Plant protein-lipid interfaces studied by molecular dynamics simulations.

The delineation of protein-lipid interfaces is essential for understanding the mechanisms of various membrane-associated processes crucial to plant development and growth, including signalling, trafficking, and membrane transport. Due to their highly dynamic nature, the precise characterization of lipid-protein interactions is challenging by experimental techniques. Molecular dynamics (MD) simulations provide a powerful computational alternative with a spatial-temporal resolution allowing the atomistic-level description. In this review, we aim to introduce plant scientists to the MD simulations. We describe different steps of performing the MD simulations and provide a broad survey of the MD studies investigating plant protein-lipid interfaces. Our aim is also to illustrate that combining the MD simulations with artificial intelligence-based protein structure determination opens unprecedented possibilities for future investigations of dynamic plant protein-lipid interfaces.

Efficient determination of the accessible conformation space of multi-domain complexes based on EPR PELDOR data.

Many proteins can adopt multiple conformations which are important for their function. This is also true for proteins and domains that are covalently linked to each other. One important example is ubiquitin, which can form chains of different conformations depending on which of its lysine side chains is used to form an isopeptide bond with the C-terminus of another ubiquitin molecule. Similarly, ubiquitin gets covalently attached to active-site residues of E2 ubiquitin-conjugating enzymes. Due to weak interactions between ubiquitin and its interaction partners, these covalent complexes adopt multiple conformations. Understanding the function of these complexes requires the characterization of the entire accessible conformation space and its modulation by interaction partners. Long-range (1.8-10 nm) distance restraints obtained by EPR spectroscopy in the form of probability distributions are ideally suited for this task as not only the mean distance but also information about the conformation dynamics is encoded in the experimental data. Here we describe a computational method that we have developed based on well-established structure determination software using NMR restraints to calculate the accessible conformation space using PELDOR/DEER data.

RootSlice-A novel functional-structural model for root anatomical phenotypes.

Root anatomy is an important determinant of root metabolic costs, soil exploration, and soil resource capture. Root anatomy varies substantially within and among plant species. RootSlice is a multicellular functional-structural model of root anatomy developed to facilitate the analysis and understanding of root anatomical phenotypes. RootSlice can capture phenotypically accurate root anatomy in three dimensions of different root classes and developmental zones, of both monocotyledonous and dicotyledonous species. Several case studies are presented illustrating the capabilities of the model. For maize nodal roots, the model illustrated the role of vacuole expansion in cell elongation; and confirmed the individual and synergistic role of increasing root cortical aerenchyma and reducing the number of cortical cell files in reducing root metabolic costs. Integration of RootSlice for different root zones as the temporal properties of the nodal roots in the whole-plant and soil model OpenSimRoot/maize enabled the multiscale evaluation of root anatomical phenotypes, highlighting the role of aerenchyma formation in enhancing the utility of cortical cell files for improving plant performance over varying soil nitrogen supply. Such integrative in silico approaches present avenues for exploring the fitness landscape of root anatomical phenotypes.

What defines a chemokine? - The curious case of CXCL17.

Chemotactic cytokines (chemokines) are a group of around 40 small proteins which share a similar protein fold and are well known for their ability to direct the migration of leukocytes to a variety of tissue locations. CXCL17 was the last member of the chemokine family to be assigned and was admitted to the family based on theoretical modelling of the CXCL17 structure and chemotactic activity for monocytes and dendritic cells. Of Interest, CXCL17 expression appears to be restricted to mucosal tissues such as the tongue, stomach and lung, suggestive of specific roles at these locations. A putative CXCL17 receptor, GPR35 was reportedly identified and mice deficient in CXCL17 were generated and characterised. More recently, however, some apparent contradictions regarding aspects of CXCL17 biology have been raised by ourselves and others. Notably, GPR35 appears to be a receptor for the serotonin metabolite 5-hydroxyindoleacetic acid rather than for CXCL17 and modelling of CXCL17 using a variety of platforms fails to identify a chemokine-like fold. In this article, we summarize the discovery of CXCL17 and discuss key papers describing the subsequent characterisation of this protein. Ultimately, we pose the question, 'What defines a chemokine?' (185 words).

Isolation, purification and biochemical characterization of Conopomorpha cramerella farnesol dehydrogenase.

