bims-mecosi 2022-05-15 papers

bims-mecosi

Biomed News

on Membrane contact sites

Issue of 2022‒05‒15
thirteen papers selected by
Verena Kohler

A Novel Peroxin is Required for Mitochondrial Morphology: Implications for Resolving a New Peroxisome-Mitochondrial Contact Site.
Formation of Nascent Lipid Droplets at Specialized ER subdomains.
Dysregulation of organelle membrane contact sites in neurological diseases.
Actin assembly at sites of contact between the cortical ER and endocytic pits promotes ER autophagy.
Control of the Cellular Lipid Landscape by Inositol Lipids.
Inositol-requiring enzyme 1 alpha (IRE1α) regulates astrocyte metabolic and inflammatory phenotypes: Implications in (METH)amphetamine use disorder and HIV-associated neurocognitive disorders.
Key tethering function of Atg11 autophagy scaffold protein in formation of virus-induced membrane contact sites during tombusvirus replication.
Regulation of PIP2 homeostasis at ER-plasma membrane contacts by Nir proteins.
Roles for inter-organelle contacts in organizing metabolism.
Novel pathways of intracellular membrane lipid transport and neurodegenerative diseases.
GRP75-faciliated Mitochondria-associated ER Membrane (MAM) Integrity controls Cisplatin-resistance in Ovarian Cancer Patients.
Inhalation Exposure to Airborne PM2.5 Induces Integrated Organelle Stress Response in the Liver.
Arsenic induces ferroptosis and acute lung injury through mtROS-mediated mitochondria-associated endoplasmic reticulum membrane dysfunction.

FASEB J. 2022 May;36 Suppl 1

A Novel Peroxin is Required for Mitochondrial Morphology: Implications for Resolving a New Peroxisome-Mitochondrial Contact Site.

Emily W Knight, Andrew Gianos, Logan Crowe, Meredith T Morris.

Peroxisomes are dynamic and ubiquitous organelles that house many metabolic pathways and interact with other organelles such as the endoplasmic reticulum, lipid droplets, and mitochondria. One mechanism for organelle interaction is through membrane contact sites. While contact sites between multiple organelles have been identified, little is known about the proteins that serve as molecular tethers in such sites. We study organelle dynamics using peroxisome-like organelles called glycosomes in the early diverging organism Trypanosoma brucei and have identified a novel peroxin (protein involved in peroxisome biogenesis) that is essential for mitochondrial morphology. Silencing this protein leads to a significant growth defect and swollen mitochondria. Multiple mitochondrial membrane transport channels have been identified in immunoprecipitation studies. Based on these findings, we hypothesize that this protein that we have named a putative peroxisome-mitochondrial contact protein (PPMCP), localizes to glycosomes and mitochondria at contact points, which facilitate the transfer of metabolites between the two organelles. Disruption of this connection results in "leaky" mitochondria and cell death. Current work is focused on resolving the metabolic defects in PPMCP-deficient cells and identifying additional molecular components of these contact sites. This work forwards our understanding of how contact sites are established and the role they play in interorganelle communication.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R2441
FASEB J. 2022 May;36 Suppl 1

Formation of Nascent Lipid Droplets at Specialized ER subdomains.

Amit Joshi, Joey Ragusa, William Prinz, Sarah Cohen.

Lipid droplets (LDs) are neutral lipid containing organelles enclosed in a single monolayer of phospholipids. LD formation begins with the accumulation of neutral lipids within the bilayer of the endoplasmic reticulum (ER) membrane. It is not known how the sites of formation of nascent LDs in the ER membrane are determined. Here we show that multiple C2 domain-containing transmembrane proteins, MCTP1 and MCTP2, are at sites of LD formation in specialized ER subdomains. We show that the transmembrane domain of these proteins is similar to a reticulon homology domain. Like reticulons, MCTPs tubulate the ER membrane and favor highly curved regions of the ER. Our data indicate that the transmembrane domains (TMD) of MCTP promote LD biogenesis by increasing LD number. MCTPs colocalize with seipin, a protein involved in LD biogenesis, but form more stable microdomains in the ER. The MCTP C2 domains bind charged lipids and regulate LD size, likely by mediating ER-LD contact sites. Together, our data indicate that MCTPs form specialized subdomains within ER tubules that regulate LD biogenesis, size, and ER-LD contacts. Interestingly, MCTP punctae are associated with other organelles as well, suggesting that these proteins may play a more general role in linking tubular ER to organelle contact sites.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.0R300
Neuron. 2022 May 06. pii: S0896-6273(22)00365-8. [Epub ahead of print]

Dysregulation of organelle membrane contact sites in neurological diseases.

