Small Interfering RNA Mediated Messenger RNA Knockdown in the Amphibian Pathogen Batrachochytrium dendrobatidis.

RNA interference (RNAi) has not been tested in the pandemic amphibian pathogen, Batrachochytrium dendrobatidis, but developing this technology could be useful to elucidate virulence mechanisms, identify therapeutic targets, and may present a novel antifungal treatment option for chytridiomycosis. To manipulate and decipher gene function, rationally designed small interfering RNA (siRNA) can initiate the destruction of homologous messenger RNA (mRNA), resulting in the "knockdown" of target gene expression. Here, we investigate whether siRNA can be used to manipulate gene expression in B. dendrobatidis via RNAi using differing siRNA strategies to target genes involved in glutathione and ornithine synthesis. To determine the extent and duration of mRNA knockdown, target mRNA levels were monitored for 24-48 h after delivery of siRNA targeting glutamate-cysteine ligase, with a maximum of ~56% reduction in target transcripts occurring at 36 h. A second siRNA design targeting glutamate-cysteine ligase also resulted in ~53% knockdown at this time point. siRNA directed toward a different gene target, ornithine decarboxylase, achieved 17% reduction in target transcripts. Although no phenotypic effects were observed, these results suggest that RNAi is possible in B. dendrobatidis, and that gene expression can be manipulated in this pathogen. We outline ideas for further optimization steps to increase knockdown efficiency to better harness RNAi techniques for control of B. dendrobatidis.

Tumour-derived Extracellular Vesicle and Particle Reprogramming of Interstitial Macrophages in the Lung Pre-Metastatic Niche Enhances Vascular Permeability and Metastatic Potential.

Extracellular vesicles and particles (EVPs) are pivotal mediators of pre-metastatic niche formation and cancer progression, including induction of vascular permeability, which facilitates tumor cell extravasation and metastasis. However, the mechanisms through which EVPs exert this effect remain poorly understood. Here, we elucidate a novel mechanism by which tumor EVPs enhance endothelial cell permeability, tumor extravasation, and lung metastasis to different degrees, depending on tumor type. Strikingly, vascular leakiness is observed within 48h following tumor implantation and as early as one hour following intravenous injection of tumour-derived EVPs in naïve mice. Surprisingly, rather than acting directly on endothelial cells, EVPs first activate interstitial macrophages (IMs) leading to activation of JAK/STAT signaling and IL-6 secretion in IMs which subsequently promote endothelial permeability. Depletion of IMs significantly reduces tumour-derived EVP-dependent vascular leakiness and metastatic potential. Tumour EVPs that strongly induce vascular leakiness express high levels of ITGα5, and ITGα5 ablation impairs IM activation, cytokine secretion, and subsequently vascular permeability and metastasis. Importantly, IL-6 expression is elevated in IMs from non-involved tumor-adjacent lung tissue compared to distal lung tissue in lung cancer patients, highlight the clinical relevance of our discovery. Our findings identify a key role for IM activation as an initiating step in tumor type-specific EVP-driven vascular permeability and metastasis, offering promising targets for therapeutic intervention.

Glutathione-Mediated Metal Tolerance in an Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis).

The spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis, has resulted in amphibian declines and extinctions worldwide. Some susceptible amphibian species can persist in contaminated habitats, prompting the hypothesis that B. dendrobatidis might be sensitive to heavy metals. We tested a panel of 12 metals to rank their toxicity to B. dendrobatidis zoospores and zoosporangia during a 6-h exposure. To better understand the mechanism for metal detoxification, we also evaluated whether glutathione is required for metal tolerance by depleting cellular glutathione before metal exposure. In addition, we investigated whether prior exposure to low metal concentrations impacted tolerance of subsequent exposure, as well as identifying metal combinations that may act synergistically. Silver (Ag), cadmium (Cd), and copper (Cu) were particularly toxic to B. dendrobatidis, with zoospore minimum lethal concentration values of 0.01 mM (Ag), 0.025 mM (Cd), and 0.5 mM (Cu). These three metals along with zinc (Zn) were also inhibitory to zoosporangia, with minimum inhibitory concentration values of 0.005 mM (Ag), 0.04 mM (Cd), 0.075 mM (Cu), and 0.04 mM (Zn). The fungicidal effects of several metals was reduced when assays were conducted in nutrient medium compared with synthetic pond water, highlighting the need for careful in vitro assay design and interpretation. Glutathione depletion strongly influenced tolerance of Cd and Ag (85% and 75% less growth, respectively) and moderately influenced tolerance of Cu, Zn, and lead (37%, 18%, and 14% less growth, respectively), indicating the importance of glutathione for metal detoxification. In general, the minimum metal concentrations that inhibited growth of B. dendrobatidis far exceeded values detected in contaminated amphibian habitats in Australia, suggesting that metal contamination alone may not have a strong protective effect against chytridiomycosis. We discuss future research directions to futher understand the potential for dissolved metals to create chytrid refuges. Environ Toxicol Chem 2024;43:1583-1591. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Glutathione is required for growth and cadmium tolerance in the amphibian chytrid fungus, Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd) is a lethal amphibian pathogen, partly due to its ability to evade the immune system of susceptible frog species. In many pathogenic fungi, the antioxidant glutathione is a virulence factor that helps neutralise oxidative stressors generated from host immune cells, as well as other environmental stressors such as heavy metals. The role of glutathione in stress tolerance in Bd has not been investigated. Here, we examine the changes in the glutathione pool after stress exposure and quantify the effect of glutathione depletion on cell growth and stress tolerance. Depletion of glutathione repressed growth and release of zoospores, suggesting that glutathione is essential for life cycle completion in Bd. Supplementation with <2 mM exogenous glutathione accelerated zoospore development, but concentrations >2 mM were strongly inhibitory to Bd cells. While hydrogen peroxide exposure lowered the total cellular glutathione levels by 42 %, glutathione depletion did not increase the sensitivity to hydrogen peroxide. Exposure to cadmium increased total cellular glutathione levels by 93 %. Glutathione-depleted cells were more sensitive to cadmium, and this effect was attenuated by glutathione supplementation, suggesting that glutathione plays an important role in cadmium tolerance. The effects of heat and salt were exacerbated by the addition of exogenous glutathione. The impact of glutathione levels on Bd stress sensitivity may help explain differences in host susceptibility to chytridiomycosis and may provide opportunities for synergistic therapeutics.

Perioperative escape from dormancy of spontaneous micro-metastases: A role for malignant secretion of IL-6, IL-8, and VEGF, through adrenergic and prostaglandin signaling.

We recently showed that a minimally-invasive removal of MDA-MB-231HM primary tumors (PTs) and elimination of their secreted factors (including IL-6, IL-8, VEGF, EGF, PDGF-aa, MIF, SerpinE1, and M-CSF), caused regression of spontaneous micro-metastases into a non-growing dormant state. To explore the underlying mechanisms and potential clinical ramifications of this phenomenon, we herein used the MDA-MB-231HM human breast cancer cell-line, in-vitro, and in vivo following orthotopic implantation in immune-deficient BALB/C nu/nu mice. Employing bioluminescence imaging, we found that adding laparotomy to minimally-invasive removal of the PT caused an outbreak of micro-metastases. However, perioperative ß-adrenergic and COX-2 inhibition, using propranolol + etodolac, maintained metastatic dormancy following laparotomy. In-vitro, ß-adrenergic agonists (epinephrine or metaproterenol) and prostaglandin-E2 markedly increased MDA-MB-231HM secretion of the pro-metastatic factors IL-6, IL-8, and VEGF, whereas cortisol reduced their secretion, effects that were maintained even 12 h after the washout of these agonists. In-vivo, laparotomy elevated IL-6 and IL-8 levels in both plasma and ex-vivo PT spontaneous secretion, whereas perioperative propranolol + etodolac administration blocked these effects. Similar trends were evident for EGF and MIF. Promoter-based bioinformatics analyses of excised PT transcriptomes implicated elevated NF-kB activity and reduced IRF1 activity in the gene regulatory effects of laparotomy, and these effects were inhibited by pre-surgical propranolol + etodolac. Taken together, our findings suggest a novel mechanism of post-operative metastatic outbreak, where surgery-induced adrenergic and prostanoid signaling increase the secretion of pro-metastatic factors, including IL-6, IL-8, and VEGF, from PT and possibly residual malignant tissue, and thereby prevent residual disease from entering dormancy.

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis.

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

A case of benign osteogenic tumour in Homo naledi: Evidence for peripheral osteoma in the U.W. 101-1142 mandible.

The reported incidence of neoplasia in the extinct hominin record is rare. We describe here the first palaeopathological analysis of an osteogenic lesion in the extinct hominin Homo naledi from Dinaledi Cave (Rising Star), South Africa. The lesion presented as an irregular bony growth, found on the right lingual surface of the body of the adult mandible U.W. 101-1142. The growth was macroscopically evaluated and internally imaged using micro-focus x-ray computed tomography (µCT). A detailed description and differential diagnosis were undertaken using gross and micromorphology, and we conclude that the most probable diagnosis is peripheral osteoma - a benign osteogenic neoplasia. These tumours are cryptic in clinical expression, though they may present localised discomfort and swelling. It has been suggested that muscle traction may play a role in the development and expression of these tumours. The impact of this lesion on the individual affected is unknown. This study adds to the growing corpus of palaeopathological data from the South African fossil record, which suggests that the incidence of neoplastic disease in deep prehistory was more prevalent than traditionally accepted. The study also highlights the utility of micro-computed tomography in assisting accurate diagnoses of ancient pathologies.

