A comprehensive transcriptomic analysis of the bisphenol A affected kidney in mice.

Introduction: Bisphenol A (BPA) is a substance belonging to the endocrine-disrupting chemicals, globally used in the production of polycarbonate plastics. It has been found that BPA enhances carcinogenesis, triggers obesity and exerts a pathogenic effect in several disorders, such as type 2 diabetes, asthma, or increased blood pressure. Recent studies have revealed, that BPA has a harmful impact on the kidneys function, therefore, the current research aimed to explore the specific molecular changes triggered in these organs after oral BPA exposure in mice. Materials and Methods: The experiment was carried out on 12 (3-month-old) female mice. Six mice served as controls. The other 6 mice were treated with BPA in the drinking water at a dose of 50 mg/kg b. w. for 3 months. Then animals were euthanized, the kidneys were collected, and extracted RNA was used to perform RNA-seq. Results: Applied multistep bioinformatics revealed 433 differentially expressed genes (DEGs) in the BPA-treated kidneys (232 upregulated and 201 downregulated). Additionally, 95 differentially expressed long-noncoding RNAs (DELs) were revealed in BPA samples. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations indicated that BPA exposure resulted in profound changes in several essential processes, such as oxidative phosphorylation, mitochondrial and ribosome function, or chemical carcinogenesis. Conclusion: The obtained novel results suggest that BPA has a harmful impact on the fundamental processes of the kidney and significantly impairs its function by inducing mitochondrial dysfunction leading to oxidative stress and reactive oxygen species production.

The Carcinogenic Potential of Bisphenol A in the Liver Based on Transcriptomic Studies.

Bisphenol A (BPA) is an environmental toxin widely used in the production of polycarbonate plastics. A correlation exists between BPA tissue contamination and the occurrence of pathological conditions, including cancer. First-passage detoxification of high BPA amounts in the liver promotes hepatotoxicity and morphological alterations of this organ, but there is a lack of knowledge about the molecular mechanisms underlying these phenomena. This prompted us to investigate changes in the liver transcriptomics of 3-month-old female mice exposed to BPA (50 mg/kg) in drinking water for 3 months. Five female mice served as controls. The animals were euthanized, the livers were collected, and RNA was extracted to perform RNA-seq analysis. The multistep transcriptomic bioinformatics revealed 120 differentially expressed genes (DEGs) in the BPA-exposed samples. Gene Ontology (GO) annotations indicated that DEGs have been assigned to many biological processes, including "macromolecule modification" and "protein metabolic process". Several of the revealed DEGs have been linked to the pathogenesis of severe metabolic liver disorders and malignant tumors, in particular hepatocellular carcinoma. Data from this study suggest that BPA has a significant impact on gene expression in the liver, which is predictive of the carcinogenic potential of this compound in this organ.

Changes Caused by Bisphenols in the Chemical Coding of Neurons of the Enteric Nervous System of Mouse Stomach.

Bisphenol A (BPA), an organic chemical compound which is widely used in the production of plastics, can severely damage live organisms. Due to these findings, the plastic industry has started to replace it with other substances, most often with bisphenol S (BPS). Therefore, during the present investigation, with the use of double immunofluorescence labeling, we compared the effect of BPA and BPS on the enteric nervous system (ENS) in the mouse corpus of the stomach. The obtained results show that both studied toxins impact the amount of nerve cells immunoreactive to substance P (SP), galanin (GAL), vesicular acetylcholine transporter (VAChT is used here as a marker of cholinergic neurons) and vasoactive intestinal polypeptide (VIP). Changes observed under the impact of both bisphenols depended on the neuronal factor, the type of the enteric ganglion and the doses of bisphenols studied. Generally, the increase in the percentage of neurons immunoreactive to SP, GAL and/or VIP, and the decrease in the percentage of VAChT-positive neurons, was noted. Severity of changes was more visible after BPA administration. However, the study has shown that long time exposure to BPS also significantly affects the ENS.

Changes in the Enteric Neurons Containing Selected Active Substances in the Porcine Descending Colon after the Administration of Bisphenol A (BPA).