Soojin Kim, Robert Coukos, Fanding Gao, Dimitri Krainc.

The defining evolutionary feature of eukaryotic cells is the emergence of membrane-bound organelles. Compartmentalization allows each organelle to maintain a spatially, physically, and chemically distinct environment, which greatly bolsters individual organelle function. However, the activities of each organelle must be balanced and are interdependent for cellular homeostasis. Therefore, properly regulated interactions between organelles, either physically or functionally, remain critical for overall cellular health and behavior. In particular, neuronal homeostasis depends heavily on the proper regulation of organelle function and cross talk, and deficits in these functions are frequently associated with diseases. In this review, we examine the emerging role of organelle contacts in neurological diseases and discuss how the disruption of contacts contributes to disease pathogenesis. Understanding the molecular mechanisms underlying the formation and regulation of organelle contacts will broaden our knowledge of their role in health and disease, laying the groundwork for the development of new therapies targeting interorganelle cross talk and function.

DOI: https://doi.org/10.1016/j.neuron.2022.04.020
Autophagy. 2022 May 09.

Actin assembly at sites of contact between the cortical ER and endocytic pits promotes ER autophagy.

Dongmei Liu, Susan Ferro-Novick, Peter Novick.

  A recent screen of the Saccharomyces cerevisiae deletion library implicated End3 in autophagy of the endoplasmic reticulum (ER). Together with Pan1, End3 coordinates endocytic site initiation with the localized assembly of branching actin filaments that promotes invagination of endocytic pits. Oxysterol binding proteins function as an inter-organelle bridge by interacting with VAP proteins on the cortical ER and type I myosins on the endocytic pit. These proteins not only promote localized actin assembly at contact sites, they are required for ER autophagy as well. We propose that localized actin polymerization can push the edge of an ER sheet from the cell cortex towards the site of autophagosome assembly near the vacuole.

Keywords:  Actin assembly; End3-Pan1; Myo3/Myo5; Osh2/Osh3; Scs2/Scs22; contact sites; endocytic pits; endoplasmic reticulum; reticulophagy

DOI:  https://doi.org/10.1080/15548627.2022.2074614
FASEB J. 2022 May;36 Suppl 1

Control of the Cellular Lipid Landscape by Inositol Lipids.

Tamas Balla, Amrita Mandal, Yeun Ju Kim, Joshua Pemberton.

Cellular organization of eukaryotic cells relies upon compartmentalization of their signaling nodes on special membrane platforms forming the various organelles. Unique lipid composition of the different organelle membranes not only defines their identity but also is critical for the proper assembly and functioning of the protein signaling complexes associated with them. Inositol phospholipids (PPIns), a class of regulatory lipids, play a critical role in defining membrane identity and forming membrane microdomains with unique signaling properties. Recent developments in lipid membrane biology revealed that phosphatidylinositol (PI) 4-phosphate (PI4P) gradients and the PI 4-kinases that form them drive non-vesicular transport of several structural lipids against their concentration gradients at membrane contact sites (MCSs). These processes clearly depend on the delivery of PI from its site of synthesis in the ER to the membranes where PI4Ks convert them to PI4P. Therefore, our recent efforts have been focused on the question of how PI synthesis and PI transport systems provide the means of proper PI delivery to their other membrane destinations. In this presentation we will review our recent data on the generation and use of molecular tools to visualize and manipulate PI metabolism and delivery and demonstrate, how they can help us better understand the central role of PPIns in defining the overall lipid landscape of eukaryotic cells.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.0I182
FASEB J. 2022 May;36 Suppl 1

Inositol-requiring enzyme 1 alpha (IRE1α) regulates astrocyte metabolic and inflammatory phenotypes: Implications in (METH)amphetamine use disorder and HIV-associated neurocognitive disorders.

