Characterization of a glycan-binding complex of minor pilins completes the analysis of Streptococcus sanguinis type 4 pili subunits.

Type 4 filaments (T4F)-of which type 4 pili (T4P) are the archetype-are a superfamily of nanomachines nearly ubiquitous in prokaryotes. T4F are polymers of one major pilin, which also contain minor pilins whose roles are often poorly understood. Here, we complete the structure/function analysis of the full set of T4P pilins in the opportunistic bacterial pathogen Streptococcus sanguinis. We determined the structure of the minor pilin PilA, which is unexpectedly similar to one of the subunits of a tip-located complex of four minor pilins, widely conserved in T4F. We found that PilA interacts and dramatically stabilizes the minor pilin PilC. We determined the structure of PilC, showing that it is a modular pilin with a lectin module binding a subset of glycans prevalent in the human glycome, the host of S. sanguinis. Altogether, our findings support a model whereby the minor pilins in S. sanguinis T4P form a tip-located complex promoting adhesion to various host receptors. This has general implications for T4F.

PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion.

Type IV pili (T4P) are functionally versatile filamentous nanomachines, nearly ubiquitous in prokaryotes. They are predominantly polymers of one major pilin but also contain minor pilins whose functions are often poorly defined and likely to be diverse. Here, we show that the minor pilin PilB from the T4P of Streptococcus sanguinis displays an unusual bimodular three-dimensional structure with a bulky von Willebrand factor A-like (vWA) module "grafted" onto a small pilin module via a short loop. Structural modeling suggests that PilB is only compatible with a localization at the tip of T4P. By performing a detailed functional analysis, we found that 1) the vWA module contains a canonical metal ion-dependent adhesion site, preferentially binding Mg2+ and Mn2+, 2) abolishing metal binding has no impact on the structure of PilB or piliation, 3) metal binding is important for S. sanguinis T4P-mediated twitching motility and adhesion to eukaryotic cells, and 4) the vWA module shows an intrinsic binding ability to several host proteins. These findings reveal an elegant yet simple evolutionary tinkering strategy to increase T4P functional versatility by grafting a functional module onto a pilin for presentation by the filaments. This strategy appears to have been extensively used by bacteria, in which modular pilins are widespread and exhibit an astonishing variety of architectures.

Cognitive remediation in residential substance use treatment: A randomized stepped-wedge trial.

ABSTRACTExecutive dysfunction is common in individuals with substance use disorder (SUD) and presents a barrier to treatment engagement. The study aimed to investigate the effectiveness of cognitive remediation (CR) for improving executive functioning and treatment retention in patients with SUD, using a stepped-wedge cluster randomized controlled trial. The sample included 527 adults enrolled across ten residential SUD treatment providers in NSW, Australia. The intervention consisted of 12 hours of CR delivered over six weeks in a group format. The comparator was treatment-as-usual (TAU). Primary outcomes included self-reported executive functioning and proportion of treatment completed (PoTC), measured as the number of days in treatment divided by the planned treatment duration. Intention-to-treat analysis did not find significant differences for self-reported executive functioning (mean difference = -2.49, 95%CI [-5.07, 0.09], p = .059) or PoTC (adjusted mean ratio = 1.09, 95%CI [0.88, 1.36], p = .442). Due to high dropout from the intention-to-treat sample (56%) a post-hoc analysis was conducted using a per-protocol approach, in which CR was associated with improved self-reported executive functioning (mean difference = -3.33, 95%CI [-6.10, -0.57], p = .019) and improved likelihood of treatment graduation (adjusted odds ratio = 2.43, 95%CI [1.43, 4.11], p < .001). More research is required to develop a CR approach that results in service-wide treatment effectiveness.

Cognitive Remediation as an Adjunct Treatment for Substance Use Disorders: A Systematic Review.

