis.

Colorectal cancer (CRC) presents significant mortality risks, underscoring the urgency of timely diagnosis and intervention. Advanced stages of CRC are managed through chemotherapy, targeted therapy, immunotherapy, radiotherapy, and surgery. Immunotherapy, while effective in bolstering the immune system against cancer cells, often carries toxic side effects, including colitis. This study aimed to evaluate the incidence of colitis in patients with metastatic CRC (mCRC) undergoing various immunotherapy treatments. Through a systematic search of Google Scholar and PubMed databases from inception until November 2023, nine relevant studies were identified. Subgroup analyses revealed a higher incidence of colitis, particularly in patients treated with anti-cytotoxic T-lymphocyte-associated molecule-4 (anti-CTLA-4) and combination therapies compared to monotherapy with programmed cell death receptor-1 (PD-1) or programmed cell death ligand receptor-1 (PDL-1) inhibitors. Notably, naive-treated metastatic CRC patients exhibited elevated colitis incidences compared to those previously treated. In conclusion, anti-CTLA-4 and combination therapies, such as nivolumab plus ipilimumab, were associated with increased colitis occurrences in metastatic CRC patients, highlighting the need for vigilant monitoring and management strategies, especially in immunotherapy-naive individuals.

Prediction of developmental toxic effects of fine particulate matter (PM2.5) water-soluble components via machine learning through observation of PM2.5 from diverse urban areas.

The global health implications of fine particulate matter (PM2.5) underscore the imperative need for research into its toxicity and chemical composition. In this study, zebrafish embryos exposed to the water-soluble components of PM2.5 from two cities (Harbin and Hangzhou) with differences in air quality, underwent microscopic examination to identify primary target organs. The Harbin PM2.5 induced dose-dependent organ malformation in zebrafish, indicating a higher level of toxicity than that of the Hangzhou sample. Harbin PM2.5 led to severe deformities such as pericardial edema and a high mortality rate, while the Hangzhou sample exhibited hepatotoxicity, causing delayed yolk sac absorption. The experimental determination of PM2.5 constituents was followed by the application of four algorithms for predictive toxicological assessment. The random forest algorithm correctly predicted each of the effect classes and showed the best performance, suggesting that zebrafish malformation rates were strongly correlated with water-soluble components of PM2.5. Feature selection identified the water-soluble ions F- and Cl- and metallic elements Al, K, Mn, and Be as potential key components affecting zebrafish development. This study provides new insights into the developmental toxicity of PM2.5 and offers a new approach for predicting and exploring the health effects of PM2.5.

MCF-7 cell - derived exosomes were involved in protecting source cells from the damage caused by tributyltin chloride via transport function.

Tributyltin chloride (TBTC) is a ubiquitous environmental pollutant with various adverse effects on human health. Exosomes are cell - derived signaling and substance transport vesicles. This investigation aimed to explore whether exosomes could impact the toxic effects caused by TBTC via their transport function. Cytotoxicity, DNA and chromosome damage caused by TBTC on MCF-7 cells were analyzed with CCK-8, flow cytometry, comet assay and micronucleus tests, respectively. Exosomal characterization and quantitative analysis were performed with ultracentrifugation, transmission electron microscope (TEM) and bicinchoninic acid (BCA) methods. TBTC content in exosomes was detected with Liquid Chromatography-Mass Spectrometry (LC-MS). The impacts of exosomal secretion on the toxic effects of TBTC were analyzed. Our data indicated that TBTC caused significant cytotoxicity, DNA and chromosome damage effects on MCF-7 cells, and a significantly increased exosomal secretion. Importantly, TBTC could be transported out of MCF-7 cells by exosomes. Further, when exosomal secretion was blocked with GW4869, the toxic effects of TBTC were significantly exacerbated. We concluded that TBTC promoted exosomal secretion, which in turn transported TBTC out of the source cells to alleviate its toxic effects. This investigation provided a novel insight into the role and mechanism of exosomal release under TBTC stress.

Advances of microplastics ingestion on the morphological and behavioral conditions of model zebrafish: A review.

Concerns have been conveyed regarding the availability and hazards of microplastics (MPs) in aquatic biota due to their widespread presence in aquatic habitats. Zebrafish (Danio rerio) are widely used as a model organism to study the adverse impacts of MPs due to their several compelling advantages, such as their small size, ease of breeding, inexpensive maintenance, short life cycle, year-round spawning, high fecundity, fewer legal restrictions, and genetic resemblances to humans. Exposure of organisms to MPs produces physical and chemical toxic effects, including abnormal behavior, oxidative stress, neurotoxicity, genotoxicity, immune toxicity, reproductive imbalance, and histopathological effects. But the severity of the effects is size and concentration-dependent. It has been demonstrated that smaller particles could reach the gut and liver, while larger particles are only confined to the gill, the digestive tract of adult zebrafish. This thorough review encapsulates the current body of literature concerning research on MPs in zebrafish and demonstrates an overview of MPs size and concentration effects on the physiological, morphological, and behavioral characteristics of zebrafish. Finding gaps in the literature paves the way for further investigation.