Jessica M Proulx, Kathleen Borgmann.

Human immunodeficiency virus 1 (HIV-1) invades the central nervous system (CNS) early during infection and can persist in the CNS for life despite effective antiretroviral treatment. Infection and activation of residential glial cells leads to low viral replication and chronic inflammation, which damage neurons contributing to a spectrum of HIV-associated neurocognitive disorders (HAND). Astrocytes are the most numerous glial cells in the CNS and provide essential support to neurons. During a neuropathological challenge, such as HIV-1 infection, astrocytes can shift their neurotrophic functions to become neurotoxic and even serve as latent reservoirs for HIV-1 infection. Notably, substance use disorders, including methamphetamine (METH) are disproportionately elevated among people living with HIV-1. METH use can induce neurotoxic and neurodegenerative consequences, which can increase one's risk and severity of HAND. Thus, a better understanding of HIV-1 infection and METH exposure both alone and in combination on astrocyte function could help identify key cellular or molecular targets that can regulate astrocyte neuroprotective versus neurotoxic phenotypes to optimize astrocyte and neuronal coupling and combat CNS pathology. Direct contact sites between the endoplasmic reticulum (ER) and the mitochondria, termed mitochondria-associated ER membranes (MAMs), are central hubs for regulating several cellular processes, including inflammation and mitochondrial function and dynamics. In fact, the transfer of calcium from the ER to mitochondria is essential for mitochondrial bioenergetics. Interestingly, increasing evidence supports that the three arms of the unfolded protein response (UPR) are key cell signaling messengers within the ER-mitochondrial interface, beyond their classical ER stress functions. Briefly, protein kinase RNA-like endoplasmic reticulum kinase (PERK) has been determined as a regulator for MAM tethering and mitochondrial morphology. Inositol-requiring enzyme 1 alpha (IRE1α) is implicated in regulating MAM-mediated calcium transfer. Activating transcription factor 6 (ATF6) is suspected to participate in MAM formation as it is known to mediate ER elongation and lipid homeostasis. However, these regulatory mechanisms have not yet been fully elucidated. Our studies specifically highlight IRE1α as a key regulator of astrocyte metabolic and inflammatory phenotypes. Using primary human astrocytes infected with pseudotyped HIV and/or exposed to low doses of METH for seven days, astrocytes have increased protein expression of select UPR/MAM mediators. Under the same paradigms, we see increased cytosolic calcium flux and mitochondrial oxygen consumption rate, which were associated with increased mitochondria calcium uptake. Manipulation of IRE1α using both pharmacological inhibitors and an overexpression plasmid, confirms IRE1α modulates astrocyte calcium signaling, metabolic activity, glutamate clearance, and cytokine release. These findings identify a novel target for regulating astrocyte metabolic and inflammatory phenotypes, which could help combat astrocyte-mediated neurotoxicity and potentially promote a neurotrophic phenotype during CNS pathologies.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R3764
Virology. 2022 Apr 29. pii: S0042-6822(22)00071-X. [Epub ahead of print]572 1-16

Key tethering function of Atg11 autophagy scaffold protein in formation of virus-induced membrane contact sites during tombusvirus replication.

Yuanrong Kang, Wenwu Lin, Yuyan Liu, Peter D Nagy.

Positive-strand RNA viruses induce the biogenesis of viral replication organelles (VROs), which support viral replication in infected cells. VRO formation requires viral replication proteins, co-opted host factors and intracellular membranes. Here, we show that the conserved Atg11 autophagy scaffold protein is co-opted by Tomato bushy stunt virus (TBSV) via direct interactions with the viral replication proteins. Deletion of ATG11 in yeast or knockdown of the homologous Atg11 in plants led to reduced tombusvirus replication, thus indicating pro-viral function for Atg11. Based on co-purification, BiFC and proximity-labeling experiments, we find that Atg11 is co-opted to stabilize virus-induced membrane contact sites (vMCS) within VROs. We propose that the tethering and scaffold function of Atg11 is critical in vMCSs for lipid enrichment. Absence of Atg11 interferes with sterols enrichment in VROs, rendering VROs RNAi-sensitive. Altogether, the expanding roles of co-opted host proteins with tethering functions suggest that the tombusvirus VROs are elaborate structures.

