Semi-continuous biomanufacturing for maximizing the production of complex chemicals and fuels: a case study of amorpha-4,11-diene.

Biomanufacturing is emerging as a key technology for the sustainable production of chemicals, materials, and food ingredients using engineered microbes. However, despite billions of dollars of investment, few processes have been successfully commercialized due to a lack of attention on industrial-scale bioprocess design and innovation. In this study, we address this challenge through the development of a novel semi-continuous bioprocess for the production of the terpene amorpha-4,11-diene (AMD4,11) using engineered Escherichia coli. Using a hydrophilic membrane for product and biomass retention, we successfully decoupled production at low growth rates (~0.01 1/h) and improved reactor productivity up to 166 mg/lReactor h, threefold compared with traditional fed-batch fermentations. When cell recycling was implemented, we showed sustained production at the highest conversion yield and production rate for up to three cycles, demonstrating the robustness of both the strain and the process and highlighting the potential for new bioprocess strategies to improve the economic viability of industrial biomanufacturing.

Ileus at Altitude.

Autoimmune myasthenia gravis (MG) is a condition commonly caused by the production of antibodies that block acetylcholine receptors. Until recently, gastrointestinal (GI) symptoms were considered rare in myasthenia gravis, but are now being proposed as an early identification tool. Presented here is a case study that exemplifies GI symptoms in MG, exacerbated by low barometric pressure at altitude. This illustrates the need to identify GI symptoms earlier in MG patients, as well as the need for providers at high altitude to be aware of these manifestations of MG.

How do we operate a large monthly red blood cell exchange program.

BACKGROUND: Red blood cell (RBC) exchange for sickle cell disease presents unique difficulties due to RBC phenotyping, complex antibody work-ups, large number of RBC units required, and vascular access considerations, any of which can delay the procedure. Multidisciplinary coordination and systemic processes ensure that monthly appointments remain on schedule. STUDY DESIGN AND METHODS: A high-volume chronic RBC exchange program is described, highlighting the importance of multidisciplinary coordination and process improvement strategies involving initial referral, vascular access, order sets, and allocation of antigen-negative or phenotypically matched RBCs. RESULTS: Approximately 50 outpatient RBC exchanges are performed each month with an 82% kept-appointment rate. Specific factors for program success include open communication across services and improvements to referrals and standardized order sets. CONCLUSION: A combination of multidisciplinary coordination and process improvement can ensure the success of a high volume RBC exchange program. Frequent communication of upcoming appointments between the referring hematologists, the hemapheresis clinic, transfusion service, and interventional radiology is critical. Advance notice to the immunohematology reference lab of upcoming appointments is needed to allow enough time for allocating antigen-negative RBCs. Order sets can be leveraged to standardize and streamline RBC exchanges. Lastly, numerous mechanisms help patients compensate for the cognitive sequelae of stroke.

Frontal Arteriovenous Malformation Presenting as Painful Unilateral Conjunctiva Injection.

CASE PRESENTATION: Arteriovenous malformations (AVM) have a variety of clinically significant manifestations. This report details a patient who presented with unilateral conjunctiva injection, which was found to be due to an atypical manifestation of an AVM with a large draining vein mimicking carotid cavernous fistula. DISCUSSION: While imaging for patients presenting with eye pain and unilateral conjunctiva injection is not always warranted, emergency physicians should keep their differential diagnosis broad and pursue additional workup when warning signs of more sinister pathology present.

Enabling commercial success of industrial biotechnology.

Commercial-scale research translation has been muted.

Pediatric Coping During Venipuncture With Virtual Reality: Pilot Randomized Controlled Trial.

