Parageobacillus thermoglucosidasius Strain Engineering Using a Theophylline Responsive RiboCas for Controlled Gene Expression.

The relentless increase in atmospheric greenhouse gas concentrations as a consequence of the exploitation of fossil resources compels the adoption of sustainable routes to chemical and fuel manufacture based on biological fermentation processes. The use of thermophilic chassis in such processes is an attractive proposition; however, their effective exploitation will require improved genome editing tools. In the case of the industrially relevant chassis Parageobacillus thermoglucosidasius, CRISPR/Cas9-based gene editing has been demonstrated. The constitutive promoter used, however, accentuates the deleterious nature of Cas9, causing decreased transformation and low editing efficiencies, together with an increased likelihood of off-target effects or alternative mutations. Here, we rectify this issue by controlling the expression of Cas9 through the use of a synthetic riboswitch that is dependent on the nonmetabolized, nontoxic, and cheap inducer, theophylline. We demonstrate that the riboswitches are dose-dependent, allowing for controlled expression of the target gene. Through their use, we were then able to address the deleterious nature of Cas9 and produce an inducible system, RiboCas93. The benefits of RiboCas93 were demonstrated by increased transformation efficiency of the editing vectors, improved efficiency in mutant generation (100%), and a reduction of Cas9 toxicity, as indicated by a reduction in the number of single nucleotide polymorphisms (SNPs) observed. This new system provides a quick and efficient way to produce mutants in P. thermoglucosidasius.

An Experimental and Computational Approach to Development of Mixing Processes for Miscible Liquids in Unbaffled Tanks.

PURPOSE: Mixing of liquids is a critical unit operation in the biopharmaceutical drug product manufacturing. It commonly consists of mixing miscible liquids to dilute bulk drug substance (DS) or pool multiple lots of drug substance. In the past, at-scale mixing studies have been conducted to determine the mixing parameters, namely mixing speed, and mixing time. At-scale studies have historically been utilized to overcome the challenges associated with geometric dissimilarity of mixing systems found when scaling up. In addition, such studies are quite costly, as they often use actual DS to overcome a lack of representativeness associated with simple salt trace models often employed. As a result, there is a significant need for alternative cost-effective methods that can predict mixing parameters with close agreement to actual experiments and operations. METHOD: At-scale mixing experiments were conducted using full-sized tanks and surrogate solutions. Several computational fluid dynamic (CFD) simulation methods were conducted and compared with the experiments to determine the most reliable computational techniques. RESULTS: The experiments demonstrate that surrogate solutions can be used reliably to determine mixing parameters in at-scale studies instead of the valuable drug products. Studying different CFD methods also showed that transient simulations that use a large eddy simulation (LES) viscous model and a sliding mesh can correctly predict the mixing parameters. CONCLUSION: Results of this study establish a practical and reliable methodology to perform mixing studies for miscible liquids with different kinematic viscosities. The methods discussed herein greatly reduce the routine mixing study costs in the biopharmaceutical industry and increase efficiency and accuracy of the results, allowing proactive scale-up of mixing operations.

Metabolic engineering for the production of acetoin and 2,3-butanediol at elevated temperature in Parageobacillus thermoglucosidasius NCIMB 11955.

The current climate crisis has emphasised the need to achieve global net-zero by 2050, with countries being urged to set considerable emission reduction targets by 2030. Exploitation of a fermentative process that uses a thermophilic chassis can represent a way to manufacture chemicals and fuels through more environmentally friendly routes with a net reduction in greenhouse gas emissions. In this study, the industrially relevant thermophile Parageobacillus thermoglucosidasius NCIMB 11955 was engineered to produce 3-hydroxybutanone (acetoin) and 2,3-butanediol (2,3-BDO), organic compounds with commercial applications. Using heterologous acetolactate synthase (ALS) and acetolactate decarboxylase (ALD) enzymes, a functional 2,3-BDO biosynthetic pathway was constructed. The formation of by-products was minimized by the deletion of competing pathways surrounding the pyruvate node. Redox imbalance was addressed through autonomous overexpression of the butanediol dehydrogenase and by investigating appropriate aeration levels. Through this, we were able to produce 2,3-BDO as the predominant fermentation metabolite, with up to 6.6 g/L 2,3-BDO (0.33 g/g glucose) representing 66% of the theoretical maximum at 50°C. In addition, the identification and subsequent deletion of a previously unreported thermophilic acetoin degradation gene (acoB1) resulted in enhanced acetoin production under aerobic conditions, producing 7.6 g/L (0.38 g/g glucose) representing 78% of the theoretical maximum. Furthermore, through the generation of a ΔacoB1 mutant and by testing the effect of glucose concentration on 2,3-BDO production, we were able to produce 15.6 g/L of 2,3-BDO in media supplemented with 5% glucose, the highest titre of 2,3-BDO produced in Parageobacillus and Geobacillus species to date.