Bisphenol A (BPA) is an endocrine disruptor widely distributed in the environment due to its common use in the plastics industry. It is known that it has a strong negative effect on human and animal organisms, but a lot of aspects of this impact are still unexplored. This includes the impact of BPA on the enteric nervous system (ENS) in the large intestine. Therefore, the aim of the study was to investigate the influence of various doses of BPA on the neurons located in the descending colon of the domestic pig, which due to similarities in the organization of intestinal innervation to the human gastrointestinal tract is a good animal model to study processes occurring in human ENS. During this study, the double immunofluorescence technique was used. The obtained results have shown that BPA clearly affects the neurochemical characterization of the enteric neurons located in the descending colon. The administration of BPA caused an increase in the number of enteric neurons containing substance P (SP) and vasoactive intestinal polypeptide (VIP) with a simultaneously decrease in the number of neurons positive for galanin (GAL) and vesicular acetylcholine transporter (VAChT used as a marker of cholinergic neurons). Changes were noted in all types of the enteric plexuses, i.e., the myenteric plexus, outer submucous plexus and inner submucous plexus. The intensity of changes depended on the dose of BPA and the type of enteric plexus studied. The results have shown that BPA may affect the descending colon through the changes in neurochemical characterization of the enteric neurons located in this segment of the gastrointestinal tract.

Endocrine Disruptor Bisphenol a Affects the Neurochemical Profile of Nerve Fibers in the Aortic Arch Wall in the Domestic Pig.

Bisphenol A (BPA) is a synthetic compound utilized in industry for the production of various plastics. BPA penetrates into the environment and adversely affects living organisms. Therefore, the influence of various BPA dosages on the neurochemical characteristics of nerve fibers located in the aortic branch wall was investigated in this study utilizing a double immunofluorescence method. It was found that BPA in concentration of 0.5 mg/kg body weight/day causes a clear increase in the density of nerves within aortic branch walls immunoreactive to cocaine- and amphetamine-regulated transcript (CART), calcitonin gene-related peptide (CGRP), neuronal isoform of nitric oxide synthase (nNOS), pituitary adenylate cyclase-activating peptide (PACAP), and vasoactive intestinal polypeptide (VIP). Nerves containing galanin (GAL) and/or somatostatin (SOM) did not change when BPA was introduced into the system. Changes noted after administration of BPA at a dose of 0.05 mg/kg body weight/day were less visible and concerned fibers immunoreactive to CART, CGRP, and/or PACAP. The obtained results show that BPA affects the neurochemical coding of nerves in the aortic branch wall. These fluctuations may be the first signs of the influence of this substance on blood vessels and may also be at the root of the disturbances in the cardiovascular system.

The Comparison of the Influence of Bisphenol A (BPA) and Its Analogue Bisphenol S (BPS) on the Enteric Nervous System of the Distal Colon in Mice.

Bisphenol A (BPA), commonly used as a plasticizer in various branches of industry has a strong negative effect on living organisms. Therefore, more and more often it is replaced in production of plastics by other substances. One of them is bisphenol S (BPS). This study for the first time compares the impact of BPA and BPS on the enteric neurons using double immunofluorescence technique. It has been shown that both BPA and BPS affect the number of enteric neurons containing substance P (SP), galanin (GAL), vasoactive intestinal polypeptide (VIP), neuronal isoform of nitric oxide synthase (nNOS-a marker of nitrergic neurons) and/or vesicular acetylcholine transporter (VAChT- a marker of cholinergic neurons). The changes noted under the impact of both bisphenols are similar and consisted of an increase in the number of enteric neurons immunoreactive to all neuronal factors studied. The impact of BPS on some populations of neurons was stronger than that noted under the influence of BPA. The obtained results clearly show that BPS (similarly to BPA) administered for long time is not neutral for the enteric neurons even in relatively low doses and may be more potent than BPA for certain neuronal populations.

The Influence of Bisphenol A (BPA) on the Occurrence of Selected Active Substances in Neuregulin 1 (NRG1)-Positive Enteric Neurons in the Porcine Large Intestine.

