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Bacterial mRNAs are organized into operons consisting of discrete open reading frames (ORFs) in a single polycistronic mRNA. Individual ORFs on the mRNA are differentially translated, with rates varying as much as 100-fold. The signals controlling differential translation are poorly understood. Our genome-wide mRNA secondary structure analysis indicated that operonic mRNAs are comprised of ORF-wide units of secondary structure that vary across ORF boundaries such that adjacent ORFs on the same mRNA molecule are structurally distinct. ORF translation rate is strongly correlated with its mRNA structure in vivo, and correlation persists, albeit in a reduced form, with its structure when translation is inhibited and with that of in vitro refolded mRNA. These data suggest that intrinsic ORF mRNA structure encodes a rough blueprint for translation efficiency. This structure is then amplified by translation, in a self-reinforcing loop, to provide the structure that ultimately specifies the translation of each ORF. Proteins make up much of the biological machinery inside cells and perform the essential tasks needed to keep each cell alive. Cells contain thousands of different proteins and the instructions needed to build each protein are encoded in genes. However, these instructions cannot be used directly to manufacture the proteins. Instead, a messenger molecule called mRNA is needed to carry the information stored within genes to the parts of the cell where proteins are made. In bacteria, one mRNA molecule can include information from several genes. This group of genes is called an operon and produces a set of proteins that perform a shared task. Although these proteins work together, some of them are needed in greater numbers than others. Because they are all made using information from the same mRNA, some instructions on the mRNA must be read more times than others. It is unclear how bacterial cells control how many proteins are produced from each part of one mRNA but it is thought to relate to the three-dimensional shape of the molecule itself. Burkhardt, Rouskin, Zhang et al. have now examined the production of proteins from mRNAs in the commonly studied bacterium, Escherichia coli. The results showed that each set of instructions on the mRNA formed a three-dimensional structure that corresponds to the amount of protein produced from that portion of the mRNA. When this three-dimensional structure is more stable or rigid, the corresponding instructions tended to produce fewer proteins than if the structure was relatively simple and unstable. Further investigation showed that these three-dimensional mRNA structures could form spontaneously outside of cells, suggesting that molecules other than the mRNA itself have a relatively small role in controlling the number of proteins produced. This also suggests that the entire structure of each mRNA is important and is likely to be essential for cell survival. The next step is to understand why bacteria organise their genes in this way and how the different mRNA structures control how proteins are produced. Moreover, because many bacteria are used like biological factories to produce a variety of commercially useful molecules, these new insights have the potential to enhance a number of manufacturing processes.

Cells react to their environment through gene regulatory networks. Network integrity requires minimization of undesired crosstalk between their biomolecules. Similar constraints also limit the use of regulators when building synthetic circuits for engineering applications. Here, we mapped the promoter specificities of extracytoplasmic function (ECF) σ s as well as the specificity of their interaction with anti‐ σ s. DNA synthesis was used to build 86 ECF σ s (two from every subgroup), their promoters, and 62 anti‐ σ s identified from the genomes of diverse bacteria. A subset of 20 σ s and promoters were found to be highly orthogonal to each other. This set can be increased by combining the −35 and −10 binding domains from different subgroups to build chimeras that target sequences unrepresented in any subgroup. The orthogonal σ s, anti‐ σ s, and promoters were used to build synthetic genetic switches in Escherichia coli . This represents a genome‐scale resource of the properties of ECF σ s and a resource for synthetic biology, where this set of well‐characterized regulatory parts will enable the construction of sophisticated gene expression programs. The interaction specificities of extracytoplasmic function (ECF) sigma (σ) factors with promoters and their negative regulators (anti‐σs) were mapped to identify non‐crossreacting parts. These orthogonal sets represent a synthetic biology toolbox of genetic switches. Synopsis The interaction specificities of extracytoplasmic function (ECF) sigma (σ) factors with promoters and their negative regulators (anti‐σs) were mapped to identify non‐crossreacting parts. These orthogonal sets represent a synthetic biology toolbox of genetic switches. Part mining was applied to characterize 86 extracytoplasmic function (ECF) σs, their promoters, and 62 anti‐σs identified from the genomes of diverse bacteria. A subset of 20 σs and promoters were found to be highly orthogonal to each other and can be used to build non‐crossreacting switches in single cells. The N‐ and C‐terminal domains from σs from different subgroups can be recombined and recognize the corresponding chimeric promoter. These parts functioned off‐the‐shelf in an E. coli host with minimal re‐engineering and minimally affected host growth and gene expression.