Substance use disorders are associated with diverse neuropsychological impairments, with deficits in memory and executive functioning commonly observed. Cognitive remediation has been shown to be effective in other populations with cognitive impairments in these domains, including those with psychiatric disorders and acquired brain injuries, and it has been hypothesised to be similarly effective for those in treatment for substance use disorders. We aimed to systematically review the evidence for cognitive remediation interventions administered as an adjunct treatment to substance use rehabilitation. Studies were included if participants were receiving substance use treatment, if improving cognitive functioning was the main focus of the intervention and if they used an experimental design with a control condition receiving treatment-as-usual or an active control intervention. Two independent reviewers agreed on the final selection of 32 studies, encompassing cognitive remediation for working memory, memory, executive functioning and general cognition. Significant differences between intervention and control groups for cognitive test results and treatment outcomes were extracted and compared across treatment approaches. The review found considerable heterogeneity across studies, including in the types of interventions, the nature of participants and the outcome measures used. Further, a lack of quality studies with sufficient power meant that limited conclusions could be drawn, highlighting a need for further replication and research. However, findings indicate that cognitive remediation remains a promising potential avenue for improving cognition and treatment outcomes for those in treatment for substance use disorders. Protocol submitted prospectively to PROSPERO 30.09.2019, CRD42020150978.

The major subunit of widespread competence pili exhibits a novel and conserved type IV pilin fold.

Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, Streptococcus sanguinis (ComGCSS) and Streptococcus pneumoniae (ComGCSP), revealing that this pilin displays extensive structural conservation. Strikingly, ComGCSS and ComGCSP exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.

Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis.

Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, Streptococcus sanguinis In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in S. sanguinis We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins, i.e. a methylated N terminus; (iii) are found in the same heteropolymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin-fold with a highly unusual flexible C terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff than bona fide Tfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show that S. sanguinis Tfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.

A stepped wedge cluster randomised trial of a cognitive remediation intervention in alcohol and other drug (AOD) residential treatment services.

BACKGROUND: Executive functioning impairment is common in substance use disorder and is a major risk factor for poor treatment outcomes, including treatment drop-out and relapse. Cognitive remediation interventions seek to improve executive functioning and offer a promising approach to increase the efficacy of alcohol and other drug (AOD) treatments and improve long-term therapeutic outcomes. This protocol describes a study funded by the NSW Agency for Clinical Innovation that assesses the effectiveness of delivering a six-week group-based intervention of cognitive remediation in an ecologically valid sample of people attending residential AOD treatment services. We primarily aim to investigate whether cognitive remediation will be effective in improving executive functioning and treatment retention rates. We will also evaluate if cognitive remediation may reduce long-term AOD use and rates of health service utilisation, as well as improve personal goal attainment, quality of life, and client satisfaction with treatment. In addition, the study will involve an economic analysis of the cost of delivering cognitive remediation. METHODS/DESIGN: The study uses a stepped wedge cluster randomised design, where randomisation will occur at the cluster level. Participants will be recruited from ten residential AOD treatment services provided by the non-government sector. The intervention will be delivered in 12 one-hour group-based sessions over a period of six weeks. All participants who are expected to receive treatment for the duration of the six-week intervention will be asked to participate in the study. The clusters of participants who are randomly assigned to the treatment condition will complete cognitive remediation in addition to treatment as usual (TAU). Primary and secondary outcome assessments will be conducted at pre-cognitive remediation/TAU phase, post-cognitive remediation/TAU phase, two-month follow-up, four-month follow-up, six-month follow-up, and eight-month follow-up intervals. DISCUSSION: This study will provide comprehensive data on the effect of delivering a cognitive remediation intervention within residential AOD treatment services. If shown to be effective, cognitive remediation may be incorporated as an adjunctive intervention in current treatment programs. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Register (ANZCTR): ACTRN12618001190291 . Prospectively registered 17th July 2018.

Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology.

Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)-exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ. We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the prepilin peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable.

Functional analysis of the interdependence between DNA uptake sequence and its cognate ComP receptor during natural transformation in Neisseria species.

Natural transformation is the widespread biological process by which "competent" bacteria take up free DNA, incorporate it into their genomes, and become genetically altered or "transformed". To curb often deleterious transformation by foreign DNA, several competent species preferentially take up their own DNA that contains specific DUS (DNA uptake sequence) watermarks. Our recent finding that ComP is the long sought DUS receptor in Neisseria species paves the way for the functional analysis of the DUS-ComP interdependence which is reported here. By abolishing/modulating ComP levels in Neisseria meningitidis, we show that the enhancement of transformation seen in the presence of DUS is entirely dependent on ComP, which also controls transformation in the absence of DUS. While peripheral bases in the DUS were found to be less important, inner bases are essential since single base mutations led to dramatically impaired interaction with ComP and transformation. Strikingly, naturally occurring DUS variants in the genomes of human Neisseria commensals differing from DUS by only one or two bases were found to be similarly impaired for transformation of N. meningitidis. By showing that ComPsub from the N. subflava commensal specifically binds its cognate DUS variant and mediates DUS-enhanced transformation when expressed in a comP mutant of N. meningitidis, we confirm that a similar mechanism is used by all Neisseria species to promote transformation by their own, or closely related DNA. Together, these findings shed new light on the molecular events involved in the earliest step in natural transformation, and reveal an elegant mechanism for modulating horizontal gene transfer between competent species sharing the same niche.