BACKGROUND: Virtual reality (VR) has shown promise in reducing children's pain and anxiety during venipuncture, but studies on VR lack objective observations of pediatric coping. Notably, the process of capturing objective behavioral coping data can be labor- and personnel-intensive. OBJECTIVE: The primary aims of this pilot trial were to assess the feasibility of conducting a trial of VR in a pediatric emergency department and the feasibility of documenting observed coping behaviors during pediatric procedures. Secondarily, this study examined whether VR affects child and caregiver coping and distress during venipuncture in the pediatric emergency department. METHODS: This stratified, randomized, controlled pilot trial compared coping and distress between child life-supported VR engagement and child life specialist support without VR during painful procedures in children aged 7-22 years in the pediatric emergency department. An external control (reference group) received no standardized support. Primary feasibility outcomes included rates of recruitment, rates of withdrawal from VR, and rates of completed Child Adult Medical Procedure Interaction Scale-Short Form (CAMPIS-SF) observations. Secondary clinical outcomes were applied to venipuncture procedures and included CAMPIS-SF coping and distress (range 0-1.0), pain and anxiety on a visual analog scale (range 0-10), and cybersickness symptoms. RESULTS: Overall recruitment was 93% (66/71), VR withdrawal rate was 27% (4/15), and of the completed procedures, 100% (63/63) CAMPIS-SF observations were completed. A total of 55 patients undergoing venipuncture in the pediatric emergency department were included in the analyses of clinical outcomes: 15 patients (15 caregivers) randomized to VR, 20 patients (15 caregivers) randomized to child life specialist support, and 20 patients (17 caregivers) in the reference group. Patient coping differed across groups with higher coping in the VR group and child life specialist group than in the reference group (P=.046). There were no significant differences in the distress and pain ratings for patients and caregivers between the groups. Caregivers rated the lowest perceived anxiety in the child life specialist group (P=.03). There was no apparent change in cybersickness symptoms before and after VR use (P=.37). CONCLUSIONS: Real-time documentation of observed behaviors in patients and caregivers was feasible during medical procedures in which VR was utilized, particularly with the availability of research staff. VR and child life specialists improved coping in children during venipuncture procedures. Given the high participation rate, future studies to evaluate the efficacy of VR are recommended to determine whether an off-the-shelf VR headset can be a low-cost and low-risk tool to improve children's coping during venipuncture or other related procedures. TRIAL REGISTRATION: ClinicalTrials.gov NCT03686176; https://clinicaltrials.gov/ct2/show/NCT03686176.

Resource Use During Pediatric Venipuncture With Virtual Reality: Secondary Analysis of a Randomized Controlled Pilot Trial.

OBJECTIVES: Virtual reality (VR) therapy is growing in use and popularity during pediatric medical procedures. Currently, data that describe the hospital resources used during pediatric procedures with off-the-shelf VR games that are not tailored to medical procedures are lacking. In this study, we aimed to characterize procedural resources associated with VR use during venipuncture in a pediatric emergency department. METHODS: This was a secondary analysis of a 2-arm randomized, controlled pilot trial with an external group. Resource use was evaluated in 3 groups: child life (CL)-supported VR engagement, CL support without VR, and a reference group that received no intervention (ie, no CL and no VR). RESULTS: The study sample (N = 55) included the following: 15 patients randomly assigned to VR, 20 patients randomly assigned to CL, and 20 patients in the reference group. There was a significant difference in procedure duration, with the VR group exhibiting the longest duration compared with the CL and reference groups (P = .01). CONCLUSIONS: Longer procedure times associated with VR use during venipunctures (4-6 minutes on average) may be attributed to pauses to troubleshoot VR games not tailored for medical procedures. Although they are inexpensive and accessible, nontailored VR games may warrant the need for dedicated staff to provide restraint and/or assistance to navigate the VR application. In this study, we offer a protocol on the application of nontailored VR games for pediatric procedures. For those considering a VR program in an inpatient setting, the benefits of pain/anxiety reduction must be weighed against the resources needed, including device costs, staff availability, and increased procedure duration.

Pediatric Otoscopy Video Screening With Shift Contrastive Anomaly Detection.

Ear related concerns and symptoms represent the leading indication for seeking pediatric healthcare attention. Despite the high incidence of such encounters, the diagnostic process of commonly encountered diseases of the middle and external presents a significant challenge. Much of this challenge stems from the lack of cost effective diagnostic testing, which necessitates the presence or absence of ear pathology to be determined clinically. Research has, however, demonstrated considerable variation among clinicians in their ability to accurately diagnose and consequently manage ear pathology. With recent advances in computer vision and machine learning, there is an increasing interest in helping clinicians to accurately diagnose middle and external ear pathology with computer-aided systems. It has been shown that AI has the capacity to analyze a single clinical image captured during the examination of the ear canal and eardrum from which it can determine the likelihood of a pathognomonic pattern for a specific diagnosis being present. The capture of such an image can, however, be challenging especially to inexperienced clinicians. To help mitigate this technical challenge, we have developed and tested a method using video sequences. The videos were collected using a commercially available otoscope smartphone attachment in an urban, tertiary-care pediatric emergency department. We present a two stage method that first, identifies valid frames by detecting and extracting ear drum patches from the video sequence, and second, performs the proposed shift contrastive anomaly detection (SCAD) to flag the otoscopy video sequences as normal or abnormal. Our method achieves an AUROC of 88.0% on the patient level and also outperforms the average of a group of 25 clinicians in a comparative study, which is the largest of such published to date. We conclude that the presented method achieves a promising first step toward the automated analysis of otoscopy video.