Bisphenol A (BPA) is a substance used in the manufacture of plastics which shows multidirectional adverse effects on living organisms. Since the main path of intoxication with BPA is via the gastrointestinal (GI) tract, the stomach and intestine are especially vulnerable to the impact of this substance. One of the main factors participating in the regulation of intestinal functions is the enteric nervous system (ENS), which is characterized by high neurochemical diversity. Neuregulin 1 (NRG1) is one of the lesser-known active substances in the ENS. During the present study (performed using the double immunofluorescence method), the co-localization of NRG1 with other neuronal substances in the ENS of the caecum and the ascending and descending colon has been investigated under physiological conditions and after the administration of BPA. The obtained results indicate that NRG1-positive neurons also contain substance P, vasoactive intestinal polypeptide, a neuronal isoform of nitric oxide synthase and galanin and the degree of each co-localization depend on the type of enteric plexus and the particular fragment of the intestine. Moreover, it has been shown that BPA generally increases the degree of co-localization of NRG1 with other substances.

Bisphenol A affects vipergic nervous structures in the porcine urinary bladder trigone.

Bisphenol A (BPA) is used in the production of plastics approved for contact with feed and food. Upon entering living organisms, BPA, as a potent endocrine disruptor, negatively affects various internal organs and regulatory systems, especially in young individuals. Although previous studies have described the neurotoxic effects of BPA on various tissues, it should be underlined that the putative influence of this substance on the chemical architecture of the urinary bladder intrinsic innervation has not yet been studied. One of the most important neuronal substances involved in the regulation of urinary bladder functions is vasoactive intestinal polypeptide (VIP), which primarily participates in the regulation of muscular activity and blood flow. Therefore, this study aimed to determine the influence of various doses of BPA on the distribution pattern of VIP-positive neural structures located in the wall of the porcine urinary bladder trigone using the double-immunofluorescence method. The obtained results show that BPA influence leads to an increase in the number of both neurons and nerve fibres containing VIP in the porcine urinary bladder trigone. This may indicate that VIP participates in adaptive processes of the urinary bladder evoked by BPA.

Alfred Biesiadecki (1839-1889) and his place in the history of dermatology.

Modern dermatology, as a separate branch of medicine, came into being in the 19th century. Alfred Biesiadecki was an outstanding anatomic pathologist and the pioneer in the histopathologic examination of the skin. Biesiadecki was the first to describe the exact distribution of lymphatic vessels in the skin. He dealt with the histopathologic mechanisms of dermatitis and skin grafts, and his work was the forerunner of studies of neoplasms in the skin vasculature. His scientific achievements secured him a prominent place in the history of dermatology in Europe and around the globe. The present work outlines a biography of Alfred Biesiadecki and his most important studies of both dermatology and other branches of medicine.

Vasoactive Intestinal Polypeptide (VIP) in the Intestinal Mucosal Nerve Fibers in Dogs with Inflammatory Bowel Disease.

Canine inflammatory bowel disease (IBD) is a group of enteropathies with nonspecific chronic symptoms and poorly understood etiology. Many aspects connected with IBD are not understood. One of them is the participation of the intestinal nervous system in the development of pathological processes. Thus, this study aimed to demonstrate changes in the density of intramucosal nerve fibers containing vasoactive intestinal polypeptide (VIP)-one of the most important intestinal nervous factors caused by the various stages of IBD development. Mucosal biopsy specimens collected from the duodenum, jejunum and descending colon of healthy dogs and dogs with varied severity of IBD were included in the experiment. The density of VIP-like immunoreactive (VIP-LI) nerves was determined by a single immunofluorescence technique and a semi-quantitative method consisting in VIP-LI fiber counts in the field of view (0.1 mm2). The obtained results indicate that IBD induces changes in the density of mucosal VIP-LI nerve fibers in the canine gastrointestinal tract. The initial decrease is followed by an increase in VIP-like immunoreactivity in successive stages of the disease. These observations show that VIP is a neuronal factor that participates in the pathological processes connected with canine IBD. The observed changes probably result from the neuroprotective and/or adaptive properties of VIP. Protective and adaptive reactions induced by inflammation aim to protect the GI tract against damage by proinflammatory factors and ensure the homeostasis in the enteric nervous system (ENS) under the conditions changed by the disease process.

Changes in the expression of substance P in nerve fibres of the colonic mucosa in dogs suffering from inflammatory bowel disease.