Structure and assembly of a trans-periplasmic channel for type IV pili in Neisseria meningitidis.

Type IV pili are polymeric fibers which protrude from the cell surface and play a critical role in adhesion and invasion by pathogenic bacteria. The secretion of pili across the periplasm and outer membrane is mediated by a specialized secretin protein, PilQ, but the way in which this large channel is formed is unknown. Using NMR, we derived the structures of the periplasmic domains from N. meningitidis PilQ: the N-terminus is shown to consist of two ß-domains, which are unique to the type IV pilus-dependent secretins. The structure of the second ß-domain revealed an eight-stranded ß-sandwich structure which is a novel variant of the HSP20-like fold. The central part of PilQ consists of two α/ß fold domains: the structure of the first of these is similar to domains from other secretins, but with an additional α-helix which links it to the second α/ß domain. We also determined the structure of the entire PilQ dodecamer by cryoelectron microscopy: it forms a cage-like structure, enclosing a cavity which is approximately 55 Å in internal diameter at its largest extent. Specific regions were identified in the density map which corresponded to the individual PilQ domains: this allowed us to dock them into the cryoelectron microscopy density map, and hence reconstruct the entire PilQ assembly which spans the periplasm. We also show that the C-terminal domain from the lipoprotein PilP, which is essential for pilus assembly, binds specifically to the first α/ß domain in PilQ and use NMR chemical shift mapping to generate a model for the PilP:PilQ complex. We conclude that passage of the pilus fiber requires disassembly of both the membrane-spanning and the ß-domain regions in PilQ, and that PilP plays an important role in stabilising the PilQ assembly during secretion, through its anchorage in the inner membrane.

Neurocognition of females with substance use disorder and comorbid personality disorder: Divergence in subjective and objective cognition.

At least one in four patients with substance use disorder (SUD) meet criteria for personality disorder and overlapping neurocognitive deficits may reflect shared neurobiological mechanisms. We studied neurocognition in females attending residential SUD treatment by comparing SUD with (n = 20) or without (n = 30) comorbid personality disorder. Neuropsychological testing included working memory, inhibition, shifting, verbal fluency, design fluency, psychomotor speed, immediate and delayed verbal memory, processing speed, premorbid functioning, cognitive screening, and self-reported executive function. As expected, whole-sample deficits included working memory (d = -.91), self-reported executive function (d = -.87), processing speed (d = -.40), delayed verbal memory recall (d = -.39), premorbid functioning (d = -.51), and cognitive screening performance (d = -.61). Importantly, the comorbid personality disorder group showed greater self-reported executive dysfunction (d = -.67) and poorer shifting performance (d = -.65). However, they also evidenced better working memory (d = .84), immediate (d = .95) and delayed (d = .83) verbal memory, premorbid functioning (d = .90), and cognitive screening performance (d = .77). Overall executive dysfunction deficits were concordant with those observed in previous SUD studies. Surprisingly, comorbid personality disorder was associated with a pattern indicating poorer subjective (self-report) but better objective performance on a number of tasks, apart from shifting deficits that may relate to emotion dysregulation. Subjective emotional dysfunction may influence the cognitive deficits observed in the personality disorder group.

Cognitive training and remediation interventions for substance use disorders: a Delphi consensus study.

