Cognitive processing biases of social anxiety in adults who do and do not stutter.

PURPOSE: Adults who stutter are at risk of developing high levels of social anxiety, leading to negative outcomes and contributing towards stuttering relapse post treatment. To ensure that psychological treatments for social anxiety in stuttering adults are relevant and effective, a broader empirical understanding of the mechanisms of social anxiety in stuttering populations is required. Four key cognitive processing biases identified as maintenance factors in cognitive behavioral models of social anxiety were examined: self-focused attention, safety behavior use, negative self-imagery, and interpretation bias. METHODS: Adults who stutter and non-stuttering adults (N = 186) were assessed via an online survey. Participants were categorised into two groups based on stuttering: formally diagnosed stuttering and non-stuttering. Within those groups, participants were further categorised as having low or high levels of social anxiety. Cognitive processing bias was assessed in response to two hypothetical social scenarios (i.e., social interaction and social performance). RESULTS: As predicted, in both adults who stutter and who are non-stuttering, high social anxiety was related to greater self-focused attention, safety behavior use and negative self-imagery compared to low social anxiety. No significant effect of social anxiety or stuttering was found in relation to interpretation bias. A significant effect of social anxiety upon negative self-imagery was observed in formally-diagnosed adults who stutter compared to non-stuttering adults. CONCLUSION: The results support the use of cognitive behavioral models of social anxiety as a framework for research and treatment development within stuttering populations.

53BP1-the 'Pandora's box' of genome integrity.

53BP1 has several functions in the maintenance of genome integrity. It functions as a key mediator involved in double-strand break (DSB) repair, which functions to maintain a balance in the repair pathway choices and in preserving genomic stability. While its DSB repair functions are relatively well-characterized, its role in DNA replication and replication fork protection is less understood. In response to replication stress, 53BP1 contributes to fork protection by regulating fork reversal and restart. It helps maintain replication fork stability and speed, with 53BP1 loss leading to defective fork progression and increased sensitivity to replication stress agents. However, 53BP1's precise role in fork protection remains debated, as some studies have not observed protective effects. Therefore, it is critical to determine the role of 53BP1 in replication to better understand when it promotes replication fork protection, and the underlying mechanisms involved. Moreover, 53BP1's function in replication stress extends beyond its activity at active replication forks; it also forms specialized nuclear bodies (NBs) which protect stretches of under-replicated DNA (UR-DNA) transmitted from a previous cell cycle to daughter cells through mitosis. The mechanism of 53BP1 NBs in the coordination of replication and repair events at UR-DNA loci is not fully understood and warrants further investigation. The present review article focuses on elucidating 53BP1's functions in replication stress (RS), its role in replication fork protection, and the significance of 53BP1 NBs in this context to provide a more comprehensive understanding of its less well-established role in DNA replication.

NEAT1 modulates the TIRR/53BP1 complex to maintain genome integrity.

Tudor Interacting Repair Regulator (TIRR) is an RNA-binding protein (RBP) that interacts directly with 53BP1, restricting its access to DNA double-strand breaks (DSBs) and its association with p53. We utilized iCLIP to identify RNAs that directly bind to TIRR within cells, identifying the long non-coding RNA NEAT1 as the primary RNA partner. The high affinity of TIRR for NEAT1 is due to prevalent G-rich motifs in the short isoform (NEAT1_1) region of NEAT1. This interaction destabilizes the TIRR/53BP1 complex, promoting 53BP1's function. NEAT1_1 is enriched during the G1 phase of the cell cycle, thereby ensuring that TIRR-dependent inhibition of 53BP1's function is cell cycle-dependent. TDP-43, an RBP that is implicated in neurodegenerative diseases, modulates the TIRR/53BP1 complex by promoting the production of the NEAT1 short isoform, NEAT1_1. Together, we infer that NEAT1_1, and factors regulating NEAT1_1, may impact 53BP1-dependent DNA repair processes, with implications for a spectrum of diseases.

A theory building critical realist evaluation of an integrated cognitive-behavioural fluency enhancing stuttering treatment for school-age children. Part 1: Development of a preliminary program theory from expert speech-language pathologist data.

