David J. Kennedy

Excessive cyanobacterial proliferation and toxin production increasingly threaten freshwater ecosystems, drinking water systems, and public health. Among cyanotoxins, microcystin-LR (MC-LR) is one of the most prevalent variants and is recognized for its hepatotoxicity, with evidence showing it can also alter gut microbiota composition. Previous studies, including our own, demonstrate that MC exposure can induce inflammation, oxidative stress, and changes in gene expression associated with immune responses, even at concentrations below guideline limits. This study investigated the protective effect of an MC-degrading bacterium, Sphingopyxis sp. IM1 (IM1) with a known enzymatic MC degradation pathway, against MC-LR-induced hepatotoxicity under in vitro conditions. Human Hep3B hepatocytes were pretreated with varying ratios of IM1 bacteria and subsequently exposed to 9.95 ppm of MC-LR for 24 h. RT-qPCR analysis demonstrated that MC-LR exposure strongly increased the expression of the inflammatory markers TNFα and TGF-β1, whereas pretreatment with IM1 significantly attenuated these responses. Mass spectrometric analysis of cell pellets and spent culture media demonstrated reduced concentrations of intact MC-LR in IM1-pretreated samples compared to MC-LR-only controls, accompanied by increased detection of tetrapeptide degradation products generated during mlr-mediated MC degradation. These results demonstrate that IM1-mediated degradation of MC-LR attenuates toxin-induced hepatotoxic and inflammatory responses, highlighting the potential of a novel microbial-based therapeutic approach to mitigate MC-induced toxicity.

The Na ⁺ /K ⁺ -ATPase (NKA) regulates ion balance in the kidney and influences cellular processes like proliferation and apoptosis through its signal transduction. The endogenous ligand 20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to inflammation and fibrosis in chronic kidney disease (CKD) and inhibits NKA activity in renal tubules. However, the molecular mechanism of this interaction remains unclear. In this study, we used in-silico approach to investigate the potential interaction between 20-HETE and NKA. Various ligands, including known NKA ligands such as cardiotonic steroids (CTS), 20-HETE, and negative controls, were docked using rigid and Induced Fit Docking to predict the affinity of the ligands toward NKA. Binding free energy calculations with the Prime Molecular mechanics with generalized Born and surface area (Prime MM/GBSA) tools were used to confirm the involvement of key amino acids in ligand-receptor interactions. The docking analyses revealed that 20-HETE exhibited a binding affinity comparable to negative control, with some differences between rigid and induced fit docking. Binding free energy data highlighted key amino acids in the 20-HETE and NKA interaction. Interaction fingerprint and mutations such as Ala330Gly and Val329Ala significantly reduced binding free energy, while Thr804Ala showed a notable decrease, underscoring the potential importance of these amino acids in ligand stabilization. These findings provide computational evidence supporting potential direct interaction between 20-HETE and NKA and identify candidate residues for future experimental validation.

Microcystin-LR (MC-LR) is a potent hepatotoxin that has been shown to cause liver damage even at doses lower than the established Low Observable Adverse Effect Level (LOAEL) of 200 μg/kg in animal models. We have previously observed that low-dose exposure to MC-LR in animals with diet-induced Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) and subsequent treatment with antioxidants like N-acetylcysteine (NAC) and the Na⁺/K⁺ ATPase-Src kinase inhibitor pNaKtide significantly alleviated hepatic infiltration of immune cells, downregulated markers of inflammation and hepatotoxicity, increased the breakdown of the toxin molecule, and restored phase I and II drug metabolism pathways, including the glutathione pathway. Because the liver is composed of heterogeneous cell types, this study aimed to determine the specific role of hepatocytes in the uptake and metabolism of MC-LR, especially in the setting of MASLD. To address this, we used two well-established hepatocyte cell lines—AML-12 murine hepatocytes and human Hep3B hepatocytes. Preliminary dose comparison studies with AML-12 cells showed that MC-LR at 10 μM concentration showed a significant upregulation in the genetic expression of the markers of hepatotoxicity—OSMR (p ≤ 0.01) and SerpinE (p ≤ 0.0001)—in comparison to Vehicle. Treatment with pNaKtide (1 µM) and/or NAC (10 mM) in the presence of MC-LR significantly reduced the expression of both OSMR (p ≤ 0.0001) and SerpinE (p ≤ 0.01 and p ≤ 0.0001, respectively). To model steatotic hepatocytes characteristic of the MASLD phenotype, Hep3B hepatocytes were first treated with 500 µM of oleic acid (OA) before exposing them to the toxin in the presence and absence of antioxidants. MC-LR exposure, induced markers of inflammation and hepatotoxicity to be elevated significantly in the presence of OA as compared to MC-LR exposure alone. This elevation of the genetic markers of inflammation and hepatotoxicity was significantly attenuated on treatment with pNaKtide (1 µM) and NAC (10 mM). Quantification of human SERPINE1 (PAI1) and 8-OHdG, a stable marker of oxidative stress, in the spent media of Hep3B cells corroborated the trends observed in the genetic markers of hepatotoxicity. These observations support the central role that hepatocytes play in the uptake and metabolism of MC-LR, which is complicated by the presence of MASLD-like conditions and can help in the development of future therapeutic strategies.

