Association of general anxiety and pediatric dental preventive examination utilization, National Survey of Children's Health, 2021.

INTRODUCTION: Many factors influence preventive dental health service access for children. The objective of this research was to examine one factor, general anxiety, in accessing at least one preventive dental examination in the past 12 months in children with special healthcare needs (CSHCN) and children without special healthcare needs (CWSHCN). METHODS: National Survey of Children's Health (NSCH) 2021 were obtained for this cross-sectional research. Chi-square and logistic regression analyses were used to determine association of anxiety and past 12-month preventive dental examinations. RESULTS: The sample included 10 493 CSHCN, and 35 675 CWSHCN. Overall, 72.7% had past 12-month preventive dental examinations, and 9.9% had a healthcare provider indicate they had general anxiety. CSHCN with anxiety, CWSHCN with anxiety, and CSHCN without anxiety were more likely to have a past 12-month preventive dental examination visit than CWSHCN without anxiety (Adjusted Odds Ratios: 1.86, 1.39, 1.32, respectively). CONCLUSION: Our results suggest children with general anxiety (both CSHCN and CWSHCN) are more likely to have had at least one regular preventive dental visit within the past 12 months than CWSHCN and without general anxiety. There is a need for further understanding the relationship of general anxiety and dental health to improve the health of all children. PRACTICAL IMPLICATIONS: CWSHCN without anxiety need individualized, comprehensive care with enough time, attention, instruction, and rewards to demonstrate to parents/guardians the importance of making routine preventive dental examinations a priority for their child.

Slings and arrows: sRNAs mediate intragenomic competition.

Bacterial genomes are littered with exogenous: competing DNA elements. Here, Sprenger et al. demonstrate that the Vibrio cholerae prophage VP882 modulates host functions via production of regulatory sRNAs to promote phage development. Alternatively, host sRNAs inhibit the VP882 lytic phase by specifically regulating phage genes.

Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases 2023.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade increasing evidence from preclinical models suggests that cells, which are not normally resident in the lung can be utilized to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathologic remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "-omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

A theory of oral healthcare decision-making in Appalachia.

INTRODUCTION: People make oral healthcare decisions regardless of having partial information, misinformation, sources that deliberately mislead, or information that is culturally influenced. This is particularly true in the Appalachian culture where oral healthcare decision-making practices are not well understood by researchers and dental professionals. Despite efforts to improve dental care utilization, the Appalachia region remains low in oral healthcare utilization. There is a need for a theory to identify concepts in decision-making when seeking oral healthcare. The theory could be useful in creating oral health interventions. The study objective is to develop a theory to identify concepts that influence oral healthcare decision-making in Appalachia (OHDA). METHODS: The researchers used a grounded theory qualitative study design to explain data for a theory of OHDA. Participants from Appalachia, in 20-minute interviews, provided insights into concepts that influence OHDA from August 22, 2017 to May 26, 2022. Notes/memos were written during and after the interviews and coding was conducted after the interviews. Open coding categories emerged through constant comparison of responses. RESULTS: Five overarching concepts that embody OHDA were discovered: Affect (Level of Pain/Emotion/Stress involvement), Awareness, Trust/belief, Resources, and Risk Perception. All participants discussed the impact of social media toward these concepts. CONCLUSION: To influence a person's OHDA, public health officials and researchers need to address the person's affect, level of awareness, trust/belief, available resources, and risk perception. Social media is very important in awareness concerning oral health information. These factors are important to consider for similar research in oral healthcare utilization at the population level.

A Vibrio cholerae Type IV restriction system targets glucosylated 5-hydroxyl methyl cytosine to protect against phage infection.

A major challenge faced by Vibrio cholerae is constant predation by bacteriophage (phage) in aquatic reservoirs and during infection of human hosts. To overcome phage predation, V. cholerae has evolved a myriad of phage defense systems. Although several novel defense systems have been discovered, we hypothesized more were encoded in V. cholerae given the relative paucity of phage that have been isolated which infect this species. Using a V. cholerae genomic library, we identified a Type IV restriction system consisting of two genes within a 16kB region of the Vibrio pathogenicity island-2 that we name TgvA and TgvB (Type I-embedded gmrSD-like system of VPI-2). We show that both TgvA and TgvB are required for defense against T2, T4, and T6 by targeting glucosylated 5-hydroxymethylcytosine (5hmC). T2 or T4 phages that lose the glucose modification are resistant to TgvAB defense but exhibit a significant evolutionary tradeoff becoming susceptible to other Type IV restriction systems that target unglucosylated 5hmC. We show that additional phage defense genes are encoded in VPI-2 that protect against other phage like T3, secΦ18, secΦ27 and λ. Our study uncovers a novel Type IV restriction system in V. cholerae, increasing our understanding of the evolution and ecology of V. cholerae while highlighting the evolutionary interplay between restriction systems and phage genome modification.