In Southeast Asia, Conopomorpha cramerella (Snellen) which is commonly known as the cocoa pod borer (CPB) moth has been identified as the most detrimental pest of Theobroma cacao L. Apart from the various side effects on human health and non-target organisms, heavily relying on synthetic pyrethroid insecticides to control CPB infestations also increases the environmental contamination risks. Thus, developing biorational insecticides that minimally affect the non-target organism and environment by targeting the insect growth regulation process is needed to manage the pest population. In insects, juvenile hormones (JH) regulate critical biological events, especially metamorphosis, development and reproduction. Since the physiological roles of JH III vary among different organisms, the biochemical properties, especially substrate specificity and analogue inhibition, may also be different. Therefore, studies on the JH III biosynthetic pathway enzymes in both plants and insects are beneficial to discover more effective analogues. Bioinformatic analysis and biochemical characterization of a NADP+ -dependent farnesol dehydrogenase, an intermediate enzyme of the JH III pathway, from C. cramerella (CcFolDH), were described in this study. In addition, the farnesol analogues that may act as a potent analogue inhibitor for CcFolDH ware determined using in vitro enzymatic study. The phylogenetic analysis indicated that CcFolDH shared a close phylogenetic relationship to the honeybee's short-chain dehydrogenase/reductase. The 27 kDa CcFolDH has an NADP(H) binding domain with a typical Rossmann fold and is likely a homotetrameric protein in the solution. The enzyme had a greater preference for substrate trans, trans-farnesol and coenzyme NADP+ . In terms of analogue inhibitor inhibition, hexahydroxyfarnesyl acetone showed the highest inhibition (the lowest Ki ) compared to other farnesol analogues. Thus, hexahydroxyfarnesyl acetone would serve as the most potent active ingredient for future biorational pesticide management for C. cramerella infestation. Based on the bioinformatic analyses and biochemical characterizations conducted in this research, we proposed that rCcFolDH differs slightly from other reported farnesol dehydrogenases in terms of molecular weight, substrate preference, coenzymes utilization and analogue inhibitors selection.

Modelling 3D supramolecular structure from sparse single-molecule localisation microscopy data.

Single-molecule localisation microscopy (SMLM) has the potential to reveal the underlying organisation of specific molecules within supramolecular complexes and their conformations, which is not possible with conventional microscope resolution. However, the detection efficiency for fluorescent molecules in cells can be limited in SMLM, even to below 1% in thick and dense samples. Segmentation of individual complexes can also be challenging. To overcome these problems, we have developed a software package termed PERPL: Pattern Extraction from Relative Positions of Localisations. This software assesses the relative likelihoods of models for underlying patterns behind incomplete SMLM data, based on the relative positions of pairs of localisations. We review its principles and demonstrate its use on the 3D lattice of Z-disk proteins in mammalian cardiomyocytes. We find known and novel features at ~20 nm with localisations of less than 1% of the target proteins, using mEos fluorescent protein constructs.

Identification of Toxoplasma gondii adhesins through a machine learning approach.

Toxoplasma gondii, as other apicomplexa, employs adhesins transmembrane proteins for binding and invasion to host cells. Search and characterization of adhesins is pivotal in understanding Apicomplexa invasion mechanisms and targeting new druggable candidates. This work developed a machine learning software called ApiPredictor UniQE V2.0, based on two approaches: support vector machines and multilayer perceptron, to predict adhesins proteins from amino acid sequences. By using ApiPredictor UniQE V2.0, five SAG-Related Sequences (SRSs) were identified within the Toxoplasma gondii proteome. One of those candidates, TgSRS12B, was cloned in plasmid pEXP5-CT/TOPO and expressed in E. coli BL21 DE3. The resulting recombinant protein was purified via affinity chromatography. Co-precipitation assays in CaCo and Muller cells showed interactions between TgSRS12B-His-tagged and the membrane fractions from both human cell lines. In conclusion, we demonstrated that ApiPredictor UniQE V2.0, a bioinformatic free software, was able to identify TgSRS12B as a new adhesin protein.

Modelling Causal Factors of Unintentional Electromagnetic Emanations Compromising Information Technology Equipment Security.

