Cross-kingdom expression of synthetic genetic elements promotes discovery of metabolites in the human microbiome.

Small molecules encoded by biosynthetic pathways mediate cross-species interactions and harbor untapped potential, which has provided valuable compounds for medicine and biotechnology. Since studying biosynthetic gene clusters in their native context is often difficult, alternative efforts rely on heterologous expression, which is limited by host-specific metabolic capacity and regulation. Here, we describe a computational-experimental technology to redesign genes and their regulatory regions with hybrid elements for cross-species expression in Gram-negative and -positive bacteria and eukaryotes, decoupling biosynthetic capacity from host-range constraints to activate silenced pathways. These synthetic genetic elements enabled the discovery of a class of microbiome-derived nucleotide metabolites-tyrocitabines-from Lactobacillus iners. Tyrocitabines feature a remarkable orthoester-phosphate, inhibit translational activity, and invoke unexpected biosynthetic machinery, including a class of "Amadori synthases" and "abortive" tRNA synthetases. Our approach establishes a general strategy for the redesign, expression, mobilization, and characterization of genetic elements in diverse organisms and communities.

Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes.

We describe a multiplex genome engineering technology in Saccharomyces cerevisiae based on annealing synthetic oligonucleotides at the lagging strand of DNA replication. The mechanism is independent of Rad51-directed homologous recombination and avoids the creation of double-strand DNA breaks, enabling precise chromosome modifications at single base-pair resolution with an efficiency of >40%, without unintended mutagenic changes at the targeted genetic loci. We observed the simultaneous incorporation of up to 12 oligonucleotides with as many as 60 targeted mutations in one transformation. Iterative transformations of a complex pool of oligonucleotides rapidly produced large combinatorial genomic diversity >105. This method was used to diversify a heterologous ß-carotene biosynthetic pathway that produced genetic variants with precise mutations in promoters, genes, and terminators, leading to altered carotenoid levels. Our approach of engineering the conserved processes of DNA replication, repair, and recombination could be automated and establishes a general strategy for multiplex combinatorial genome engineering in eukaryotes.

Metagenomics harvested genus-specific single-stranded DNA-annealing proteins improve and expand recombineering in Pseudomonas species.

The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications. .

Tuning protein half-life in mouse using sequence-defined biopolymers functionalized with lipids.

The use of biologics in the treatment of numerous diseases has increased steadily over the past decade due to their high specificities, low toxicity, and limited side effects. Despite this success, peptide- and protein-based drugs are limited by short half-lives and immunogenicity. To address these challenges, we use a genomically recoded organism to produce genetically encoded elastin-like polypeptide-protein fusions containing multiple instances of para-azidophenylalanine (pAzF). Precise lipidation of these pAzF residues generated a set of sequence-defined synthetic biopolymers with programmable binding affinity to albumin without ablating the activity of model fusion proteins, and with tunable blood serum half-lives spanning 5 to 94% of albumin's half-life in a mouse model. Our findings present a proof of concept for the use of genetically encoded bioorthogonal conjugation sites for multisite lipidation to tune protein stability in mouse serum. This work establishes a programmable approach to extend and tune the half-life of protein or peptide therapeutics and a technical foundation to produce functionalized biopolymers endowed with programmable chemical and biophysical properties with broad applications in medicine, materials science, and biotechnology.

Hydrogel-based biocontainment of bacteria for continuous sensing and computation.

Genetically modified microorganisms (GMMs) can enable a wide range of important applications including environmental sensing and responsive engineered living materials. However, containment of GMMs to prevent environmental escape and satisfy regulatory requirements is a bottleneck for real-world use. While current biochemical strategies restrict unwanted growth of GMMs in the environment, there is a need for deployable physical containment technologies to achieve redundant, multi-layered and robust containment. We developed a hydrogel-based encapsulation system that incorporates a biocompatible multilayer tough shell and an alginate-based core. This deployable physical containment strategy (DEPCOS) allows no detectable GMM escape, bacteria to be protected against environmental insults including antibiotics and low pH, controllable lifespan and easy retrieval of genomically recoded bacteria. To highlight the versatility of DEPCOS, we demonstrated that robustly encapsulated cells can execute useful functions, including performing cell-cell communication with other encapsulated bacteria and sensing heavy metals in water samples from the Charles River.