Development of Fluorescent Turn-On Probes for CAG-RNA Repeats.

Fluorescent sensing of nucleic acids is a highly sensitive and efficient bioanalytical method for their study in cellular processes, detection and diagnosis in related diseases. However, the design of small molecule fluorescent probes for the selective binding and detection of RNA of a specific sequence is very challenging because of their diverse, dynamic, and flexible structures. By modifying a bis(amidinium)-based small molecular binder that is known to selectively target RNA with CAG repeats using an environment-sensitive fluorophore, a turn-on fluorescent probe featuring aggregation-induced emission (AIE) is successfully developed in this proof-of-concept study. The probe (DB-TPE) exhibits a strong, 19-fold fluorescence enhancement upon binding to a short CAG RNA, and the binding and fluorescence response was found to be specific to the overall RNA secondary structure with A·A mismatches. These promising analytical performances suggest that the probe could be applied in pathological studies, disease progression monitoring, as well as diagnosis of related neurodegenerative diseases due to expanded CAG RNA repeats.

pMTL60000: A modular plasmid vector series for Parageobacillus thermoglucosidasius strain engineering.

Parageobacillus thermoglucosidasius is a promising chassis for producing chemicals and fuels. Here we designed, built and tested the pMTL60000 modular plasmids containing standardised Gram-positive and Gram-negative replicons, selectable markers and application-specific modules. The pMTL60000 modular plasmids were characterised with regard to transformation efficiency, segregational stability, copy number and compatibility.

A large-scale study on research code quality and execution.

This article presents a study on the quality and execution of research code from publicly-available replication datasets at the Harvard Dataverse repository. Research code is typically created by a group of scientists and published together with academic papers to facilitate research transparency and reproducibility. For this study, we define ten questions to address aspects impacting research reproducibility and reuse. First, we retrieve and analyze more than 2000 replication datasets with over 9000 unique R files published from 2010 to 2020. Second, we execute the code in a clean runtime environment to assess its ease of reuse. Common coding errors were identified, and some of them were solved with automatic code cleaning to aid code execution. We find that 74% of R files failed to complete without error in the initial execution, while 56% failed when code cleaning was applied, showing that many errors can be prevented with good coding practices. We also analyze the replication datasets from journals' collections and discuss the impact of the journal policy strictness on the code re-execution rate. Finally, based on our results, we propose a set of recommendations for code dissemination aimed at researchers, journals, and repositories.

Tree genotypes affect rock lichens and understory plants: examples of trophic-independent interactions.