Sex differences are well recognized in thrombotic diseases, but the underlying mechanisms remain unclear. The sodium/potassium ATPase (NKA), composed of α and β subunits, regulates ion homeostasis and plays a key role in cardiovascular function. We investigated whether the NKA α1 subunit influences platelet activation and thrombosis. Using the ferric chloride (FeCl3)–induced carotid artery injury thrombosis model in wild-type (WT; α1+/+) and NKA α1 heterozygous (α1+/−) mice, we found that NKA α1 haploinsufficiency significantly inhibited thrombosis in males but not in females, without affecting hemostasis. Platelet NKA α1 expression was halved in α1+/− mice, but sodium homeostasis remained unchanged. Transfusion of α1+/− platelets into thrombocytopenic WT mice prolonged the time to occlusive thrombus formation. Low-dose ouabain or marinobufagenin, which bind NKA α1, suppressed thrombosis. Mechanistically, α1 interacted with P2Y12, and this interaction was disrupted by a leucine-glycine-leucine (LGL)→serine-phenylalanine-threonine mutation in either partner or by the LGL peptide. NKA α1 haploinsufficiency, ouabain, and LGL peptide treatment all reduced ADP-induced platelet aggregation. Female mice exhibited higher platelet α1 expression and shorter thrombosis times than males. Gonadectomy had no effect in females but abolished the antithrombotic phenotype in α1+/– males, whereas orchiectomy increased platelet α1 expression. Although α1 haploinsufficiency did not affect thrombosis in the 10% FeCl3 model, it prolonged thrombosis time in mice treated with low-dose clopidogrel or prasugrel, which alone had no effect. These findings identify NKA α1 as a key regulator of sex-specific platelet activation and thrombosis, suggesting its potential as a biomarker for thrombotic risk and a therapeutic target for antiplatelet and antithrombotic therapy.

Microcystin-LR (MC-LR) is one of a large family of cyanotoxins which are naturally produced by cyanobacteria within harmful algal blooms occurring in bodies of water globally. Early findings of the toxicity of such blooms stemmed from fatalities of livestock drinking from affected water. Since then, various toxins have been identified such as the microcystins. Microcystin-LR has been studied as a representative congener due to its abundance and toxicity. While there have been extensive studies of microcystin-LR by oral route exposure, we have turned our attention to inhalation route exposure due to the recent findings of microcystin-containing lake and sea-spray aerosol. We have shown inflammatory outcomes in the airways of mice and in human cell culture models after microcystin aerosol exposure, and have found a consistent molecular patterns similar to those of Type 1/Type 17 driven neutrophilic asthma. Here we address the hypothesis that MC-LR will increase the inflammatory mediators of neutrophilic asthma leading to worsening symptoms. This is tested and characterized in both in vitro and in vivo models. We found that asthma symptoms and molecular signatures of inflammation are both worsened by MC-LR exposure in a mouse model of neutrophilic asthma. We found that 3D human airway cell culture models reconstructed from asthmatic donor cells are similarly affected, however healthy donor cells are nearly unaltered by comparison. Aggregating these findings with RNA sequencing data from all models, we developed a hypothetical molecular mechanism which relies on MC-LR mediated amplification of existing inflammatory signaling. We test this in a human reporter cell line of NF-κB activity and further demonstrate the mechanism by inhibitor testing. This study sheds light on the risk to asthmatic patients living near or recreating on affected bodies of water. Beyond asthma, we believe this study provides crucial insight into the findings over the last 40 years concerning disparate outcomes of MC-LR exposure as the result of exposure will be dependent on the signaling state of the tissue upon exposure.