Genomes by design.

Next-generation DNA sequencing has revealed the complete genome sequences of numerous organisms, establishing a fundamental and growing understanding of genetic variation and phenotypic diversity. Engineering at the gene, network and whole-genome scale aims to introduce targeted genetic changes both to explore emergent phenotypes and to introduce new functionalities. Expansion of these approaches into massively parallel platforms establishes the ability to generate targeted genome modifications, elucidating causal links between genotype and phenotype, as well as the ability to design and reprogramme organisms. In this Review, we explore techniques and applications in genome engineering, outlining key advances and defining challenges.

External validation of models to predict the outcome of pregnancies of unknown location: a multicentre cohort study.

OBJECTIVE: To validate externally five approaches to predict ectopic pregnancy (EP) in pregnancies of unknown location (PUL): the M6P and M6NP risk models, the two-step triage strategy (2ST, which incorporates M6P), the M4 risk model, and beta human chorionic gonadotropin ratio cut-offs (BhCG-RC). DESIGN: Secondary analysis of a prospective cohort study. SETTING: Eight UK early pregnancy assessment units. POPULATION: Women presenting with a PUL and BhCG >25 IU/l. METHODS: Women were managed using the 2ST protocol: PUL were classified as low risk of EP if presenting progesterone ≤2 nmol/l; the remaining cases returned 2 days later for triage based on M6P. EP risk ≥5% was used to classify PUL as high risk. Missing values were imputed, and predictions for the five approaches were calculated post hoc. We meta-analysed centre-specific results. MAIN OUTCOME MEASURES: Discrimination, calibration and clinical utility (decision curve analysis) for predicting EP. RESULTS: Of 2899 eligible women, the primary analysis excluded 297 (10%) women who were lost to follow up. The area under the ROC curve for EP was 0.89 (95% CI 0.86-0.91) for M6P, 0.88 (0.86-0.90) for 2ST, 0.86 (0.83-0.88) for M6NP and 0.82 (0.78-0.85) for M4. Sensitivities for EP were 96% (M6P), 94% (2ST), 92% (N6NP), 80% (M4) and 58% (BhCG-RC); false-positive rates were 35%, 33%, 39%, 24% and 13%. M6P and 2ST had the best clinical utility and good overall calibration, with modest variability between centres. CONCLUSIONS: 2ST and M6P performed best for prediction and triage in PUL. TWEETABLE ABSTRACT: The M6 model, as part of a two-step triage strategy, is the best approach to characterise and triage PULs.

Differences in post-traumatic stress, anxiety and depression following miscarriage or ectopic pregnancy between women and their partners: multicenter prospective cohort study.

OBJECTIVES: To investigate and compare post-traumatic stress (PTS), depression and anxiety in women and their partners over a 9-month period following miscarriage or ectopic pregnancy. METHODS: This was a prospective cohort study. Consecutive women and their partners were approached in the early pregnancy units of three hospitals in central London. At 1, 3 and 9 months after early pregnancy loss, recruits were e-mailed links to surveys containing the Hospital Anxiety and Depression Scale and the Post-traumatic Stress Diagnostic Scale. The proportion of participants meeting the screening criteria for moderate or severe anxiety or depression and PTS was assessed. Mixed-effects logistic regression was used to analyze differences between women and their partners and their evolution over time. RESULTS: In total, 386 partners were approached after the woman in whom the early pregnancy loss had been diagnosed consented to participate, and 192 couples were recruited. All partners were male. Response rates were 60%, 48% and 39% for partners and 78%, 70% and 59% for women, at 1, 3 and 9 months, respectively. Of the partners, 7% met the criteria for PTS at 1 month, 8% at 3 months and 4% at 9 months, compared with 34%, 26% and 21% of women, respectively. Partners also experienced lower rates of moderate/severe anxiety (6% vs 30% at 1 month, 9% vs 25% at 3 months and 6% vs 22% at 9 months) and moderate/severe depression (2% vs 10% at 1 month, 5% vs 8% at 3 months and 1% vs 7% at 9 months). The odds ratios for psychological morbidity in partners vs women after 1 month were 0.02 (95% CI, 0.004-0.12) for PTS, 0.05 (95% CI, 0.01-0.19) for moderate/severe anxiety and 0.15 (95% CI, 0.02-0.96) for moderate/severe depression. Morbidity for each outcome decreased modestly over time, without strong evidence of a different evolution between women and their partners. CONCLUSIONS: Some partners report clinically relevant levels of PTS, anxiety and depression after pregnancy loss, though to a far lesser extent than women physically experiencing the loss. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Recoded organisms engineered to depend on synthetic amino acids.

Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems, which include bioremediation and probiotics. Although safeguards have been designed to control cell growth by essential gene regulation, inducible toxin switches and engineered auxotrophies, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations. Here we describe the construction of a series of genomically recoded organisms (GROs) whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase pair into the chromosome of a GRO derived from Escherichia coli that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering, we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harbouring three TAG codons in conserved functional residues of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼10(11) cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches. We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternative genetic codes that impart genetic isolation by impeding horizontal gene transfer and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.

DNA-Origami-Based Fluorescence Brightness Standards for Convenient and Fast Protein Counting in Live Cells.

Fluorescence microscopy has been one of the most discovery-rich methods in biology. In the digital age, the discipline is becoming increasingly quantitative. Virtually all biological laboratories have access to fluorescence microscopes, but abilities to quantify biomolecule copy numbers are limited by the complexity and sophistication associated with current quantification methods. Here, we present DNA-origami-based fluorescence brightness standards for counting 5-300 copies of proteins in bacterial and mammalian cells, tagged with fluorescent proteins or membrane-permeable organic dyes. Compared to conventional quantification techniques, our brightness standards are robust, straightforward to use, and compatible with nearly all fluorescence imaging applications, thereby providing a practical and versatile tool to quantify biomolecules via fluorescence microscopy.

First-trimester intrauterine hematoma and pregnancy complications.

OBJECTIVE: To assess whether sonographic diagnosis of intrauterine hematoma (IUH) in the first trimester of pregnancy is associated with first-trimester miscarriage and antenatal, delivery and neonatal complications. METHODS: This was a prospective observational cohort study of women with an intrauterine singleton pregnancy between 5 and 14 weeks' gestation recruited at Queen Charlotte's and Chelsea Hospital, London, UK, between March 2014 and March 2016. Participants underwent serial ultrasound examinations in the first trimester, and the presence, location, size and persistence of any IUH was evaluated. First-trimester miscarriage was defined as pregnancy loss before 14 weeks' gestation. Clinical symptoms, including pelvic pain and vaginal bleeding, were recorded at each visit using validated symptom scores. Antenatal, delivery and neonatal outcomes were obtained from hospital records. Logistic regression analysis and the chi-square test were used to assess the association between the presence and features of IUH and the incidence of adverse pregnancy outcome. Odds ratios (OR) were first adjusted for maternal age (aOR) and then further adjusted for the presence of vaginal bleeding or pelvic pain in the first trimester. RESULTS: Of 1003 women recruited to the study, 946 were included in the final analysis and of these, 268 (28.3%) were diagnosed with an IUH in the first trimester. The presence of IUH was associated with the incidence of preterm birth (aOR, 1.94 (95% CI, 1.07-3.52)), but no other individual or overall antenatal, delivery or neonatal complications. No association was found between the presence of IUH in the first trimester and first-trimester miscarriage (aOR, 0.81 (95% CI, 0.44-1.50)). These findings were independent of the absolute size of the hematoma and the presence of vaginal bleeding or pelvic pain in the first trimester. When IUH was present in the first trimester, there was no association between its size, content or position in relation to the gestational sac and overall antenatal, delivery and neonatal complications. Diagnosis of a retroplacental IUH was associated with an increased risk of overall antenatal complications (P = 0.04). CONCLUSIONS: Our findings demonstrate that there is no association between the presence of IUH in the first trimester and first-trimester miscarriage. However, an association with preterm birth, independently of the presence of symptoms of pelvic pain and/or vaginal bleeding, is evident. Women diagnosed with IUH in the first trimester should be counseled about their increased risk of preterm birth and possibly be offered increased surveillance during the course of their pregnancy. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Triaging women with pregnancy of unknown location using two-step protocol including M6 model: clinical implementation study.

