A genome-scale metabolic model of Cupriavidus necator H16 integrated with TraDIS and transcriptomic data reveals metabolic insights for biotechnological applications.

Exploiting biological processes to recycle renewable carbon into high value platform chemicals provides a sustainable and greener alternative to current reliance on petrochemicals. In this regard Cupriavidus necator H16 represents a particularly promising microbial chassis due to its ability to grow on a wide range of low-cost feedstocks, including the waste gas carbon dioxide, whilst also naturally producing large quantities of polyhydroxybutyrate (PHB) during nutrient-limited conditions. Understanding the complex metabolic behaviour of this bacterium is a prerequisite for the design of successful engineering strategies for optimising product yields. We present a genome-scale metabolic model (GSM) of C. necator H16 (denoted iCN1361), which is directly constructed from the BioCyc database to improve the readability and reusability of the model. After the initial automated construction, we have performed extensive curation and both theoretical and experimental validation. By carrying out a genome-wide essentiality screening using a Transposon-directed Insertion site Sequencing (TraDIS) approach, we showed that the model could predict gene knockout phenotypes with a high level of accuracy. Importantly, we indicate how experimental and computational predictions can be used to improve model structure and, thus, model accuracy as well as to evaluate potential false positives identified in the experiments. Finally, by integrating transcriptomics data with iCN1361 we create a condition-specific model, which, importantly, better reflects PHB production in C. necator H16. Observed changes in the omics data and in-silico-estimated alterations in fluxes were then used to predict the regulatory control of key cellular processes. The results presented demonstrate that iCN1361 is a valuable tool for unravelling the system-level metabolic behaviour of C. necator H16 and can provide useful insights for designing metabolic engineering strategies.

Effect of bilateral scalp nerve blocks on postoperative pain and discharge times in patients undergoing supratentorial craniotomy and general anesthesia: a randomized-controlled trial.

PURPOSE: Post-craniotomy pain is a common clinical issue and its optimal management remains incompletely studied. Utilization of a regional scalp block has the potential advantage of reducing perioperative pain and opioid consumption, thereby facilitating optimal postoperative neurologic assessment. The purpose of this study was to assess the efficacy of regional scalp block on post-craniotomy pain and opioid consumption. METHODS: We performed a prospective randomized-controlled trial in adults scheduled to undergo elective supratentorial craniotomy under general anesthesia to assess the efficacy of postoperative bilateral scalp block with 0.5% bupivacaine with 1:200,000 epinephrine compared with placebo on postoperative pain and opioid consumption. The primary outcome was the visual analogue scale (VAS) for pain at 24 hr postoperatively. RESULTS: Eighty-nine patients were enrolled (n = 44 in block group; n = 45 in control group). There was no difference in the mean (standard deviation) VAS score at 24 hr postoperatively between the treatment group and the control group [31.2 (21.4) mm vs 23.0 (19.2) mm, respectively; mean difference, 6.6; 95% confidence interval, -2.3, 15.5; P = 0.15]. There was also no significant difference in postoperative opioid consumption. Distribution of individual VAS score and opioid consumption revealed that postoperative pain was highly variable following craniotomy. Time to hospital discharge was not different between treatment and placebo groups. No adverse events associated with scalp block were identified. CONCLUSION: These data show that bilateral scalp blocks using bupivacaine with epinephrine did not reduce mean postoperative VAS score or overall opioid consumption at 24 hr nor the time-to-discharge from the postanesthesia care unit or from hospital. TRIAL REGISTRATION: www.ClinicalTrials.gov, NCT00972790; registered 9 September, 2009.

Applying asymptotic methods to synthetic biology: Modelling the reaction kinetics of the mevalonate pathway.

