Novel insecticidal proteins from ferns resemble insecticidal proteins from Bacillus thuringiensis.

Lepidopterans affect crop production worldwide. The use of transgenes encoding insecticidal proteins from Bacillus thuringiensis (Bt) in crop plants is a well-established technology that enhances protection against lepidopteran larvae. Concern about widespread field-evolved resistance to Bt proteins has highlighted an urgent need for new insecticidal proteins with different modes or sites of action. We discovered a new family of insecticidal proteins from ferns. The prototype protein from Pteris species (Order Polypodiales) and variants from two other orders of ferns, Schizaeales and Ophioglossales, were effective against important lepidopteran pests of maize and soybean in diet-based assays. Transgenic maize and soybean plants producing these proteins were more resistant to insect damage than controls. We report here the crystal structure of a variant of the prototype protein to 1.98 Å resolution. Remarkably, despite being derived from plants, the structure resembles the 3-domain Cry proteins from Bt but has only two out of three of their characteristic domains, lacking the C-terminal domain which is typically required for their activities. Two of the fern proteins were effective against strains of fall armyworm that were resistant to Bt 3-domain Cry proteins Cry1Fa or Cry2A.127. This therefore represents a novel family of insecticidal proteins that have the potential to provide future tools for pest control.

Structural journey of an insecticidal protein against western corn rootworm.

The broad adoption of transgenic crops has revolutionized agriculture. However, resistance to insecticidal proteins by agricultural pests poses a continuous challenge to maintaining crop productivity and new proteins are urgently needed to replace those utilized for existing transgenic traits. We identified an insecticidal membrane attack complex/perforin (MACPF) protein, Mpf2Ba1, with strong activity against the devastating coleopteran pest western corn rootworm (WCR) and a novel site of action. Using an integrative structural biology approach, we determined monomeric, pre-pore and pore structures, revealing changes between structural states at high resolution. We discovered an assembly inhibition mechanism, a molecular switch that activates pre-pore oligomerization upon gut fluid incubation and solved the highest resolution MACPF pore structure to-date. Our findings demonstrate not only the utility of Mpf2Ba1 in the development of biotechnology solutions for protecting maize from WCR to promote food security, but also uncover previously unknown mechanistic principles of bacterial MACPF assembly.

Quantitative Analysis of Extracellular Vesicle Release Using Artificial MicroRNAs.

Despite the widely used concept of extracellular vesicle (EV)-mediated intercellular communication, we are still far from understanding what is the exact role of such nanosized vesicles in human physiology and disease. Thus, development of new methods and tools that enable the study of fundamental EV biology is valuable for advancing the field. Typically, EV production and release are monitored using approaches that rely on either antibody-based FACS assays or genetically encoded fluorescent proteins. We previously devised artificially barcoded exosomal microRNAs (bEXOmiRs) that were used as high-throughput reporters of EV release. In the first part of this protocol, basic steps and considerations for the design and cloning of bEXOmiRs are explained in detail. Next, analysis of bEXOmiR expression and abundance in cells and isolated EVs is described.

Ca2+ and Annexins - Emerging Players for Sensing and Transferring Cholesterol and Phosphoinositides via Membrane Contact Sites.

Maintaining lipid composition diversity in membranes from different organelles is critical for numerous cellular processes. However, many lipids are synthesized in the endoplasmic reticulum (ER) and require delivery to other organelles. In this scenario, formation of membrane contact sites (MCS) between neighbouring organelles has emerged as a novel non-vesicular lipid transport mechanism. Dissecting the molecular composition of MCS identified phosphoinositides (PIs), cholesterol, scaffolding/tethering proteins as well as Ca2+ and Ca2+-binding proteins contributing to MCS functioning. Compelling evidence now exists for the shuttling of PIs and cholesterol across MCS, affecting their concentrations in distinct membrane domains and diverse roles in membrane trafficking. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the plasma membrane (PM) not only controls endo-/exocytic membrane dynamics but is also critical in autophagy. Cholesterol is highly concentrated at the PM and enriched in recycling endosomes and Golgi membranes. MCS-mediated cholesterol transfer is intensely researched, identifying MCS dysfunction or altered MCS partnerships to correlate with de-regulated cellular cholesterol homeostasis and pathologies. Annexins, a conserved family of Ca2+-dependent phospholipid binding proteins, contribute to tethering and untethering events at MCS. In this chapter, we will discuss how Ca2+ homeostasis and annexins in the endocytic compartment affect the sensing and transfer of cholesterol and PIs across MCS.

