Genome-wide analysis of mitochondrial DNA copy number reveals loci implicated in nucleotide metabolism, platelet activation, and megakaryocyte proliferation.

Mitochondrial DNA copy number (mtDNA-CN) measured from blood specimens is a minimally invasive marker of mitochondrial function that exhibits both inter-individual and intercellular variation. To identify genes involved in regulating mitochondrial function, we performed a genome-wide association study (GWAS) in 465,809 White individuals from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the UK Biobank (UKB). We identified 133 SNPs with statistically significant, independent effects associated with mtDNA-CN across 100 loci. A combination of fine-mapping, variant annotation, and co-localization analyses was used to prioritize genes within each of the 133 independent sites. Putative causal genes were enriched for known mitochondrial DNA depletion syndromes (p = 3.09 × 10-15) and the gene ontology (GO) terms for mtDNA metabolism (p = 1.43 × 10-8) and mtDNA replication (p = 1.2 × 10-7). A clustering approach leveraged pleiotropy between mtDNA-CN associated SNPs and 41 mtDNA-CN associated phenotypes to identify functional domains, revealing three distinct groups, including platelet activation, megakaryocyte proliferation, and mtDNA metabolism. Finally, using mitochondrial SNPs, we establish causal relationships between mitochondrial function and a variety of blood cell-related traits, kidney function, liver function and overall (p = 0.044) and non-cancer mortality (p = 6.56 × 10-4).

Fontan-associated liver disease and total cavopulmonary anatomical flow effectors.

OBJECTIVE: We investigated a relationship between a composite index comprised of Fontan-circuit anatomical features and hepatic fibrosis scores from biopsy. METHODS: We identified living extracardiac Fontan patients, ≥7 years old and ≥5 but <20 years postoperative, that underwent cardiac catheterization and transvenous liver biopsy between March 2012 and September 2020. We divided patients into anatomical groups and applied a risk score to each patient. We compared average anatomical risk scores with average hepatic total fibrosis scores by group. RESULTS: We identified 111 patients that met inclusion criteria. After excluding four patients, we assigned 107 to one of 12 anatomical variant groups (n ≥ 3). For the 107, the average age at liver biopsy was 14 ± 6 years old. Of the 107, 105 (98%) were New York Heart Association Class 1. We found average anatomical risk scores by group correlated with average total fibrosis scores by group (R = 0.8; p = .005). An average Fontan duration to biopsy of 10 ± 1 years was similar for all 12 anatomical groups. We found no other clinical variables, laboratory, or hemodynamic values that trended with anatomical risk scores or hepatic total fibrosis scores. CONCLUSIONS: In a cohort of relatively young, stable extracardiac Fontan patients, average composite anatomical risk scores strongly correlated with average hepatic total fibrosis scores by anatomical group. These findings suggest that some anatomical variants in extracardiac Fontan patients are associated with higher Fontan-associated liver disease (FALD)-related hepatic total fibrosis scores than others, despite similar Fontan durations.

The long-term maintenance of a resistance polymorphism through diffuse interactions.

Plant resistance (R) genes are a crucial component in plant defence against pathogens. Although R genes often fail to provide durable resistance in an agricultural context, they frequently persist as long-lived balanced polymorphisms in nature. Standard theory explains the maintenance of such polymorphisms through a balance of the costs and benefits of resistance and virulence in a tightly coevolving host-pathogen pair. However, many plant-pathogen interactions lack such specificity. Whether, and how, balanced polymorphisms are maintained in diffusely interacting species is unknown. Here we identify a naturally interacting R gene and effector pair in Arabidopsis thaliana and its facultative plant pathogen, Pseudomonas syringae. The protein encoded by the R gene RPS5 recognizes an AvrPphB homologue (AvrPphB2) and exhibits a balanced polymorphism that has been maintained for over 2 million years (ref. 3). Consistent with the presence of an ancient balanced polymorphism, the R gene confers a benefit when plants are infected with P. syringae carrying avrPphB2 but also incurs a large cost in the absence of infection. RPS5 alleles are maintained at intermediate frequencies in populations globally, suggesting ubiquitous selection for resistance. However, the presence of P. syringae carrying avrPphB is probably insufficient to explain the RPS5 polymorphism. First, avrPphB homologues occur at very low frequencies in P. syringae populations on A. thaliana. Second, AvrPphB only rarely confers a virulence benefit to P. syringae on A. thaliana. Instead, we find evidence that selection for RPS5 involves multiple non-homologous effectors and multiple pathogen species. These results and an associated model suggest that the R gene polymorphism in A. thaliana may not be maintained through a tightly coupled interaction involving a single coevolved R gene and effector pair. More likely, the stable polymorphism is maintained through complex and diffuse community-wide interactions.

