Fungi and arsenic. Tolerance and bioaccumulation by soil saprotrophic species

Increasing arsenic environmental concentrations are raising worldwide concern for its impacts on human health and ecosystem functionality. In order to cope with arsenic contamination, bioremediation using fungi can represent an efficient, sustainable, and cost-effective technological solution. Fungi can mitigate arsenic contamination through different mechanisms including bioaccumulation. In this work, four soil saprotrophic fungi Absidia spinosa, Purpureocillium lilacinum, Metarhizium marquandii, and Cephalotrichum nanum, isolated from soils with naturally high arsenic concentrations, were tested for their ability to tolerate different sodium arsenite concentrations and accumulate As in different cultural conditions. pH medium after fungal growth was measured to study pH variation and metabolic responses. Arsenic bioaccumulation and its influence on the uptake of other elements were investigated through multi-elemental analysis using hydride generation atomic fluorescence spectrometry (HG-AFS), inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectroscopy (ICP-OES). Considering the increasing interest in siderophore application for metal bioremediation, the production of siderophores and their affinity for both Fe and As were also evaluated. All species were able to tolerate and accumulate As in their biomass in all of the tested conditions and produced siderophores with different affinities for Fe and As. The results suggest that the tested fungi are attractive potential candidates for the bioremediation of As contaminated soil and worthy of further investigation

Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles

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Frataxin deficiency shifts metabolism to promote reactive microglia via glucose catabolism

Immunometabolism investigates the intricate relationship between the immune system and cellular metabolism. This study delves into the consequences of mitochondrial frataxin (FXN) depletion, the primary cause of Friedreich's ataxia (FRDA), a debilitating neurodegenerative condition characterized by impaired coordination and muscle control. By using single-cell RNA sequencing, we have identified distinct cellular clusters within the cerebellum of an FRDA mouse model, emphasizing a significant loss in the homeostatic response of microglial cells lacking FXN. Remarkably, these microglia deficient in FXN display heightened reactive responses to inflammatory stimuli. Furthermore, our metabolomic analyses reveal a shift towards glycolysis and itaconate production in these cells. Remarkably, treatment with butyrate counteracts these immunometabolic changes, triggering an antioxidant response via the itaconate-Nrf2-GSH pathways and suppressing the expression of inflammatory genes. Furthermore, we identify Hcar2 (GPR109A) as a mediator involved in restoring the homeostasis of microglia without FXN. Motor function tests conducted on FRDA mice underscore the neuroprotective attributes of butyrate supplementation, enhancing neuromotor performance. In conclusion, our findings elucidate the role of disrupted homeostatic function in cerebellar microglia in the pathogenesis of FRDA. Moreover, they underscore the potential of butyrate to mitigate inflammatory gene expression, correct metabolic imbalances, and improve neuromotor capabilities in FRDA

Lipocalin-2 promotes adipose-macrophage interactions to shape peripheral and central inflammatory responses in experimental autoimmune encephalomyelitis

Objective: Accumulating evidence suggests that dysfunctional adipose tissue (AT) plays a major role in the risk of developing multiple sclerosis (MS), the most common immune-mediated and demyelinating disease of the central nervous system. However, the contribution of adipose tissue to the etiology and progression of MS is still obscure. This study aimed at deciphering the responses of AT in experimental autoimmune encephalomyelitis (EAE), the best characterized animal model of MS. Results and methods: We observed a significant AT loss in EAE mice at the onset of disease, with a significant infiltration of M1-like macrophages and fibrosis in the AT, resembling a cachectic phenotype. Through an integrative and multilayered approach, we identified lipocalin2 (LCN2) as the key molecule released by dysfunctional adipocytes through redox-dependent mechanism. Adipose-derived LCN2 shapes the pro-inflammatory macrophage phenotype, and the genetic deficiency of LCN2 specifically in AT reduced weight loss as well as inflammatory macrophage infiltration in spinal cord in EAE mice. Mature adipocytes downregulating LCN2 reduced lipolytic response to inflammatory stimuli (e.g. TNFα) through an ATGL-mediated mechanism. Conclusions: Overall data highlighted a role LCN2 in exacerbating inflammatory phenotype in EAE model, suggesting a pathogenic role of dysfunctional AT in MS