Due to its difficult diagnosis and complicated treatment, inflammatory bowel disease (IBD) in dogs is a challenge for the veterinarian. Several aspects connected with pathological changes during IBD still remain unknown. Since one of these aspects is the participation of intestinal innervation in the evolution of the disease, the aim of this study was to demonstrate changes in the number and distribution of intramucosal colonic nerve fibres immunoreactive to substance P (SP) arising as the disease progresses. SP is one of the most important neuronal factors in intestinal innervation which, among other tasks, takes part in the conduction of pain stimuli. Using routine immunofluorescence technique, the density of nerve fibres containing SP was evaluated within mucosal biopsy specimens collected from the descending colon of healthy dogs and animals suffering from IBD of varying severity. The results of the study indicate that during severe IBD the number of nerve fibres containing SP located in the colonic mucosal layer increases in comparison to control animals. The number of SP-positive intramucosal nerves amounted to 10.99 ± 2.11 nerves per observation field in healthy dogs, 14.62 ± 2.86 in dogs with mild IBD, 14.80 ± 0.91 in dogs with moderate IBD and 19.03 ± 6.11 in animals with severe IBD. The observed changes were directly proportional to the intensity of the disease process. These observations may suggest a role of this neuronal substance in pathological processes occurring during IBD. Although the exact mechanism of the observed changes has not been completely explained, the results obtained in this investigation may contribute to improving the diagnosis and treatment of this disease, as well as the staging of canine IBD in veterinary practice.

Vasoactive Intestinal Polypeptide in the Carotid Body-A History of Forty Years of Research. A Mini Review.

Vasoactive intestinal polypeptide (VIP) consists of 28 amino acid residues and is widespread in many internal organs and systems. Its presence has also been found in the nervous structures supplying the carotid body not only in mammals but also in birds and amphibians. The number and distribution of VIP in the carotid body clearly depends on the animal species studied; however, among all the species, this neuropeptide is present in nerve fibers around blood vessels and between glomus cell clusters. It is also known that the number of nerves containing VIP located in the carotid body may change under various pathological and physiological factors. The knowledge concerning the functioning of VIP in the carotid body is relatively limited. It is known that VIP may impact the glomus type I cells, causing changes in their spontaneous discharge, but the main impact of VIP on the carotid body is probably connected with the vasodilatory effects of this peptide and its influence on blood flow and oxygen delivery. This review is a concise summary of forty years of research concerning the distribution of VIP in the carotid body.

The Influence of Bisphenol a on the Nitrergic Nervous Structures in the Domestic Porcine Uterus.

Bisphenol A (BPA) is one of the most common environmental pollutants among endocrine disruptors. Due to its similarity to estrogen, BPA may affect estrogen receptors and show adverse effects on many internal organs. The reproductive system is particularly vulnerable to the impact of BPA, but knowledge about BPA-induced changes in the innervation of the uterus is relatively scarce. Therefore, this study aimed to investigate the influence of various doses of BPA on nitrergic nerves supplying the uterus with the double immunofluorescence method. It has been shown that even low doses of BPA caused an increase in the number of nitrergic nerves in the uterine wall and changed their neurochemical characterization. During the present study, changes in the number of nitrergic nerves simultaneously immunoreactive to substance P, vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating peptide, and/or cocaine- and amphetamine-regulated transcript were found under the influence of BPA. The obtained results strongly suggest that nitrergic nerves in the uterine wall participate in adaptive and/or protective processes aimed at homeostasis maintenance in the uterine activity under the impact of BPA.

Bisphenol S in Food Causes Hormonal and Obesogenic Effects Comparable to or Worse than Bisphenol A: A Literature Review.

In recent years, bisphenol analogues such as bisphenol S (BPS) have come to replace bisphenol A in food packaging and food containers, since bisphenol A (BPA) has been shown to leach into food and water, causing numerous negative health effects. Unfortunately, little or no research was done to determine the safety of these BPA-free products before they were marketed to the public as a healthier alternative. The latest studies have shown that some of these bisphenol analogues may be even more harmful than the original BPA in some situations. This article used a literature survey to investigate the bisphenol analogue BPS and compare it to BPA and other analogues with regards to increased obesity, metabolic disorders, cancer, and reproductive defects; among others. It was found that BPS works via different pathways than does BPA while causing equivalent obesogenic effects, such as activating preadipocytes, and that BPS was correlated with metabolic disorders, such as gestational diabetes, that BPA was not correlated with. BPS was also shown to be more toxic to the reproductive system than BPA and was shown to hormonally promote certain breast cancers at the same rate as BPA. Therefore, a strong argument may be made to regulate BPS in exactly the same manner as BPA.