PURPOSE: This study initiated a program of research that aims to develop a program theory underlying integrated cognitive-behavioural fluency enhancing stuttering treatments for school-age children. This research asks, what in the treatment program works (or does not work), for whom, in what contexts, and why. METHODS: Using a critical realist evaluation approach, seven speech-language pathologists (SLPs) with extensive experience in treating children who stutter were asked about barriers and facilitators of optimal treatment outcomes within the context of the Comprehensive Stuttering Program - School-aged Children (CSP-SC). From these data discrete resource mechanisms, contexts, within child reasoning mechanisms, and outcomes were derived and a preliminary program theory was proposed. RESULTS: Facilitating and impeding child physiology, treatment and SLP resource mechanisms, family and school contexts, and within-child mechanisms were identified. Facilitating mechanisms included motivation, personality/psychological characteristics, understanding and trust of the treatment process, experience of speaking with less effort, and self-efficacy. Impeding mechanisms included reduced motivation, impeding personality/psychological characteristics, lack of buy-in, and, for some children, a prohibitive cost of effort in using learned strategies. CONCLUSION: A preliminary program theory was hypothesized which will be further developed in future analysis of data obtained from children and parents who participated in the CSP-SC at the same centre from which the SLPs came. Subsequent research with new cohorts of SLPs, children, and parents from other treatment programs and centres will be needed to establish the generalizability of the program theory generated in this program of research.

Targeting the Cascade Amplification of Macrophage Colony-stimulating Factor to Alleviate the Immunosuppressive Effects Following Radiotherapy.

Radiotherapy (RT) serves as the primary treatment for solid tumors. Its potential to incite an immune response against tumors both locally and distally profoundly impacts clinical outcomes. However, RT may also promote the accumulation of immunosuppressive cytokines and immunosuppressive cells, greatly impeding the activation of antitumor immune responses and substantially limiting the effectiveness of RT. Therefore, regulating post-RT immunosuppression to steer the immune milieu toward heightened activation potentially enhances RT's therapeutic potential. Cytokines, potent orchestrators of diverse cellular responses, play a pivotal role in regulating this immunosuppressive response. Identifying and promptly neutralizing early released immunosuppressive cytokines are a crucial development in augmenting RT's immunomodulatory effects. To this end, we conducted a screen of immunosuppressive cytokines following RT and identified macrophage colony-stimulating factor (MCSF) as an early up-regulated and persistent immune suppressor. Single-cell sequencing revealed that the main source of up-regulated MCSF derived from tumor cells. Mechanistic exploration revealed that irradiation-dependent phosphorylation of the p65 protein facilitated its binding to the MCSF gene promoter, enhancing transcription. Knockdown and chemical inhibitor experiments conclusively demonstrated that suppressing tumor cell-derived MCSF amplifies RT's immune-activating effects, with optimal results achieved by early MCSF blockade after irradiation. Additionally, we validated that MCSF acted on macrophages, inducing the secretion of a large number of inhibitory cytokines. In summary, we propose a novel approach to enhance the immune activation effects of RT by blocking the MCSF-CSF1R signaling pathway early after irradiation.

In situ vaccination caused by diverse irradiation-driven cell death programs.

Interest surrounding the effect of irradiation on immune activation has exponentially grown within the last decade. This includes work regarding mechanisms of the abscopal effect and the success achieved by combination of radiotherapy and immunotherapy. It is hypothesized that irradiation triggers the immune system to eliminate tumors by inducing tumor cells immunogenic cell death (ICD) in tumor cells. Activation of the ICD pathways can be exploited as an in situ vaccine. In this review, we provide fundamental knowledge of various forms of ICD caused by irradiation, describe the relationship between various cell death pathways and the immune activation effect driven by irradiation, and focus on the therapeutic value of exploiting these cell death programs in the context of irradiation. Furthermore, we summarize the immunomodulatory effect of different cell death programs on combinative radiotherapy and immunotherapy. In brief, differences in cell death programs significantly impact the irradiation-induced immune activation effect. Evaluating the transition between them will provide clues to develop new strategies for radiotherapy and its combination with immunotherapy.

Engineering irradiated tumor-derived microparticles as personalized vaccines to enhance anti-tumor immunity.