Information technology equipment (ITE) processing sensitive information can have its security compromised by unintentional electromagnetic radiation. Appropriately assessing likelihood of a potential compromise relies on radio frequency (RF) engineering expertise-specifically, requiring knowledge of the associated causal factors and their interrelationships. Several factors that can cause unintentional electromagnetic emanations that can lead to the compromise of ITE have been found in the literature. This paper confirms the list of causal factors reported in previous work, categorizes the factors as belonging to threat, vulnerability, or impact, and develops an interpretive structural model of the vulnerability factors. A participatory modelling approach was used consisting of focus groups of RF engineers. The resulting hierarchical structural model shows the relationships between factors and illustrates their relative significance. The paper concludes that the resulting model can motivate a deeper understanding of the structural relationship of the factors that can be incorporated in the RF engineers' assessment process. Areas of future work are suggested.

Identifying barriers to sustainable apple production: A stakeholder perspective.

Apple is one of the most important cash crops in China. However, negative economic, environmental and social impacts are associated with its production. This study aims to apply a holistic systems perspective to understand existing problems associated with apple production in China and use this information to improve its sustainability. A structured survey was administered to farmers (n = 245) in Shandong and Shanxi provinces, combined with semi-structured interviews with apple supply chain stakeholders (n = 25). Themes, dimensions and relationships were identified based on an inductive thematic analysis of interview data, and then triangulated against the survey data. Interpretive Structural Modelling and Cross-Impact Matrix Multiplication Applied to Classification methods were applied to investigate interrelationships and effects of the elicited elements within the system. The results indicated that various environmental, economic and social problems are associated with apple production in China, including environmental and health risks associated with synthetic input applications, yield instability, deterioration of apple quality, farmers' uncertainty about accessing routes to market, and the ageing farming workforce. The interaction of socio-economic and supply chain issues has contributed to the system "lock-in" to unsustainable practices within the apple production system. Existing agricultural policies were ineffective as they did not include policy leverage to mitigate the multiple factors driving lock-in to unsustainable practices within the system. The research has provided evidence to enable policymakers to develop effective and targeted strategies to facilitate sustainable production within the apple production system. In particular, the future policy mix should consider the entirety of the food system including perspectives and requirements of different stakeholders. The three-stage approach applied has demonstrated its feasibility of investigating sustainability issues facing a particular industry within a specific cultural and policy context.

Purification and Characterization of a Novel Extracellular Haloprotease Vpr from Bacillus licheniformis Strain KB111.

Research background: Haloalkaline proteases are one of the most interesting types of commercial enzymes in various industries due to their high specific activity and stability under extreme conditions. Biochemical characterization of enzymes is an important requirement for determining their potential for application in industrial fields. Most of microbial proteases have been isolated from Bacillus spp. In this study, the purification and characterization of an extracellular haloprotease produced from Bacillus sp. KB111 strain, which was previously isolated from mangrove forest sediments, are investigated for industrial applications. Experimental approach: The whole genome of KB111 strain was identified by DNA sequencing. Its produced protease was purified by salting out and anion-exchange chromatography, characterized based on protease activity and stability using a peptide substrate, and identified by LC-MS/MS. Results and conclusions: The strain KB111 was identified as Bacillus licheniformis. The molecular mass of its extracellular protease, termed KB-SP, was estimated to be 70 kDa. The optimal pH and temperature for the activity of this protease were 7 and 50 °C, respectively, while the enzyme exhibited maximal activity in the broad salinity range of 2-4 M NaCl. It was fully stable at an alkaline pH range of 7-11 at 50 °C with a half-life of 90 min. Metal ions such as K+, Ca2+ and Mg2+ could enhance the enzyme activity. Therefore, this protease indicates a high potential for the applications in the food and feed industry, as well as the waste management since it can hydrolyse protein at high alkaline pH and salt concentrations. The amino acid profiles of the purified KB-SP determined by LC-MS/MS analysis showed high score matching with the peptidase S8 of B. licheniformis LMG 17339, corresponding to the mature domain of a minor extracellular protease (Vpr). Amino acid sequence alignment and 3D structure modelling of KB-SP showed a conserved catalytic domain, a protease-associated (PA) domain and a C-terminal domain. Novelty and scientific contribution: A novel extracellular haloprotease from B. licheniformis was purified, characterized and identified. The purified protease was identified as being a minor extracellular protease (Vpr) and this is the first report on the halotolerance of Vpr. This protease has the ability to work in harsh conditions, with a broad alkaline pH and salinity range. Therefore, it can be useful in various applications in industrial fields.

Structure-function analysis of ZAR1 immune receptor reveals key molecular interactions for activity.