Cellular protein levels are dictated by the balance between gene transcription, mRNA translation and protein degradation, among other factors. Cells must manage their proteomes during stress; one way in which they may do so, in principle, is by differential translation. We used ribosome profiling to directly monitor translation in E. coli at 30 C and investigate how this changes after 10-20 minutes of heat shock at 42 C. Translation is controlled by the interplay of several RNA hybridization processes, which are expected to be temperature sensitive. However, translation efficiencies are robustly maintained after thermal heat shock and after mimicking the heat shock response transcriptional program at 30 C. Several gene-specific parameters correlated with translation efficiency, including predicted mRNA structure and whether a gene is cotranslationally translocated into the inner membrane. Genome-wide predictions of the temperature dependence of mRNA structure suggest that relatively few genes show a melting transition between 30 C and 42 C, consistent with our observations. A linear model with five parameters can predict 33% of the variation in translation efficiency between genes, which may be useful in interpreting transcriptome data.

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19 − disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies. A bedside-to-bench analysis identifies single-chain variable fragment linker length as an important component of chimeric antigen receptor (CAR) structure and suggests that, in contrast to CD28-based CAR T cells, tonic signaling can be beneficial for 4-1BB-based CAR T cell function.

Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.

BackgroundMoyamoya is a chronic occlusive cerebrovascular disease of unknown etiology causing neovascularization of the lenticulostriate collaterals at the base of the brain. Although revascularization surgery is the most effective treatment for moyamoya, there is still no consensus on the best surgical treatment modality as different studies provide different outcomes.ObjectiveIn this large case series, we compare the outcomes of direct (DR) and indirect revascularisation (IR) and compare our results to the literature in order to reflect on the best revascularization modality for moyamoya.MethodsWe conducted a multicenter retrospective study in accordance with the Strengthening the Reporting of Observational studies in Epidemiology guidelines of moyamoya affected hemispheres treated with DR and IR surgeries across 13 academic institutions predominantly in North America. All patients who underwent surgical revascularization of their moyamoya-affected hemispheres were included in the study. The primary outcome of the study was the rate of symptomatic strokes.ResultsThe rates of symptomatic strokes across 515 disease-affected hemispheres were comparable between the two cohorts (11.6% in the DR cohort vs 9.6% in the IR cohort, OR 1.238 (95% CI 0.651 to 2.354), p=0.514). The rate of total perioperative strokes was slightly higher in the DR cohort (6.1% for DR vs 2.0% for IR, OR 3.129 (95% CI 0.991 to 9.875), p=0.052). The rate of total follow-up strokes was slightly higher in the IR cohort (8.1% vs 6.6%, OR 0.799 (95% CI 0.374 to 1.709) p=0.563).ConclusionSince both modalities showed comparable rates of overall total strokes, both modalities of revascularization can be performed depending on the patient’s risk assessment.

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T-cell compartments by supporting homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain incompletely understood. To evaluate homeostasis of the mature B-cell pool during lymphopenia, we turned to an adoptive transfer model of purified follicular B-cells into Rag2-/- mouse recipients. Highly purified follicular B-cells transdifferentiated into marginal zone-like B-cells when transferred into Rag2-/- lymphopenic hosts, but not into wild-type hosts. In lymphopenic spleens, transferred B-cells gradually lost their follicular phenotype and acquired characteristics of marginal zone B-cells, as judged by cell surface phenotype, expression of integrins and chemokine receptors, positioning close to the marginal sinus, and an ability to rapidly generate functional plasma cells. Initiation of follicular to marginal zone B-cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B-cells and expression of Delta-like1 Notch ligands by splenic Ccl19-Cre+ fibroblastic stromal cells. Gene expression analysis showed rapid induction of Notch-regulated transcripts followed by upregulated Myc expression and acquisition of broad transcriptional features of marginal zone B-cells. Thus, naïve mature B-cells are endowed with plastic transdifferentiation potential in response to increased stromal Notch ligand availability during lymphopenia.

