Cholesterol is a type of fat that is essential for many processes in the body, such as repairing damaged cells and producing certain hormones. Normally, cholesterol enters cells from the bloodstream and is then moved to the parts of the cell that need it via a process known as ‘trafficking’.

When cholesterol trafficking goes wrong, abnormally large amounts of cholesterol and other fats accumulate within the cell. Over time, these fatty deposits become toxic to cells and eventually damage the affected tissues. Niemann-Pick type C disease (NPC) is a severe genetic disorder affecting cholesterol trafficking. It is characterized by cholesterol build-up in multiple tissues, including the brain, which ultimately causes degeneration and death of nerve cells.

Two proteins, NPC1 and NPC2, are involved in NPC disease. Both proteins normally help move cholesterol out of important trafficking compartments (known as the endosomal and lysosomal compartments) to other areas of the cell where it is needed. Patients with the disease can have mutations in either the gene for NPC1 or the gene for NPC2. This means that cells from NPC1 patients do not make enough functional NPC1 protein (but contain working NPC2), and vice versa.

Previous studies had shown that giving cells with NPC1 mutations large amounts of the small molecule lysobisphosphatidic acid (LBPA for short) could compensate for the loss of NPC1, and stop the toxic build-up of cholesterol. McCauliff, Langan, Li et al. therefore wanted to explore exactly how LBPA was doing this. They had shown that LBPA dramatically increased the ability of purified NPC2 protein to transport cholesterol, and wondered if the effect of LBPA in the cells without NPC1 depended on NPC2. They predicted that boosting LBPA levels would not work in cells lacking NPC2.

Biochemical experiments using purified protein showed that LBPA and NPC2 did indeed interact directly with each other. Systematically changing different building blocks of NPC2 revealed that a single region of the protein is sensitive to LBPA, and when this region was altered, LBPA could no longer interact with NPC2. Since LBPA is naturally produced by cells, they then stimulated cells grown in the laboratory to generate more LBPA using its precursor phosphatidylglycerol. They used cells from patients with mutations in either NPC1 or NPC2 and demonstrated that LBPA’s ability to reverse the accumulation of cholesterol was dependent on its interaction with NPC2. Thus, increasing LBPA levels in cells from patients with NPC1 mutations was beneficial, but had no effect on cells from patients with NPC2 mutations.

These results shed new light not only on how cells transport cholesterol, but also on potential methods to combat disorders of cellular cholesterol trafficking. In the future, LBPA could be developed as a genetically tailored, patient-specific therapy for diseases like NPC.

Cholesterol is a small, hydrophobic molecule that is a vital building block of cell membranes and a precursor for steroid hormones, bile salts, vitamin D, and oxysterol ligands for transcription factors. Intracellular transport of cholesterol is a highly regulated but, as yet, incompletely understood process. Perturbations can lead to detrimental outcomes such as in the lysosomal storage disorder Niemann Pick Type C (NPC) disease, where LDL-derived cholesterol becomes trapped within the late endosomal/lysosomal (LE/LY) system. The sterol particularly enriches LE/LY inner membranes which develop during endosome maturation as a means of compartmentalizing its contents (Gruenberg, 2001; Gruenberg, 2003; Matsuo et al., 2004). The accumulation of cholesterol in NPC disease is associated with amassing of other lipids in the LE/LY, disruption of post-lysosomal cholesterol metabolism, and ultimately clinical manifestations including organomegaly and neurological deterioration. In 95% of NPC cases, mutations in the large LE/LY transmembrane protein, NPC1, prevent proper export of cholesterol from the LE/LY to other cellular compartments. The remaining 5% of cases are caused by mutations in the small, 132-amino acid, soluble LE/LY protein, NPC2 (Peake and Vance, 2010; Vanier, 2010).

