Negative feedback of cyclic di-GMP levels optimizes switching between sessile and motile lifestyles in Vibrio cholerae.

The signaling molecule cyclic di-GMP (cdG) controls the switch between bacterial motility and biofilm production, and fluctuations in cellular levels of cdG have been implicated in Vibrio cholerae pathogenesis. Intracellular concentrations of cdG are controlled by the interplay of diguanylate cyclase (DGC) enzymes, which synthesize cdG to promote biofilms, and phosphodiesterase (PDE) enzymes, which hydrolyse cdG to drive motility. To track the complete regulatory logic of how V. cholerae responds to changing cdG levels, we followed a timecourse of overexpression of either the V. harveyi diguanylate cyclase QrgB or a variant of QrgB lacking catalytic activity (QrgB*). We find that QrgB increases cdG levels relative to QrgB* for 30 minutes after overexpression, but the effect of QrgB on cdG levels plateaus at 30 minutes, indicating tight adaptive control of cdG levels. In contrast, loss of VpsR, a master regulator activating biofilm formation upon binding to cdG, leads to higher baseline levels of cdG and continuously increasing cdG through 60 minutes after QrgB induction, revealing the existence of a negative feedback loop on cdG levels operating through VpsR. Through a combination of RNA polymerase ChIP-seq, RNA-seq, and genetic approaches, we show that transcription of a gene encoding a PDE, cdgC, is activated by VpsR at high cdG concentrations, mediating this negative feedback on cdG levels. We further identify a transcript encoded within, and antisense to, the cdgC open reading frame which we name sRNA negative regulator of CdgC (SnrC). RNA polymerase ChIP-seq and RNA-seq demonstrate SnrC to be expressed specifically under conditions of high cdG in the absence of VpsR. Ectopic SnrC expression increases cdG levels in a manner dependent on CdgC, demonstrating that its effect on cdG levels is likely through interference with CdgC production. Further, although cells lacking cdgC exhibit enhanced biofilm formation, these mutants are outcompeted by wild type V. cholerae in colonization assays that reward a combination of attachment, dispersal, and motility behaviors. These results underscore the importance of negative feedback regulation of cdG to maintain appropriate homeostatic levels for efficient transitioning between biofilm formation and motility, both of which are necessary over the course of the V. cholerae infection cycle.

A Vibrio cholerae Type IV restriction system targets glucosylated 5-hydroxymethylcytosine 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 acquired and/or evolved a myriad of phage defense systems. Although several novel defense systems have been discovered, we hypothesized that more were encoded in V. cholerae given the low diversity of phages 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 16-kB region of the Vibrio pathogenicity island-2, which 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 modifications are resistant to TgvAB defense but exhibit a significant evolutionary tradeoff, becoming susceptible to other Type IV restriction systems that target unglucosylated 5hmC. We also show that the Type I restriction-modification system that embeds the tgvAB genes protects against phage T3, secΦ18, secΦ27, and λ, suggesting that this region is a phage defense island. 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.IMPORTANCEBacteria are constantly being predated by bacteriophage (phage). To counteract this predation, bacteria have evolved a myriad of defense systems. Some of these systems specifically digest infecting phage by recognizing unique base modifications present on the phage DNA. In this study, we discover a Type IV restriction system encoded in V. cholerae, which we name TgvAB, and demonstrate it recognizes and restricts phage that have 5-hydroxymethylcytosine glucosylated DNA. Moreover, the evolution of resistance to TgvAB render phage susceptible to other Type IV restriction systems, demonstrating a significant evolutionary tradeoff. These results enhance our understanding of the evolution of V. cholerae and more broadly how bacteria evade phage predation.

Combinatorial control of Pseudomonas aeruginosa biofilm development by quorum-sensing and nutrient-sensing regulators.

The human pathogen Pseudomonas aeruginosa, a leading cause of hospital-acquired infections, inhabits and forms sessile antibiotic-resistant communities called biofilms in a wide range of biotic and abiotic environments. In this study, we examined how two global sensory signaling pathways-the RhlR quorum-sensing system and the CbrA/CbrB nutritional adaptation system-intersect to control biofilm development. Previous work has shown that individually these two systems repress biofilm formation. Here, we used biofilm analyses, RNA-seq, and reporter assays to explore the combined effect of information flow through RhlR and CbrA on biofilm development. We find that the ΔrhlRΔcbrA double mutant exhibits a biofilm morphology and an associated transcriptional response distinct from wildtype and the parent ΔrhlR and ΔcbrA mutants indicating codominance of each signaling pathway. The ΔrhlRΔcbrA mutant gains suppressor mutations that allow biofilm expansion; these mutations map to the crc gene resulting in loss of function of the carbon catabolite repression protein Crc. Furthermore, the combined absence of RhlR and CbrA leads to a drastic reduction in the abundance of the Crc antagonist small RNA CrcZ. Thus, CrcZ acts as the molecular convergence point for quorum- and nutrient-sensing cues. We find that in the absence of antagonism by CrcZ, Crc promotes the expression of biofilm matrix components-Pel exopolysaccharide, and CupB and CupC fimbriae. Therefore, this study uncovers a regulatory link between nutritional adaption and quorum sensing with potential implications for anti-biofilm targeting strategies.IMPORTANCEBacteria often form multicellular communities encased in an extracytoplasmic matrix called biofilms. Biofilm development is controlled by various environmental stimuli that are decoded and converted into appropriate cellular responses. To understand how information from two distinct stimuli is integrated, we used biofilm formation in the human pathogen Pseudomonas aeruginosa as a model and studied the intersection of two global sensory signaling pathways-quorum sensing and nutritional adaptation. Global transcriptomics on biofilm cells and reporter assays suggest parallel regulation of biofilms by each pathway that converges on the abundance of a small RNA antagonist of the carbon catabolite repression protein, Crc. We find a new role of Crc as it modulates the expression of biofilm matrix components in response to the environment. These results expand our understanding of the genetic regulatory strategies that allow P. aeruginosa to successfully develop biofilm communities.

Identifying FUS amyotrophic lateral sclerosis disease signatures in patient dermal fibroblasts.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, highly heterogeneous neurodegenerative disease, underscoring the importance of obtaining information to personalize clinical decisions quickly after diagnosis. Here, we investigated whether ALS-relevant signatures can be detected directly from biopsied patient fibroblasts. We profiled familial ALS (fALS) fibroblasts, representing a range of mutations in the fused in sarcoma (FUS) gene and ages of onset. To differentiate FUS fALS and healthy control fibroblasts, machine-learning classifiers were trained separately on high-content imaging and transcriptional profiles. "Molecular ALS phenotype" scores, derived from these classifiers, captured a spectrum from disease to health. Interestingly, these scores negatively correlated with age of onset, identified several pre-symptomatic individuals and sporadic ALS (sALS) patients with FUS-like fibroblasts, and quantified "movement" of FUS fALS and "FUS-like" sALS toward health upon FUS ASO treatment. Taken together, these findings provide evidence that non-neuronal patient fibroblasts can be used for rapid, personalized assessment in ALS.

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 mesenchymal stromal cells, which are not normally resident in the lung, can be used 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 pathological 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.

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.

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.

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.

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.

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.

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.

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.

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.