The inadequate activation of antigen-presenting cells, the entanglement of T cells, and the highly immunosuppressive conditions in the tumor microenvironment (TME) are important factors that limit the effectiveness of cancer vaccines. Studies show that a personalized and broad antigen repertoire fully activates anti-tumor immunity and that inhibiting the function of transforming growth factor (TGF)-ß facilitates T cell migration. In our study, we introduce a vaccine strategy by engineering irradiated tumor cell-derived microparticles (RT-MPs), which have both personalized and broad antigen repertoire, to induce comprehensive anti-tumor effects. Encouraged by the proinflammatory effects of the spike protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the high affinity between TGF-ß receptor 2 (TGFBR2) and TGF-ß, we develop RT-MPs with the SARS-CoV-2 spike protein and TGFBR2. This spike protein and high TGFBR2 expression induce the innate immune response and ameliorate the immunosuppressive TME, thereby promoting T cell activation and infiltration and ultimately inhibiting tumor growth. Our study provides a strategy for producing an effective personalized anti-tumor vaccine.

Dynamics of the DYNLL1-MRE11 complex regulate DNA end resection and recruitment of Shieldin to DSBs.

The extent and efficacy of DNA end resection at DNA double-strand breaks (DSB) determine the repair pathway choice. Here we describe how the 53BP1-associated protein DYNLL1 works in tandem with the Shieldin complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1, where it limits end resection by binding and disrupting the MRE11 dimer. The Shieldin complex is recruited to a fraction of 53BP1-positive DSBs hours after DYNLL1, predominantly in G1 cells. Shieldin localization to DSBs depends on MRE11 activity and is regulated by the interaction of DYNLL1 with MRE11. BRCA1-deficient cells rendered resistant to PARP inhibitors by the loss of Shieldin proteins can be resensitized by the constitutive association of DYNLL1 with MRE11. These results define the temporal and functional dynamics of the 53BP1-centric DNA end resection factors in cells.

Dynamics of the DYNLL1/MRE11 complex regulates DNA end resection and recruitment of the Shieldin complex to DSBs.

Extent and efficacy of DNA end resection at DNA double strand break (DSB)s determines the choice of repair pathway. Here we describe how the 53BP1 associated protein DYNLL1 works in tandem with Shieldin and the CST complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1 where it limits end resection by binding and disrupting the MRE11 dimer. The Shieldin complex is recruited to a fraction of 53BP1-positive DSBs hours after DYNLL1 predominantly in the G1 cells. Shieldin localization to DSBs is dependent on MRE11 activity and is regulated by the interaction of DYNLL1 with MRE11. BRCA1-deficient cells rendered resistant to PARP inhibitors by the loss of Shieldin proteins can be re-sensitized by the constitutive association of DYNLL1 with MRE11. These results define the temporal and functional dynamics of the 53BP1-centric DNA end resection factors in cells.

Transcription factor Sp1 regulates mitotic chromosome assembly and segregation.

Aneuploidy is a pervasive feature of cancer cells that results from chromosome missegregation. Several transcription factors have been associated with aneuploidy; however, no studies to date have demonstrated that mammalian transcription factors directly regulate chromosome segregation during mitosis. Here, we demonstrate that the ubiquitously expressed transcription factor specificity protein 1 (Sp1), which we have previously linked to aneuploidy, has a mitosis-specific role regulating chromosome segregation. We find that Sp1 localizes to mitotic centromeres and auxin-induced rapid Sp1 degradation at mitotic onset results in chromosome segregation errors and aberrant mitotic progression. Furthermore, rapid Sp1 degradation results in anomalous mitotic chromosome assembly characterized by loss of condensin complex I localization to mitotic chromosomes and chromosome condensation defects. Consistent with these defects, Sp1 degradation results in reduced chromosome passenger complex activity and histone H3 serine 10 phosphorylation during mitosis, which is essential for condensin complex I recruitment and chromosome condensation. Together, these data provide the first evidence of a mammalian transcription factor acting specifically during mitosis to regulate chromosome segregation.

DNA damage-induced sumoylation of Sp1 induces its interaction with RNF4 and degradation in S phase to remove 53BP1 from DSBs and permit HR.