Similarities in the cellular and clinical phenotypes resulting from either NPC1 or NPC2 deficiency led to the suggestion that these two proteins function cooperatively in normal LE/LY cholesterol trafficking (Kwon et al., 2009; Sleat et al., 2004); a proposed model shows cholesterol directly transferred from NPC2, in the LE/LY lumen, to NPC1, located in the limiting membrane of the compartment (Estiu et al., 2013; Wang et al., 2010). Recent tertiary structural analyses identifying a potential NPC2 interacting domain on the NPC1 protein support this mode of cholesterol egress from the LE/LY compartment (Zhao et al., 2016). It is further proposed that cholesterol transfer from NPC2 to the luminally localized N-terminal domain of NPC1 allows sterol passage through the glycocalyx found at the luminal surface of the LE/LY limiting membrane, via concerted effort from membrane glycoproteins (Li et al., 2016).

NPC2 binds cholesterol with a 1:1 stoichiometry (Xu et al., 2007). Ko et al. (2003) showed that cholesterol binding was necessary but not sufficient for its cholesterol efflux function, and we later demonstrated that cholesterol transport was the second critical component of NPC2 functionality (Cheruku et al., 2006; Xu et al., 2008). We further showed that cholesterol transfer by NPC2 occurs via protein-membrane interactions (Cheruku et al., 2006; Xu et al., 2008). Using model membranes, we demonstrated a remarkable, order of magnitude stimulation in wild type NPC2 cholesterol transfer rates by the incorporation of lysobisphosphatidic acid (LBPA), also known as bis-monoacylglycerol phosphate (Cheruku et al., 2006; Xu et al., 2008). LBPA is a structural isomer of phosphatidylglyerol with an atypical phospholipid stereoconfiguration. It is localized primarily to inner LE/LY membranes and is thought to be involved not only in the formation of these internal membranes and their architecture, but also in the sorting and efflux of LE/LY components, including cholesterol (Gruenberg, 2003; Hullin-Matsuda et al., 2007; Kobayashi et al., 1998; Kobayashi et al., 1999; Möbius et al., 2003). Incubation of BHK cells and macrophages with an anti-LBPA monoclonal antibody results in cholesterol accumulation in the LE/LY compartment, resembling the NPC phenotype (Delton-Vandenbroucke et al., 2007; Kobayashi et al., 1999).

Interestingly, Chevallier et al. (2008) showed that by enriching NPC1-deficient cells with exogenously added LBPA, the cholesterol accumulation was reversed ; the underlying molecular mechanism for LBPA stimulated LE/LY cholesterol egress remains unknown. Based on the dramatic effects of LBPA on NPC2-mediated cholesterol transfer, we hypothesized that the mechanism of LBPA action involves its specific functional interaction with NPC2, such that LBPA enrichment of cells deficient in NPC2 would not reverse cholesterol accumulation, in contrast to what had been found in NPC1-deficient cells.

In the present studies we demonstrate that, indeed, LBPA enrichment does not lead to the clearance of cholesterol in NPC2-deficient cells despite the presence of functional NPC1. We show for the first time that the mechanism involves an obligate direct interaction of NPC2 with LBPA, identify the LBPA-sensitive domain on the NPC2 surface, and establish the essential functional nature of NPC2-LBPA interactions in cholesterol egress from the LE/LY compartment. The results identify a novel, heretofore unknown step in LE/LY cholesterol egress which is dependent upon LBPA interaction with the hydrophobic knob of NPC2 protein. This in turn suggests that LBPA enrichment may be used to effect cholesterol egress in cells with defective NPC1 but functional WT NPC2, and NPC2 with disease-causing mutations outside the hydrophobic knob domain.

LBPA was proposed to be involved in intracellular cholesterol trafficking based on the sterol accumulation which accompanies incubation of cells with an anti-LBPA antibody (Kobayashi et al., 1999), however the mechanism of LBPA action has remained unknown. In this study we have, for the first time, shown that LBPA function in cholesterol trafficking is obligately dependent upon its interaction with the NPC2 protein within the endo-lysosomal system. At the molecular level, we show that this novel step in intracellular cholesterol trafficking involves direct interaction of LBPA with the hydrophobic knob domain on NPC2. This surface domain is located near the cholesterol binding pocket, thus its insertion into the bilayer would position the protein to efficiently exchange cholesterol with the membrane. Recent molecular dynamic simulations show that LBPA, but not other phospholipids, may position NPC2 in an orientation that could promote protein-membrane sterol exchange (Enkavi et al., 2017), potentially inducing a conformational change in the binding pocket of NPC2 such that the off-rate of cholesterol is markedly increased, allowing for rapid transfer of cholesterol between protein and LBPA-enriched membranes.