OBJECTIVE: The M6 risk-prediction model was published as part of a two-step protocol using an initial progesterone level of ≤ 2 nmol/L to identify probable failing pregnancies (Step 1) followed by the M6 model (Step 2). The M6 model has been shown to have good triage performance for stratifying women with a pregnancy of unknown location (PUL) as being at low or high risk of harboring an ectopic pregnancy (EP). This study validated the triage performance of the two-step protocol in clinical practice by evaluating the number of protocol-related adverse events and how effectively patients were triaged. METHODS: This was a prospective multicenter interventional study of 3272 women with a PUL, carried out between January 2015 and January 2017 in four district general hospitals and four university teaching hospitals in the UK. The final pregnancy outcome was defined as: a failed PUL (FPUL), an intrauterine pregnancy (IUP) or an EP (including persistent PUL (PPUL)). FPUL and IUP were grouped as low-risk and EP/PPUL as high-risk PUL. Serum progesterone and human chorionic gonadotropin (hCG) levels were measured at presentation in all patients. If the initial progesterone level was ≤ 2 nmol/L, patients were discharged and were asked to have a follow-up urine pregnancy test in 2 weeks to confirm a negative result. If the progesterone level was > 2 nmol/L or a measurement had not been taken, hCG level was measured again at 48 h and results were entered into the M6 model. Patients were managed according to the outcome predicted by the protocol. Those classified as 'low risk, probable FPUL' were advised to perform a urine pregnancy test in 2 weeks and those classified as 'low risk, probable IUP' were invited for a scan a week later. When a woman with a PUL was classified as high risk (i.e. risk of EP ≥ 5%) she was reviewed clinically within 48 h. One center used a progesterone cut-off of ≤ 10 nmol/L and its data were analyzed separately. If the recommended management protocol was not adhered to, this was recorded as a protocol deviation and classified as: unscheduled visit for clinician reason, unscheduled visit for patient reason or incorrect timing of blood test or ultrasound scan. The classifications outlined in the UK Good Clinical Practice (GCP) guidelines were used to evaluate the incidence of adverse events. Data were analyzed using descriptive statistics. RESULTS: Of the 3272 women with a PUL, 2625 were included in the final analysis (317 met the exclusion criteria or were lost to follow-up, while 330 were evaluated using a progesterone cut-off of ≤ 10 nmol/L). Initial progesterone results were available for 2392 (91.1%) patients. In Step 1, 407 (15.5%) patients were classified as low risk (progesterone ≤ 2 nmol/L), of whom seven (1.7%) were ultimately diagnosed with an EP. In 279 of the remaining 2218 women with a PUL, the M6 model was not applied owing to protocol deviation or because the outcome was already known (usually on the basis of an ultrasound scan) before a second hCG reading was taken; of these patients, 30 were diagnosed with an EP. In Step 2, 1038 women with a PUL were classified as low risk, of whom eight (0.8%) had a final outcome of EP. Of 901 women classified as high risk at Step 2, 275 (30.5%) had an EP. Therefore, 275/320 (85.9%) EPs were correctly classified as high risk. Overall, 1445/2625 PUL (55.0%) were classified as low risk, of which 15 (1.0%) were EP. None of these cases resulted in a ruptured EP or significant clinical harm. Sixty-two women participating in the study had an adverse event, but no woman had a serious adverse event as defined in the UK GCP guidelines. CONCLUSIONS: This study has shown that the two-step protocol incorporating the M6 model effectively triaged the majority of women with a PUL as being at low risk of an EP, minimizing the follow-up required for these patients after just two visits. There were few misclassified EPs and none of these women came to significant clinical harm or suffered a serious adverse clinical event. The two-step protocol incorporating the M6 model is an effective and clinically safe way of rationalizing the management of women with a PUL. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Active Targeting of Cancer Cells by Nanobody Decorated Polypeptide Micelle with Bio-orthogonally Conjugated Drug.