AIMS: Substance use disorders (SUD) are associated with cognitive deficits that are not always addressed in current treatments, and this hampers recovery. Cognitive training and remediation interventions are well suited to fill the gap for managing cognitive deficits in SUD. We aimed to reach consensus on recommendations for developing and applying these interventions. DESIGN, SETTING AND PARTICIPANTS: We used a Delphi approach with two sequential phases: survey development and iterative surveying of experts. This was an on-line study. During survey development, we engaged a group of 15 experts from a working group of the International Society of Addiction Medicine (Steering Committee). During the surveying process, we engaged a larger pool of experts (n = 54) identified via recommendations from the Steering Committee and a systematic review. MEASUREMENTS: Survey with 67 items covering four key areas of intervention development: targets, intervention approaches, active ingredients and modes of delivery. FINDINGS: Across two iterative rounds (98% retention rate), the experts reached a consensus on 50 items including: (i) implicit biases, positive affect, arousal, executive functions and social processing as key targets of interventions; (ii) cognitive bias modification, contingency management, emotion regulation training and cognitive remediation as preferred approaches; (iii) practice, feedback, difficulty-titration, bias modification, goal-setting, strategy learning and meta-awareness as active ingredients; and (iv) both addiction treatment work-force and specialized neuropsychologists facilitating delivery, together with novel digital-based delivery modalities. CONCLUSIONS: Expert recommendations on cognitive training and remediation for substance use disorders highlight the relevance of targeting implicit biases, reward, emotion regulation and higher-order cognitive skills via well-validated intervention approaches qualified with mechanistic techniques and flexible delivery options.

Psychometric Properties of a New Decision-Making Capacity Assessment Tool for People with Substance Use Disorder: The CAT-CAT.

Individuals with substance use disorder (SUD) often present with cognitive impairments, which may impede their ability to make decisions for themselves, including treatment-related decisions. It is therefore important to assess whether individuals with SUD have adequate decision-making capacity. Indeed, there have not been any capacity assessment tools tailored for use with SUD populations that demonstrate adequate psychometric properties or that have the strong ethical foundation that is required of capacity assessment tools. The Compulsory Assessment and Treatment-Capacity Assessment Tool (CAT-CAT) was designed to fill this gap in the literature. Therefore, the aim of this study was to establish the interrater and test-retest reliability, and discriminative validity of the CAT-CAT. The first of this two-part study recruited healthcare professionals in New Zealand and asked them to conduct a capacity assessment on two hypothetical clients. Generally, excellent interrater reliability (ρ = .998 overall) and test-retest reliability (ρ = .996 overall) was found. The CAT-CAT has exhibited reliability that was at least comparable to widely used capacity assessment tools for other pathologies. The second part of this study involved cognitively normal individuals undergoing capacity assessments to investigate the hypothesis that individuals that do not lack capacity will obtain scores significantly higher than 50% in each section of the CAT-CAT. This hypothesis was met with highly significant results. To conclude, preliminary data suggest that the CAT-CAT has excellent reliability and correctly classifies those with capacity.

The Alcohol and Drug Cognitive Enhancement (ACE) Screening Tool: A simple and brief questionnaire to screen for cognitive impairment in substance use disorder treatment services.

The objective of this study was to determine the test-retest reliability; construct and criterion validity; and test operating characteristics of a newly developed cognitive impairment risk factor screening instrument, the Alcohol and Drug Cognitive Enhancement (ACE) Screening Tool. Participants in the validation study were 129 adults with substance use disorder (SUD) enrolled in residential SUD treatment services and 209 normal controls. Test and retest data were available for 36 participants with SUD and 40 normal control individuals on the ACE Screening Tool. Test-retest reliability was excellent (ICC = 0.97). The ACE Screening Tool was significantly correlated with the Montreal Cognitive Assessment (MoCA), Behavior Rating Inventory of Executive Functioning-Adult Version (BRIEF-A), Test of Premorbid Functioning (TOPF) and Five Point Test, establishing construct validity. Criterion validity was established using a ternary severity variable constructed using results obtained on the MoCA and BRIEF-A. Test operating characteristics analysis showed 93% sensitivity, 46% specificity, 33% positive predictive power, and 96% negative predictive power using a cut-score of >3. Those high levels of sensitivity and negative predictive power indicated that the tool would likely detect cognitive impairment when present and should therefore be considered suitable as an initial screening tool for cognitive impairment in individuals attending SUD services.

Mental contrasting with implementation intentions increases study time for university students.