A decade ago, the Gruenberg laboratory reported that viral-mediated supplementation of NPC1-deficient cells with exogenous LBPA reversed cholesterol accumulation in the diseased cells (Chevallier et al., 2008). Here we show that LBPA enrichment is, conversely, completely ineffective in cells expressing NPC1 but lacking NPC2, underscoring the required functional interaction of LBPA with the NPC2 protein. In our efforts to establish the molecular basis of this interaction, we found that several NPC2 mutants that could not reverse cholesterol accumulation in unsupplemented NPC2-deficient cells, became effective if the cells were first enriched with LBPA. The NPC2 mutations which were ‘rescued’ by LBPA enrichment are all localized outside of the hydrophobic knob domain. By contrast, mutations within the hydrophobic knob were insensitive to LBPA enrichment apart from H56A, which may be on the limiting edge of this region. Thus, LBPA is able to normalize deficient transfer by mutant NPC2 as long as the mutation is outside of the hydrophobic knob. Interestingly, direct interaction between NPC2 and NPC1, residing in the limiting LE/LY membrane, has also been identified as involving the hydrophobic knob region on NPC2 (Li et al., 2016; Wang et al., 2010). This highlights further the critical nature of this domain in intracellular cholesterol trafficking, such that the NPC2 hydrophobic knob is involved in both LBPA-dependent binding of cholesterol by NPC2 at inner-LE/LY membranes, and the delivery of cholesterol by NPC2 to the NPC1 N-terminal domain. Primary amino acid sequence comparisons reveal a high degree of conservation within the NPC2 hydrophobic knob domain for all species with the exception of yeast; interestingly, yeast lacks the LBPA phospholipid, indirectly supporting a functional interaction between the mammalian NPC2 hydrophobic knob and LBPA. Thus, we propose that the hydrophobic knob on NPC2 interacts with LBPA in the inner LE/LY membranes, leading to cholesterol extraction, with NPC2 then subsequently involved in the ‘handoff’ of the sterol to NPC1 in the limiting membrane of these organelles (Deffieu and Pfeffer, 2011; Infante et al., 2008; Li et al., 2016; Wang et al., 2010).

In healthy cells, the enrichment of inner LE/LY membranes with LBPA and concurrent decline in cholesterol content may be reflective of the efficient role LBPA plays in normal LE/LY cholesterol efflux. If direct interactions between LBPA and NPC2 are integral to this mechanism of efflux, a rate determining step may be their frequency of interaction and, thus, be partially dependent upon the concentration of both phospholipid and protein in the LE/LY compartment. While the concentration of LE/LY LBPA has been shown to increase in parallel with cholesterol and other lipids in NPC disease (Davidson et al., 2009; Sleat et al., 2004; Vanier, 1983), as also found here, it is possible that LBPA levels nevertheless remain too low, as previously suggested (Chevallier et al., 2008), to provide support for NPC2-mediated transport of the elevated cholesterol load. Our results using PG supplementation to augment LBPA levels further indicate that LBPA may become limiting in NPC1 disease. Importantly, we show here that LBPA enrichment cannot reverse cholesterol accumulation caused by NPC2 deficiency unless the mutation is outside the hydrophobic knob domain in which case PG supplementation is effective, as it is in NPC1-deficient cells. Interestingly, Chen et al. observed a pronounced association of the NPC2 protein with inner LE/LY membranes in cells of Npc1^-/- mice (Balb/c Npc1^nih), relative to WT (Chen et al., 2005), suggesting that increased recruitment of NPC2 to inner LE/LY membranes in NPC1 disease, where LBPA concentration is high, may be a compensatory mechanism to alleviate sterol accumulation.