IPD072Aa from Pseudomonas chlororaphis Targets Midgut Epithelial Cells in Killing Western Corn Rootworm (Diabrotica virgifera virgifera).

IPD072Aa from Pseudomonas chlororaphis is a new insecticidal protein that has been shown to have high activity against western corn rootworm (WCR). IPD072 has no sequence signatures or predicted structural motifs with any known protein revealing little insight into its mode of action using bioinformatic tools. As many bacterially derived insecticidal proteins are known to act through mechanisms that lead to death of midgut cells, we evaluated whether IPD072Aa also acts by targeting the cells of WCR midgut. IPD072Aa exhibits specific binding to brush border membrane vesicles (BBMVs) prepared from WCR guts. The binding was found to occur at binding sites that are different than those recognized by Cry3A or Cry34Ab1/Cry35Ab1, proteins expressed by current maize traits that target WCR. Using fluorescence confocal microscopy, immuno-detection of IPD072Aa in longitudinal sections from whole WCR larvae that were fed IPD072Aa revealed the association of the protein with the cells that line the gut. High-resolution scanning electron microscopy of similar whole larval sections revealed the disruption of the gut lining resulting from cell death caused by IPD072Aa exposure. These data show that the insecticidal activity of IPD072Aa results from specific targeting and killing of rootworm midgut cells. IMPORTANCE Transgenic traits targeting WCR based on insecticidal proteins from Bacillus thuringiensis have proven effective in protecting maize yield in North America. High adoption has led to WCR populations that are resistant to the trait proteins. Four proteins have been developed into commercial traits, but they represent only two modes of action due to cross-resistance among three. New proteins suited for trait development are needed. IPD072Aa, identified from the bacterium Pseudomonas chlororaphis, was shown to be effective in protecting transgenic maize against WCR. To be useful, IPD072Aa must work through binding to different receptors than those utilized by current traits to reduce risk of cross-resistance and understanding its mechanism of toxicity could aid in countering resistance development. Our results show that IPD072Aa binds to receptors in WCR gut that are different than those utilized by current commercial traits and its targeted killing of midgut cells results in larval death.

Endolysosomal cholesterol export: More than just NPC1.

NPC1 plays a central role in cholesterol egress from endolysosomes, a critical step for maintaining intracellular cholesterol homeostasis. Despite recent advances in the field, the full repertoire of molecules and pathways involved in this process remains unknown. Emerging evidence suggests the existence of NPC1-independent, alternative routes. These may involve vesicular and non-vesicular mechanisms, as well as release of extracellular vesicles. Understanding the underlying molecular mechanisms that bypass NPC1 function could have important implications for the development of therapies for lysosomal storage disorders. Here we discuss how cholesterol may be exported from lysosomes in which NPC1 function is impaired.

A neural network solves, explains, and generates university math problems by program synthesis and few-shot learning at human level.

