Improving Cell-Free Expression of Model Membrane Proteins by Tuning Ribosome Cotranslational Membrane Association and Nascent Chain Aggregation.

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than that for soluble proteins. In this paper, we study the CFE of membrane proteins and develop a quantitative kinetic model. We identify that ribosome stalling during the translation of membrane proteins is a strong predictor of membrane protein synthesis due to aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes, which incorporate protein nascent chains and, therefore, kinetically compete with aggregation. We show that the balance between peptide-membrane association and peptide aggregation rates determines the yield of the synthesized membrane protein. We define a membrane protein expression score that can be used to rationalize the engineering of lipid composition and the N-terminal domain of a native and computationally designed membrane proteins produced through CFE.

Traumatic Life Events and Association With Depression, Anxiety, and Somatization Symptoms in Female Refugees.

Importance: Different types of traumatic life events have varying impacts on symptoms of depression, anxiety, and somatization. For women from areas of the world experiencing war and humanitarian crises, who have experienced cumulative trauma exposure during war and forced migration, it is not known whether cumulative trauma or particular events have the greatest impact on symptoms. Objective: To examine which traumatic life events are associated with depression, anxiety, and somatization symptoms, compared with the cumulative amount, in a sample of female refugees. Design, Setting, and Participants: For this cross-sectional study, data were collected in 2016 as a part of The Study on Female Refugees. The current analysis was conducted in 2022 to 2023. This multicenter study covered 5 provinces in Germany. Participants were recruited at reception centers for refugees. Women volunteered to participate and to be interviewed after information seminars at the different centers. Exposure: Traumatic life events experienced by refugees from areas of the world experiencing war and humanitarian crises. Main Outcomes and Measures: Demographic variables (age, country of origin, religion, education, relationship status, and children), traumatic and adverse life events, and self-reported depression, anxiety, and somatization symptoms were measured. Random forest regressions simultaneously examined the importance of these variables on symptom scores. Follow-up exploratory mediation models tested potential associative pathways between the identified variables of importance. Results: For the final sample of 620 refugee women (mean [SD] age, 32.34 [10.35] years), family violence was most associated with depression (mean [SD] variable of importance [VIM], 2.93 [0.09]), anxiety (mean [SD] VIM, 4.15 [0.11]), and somatization (mean [SD] VIM, 3.99 [0.15]), even though it was less common than other traumatic experiences, including war, accidents, hunger, or lack of housing. Other factors, such as childhood sexual abuse, injury, near-death experiences, and lack of access to health care, were also important. Follow-up analyses showed partial mediation effects between these factors in their association with symptoms, supporting the unique importance of family violence in understanding mental health. Conclusions and Relevance: The findings of this cross-sectional study of refugee women who experienced multiple severe traumas related to war in their home countries and danger encountered during their migration suggest that family violence was key to their current mental health problems. Culturally sensitive assessment and treatment need to place special emphasis on these family dynamics.

A rapid cell-free expression and screening platform for antibody discovery.

Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly.

Community science designed ribosomes with beneficial phenotypes.

Functional design of ribosomes with mutant ribosomal RNA (rRNA) can expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts are hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we develop an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We apply our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides insights into rRNA sequence-function relationships and has implications for synthetic biology.

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.

Ribosome-mediated incorporation of fluorescent amino acids into peptides in vitro.

We report the design, chemical synthesis, and flexizyme-catalyzed transfer RNA (tRNA) acylation of a variety of fluorescent amino acids (FAAs). The fluorescent groups include pyrene, coumarin, nitrobenzoxadiazole, and fluorescein variants. We further demonstrate site-specific incorporation of the FAAs into peptides by the ribosome in vitro through genetic code reprogramming.

Optimizing Antibiotic Prescribing for Acute Respiratory Tract Infection in German Primary Care: Study Protocol for Evaluation of the RESIST Program.