Beyond its obligate interaction with NPC2 within the LE/LY, it is not yet clear how increased LBPA leads to the efflux of LE/LY cholesterol in the absence of NPC1 protein, although in recent studies we have found that increased concentrations of cellular LBPA increase macroautophagy in NPC1 deficient cells (data not shown). It is well known that impaired autophagy is present in NPC disease (Liao et al., 2007; Lieberman et al., 2012; Seranova et al., 2017), and stabilization of this pathway has been shown to reduce cholesterol accumulation in NPC1 disease (Dai et al., 2017; Ordonez et al., 2012; Sarkar et al., 2013). LBPA also displays a variety of other unique functions within the endo/lysosomal system that could potentially promote cholesterol efflux. Studies have demonstrated, for instance, that its transient interaction with the ESCRT-associated protein, ALIX, is required for the proper formation of multivesicular structures within the LE/LY (Matsuo et al., 2004), where cholesterol is localized (Fivaz, 2002); it is also found, along with cholesterol, on exosomes derived from fusion of inner lamellae with the limiting LE/LY membrane (Matsuo et al., 2004). Furthermore, it has recently been proposed that LBPA may influence cholesterol homeostasis beyond the confines of late endosomes and lysosomes, having been shown to be necessary for lipid droplet formation via Wnt signaling within the endoplasmic reticulum, where cholesteryl esters are synthesized (Scott et al., 2015). Within the endo/lysosomal system, however, our results demonstrating that cellular enrichment of LBPA cannot reverse cholesterol accumulation in cells lacking functional NPC2 protein indicate that the cholesterol efflux mechanism utilized by LBPA is dependent upon its interaction with NPC2.

LBPA is reported to comprise only 1% of total cellular phospholipids, but about 15 mol% of total phospholipids in the LE/LY (Chevallier et al., 2000; Kobayashi et al., 1998; Kobayashi et al., 2002). There is no agreement regarding which isomeric form of LBPA is predominant in cells, as the fatty acids can be linked at the sn-2 or sn-3 positions of each glycerol in the S or R conformation, though the sn-2, sn-2’ positions are currently favored (Chevallier et al., 2000; Kobayashi et al., 1998; Kobayashi et al., 2002; Mason et al., 1972; Matsuo et al., 2004). The 2,2’-LBPA was shown to be quite effective at mobilizing cholesterol in NPC1 disease cells while the 3,3’- and semi-LBPA isoforms were unable to promote sterol efflux. No difference in efficacy between S,S, S,R, and R,R LBPA isomers were noted, however, suggesting that the conformation of LBPA has no bearing on its ability to reverse cholesterol accumulation in NPC1 deficient cells (Chevallier et al., 2008). In agreement with this observation, we previously showed that the S,S, S,R, and R,R configurations of LBPA had little to no effect on in vitro cholesterol transfer rates by NPC2 protein (Xu et al., 2008). Similarly, in the present studies we observed little variation in NPC2 binding to these different LBPA stereoisomers. Regardless, we chose to use the presumed precursor and structural isomer of LBPA, phosphatidylglycerol, to increase the cellular LBPA content in these studies, obviating any potential concern about the LBPA isoform as the cells presumably generate the physiologically accurate form. PG is thought to convert to LBPA along the endo/lysosomal system (Hullin-Matsuda et al., 2007; Poorthuis and Hostetler, 1978), and studies have demonstrated that exogenously administered PG can be converted to LBPA in vivo (Somerharju and Renkonen, 1980), although the anabolic pathway of this conversion remains unknown. The present demonstration that PG supplementation specifically increases the LBPA content of all cells tested supports the hypothesis that PG is a precursor to LBPA. Cellular conversion of PG to LBPA has also been demonstrated in mammalian alveolar macrophages (Waite et al., 1987), lymphoblasts (Hullin-Matsuda et al., 2007) and RAW macrophages (Bouvier et al., 2009).