Genetic variation in foundation tree species can strongly influence communities of trophic-dependent organisms, such as herbivorous insects, pollinators, and mycorrhizal fungi. However, the extent and manner in which this variation results in unexpected interactions that reach trophic-independent organisms remains poorly understood, even though these interactions are essential to understanding complex ecosystems. In pinyon-juniper woodland at Sunset Crater (Arizona, USA), we studied pinyon (Pinus edulis) that were either resistant or susceptible to stem-boring moths (Dioryctria albovittella). Moth herbivory alters the architecture of susceptible trees, thereby modifying the microhabitat beneath their crowns. We tested the hypothesis that this interaction between herbivore and tree genotype extends to affect trophic-independent communities of saxicolous (i.e., growing on rocks) lichens and bryophytes and vascular plants beneath their crowns. Under 30 pairs of moth-resistant and moth-susceptible trees, we estimated percent cover of lichens, bryophytes, and vascular plants. We also quantified the cover of leaf litter and rocks as well as light availability. Four major findings emerged. (1) Compared to moth-resistant trees, which exhibited monopodial architecture, the microhabitat under the shrub-like susceptible trees was 60% darker and had 21% more litter resulting in 68% less rock exposure. (2) Susceptible trees had 56% and 87% less cover, 42% and 80% less richness, and 38% and 92% less diversity of saxicolous and plant communities, respectively, compared to resistant trees. (3) Both saxicolous and plant species accumulated at a slower rate beneath susceptible trees, suggesting an environment that might inhibit colonization and/or growth. (4) Both saxicolous and plant communities were negatively affected by the habitat provided by susceptible trees. The results suggest that herbivory of moth-susceptible trees generated litter at high enough rates to reduce rock substrate availability, thereby suppressing the saxicolous communities. However, our results did not provide a causal pathway explaining the suppression of vascular plants. Nonetheless, the cascading effects of genetic variation in pinyon appear to extend beyond trophic-dependent moths to include trophic-independent saxicolous and vascular plant communities that are affected by specific tree-herbivore interactions that modify the local environment. We suggest that such genetically based interactions are common in nature and contribute to the evolution of complex communities.

Development of a Suite of Tools for Genome Editing in Parageobacillus thermoglucosidasius and Their Use to Identify the Potential of a Native Plasmid in the Generation of Stable Engineered Strains.

The relentless rise in the levels of atmospheric greenhouse gases caused by the exploitation of fossil fuel necessitates the development of more environmentally friendly routes to the manufacture of chemicals and fuels. The exploitation of a fermentative process that uses a thermophilic chassis represents an attractive option. Its use, however, is hindered by a dearth of genetic tools. Here we expand on those available for the engineering of the industrial chassis Parageobacillus thermoglucosidasius through the assembly and testing of a range of promoters, ribosome binding sites, reporter genes, and the implementation of CRISPR/Cas9 genome editing based on two different thermostable Cas9 nucleases. The latter were used to demonstrate that the deletion of the two native plasmids carried by P. thermoglucosidasius, pNCI001 and pNCI002, either singly or in combination, had no discernible effects on the overall phenotypic characteristics of the organism. Through the CRISPR/Cas9-mediated insertion of the gene encoding a novel fluorescent reporter, eCGP123, we showed that pNCI001 exhibited a high degree of segregational stability. As the relatively higher copy number of pNCI001 led to higher levels of eCGP123 expression than when the same gene was integrated into the chromosome, we propose that pNCI001 represents the preferred option for the integration of metabolic operons when stable commercial strains are required.

Glutathione S-transferase A2 promotes hepatocellular carcinoma recurrence after liver transplantation through modulating reactive oxygen species metabolism.

Hepatocellular carcinoma (HCC) recurrence after liver transplantation remains a significant clinical problem. Ischemia-reperfusion injury (IRI) occurred inevitably at the early phase after liver transplantation (LT) spawns a significant risk of HCC recurrence. However, their linkage and IRI-derived risk factors for HCC recurrence remain exclusive. Understanding the mechanism of post-transplantation hepatic injury could provide new strategies to prevent the later event of HCC recurrence. We demonstrated that glutathione S-transferase A2 (GSTA2) expression was significantly associated with early phase hepatic and systemic injury and ROS level after liver transplantation. Early phase circulating GSTA2 (EPCGSTA2) protein was a significant predictor of HCC recurrence and survival. Heterogeneous single nucleotide polymorphism at G335C of GSTA2 was significantly associated with poor survival of HCC recipients. Enhancement of GSTA2 could protect HCC cells against H2O2-induced cell death by compensating for the elevated ROS stress. We also demonstrated that GSTA2 played crucial roles in regulating the ROS-associated JNK and AKT signaling pathways and ROS metabolism in HCCs in responding to a dynamic ROS environment. Functionally, endogenous or exogenous upregulation of GSTA2 could promote HCC growth and invasion through activating the epithelial-mesenchymal-transition process. Targeted inhibition of GSTA2 could suppress HCC growth and metastasis. In conclusion, GSTA2 could be a novel prognostic and therapeutic target to combat HCC recurrence after liver transplantation.