The factors involved in DNA damage recognition and repair are tightly regulated to ensure proper repair pathway choice. The mechanism(s) that determines the cell cycle-dependent turnover of these DNA damage repair factors remains unclear. Here, we show that Sp1, which regulates double-strand break (DSB) repair pathway choice through localization of 53BP1, is sumoylated at Lys16 following DNA damage; Sp1 sumoylation is required for its degradation and the removal of both Sp1 and 53BP1 from DSB sites. Induction of DNA DSBs induces Sp1 phosphorylation at DSBs by ATM, which is necessary for the subsequent sumoylation of Sp1. In addition to this damage-induced ATM-dependent phosphorylation and sumoylation, phosphorylation of Sp1 at Ser59 by Cyclin A/cdk2 upon entry into S phase is necessary for recognition, ubiquitination and degradation by the SUMO-targeted E3 ubiquitin ligase, RNF4. Eliminating Sp1 sumoylation by mutation of Sp1 at Lys16 (K16R) precluded removal of both Sp1 and 53BP1 from DSBs in S phase, resulting in decreased BRCA1 recruitment and defective homologous recombination (HR). Like BRCA1 deficient cells, cells expressing Sp1K16R are sensitive to PARP inhibition due to failure to degrade Sp1 and recruit BRCA1 resulting in defective HR that is rescued by knockdown of 53BP1. These results reveal the dynamic regulation of Sp1 and its role in the assembly and disassembly of DNA repair factors at DSBs.

DNA damage-induced degradation of Sp1 promotes cellular senescence.

Persistent DNA damage (genotoxic stress) triggers signaling cascades that drive cells into apoptosis or senescence to avoid replicating a damaged genome. Sp1 has been found to play a role in double strand break (DSB) repair, and a link between Sp1 and aging has also been established, where Sp1 protein, but not RNA, levels decrease with age. Interestingly, inhibition ATM reverses the age-related degradation of Sp1, suggesting that DNA damage signaling is involved in senescence-related degradation of Sp1. Proteasomal degradation of Sp1 in senescent cells is mediated via sumoylation, where sumoylation of Sp1 on lysine 16 is increased in senescent cells. Taking into consideration our previous findings that Sp1 is phosphorylated by ATM in response to DNA damage and that proteasomal degradation of Sp1 at DSBs is also mediated by its sumoylation and subsequent interaction with RNF4, we investigated the potential contribution of Sp1's role as a DSB repair factor in mediating cellular senescence. We report here that Sp1 expression is decreased with a concomitant increase in senescence markers in response to DNA damage. Mutation of Sp1 at serine 101 to create an ATM phospho-null mutant, or mutation of lysine 16 to create a sumo-null mutant, prevents the sumoylation and subsequent proteasomal degradation of Sp1 and results in a decrease in senescence. Conversely, depletion of Sp1 or mutation of Sp1 to create an ATM phosphomimetic results in premature degradation of Sp1 and an increase in senescence markers. These data link a loss of genomic stability with senescence through the action of a DNA damage repair factor.

Sp1-dependent recruitment of the histone acetylase p300 to DSBs facilitates chromatin remodeling and recruitment of the NHEJ repair factor Ku70.

In response to DNA damage, most factors involved in damage recognition and repair are tightly regulated to ensure proper repair pathway choice. Histone acetylation at DNA double strand breaks (DSBs) by p300 histone acetyltransferase (HAT) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that phosphorylation of Sp1 by ATM increases its interaction with p300 and that Sp1-dependent recruitment of p300 to DSBs is necessary to modify the histones associated with p300 activity and NHEJ repair factor recruitment and repair. p300 is known to acetylate multiple residues on histones H3 and H4 necessary for NHEJ. Acetylation of H3K18 by p300 is associated with the recruitment of the SWI/SNF chromatin remodeling complex and Ku70 to DSBs for NHEJ repair. Depletion of Sp1 results in decreased acetylation of lysines on histones H3 and H4. Specifically, cells depleted of Sp1 display defects in the acetylation of H3K18, resulting in defective SWI/SNF and Ku70 recruitment to DSBs. These results shed light on mechanisms by which chromatin remodelers are regulated to ensure activation of the appropriate DSB repair pathway.

DSB repair pathway choice is regulated by recruitment of 53BP1 through cell cycle-dependent regulation of Sp1.

Although many of the factors, epigenetic changes, and cell cycle stages that distinguish repair of double-strand breaks (DSBs) by homologous recombination (HR) from non-homologous end joining (NHEJ) are known, the underlying mechanisms that determine pathway choice are incompletely understood. Previously, we found that the transcription factor Sp1 is recruited to DSBs and is necessary for repair. Here, we demonstrate that Sp1 localizes to DSBs in G1 and is necessary for recruitment of the NHEJ repair factor, 53BP1. Phosphorylation of Sp1-S59 in early S phase evicts Sp1 and 53BP1 from the break site; inhibition of that phosphorylation results in 53BP1 and Sp1 remaining at DSBs in S phase cells, precluding BRCA1 binding and suppressing HR. Expression of Sp1-S59A increases sensitivity of BRCA1+/+ cells to poly (ADP-ribose) polymerase (PARP) inhibition similar to BRCA1 deficiency. These data demonstrate how Sp1 integrates the cell cycle and DSB repair pathway choice to favor NHEJ.