The mevalonate pathway is normally found in eukaryotes, and allows for the production of isoprenoids, a useful class of organic compounds. This pathway has been successfully introduced to Escherichia coli, enabling a biosynthetic production route for many isoprenoids. In this paper, we develop and solve a mathematical model for the concentration of metabolites in the mevalonate pathway over time, accounting for the loss of acetyl-CoA to other metabolic pathways. Additionally, we successfully test our theoretical predictions experimentally by introducing part of the pathway into Cupriavidus necator. In our model, we exploit the natural separation of time scales as well as of metabolite concentrations to make significant asymptotic progress in understanding the system. We confirm that our asymptotic results agree well with numerical simulations, the former enabling us to predict the most important reactions to increase isopentenyl diphosphate production whilst minimizing the levels of HMG-CoA, which inhibits cell growth. Thus, our mathematical model allows us to recommend the upregulation of certain combinations of enzymes to improve production through the mevalonate pathway.

Awake craniotomy using dexmedetomidine and scalp blocks: a retrospective cohort study.

PURPOSE: Anesthetic and surgical considerations for awake craniotomy (AC) include airway patency, patient comfort, and optimization of real-time brain mapping. The purpose of this study is to report our experience of using dexmedetomidine and scalp blocks, without airway intervention, as a means to facilitate and optimize intraoperative brain mapping and brain tumour resection during AC. METHODS: We conducted a retrospective cohort study of 55 patients who underwent AC from March 2012 to September 2016. The incidence of critical airway outcomes, perioperative complications, and successful intraoperative mapping was determined. The primary outcome was the incidence of a failed AC anesthetic technique as defined by the need to convert to general anesthesia with a secured airway prior to (or during) brain mapping and brain tumour resection. Secondary outcomes were the intraoperative incidence of: 1) altered surgical management due to information acquired through real-time brain mapping, 2) interventions to restore airway patency or rescue the airway, 3) hemodynamic instability (> 20% from baseline), 4) nausea and vomiting, 5) new onset neurologic deficits, and 6) seizure activity. RESULTS: There were no anesthesia-related critical events and no patients required airway manipulation or conversion to a general anesthetic. Multimodal language, motor, and sensory assessment with direct cortical electrical stimulation was successfully performed in 100% of cases. In 24% (13/55) of patients, data acquired during intraoperative brain mapping influenced surgical decision-making regarding the extent of tumour resection. Nine (16%) patients had intraoperative seizures. CONCLUSIONS: Dexmedetomidine-based anesthesia and scalp block facilitated AC surgery without any requirement for urgent airway intervention or unplanned conversion to a full general anesthetic. This approach can enable physiologic testing before and during tumour resection facilitating real-time surgical decision-making based on intraoperative brain mapping with patients awake thereby minimizing the risk of neurologic deficit and increasing the opportunity for optimal surgical resection.

Synthetic Biology Toolbox, Including a Single-Plasmid CRISPR-Cas9 System to Biologically Engineer the Electrogenic, Metal-Resistant Bacterium Cupriavidus metallidurans CH34.

Cupriavidus metallidurans CH34 exhibits extraordinary metabolic versatility, including chemolithoautotrophic growth; degradation of BTEX (benzene, toluene, ethylbenzene, xylene); high resistance to numerous metals; biomineralization of gold, platinum, silver, and uranium; and accumulation of polyhydroxybutyrate (PHB). These qualities make it a valuable host for biotechnological applications such as bioremediation, bioprocessing, and the generation of bioelectricity in microbial fuel cells (MFCs). However, the lack of genetic tools for strain development and studying its fundamental physiology represents a bottleneck to boosting its commercial applications. In this study, inducible and constitutive promoter libraries were built and characterized, providing the first comprehensive list of biological parts that can be used to regulate protein expression and optimize the CRISPR-Cas9 genome editing tools for this host. A single-plasmid CRISPR-Cas9 system that can be delivered by both conjugation and electroporation was developed, and its efficiency was demonstrated by successfully targeting the pyrE locus. The CRISPR-Cas9 system was next used to target candidate genes encoding type IV pili, hypothesized by us to be involved in extracellular electron transfer (EET) in this organism. Single and double deletion strains (ΔpilA, ΔpilE, and ΔpilAE) were successfully generated. Additionally, the CRISPR-Cas9 tool was validated for constructing genomic insertions (ΔpilAE::gfp and ΔpilAE::λPrgfp). Finally, as type IV pili are believed to play an important role in extracellular electron transfer to solid surfaces, C. metallidurans CH34 ΔpilAE was further studied by means of cyclic voltammetry using disposable screen-printed carbon electrodes. Under these conditions, we demonstrated that C. metallidurans CH34 could generate extracellular currents; however, no difference in the intensity of the current peaks was found in the ΔpilAE double deletion strain when compared to the wild type. This finding suggests that the deleted type IV pili candidate genes are not involved in extracellular electron transfer under these conditions. Nevertheless, these experiments revealed the presence of different redox centers likely to be involved in both mediated electron transfer (MET) and direct electron transfer (DET), the first interpretation of extracellular electron transfer mechanisms in C. metallidurans CH34.