Plasmid-free cheater cells commonly evolve during laboratory growth.

It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, extracellular beta-lactamases produced by resistant cells that subsequently degrade penicillin and related antibiotics allow neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show in multiple bacterial species that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface-grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss was still observed. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated.IMPORTANCEPlasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid-containing cell is grown in the presence of antibiotic. Here, we find that growth of plasmid-containing bacteria on a surface and to a lesser extent in liquid culture in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation.

Beta-defensin index: A functional biomarker for oral cancer detection.

There is an unmet clinical need for a non-invasive and cost-effective test for oral squamous cell carcinoma (OSCC) that informs clinicians when a biopsy is warranted. Human beta-defensin 3 (hBD-3), an epithelial cell-derived anti-microbial peptide, is pro-tumorigenic and overexpressed in early-stage OSCC compared to hBD-2. We validate this expression dichotomy in carcinoma in situ and OSCC lesions using immunofluorescence microscopy and flow cytometry. The proportion of hBD-3/hBD-2 levels in non-invasively collected lesional cells compared to contralateral normal cells, obtained by ELISA, generates the beta-defensin index (BDI). Proof-of-principle and blinded discovery studies demonstrate that BDI discriminates OSCC from benign lesions. A multi-center validation study shows sensitivity and specificity values of 98.2% (95% confidence interval [CI] 90.3-99.9) and 82.6% (95% CI 68.6-92.2), respectively. A proof-of-principle study shows that BDI is adaptable to a point-of-care assay using microfluidics. We propose that BDI may fulfill a major unmet need in low-socioeconomic countries where pathology services are lacking.

pGpG-signaling regulates virulence and global transcriptomic targets in Erwinia amylovora.

Cyclic-di-GMP (c-di-GMP) is a critical bacterial second messenger that enables the physiological phase transition in Erwinia amylovora, the phytopathogenic bacterium that causes fire blight disease. C-di-GMP generation is dependent on diguanylate cyclase enzymes while the degradation of c-di-GMP can occur through the action of phosphodiesterase (PDE) enzymes that contain an active EAL and/or a HD-GYP domain. The HD-GYP-type PDEs, which are absent in E. amylovora, can directly degrade c-di-GMP into two GMP molecules. PDEs that contain an active EAL domain, as found in all active PDEs in E. amylovora, degrade c-di-GMP into pGpG. The signaling function of pGpG is not fully understood in bacterial systems. A transcriptomic approach revealed that elevated levels of pGpG in E. amylovora impacted several genes involved in metabolic and regulatory functions including several type III secretion and extracellular appendage related genes. The heterologous overexpression of an EAL or HD-GYP-type PDE in different background E. amylovora strains with varying c-di-GMP levels revealed that in contrast to the generation of pGpG, the direct breakdown of c-di-GMP into GMP by the HD-GYP-type PDE led to an elevation in amylovoran production and biofilm formation despite a decrease in c-di-GMP levels. The breakdown of c-di-GMP into pGpG (as opposed to GTP) also led to a decrease in virulence in apple shoots. The expression of hrpS was significantly increased in response to the breakdown of c-di-GMP into pGpG. Further, our model suggests that a balance in the intracellular ratio of pGpG and c-di-GMP is essential for biofilm regulation in E. amylovora.

Two-pore potassium channel TREK-1 (K2P2.1) regulates NLRP3 inflammasome activity in macrophages.

Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages (AMs) and bone marrow-derived macrophages (BMDMs) from wild-type (wt) and TREK-1-/- mice, we measured responses to inflammasome priming [using lipopolysaccharide (LPS)] and activation (LPS + ATP). We measured IL-1ß, caspase-1, and NLRP3 via ELISA and Western blot. A membrane-permeable potassium indicator was used to measure potassium efflux during ATP exposure, and a fluorescence-based assay was used to assess changes in membrane potential. Inflammasome activation induced by LPS + ATP increased IL-1ß secretion in wt AMs, whereas activation was significantly reduced in TREK-1-/- AMs. Priming of BMDMs using LPS was not affected by either genetic deficiency or pharmacological inhibition of TREK-1 with Spadin. Cleavage of caspase-1 following LPS + ATP treatment was significantly reduced in TREK-1-/- BMDMs. The intracellular potassium concentration in LPS-primed wt BMDMs was significantly lower compared with TREK-1-/- BMDMs or wt BMDMs treated with Spadin. Conversely, activation of TREK-1 with BL1249 caused a decrease in intracellular potassium in wt BMDMs. Treatment of LPS-primed BMDMs with ATP caused a rapid reduction in intracellular potassium levels, with the largest change observed in TREK-1-/- BMDMs. Intracellular K+ changes were associated with changes in the plasma membrane potential (Em), as evidenced by a more depolarized Em in TREK-1-/- BMDMs compared with wt, and Em hyperpolarization upon TREK-1 channel opening with BL1249. These results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.NEW & NOTEWORTHY Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages and bone marrow-derived macrophages from wild-type and TREK-1-/- mice, we measured responses to inflammasome priming (using LPS) and activation (LPS + ATP). Our results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.

Deployment of a Vibrio cholerae ordered transposon mutant library in a quorum-competent genetic background.

Vibrio cholerae, the causative agent of cholera, has sparked seven pandemics in recent centuries, with the current one being the most prolonged. V. cholerae's pathogenesis hinges on its ability to switch between low and high cell density gene regulatory states, enabling transmission between host and the environment. Previously, a transposon mutant library for V. cholerae was created to support investigations aimed toward uncovering the genetic determinants of its pathogenesis. However, subsequent sequencing uncovered a mutation in the gene luxO of the parent strain, rendering mutants unable to exhibit high cell density behaviors. In this study, we used chitin-independent natural transformation to move transposon insertions from these low cell density mutants into a wildtype genomic background. Library transfer was aided by a novel gDNA extraction we developed using thymol, which also showed high lysis-specificity for Vibrio. The resulting Grant Library comprises 3,102 unique transposon mutants, covering 79.8% of V. cholerae's open reading frames. Whole genome sequencing of randomly selected mutants demonstrates 100% precision in transposon transfer to cognate genomic positions of the recipient strain. Notably, in no instance did the luxO mutation transfer into the wildtype background. Our research uncovered density-dependent epistasis in growth on inosine, an immunomodulatory metabolite secreted by gut bacteria that is implicated in enhancing gut barrier functions. Additionally, Grant Library mutants retain the plasmid that enables rapid, scarless genomic editing. In summary, the Grant Library reintroduces organismal relevant genetic contexts absent in the low cell density locked library equivalent.

Deficiency of Acute-Phase Serum Amyloid A Exacerbates Sepsis-Induced Mortality and Lung Injury in Mice.

Serum amyloid A (SAA) is a family of proteins, the plasma levels of which may increase >1000-fold in acute inflammatory states. We investigated the role of SAA in sepsis using mice deficient in all three acute-phase SAA isoforms (SAA-TKO). SAA deficiency significantly increased mortality rates in the three experimental sepsis mouse models: cecal ligation and puncture (CLP), cecal slurry (CS) injection, and lipopolysaccharide (LPS) treatments. SAA-TKO mice had exacerbated lung pathology compared to wild-type (WT) mice after CLP. A bulk RNA sequencing performed on lung tissues excised 24 h after CLP indicated significant enrichment in the expression of genes associated with chemokine production, chemokine and cytokine-mediated signaling, neutrophil chemotaxis, and neutrophil migration in SAA-TKO compared to WT mice. Consistently, myeloperoxidase activity and neutrophil counts were significantly increased in the lungs of septic SAA-TKO mice compared to WT mice. The in vitro treatment of HL-60, neutrophil-like cells, with SAA or SAA bound to a high-density lipoprotein (SAA-HDL), significantly decreased cellular transmigration through laminin-coated membranes compared to untreated cells. Thus, SAA potentially prevents neutrophil transmigration into injured lungs, thus reducing exacerbated tissue injury and mortality. In conclusion, we demonstrate for the first time that endogenous SAA plays a protective role in sepsis, including ameliorating lung injury.