Bisphenol A (BPA) Affects the Enteric Nervous System in the Porcine Stomach.

Bisphenol A (BPA) is widely utilized in plastic production process all over the world. Previous studies have shown that BPA, with its similarity to estrogen, may negatively affect living organisms. It is acknowledged that BPA distorts the activity of multiple internal systems, including the nervous, reproductive, urinary, and endocrine systems. BPA also affects the gastrointestinal tract and enteric nervous system (ENS), which is placed throughout the wall from the esophagus to the rectum. Contrary to the intestine, the influence of BPA on the ENS in the stomach is still little known. This study, performed using the double immunofluorescence method, has revealed that BPA affects the number of nervous structures in the porcine gastric wall immunoreactive to vesicular acetylcholine transporter (VAChT, a marker of cholinergic neurons), substance P (SP), vasoactive intestinal polypeptide (VIP), galanin (GAL) and cocaine- and amphetamine-regulated transcript peptide (CART). The character and severity of noted alterations depended on the part of the ENS, the BPA dose, and the type of neuronal substance. Administration of BPA resulted in an increase in the number of nervous structures containing SP, GAL, and/or CART, and a decrease in the number of cholinergic neurons in all parts of the gastric wall. The number of VIP-positive nervous structures increased in the enteric myenteric ganglia, along with the muscular and mucosal layers, whilst it decreased in the submucous ganglia. The exact mechanism of noted changes was not absolutely obvious, but they were probably related to the neuroprotective and adaptive processes constituting the response to the impact of BPA.

Neurochemical characterization of the enteric neurons within the porcine jejunum in physiological conditions and under the influence of bisphenol A (BPA).

BACKGROUND: Bisphenol A (BPA) is commonly used in the production of plastics and has multidirectional, negative effects on the living organisms. It may also affect the enteric nervous system (ENS) located in the wall of the gastrointestinal tract. Enteric neurons express many active substances, which regulate majority of intestinal activities not only in physiological conditions but also under the impact of pathological factors. METHODS: The influence of various doses of BPA on the ENS of jejunum has been investigated using the double immunofluorescence technique. The commercial antibodies against substance P (SP), vasoactive intestinal polypeptide (VIP), galanin (GAL), vesicular acetylcholine transporter (VAChT), and cocaine- and amphetamine-regulated transcript peptide (CART) were used. KEY RESULTS: Both doses of BPA studied changed the number of the enteric neurons immunoreactive to SP, VIP, GAL, VAChT, and CART, and the intensity of fluctuations depended on the BPA dose and on the type of the enteric plexus. Bisphenol A causes the increase in the number of neurons immunoreactive to the majority of substances studied. The only exception was VAChT-positive neurons, the number of which was lower under the impact of BPA in the comparison with physiological conditions. CONCLUSIONS & INFERENCES: Even low doses of BPA cause the changes in neurochemical characterization of the enteric neurons in the jejunum. These changes may be the first sign of subclinical BPA intoxication. The mechanisms of observed changes are probably connected with neurotoxic and/or pro-inflammatory activity of BPA, but their exact mechanisms are not fully explained.

Cocaine- and amphetamine-regulated transcript (CART) peptide in the enteric nervous system of the porcine esophagus.

Cocaine- and amphetamine-regulated transcript peptide (CART) is widely distributed within the central and peripheral nervous system. In the brain, CART is considered as the main anorectic peptide involved in the regulation of food intake. Contrary to the central nervous system, a lot of aspects connected with the distribution and functions of CART within the enteric nervous system (ENS) still remain unknown. The aim of the present study was to investigate, for the first time, the population of CART-like immunoreactive (CART-LI) neurons within the porcine esophagus and the denotation of their neurochemical coding. During this experiment, the distribution of CART-LI neurons and the colocalization of CART with other neuronal active substances were examined using standard double- and triple-immunofluorescence techniques in enteric plexuses of cervical, thoracic, and abdominal esophagus fragments. The obtained results showed that CART is present in a relatively high percentage of esophageal neurons (values fluctuated from 45.2±0.9% in the submucous plexus of the thoracic esophagus to 58.1±5.0% in the myenteric plexus of the same fragment of the esophagus). Moreover, CART colocalized with a wide range of other active neuronal substances, mainly with the vesicular acetylcholine transporter (VAChT, a marker of cholinergic neurons), neuronal isoform of nitric oxide synthase (nNOS, a marker of nitrergic neurons), vasoactive intestinal polypeptide (VIP) and galanin (GAL). The number of CART-positive neuronal cells and their neurochemical coding clearly depended on the fragment of esophagus studied and the type of enteric plexus. The obtained results suggest that CART may play important and multidirectional roles in the neuronal regulation of esophageal functions.