A novel tablet computer platform for advanced language mapping during awake craniotomy procedures.

A computerized platform has been developed to enhance behavioral testing during intraoperative language mapping in awake craniotomy procedures. The system is uniquely compatible with the environmental demands of both the operating room and preoperative functional MRI (fMRI), thus providing standardized testing toward improving spatial agreement between the 2 brain mapping techniques. Details of the platform architecture, its advantages over traditional testing methods, and its use for language mapping are described. Four illustrative cases demonstrate the efficacy of using the testing platform to administer sophisticated language paradigms, and the spatial agreement between intraoperative mapping and preoperative fMRI results. The testing platform substantially improved the ability of the surgeon to detect and characterize language deficits. Use of a written word generation task to assess language production helped confirm areas of speech apraxia and speech arrest that were inadequately characterized or missed with the use of traditional paradigms, respectively. Preoperative fMRI of the analogous writing task was also assistive, displaying excellent spatial agreement with intraoperative mapping in all 4 cases. Sole use of traditional testing paradigms can be limiting during awake craniotomy procedures. Comprehensive assessment of language function will require additional use of more sophisticated and ecologically valid testing paradigms. The platform presented here provides a means to do so.

Sources of Variation Influencing Concordance between Functional MRI and Direct Cortical Stimulation in Brain Tumor Surgery.

Object: Preoperative functional magnetic resonance imaging (fMRI) remains a promising method to aid in the surgical management of patients diagnosed with brain tumors. For patients that are candidates for awake craniotomies, surgical decisions can potentially be improved by fMRI but this depends on the level of concordance between preoperative brain maps and the maps provided by the gold standard intraoperative method, direct cortical stimulation (DCS). There have been numerous studies of the concordance between fMRI and DCS using sensitivity and specificity measures, however the results are variable across studies and the key factors influencing variability are not well understood. Thus, the present work addresses the influence of technical factors on fMRI and DCS concordance. Methods: Motor and language mapping data were collected for a group of glioma patients (n = 14) who underwent both preoperative fMRI and intraoperative DCS in an awake craniotomy procedure for tumor removal. Normative fMRI data were also acquired in a healthy control group (n = 12). The fMRI and DCS mapping data were co-registered; true positive (TP), true negative (TN), false positive (FP), and false negative (FN) occurrences were tabulated over the exposed brain surface. Sensitivity and specificity were measured for the total group, and for the motor and language sub-groups. The influence of grid placement, fMRI statistical thresholding, and task standardization were assessed. Correlations between proportions of agreement and error were also carefully scrutinized to evaluate concordance in more detail. Results: Concordance was significantly better for motor vs. language mapping. There was an inverse relationship between TP and TN with increasing statistical threshold, and FP dominated the total error. Sensitivity and specificity were reduced when tasks were not standardized across fMRI and DCS. Conclusions: Although the agreement between fMRI and DCS is good, variability is introduced by technical factors that can diminish the quality of patient data. Neurosurgeons should evaluate the usefulness of fMRI data while considering that (a) discordance arises primarily from FP fMRI results; (b) there is an inherent trade-off between sensitivity and specificity with fMRI statistical threshold; and

Airway Management of Incidental Vallecular Cysts in Adults.