DOI: https://doi.org/10.1016/j.virol.2022.04.007
FASEB J. 2022 May;36 Suppl 1

Regulation of PIP2 homeostasis at ER-plasma membrane contacts by Nir proteins.

Jen Liou.

Phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane is a critical lipid molecule that regulates nearly all functional processes occurring at the cell surface. Additionally, PIP2 is hydrolyzed following receptor-induced activation of phospholipase C to generate second messengers for signal transduction. It is crucial to quickly replenish PIP2 in the plasma membrane following receptor-induced hydrolysis to sustain signaling outputs and to maintain PIP2-dependent cellular functions. Replenishment of plasma membrane PIP2 in receptor-stimulated cells involves transport of phosphatidylinositol (PI) from the endoplasmic reticulum (ER) to the plasma membrane for PIP2 re-synthesis. ER-plasma membrane contacts are subcellular loci characterized by the close apposition of the ER to the plasma membrane. Recent findings revealed that the PI transfer proteins, Nir2 and Nir3, dynamically localize at ER-plasma membrane contacts to mediate replenishment of PIP2 hydrolyzed following receptor stimulation. Deficiency in Nir2 and Nir3 suppressed replenishment of PIP2 in the plasma membrane and Ca2+ signaling in receptor-stimulated cells. A third mammalian Nir protein, Nir1, has been linked to several human diseases including retinal dystrophy. Unlike Nir2 and Nir3, Nir1 lacks a PI transfer protein domain and readily localizes at ER-plasma membrane contacts in resting cells. The presence of Nir1 promotes Nir2 localization at ER-plasma membrane contacts to mediate PIP2 replenishment following receptor stimulation. Together, Nir proteins maintain plasma membrane PIP2 homeostasis at ER-plasma membrane contacts to support cell signaling and plasma membrane functions in receptor-stimulated cells.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.0I150
FASEB J. 2022 May;36 Suppl 1

Roles for inter-organelle contacts in organizing metabolism.

W M Henne.

When faced with nutrient shortage, cells adapt by remodeling their metabolic pathways and organelles. I will discuss our work using budding yeast to dissect how nutrient shortage impacts the spatial organization of metabolism and lipid droplets. We find that in response to glucose restriction, yeast remodel their mevalonate pathway by spatially compartmentalizing its rate-limiting enzyme, HMG-CoA Reductase (HMGCR). HMGCR spatial compartmentalization occurs at a unique inter-organelle contact site called the nucleus-vacuole junction (NVJ). This spatial partitioning enhances HMGCR activity, driving mevalonate synthesis to enable cellular adaptation. Our work suggests a new use for an inter-organelle contact site in the fine-tuning of mevalonate metabolism during nutrient stress. Remarkably, we also find that glucose restriction drives the phase transition of lipids within lipid droplets (LDs), causing them to convert from a disordered to liquid-crystalline phase. Mechanistically, we find that these liquid crystalline lattices (LCLs) within LDs require triglyceride lipolysis. We also find that LCL-LDs exhibit changes to the LD surface proteome. Several known LD proteins redistribute from LDs to the ER network, whereas others remain on LCL-LDs, suggesting phase transitions of LD lipids influences LD protein targeting. Global proteomics also reveals that triglycerides harvested from LDs fuel cellular energetics at peroxisomes and mitochondria. This indicates that glucose starvation induces inter-organelle lipid flux while promoting lipid phase transitions within LDs.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.0I151
FASEB J. 2022 May;36 Suppl 1

Novel pathways of intracellular membrane lipid transport and neurodegenerative diseases.

Pietro De Camilli.

Membrane lipids move from one compartment to another within cells as part of the membranes of vesicular carriers. However, this mode of lipid traffic is complemented by the action of lipid transport proteins that often act at membrane contact sites. Typically, lipid transport by these proteins is achieved by lipid binding modules that shuttle back and forth between the participating membranes. However, recently, studies of VPS13 family proteins (which also comprise the autophagy factor ATG2) have revealed a new mechanism of lipid transfer mediated by rod-like proteins that bridge two adjacent bilayers and harbor a hydrophobic groove that runs along their entire length. Most interestingly, loss of function mutations of members of this protein family result in neurodegenerative or neurodevelopmental diseases, including Parkinson's disease (VPS13C) and a Huntington-like syndrome called chorea acanthocytosis (VPS13A). The talk will provide an overview of the known properties and functions of mammalian VPS13 family proteins and of potential roles of their dysfunction in disease (Leonzino M, Reinisch KM and De Camilli P. 2021. Insights into VPS13 properties and function reveal a new mechanism of eukaryotic lipid transport. PMID: 34216812).