BACKGROUND: The emergence and increased spread of microbial resistance is a major challenge to all health care systems worldwide. In primary care, acute respiratory tract infection (ARTI) is the health condition most strongly related to antibiotic overuse. OBJECTIVE: The RESIST program aims at optimizing antibiotic prescribing for ARTI in German primary care. By completing a problem-orientated online training course, physicians are motivated and empowered to utilize patient-centered doctor-patient communication strategies, including shared decision making, in the treatment of patients with ARTI. METHODS: RESIST will be evaluated in the form of a nonrandomized controlled trial. Approximately 3000 physicians of 8 (out of 16) German federal states can participate in the program. Patient and physician data are retrieved from routine health care data. Physicians not participating in the program serve as controls, either among the 8 participating regional Associations of Statutory Health Insurance Physicians (control group 1) or among the remaining associations not participating in RESIST (control group 2). Antibiotic prescription rates before the intervention (T0: 2016, 1st and 2nd quarters of 2017) and after the intervention (T1: 3rd quarter of 2017 until 1st quarter of 2019) will be compared. The primary outcome measure is the overall antibiotic prescription rate for all patients insured with German statutory health insurance before and after provision of the online course. The secondary outcome is the antibiotic prescription rate for coded ARTI before and after the intervention. RESULTS: RESIST is publicly funded by the Innovations funds of the Federal Joint Committee in Germany and was approved in December 2016. Recruitment of physicians is now completed, and a total of 2460 physicians participated in the intervention. Data analysis started in February 2020. CONCLUSIONS: With approximately 3000 physicians participating in the program, RESIST is among the largest real-world interventions aiming at reducing inadequate antibiotic prescribing for ARTI in primary care. Long-term follow up of up to 21 months will allow for investigating the sustainability of the intervention. TRIAL REGISTRATION: ISRCTN Registry ISRCTN13934505; http://www.isrctn.com/ISRCTN13934505. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR1-10.2196/18648.

Cell-free styrene biosynthesis at high titers.

Styrene is an important petroleum-derived molecule that is polymerized to make versatile plastics, including disposable silverware and foamed packaging materials. Finding more sustainable methods, such as biosynthesis, for producing styrene is essential due to the increasing severity of climate change as well as the limited supply of fossil fuels. Recent metabolic engineering efforts have enabled the biological production of styrene in Escherichia coli, but styrene toxicity and volatility limit biosynthesis in cells. To address these limitations, we have developed a cell-free styrene biosynthesis platform. The cell-free system provides an open reaction environment without cell viability constraints, which allows exquisite control over reaction conditions and greater carbon flux toward product formation rather than cell growth. The two biosynthetic enzymes required for styrene production were generated via cell-free protein synthesis and mixed in defined ratios with supplemented L-phenylalanine and buffer. By altering the time, temperature, pH, and enzyme concentrations in the reaction, this approach increased the cell-free titer of styrene from 5.36 ± 0.63 mM to 40.33 ± 1.03 mM, the highest amount achieved using biosynthesis without process modifications and product removal strategies. Cell-free systems offer a complimentary approach to cellular synthesis of small molecules, which can provide particular benefits for producing toxic molecules.

Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways.

Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic part function, gene expression patterns, and biosynthetic pathway performance in vitro before implementing designs in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 µg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map aerobic cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.

Mutational characterization and mapping of the 70S ribosome active site.

The synthetic capability of the Escherichia coli ribosome has attracted efforts to repurpose it for novel functions, such as the synthesis of polymers containing non-natural building blocks. However, efforts to repurpose ribosomes are limited by the lack of complete peptidyl transferase center (PTC) active site mutational analyses to inform design. To address this limitation, we leverage an in vitro ribosome synthesis platform to build and test every possible single nucleotide mutation within the PTC-ring, A-loop and P-loop, 180 total point mutations. These mutant ribosomes were characterized by assessing bulk protein synthesis kinetics, readthrough, assembly, and structure mapping. Despite the highly-conserved nature of the PTC, we found that >85% of the PTC nucleotides possess mutational flexibility. Our work represents a comprehensive single-point mutant characterization and mapping of the 70S ribosome's active site. We anticipate that it will facilitate structure-function relationships within the ribosome and make possible new synthetic biology applications.

Strategies for in vitro engineering of the translation machinery.

Engineering the process of molecular translation, or protein biosynthesis, has emerged as a major opportunity in synthetic and chemical biology to generate novel biological insights and enable new applications (e.g. designer protein therapeutics). Here, we review methods for engineering the process of translation in vitro. We discuss the advantages and drawbacks of the two major strategies-purified and extract-based systems-and how they may be used to manipulate and study translation. Techniques to engineer each component of the translation machinery are covered in turn, including transfer RNAs, translation factors, and the ribosome. Finally, future directions and enabling technological advances for the field are discussed.