HDAC6 inhibition promotes α-tubulin acetylation and ameliorates CMT2A peripheral neuropathy in mice.

Charcot-Marie-Tooth type 2A (CMT2A) peripheral neuropathy, the most common axonal form of CMT, is caused by dominantly inherited point mutations in the Mitofusin 2 (Mfn2) gene. It is characterized by progressive length-dependent degeneration of motor and sensory nerves with corresponding clinical features of motor and sensory impairment. There is no cure for CMT, and therapeutic approaches are limited to physical therapy, orthopedic devices, surgery, and analgesics. In this study we focus on histone deacetylase 6 (HDAC6) as a therapeutic target in a mouse model of mutant MFN2 (MFN2R94Q)-induced CMT2A. We report that these mice display progressive motor and sensory dysfunction as well as a significant decrease in α-tubulin acetylation in distal segments of long peripheral nerves. Treatment with a new, highly selective HDAC6 inhibitor, SW-100, was able to restore α-tubulin acetylation and ameliorate motor and sensory dysfunction when given either prior to or after the onset of symptoms. To confirm HDAC6 is the target for ameliorating the CMT2A phenotype, we show that genetic deletion of Hdac6 in CMT2A mice prevents the development of motor and sensory dysfunction. Our findings suggest α-tubulin acetylation defects in distal parts of nerves as a pathogenic mechanism and HDAC6 as a therapeutic target for CMT2A.

Hereditary spastic paraplegia: gain-of-function mechanisms revealed by new transgenic mouse.

Mutations of the SPAST gene, which encodes the microtubule-severing protein spastin, are the most common cause of hereditary spastic paraplegia (HSP). Haploinsufficiency is the prevalent opinion as to the mechanism of the disease, but gain-of-function toxicity of the mutant proteins is another possibility. Here, we report a new transgenic mouse (termed SPASTC448Y mouse) that is not haploinsufficient but expresses human spastin bearing the HSP pathogenic C448Y mutation. Expression of the mutant spastin was documented from fetus to adult, but gait defects reminiscent of HSP (not observed in spastin knockout mice) were adult onset, as is typical of human patients. Results of histological and tracer studies on the mouse are consistent with progressive dying back of corticospinal axons, which is characteristic of the disease. The C448Y-mutated spastin alters microtubule stability in a manner that is opposite to the expectations of haploinsufficiency. Neurons cultured from the mouse display deficits in organelle transport typical of axonal degenerative diseases, and these deficits were worsened by depletion of endogenous mouse spastin. These results on the SPASTC448Y mouse are consistent with a gain-of-function mechanism underlying HSP, with spastin haploinsufficiency exacerbating the toxicity of the mutant spastin proteins. These findings reveal the need for a different therapeutic approach than indicated by haploinsufficiency alone.

α-Tubulin Acetyltransferase Is a Novel Target Mediating Neurite Growth Inhibitory Effects of Chondroitin Sulfate Proteoglycans and Myelin-Associated Glycoprotein.

Damage to the CNS results in neuronal and axonal degeneration, and subsequent neurological dysfunction. Endogenous repair in the CNS is impeded by inhibitory chemical and physical barriers, such as chondroitin sulfate proteoglycans (CSPGs) and myelin-associated glycoprotein (MAG), which prevent axon regeneration. Previously, it has been demonstrated that the inhibition of axonal histone deacetylase-6 (HDAC6) can promote microtubule α-tubulin acetylation and restore the growth of CSPGs- and MAG-inhibited axons. Since the acetylation of α-tubulin is regulated by two opposing enzymes, HDAC6 (deacetylation) and α-tubulin acetyltransferase-1 (αTAT1; acetylation), we have investigated the regulation of these enzymes downstream of a growth inhibitory signal. Our findings show that exposure of primary mouse cortical neurons to soluble CSPGs and MAG substrates cause an acute and RhoA-kinase-dependent reduction in α-tubulin acetylation and αTAT1 protein levels, without changes to either HDAC6 levels or HDAC6 activity. The CSPGs- and MAG-induced reduction in αTAT1 occurs primarily in the distal and middle regions of neurites and reconstitution of αTAT1, either by Rho-associated kinase (ROCK) inhibition or lentiviral-mediated αTAT1 overexpression, can restore neurite growth. Lastly, we demonstrate that CSPGs and MAG signaling decreases αTAT1 levels posttranscriptionally via a ROCK-dependent increase in αTAT1 protein turnover. Together, these findings define αTAT1 as a novel potential therapeutic target for ameliorating CNS injury characterized by growth inhibitory substrates that are prohibitive to axonal regeneration.