In addition to variations in stereochemistry, the cellular fatty acyl chain components of LBPA have been found to vary (Bouvier et al., 2009), with oleic acid and docosahexaenoic acid (DHA) reported to be selectively incorporated (Besson et al., 2006; Luquain et al., 2001). Here we found acyl chain-dependent differences in NPC2-LBPA interactions, with reduced binding to the 14-carbon saturated dimyristoyl-LBPA species relative to the 18-carbon monounsaturated dioleoyl-LBPA species. In prior work we showed that cholesterol transfer from NPC2 to membranes containing dioleoyl-LBPA was >2 fold faster than transfer to vesicles with dimyristoyl-LBPA (Xu et al., 2008). Taken together, the results suggest that the acyl composition but not the steroconfiguration of LBPA may be important in normal LE/LY cholesterol efflux. Ongoing studies focused on identifying the specific acyl-chain species that are most effective at stimulating LE/LY cholesterol clearance in an NPC phenotype, for instance, should inform both our understanding of intracellular cholesterol transport and the development of therapies for lysosomal storage disorders.

Based on the present results, we propose that the NPC2 hydrophobic knob domain inserts into LBPA enriched inner LE/LY membranes, interacting directly with the phospholipid. Given the ability of NPC2 to promote membrane-membrane interactions (Abdul-Hammed et al., 2010; McCauliff et al., 2011; McCauliff et al., 2015), we speculate that this interaction between LBPA and NPC2 is involved in the formation of membrane contact sites which could potentially exist between closely apposed inner LE/LY membranes and facilitate rapid transfer of sterol. Membrane contact sites have been shown to be important in intermembrane lipid transfer (Helle et al., 2013; Holthuis and Levine, 2005; Prinz, 2014), although none have yet been specifically described within the multivesicular LE/LY. Our results show that NPC2 promotes membrane-membrane interaction, and further indicate the ability of NPC2 to bind to LBPA; this interaction may represent one membrane contact point on inner LE/LY membranes. Our previous kinetic studies suggested that NPC2 interacts with other membrane phospholipids as well, and preliminary molecular dynamics simulations indicate that unlike LBPA, where NPC2 interacts at the hydrophobic knob, these interactions occur at NPC2 surface sites other than the knob domain (data not shown); such interactions could represent a second contact point within the inner LE/LY membranes. Interestingly, it has now been demonstrated that NPC2 also binds directly to NPC1 which resides in the limiting LE/LY membrane, and possibly also with LAMP proteins in these same membranes (Li et al., 2016). These could also be potential tether points for the NPC2 and would effectively bring inner LE/LY cholesterol-laden membranes in closer proximity to the limiting LE/LY membrane, which cholesterol must ultimately cross to exit the compartment.

The key finding of the present studies is that the primary mechanism by which LBPA stimulates LE/LY cholesterol efflux is critically dependent upon its interaction with functional NPC2. LBPA first appears on the internal vesicles and membranes of cholesterol rich multivesicular bodies characteristic of late endosomes (Gruenberg, 2003; Möbius et al., 2003) and increases in concentration through the pathway to cholesterol depleted lysosomes (Möbius et al., 2003). This localization overlaps with NPC2, targeted to late endosomes/lysosomes via the mannose-6-phosphate receptor (Naureckiene et al., 2000). While NPC1 is also targeted to late endosomes (Garver et al., 2000; Higgins et al., 1999; Neufeld et al., 1999), Blanchette-Mackie and colleagues observed that most NPC1 localizes to a specific set of LAMP2 positive, mannose 6-phosphate receptor negative vesicles that are distinct from cholesterol enriched LAMP2 positive lysosomes, where LBPA and NPC2 reside, suggesting that transient interactions between NPC1 positive organelles and cholesterol rich lysosomes occur to effect normal cholesterol egress via NPC2-NPC1 interaction (Neufeld et al., 1999). While several groups have reported NPC1-independent egress of cholesterol from the LE/LY (Boadu et al., 2012; Goldman and Krise, 2010; Kennedy et al., 2012), it is not clear whether these function in the normal situation, or whether they are manifested secondary to NPC1 dysfunction.