Low catalytic activity is insufficient to induce disease pathology in triosephosphate isomerase deficiency.

Triosephosphate isomerase (TPI) deficiency is a fatal genetic disorder characterized by hemolytic anemia and neurological dysfunction. Although the enzyme defect in TPI was discovered in the 1960s, the exact etiology of the disease is still debated. Some aspects indicate the disease could be caused by insufficient enzyme activity, whereas other observations indicate it could be a protein misfolding disease with tissue-specific differences in TPI activity. We generated a mouse model in which exchange of a conserved catalytic amino acid residue (isoleucine to valine, Ile170Val) reduces TPI specific activity without affecting the stability of the protein dimer. TPIIle170Val/Ile170Val mice exhibit an approximately 85% reduction in TPI activity consistently across all examined tissues, which is a stronger average, but more consistent, activity decline than observed in patients or symptomatic mouse models that carry structural defect mutant alleles. While monitoring protein expression levels revealed no evidence for protein instability, metabolite quantification indicated that glycolysis is affected by the active site mutation. TPIIle170Val/Ile170Val mice develop normally and show none of the disease symptoms associated with TPI deficiency. Therefore, without the stability defect that affects TPI activity in a tissue-specific manner, a strong decline in TPI catalytic activity is not sufficient to explain the pathological onset of TPI deficiency.

Near-death experiences, attacks by family members, and absence of health care in their home countries affect the quality of life of refugee women in Germany: a multi-region, cross-sectional, gender-sensitive study.

BACKGROUND: The year 2016 has marked the highest number of displaced people worldwide on record. A large number of these refugees are women, yet little is known about their specific situation and the hurdles they have to face during their journey. Herein, we investigated whether sociodemographic characteristics and traumatic experiences in the home country and during the flight affected the quality of life of refugee women arriving in Germany in 2015-2016. METHODS: Six hundred sixty-three women from six countries (Afghanistan, Syria, Iran, Iraq, Somalia, and Eritrea) living in shared reception facilities in five distinct German regions were interviewed by native speakers using a structured questionnaire. Sociodemographic data and information about reasons for fleeing, traumatic experiences, symptoms, quality of life, and expectations towards their future were elicited. All information was stored in a central database in Berlin. Descriptive analyses, correlations, and multivariate analyses were performed. RESULTS: The most frequent reasons cited for fleeing were war, terror, and threat to one's life or the life of a family member. Eighty-seven percent of women resorted to smugglers to make the journey to Europe, and this significantly correlated to residence in a war zone (odds ratio (OR) = 2.5, 95% confidence interval (CI) = 1.4-4.6, p = 0.003) and homelessness prior to fleeing (OR = 2.1, 95% CI = 1-4.3, p = 0.04). Overall the described quality of life by the women was moderate (overall mean = 3.23, range of 1-5) and slightly worse than that of European populations (overall mean = 3.68, p < 0.0001). The main reasons correlating with lower quality of life were older age, having had a near-death experience, having been attacked by a family member, and absence of health care in case of illness. CONCLUSIONS: Refugee women experience multiple traumatic experiences before and/or during their journey, some of which are gender-specific. These experiences affect the quality of life in their current country of residence and might impact their integration. We encourage the early investigation of these traumatic experiences to rapidly identify women at higher risk and to improve health care for somatic and mental illness.

[Costs of Mental Health Care in Patients with Posttraumatic Stress Disorder Related to Sexual Abuse One Year Before and After Inpatient DBT-PTSD]. / Psychiatrische Behandlungskosten von Patientinnen mit Posttraumatischer Belastungsstörung nach sexuellem Missbrauch vor und nach stationärer DBT-PTSD.

Objective In Germany, patients with posttraumatic stress disorder (PTSD) related to childhood sexual abuse (CSA) often receive inpatient treatment. However, data on utilization and costs of mental health care as well as on the impact of trauma-focused treatment are missing. Methods Within the context of a randomized controlled trial mental health service utilization was assessed in female patients with PTSD related to CSA. Data on psychiatric-psychotherapeutic inpatient and outpatient treatment and psychotropic medication was obtained for the year before and after inpatient DBT-PTSD. Results The mean total costs of utilization of psychiatric-psychotherapeutic care and use of psychotropics were €â€Š18.100 per patient in the year before and €â€Š7.233 in the year after DBT-PTSD. The significant cost decrease was due to large reductions in inpatient treatment days (on average 57 days before and 14 days after DBT-PTSD), while outpatient treatment and psychotropic medication remained unchanged. Conclusion PTSD related to CSA is associated with high utilization and costs of mental health care. The results suggest that DBT-PTSD might contribute to reducing the mental health care costs.