A fungal solution to a fungal problem: Chaetomium globosum and Minimedusa polyspora potential in the biocontrol of plant pathogenic fungi

Plant diseases, resulting in an annual estimated loss of 10–15% of world's major crops, represent a major threat to global crops production and social and political stability of nations [1]. About 70–80% of these diseases are caused by pathogenic fungi, numbers that are expected to increase in future years due to the effect of climate change on plant-pathogens interactions [2,3]. In the effort to transition to a more sustainable and resilient agriculture, the application of biological control agents and their secondary metabolites represent a promising option to support the achievement of food security, without further compromise ecosystems’ health [4,5]. Therefore, it is important deepening the potential of known fungal biocontrol agents against the existing fungal pathogens, shedding further light on their action mechanisms and discovering new efficient fungal strains suitable for biotechnological applications. In vitro screenings, despite presenting several limitations, constitute valuable methods for the identification of potential biocontrol agents [6]. Therefore, this study, through an array of in vitro plate assays, aimed at evaluating Minimedusa polyspora (Hotson) Weresub & P. M. LeClair and Chaetomium globosum Kunze ability to inhibit the growth of Alternaria alternata (Fr.) Keissl., Berkeleyomyces basicola (Berk. & Broome) W.J. Nel, Z.W. de Beer, and Botrytis cinerea Pers.. Furthermore, this study aimed also at gaining insights on possible antimicrobial mechanism/s involved in their biological control action. More specifically, a dual culture assay, a dual culture for volatile antimicrobial compounds (performed in two different conditions), and a culture filtrate antifungal activity assay were designed to try to discriminate the impact of direct and indirect biological control mechanisms. This study’s results show that both M. polyspora and C. globosum were able to inhibit, to a different extent, all the pathogens’ growth in the dual culture assay, suggesting a mechanism of biocontrol involving competition for nutrients and space. M. polyspora, based on the culture filtrate antifungal activity assay, was found to exert its inhibition on all the pathogens thanks also to an antibiosis mechanism through the release of diffusible compounds. Moreover, M. polyspora culture filtrate resulted to be particularly effective especially against B. basicola whose growth was completely inhibited; furthermore, its high inhibition effect against this species was also observed in the dual culture for volatile antimicrobial compounds assay, suggesting that M. polyspora antagonism against B. basicola occurs through multiple or mixed mechanisms. Therefore, based on this preliminary study’s results M. polyspora and C. globosum are promising biocontrol agents of three fungal phytopathogens of economical and agronomical relevance, and consequently species of interest for further studies in this area aimed at validating their potential as antagonists in in vivo conditions. 1) J.B. Ristaino, P.K. Anderson, D.P. Bebber, K.A. Brauman, N.J.Cunniffe, N.V. Fedoroff, C. Finegold, K.A. Garrett, C.A. Gilligan, C.M. Jones, et al. (2021). PNAS 118, 23 e2022239118. 2) A.C. Velásquez, C.D.M. Castroverde, S.Y. He (2018) Current Biology 28, 619–634. 3) S. Sarrocco, G. Vannacci (2018) Crop Protection 110, 160–170. 4) R.A.A. Khan, S. Najeeb, S. Hussain, B. Xie, Y. Li (2020) Microorganisms 8, 817. 5) Y. Peng, S.J. Li, J. Yan, Y. Tang, J.P. Cheng, A.J. Gao, X. Yao, J.J. Ruan, B.L. Xu (2021) Frontiers in Microbiology 12, 670135. 6) K. Raymaekers, L. Ponet, D. Holtappels, B. Berckmans, B.P.A. Cammue (2020) Biological Control 144, 104240

Fungal diversity for bioremediation: Tackling co-contaminations in a decomissioned military site