Under normal conditions it is likely that the critical functional interaction between NPC2 and LBPA, demonstrated in the present studies, is followed by an interaction between NPC2 and NPC1 at the limiting lysosomal membrane, allowing for cholesterol to egress from the endolysosomal system. However, our studies have also implied that in the presence of intact NPC2, bypassing dysfunctional NPC1 may be achieved via PG-mediated or direct LBPA enrichment. LBPA enrichment may also be effective in NPC2 cases where the mutation is in a residue outside of the hydrophobic knob. For example, a human mutation in D72 has been reported to be disease causing (Biesecker et al., 2009), and we found here that PG supplementation/LBPA enrichment allowed NPC2 deficient cells to be effectively cleared by the D72A protein; supplementation with D72A-NPC2 was entirely ineffective prior to LBPA enrichment. In a recent high-throughput screen for drugs that raise LBPA levels, (Moreau et al., 2019) showed that a compound which increased LBPA levels cleared cholesterol from an NPC2 patient cell line; the mutated residue in this instance, C93, lies outside the hydrophobic knob. Thus, the in vitro evidence strongly suggests that LBPA enrichment can affectively ameliorate cellular cholesterol accumulation in the majority of NPC disease cases, including potentially all NPC1 mutations as well as NPC2 mutations outside the hydrophobic knob.

Currently there is no cure for NPC disease. Pharmacological options are limited, and palliative care remains the standard for treatment of the disease, focusing on increasing the length and quality of life for affected patients. Based on the existing in vitro evidence, we propose that cellular LBPA enrichment is worth exploring as a possible therapy. Aerosolized phospholipids such as dipalmitoyl phosphatidylcholine are widely used to enhance pulmonary drug delivery (Duret et al., 2014), and can be adequately nebulized without losing compositional integrity (Schreier et al., 1994). PG itself, administered intranasally, has been used as a therapy by the Voelker group to effectively inhibit respiratory syncytial virus infection (Numata et al., 2010; Numata et al., 2013) and influenza A virus (Numata et al., 2012). Moreover, lipid based colloidal carriers are able to cross the blood brain barrier (BBB) when administered intranasally (Ganesan et al., 2018; Mittal et al., 2014; Patel and Patel, 2017; Tapeinos et al., 2017) and have recently been shown to be efficient drug delivery vehicles in the treatment of intrinsic brain tumors (van Woensel et al., 2013) and neurodegenerative diseases including Alzheimer’s (Agrawal et al., 2018; Tapeinos et al., 2017) and Parkinson’s (Tapeinos et al., 2017; Yang et al., 2016). Given the historical difficulties in treating the neurological effects of NPC disease, the development of a minimally invasive, effective treatment with intrinsic ability to cross the BBB is of interest.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Leslie A McCauliff

   1. Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
   2. Rutgers Center for Lipid Research, Rutgers University, New Brunswick, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Annette Langan and Ran Li

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5744-2737

2. Annette Langan

   1. Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
   2. Rutgers Center for Lipid Research, Rutgers University, New Brunswick, United States

   Contribution

   Formal analysis, Investigation, Visualization, Writing—original draft, Writing—review and editing

   Contributed equally with

   Leslie A McCauliff and Ran Li

   Competing interests

   No competing interests declared

3. Ran Li

   1. Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
   2. Rutgers Center for Lipid Research, Rutgers University, New Brunswick, United States

   Contribution

   Formal analysis, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Leslie A McCauliff and Annette Langan

   Competing interests

   No competing interests declared

4. Olga Ilnytska

   1. Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
   2. Rutgers Center for Lipid Research, Rutgers University, New Brunswick, United States

   Contribution

   Investigation, Visualization, Methodology, Writing—review and editing, Funding acquisition

   Competing interests

   No competing interests declared

5. Debosreeta Bose

   1. Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
   2. Rutgers Center for Lipid Research, Rutgers University, New Brunswick, United States

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

6. Miriam Waghalter

   Department of Nutritional Sciences, Rutgers University, New Brunswick, United States

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

7. Kimberly Lai

   Department of Nutritional Sciences, Rutgers University, New Brunswick, United States

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

8. Peter C Kahn

   Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, United States

   Contribution

   Supervision, Writing—review and editing

   Competing interests

   No competing interests declared

9. Judith Storch

   1. Department of Nutritional Sciences, Rutgers University, New Brunswick, United States
   2. Rutgers Center for Lipid Research, Rutgers University, New Brunswick, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   storch@sebs.rutgers.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5482-1777

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