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.0I152
Int J Biol Sci. 2022 ;18(7): 2914-2931

GRP75-faciliated Mitochondria-associated ER Membrane (MAM) Integrity controls Cisplatin-resistance in Ovarian Cancer Patients.

Jing Li, Fangzheng Qi, Huishan Su, Chuanshan Zhang, Qing Zhang, Ying Chen, Ping Chen, Linjia Su, Yanan Chen, Yuqi Yang, Zhesheng Chen, Sihe Zhang.

  Background: Control of ER-mitochondrial Ca2+ fluxes is a critical checkpoint to determine cell fate under stress. The 75-kDa glucose-regulated protein (GRP75) is a key tether protein facilitating mitochondria-associated ER membrane (MAM) formation through the IP3R-GRP75-VDAC1 complex. Although GRP75 contributes to cisplatin (CP)-resistance of ovarian cancer (OC), the underlying mechanisms are not clear. Methods: CP-resistant and -sensitive OC cell lines with GRP75 stable modulation were established. Confocal, PLA, co-IP, and TEM analysis were utilized to detect MAM integrity. Live cell Ca2+ imaging, intracellular ATP, ROS, and NAD+ assays were utilized to investigate ER-to-mitochondrial Ca2+ transfer and mitochondrial bioenergetics. Western blot, flow cytometry, CCK-8, Δψm, and mPTP assays were utilized to examine apoptotic cell death. Bioinformatics, patient's specimens, and immunohistochemistry were conducted to obtain the clinical relevance for GRP75-facilitated MAM formation. Results: GRP75-faciliated MAM formation was enriched in CP-resistant OC cells. CP-exposure only increased MAM formation in CP-sensitive OC cells, and enrichment of GRP75 and VDAC1 at MAMs is indispensable to CP-resistance. Diminishing MAM integrity by GRP75-deficiency reduced ER-to-mitochondria Ca2+ transfer, accelerated CP-induced mitochondrial dysfunction, provoked catastrophic ROS, and enhanced CP-triggered apoptotic cell death in OC cells. Clinical investigations confirmed the enrichment of GRP75-faciliated MAM formation in relapsed OC patients, and such enrichment was associated with the CP-resistance phenotype. Conclusion: GRP75-overexpression confers CP-resistance by distinctively managing MAM-facilitated Ca2+ fluxes and the pro-survival ROS signal, whereas GRP75-deficiency induces cell death via bioenergetic crisis and apoptotic ROS accumulation in OC cells. Our results show that GRP75-faciliated MAM formation is a potential target to overcome CP-resistance of OC.

Keywords:  Ca2+ fluxes; cisplatin-resistance; glucose-regulated protein; mitochondria-associated ER membrane; ovarian cancer

DOI:  https://doi.org/10.7150/ijbs.71571
FASEB J. 2022 May;36 Suppl 1

Inhalation Exposure to Airborne PM2.5 Induces Integrated Organelle Stress Response in the Liver.

Kezhong Zhang, Lawrence I Grossman, Paul M Stemmer, Hyunbae Kim, Nicholas Carruthers.