Military sites, both active and decommissioned, represent a unique situation in which both biodiversity loss and protection may occur, depending on how those sites are managed. Exclusion zones and good practices such those provided in Natura 2000 report for military sites — can maintain or even increase the detected biodiversity. However, potential and identified contaminations in military zones are soil threats of increasing interest, especially when co-contaminations by organic and inorganic pollutants are established. Remediating such complex contaminations through conventional techniques is not only economically unsustainable but can also impact soil biodiversity. Novel methods to tackle co-contaminations may be found in biotechnological applications of the bioresources isolated from those same contaminated sites, even though there is a knowledge gap to be filled regarding the potentialities of autochthonous microbial communities. Therefore, this study aimed at gaining insight on the culturable fungal community of a decommissioned military site in Italy and the potentialities in fungal bioremediation. To reach the aims of the study, soil samples were collected in six sampling plots and the fungal communities were isolated. Furthermore, the fungal community associated with the rhizosphere of a specimen of Plantago lanceolata L., a wild herb largely distributed in the site, was similarly studied for the same purposes. The results showed high differences in species' abundances among samples with Penicillium, Aspergillus and Trichoderma as the most abundant genera. The analysis of alpha diversity and evenness revealed that the samples with the lowest abundance in Colony Forming Units (CFUs) showed the highest values of Shannon’s Diversity Index (H’) and Simpson Diversity Index (D1), pointing to a lack of a dominant species among the isolates from those samples, while in samples with higher CFU values a more varied situation arises. In fact, sample S28 had the lowest diversity indexes but also the second highest CFU abundance, pointing to a dominance of few species, especially Penicillium S28A5, which was the most abundant species isolated from all samples. The analysis of genera alpha diversity revealed a similar situation, except for sample S22, whose diversity of genera showed to be the lowest among all samples, with a clear dominance of the Penicillium genus. Focusing on the rhizosphere sample, the results of alpha revealed a highly diverse community of culturable fungi, in both species and genera. The analysis of beta diversity, using Sørensen’s index, showed that while the samples shared few common species, the isolated genera mostly overlapped. To evaluate the potentialities of the isolated species, a set of screenings were performed on a selected group of 30 species, which included the most abundant species and a species for each genus isolated. The assays performed in this study were the Remazol Brilliant Blue R (RBBR) and Fe-Chromeazurol S (Fe-CAS) decolorization assay, which are reported in literature as proxy test to investigate the ability to degrade complex organic compounds and to produce siderophores in response to metallic and non-metallic elements. The species showing the best performances were further tested to assess their tolerance to zinc (Zn), lead (Pb), Polycyclic Aromatic Hydrocarbons (PAHs) and mixtures of organic and inorganic pollutants (Zn-PAH and Pb-PAH) in in-vitro assays. The results of the screenings pointed to Gliomastix S28RE2 and Westerdykella S28RA1 having high capacities of degrading RBBR, while Acremonium S76A16, Aspergillus S56C4 and the aforementioned Gliomastix species showed to be able to produce high quantities of siderophores. Eleven species were chosen to be tested in the tolerance tests on Zn and PAHs, while four strains isolated from a Pb contaminated sample were exposed to Pb and PAHs. Overall, Penicillium S56C6 showed the best results in both test conditions, retaining more than 70% of its growth rate when compared with control, while Mucor S56E4 showed no change in growth rate, but suffered a loss in mycelium density. To conclude, several strains isolated from this decommissioned military site showed promising potentialities for possible application in bioremediation and further studies are currently underway to develop microbial consortia to enhance their performance

Potential in mycoremediation of soil saprotrophic fungi: arsenic uptake and tolerance in different nutritional conditions