The Rate of Hepatic Fibrosis Progression in Patients Post-Fontan.

This investigation analyzed the rate of hepatic fibrosis progression in post-Fontan patients that underwent hepatic biopsy. The study cohort comprised post-Fontan patients that underwent cardiac catheterization and transvenous liver biopsy between March 2012 and September 2019. We identified 126 patients that met inclusion criteria. Of the 126, 27 (21%) had a lateral tunnel Fontan, and 99 (79%) had an extracardiac Fontan. For the 27 lateral tunnel Fontan patients, age at Fontan was 4 ± 2 years, and for the 99 extracardiac Fontan patients age at Fontan was 4 ± 2 years (p = 0.98). For the 27 lateral tunnel Fontan patients, the average total fibrosis score was 3.0 ± 1.5; and for the 99 extracardiac Fontan patients, the average total fibrosis was 2.7 ± 1.7 (p = 0.48). For the lateral tunnel Fontan patients, the average Fontan duration was 20 ± 6 years; and for the 99 extracardiac Fontan patients, the average Fontan duration was 11 ± 5 years (p < 0.001). For the 27 lateral tunnel Fontan patients, the average rate of fibrosis progression was 0.16 ± 0.10 total fibrosis score/year; and for the 99 extracardiac Fontan patients, the average rate of fibrosis progression was 0.30 ± 0.23 total fibrosis score/year (p < 0.001). In conclusion, our findings suggest that those with extracardiac Fontans have a faster rate of hepatic fibrosis progression than those with lateral tunnel Fontans. More extensive or multi-institutional studies will be needed to confirm these findings and define the clinical significance of discrepant rates of hepatic fibrosis in post-Fontan patients.

Fontan venovenous collaterals and hepatic fibrosis.

OBJECTIVE: We hypothesized that a relationship might exist between angiographically demonstrable, post-Fontan venovenous collaterals, and hepatic fibrosis. METHODS: We analyzed data from post-Fontan patients that underwent cardiac catheterization and transvenous-hepatic biopsy procedures between March 2012 and March 2020. From innominate vein angiography, we determined those that either had or lacked venovenous collaterals. Additionally, we examined data from post-Fontan patients that underwent hepatic ultrasound, shear-wave elastography between January 2017 and March 2020. RESULTS: We identified 164 patients that met inclusion criteria. Of the 164, 101 (62%) had venovenous collaterals. Of the 101 with collaterals, average total fibrosis score (TFS) was 3.2 and the average rate of fibrosis progression was 0.28 vs an average TFS of 2.1 and an average fibrosis progression rate of 0.22 for those without collaterals (P = .00001 and P = .01, respectively). Of the 101 with collaterals, oxygen saturation was 91% ± 4% vs 93% ± 3% (P = .048) without collaterals. Of the 164, 86 (52%) underwent ultrasound shear-wave elastography. Of the 86 patients undergoing elastography, 50 (58%) were performed in those with collaterals, and 36 (42%) in those without collaterals. For the 50 with collaterals, average elastography values were 13.3 vs 11.2 kPa for the 36 without collaterals (P = .006). We found no statistically significant differences for age at biopsy, Fontan duration, Fontan-type, type of functional univentricle, laboratory, clinical, or hemodynamic values between those with or without collaterals. CONCLUSIONS: The presence of angiographically demonstrated venovenous collaterals was associated with statistically, significantly more advanced liver fibrosis than those without collaterals.