We demonstrate that a neural network pretrained on text and fine-tuned on code solves mathematics course problems, explains solutions, and generates questions at a human level. We automatically synthesize programs using few-shot learning and OpenAI's Codex transformer and execute them to solve course problems at 81% automatic accuracy. We curate a dataset of questions from Massachusetts Institute of Technology (MIT)'s largest mathematics courses (Single Variable and Multivariable Calculus, Differential Equations, Introduction to Probability and Statistics, Linear Algebra, and Mathematics for Computer Science) and Columbia University's Computational Linear Algebra. We solve questions from a MATH dataset (on Prealgebra, Algebra, Counting and Probability, Intermediate Algebra, Number Theory, and Precalculus), the latest benchmark of advanced mathematics problems designed to assess mathematical reasoning. We randomly sample questions and generate solutions with multiple modalities, including numbers, equations, and plots. The latest GPT-3 language model pretrained on text automatically solves only 18.8% of these university questions using zero-shot learning and 30.8% using few-shot learning and the most recent chain of thought prompting. In contrast, program synthesis with few-shot learning using Codex fine-tuned on code generates programs that automatically solve 81% of these questions. Our approach improves the previous state-of-the-art automatic solution accuracy on the benchmark topics from 8.8 to 81.1%. We perform a survey to evaluate the quality and difficulty of generated questions. This work automatically solves university-level mathematics course questions at a human level and explains and generates university-level mathematics course questions at scale, a milestone for higher education.

RNAi for Western Corn Rootworm Management: Lessons Learned, Challenges, and Future Directions.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is considered one of the most economically important pests of maize (Zea mays L.) in the United States (U.S.) Corn Belt with costs of management and yield losses exceeding USD ~1-2 billion annually. WCR management has proven challenging given the ability of this insect to evolve resistance to multiple management strategies including synthetic insecticides, cultural practices, and plant-incorporated protectants, generating a constant need to develop new management tools. One of the most recent developments is maize expressing double-stranded hairpin RNA structures targeting housekeeping genes, which triggers an RNA interference (RNAi) response and eventually leads to insect death. Following the first description of in planta RNAi in 2007, traits targeting multiple genes have been explored. In June 2017, the U.S. Environmental Protection Agency approved the first in planta RNAi product against insects for commercial use. This product expresses a dsRNA targeting the WCR snf7 gene in combination with Bt proteins (Cry3Bb1 and Cry34Ab1/Cry35Ab1) to improve trait durability and will be introduced for commercial use in 2022.

Annexin A6 and NPC1 regulate LDL-inducible cell migration and distribution of focal adhesions.

Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.

Sorting (Nexin-13) out Novel Insights into Endolysosomal Cholesterol Export.

Transport in and out of the endolysosomal compartment represents a key step in the regulation of cellular cholesterol homeostasis. Despite important recent advances, how LDL-derived, free cholesterol is exported from the lumen of endolysosomes to other organelles is still a matter of debate. We recently devised a CRISPR/Cas9 genome-scale strategy to uncover genes involved in the regulation of endolysosomal cholesterol homeostasis and the functionally linked phospholipid, bis(monoacylglycerol)-phosphate. This approach confirmed known genes and pathways involved in this process, and more importantly revealed previously unrecognized roles for new players, such as Sorting Nexin-13 (SNX13). Here we discuss the unexpected regulatory role of SNX13 in endolysosomal cholesterol export.

CRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export.

We report here two genome-wide CRISPR screens performed to identify genes that, when knocked out, alter levels of lysosomal cholesterol or bis(monoacylglycero)phosphate. In addition, these screens were also performed under conditions of NPC1 inhibition to identify modifiers of NPC1 function in lysosomal cholesterol export. The screens confirm tight coregulation of cholesterol and bis(monoacylglycero)phosphate in cells and reveal an unexpected role for the ER-localized SNX13 protein as a negative regulator of lysosomal cholesterol export and contributor to ER-lysosome membrane contact sites. In the absence of NPC1 function, SNX13 knockdown redistributes lysosomal cholesterol and is accompanied by triacylglycerol-rich lipid droplet accumulation and increased lysosomal bis(monoacylglycero)phosphate. These experiments provide unexpected insight into the regulation of lysosomal lipids and modification of these processes by novel gene products.

The healthier healthcare management models for COVID-19.