Vallecular cysts, largely asymptomatic in adults, are typically described only on incidental discovery during laryngoscopy, where they may present a challenge in airway management. The current literature is limited to case reports despite the potential for life-threatening complications. We describe management of such a case complicated by cyst rupture and intensive care unit admission. A literature review of eligible case reports was conducted, demonstrating an association between incidental vallecular cysts and difficult bag-mask ventilation and laryngoscopy with intraoperative otolaryngology consultation and intervention being common. Anesthetic management recommendations are thus presented, highlighting any conflicts with current difficult airway algorithms.

Clostridium difficile recurrent infection: possible implication of TA systems.

Clostridium difficile is an important nosocomial pathogen associated with antibiotic treatments. C. difficile's ability to survive antimicrobial therapy and transition from inert colonization to active infection is one of the most perplexing aspects of C. difficile infections and suggests that additional mechanisms are involved in persistence. In this regard, novel mechanisms linked with pathogenesis and persistence of C. difficile such as toxin-antitoxin systems might significantly contribute to biofilm formation and persistent infection. This review will focus on advances of toxin-antitoxin systems in C. difficile and their putative roles will be discussed.

Anesthetic approach to high-risk patients and prolonged awake craniotomy using dexmedetomidine and scalp block.

BACKGROUND: Awake craniotomy with intraoperative speech or motor testing is relatively contraindicated in cases requiring prolonged operative times and in patients with severe medical comorbidities including anxiety, anticipated difficult airway, obesity, large tumors, and intracranial hypertension. The anesthetic management of neurosurgical patients who possess these contraindications but would be optimally treated by an awake procedure remains unclear. METHODS: We describe a new anesthetic approach for awake craniotomy that did not require any airway manipulation, utilizing a bupivacaine-based scalp nerve block, and dexmedetomidine as the primary hypnotic-sedative agent. Using this technique, we provided optimal operative conditions to perform awake craniotomy facilitating safe tumor resection, while utilizing intraoperative electrocorticography for motor and speech mapping in a cohort of 10 patients at a high risk for airway compromise and complications associated with patient comorbidities. RESULTS: All patients underwent successful awake craniotomy, intraoperative mapping, and tumor resection with adequate sedation for up to 9 hours (median 3.5 h, range 3 to 9 h) without any loss of neurological function, airway competency, or the need to provide any active rescue airway management. We report 4 of these cases that highlight our experience: 1 case required prolonged surgery because of the complexity of tumor resection and 3 patients had important medical comorbidities and/or relative contraindication for an awake procedure. CONCLUSIONS: Dexmedetomidine, with concurrent scalp block, is an effective and safe anesthetic approach for awake craniotomy. Dexmedetomidine facilitates the extension procedure complexity and duration in patients who might traditionally not be considered to be candidates for this procedure.

The pyrimidine nucleotide biosynthetic pathway modulates production of biofilm determinants in Escherichia coli.

Bacteria are often found in multicellular communities known as biofilms, which constitute a resistance form against environmental stresses. Extracellular adhesion and cell aggregation factors, responsible for bacterial biofilm formation and maintenance, are tightly regulated in response to physiological and environmental cues. We show that, in Escherichia coli, inactivation of genes belonging to the de novo uridine monophosphate (UMP) biosynthetic pathway impairs production of curli fibers and cellulose, important components of the bacterial biofilm matrix, by inhibiting transcription of the csgDEFG operon, thus preventing production of the biofilm master regulator CsgD protein. Supplementing growth media with exogenous uracil, which can be converted to UMP through the pyrimidine nucleotide salvage pathway, restores csgDEFG transcription and curli production. In addition, however, exogenous uracil triggers cellulose production, particularly in strains defective in either carB or pyrB genes, which encode enzymes catalyzing the first steps of de novo UMP biosynthesis. Our results indicate the existence of tight and complex links between pyrimidine metabolism and curli/cellulose production: transcription of the csgDEFG operon responds to pyrimidine nucleotide availability, while cellulose production is triggered by exogenous uracil in the absence of active de novo UMP biosynthesis. We speculate that perturbations in the UMP biosynthetic pathways allow the bacterial cell to sense signals such as starvation, nucleic acids degradation, and availability of exogenous pyrimidines, and to adapt the production of the extracellular matrix to the changing environmental conditions.