The Influence of High and Low Doses of Bisphenol A (BPA) on the Enteric Nervous System of the Porcine Ileum.

Bisphenol A, used in the production of plastic, is able to leach from containers into food and cause multidirectional adverse effects in living organisms, including neurodegeneration and metabolic disorders. Knowledge of the impact of BPA on enteric neurons is practically non-existent. The destination of this study was to investigate the influence of BPA at a specific dose (0.05 mg/kg body weight/day) and at a dose ten times higher (0.5 mg/kg body weight/day), given for 28 days, on the porcine ileum. The influence of BPA on enteric neuron immunoreactive to selected neuronal active substances, including substance P (SP), vasoactive intestinal polypeptide (VIP), galanin (GAL), vesicular acetylcholine transporter (VAChT-used here as a marker of cholinergic neurons), and cocaine- and amphetamine-regulated transcript peptide (CART), was studied by the double immunofluorescence method. Both doses of BPA affected the neurochemical characterization of the enteric neurons. The observed changes depended on the type of enteric plexus but were generally characterized by an increase in the number of cells immunoreactive to the particular substances. More visible fluctuations were observed after treatment with higher doses of BPA. The results confirm that even low doses of BPA may influence the neurochemical characterization of the enteric neurons and are not neutral for living organisms.

The Impact of T-2 Toxin on Vasoactive Intestinal Polypeptide-Like Immunoreactive (VIP-LI) Nerve Structures in the Wall of the Porcine Stomach and Duodenum.

T-2 toxin is a secondary metabolite of some Fusarium species. It is well-known that this substance can harmfully impact living organisms. Among others, thanks to the ability of crossing the blood-brain barrier, T-2 toxin can affect the central nervous system. Mycotoxins mostly get into the organism through the digestive tract; therefore, first of all they have to break the intestinal barrier, wherein the important component is the enteric nervous system (ENS). However, knowledge about the impact of T-2 toxin on the ENS is rather scant. As a result of the influence of various physiological and pathological agents, ENS can undergo adaptive and reparative processes which manifest as changes in the immunoreactivity of perikaryons for neuronal active substances. So, the aim of the present investigation was to study how low doses of T-2 toxin affect vasoactive intestinal polypeptide-like immunoreactive (VIP-LI) nervous structures in the ENS of the porcine stomach and duodenum. Obtained results have shown that T-2 toxin causes an percentage increase of VIP-LI nerve cells and nerve fibers in every enteric plexus in both fragments of gastrointestinal tract studied. This shows that even low doses of T-2 toxin can have an influence on living organisms.

Bisphenol A-Induced changes in the enteric nervous system of the porcine duodenum.

Bisphenol A (BPA) is an organic compound from the phenolic group commonly used for the production of plastics. The use of BPA in food and drinking water containers carries a significant risk to human health since BPA can be washed out and enter consumables. BPA entering the human body with food shows a multi-directional effect and causes disorders in the functioning of many systems and organs. There is no current knowledge about the effects of BPA on the enteric nervous system. The purpose of the present study was to verify the influence of BPA on tolerable daily intake (TDI) dose (0.05 mg/kg body weight/day) and a dose ten times higher than TDI (0.5 mg/kg body weight/day) administered for 28 days on the porcine duodenum. The neurochemical characterization of the enteric neurons to five active neuronal substances was then investigated: substance P (SP), vasoactive intestinal polypeptide (VIP), galanin (GAL), vesicular acetylcholine transporter (VAChT) or cocaine- and amphetamine-regulated transcript peptide (CART) with double immunofluorescence method. Both doses of BPA caused visible changes in duodenal immunoreactivity to the majority of neuronal factors studied and the obtained results show that even TDI dose may affect the living organism.