Polypeptides are promising carriers for chemotherapeutics: they have minimal toxicity, can be recombinantly synthesized with precise control over molecular weight, and enhance drug pharmacokinetics as self-assembled nanoparticles. Polypeptide-based systems also provide the ability to achieve active targeting with genetically encoded targeting ligands. While passive targeting promotes accumulation of nanocarriers in solid tumors, active targeting provides an additional layer of tunable control and widens the therapeutic window. However, fusion of most targeting proteins to polypeptide carriers exposes the limitations of this approach: the residues that are used for drug attachment are also promiscuously distributed on protein surfaces. We present here a universal methodology to solve this problem by the site-specific attachment of extrinsic moieties to polypeptide drug delivery systems without cross-reactivity to fused targeting domains. We incorporate an unnatural amino acid, p-acetylphenylalanine, to provide a biorthogonal ketone for attachment of doxorubicin in the presence of reactive amino acids in a nanobody-targeted, elastin-like polypeptide nanoparticle. These nanoparticles exhibit significantly greater cytotoxicity than nontargeted controls in multiple cancer cell lines.

Engineering an allosteric transcription factor to respond to new ligands.

Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl ß-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.

Early-pregnancy events and subsequent antenatal, delivery and neonatal outcomes: prospective cohort study.

OBJECTIVE: To assess prospectively the association between pelvic pain, vaginal bleeding, and nausea and vomiting occurring in the first trimester of pregnancy and the incidence of later adverse pregnancy outcomes. METHODS: This was a prospective observational cohort study of consecutive women with confirmed intrauterine singleton pregnancy between 5 and 14 weeks' gestation recruited at Queen Charlotte's & Chelsea Hospital, London, UK, from March 2014 to March 2016. Serial ultrasound scans were performed in the first trimester. Participants completed validated symptom scores for vaginal bleeding, pelvic pain, and nausea and vomiting. The key symptom of interest was any pelvic pain and/or vaginal bleeding during the first trimester. Pregnancies were followed up until the final outcome was known. Antenatal, delivery and neonatal outcomes were obtained from hospital records. Logistic regression analysis was used to assess the association between first-trimester symptoms and pregnancy complications by calculating adjusted odds ratios (aOR) with correction for maternal age. RESULTS: Of 1003 women recruited, 847 pregnancies were included in the final analysis following exclusion of cases due to first-trimester miscarriage (n = 99), termination of pregnancy (n = 20), loss to follow-up (n = 32) or withdrawal from the study (n = 5). Adverse antenatal complications were observed in 166/645 (26%) women with pelvic pain and/or vaginal bleeding in the first trimester (aOR = 1.79; 95% CI, 1.17-2.76) and in 30/181 (17%) women with no symptoms. Neonatal complications were observed in 66/634 (10%) women with and 11/176 (6%) without pelvic pain and/or vaginal bleeding (aOR = 1.73; 95% CI, 0.89-3.36). Delivery complications were observed in 402/615 (65%) women with and 110/174 (63%) without pelvic pain and/or vaginal bleeding during the first trimester (aOR = 1.16; 95% CI, 0.81-1.65). For 18 of 20 individual antenatal complications evaluated, incidence was higher among women with pelvic pain and/or vaginal bleeding, despite the overall incidences being low. Nausea and vomiting in pregnancy showed little association with adverse pregnancy outcomes. CONCLUSIONS: Our study suggests that there is an increased incidence of antenatal complications in women experiencing pelvic pain and/or vaginal bleeding in the first trimester. This should be considered when advising women attending early-pregnancy units. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli.

The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 "recalcitrant" AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional "safe replacement zone" (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.