Arsenic (As) is a metalloid, naturally occurring in the earth crust. Anthropogenic sources, such as processing of arsenic-bearing minerals, are mainly responsible for environmental contamination, arising concern for toxic effects of As on human health and ecosystems. Fungi play key roles in fundamental biogeochemical cycles of essential and toxic elements and soil formation. Several studies reported that fungi can tolerate and accumulate high concentrations of arsenic and mediate processes of biovolatilisation through methylation. Our research aimed to evaluate As tolerance and bioaccumulation of four species of soil saprotrophic fungi and how nutritional conditions influence them. Absidia spinosa, Purpureocillium lilacinum, Metarhizium marquandii and Cephalotrichum nanum, previously isolated from areas with high natural As concentrations, were tested in two cultural media (MEA and Czapek-Dox). Results revealed high tolerance to all tested As concentrations (10, 20 and 50 mg/L sodium arsenite). Growth responses and tolerance to As have been investigated by tolerance indices (Rt:Rc (%) and T.I. (%)), based on growth data (diametric extension and dry weight). Most of these species resulted tolerant to all tested As concentrations, with growth responses also varying according to cultural media. pH medium after fungal growth was measured to study pH variation and metabolic responses. Chemical analyses by HG-AFS revealed As bioaccumulation. Elemental chemical composition of fungal biomass for all tested species was determined by ICP-MS and ICP-AES to assess how As occurrence and fungal responses to it influence the elements’ uptake. Results shed further light on fungal geoactive roles in the environmental fate of As and provide potential applications in bioremediation

Tackling co-contaminations: potentialities of soil fungi isolated from a decommissioned military site.

Approximately 250 000 sites in Europe are thought to be contaminated and in need of remediation, mostly due to industries and waste processing, with a rising concern over the underestimation of current and past military activities 1 . A recognized common trend is the soil co-contamination by both organic and inorganic contaminants caused by the production and use of ammunitions and weaponry and vehicle maintenance 2–4 . Subsequently, restoring military sites should be a primary objective, aiming to preserve the untouched habitats found within exclusion zones 5,6 and return decommissioned sites to local communities. Unfortunately, the remediation of such contaminations, especially through conventional methods, comes with many drawbacks, both economically and environmentally. Hence, in recent years the potential of biotechnological applications of biological resources, such as fungi, bacteria and plants, has garnered rising interest. Particularly with a focus on organisms isolated from contaminated environments, who may possess useful traits, such as increased tolerance and ability to degrade/detoxify pollutants 7,8 . Therefore, in this study, the microbial community of a decommissioned military site, previously identified as co-contaminated, has been isolated. Consequently, a preliminary screening has been implemented in order to evaluate the potentialities of fungal isolates in the bioremediation of co-contaminated soils. Furthermore, this study also focused on evaluating the potentialities of the rhizospherical fungal community of a specimen of Plantago lanceolata L., a wild herb widely distributed in the site. This study’s results highlighted a high variability in Colony Forming Unit (CFU) abundance among the isolates of the 6 samples collected from the site. The most abundant genera were found to be Penicillium, Aspergillus and Trichoderma, with the strain labelled Penicillium S28A5 being the most represented among the samples. To study the potentialities of the isolates, a representative subset of 30 strains, including the most represented strains and at least a strain for each identified genera, were tested in a two-phase screening. The first phase included the Remazol Brilliant Blue R (RBBR) and the Fe-Chromeazurol S (Fe-CAS) decolorization assays to pinpoint the best candidates for the subsequent in-vitro tolerance tests. The selected strains were tested for their tolerance against Zn (an inorganic contaminant detected in all samples) and a mixture of polycyclic aromatic hydrocarbons (PAHs), in single contaminant and co-presence conditions. The decolorization assays showed that Gliomastix S28RE2 and Westerdykella S28RA1 showed large decolorization halos in the RBBR assay, pointing to a strong capacity of degrading complex organic compounds. Meanwhile, in the Fe-CAS test, Gliomastix S28RE2 as well as Acremonium S76A16 and Aspergillus S56C4 showed the ability to produce high amounts of siderophores, similar to a strong chelating agent used as positive control. Based on the results of the first phase of screenings 11 strains were chosen for the subsequent tolerance test on Zn and PAHs. Based on the analysis performed on the soil samples, a second tolerance test, replacing Zn with Pb, were performed involving 4 strains, isolated from a Pb-contaminated area. These tests revealed that Penicillium S56C6 tolerated both the Zn-PAH and the Pb-PAH mixture, with a tolerance index of more than 70%, while Mucor S56E4 showed to be unaffected by co-presence of Pb and PAH in the growth medium, although showing a less dense mycelium. Overall, several strains isolated from this contaminated site showed promising abilities for application as bioresources in the bioremediation of co-contaminated soils. Further studies are currently ongoing to evaluate the application of those strains in consortia. 1) Contamination from local sources — EEA. https://www.eea.europa.eu/themes/soil/soil-threats. 2) P. Pereira, Barceló, D. & Panagos, P. (2020) Environ. Res. 186, 109501. 3) Stolte, J. et al. EUR 27607. (2016) JRC Scientific and Technical Reports. 4) Siles, J. A. & Margesin, R. (2018) Appl. Microbiol. Biotechnol. 102, 4409–4421. 5) Ellwanger, G., Müller, C., Ssymank, A., Vischer-Leopold, M. & Paulsch, C. (2016). Naturschutz und Biologische Viefalt, 152 6) European Commission (2005) Natura 2000 and the military. http://europa.eu.int/comm/environment/life/home.htm. 7) Ye, S. et al. (2017) Crit. Rev. Biotechnol. 37, 1062–1076. 8) Harms, H., Schlosser, D. & Wick, L. Y. (2011) Nat. Rev. Microbiol. 9, 177–192