The worldwide pandemic situation of COVID-19 generates a situation in which healthcare resources such as diagnostic kits, drugs and basic healthcare infrastructure were on shortage throughout the period, along with negative impact on socio-economic system. Standardized public healthcare models were missing in pandemic situation, covering from hospitalized patient care to local resident's healthcare managements in terms of monitoring, assess to diagnosis and medicines. This exploratory and intervention-based study with the objective of proposing COVID-19 Care Management Model representing comprehensive care of society including patients (COVID-19 and other diseases) and healthy subjects under integrated framework of healthier management model. Shifting policy towards technology-oriented models with well-aligned infrastructure can achieve better outcomes in COVID-19 prevention and care. The planned development of technical healthcare models for prognosis and improved treatment outcomes that take into account not only genomics, proteomics, nanotechnology, materials science perspectives but also the possible contribution of advanced digital technologies is best strategies for early diagnosis and infections control. In view of current pandemic, a Healthier Healthcare Management Model is proposed here as a source of standardized care having technology support, medical consultation, along with public health model of sanitization, distancing and contact less behaviours practices. Effective healthcare managements have been the main driver of healthier society where, positive action at identified research, technology and management segment more specifically public health, patient health, technology selection and political influence has great potential to enhanced the global response to COVID-19. The implementation of such practices will deliver effective diagnosis and control mechanism and make healthier society.

Coordinated binding of a two-component insecticidal protein from Alcaligenes faecalis to western corn rootworm midgut tissue.

AfIP-1A/1B is a two-component insecticidal protein identified from the soil bacterium Alcaligenes faecalis that has high activity against western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). Previous results revealed that AfIP-1A/1B is cross-resistant to the binary protein from Bacillus thuringiensis (Bt), Cry34Ab1/Cry35Ab1 (also known as Gpp34Ab1/Tpp35Ab1; Crickmore et al., 2020), which was attributed to shared binding sites in WCR gut tissue (Yalpani et al., 2017). To better understand the interaction of AfIP-1A/1B with its receptor, we have systematically evaluated the binding of these proteins with WCR brush border membrane vesicles (BBMVs). Our findings show that AfIP-1A binds directly to BBMVs, while AfIP-1B does not; AfIP-1B binding only occurred in the presence of AfIP-1A which was accompanied by the presence of stable, high molecular weight oligomers of AfIP-1B observed on denaturing protein gels. Additionally, we show that AfIP-1A/1B forms pores in artificial lipid membranes. Finally, binding of AfIP-1A/1B was found to be reduced in BBMVs from Cry34Ab1/Cry35Ab1-resistant WCR where Cry34Ab1/Cry35Ab1 binding was also reduced. The reduced binding of both proteins is consistent with recognition of a shared receptor that has been altered in the resistant strain. The coordination of AfIP-1B binding by AfIP-1A, the similar structures between AfIP-1A and Cry34Ab1, along with their shared binding sites and cross-resistance, suggest a similar role for AfIP1A and Cry34Ab1 in receptor recognition and docking site for their cognate partners, AfIP-1B and Cry35Ab1, respectively.

Annexins Bridging the Gap: Novel Roles in Membrane Contact Site Formation.

Membrane contact sites (MCS) are specialized small areas of close apposition between two different organelles that have led researchers to reconsider the dogma of intercellular communication via vesicular trafficking. The latter is now being challenged by the discovery of lipid and ion transfer across MCS connecting adjacent organelles. These findings gave rise to a new concept that implicates cell compartments not to function as individual and isolated entities, but as a dynamic and regulated ensemble facilitating the trafficking of lipids, including cholesterol, and ions. Hence, MCS are now envisaged as metabolic platforms, crucial for cellular homeostasis. In this context, well-known as well as novel proteins were ascribed functions such as tethers, transporters, and scaffolds in MCS, or transient MCS companions with yet unknown functions. Intriguingly, we and others uncovered metabolic alterations in cell-based disease models that perturbed MCS size and numbers between coupled organelles such as endolysosomes, the endoplasmic reticulum, mitochondria, or lipid droplets. On the other hand, overexpression or deficiency of certain proteins in this narrow 10-30 nm membrane contact zone can enable MCS formation to either rescue compromised MCS function, or in certain disease settings trigger undesired metabolite transport. In this "Mini Review" we summarize recent findings regarding a subset of annexins and discuss their multiple roles to regulate MCS dynamics and functioning. Their contribution to novel pathways related to MCS biology will provide new insights relevant for a number of human diseases and offer opportunities to design innovative treatments in the future.