NLR (nucleotide-binding [NB] leucine-rich repeat [LRR] receptor) proteins are critical for inducing immune responses in response to pathogen proteins, and must be tightly modulated to prevent spurious activation in the absence of a pathogen. The ZAR1 NLR recognizes diverse effector proteins from Pseudomonas syringae, including HopZ1a, and Xanthomonas species. Receptor-like cytoplasmic kinases (RLCKs) such as ZED1, interact with ZAR1 and provide specificity for different effector proteins, such as HopZ1a. We previously developed a transient expression system in Nicotiana benthamiana that allowed us to demonstrate that ZAR1 function is conserved from the Brassicaceae to the Solanaceae. Here, we combined structural modelling of ZAR1, with molecular and functional assays in our transient system, to show that multiple intramolecular and intermolecular interactions modulate ZAR1 activity. We identified determinants required for the formation of the ZARCC oligomer and its activity. Lastly, we characterized intramolecular interactions between ZAR1 subdomains that participate in keeping ZAR1 immune complexes inactive. This work identifies molecular constraints on immune receptor function and activation.

Topological analysis of a bacterial DedA protein associated with alkaline tolerance and antimicrobial resistance.

Maintaining membrane integrity is of paramount importance to the survival of bacteria as the membrane is the site of multiple crucial cellular processes including energy generation, nutrient uptake and antimicrobial efflux. The DedA family of integral membrane proteins are widespread in bacteria and are associated with maintaining the integrity of the membrane. In addition, DedA proteins have been linked to resistance to multiple classes of antimicrobials in various microorganisms. Therefore, the DedA family are attractive targets for the development of new antibiotics. Despite DedA family members playing a key physiological role in many bacteria, their structure, function and physiological role remain unclear. To help illuminate the structure of the bacterial DedA proteins, we performed substituted cysteine accessibility method (SCAM) analysis on the most comprehensively characterized bacterial DedA protein, YqjA from Escherichia coli. By probing the accessibility of 15 cysteine residues across the length of YqjA using thiol reactive reagents, we mapped the topology of the protein. Using these data, we experimentally validated a structural model of YqjA generated using evolutionary covariance, which consists of an α-helical bundle with two re-entrant hairpin loops reminiscent of several secondary active transporters. In addition, our cysteine accessibility data suggest that YqjA forms an oligomer wherein the protomers are arranged in a parallel fashion. This experimentally verified model of YqjA lays the foundation for future work in understanding the function and mechanism of this interesting and important family.

Predicting the zoonotic capacity of mammals to transmit SARS-CoV-2.

Back and forth transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) between humans and animals will establish wild reservoirs of virus that endanger long-term efforts to control COVID-19 in people and to protect vulnerable animal populations. Better targeting surveillance and laboratory experiments to validate zoonotic potential requires predicting high-risk host species. A major bottleneck to this effort is the few species with available sequences for angiotensin-converting enzyme 2 receptor, a key receptor required for viral cell entry. We overcome this bottleneck by combining species' ecological and biological traits with three-dimensional modelling of host-virus protein-protein interactions using machine learning. This approach enables predictions about the zoonotic capacity of SARS-CoV-2 for greater than 5000 mammals-an order of magnitude more species than previously possible. Our predictions are strongly corroborated by in vivo studies. The predicted zoonotic capacity and proximity to humans suggest enhanced transmission risk from several common mammals, and priority areas of geographic overlap between these species and global COVID-19 hotspots. With molecular data available for only a small fraction of potential animal hosts, linking data across biological scales offers a conceptual advance that may expand our predictive modelling capacity for zoonotic viruses with similarly unknown host ranges.

Substitution of histidine 30 by asparagine in manganese superoxide dismutase alters biophysical properties and supports proliferation in a K562 leukemia cell line.

We have generated a mutant of C. elegans manganese superoxide dismutase at histidine 30 by site-directed mutagenesis. The structure was solved at a resolution of 1.52 Å by X-ray crystallography (pdb: 6S0D). His30 was targeted, as it forms as a gateway residue at the top of the solvent access funnel to the active site, together with Tyr34. In the wild-type protein, these gateway residues are involved in the hydrogen-bonding network providing the protons necessary for the catalytic reaction at the metal center. However, biophysical characterization and cell viability experiments reveal that a mutation from histidine to asparagine in the H30N mutant modifies metal selectivity in the protein, favoring the uptake of iron over manganese in minimal media conditions, alters active-site coordination from the characteristic trigonal bipyramidal to octahedral geometry, and encourages cellular proliferation in K562 cells, when added exogenously to the cells.