Novel molecular biomarkers and diagnosis of acute appendicitis in children.

Reliable and efficient diagnosis of pediatric appendicitis is essential for the establishment of a clinical management plan and improvement of patient outcomes. Current strategies used to diagnose a child presenting with a suspected appendicitis include laboratory studies, clinical scores and diagnostic imaging. Although these modalities work in conjunction with each other, one optimal diagnostic strategy has yet to be agreed upon. The recent introduction of precision medicine techniques such as genomics, transcriptomics, proteomics and metabolomics has increased both the diagnostic sensitivity and specificity of appendicitis. Using these novel strategies, the integration of precision medicine into clinical practice via point-of-care technologies is a plausible future. These technologies would assist in the screening, diagnosis and prognosis of pediatric appendicitis.

CAG RNAs induce DNA damage and apoptosis by silencing NUDT16 expression in polyglutamine degeneration.

DNA damage plays a central role in the cellular pathogenesis of polyglutamine (polyQ) diseases, including Huntington's disease (HD). In this study, we showed that the expression of untranslatable expanded CAG RNA per se induced the cellular DNA damage response pathway. By means of RNA sequencing (RNA-seq), we found that expression of the Nudix hydrolase 16 (NUDT16) gene was down-regulated in mutant CAG RNA-expressing cells. The loss of NUDT16 function results in a misincorporation of damaging nucleotides into DNAs and leads to DNA damage. We showed that small CAG (sCAG) RNAs, species generated from expanded CAG transcripts, hybridize with CUG-containing NUDT16 mRNA and form a CAG-CUG RNA heteroduplex, resulting in gene silencing of NUDT16 and leading to the DNA damage and cellular apoptosis. These results were further validated using expanded CAG RNA-expressing mouse primary neurons and in vivo R6/2 HD transgenic mice. Moreover, we identified a bisamidinium compound, DB213, that interacts specifically with the major groove of the CAG RNA homoduplex and disfavors the CAG-CUG heteroduplex formation. This action subsequently mitigated RNA-induced silencing complex (RISC)-dependent NUDT16 silencing in both in vitro cell and in vivo mouse disease models. After DB213 treatment, DNA damage, apoptosis, and locomotor defects were rescued in HD mice. This work establishes NUDT16 deficiency by CAG repeat RNAs as a pathogenic mechanism of polyQ diseases and as a potential therapeutic direction for HD and other polyQ diseases.

Insights into the choice between intravenous infusion and subcutaneous injection: physician and patient characteristics driving treatment in SLE.

INTRODUCTION/OBJECTIVES: Multiple modes of administration are available for systemic lupus erythematosus (SLE) treatments. This study examined patient and physician characteristics associated with the choice of weekly subcutaneous (SC) injection or monthly intravenous (IV) infusion for an unspecified SLE treatment. METHODS: This was a cross-sectional, US web-based survey using a direct elicitation, stated-preference methodology (HO-16-16706). Two hundred patients and 200 physicians were asked to choose between IV or SC administration in a hypothetical scenario. Pairwise and multivariate analyses estimated the odds ratio (OR) for the likelihood of choosing SC over IV for respondent characteristics. RESULTS: Among patients, taking non-steroidal anti-inflammatory drugs increased the likelihood of choosing SC injection (OR 3.884), whilst having SLE-related skin problems, a fear of needles or self-injection, and never needing help around the house decreased the likelihood (OR 0.28, 0.13, 0.12, respectively; all p ≤ 0.05). Among physicians, > 95% recommended SC injection for patients who live or work far from an infusion center, prefer SC administration, and never or rarely miss medication doses. Physician characteristics including age and treatment practice also influenced choice. CONCLUSIONS: Patient and physician characteristics influence choice of SC versus IV therapy for SLE. These findings might inform shared decision-making, which could lead to improved patient outcomes. Key Points • Data regarding patient and physician preference for different modes of administration of SLE therapy are sparse. • This cross-sectional, US web-based study showed that patient and physician characteristics influence choice of SC versus IV therapy for SLE. • A degree of disconnect exists between how factors influence patients' choice and how those characteristics influence physicians' choice of SLE treatment mode of administration. • The findings from this study might inform shared decision-making, which could improve alignment between treatment choice and patient preferences, treatment satisfaction, adherence, and improved patient outcomes.