BACKGROUND: Goal setting is a core aspect of human behaviour that drives action. The intention to achieve one's goals, however, does not necessarily translate into desired outcomes. Although the mental contrasting with implementation intentions (MCII) strategy has demonstrated strong efficacy, limited investigations have been conducted in a university academic goal-setting context. AIMS: The current study sought to investigate the efficacy of MCII in facilitating academic goal attainment in university students. METHOD: Fifty-eight students from Macquarie University, Australia, were randomly allocated to either MCII or stress management training, and were assessed on their progress towards the target goal of increased hours of study four weeks later. Goal attainment scaling (GAS) facilitated the generation of tailored specific goals and was the primary outcome measure. RESULTS: An analysis of covariance indicated that students trained in MCII achieved significantly better goal outcomes than those trained in stress management for both broad (p = .038) and course- or unit-specific (p = .005) study goals. CONCLUSIONS: Results highlight the efficacy of using MCII and GAS in combination to promote increased study time for university students.

Structural determinants of lipid specificity within Ups/PRELI lipid transfer proteins.

Conserved lipid transfer proteins of the Ups/PRELI family regulate lipid accumulation in mitochondria by shuttling phospholipids in a lipid-specific manner across the intermembrane space. Here, we combine structural analysis, unbiased genetic approaches in yeast and molecular dynamics simulations to unravel determinants of lipid specificity within the conserved Ups/PRELI family. We present structures of human PRELID1-TRIAP1 and PRELID3b-TRIAP1 complexes, which exert lipid transfer activity for phosphatidic acid and phosphatidylserine, respectively. Reverse yeast genetic screens identify critical amino acid exchanges that broaden and swap their lipid specificities. We find that amino acids involved in head group recognition and the hydrophobicity of flexible loops regulate lipid entry into the binding cavity. Molecular dynamics simulations reveal different membrane orientations of PRELID1 and PRELID3b during the stepwise release of lipids. Our experiments thus define the structural determinants of lipid specificity and the dynamics of lipid interactions by Ups/PRELI proteins.

The FapF Amyloid Secretion Transporter Possesses an Atypical Asymmetric Coiled Coil.

Gram-negative bacteria possess specialized biogenesis machineries that facilitate the export of amyloid subunits, the fibers of which are key components of their biofilm matrix. The secretion of bacterial functional amyloid requires a specialized outer-membrane protein channel through which unfolded amyloid substrates are translocated. We previously reported the crystal structure of the membrane-spanning domain of the amyloid subunit transporter FapF from Pseudomonas. However, the structure of the periplasmic domain, which is essential for amyloid transport, is yet to be determined. Here, we present the crystal structure of the N-terminal periplasmic domain at 1.8-Å resolution. This domain forms a novel asymmetric trimeric coiled coil that possesses a single buried tyrosine residue as well as an extensive hydrogen-bonding network within a glutamine layer. This new structural insight allows us to understand this newly described functional amyloid secretion system in greater detail.

Cognitive remediation improves executive functions, self-regulation and quality of life in residents of a substance use disorder therapeutic community.

BACKGROUND: Executive dysfunction is common in substance use disorder (SUD) populations and hinders treatment. We previously found that 50% of residents in SUD therapeutic communities had been hospitalized for head injuries; this was a significant determinant of cognitive impairment. The current study aimed to establish whether cognitive remediation improves executive functions (EFs) and self-regulation in an ecologically valid sample of female residents attending SUD therapeutic community treatment, including those with past head injuries and psychiatric comorbidities. METHODS: Controlled sequential groups design with residents (N=33, all female) receiving treatment as usual (TAU). The intervention group (n=16) completed four weeks of cognitive remediation (CR) and the control, TAU only (n=17). Outcome measures assessed pre- and post-intervention included both performance- and inventory-based measures of EFs, and self-reported self-regulation and quality of life. RESULTS: CR relative to TAU significantly improved performance-based assessment of inhibition (Color-Word Interference Test; F=4.29, p=0.047), inventory-based assessment of EFs (Behavior Rating Inventory of Executive Function - Adult Version: Global Executive Composite; F=6.38, p=0.017), impulsivity (Barratt Impulsiveness Scale; F=4.61, p=0.040), self-control (Brief Self-Control Scale; F=5.53, p=0.026), and quality of life (Quality of Life Enjoyment and Satisfaction Questionnaire - Short Form; F=7.68, p=0.010). CONCLUSIONS: Findings suggest that CR improves EFs in a heterogeneous sample of female residents in therapeutic community SUD treatment. Future research may explore the possibility of tailoring CR interventions for various SUD subgroups.