Synthetic biology approaches to biological containment: pre-emptively tackling potential risks.

Biocontainment comprises any strategy applied to ensure that harmful organisms are confined to controlled laboratory conditions and not allowed to escape into the environment. Genetically engineered microorganisms (GEMs), regardless of the nature of the modification and how it was established, have potential human or ecological impact if accidentally leaked or voluntarily released into a natural setting. Although all evidence to date is that GEMs are unable to compete in the environment, the power of synthetic biology to rewrite life requires a pre-emptive strategy to tackle possible unknown risks. Physical containment barriers have proven effective but a number of strategies have been developed to further strengthen biocontainment. Research on complex genetic circuits, lethal genes, alternative nucleic acids, genome recoding and synthetic auxotrophies aim to design more effective routes towards biocontainment. Here, we describe recent advances in synthetic biology that contribute to the ongoing efforts to develop new and improved genetic, semantic, metabolic and mechanistic plans for the containment of GEMs.

What mediates the link between childhood maltreatment and depression? The role of emotion dysregulation, attachment, and attributional style.

BACKGROUND: Childhood maltreatment (CM) has been shown to be related to a severe and/or chronic course of depression. This study investigated which psychological processes mediate this relationship. METHOD: A large sample of acute or recovered depressed individuals (N=340) participated in an online survey assessing characteristics of depression, trauma exposure, and potential mediators (emotion regulation difficulties, attributional style, and attachment). RESULTS: The experience of CM was related to more severe depression and more depressive episodes. In multiple mediation models, emotion dysregulation, a depressogenic attributional style, and avoidance in close relationships conjointly mediated the relationship between CM and depression severity as well as number of depressive episodes. However, a significant direct path between CM and depression characteristics remained. Exploratory analyses suggested that posttraumatic stress disorder symptom severity was an important additional mediator in our sample. CONCLUSIONS: Our findings provide preliminary evidence for psychological mediators between CM and depression that may be promising targets for interventions tailored for the treatment of depression in this subgroup.

The metabolic background is a global player in Saccharomyces gene expression epistasis.

The regulation of gene expression in response to nutrient availability is fundamental to the genotype-phenotype relationship. The metabolic-genetic make-up of the cell, as reflected in auxotrophy, is hence likely to be a determinant of gene expression. Here, we address the importance of the metabolic-genetic background by monitoring transcriptome, proteome and metabolome in a repertoire of 16 Saccharomyces cerevisiae laboratory backgrounds, combinatorially perturbed in histidine, leucine, methionine and uracil biosynthesis. The metabolic background affected up to 85% of the coding genome. Suggesting widespread confounding, these transcriptional changes show, on average, 83% overlap between unrelated auxotrophs and 35% with previously published transcriptomes generated for non-metabolic gene knockouts. Background-dependent gene expression correlated with metabolic flux and acted, predominantly through masking or suppression, on 88% of transcriptional interactions epistatically. As a consequence, the deletion of the same metabolic gene in a different background could provoke an entirely different transcriptional response. Propagating to the proteome and scaling up at the metabolome, metabolic background dependencies reveal the prevalence of metabolism-dependent epistasis at all regulatory levels. Urging a fundamental change of the prevailing laboratory practice of using auxotrophs and nutrient supplemented media, these results reveal epistatic intertwining of metabolism with gene expression on the genomic scale.

The return of metabolism: biochemistry and physiology of the pentose phosphate pathway.