Multi-omics tools for studying microbial biofilms: current perspectives and future directions.

The advent of omics technologies has greatly improved our understanding of microbial biology, particularly in the last two decades. The field of microbial biofilms is, however, relatively new, consolidated in the 1980s. The morphogenic switching by microbes from planktonic to biofilm phenotype confers numerous survival advantages such as resistance to desiccation, antibiotics, biocides, ultraviolet radiation, and host immune responses, thereby complicating treatment strategies for pathogenic microorganisms. Hence, understanding the mechanisms governing the biofilm phenotype can result in efficient treatment strategies directed specifically against molecular markers mediating this process. The application of omics technologies for studying microbial biofilms is relatively less explored and holds great promise in furthering our understanding of biofilm biology. In this review, we provide an overview of the application of omics tools such as transcriptomics, proteomics, and metabolomics as well as multi-omics approaches for studying microbial biofilms in the current literature. We also highlight how the use of omics tools directed at various stages of the biological information flow, from genes to metabolites, can be integrated via multi-omics platforms to provide a holistic view of biofilm biology. Following this, we propose a future artificial intelligence-based multi-omics platform that can predict the pathways associated with different biofilm phenotypes.

The Role of Dentists in COVID-19 Is Beyond Dentistry: Voluntary Medical Engagements and Future Preparedness.

The emergence of the highly infectious novel coronavirus SARS-CoV-2 has led to a global COVID-19 pandemic. Since the outbreak of COVID-19, worldwide healthcare systems have been severely challenged. The rapid and explosive surge of positive cases has significantly increased the demand for medical care. Herein we provide a perspective on the role dentists can play in voluntary medical assistance and future preparedness for a similar pandemic. Though dentists and physicians have different scopes of practice, their trainings share many similarities. Hence, dental professionals, with their knowledge of basic human science and sterile surgical techniques, are an invaluable resource in the COVID-19 pandemic response. Overall, it is commendable that many dentists have risen to the challenge in the fight against COVID-19. For example, in Singapore, National Dental Centre Singapore (NDCS) deployed dental clinicians as well as volunteers from research laboratories to screen for suspected cases, provide consultations as well as conduct swabbing operations. Dental practice will be considerably changed in the post-COVID-19 era. There is a greater need to have refresher courses for practicing dentists on new infection control strategies. Moreover, the curriculum in dental schools should be expanded to include competencies in pandemic and disaster relief. In addition, voluntary medical work should be made a part of the community dentistry curriculum. This volunteerism will leave a positive impact on developing the careers of young dentists. Hence, the contribution of dentists beyond dental practice in this pandemic situation will be appreciated by future generations.

Clinical significance and functional role of transmembrane protein 47 (TMEM47) in chemoresistance of hepatocellular carcinoma.

Chemoresistance is the main cause of chemotherapy failure in patients with hepatocellular carcinoma (HCC). The gene encoding transmembrane protein 47 (TMEM47) was previously identified to be significantly upregulated in HCC cell lines with acquired chemoresistance. The aim of the present study was to characterize the clinical significance and function of TMEM47 in HCC chemoresistance. The results demonstrated that the TMEM47 expression levels in the tumors of patients not responding to cisplatin­based transarterial chemoembolization (TACE) treatment was significantly higher compared with those in patients who responded to TACE treatment. Moreover, analyses from clinical samples and HCC cell lines indicated that TMEM47 expression may be upregulated in HCC in response to cisplatin treatment. Furthermore, the TMEM47 mRNA expression levels were positively correlated with the degree of cisplatin resistance of HCC cells. Overexpression of TMEM47 in HCC cells significantly promoted cisplatin resistance. The present study also demonstrated that targeted inhibition of TMEM47 could significantly reduce cisplatin resistance of cisplatin­resistant HCC cells via enhancing caspase­mediated apoptosis. In addition, targeted inhibition of TMEM47 enhanced the sensitivity of cisplatin­resistant cells to cisplatin via suppressing cisplatin­induced activation of the genes involved in drug efflux and metabolism. The present study also validated that TMEM47 expression was significantly correlated with multidrug resistance­associated protein 1 in patients with HCC who received TACE treatment. In conclusion, the findings of the present study demonstrated that TMEM47 may be a useful biomarker for predicting the response to chemotherapy and a potential therapeutic target for overcoming HCC chemoresistance.