A DNA methylation biomarker of alcohol consumption.

The lack of reliable measures of alcohol intake is a major obstacle to the diagnosis and treatment of alcohol-related diseases. Epigenetic modifications such as DNA methylation may provide novel biomarkers of alcohol use. To examine this possibility, we performed an epigenome-wide association study of methylation of cytosine-phosphate-guanine dinucleotide (CpG) sites in relation to alcohol intake in 13 population-based cohorts (ntotal=13 317; 54% women; mean age across cohorts 42-76 years) using whole blood (9643 European and 2423 African ancestries) or monocyte-derived DNA (588 European, 263 African and 400 Hispanic ancestry) samples. We performed meta-analysis and variable selection in whole-blood samples of people of European ancestry (n=6926) and identified 144 CpGs that provided substantial discrimination (area under the curve=0.90-0.99) for current heavy alcohol intake (⩾42 g per day in men and ⩾28 g per day in women) in four replication cohorts. The ancestry-stratified meta-analysis in whole blood identified 328 (9643 European ancestry samples) and 165 (2423 African ancestry samples) alcohol-related CpGs at Bonferroni-adjusted P<1 × 10-7. Analysis of the monocyte-derived DNA (n=1251) identified 62 alcohol-related CpGs at P<1 × 10-7. In whole-blood samples of people of European ancestry, we detected differential methylation in two neurotransmitter receptor genes, the γ-Aminobutyric acid-A receptor delta and γ-aminobutyric acid B receptor subunit 1; their differential methylation was associated with expression levels of a number of genes involved in immune function. In conclusion, we have identified a robust alcohol-related DNA methylation signature and shown the potential utility of DNA methylation as a clinically useful diagnostic test to detect current heavy alcohol consumption.

Starting the fight in the tumor: expert recommendations for the development of human intratumoral immunotherapy (HIT-IT).

A European Society for Medical Oncology (ESMO)-sponsored expert meeting was held in Paris on 8 March 2018 which comprised 11 experts from academia, 11 experts from the pharmaceutical industry and 2 clinicians who were representatives of ESMO. The focus of the meeting was exclusively on the intratumoral injection/delivery of immunostimulatory agents with the aim of harmonizing the standard terms and methodologies used in the reporting of human intratumoral immunotherapy (HIT-IT) clinical trials to ensure quality assurance and avoid a blurring of the data reported from different studies. The goal was to provide a reference document, endorsed by the panel members that could provide guidance to clinical investigators, pharmaceutical companies, ethics committees, independent review boards, patient advocates and the regulatory authorities and promote an increase in the number and quality of HIT-IT clinical trials in the future. Particular emphasis was placed not only on the development of precise definitions to facilitate a better understanding between investigators but also on the importance of systematic serial biopsies as a driver for translational research and the need for the recording and reporting of data, to facilitate a better understanding of the key processes involved.

Coat Protein Mutations That Alter the Flux of Morphogenetic Intermediates through the ϕX174 Early Assembly Pathway.

Two scaffolding proteins orchestrate ϕX174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine, and methionine substitutions were phenotypically indistinguishable from the wild type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperature-sensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, cloned-gene expression compensated for inefficient B protein binding, as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wild-type levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability.IMPORTANCE Genetic analyses have been instrumental in deciphering the temporal events of many biochemical pathways. However, pleiotropic effects can complicate analyses. Vis-à-vis virion morphogenesis, an improper protein-protein interaction within an early assembly intermediate can influence the efficiency of all subsequent reactions. Consequently, the flux of assembly intermediates cumulatively decreases as the pathway progresses. During morphogenesis, ϕX174 coat protein participates in at least four well-defined reactions, each one characterized by an interaction with a scaffolding or structural protein. In this study, genetic analyses, biochemical characterizations, and physiological assays, i.e., elevating the protein levels with which the coat protein interacts, were used to elucidate pleiotropic effects that may alter the flux of intermediates through a morphogenetic pathway.