Lack of Annexin A6 Exacerbates Liver Dysfunction and Reduces Lifespan of Niemann-Pick Type C Protein-Deficient Mice.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by cholesterol accumulation caused by loss-of-function mutations in the Npc1 gene. NPC disease primarily affects the brain, causing neuronal damage and affecting motor coordination. In addition, considerable liver malfunction in NPC disease is common. Recently, we found that the depletion of annexin A6 (ANXA6), which is most abundant in the liver and involved in cholesterol transport, ameliorated cholesterol accumulation in Npc1 mutant cells. To evaluate the potential contribution of ANXA6 in the progression of NPC disease, double-knockout mice (Npc1-/-/Anxa6-/-) were generated and examined for lifespan, neurologic and hepatic functions, as well as liver histology and ultrastructure. Interestingly, lack of ANXA6 in NPC1-deficient animals did not prevent the cerebellar degeneration phenotype, but further deteriorated their compromised hepatic functions and reduced their lifespan. Moreover, livers of Npc1-/-/Anxa6-/- mice contained a significantly elevated number of foam cells congesting the sinusoidal space, a feature commonly associated with inflammation. We hypothesize that ANXA6 deficiency in Npc1-/- mice not only does not reverse neurologic and motor dysfunction, but further worsens overall liver function, exacerbating hepatic failure in NPC disease.

Author Correction: Characterization of DvSSJ1 transcripts targeting the smooth septate junction (SSJ) of western corn rootworm (Diabrotica virgifera virgifera).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Functional validation of DvABCB1 as a receptor of Cry3 toxins in western corn rootworm, Diabrotica virgifera virgifera.

Western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is a serious insect pest in the major corn growing areas of North America and in parts of Europe. WCR populations with resistance to Bacillus thuringiensis (Bt) toxins utilized in commercial transgenic traits have been reported, raising concerns over their continued efficacy in WCR management. Understanding the modes of action of Bt toxins is important for WCR control and resistance management. Although different classes of proteins have been identified as Bt receptors for lepidopteran insects, identification of receptors in WCR has been limited with no reports of functional validation. Our results demonstrate that heterologous expression of DvABCB1 in Sf9 and HEK293 cells conferred sensitivity to the cytotoxic effects of Cry3A toxins. The result was further validated using knockdown of DvABCB1 by RNAi which rendered WCR larvae insensitive to a Cry3A toxin. However, silencing of DvABCB2 which is highly homologous to DvABCB1 at the amino acid level, did not reduce the sensitivity of WCR larvae to a Cry3A toxin. Furthermore, our functional studies corroborate different mode-of-actions for other insecticidal proteins including Cry34Ab1/35Ab1, Cry6Aa1, and IPD072Aa against WCR. Finally, reduced expression and alternatively spliced transcripts of DvABCB1 were identified in a mCry3A-resistant strain of WCR. Our results provide the first clear demonstration of a functional receptor in the molecular mechanism of Cry3A toxicity in WCR and confirmed its role in the mechanism of resistance in a mCry3A resistant strain of WCR.

Characterization of DvSSJ1 transcripts targeting the smooth septate junction (SSJ) of western corn rootworm (Diabrotica virgifera virgifera).