A PINK1 input threshold arises from positive feedback in the PINK1/Parkin mitophagy decision circuit.

Mechanisms that prevent accidental activation of the PINK1/Parkin mitophagy circuit on healthy mitochondria are poorly understood. On the surface of damaged mitochondria, PINK1 accumulates and acts as the input signal to a positive feedback loop of Parkin recruitment, which in turn promotes mitochondrial degradation via mitophagy. However, PINK1 is also present on healthy mitochondria, where it could errantly recruit Parkin and thereby activate this positive feedback loop. Here, we explore emergent properties of the PINK1/Parkin circuit by quantifying the relationship between mitochondrial PINK1 concentrations and Parkin recruitment dynamics. We find that Parkin is recruited to mitochondria only if PINK1 levels exceed a threshold and then only after a delay that is inversely proportional to PINK1 levels. Furthermore, these two regulatory properties arise from the input-coupled positive feedback topology of the PINK1/Parkin circuit. These results outline an intrinsic mechanism by which the PINK1/Parkin circuit can avoid errant activation on healthy mitochondria.

Sex gaps in perception of tobacco conversations between adult patients who now smoke cigarettes and oral health care providers: National Health and Nutrition Examination Survey 2017-March 2020 prepandemic.

BACKGROUND: Smoking cessation is difficult. A potential gap based on sex exists in the tobacco cessation aid that dental care professionals provide to patients. The purpose of this research was to examine whether there is a sex difference in dental patients' reports of having a direct conversation about the benefits of giving up cigarettes or other types of tobacco products with their oral health care provider. METHODS: National Health and Nutrition Examination Survey 2017-March 2020 prepandemic data were used in this cross-sectional study for participants 18 years and older who reported that they "now smoke cigarettes," had a dental visit within the previous year, self-reported their sex, and responded whether their oral health care provider had a direct conversation about the benefits of giving up cigarettes or other types of tobacco products to improve dental health at their last visit (n = 582). Multivariable logistic regression analysis was conducted to compare data according to sex. RESULTS: Overall, 50.7% of patients (59.2% of men, 42.9% of women; P = .0037) reported having a conversation about tobacco cessation at their dental visit. The odds of women reporting having no such discussion were twice those of men (odds ratio, 2.17; 95% CI, 1.10 to 4.28; P = .0270). CONCLUSIONS: One-half of the participants reported having no tobacco cessation conversation about the benefits of giving up cigarettes or other types of tobacco with their dental care provider. Women were twice as likely to report no such discussion. PRACTICAL IMPLICATIONS: Oral health care providers need to ensure that primary and secondary prevention information and intervention programs about the benefits of giving up cigarettes or other types of tobacco products are provided equitably to all patients.

Replication cycle timing determines phage sensitivity to a cytidine deaminase toxin/antitoxin bacterial defense system.

Toxin-antitoxin (TA) systems are ubiquitous two-gene loci that bacteria use to regulate cellular processes such as phage defense. Here, we demonstrate the mechanism by which a novel type III TA system, avcID, is activated and confers resistance to phage infection. The toxin of the system (AvcD) is a deoxycytidylate deaminase that converts deoxycytidines (dC) to dexoyuridines (dU), while the RNA antitoxin (AvcI) inhibits AvcD activity. We have shown that AvcD deaminated dC nucleotides upon phage infection, but the molecular mechanism that activated AvcD was unknown. Here we show that the activation of AvcD arises from phage-induced inhibition of host transcription, leading to degradation of the labile AvcI. AvcD activation and nucleotide depletion not only decreases phage replication but also increases the formation of defective phage virions. Surprisingly, infection of phages such as T7 that are not inhibited by AvcID also lead to AvcI RNA antitoxin degradation and AvcD activation, suggesting that depletion of AvcI is not sufficient to confer protection against some phage. Rather, our results support that phage with a longer replication cycle like T5 are sensitive to AvcID-mediated protection while those with a shorter replication cycle like T7 are resistant.

Activation of a Vibrio cholerae CBASS anti-phage system by quorum sensing and folate depletion.