Large-scale pharmacogenomic study of sulfonylureas and the QT, JT and QRS intervals: CHARGE Pharmacogenomics Working Group.

Sulfonylureas, a commonly used class of medication used to treat type 2 diabetes, have been associated with an increased risk of cardiovascular disease. Their effects on QT interval duration and related electrocardiographic phenotypes are potential mechanisms for this adverse effect. In 11 ethnically diverse cohorts that included 71 857 European, African-American and Hispanic/Latino ancestry individuals with repeated measures of medication use and electrocardiogram (ECG) measurements, we conducted a pharmacogenomic genome-wide association study of sulfonylurea use and three ECG phenotypes: QT, JT and QRS intervals. In ancestry-specific meta-analyses, eight novel pharmacogenomic loci met the threshold for genome-wide significance (P<5 × 10-8), and a pharmacokinetic variant in CYP2C9 (rs1057910) that has been associated with sulfonylurea-related treatment effects and other adverse drug reactions in previous studies was replicated. Additional research is needed to replicate the novel findings and to understand their biological basis.

Pharmacogenomics study of thiazide diuretics and QT interval in multi-ethnic populations: the cohorts for heart and aging research in genomic epidemiology.

Thiazide diuretics, commonly used antihypertensives, may cause QT interval (QT) prolongation, a risk factor for highly fatal and difficult to predict ventricular arrhythmias. We examined whether common single-nucleotide polymorphisms (SNPs) modified the association between thiazide use and QT or its component parts (QRS interval, JT interval) by performing ancestry-specific, trans-ethnic and cross-phenotype genome-wide analyses of European (66%), African American (15%) and Hispanic (19%) populations (N=78 199), leveraging longitudinal data, incorporating corrected standard errors to account for underestimation of interaction estimate variances and evaluating evidence for pathway enrichment. Although no loci achieved genome-wide significance (P<5 × 10-8), we found suggestive evidence (P<5 × 10-6) for SNPs modifying the thiazide-QT association at 22 loci, including ion transport loci (for example, NELL1, KCNQ3). The biologic plausibility of our suggestive results and simulations demonstrating modest power to detect interaction effects at genome-wide significant levels indicate that larger studies and innovative statistical methods are warranted in future efforts evaluating thiazide-SNP interactions.

Pattern recognition receptors: immune targets to enhance cancer immunotherapy.

Durable tumor responses and significant levels of disease control rates have been described in more than 20 advanced/metastatic cancer types with B7-family immune checkpoint-targeted anti-CTLA-4, anti-PD-1, and anti-PD-L1 monoclonal antibodies. These results and the recent approvals of ipilimumab, pembrolizumab, nivolumab and atezolizumab are currently revolutionizing the way we envision the future of cancer care. However these clinical benefits are not observed in all cancer types and in every patient. Therefore, our clinical challenge is to identify therapeutic strategies which could overcome the primary and secondary resistances to these novel cancer immunotherapies. Pattern recognition receptors (PRRs) are other critical costimulatory molecules of immune cells, notably myeloid cells (macrophages and dendritic cells). They were initially described as sensors for 'danger signals' released by pathogens (e.g. viral DNA and bacterial proteins). We know now that PRRs can also be recruited and activated upon recognition of endogenous stress signals such as molecules released upon self-cell death (e.g. ATP and HMGB1). Natural endo/exogenous or synthetic PRRs agonists have notably the ability to activate phagocytosis and antigen presentation by myeloid cells residing in the tumor micro-environment. In pre-clinical models, these PRRs agonists have also been shown to overcome the resistance to T-cell targeted immune checkpoints anti-CTLA-4 and anti-PD-1/PD-L1. This manuscript reviews the current knowledge on this major family of immune receptors and the molecules targeting them which are currently in clinical development.

MELD-XI Scores Correlate with Post-Fontan Hepatic Biopsy Fibrosis Scores.