Transgenic maize plants expressing dsRNA targeting western corn rootworm (WCR, Diabrotica virgifera virgifera) DvSSJ1 mRNA, a Drosophila snakeskin (ssk) ortholog, show insecticidal activity and significant plant protection from WCR damage. The gene encodes a membrane protein associated with the smooth sepate junction (SSJ) which is required for intestinal barrier function. To understand the active RNA form that leads to the mortality of WCR larvae by DvSSJ1 RNA interference (RNAi), we characterized transgenic plants expressing DvSSJ1 RNA transcripts targeting WCR DvSSJ1 mRNA. The expression of the silencing cassette results in the full-length transcript of 901 nucleotides containing a 210 bp inverted fragment of the DvSSJ1 gene, the formation of a double-stranded RNA (dsRNA) transcript and siRNAs in transgenic plants. Our artificial diet-feeding study indicates that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for DvSSJ1 insecticidal activity. Impact of specificity of dsRNA targeting DvSSJ1 mRNA on insecticidal activities was also evaluated in diet bioassay, which showed a single nucleotide mutation can have a significant impact or abolish diet activities against WCR. These results provide insights as to the functional forms of plant-delivered dsRNA for the protection of transgenic maize from WCR feeding damage and information contributing to the risk assessment of transgenic maize expressing insecticidal dsRNA.

Pleiotropic Roles of Calmodulin in the Regulation of KRas and Rac1 GTPases: Functional Diversity in Health and Disease.

Calmodulin is a ubiquitous signalling protein that controls many biological processes due to its capacity to interact and/or regulate a large number of cellular proteins and pathways, mostly in a Ca2+-dependent manner. This complex interactome of calmodulin can have pleiotropic molecular consequences, which over the years has made it often difficult to clearly define the contribution of calmodulin in the signal output of specific pathways and overall biological response. Most relevant for this review, the ability of calmodulin to influence the spatiotemporal signalling of several small GTPases, in particular KRas and Rac1, can modulate fundamental biological outcomes such as proliferation and migration. First, direct interaction of calmodulin with these GTPases can alter their subcellular localization and activation state, induce post-translational modifications as well as their ability to interact with effectors. Second, through interaction with a set of calmodulin binding proteins (CaMBPs), calmodulin can control the capacity of several guanine nucleotide exchange factors (GEFs) to promote the switch of inactive KRas and Rac1 to an active conformation. Moreover, Rac1 is also an effector of KRas and both proteins are interconnected as highlighted by the requirement for Rac1 activation in KRas-driven tumourigenesis. In this review, we attempt to summarize the multiple layers how calmodulin can regulate KRas and Rac1 GTPases in a variety of cellular events, with biological consequences and potential for therapeutic opportunities in disease settings, such as cancer.

Selective Degradation Permits a Feedback Loop Controlling Annexin A6 and Cholesterol Levels in Endolysosomes of NPC1 Mutant Cells.

We recently identified elevated annexin A6 (AnxA6) protein levels in Niemann-Pick-type C1 (NPC1) mutant cells. In these cells, AnxA6 depletion rescued the cholesterol accumulation associated with NPC1 deficiency. Here, we demonstrate that elevated AnxA6 protein levels in NPC1 mutants or upon pharmacological NPC1 inhibition, using U18666A, were not due to upregulated AnxA6 mRNA expression, but caused by defects in AnxA6 protein degradation. Two KFERQ-motifs are believed to target AnxA6 to lysosomes for chaperone-mediated autophagy (CMA), and we hypothesized that the cholesterol accumulation in endolysosomes (LE/Lys) triggered by the NPC1 inhibition could interfere with the CMA pathway. Therefore, AnxA6 protein amounts and cholesterol levels in the LE/Lys (LE-Chol) compartment were analyzed in NPC1 mutant cells ectopically expressing lysosome-associated membrane protein 2A (Lamp2A), which is well known to induce the CMA pathway. Strikingly, AnxA6 protein amounts were strongly decreased and coincided with significantly reduced LE-Chol levels in NPC1 mutant cells upon Lamp2A overexpression. Therefore, these findings suggest Lamp2A-mediated restoration of CMA in NPC1 mutant cells to lower LE-Chol levels with concomitant lysosomal AnxA6 degradation. Collectively, we propose CMA to permit a feedback loop between AnxA6 and cholesterol levels in LE/Lys, encompassing a novel mechanism for regulating cholesterol homeostasis in NPC1 disease.