Air pollution is a sustained problem of public health for the general population. Accumulating evidence has confirmed a significant association between exposure to fine ambient particulate matter with aerodynamic diameters < 2.5 μm (PM2.5 ) and the increase of morbidity and mortality associated with cardiovascular and metabolic diseases. Chronic inflammation and dysregulated energy metabolism have been identified as the driving force of the PM2.5 -caused pathogenesis. However, a precise understanding of the molecular and cellular mechanisms by which PM2.5 induces inflammatory stress responses and impairs energy homeostasis remains elusive. Research in our laboratory has addressed the mechanistic basis underlying the pathophysiologic effects of PM2.5 exposure on liver and adipose tissues with mouse models under inhalation exposure to environmentally relevant PM2.5 . Proteomics analysis with PM2.5 -exposed mouse liver tissues indicated that mitochondria and endoplasmic reticulum (ER) are the most sensitive organelles in transducing signaling cascades upon PM2.5 stimulation. Further investigation revealed that inhalation exposure to PM2.5 induces an integrated ER stress and mitochondrial stress response in mouse livers. Under PM2.5 exposure, the ER and mitochondria interact and build up a platform through Mitochondria-Associated Membranes (MAMs) to augment an integrated inflammatory stress response, mediated through the primary ER stress sensor IRE1α and Toll-like receptor (TLR) 2 and 4. This integrated organelle stress response pathway promoted non-alcoholic steatohepatitis (NASH), characterized by hepatic inflammation, steatosis and fibrogenesis, hepatic glycogen depletion, and insulin resistance in PM2.5 -exposed mice. Disruption of MAMs by knockdown of the MAM-residing ER chaperone Sigma-1 Receptor (Sig-1R) or mitofusin (MNF2) in Kupffer cells or hepatocytes suppressed PM2.5- induced NASH and insulin resistance in mice. Together, our studies suggested that exposure to airborne PM2.5 pollution activates an integrated ER and mitochondrial stress response in the liver, which promotes NASH, disrupts energy homeostasis, and impairs insulin signaling associated with the development of metabolic syndrome. These findings provide significant insights into the hepatic pathways underlying PM2.5 -triggered liver pathology, and have important implications in the understanding, prevention, and treatment of air pollution-associated metabolic disease.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.L7647
Ecotoxicol Environ Saf. 2022 May 04. pii: S0147-6513(22)00435-3. [Epub ahead of print]238 113595

Arsenic induces ferroptosis and acute lung injury through mtROS-mediated mitochondria-associated endoplasmic reticulum membrane dysfunction.

Meng-Die Li, Lin Fu, Bian-Bian Lv, Ying Xiang, Hui-Xian Xiang, De-Xiang Xu, Hui Zhao.

  The goal of this study was to analyze whether mitochondria-associated endoplasmic reticulum membrane (MAMs) dysfunction mediated arsenic (As)-evoked pulmonary ferroptosis and acute lung injury (ALI). As exposure led to alveolar structure damage, inflammatory cell infiltration and pulmonary function decline in mice. Ferritin, the marker of iron overload, was increased, GPX4, the index of lipid peroxidation, was decreased in As-exposed lungs and pulmonary epithelial cells (MLE-12). Pretreatment with ferrostatin-1 (Fer-1), the inhibitor of ferroptosis, alleviated As-evoked ALI. In addition, As-induced non-heme iron deposition was inhibited in Fer-1 pretreated-mice. Moreover, As-triggered mitochondria damage and ferroptosis were mitigated in Fer-1 pretreated-MLE-12 cells. Mechanistically, PERK phosphorylation and mitofusin-2 (Mfn-2) reduction was observed in As-exposed MLE-12 cells and mice lungs. Additionally, the interaction between PERK and Mfn-2 was downregulated and MAMs dysfunction was observed in As-exposed MLE-12 cells. Intriguingly, PERK inhibitor and Mfn-2-overexpression all mitigated As-induced ferroptosis in MLE-12 cells. Additionally, CLPP and mtHSP70, the markers of mitochondrial stress, were upregulated, mitochondrial ROS (mtROS) was elevated, mitochondrial membrane potential (MMP) and ATP were decreased in As-exposed MLE-12 cells. Mitoquinone mesylate (MitoQ), a novel mitochondrial-targeted antioxidant, alleviated As-induced excess mtROS, mitochondrial stress, MAMs dysfunction in pulmonary epithelial cells. Similarly, in vivo experiments indicated that MitoQ pretreatment countered As-induced pulmonary ferroptosis and ALI. These data indicated that mtROS-initiated MAMs dysfunction is, at least partially, implicated in As-evoked ferroptosis and ALI.

Keywords:  Acute lung injury; Arsenic; Ferroptosis; Mitofusin-2; MtROS; PERK

DOI:  https://doi.org/10.1016/j.ecoenv.2022.113595