IMPORTANCE: To counteract infection with phage, bacteria have evolved a myriad of molecular defense systems. Some of these systems initiate a process called abortive infection, in which the infected cell kills itself to prevent phage propagation. However, such systems must be inhibited in the absence of phage infection to prevent spurious death of the host. Here, we show that the cyclic oligonucleotide based anti-phage signaling system (CBASS) accomplishes this by sensing intracellular folate molecules and only expressing this system in a group. These results enhance our understanding of the evolution of the seventh Vibrio cholerae pandemic and more broadly how bacteria defend themselves against phage infection.

Vibrio cholerae phage ICP3 requires O1 antigen for infection.

In its natural aquatic environment, the bacterial pathogen Vibrio cholerae, the causative agent of the enteric disease cholera, is in constant competition with bacterial viruses known as phages. Following ICP3 infection, V. cholerae cultures that exhibited phage killing always recovered overnight, and clones isolated from these regrowth populations exhibited complete resistance to subsequent infections. Whole-genome sequencing of these resistant mutants revealed seven distinct mutations in genes encoding for enzymes involved in O1 antigen biosynthesis, demonstrating that the O1 antigen is a previously uncharacterized putative receptor of ICP3. To further elucidate the specificity of the resistance conferred by these mutations, they were challenged with the V. cholerae-specific phages ICP1 and ICP2. All seven O1 antigen mutants demonstrated pan-resistance to ICP1 but not ICP2, which utilizes the OmpU outer membrane protein as a receptor. We show that resistant mutations to ICP1 and ICP3 evolve at a significantly higher frequency than ICP2, but these mutations have a significant fitness tradeoff to V. cholerae and are unable to evolve in the presence of an antimicrobial that mimics host cell defensins.

The Effect of Patency Files on Apical Canal Anatomy Using SEM Imaging.

Introduction: There are many reasons to maintain apical patency during routine endodontic treatment. Thousands of canals are treated utilizing a patency file every year all around the world. The effect patency has on the apical anatomy of the root has been controversial for generations. Objective: This ex vivo descriptive study was created to show the effect patency files actually have on the apical root canal anatomy using visually detailed SEM images supported by dental radiographs. Materials and Methods: Three extracted maxillary anterior teeth that represent the multitude of canals in vivo were instrumented utilizing patency files. Two of the three maxillary anterior teeth were instrumented with hand files, the other maxillary anterior tooth with a .06 taper rotary file. The teeth were then scanned with an electron microscope to view the effect that the instruments had on the apical canal anatomy. A fourth tooth, a maxillary second molar, was shaped with an .06 taper rotary file and cone fitted. This tooth was radiographed with a gutta percha cone fitted to reveal the position of the narrowest constriction after patency was achieved. Results: The patency files, both hand files and rotary, were shown not to adversely affect the apical canal anatomy. Additionally, the SEM's revealed a precise demarcation of cementum to dentin which was at the root surface after patency was achieved. Conclusion: The patent use of greater tapered rotary files provides a clear demarcation of the CDJ which allows a precise acquisition of the narrowest constriction of the canal with the use of an electronic apex locator for establishing the ideal working length and precision placement of a gutta percha cone.

Plasmid-free cheater cells commonly evolve during laboratory growth.

It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, secretion of beta-lactamase from resistant cells, and subsequent degradation of nearby penicillin and related antibiotics, allows neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss still occurred. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated.

Dissecting the effects of GTPase and kinase domain mutations on LRRK2 endosomal localization and activity.

Parkinson's disease-causing leucine-rich repeat kinase 2 (LRRK2) mutations lead to varying degrees of Rab GTPase hyperphosphorylation. Puzzlingly, LRRK2 GTPase-inactivating mutations-which do not affect intrinsic kinase activity-lead to higher levels of cellular Rab phosphorylation than kinase-activating mutations. Here, we investigate whether mutation-dependent differences in LRRK2 cellular localization could explain this discrepancy. We discover that blocking endosomal maturation leads to the rapid formation of mutant LRRK2+ endosomes on which LRRK2 phosphorylates substrate Rabs. LRRK2+ endosomes are maintained through positive feedback, which mutually reinforces membrane localization of LRRK2 and phosphorylated Rab substrates. Furthermore, across a panel of mutants, cells expressing GTPase-inactivating mutants form strikingly more LRRK2+ endosomes than cells expressing kinase-activating mutants, resulting in higher total cellular levels of phosphorylated Rabs. Our study suggests that the increased probability that LRRK2 GTPase-inactivating mutants are retained on intracellular membranes compared to kinase-activating mutants leads to higher substrate phosphorylation.