A-to-I RNA editing occurs at over a hundred million genomic sites, located in a majority of human genes.

RNA molecules transmit the information encoded in the genome and generally reflect its content. Adenosine-to-inosine (A-to-I) RNA editing by ADAR proteins converts a genomically encoded adenosine into inosine. It is known that most RNA editing in human takes place in the primate-specific Alu sequences, but the extent of this phenomenon and its effect on transcriptome diversity are not yet clear. Here, we analyzed large-scale RNA-seq data and detected ∼1.6 million editing sites. As detection sensitivity increases with sequencing coverage, we performed ultradeep sequencing of selected Alu sequences and showed that the scope of editing is much larger than anticipated. We found that virtually all adenosines within Alu repeats that form double-stranded RNA undergo A-to-I editing, although most sites exhibit editing at only low levels (<1%). Moreover, using high coverage sequencing, we observed editing of transcripts resulting from residual antisense expression, doubling the number of edited sites in the human genome. Based on bioinformatic analyses and deep targeted sequencing, we estimate that there are over 100 million human Alu RNA editing sites, located in the majority of human genes. These findings set the stage for exploring how this primate-specific massive diversification of the transcriptome is utilized.

Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.

The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc.

Psychological morbidity associated with hyperemesis gravidarum: a systematic review and meta-analysis.

BACKGROUND: Psychological illness occurring in association with hyperemesis gravidarum (HG) has been widely reported. OBJECTIVE: To determine if there is a higher incidence of psychological morbidity in women with HG compared with women without significant nausea and vomiting in pregnancy. SEARCH STRATEGY: PubMed, MEDLINE, Embase and PsychINFO were searched up to September 2015. SELECTION CRITERIA: Articles referring to psychological morbidity in relation to HG. For meta-analysis case-control studies using numerical scales to compare psychological symptoms. DATA COLLECTION AND ANALYSIS: Articles were independently assessed for inclusion by two reviewers and methodology was appraised using the Newcastle Ottawa Scale. Comparison was made using the standard mean difference (SMD) in symptom scale scores. MAIN RESULTS: In all, 59 articles were included in the systematic review, 12 of these were used in the meta-analysis. Meta-analysis of depression scale scores demonstrated a very large effect with statistically significantly higher depression scale scores in women with HG (SMD 1.22; 95% CI 0.80-1.64; P ≤ 0.01) compared with controls. Meta-analysis of anxiety scores demonstrated a large effect with statistically significantly higher anxiety disorder scale scores in women with HG (SMD 0.86; 95% CI 0.53-1.19; P ≤ 0.01). In both analyses significant heterogeneity was identified (depression and HG I2  = 94%, P ≤ 0.01; anxiety and HG I2  = 84%, P = 0.02). CONCLUSIONS: Our systematic review and meta-analysis have shown a significantly increased frequency of depression and anxiety in women with HG. The findings should prompt service development for women with HG that includes provision of psychological care and support. TWEETABLE ABSTRACT: Meta-analysis demonstrates an increase in #PsychologicalMorbidity in women with #HyperemesisGravidarum.

Multilayered genetic safeguards limit growth of microorganisms to defined environments.

Genetically modified organisms (GMOs) are commonly used to produce valuable compounds in closed industrial systems. However, their emerging applications in open clinical or environmental settings require enhanced safety and security measures. Intrinsic biocontainment, the creation of bacterial hosts unable to survive in natural environments, remains a major unsolved biosafety problem. We developed a new biocontainment strategy containing overlapping 'safeguards'-engineered riboregulators that tightly control expression of essential genes, and an engineered addiction module based on nucleases that cleaves the host genome-to restrict viability of Escherichia coli cells to media containing exogenously supplied synthetic small molecules. These multilayered safeguards maintain robust growth in permissive conditions, eliminate persistence and limit escape frequencies to <1.3 × 10(-12). The staged approach to safeguard implementation revealed mechanisms of escape and enabled strategies to overcome them. Our safeguarding strategy is modular and employs conserved mechanisms that could be extended to clinically or industrially relevant organisms and undomesticated species.