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.

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.

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.

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.

Tpo1-mediated spermine and spermidine export controls cell cycle delay and times antioxidant protein expression during the oxidative stress response.

Cells counteract oxidative stress by altering metabolism, cell cycle and gene expression. However, the mechanisms that coordinate these adaptations are only marginally understood. Here we provide evidence that timing of these responses in yeast requires export of the polyamines spermidine and spermine. We show that during hydrogen peroxide (H2O2) exposure, the polyamine transporter Tpo1 controls spermidine and spermine concentrations and mediates induction of antioxidant proteins, including Hsp70, Hsp90, Hsp104 and Sod1. Moreover, Tpo1 determines a cell cycle delay during adaptation to increased oxidant levels, and affects H2O2 tolerance. Thus, central components of the stress response are timed through Tpo1-controlled polyamine export.

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.

Dialectical behaviour therapy for post-traumatic stress disorder after childhood sexual abuse in patients with and without borderline personality disorder: a randomised controlled trial.

BACKGROUND: Post-traumatic stress disorder (PTSD) with co-occurring severe psychopathology such as borderline personality disorder (BPD) is a frequent sequel of childhood sexual abuse (CSA). CSA-related PTSD has been effectively treated through cognitive-behavioural treatments, but it remains unclear whether success can be achieved in patients with co-occurring BPD. The aim of the present study was to determine the efficacy of a newly developed modular treatment programme (DBT-PTSD) that combines principles of dialectical behaviour therapy (DBT) and trauma-focused interventions. METHODS: Female patients (n = 74) with CSA-related PTSD were randomised to either a 12-week residential DBT-PTSD programme or a treatment-as-usual wait list. About half of the participants met the criteria for co-occurring BPD. Individuals with ongoing self-harm were not excluded. The primary outcomes were reduction of PTSD symptoms as assessed by the Clinician-Administered PTSD Scale (CAPS) and by the Posttraumatic Stress Diagnostic Scale (PDS). Hierarchical linear models were used to compare improvements across treatment groups. Assessments were carried out by blinded raters at admission, at end of treatment, and at 6 and 12 weeks post-treatment. RESULTS: Under DBT-PTSD the mean change was significantly greater than in the control group on both the CAPS (33.16 vs. 2.08) and the PDS (0.70 vs. 0.14). Between-group effect sizes were large and highly significant. Neither a diagnosis of BPD nor the severity or the number of BPD symptoms was significantly related to treatment outcome. Safety analyses indicated no increase in dysfunctional behaviours during the trial. CONCLUSION: DBT-PTSD is an efficacious treatment of CSA-related PTSD, even in the presence of severe co-occurring psychopathology such as BPD.

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.

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.

Combined activity of LACS1 and LACS4 is required for proper pollen coat formation in Arabidopsis.

Very long chain lipids are important components of the plant cuticle that establishes the boundary surface of aerial organs. In addition, these lipids were detected in the extracellular pollen coat (tryphine), where they play a crucial role in appropriate pollen-stigma communication. As such they are involved in the early interaction of pollen with the stigma. A substantial reduction in tryphine lipids was shown to compromise pollen germination and, consequently, resulted in male sterility. We investigated the role of two long-chain acyl-CoA synthetases (LACSs) in Arabidopsis with respect to their contribution to the production of tryphine lipids. LACS was shown to provide CoA-activated very long chain fatty acids (VLCFA-CoAs) to the pathways of wax biosynthesis. The allocation of sufficient quantities of VLCFA-CoA precursors should therefore be relevant to the generation of tryphine lipids. Here, we report on the identification of lacs1 lacs4 double knock-out mutant lines that were conditionally sterile and showed significant reductions in pollen coat lipids. Whereas the contributions of both LACS proteins to surface wax levels were roughly additive, their co-operation in tryphine lipid biosynthesis was clearly more complex. The inactivation of LACS4 resulted in increased levels of tryphine lipids accompanied by morphological anomalies of the pollen grains. The additional inactivation of LACS1 neutralized the morphological defects, decreased the tryphine lipids far below wild-type levels and resulted in conditionally sterile pollen.

Analysis of new type III effectors from Xanthomonas uncovers XopB and XopS as suppressors of plant immunity.

The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is dependent on type III effectors (T3Es) that are injected into plant cells by a type III secretion system and interfere with cellular processes to the benefit of the pathogen. In this study, we analyzed eight T3Es from Xcv strain 85-10, six of which were newly identified effectors. Genetic studies and protoplast expression assays revealed that XopB and XopS contribute to disease symptoms and bacterial growth, and suppress pathogen-associated molecular pattern (PAMP)-triggered plant defense gene expression. In addition, XopB inhibits cell death reactions induced by different T3Es, thus suppressing defense responses related to both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). XopB localizes to the Golgi apparatus and cytoplasm of the plant cell and interferes with eukaryotic vesicle trafficking. Interestingly, a XopB point mutant derivative was defective in the suppression of ETI-related responses, but still interfered with vesicle trafficking and was only slightly affected with regard to the suppression of defense gene induction. This suggests that XopB-mediated suppression of PTI and ETI is dependent on different mechanisms that can be functionally separated.

[Psychological treatment of posttraumatic stress disorder after sexual abuse: an overview]. / Psychotherapie der Posttraumatischen Belastungsstörung nach sexuellem Missbrauch: Ein Überblick über die Datenlage.

There is an ongoing debate how to treat posttraumatic stress disorder related to childhood sexual abuse. In Germany patients mostly receive a psychodynamically oriented treatment with a long-lasting stabilization before the use of exposure-based interventions. The number of randomized controlled trials on posttraumatic stress disorder related to childhood sexual abuse is quite limited. The results of these studies show that cognitive-behavioral trauma-focussing interventions are very efficacious with large effect sizes. 2 controlled studies on psychodynamically oriented treatment found only small improvements in posttraumatic symptoms. The high dropout rates in prolonged exposure especially in patients with co-occurring personality disorders point towards the need of a emotion regulation training before the exposure phase. Future studies should include subgroup-analyses and the assessment of adverse effects during therapy.

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.

[Dialectical behavior therapy for posttraumatic stress disorder in survivors of childhood sexual abuse]. / Dialektisch Behaviorale Therapie für Posttraumatische Belastungsstörung nach sexualisierter Gewalt in der Kindheit und Jugend (DBT-PTSD).

So far, no specifically tailored and empirically evaluated psychological treatment program exists, which is tailored for adult survivors with posttraumatic stress disorder (PTSD) after childhood sexual abuse. At the Central Institute of Mental Health, Mannheim we developed Dialectical Behavioral Therapy for PTSD (DBT-PTSD) as a residential intensive program, which is specifically tailored to the needs of these patients and which is acceptable as well as tolerable for patients and therapists. The treatment program is mainly based on the principles and methods of Dialectical Behavior Therapy according to M. Linehan and integrates methods of traumafocused cognitive-behavioural therapy. An overview is given on the treatment rational, the dynamic hierarchy of treatment focusses and the interventions used. In the internet version of this article treatment application is exemplified in a case study.

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.

[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.