Using critical realistic evaluation to support translation of research into clinical practice.

A challenge that speech-language pathologists (SLPs) face is the translation of research into clinical practice. While randomised controlled trials (RCTs) are often touted as the "gold standard" of efficacy research, much valuable information is lost through the process; RCTs by nature are designed to wash out individual client factors and contexts that might influence the outcome in order to present the "true" impact of the intervention. However, in the area of behavioural interventions, the interaction of client factors and contexts with the treatment agent can substantially influence the outcome. This paper provides an overview of the theoretical background and methods involved in critical realistic evaluation (CRE) and discusses its current and potential application to speech-language pathology. CRE is based on the premise that a behavioural intervention cannot be evaluated without considering the context in which it was provided. While the ways in which contextual aspects and treatment mechanisms interact may seem endless, CRE methodology attempts to operationalise them into hypotheses to be empirically tested. Research based on these principles has the potential to support clinical translation of research outcomes and reduce the costs of unsuccessful treatment attempts for SLPs, clients and the service provider.

Case file audit of Lidcombe program outcomes in a student-led stuttering clinic.

PURPOSE: The current study aimed to benchmark clinical outcomes for preschool-aged clients (2;0-5;11 years old) that attended a student-led clinic and undertook the Lidcombe Program. METHOD: A case file audit was undertaken for all preschool clients who attended the clinic between February 2008 and February 2013 and commenced the Lidcombe Program. Clients were grouped according to Stage 1 completion. A mixed ANOVA was used to test for differences between the groups in initial and final percentage syllables stuttered (%SS). Associations between case variable factors and treatment duration were investigated using Pearson correlations. RESULT: Clients who completed Stage 1 had final %SS and severity rating (SR) scores comparable to the literature; however, the median Stage 1 duration was greater. Over half of the clients (57%) withdrew prior to completing Stage 1. These clients had a significantly higher %SS at final treatment session than their completing peers. Initial %SS and SR scores were the only case variables associated with treatment duration. CONCLUSION: Students can achieve the same short-term treatment outcomes for children who stutter using the Lidcombe Program as the current published literature; however, treatment duration is greater and may impact completion. Implications of this for clinical education are discussed.

Parent verbal contingencies during the Lidcombe Program: Observations and statistical modeling of the treatment process.

PURPOSE: The purpose of this study was to document parent presentation of the Lidcombe Program verbal contingencies and model potential relationships between contingency provision and treatment duration. METHODS: Forty parent-child pairs undertaking the Lidcombe Program participated, 26 of whom completed Stage 1. All participants were included in the analyses. Parents completed weekly audio-recordings of treatment during practice sessions and a diary of treatment during natural conversations. The number and types of contingencies provided during practice sessions were counted for 520 recordings. Accelerated failure time modeling was used to investigate associations between contingency provision during the first 4 weeks of treatment and duration of time to complete Stage 1. RESULTS: During practice sessions 91% of contingencies were for stutter-free speech, 6.8% were for stuttering and 2.7% were incorrectly applied. Parents often combined several verbal contingencies into one. During natural conversations, the number of verbal contingencies reportedly provided across the day was low, an average of 8.5 (SD=7.82) contingencies for stutter-free speech and 1.7 (SD=2.43) for unambiguous stuttering. There was a positive, significant relationship between the number of verbal contingencies for stuttering provided during the first 4 weeks of treatment and time taken to complete Stage 1. CONCLUSION: Parents mostly provided the expected types of contingencies but the number was lower than expected. An unexpected association was found between number of verbal contingencies for stuttering and treatment duration. Further research is required to explore the relation between rates of parent verbal contingencies, treatment process duration, and treatment outcome.