Malaria elimination requires tools that interrupt parasite transmission. Here, we characterize B cell receptor responses among Malian adults vaccinated against the first domain of the cysteine-rich 230 kDa gamete surface protein Pfs230, a key protein in sexual stage development of P. falciparum parasites. Among nine Pfs230 human monoclonal antibodies (mAbs) that we generated, one potently blocks transmission to mosquitoes in a complement-dependent manner and reacts to the gamete surface; the other eight show only low or no blocking activity. The structure of the transmission-blocking mAb in complex with vaccine antigen reveals a large discontinuous conformational epitope, specific to domain 1 of Pfs230 and comprising six structural elements in the protein. The epitope is conserved, suggesting the transmission-blocking mAb is broadly functional. This study provides a rational basis to improve malaria vaccines and develop therapeutic antibodies for malaria elimination. Vaccines that interrupt malaria transmission will be important tools for malaria elimination. Here the authors identify a human monoclonal antibody from Pfs230 vaccinated individuals that blocks transmission of Plasmodium falciparum to mosquitoes in a complement-dependent manner and reacts with gamete surface.

Emerging evidence indicates the effectiveness of internet-based mobile-supported stress management interventions (iSMIs) in highly stressed employees. It is yet unclear, however, whether iSMIs are also effective without a preselection process in a universal prevention approach, which more closely resembles routine occupational health care. Moreover, evidence for whom iSMIs might be suitable and for whom not is scarce. The aim of this study was to evaluate the iSMI GET.ON Stress in a universal prevention approach without baseline inclusion criteria and to examine the moderators of the intervention effects. A total of 396 employees were randomly assigned to the intervention group or the 6-month waiting list control group. The iSMI consisted of 7 sessions and 1 booster session and offered no therapeutic guidance. Self-report data were assessed at baseline, 7 weeks, and at 6 months following randomization. The primary outcome was perceived stress. Several a priori defined moderators were explored as potential effect modifiers. Participants in the intervention group reported significantly lower perceived stress at posttreatment (d=0.71, 95% CI 0.51-0.91) and at 6-month follow-up (d=0.61, 95% CI 0.41-0.81) compared to those in the waiting list control group. Significant differences with medium-to-large effect sizes were found for all mental health and most work-related outcomes. Resilience (at 7 weeks, P=.04; at 6 months, P=.01), agreeableness (at 7 weeks, P=.01), psychological strain (at 6 months, P=.04), and self-regulation (at 6 months, P=.04) moderated the intervention effects. This study indicates that iSMIs can be effective in a broad range of employees with no need for preselection to achieve substantial effects. The subgroups that might not profit had extreme values on the respective measures and represented only a very small proportion of the investigated sample, thereby indicating the broad applicability of GET.ON Stress. German Clinical Trials Register DRKS00005699; https://www.drks.de/DRKS00005699.

Stress is highly prevalent and known to be a risk factor for a wide range of physical and mental disorders. The effectiveness of digital stress management interventions has been confirmed; however, research on its economic merits is still limited. This study aims to assess the cost-effectiveness, cost-utility, and cost-benefit of a universal digital stress management intervention for employees compared with a waitlist control condition within a time horizon of 6 months. Recruitment was directed at the German working population. A sample of 396 employees was randomly assigned to the intervention group (n=198) or the waitlist control condition (WLC) group (n=198). The digital stress management intervention included 7 sessions plus 1 booster session, which was offered without therapeutic guidance. Health service use, patient and family expenditures, and productivity losses were self-assessed and used for costing from a societal and an employer's perspective. Costs were related to symptom-free status (PSS-10 [Perceived Stress Scale] score 2 SDs below the study population baseline mean) and quality-adjusted life years (QALYs) gained. The sampling error was handled using nonparametric bootstrapping. From a societal perspective, the digital intervention was likely to be dominant compared with WLC, with a 56% probability of being cost-effective at a willingness-to-pay (WTP) of €0 per symptom-free person gained. At the same WTP threshold, the digital intervention had a probability of 55% being cost-effective per QALY gained relative to the WLC. This probability increased to 80% at a societal WTP of €20,000 per QALY gained. Taking the employer's perspective, the digital intervention showed a probability of a positive return on investment of 78%. Digital preventive stress management for employees appears to be cost-effective societally and provides a favorable return on investment for employers. German Clinical Trials Register DRKS00005699; https://drks.de/search/en/trial/DRKS00005699.