Acetylsalicylic acid inhibits intravascular coagulation during Staphylococcus aureus-induced sepsis in mice.

Antiplatelet therapies have been proposed for the treatment of sepsis, a syndrome resulting from a dysregulated immune response and inappropriate activation of coagulation. Acetylsalicylic acid (ASA) may serve as a potential therapeutic strategy to prevent infection-induced coagulopathy and associated tissue damage. Using intravital microscopy, we found that Staphylococcus aureus infection induced neutrophil recruitment, platelet aggregation, and neutrophil extracellular trap (NET) release in the liver. Mice pretreated with ASA, or animals receiving ASA 3 hours postinfection, had significantly reduced platelet aggregation and NET release. Additionally, ASA-treated mice had reduced intravascular thrombin activity and microvascular occlusion as compared with untreated S aureus-infected mice. This inhibition of coagulation was accompanied by decreased levels of alanine aminotransferase and aspartate aminotransferase in the plasma, indicating less liver damage. Finally, bacterial loads (colony-forming units per milliliter) in liver, lung, and spleen were not different between groups, and the phagocytic capacity of Kupffer cells was preserved following ASA treatment. These results suggest that ASA may serve as a therapeutic approach to sepsis through its ability to reduce the deleterious action of immunothrombi while maintaining innate immune functions.

Effect of subacute and prenatal DINCH plasticizer exposure on rat dams and male offspring hepatic function: The role of PPAR-α.

Plasticizers are compounds added to plastics to modify their physical proprieties. The most well-known class of plasticizers, the phthalates, has been shown to possess antiandrogenic and tumor promoting activities. 1,2-Cyclohexane dicarboxylic acid diisononyl ester (DINCH) was approved for use in food contact containers in 2006 and has been used as a replacement for phthalates in toys and children products. However, we reported previously that the DINCH metabolite MINCH acts on primary rat adipocytes through the peroxisome proliferator activated receptor (PPAR)-α pathway in a manner similar to phthalates. Evidence from our studies, as well as from the current bibliography on DINCH, suggests that the liver might be one of its target organs. In the present study, we collected tissues from dams exposed subacutely and progeny at postnatal day (PND) 3 and 60 exposed in utero to DINCH (1, 10 and 100 mg/kg bw/day). Exposure to DINCH drastically affected liver gene expression in all 3 age groups tested and in particular at the dose of 1 mg/kg bw/day. The PPAR-α pathway along with other metabolic and DNA replication pathways were affected by DINCH. Modifications in PPAR-α and superoxide dismutase (SOD)-1 protein levels were observed in dams at PND21, as well as male progeny at PND3 and 60. No sign of fibrosis or direct liver toxicity was observed after 8 days of stimulus with low doses of DINCH. This study provides evidence that DINCH is not a biologically inert molecule in the rat, and in the liver its actions are mediated, at least in part, by PPAR-α.

Draft Aphaenogaster genomes expand our view of ant genome size variation across climate gradients.