There is scientific evidence that supports the association between aerobic exercise capacity and the risk of developing complex metabolic diseases. The factors that determine aerobic capacity can be categorized into two groups: intrinsic and extrinsic components. While exercise capacity is influenced by both the intrinsic fitness levels of an organism and the extrinsic factors that emerge during training, physiological adaptations to exercise training can differ significantly among individuals. The interplay between intrinsic and acquired exercise capacities represents an obstacle to recognizing the exact mechanisms connecting aerobic exercise capacity and human health. Despite robust clinical associations between disease and a sedentary state or condition, the precise causative links between aerobic exercise capacity and disease susceptibility are yet to be fully uncovered. To provide clues into the intricacies of poor aerobic metabolism in an exercise‐resistant phenotype, over two decades ago a novel rat model system was developed through two‐way artificial selection and raised the question of whether large genetic differences in training responsiveness would bring about aberrant systemic disorders and closely regulate the risk factors in health and diseases. Genetically heterogeneous outbred (N/NIH) rats were used as a founder population to develop contrasting animal models of high versus low intrinsic running capacity (HCR vs. LCR) and high versus low responsiveness to endurance training (HRT vs. LRT). The underlying hypothesis was that variation in capacity for energy transfer is the central mechanistic determinant of the divide between complex disease and health. The use of the outbred, genetically heterogeneous rat models for exercise capacity aims to capture the genetic complexity of complex diseases and mimic the diversity of exercise traits among humans. Accumulating evidence indicates that epigenetic markers may facilitate the transmission of effects from exercise and diet to subsequent generations, implying that both exercise and diet have transgenerational effects on health and fitness. The process of selective breeding based on the acquired change in maximal running distance achieved during a treadmill‐running tests before and after 8 weeks of training generated rat models of high response to training (HRT) and low response to training (LRT). In an untrained state, both LRT and HRT rats exhibit comparable levels of exercise capacity and show no major differences in cardiorespiratory fitness (maximal oxygen consumption, VO_2max). However, after training, the HRT rats demonstrate significant improvements in running distance, VO_2max, as well as other classic markers of cardiorespiratory fitness. The LRT rats, on the other hand, show no gain in running distance or VO_2max upon completing the same training regime. The purpose of this article is to provide an overview of studies using LRT and HRT models with a focus on differences in neuromuscular adaptations. This review also summarizes the involved molecular and cellular signaling pathways underlying skeletal muscle adaptations in LRT models in comparison to the HRT model, which responds positively to endurance training. The LRT‐related adverse effects in neuromuscular responses seem to be primarily driven by: (i) impaired glucose tolerance or impaired insulin sensitivity, (ii) increased extracellular matrix (ECM) remodeling, (iii) loss of type I muscle fibers, (iv) mitochondrial dysfunction, and (v) intricate cellular signaling orchestrated by TGF‐ß1‐JNK and TNF‐α‐MAPK pathways. Alternatively, the HRT model demonstrates improved neurovascular and muscle remodeling responses and increased central nervous system excitability, which might reflect an inherent protective mechanism to stress events.

Micro- and nanoplastics (MPs and NPs) are pervasive environmental pollutants detected in aquatic ecosystems, with emerging evidence suggesting their presence in airborne particles generated by water body motion. Inhalation exposure to airborne MPs and NPs remains understudied despite documented links between occupational exposure to these particles and adverse respiratory outcomes, including airway inflammation, oxidative stress, and chronic respiratory diseases. This study explored the effects of acute NP exposure on a fully differentiated 3D human airway epithelial model derived from 14 healthy donors. Airway epithelium was exposed to aerosolized 50 nm polystyrene NPs at concentrations ranging from 2.5 to 2500 µg/mL for three minutes per day over three days. Functional assays revealed no significant alterations in tissue integrity, cell survival, mucociliary clearance, or cilia beat frequency, suggesting intact epithelial function post-exposure. However, cytokine and chemokine profiling identified a significant five-fold increase in CCL3 (MIP-1α), a neutrophilic chemoattractant, in NP-exposed samples compared to controls. This was corroborated by increased neutrophil chemotaxis in response to conditioned media from NP-exposed tissues, indicating a pro-inflammatory neutrophilic response. Conversely, levels of interleukins (IL-21, IL-2, IL-15), CXCL10, and TGF-β were significantly reduced, suggesting immunomodulatory effects that may impair adaptive immune responses and tissue repair mechanisms. These findings demonstrate that short-term exposure to NP-containing aerosols induces a distinct pro-inflammatory response in airway epithelium, characterized by enhanced neutrophil recruitment and reduced secretion of key immune modulators. These findings underscore the potential for aerosolized NPs to induce oxidative and inflammatory stress, raising concerns about their long-term impact on respiratory health and redox regulation.