The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. The PPP is important to maintain carbon homoeostasis, to provide precursors for nucleotide and amino acid biosynthesis, to provide reducing molecules for anabolism, and to defeat oxidative stress. The PPP shares reactions with the Entner-Doudoroff pathway and Calvin cycle and divides into an oxidative and non-oxidative branch. The oxidative branch is highly active in most eukaryotes and converts glucose 6-phosphate into carbon dioxide, ribulose 5-phosphate and NADPH. The latter function is critical to maintain redox balance under stress situations, when cells proliferate rapidly, in ageing, and for the 'Warburg effect' of cancer cells. The non-oxidative branch instead is virtually ubiquitous, and metabolizes the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate as well as sedoheptulose sugars, yielding ribose 5-phosphate for the synthesis of nucleic acids and sugar phosphate precursors for the synthesis of amino acids. Whereas the oxidative PPP is considered unidirectional, the non-oxidative branch can supply glycolysis with intermediates derived from ribose 5-phosphate and vice versa, depending on the biochemical demand. These functions require dynamic regulation of the PPP pathway that is achieved through hierarchical interactions between transcriptome, proteome and metabolome. Consequently, the biochemistry and regulation of this pathway, while still unresolved in many cases, are archetypal for the dynamics of the metabolic network of the cell. In this comprehensive article we review seminal work that led to the discovery and description of the pathway that date back now for 80 years, and address recent results about genetic and metabolic mechanisms that regulate its activity. These biochemical principles are discussed in the context of PPP deficiencies causing metabolic disease and the role of this pathway in biotechnology, bacterial and parasite infections, neurons, stem cell potency and cancer metabolism.

Sudden losses and sudden gains during a DBT-PTSD treatment for posttraumatic stress disorder following childhood sexual abuse.

BACKGROUND: Exposure-based treatment approaches are first-line interventions for patients suffering from posttraumatic stress disorder (PTSD). However, the dissemination of exposure-based treatments for PTSD is challenging, as a large proportion of clinicians report being concerned about symptoms worsening as a result of this type of intervention and are therefore reluctant to offer it to patients with PTSD. However, there is only little empirical evidence to date on the pattern of symptom worsening during exposure-based treatment for PTSD. OBJECTIVE: The goal of the present study was to explore the frequency of sudden losses and sudden gains in the course of an exposure-based treatment programme for female patients suffering from PTSD related to childhood sexual abuse who also show severe comorbidity. In addition, the relationship between sudden changes and treatment outcome was examined. METHODS: Female participants (N=74) were randomised to either a 12-week residential DBT-PTSD programme or a treatment-as-usual wait list. The pattern of symptom change was assessed via weekly assessments using the Posttraumatic Diagnostic Scale (PDS). Sudden changes were computed as suggested by the literature on sudden gains. RESULTS: During treatment, only one participant (3%) experienced a sudden loss, whereas 25% of participants experienced sudden gains. In the waiting condition, 8% of the participants experienced sudden losses and 5% experienced sudden gains during the same time period. No symptom worsening was observed in response to exposure sessions. However, sudden gains occurred during exposure and non-exposure treatment weeks. Patients with sudden gains showed better treatment outcome in the post-treatment and follow-up assessments. CONCLUSIONS: Exposure-based treatment did not lead to PTSD symptom worsening in the study sample. Results show that sudden gains occur frequently during PTSD treatment and have a prognostic value for treatment outcome.

Non-suicidal self-injury during an exposure-based treatment in patients with posttraumatic stress disorder and borderline features.

Patients with posttraumatic stress disorder (PTSD) and features of borderline personality disorder (BPD) often show non-suicidal self-injury (NSSI). However, patients with on-going NSSI are mostly excluded from PTSD treatments and NSSI during PTSD treatment has rarely been investigated. The aim of the present study was to evaluate the course of NSSI during an exposure-based PTSD treatment. This study focused on a subset (n = 34) of data from a randomised controlled trial that tested the efficacy of a residential PTSD programme (DBT-PTSD) in comparison to a treatment-as-usual wait-list. In this subset we compared a) NSSI during treatment between participants who had or had not engaged in NSSI pre-treatment and b) NSSI between treatment weeks that included exposure interventions vs. those that did not. We further compared the outcome between participants with vs. without NSSI at pre-treatment. At pre-treatment, 62% participants reported on-going NSSI. During treatment, the percentage of participants carrying out NSSI decreased to 38% (p = 0.003). The rates of NSSI were similar in treatment weeks with exposure compared to weeks without. Similar results were observed for the frequency of NSSI. At the end of treatment, participants showed comparable improvement in PTSD symptoms regardless of whether or not they had exhibited NSSI beforehand.