Given the abundance, broad distribution, and diversity of roles that ants play in many ecosystems, they are an ideal group to serve as ecosystem indicators of climatic change. At present, only a few whole-genome sequences of ants are available (19 of >16,000 species), mostly from tropical and sub-tropical species. To address this limited sampling, we sequenced genomes of temperate-latitude species from the genus Aphaenogaster, a genus with important seed dispersers. In total, we sampled seven colonies of six species: Aphaenogaster ashmeadi, Aphaenogaster floridana, Aphaenogaster fulva, Aphaenogaster miamiana, Aphaenogaster picea, and Aphaenogaster rudis. The geographic ranges of these species collectively span eastern North America from southern Florida to southern Canada, which encompasses a latitudinal gradient in which many climatic variables are changing rapidly. For the six genomes, we assembled an average of 271,039 contigs into 47,337 scaffolds. The Aphaenogaster genomes displayed high levels of completeness with 96.1% to 97.6% of Hymenoptera BUSCOs completely represented, relative to currently sequenced ant genomes which ranged from 88.2% to 98.5%. Additionally, the mean genome size was 370.5 Mb, ranging from 310.3 to 429.7, which is comparable to that of other sequenced ant genomes (212.8-396.0 Mb) and flow cytometry estimates (210.7-690.4 Mb). In an analysis of currently sequenced ant genomes and the new Aphaenogaster sequences, we found that after controlling for both spatial autocorrelation and phylogenetics ant genome size was marginally correlated with sample site climate similarity. Of all examined climate variables, minimum temperature, and annual precipitation had the strongest correlations with genome size, with ants from locations with colder minimum temperatures and higher levels of precipitation having larger genomes. These results suggest that climate extremes could be a selective force acting on ant genomes and point to the need for more extensive sequencing of ant genomes.

Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells.

Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.

External Validation of a COPD Risk Measure in a Commercial and Medicare Population: The COPD Treatment Ratio.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) exacerbations can accelerate disease progression and lead to higher health care costs. To improve patient survival and reduce cost, risk assessment measures should be developed to identify patients at risk for exacerbations and prevent future exacerbations. OBJECTIVES: To (a) externally validate the COPD treatment ratio (CTR) as a measure of COPD exacerbation risk based on predictive models previously tested and (b) assess the measure's capability to assess risk using only pharmacy claims for use in Medicare Part D programs. METHODS: This was a retrospective observational study conducted using the Humana research datasets. Separate assessments were performed using pharmacy-only models that excluded risk factors derived from medical claims. Patients were aged ≥ 40 years, with ≥ 1 inpatient hospitalization or ≥ 2 physician's office, emergency department, or urgent care visits with a COPD diagnosis. Using logistic regression models, risk factors (age, exacerbation history, COPD and concomitant medication use, and comorbidities) were assessed during the baseline period (year 1) and were used to predict the risk of exacerbation during year 2. Continuous and dichotomized CTRs were analyzed. A cut-point of 0.3 was initially used for dichotomizing CTR, and subsequently receiver operating characteristics (ROC) analysis was used to determine the optimal cut-point for CTR. RESULTS: A total of 92,496 patients were identified, the majority of which (96.2%) were Medicare members with a mean age of 69 years. During the baseline period, 14.0% and 11.2% of patients had ≥ 1 moderate or severe exacerbation, respectively. Overall, the CTR performed well in predicting future COPD exacerbations, especially severe exacerbations. ROC analysis suggested that 0.7 was the optimal cut-point for dichotomizing CTR. Patients with a CTR ≥ 0.7 had a 7.9% (OR = 0.921; 95% CI = 0.852-0.995) lower risk of a severe exacerbation, compared with those with a CTR < 0.7. Stronger effects were seen in pharmacy-only models, with patients 17% less likely to experience a severe exacerbation with a CTR ≥ 0.7 compared with patients with a CTR < 0.7. CONCLUSIONS: This study validated the use of CTR as a modifiable measure of risk of COPD exacerbation in a large commercial and Medicare population and remained a robust predictor when pharmacy-only claims data were available. A CTR of ≥ 0.7 may be a useful target to help reduce the risk of severe exacerbations, and its use by payer or quality organizations has the potential to improve COPD management. DISCLOSURES: This study was funded by GlaxoSmithKline (GSK; study number HO-15-16651). GSK had a role in the study design, collection, analysis, and interpretation of data and in the writing of the study report but did not place any restrictions on access to the data or on the statements made in the manuscript. The authors were in full editorial control of publication target journal and content and conclusions, accepted full responsibility for final approval of a manuscript describing this GSK-sponsored research, and had final responsibility for the decision to submit for publication. Stanford and Lau are employees of GSK and hold GSK stocks/shares. Li and Stemkowski are employees of Comprehensive Health Insights, which was contracted to conduct the study but did not receive funding for manuscript development. This manuscript was presented in part at the American Thoracic Society 2017 International Conference; May 19-24, 2017; Washington, DC.