Commonly used adenine-induced chronic kidney disease (CKD) murine models often employ C57BL/6 mice; however, this strain has inherent limitations due to its natural resistance to developing key pathological features of CKD, such as tubulointerstitial fibrosis and inflammation. There have been attempts to overcome these barriers by using multiple concentrations of adenine-supplemented diets or by performing prolonged experiments up to 20 weeks. Here, we demonstrate that SKH1 Elite mice develop clinically relevant CKD phenotypes (e.g., polyuria, proteinuria, inflammation, and renal fibrosis) over the course of only 6 weeks of low-dose (0.15%) adenine supplementation. As a docile, immunocompetent, and hairless strain, SKH1 Elite mice offer several logistical advantages over C57BL/6 mice, including ease of handling and the ability to study dermal conditions, which are often secondary to CKD.

Harmful algal blooms are increasing globally and pose serious health concerns releasing cyanotoxins. Microcystin-LR (MC-LR), one of the most frequently produced cyanotoxins, has recently been detected in aerosols generated by the normal motions of affected bodies of water. MC-LR aerosol exposure has been linked to a pro-inflammatory influence on the airways of mice; however, little is understood about the underlying mechanism or the potential consequences. This study aimed to investigate the pro-inflammatory effects of aerosolized MC-LR on murine airways. C57BL/6 and BALB/c mice were exposed to MC-LR aerosols, as these strains are predisposed to type 1/type 17 and type 2 immune responses, respectively. Exposure to MC-LR induced granulocytic inflammation in C57BL/6 but not BALB/c mice, as observed by increased expression of cytokines MIP-1α, CXCL1, CCL2, and GM-CSF compared with their respective vehicle controls. Furthermore, the upregulation of interleukins IL-17A and IL-12 is consistent with Th1- and Th17-driven type 1/type 17 inflammation. Histological analysis confirmed inflammation in the C57BL/6 lungs, with elevated neutrophils and macrophages in the bronchoalveolar lavage fluid and increased pro-inflammatory and pro-resolving oxidized lipids. In contrast, BALB/c mice showed no significant airway inflammation. These results highlight the ability of aerosolized MC-LR to trigger harmful airway inflammation, requiring further research, particularly into populations with predispositions to type 1/type 17 inflammation.

Microcystis spp. threaten freshwater ecosystems through the proliferation of cyanobacterial harmful algal blooms (cyanoHABs) and production of the hepatotoxin, microcystin. While microcystin and its biosynthesis pathway, encoded by the mcy genes, have been well studied for over 50 years, a recent study found that Microcystis populations in western Lake Erie contain a transcriptionally active partial mcy operon, in which the A2 domain of mcyA and mcyB-C are present but the mcyD-J genes are absent. Here, we investigate the potential biosynthetic products and the evolutionary history of this partial operon. Our results reveal two candidate tetrapeptide constructs, with an X variable position, to be produced by strains with the partial operon. The partial operon appears necessary and sufficient for tetrapeptide biosynthesis and likely evolved from a single ancestor hundreds to tens of thousands of years ago. Bioactivity screens using Hep3B cells indicate a mild elevation of some markers of hepatotoxicity and inflammation, suggesting the need to further assess the effects of these novel secondary metabolites on freshwater ecosystems and public health. The need to assess these effects is even more pressing given the detection of tetrapeptides in both culture and western Lake Erie, which is a vital source of fresh water. Results from this study emphasize previous findings in which novel bacterial secondary metabolites may be derived from the molecular evolution of existing biosynthetic machinery under different environmental forcings.