Confirmatory and bi-factor analysis of the Short Form Health Survey 8 (SF-8) scale structure in a German general population sample.

BACKGROUND: The SF-8 is a short form of the SF-36 Health Survey, which is used for generic assessment of physical and mental aspects of health-related quality of life (HRQoL). Each of the 8 dimensions of the SF-36 is covered by a single item in the SF-8. The aim of the study was to examine the latent model structure of the SF-8. METHOD: One-, two- and three dimensional as well as bi-factor structural models were defined and estimated adopting the ML- as well as the WLSMV-algorithm for ordinal data. The data were collected in a German general population sample (N = 2545 persons). RESULTS: A two- (physical and mental health) and a three-dimensional CFA structure (in addition overall health) represent the empirical data information adequately [CFI = .987/.995; SRMR = .024/.014]. If a general factor is added, the resulting bi-factor models provide a further improvement in data fit [CFI = .999/.998; SRMR = .001]. The individual items are much more highly associated with the general HRQoL factor (loadings: .698 to .908) than with the factors physical, mental, and overall health (loadings: -.206 to .566). CONCLUSIONS: In the SF-8, each item reflects mainly general HRQoL (general factor) as well as one of the three components physical, mental, and overall health. The findings suggest in particular that the evaluation of the information of the SF-8 items can be validly supplemented by a general value HRQoL.

Bovine seminal plasma osteopontin: Structural modelling, recombinant expression and its relationship with semen quality.

Osteopontin (OPN) is a multifunctional phosphoprotein that has been linked to fertility in bulls. However, the exact mechanism by which OPN contributes to fertilisation is yet unknown. The biotechnological use of OPN in bovine reproduction is promising but some gaps remain unfilled. The present work aimed: (a) to verify whether the seminal plasma OPN is associated with seminal traits and a standard breeding soundness exam; (b) to predict OPN interactions with integrins, CD44 and glycosaminoglycans through molecular docking; and (c) to develop a protocol for recombinant expression of OPN from vesicular gland cDNA. Ejaculates from top ranked bulls had higher amounts of seminal plasma OPN in comparison with bulls classified as questionable (p < .01). The structural modelling and molecular docking predictions indicated that bovine OPN binds to heparin disaccharide, hyaluronic acid and hyaluronan. In addition, docking studies described the binding complexes of OPN with CD44 and the integrin heterodimers α5ß1 and αVß3. Finally, expression of rOPN-6His was successfully obtained after 3 hr of induction with 0.5 mM IPTG at 37°C and a denaturing purification protocol resulted in efficiently purified recombinant OPN. The present results contribute to the development of biotechnological uses of OPN as a biomarker in bovine reproduction.

Improving the thermostability of a GH97 dextran glucosidase by rational design.

This study was aimed at improving the thermostability of dextran glucosidase PspAG97A, a member of the glycoside hydrolase family 97, from Pseudoalteromonas sp. K8. A total of 9 lysine residues were chosen using the TKSA-MC program based on the optimization of surface charge-charge interactions and were mutated to glutamate for shifting the enzyme's isoelectric point off its optimum pH value. Three mutants K75E, K363E and K420E showed enhanced thermostability. The triple mutant, K75E/K363E/K420E, was found to be the best with a 7.3-fold increase in half-life (t1/2) at 33 °C compared to that of the wild-type (WT). Most importantly, this mutant showed comparable enzymatic activity to that of the WT protein. Structural modelling demonstrated that increased surface charge-charge interactions and optimization of surface hydrophobic and electrostatic contacts contributed to the improved thermostability displayed by K75E/K363E/K420E.

Further corroboration of distinct functional features in SCN2A variants causing intellectual disability or epileptic phenotypes.