We tested the hypothesis that MELD-XI values correlated with hepatic total fibrosis scores obtained in 70 predominately stable, post-Fontan patients that underwent elective cardiac catheterization. We found a statistically significant correlation between MELD-XI values and total fibrosis scores (p = 0.003). Thus, serial MELD-XI values may be an additional useful clinical parameter for follow-up care in post-Fontan patients.

An Observation from Liver Biopsies Two Decades Post-Fontan.

This brief report describes an observation from liver biopsy results in nonfailing Fontan patients, currently in their second postoperative decade. In three patients, with either atriopulmonary or atrioventricular connections and functional left ventricles, we found no portal fibrosis. In contrast, we found portal fibrosis in three clinically similar, nonfailing Fontan patients with lateral tunnel connections and functional left ventricles. We recognize the results may be secondary to chance; nevertheless, we speculate about possible relevancy.

Fontan Outcomes and Pulmonary Blood Flow at Birth.

We previously noted, in a small group of post-Fontan patients, a possible association between hepatic fibrosis scores and the status of pulmonary blood flow at birth. To further explore this observation, we examined data from all Fontan patients seen in our center from July 2010 to March 2015. We identified 200 patients for analysis. Of the 200 patients, 56 underwent transvenous-hepatic biopsy. Of the 200 patients, 13 (6.5%) had protein-losing enteropathy. We divided both the 56 biopsy patients and the entire cohort of 200 patients into 4 groups: (1) unobstructed pulmonary blood flow at birth with functional left ventricles, (2) unobstructed pulmonary blood flow at birth with functional right ventricles, (3) obstructed pulmonary blood flow at birth with functional left ventricles, and (4) obstructed pulmonary blood flow at birth with functional right ventricles. Analysis of the 56 liver-biopsy patient groups showed median hepatic total-fibrosis scores for the 4 groups of 2 (0-6), 2 (0-8), 3 (2-6), and 4 (1-8), respectively, with statistical significance between groups 4 and 1 (p = 0.031). For the entire cohort of 200 patients, we analyzed the incidence of protein-losing enteropathy for each of the four groups and found protein-losing enteropathy percent occurrences of 0, 2.9, 8.8, and 16.1, respectively, with statistical significance between groups 4 and 2 (p = 0.031) and between groups 4 and 1 (p = 0.025). A history of obstructed pulmonary blood flow at birth, coupled with a functional right ventricle, may predict a poorer long-term Fontan outcome.

Fontan hepatic fibrosis and pulmonary vascular development.

Fontan patients are at risk for hepatic fibrosis; however, risk factors are unclear. We performed a multivariate analysis in a small cohort of 14 patients (7-24 years old, mean 15) with Fontan circulation, undergoing cardiac catheterization and transvenous liver biopsies, all demonstrating fibrosis. We found by stepwise regression analysis that the history of pulmonary atresia was a predictor of higher total hepatic fibrosis scores than a history of unobstructed pulmonary blood flow (p = 0.002). Other variables including age, time from Fontan, hemodynamic measurements, and laboratory values were not predictive of total fibrosis scores at p values <0.05. Hepatic fibrosis scores between those born with pulmonary atresia versus unrestricted pulmonary blood flow may reflect differences in pulmonary circulatory physiology, resulting from differences in pulmonary vascular development.

Evaluation of health literacy tools for correct prescription understanding.

OBJECTIVES: To evaluate two brief health literacy prediction tools for understanding of a prescription label and using a drug correctly. METHODS: Patients who visited the Family HealthCare Pharmacy were asked to complete the Newest Vital Sign (NVS) and Rapid Estimate of Adult Literacy in Medicine (REALM-R) and to interpret a prescription medication label. Each patient received a bottle of amoxicillin suspension with one of four prescription labels randomly varied in directions for frequency and amount. Patients were also asked to calculate the days' supply and demonstrate their ability to draw up the required dose using an oral syringe. RESULTS: A total of 150 patients consented to participate. Only the NVS score was associated with ability to fully interpret the label correctly. Neither tool was related to ability to draw up the correct amount using an oral syringe but both had some correlation to successfully calculating the days' supply provided. The NVS and REALM-R literacy scores had a modest correlation with each other. CONCLUSION: The NVS may be useful in predicting prescription label understanding and a patient's ability to do a simple dosage calculation. Neither the NVS nor the REALM-R correlated well with ability to draw an amount using an oral syringe correctly.