White blood cell profiles in amphibians help to explain disease susceptibility following temperature shifts.

Temperature variability, and in particular temperature decreases, can increase susceptibility of amphibians to infections by the fungus Batrachochytrium dendrobatidis (Bd). However, the effects of temperature shifts on the immune systems of Bd-infected amphibians are unresolved. We acclimated frogs to 16 °C and 26 °C (baseline), simultaneously transferred them to an intermediate temperature (21 °C) and inoculated them with Bd (treatment), and tracked their infection levels and white blood cell profiles over six weeks. Average weekly infection loads were consistently higher in 26°C-history frogs, a group that experienced a 5 °C temperature decrease, than in 16°C-history frogs, a group that experienced a 5 °C temperature increase, but this pattern only approached statistical significance. The 16°C-acclimated frogs had high neutrophil:lymphocyte (N:L) ratios (suggestive of a hematopoietic stress response) at baseline, which were conserved post-treatment. In contrast, the 26°C-acclimated frogs had low N:L ratios at baseline which reversed to high N:L ratios post-treatment (suggestive of immune system activation). Our results suggest that infections were less physiologically taxing for the 16°C-history frogs than the 26°C-history frogs because they had already adjusted immune parameters in response to challenging conditions (cold). Our findings provide a possible mechanistic explanation for observations that amphibians are more susceptible to Bd infection following temperature decreases compared to increases and underscore the consensus that increased temperature variability associated with climate change may increase the impact of infectious diseases.

Effects of chytridiomycosis on hematopoietic tissue in the spleen, kidney and bone marrow in three diverse amphibian species.

One of the major causes of amphibian population decline is the deadly fungal pathogen Batrachochytrium dendrobatidis, Bd Research on pathogenesis and host immunity aims to inform development of targeted conservation interventions. Studies examining global host immune responses as well as effects on lymphocytes in vitro suggest that Bd infection causes immunosuppression. However, it is unknown which hematopoietic tissues are affected and if these effects differ among host species. We investigated the effect of experimental Bd infection on three diverse amphibian species by quantifying the amount of hematopoietic tissue in the spleen, bone marrow and kidney. Upon Bd infection, hematopoietic tissue in the kidney tended to be depleted, while the spleen appeared unaffected. The bone marrow in highly susceptible species was depleted, whereas an increase in hematopoietic tissue was observed in the more resistant species. Our study demonstrates that species and hematopoietic tissues behave differently in response to Bd infection, and may be related to the species' susceptibility to infection.

Severe sparganosis in Australian tree frogs.

Spargana of Spirometra erinacei infect many vertebrate species, but severe disease from sparganosis has been reported from few host species. Information on the effects of this common, introduced tapeworm of cats on Australian frogs is lacking. Our survey to detect significant diseases in free-ranging amphibians in eastern Australia between 1993 and 2000 revealed that infection with spargana (plerocercoids) of S. erinacei occurred in 12/243 (4.9%) sick frogs. Infections occurred in skeletal muscle and subcutis, especially the thighs, of large adults of Litoria caerulea, Litoria aurea, Litoria gracilenta, and Litoria peronii. Three frogs were also infected in the coelomic cavity. Heavy burdens in seven frogs were associated with poor body condition and debilitating lesions, whereas lighter infections in five sick frogs were considered likely to be incidental to other diseases. In severe infections, a large proportion of thigh muscle was replaced with spargana and various amounts of fibrosis, and some frogs also had myonecrosis, granulomatous inflammation, hemorrhage, and skin ulceration. Concurrent infections were common. Our findings suggest sparganosis is one of a few currently recognized serious diseases affecting free-ranging frogs in Australia.

Production of polyclonal antibodies to Batrachochytrium dendrobatidis and their use in an immunoperoxidase test for chytridiomycosis in amphibians.

Polyclonal antibodies were produced for diagnosing chytridiomycosis in amphibians. Two sheep and 4 rabbits were inoculated with homogenized whole culture of Batrachochytrium dendrobatidis in Freund's complete adjuvant or triple adjuvant. Antisera from all animals reacted strongly with all stages of B. dendrobatidis and stained the walls, cytoplasm, rhizoids and zoospores in an indirect immunoperoxidase test. Significant cross-reactivity occurred only with some fungi in the Chytridiomycota, and there are no members of this phylum besides B. dendrobatidis that infect frogs. The immunoperoxidase stain is a useful screening test when combined with recognition of the morphology and infection site of B. dendrobatidis.