Balancing glucose and oxygen uptake rates to enable high amorpha-4,11-diene production in Escherichia coli via the methylerythritol phosphate pathway.

Amorpha-4,11-diene (AMD4,11) is a precursor to artemisinin, a potent antimalarial drug that is traditionally extracted from the shrubs of Artemisia annua. Despite significant prior efforts to produce artemisinin and its precursors through biotechnology, there remains a dire need for more efficient biosynthetic routes for its production. Here, we describe the optimization of key process conditions for an Escherichia coli strain producing AMD4,11 via the native methylerythritol phosphate (MEP) pathway. By studying the interplay between glucose uptake rates and oxygen demand, we were able to identify optimal conditions for increasing carbon flux through the MEP pathway by manipulating the availability of NADPH required for terpenoid production. Installation of an optimal qO2 /qglucose led to a 6.7-fold increase in product titers and a 6.5-fold increase in carbon yield.

Targeted Mass Spectrometry-Based Proteomics Tools for Strain Optimization.

The goal of strain optimization is to create high-performance strains producing compounds of interest at a high titer, yield, and volumetric productivity. The effectiveness of strain optimization relies on methodologies used to aid optimization of native or novel pathways within cells. Many different factors, including mRNA abundance, protein abundance, and enzyme activity/stability, will contribute to the strain performance, which is not often evident by simply monitoring product titers. To this end, targeted proteomics tools utilizing LC-MS-MS (liquid chromatography coupled with tandem mass spectrometry) have recently been developed and can monitor protein levels at great sensitivities. Here, we describe all relevant aspects when developing proteomics tools for strain optimization.

High-Altitude Pulmonary Edema in Mountain Community Residents.

Ebert-Santos, Christine. High-altitude pulmonary edema in mountain community residents. High Alt Med Biol. 18:278-284, 2017.-High-altitude pulmonary edema (HAPE) affects lowlanders ascending quickly to elevations above 2440 m. Mountain resident children with no travel can sometimes develop HAPE as was observed over 30 years ago (Fasules et al., 1985). This is not well known and children instead are diagnosed as having pneumonia or asthma. In our clinic at 2800 m, we see children presenting with severe hypoxemia, clinical, and radiographic findings consistent with HAPE despite no recent travel. We call this mountain resident HAPE. We reviewed records of 48 patients with pulmonary symptoms. Analysis included vital signs, pulse oximetry, laboratories, physical findings, and clinical course. We identified 33 residents with HAPE and no travel, five with reentry HAPE, two visitors with classic HAPE, six residents with pneumonia, and two with asthma. Also, 48 X-rays on hypoxemic children seen between 2006 and 2017 were reviewed. Five showed definite HAPE with follow-up X-rays within 48 hours confirming rapid clearing on oxygen, 27 showed findings consistent with HAPE or viral pneumonia and no repeat study. Children living at elevation presenting with hypoxemia are commonly misdiagnosed. Rapid improvement with oxygen and little to no improvement with bronchodilators are more consistent with HAPE, and thus, antibiotics and other treatments can be avoided.

Multivariate modular metabolic engineering for pathway and strain optimization.

Despite the potential in utilizing microbial fermentation for chemical production, the field of industrial biotechnology still lacks a standard, universally applicable principle for strain optimization. A key challenge has been in finding and applying effective ways to address metabolic flux imbalances. Strategies based on rational design require significant a priori knowledge and often fail to take a holistic view of cellular metabolism. Combinatorial approaches enable more global searches but require a high-throughput screen. Here, we present the recent advances and promises of a novel approach to metabolic pathway and strain optimization called multivariate modular metabolic engineering (MMME). In this technique, key enzymes are organized into distinct modules and simultaneously varied based on expression to balance flux through a pathway. Because of its simplicity and broad applicability, MMME has the potential to systematize and revolutionize the field of metabolic engineering and industrial biotechnology.

Engineering complex biological systems in bacteria through recombinase-assisted genome engineering.

Here we describe an advanced paradigm for the design, construction and stable implementation of complex biological systems in microbial organisms. This engineering strategy was previously applied to the development of an Escherichia coli-based platform, which enabled the use of brown macroalgae as a feedstock for the production of biofuels and renewable chemicals. In this approach, functional genetic modules are first designed in silico and constructed on a bacterial artificial chromosome (BAC) by using a recombineering-based inchworm extension technique. Stable integration into the recipient chromosome is then mediated through the use of recombinase-assisted genome engineering (RAGE). The flexibility, simplicity and speed of this method enable a comprehensive optimization of several different parameters, including module configuration, strain background, integration locus, gene copy number and intermodule compatibility. This paradigm therefore has the potential to markedly expedite most strain-engineering endeavors. Once a biological system has been designed and constructed on a BAC, its implementation and optimization in a recipient host can be carried out in as little as 1 week.

Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform.

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-L-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l(-1)) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

Implementation of stable and complex biological systems through recombinase-assisted genome engineering.

Evaluating the performance of engineered biological systems with high accuracy and precision is nearly impossible with the use of plasmids due to phenotypic noise generated by genetic instability and natural population dynamics. Minimizing this uncertainty therefore requires a paradigm shift towards engineering at the genomic level. Here, we introduce an advanced design principle for the stable installment and implementation of complex biological systems through recombinase-assisted genome engineering (RAGE). We apply this concept to the development of a robust strain of Escherichia coli capable of producing ethanol directly from brown macroalgae. RAGE significantly expedites the optimal implementation of a 34 kb heterologous pathway for alginate metabolism based on genetic background, integration locus, copy number and compatibility with two other pathway modules (alginate degradation and ethanol production). The resulting strain achieves a ~40% higher titre than its plasmid-based counterpart and enables substantial improvements in titre (~330%) and productivity (~1,200%) after 50 generations.

Rational, combinatorial, and genomic approaches for engineering L-tyrosine production in Escherichia coli.

Although microbial metabolic engineering has traditionally relied on rational and knowledge-driven techniques, significant improvements in strain performance can be further obtained through the use of combinatorial approaches exploiting phenotypic diversification and screening. Here, we demonstrate the combined use of global transcriptional machinery engineering and a high-throughput L-tyrosine screen towards improving L-tyrosine production in Escherichia coli. This methodology succeeded in generating three strains from two separate mutagenesis libraries (rpoA and rpoD) exhibiting up to a 114% increase in L-tyrosine titer over a rationally engineered parental strain with an already high capacity for production. Subsequent strain characterization through transcriptional analysis and whole genome sequencing allowed complete phenotype reconstruction from well-defined mutations and point to important roles for both the acid stress resistance pathway and the stringent response of E. coli in imparting this phenotype. As such, this study presents one of the first examples in which cell-wide measurements have helped to elucidate the genetic and biochemical underpinnings of an engineered cellular property, leading to the total restoration of metabolite overproduction from specific chromosomal mutations.

Growth hormone receptor polymorphism and growth hormone therapy response in children: a Bayesian meta-analysis.

Recombinant human growth hormone (rhGH) therapy is used in the long-term treatment of children with growth disorders, but there is considerable treatment response variability. The exon 3-deleted growth hormone receptor polymorphism (GHR(d3)) may account for some of this variability. The authors performed a systematic review (to April 2011), including investigator-only data, to quantify the effects of the GHR(fl-d3) and GHR(d3-d3) genotypes on rhGH therapy response and used a recently established Bayesian inheritance model-free approach to meta-analyze the data. The primary outcome was the 1-year change-in-height standard-deviation score for the 2 genotypes. Eighteen data sets from 12 studies (1,527 children) were included. After several prior assumptions were tested, the most appropriate inheritance model was codominant (posterior probability = 0.93). Compared with noncarriers, carriers had median differences in 1-year change-in-height standard-deviation score of 0.09 (95% credible interval (CrI): 0.01, 0.17) for GHR(fl-d3) and of 0.14 (95% CrI: 0.02, 0.26) for GHR(d3-d3). However, the between-study standard deviation of 0.18 (95% CrI: 0.10, 0.33) was considerable. The authors tested by meta-regression for potential modifiers and found no substantial influence. They conclude that 1) the GHR(d3) polymorphism inheritance is codominant, contrasting with previous reports; 2) GHR(d3) genotypes account for modest increases in rhGH effects in children; and 3) considerable unexplained variability in responsiveness remains.

An engineered microbial platform for direct biofuel production from brown macroalgae.

Prospecting macroalgae (seaweeds) as feedstocks for bioconversion into biofuels and commodity chemical compounds is limited primarily by the availability of tractable microorganisms that can metabolize alginate polysaccharides. Here, we present the discovery of a 36-kilo-base pair DNA fragment from Vibrio splendidus encoding enzymes for alginate transport and metabolism. The genomic integration of this ensemble, together with an engineered system for extracellular alginate depolymerization, generated a microbial platform that can simultaneously degrade, uptake, and metabolize alginate. When further engineered for ethanol synthesis, this platform enables bioethanol production directly from macroalgae via a consolidated process, achieving a titer of 4.7% volume/volume and a yield of 0.281 weight ethanol/weight dry macroalgae (equivalent to ~80% of the maximum theoretical yield from the sugar composition in macroalgae).