Transvenous hepatic biopsy in stable Fontan patients undergoing cardiac catheterization.

Liver pathology complicates Fontan palliation. Previous reports established that both hepatic sinusoidal and portal fibrosis occur in patients after Fontan procedures. Past studies predominantly included symptomatic patient cohorts. Thus, the authors of this study aimed to characterize hepatic pathology via transvenous hepatic biopsies in 21 asymptomatic patients at the time of elective cardiac catheterization. Seven of these patients (33 %) were accompanied by an interventional procedure. Hepatic biopsies showed evidence of either sinusoidal or portal fibrosis or both in all but one patient. The findings showed a statistically significant (p = 0.005) moderately strong positive correlation between fibrosis scores and time since Fontan surgery. Additionally, no significant correlation was found between fibrosis scores and inferior vena cava pressure, pulmonary vascular resistance, platelet counts, or serum laboratory testing of hepatic function.

Structure-function analysis of the coiled-coil and leucine-rich repeat domains of the RPS5 disease resistance protein.

The Arabidopsis (Arabidopsis thaliana) RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5) disease resistance protein mediates recognition of the Pseudomonas syringae effector protein AvrPphB. RPS5 belongs to the coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) family and is activated by AvrPphB-mediated cleavage of the protein kinase PBS1. Here, we present a structure-function analysis of the CC and LRR domains of RPS5 using transient expression assays in Nicotiana benthamiana. We found that substituting the CC domain of RPS2 for the RPS5 CC domain did not alter RPS5 specificity and only moderately reduced its ability to activate programmed cell death, suggesting that the CC domain does not play a direct role in the recognition of PBS1 cleavage. Analysis of an RPS5-super Yellow Fluorescent Protein fusion revealed that RPS5 localizes to the plasma membrane (PM). Alanine substitutions of predicted myristoylation (glycine-2) and palmitoylation (cysteine-4) residues affected RPS5 PM localization, protein stability, and function in an additive manner, indicating that PM localization is essential to RPS5 function. The first 20 amino acids of RPS5 were sufficient for directing super Yellow Fluorescent Protein to the PM. C-terminal truncations of RPS5 revealed that the first four LRR repeats are sufficient for inhibiting RPS5 autoactivation; however, the complete LRR domain was required for the recognition of PBS1 cleavage. Substitution of the RPS2 LRR domain resulted in the autoactivation of RPS5, indicating that the LRR domain must coevolve with the NBS domain. We conclude that the RPS5 LRR domain functions to suppress RPS5 activation in the absence of PBS1 cleavage and promotes RPS5 activation in its presence.

Activation of a plant nucleotide binding-leucine rich repeat disease resistance protein by a modified self protein.

Nucleotide binding-leucine rich repeat (NB-LRR) proteins function as intracellular receptors for the detection of pathogens in both plants and animals. Despite their central role in innate immunity, the molecular mechanisms that govern NB-LRR activation are poorly understood. The Arabidopsis NB-LRR protein RPS5 detects the presence of the Pseudomonas syringae effector protein AvrPphB by monitoring the status of the Arabidopsis protein kinase PBS1. AvrPphB is a cysteine protease that targets PBS1 for cleavage at a single site within the activation loop of PBS1. Using a transient expression system in the plant Nicotiana benthamiana and stable transgenic Arabidopsis plants we found that both PBS1 cleavage products are required to activate RPS5 and can do so in the absence of AvrPphB. We also found, however, that the requirement for cleavage of PBS1 could be bypassed simply by inserting five amino acids at the PBS1 cleavage site, which is located at the apex of the activation loop of PBS1. Activation of RPS5 did not require PBS1 kinase function, and thus RPS5 appears to sense a subtle conformational change in PBS1, rather than cleavage. This finding suggests that NB-LRR proteins may function as fine-tuned sensors of alterations in the structures of effector targets.