BACKGROUND: Deleterious variants in the voltage-gated sodium channel type 2 (Nav1.2) lead to a broad spectrum of phenotypes ranging from benign familial neonatal-infantile epilepsy (BFNIE), severe developmental and epileptic encephalopathy (DEE) and intellectual disability (ID) to autism spectrum disorders (ASD). Yet, the underlying mechanisms are still incompletely understood. METHODS: To further elucidate the genotype-phenotype correlation of SCN2A variants we investigated the functional effects of six variants representing the phenotypic spectrum by whole-cell patch-clamp studies in transfected HEK293T cells and in-silico structural modeling. RESULTS: The two variants p.L1342P and p.E1803G detected in patients with early onset epileptic encephalopathy (EE) showed profound and complex changes in channel gating, whereas the BFNIE variant p.L1563V exhibited only a small gain of channel function. The three variants identified in ID patients without seizures, p.R937C, p.L611Vfs*35 and p.W1716*, did not produce measurable currents. Homology modeling of the missense variants predicted structural impairments consistent with the electrophysiological findings. CONCLUSIONS: Our findings support the hypothesis that complete loss-of-function variants lead to ID without seizures, small gain-of-function variants cause BFNIE and EE variants exhibit variable but profound Nav1.2 gating changes. Moreover, structural modeling was able to predict the severity of the variant impact, supporting a potential role of structural modeling as a prognostic tool. Our study on the functional consequences of SCN2A variants causing the distinct phenotypes of EE, BFNIE and ID contributes to the elucidation of mechanisms underlying the broad phenotypic variability reported for SCN2A variants.

Key amino acid residues of the AGT1 permease required for maltotriose consumption and fermentation by Saccharomyces cerevisiae.

AIMS: The AGT1 gene encodes for a general α-glucoside-H+ symporter required for efficient maltotriose fermentation by Saccharomyces cerevisiae. In the present study, we analysed the involvement of four charged amino acid residues present in this transporter that are required for maltotriose consumption and fermentation by yeast cells. METHODS AND RESULTS: By using a knowledge-driven approach based on charge, conservation, location, three-dimensional (3D) structural modelling and molecular docking analysis, we identified four amino acid residues (Glu-120, Asp-123, Glu-167 and Arg-504) in the AGT1 permease that could mediate substrate binding and translocation. Mutant permeases were generated by site-directed mutagenesis of these charged residues, and expressed in a yeast strain lacking this permease (agt1∆). While mutating the Arg-504 or Glu-120 residues into alanine totally abolished (R504A mutant) or greatly reduced (E120A mutant) maltotriose consumption by yeast cells, as well as impaired the active transport of several other α-glucosides, in the case of the Asp-123 and Glu-167 amino acids, it was necessary to mutate both residues (D123G/E167A mutant) in order to impair maltotriose consumption and fermentation. CONCLUSIONS: Based on the results obtained with mutant proteins, molecular docking and the localization of amino acid residues, we propose a transport mechanism for the AGT1 permease. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results present new insights into the structural basis for active α-glucoside-H+ symport activity by yeast transporters, providing the molecular bases for improving the catalytic properties of this type of sugar transporters.

A bidirectional path analysis model of smoking cessation self-efficacy and concurrent smoking status: impact on abstinence outcomes.

Self-efficacy is routinely associated with abstinence in the addictions literature, and is a major component relapse-prevention models. The magnitude of this relationship has been brought into question following equivocal results in studies controlling for concurrent smoking status. The aim of our study was to clarify the relationship between cessation self-efficacy, smoking status, and cessation outcomes in a cohort of treatment-seeking smokers. Smokers participating in the FLEX trial, a randomized trial investigating the efficacy of three pharmacologic treatments for smoking cessation, completed questionnaires assessing cessation self-efficacy at baseline and at weeks 1, 3, 5 and 10 post-target quit date; smoking status was verified using expired carbon monoxide. Structural models were fit in order to ascertain the relationship between cessation self-efficacy and concurrent smoking at each time-point, and to assess the association between cessation self-efficacy, smoking and seven-day point prevalence smoking status at week 10. A total of 737 treatment-seeking smokers participated. In our path model, self-efficacy and smoking status at all time points were associated with week 10 abstinence (except week 3 self-efficacy), after controlling these values' previous time-points. All direct pathways between cessation self-efficacy and smoking were also significant, supporting a bidirectional relationship. Our results support a bidirectional and reciprocal relationship between cessation self-efficacy and concurrent smoking behavior; participants with higher confidence were more likely to be smoke-free, and concurrent smoking status predicted levels of confidence over the ensuing weeks. Both measures were associated with week 10 abstinence. Our results indicate that while correlated, both cessation self-efficacy and current smoking behavior during a cessation attempt are important independent markers of ultimate cessation success.