Biodiversity of fungi as bioresources to face diversity of soil threats

Degradation threats affect soils and ecosystems, providing fundamental services for humans and living organisms. Contamination represents a major soil threat and can impair several soil functions, such as biomass production, storage, filtration and transformation of nutrients and water, and biodiversity pool (1). Despite in a smaller measure than in the past, agriculture is one of the major drivers of soil contamination, contributing with pesticides, herbicides and fertilizers added to improve crop yield. More than 3000 different types of pesticides have been used in the European agroecosystems in the past 50 years and less than 0.1% of applied pesticide to crops are estimated to reach target pests while the rest enters the environment (1). Even if contamination can reduce soil biodiversity, in microbial communities tolerant microorganisms can develop (1). Several studies have demonstrated the efficacy of indigenous fungi as a promising tool for soil bioremediation and as bioresources to reverse contamination processes in mid-term (2). The application of fungi as bioresources can also help to prevent or at least reduce the application of agrochemicals, improving at the same time quality and quantity of the yields in the contest of sustainable agricultural practices. Indeed, several soil fungi can act as plant growth promoters, both improving nutrition and stimulating protection against pests (3). Considering the pivotal role of developing nature-based solutions in coping with future challenges (e.g. world population increase, rock phosphate exhaustion), in the last years the Fungal Biodiversity Laboratory of Sapienza University of Rome focused its research mainly on selecting suitable fungal strains as bioresources for agriculture and bioremediation. The biological characterization of historically contaminated sites by DDT and HCH, respectively in Poland and in the Czech Republic, allowed to individuate indigenous fungi suitable for integrated, sustainable and cost-effective solutions for future applications in bioremediation of persistent chlorinated compounds. While, investigations on the ability of saprotrophic fungi to solubilize inorganic phosphates allowed to screen and individuate, among several strains preserved in the culture collection of Fungal Biodiversity Laboratory, a pool of strains applicable to improve phosphorus plant nutrition (4). As appear clear from these experiences, culture collections play a pivotal role in finding and screening microorganisms, which possess interesting traits as bioresources. In fact, the conservation of organisms isolated from specific environments in fungal collections, even after years, can provide useful tools and bioresources to develop new cost-effective and environmentally friendly biotechnologies and improve new feasible practices in a context of sustainable bioeconomy. References 1. Stolte, J., Tesfai, M., Øygarden, L., Kværnø, S., Keizer, J., Verheijen, F., Panagos,P., Ballabio, C. & Hessel, R., 2015. Soil threats in Europe. 2. Ceci, A., Pinzari, F., Russo, F., Persiani, A. M. & Gadd, G. M., 2019. Appl. Microbiol. Biotechnol. 103, 53–68. 3. Owen, D., Williams, A. P., Griffith, G. W. & Withers, P. J. A., 2015. Appl. Soil Ecol. 86, 41–54. 4. Ceci, A., Pinzari, F., Russo, F., Maggi, O. & Persiani, A. M., 2018. Ambio. 47, 30–40