Cell-Free Protein Expression in Polymer Materials.

While synthetic biology has advanced complex capabilities such as sensing and molecular synthesis in aqueous solutions, important applications may also be pursued for biological systems in solid materials. Harsh processing conditions used to produce many synthetic materials such as plastics make the incorporation of biological functionality challenging. One technology that shows promise in circumventing these issues is cell-free protein synthesis (CFPS), where core cellular functionality is reconstituted outside the cell. CFPS enables genetic functions to be implemented without the complications of membrane transport or concerns over the cellular viability or release of genetically modified organisms. Here, we demonstrate that dried CFPS reactions have remarkable tolerance to heat and organic solvent exposure during the casting processes for polymer materials. We demonstrate the utility of this observation by creating plastics that have spatially patterned genetic functionality, produce antimicrobials in situ, and perform sensing reactions. The resulting materials unlock the potential to deliver DNA-programmable biofunctionality in a ubiquitous class of synthetic materials.

Supporting clinical competency in managing peripherally inserted central catheters during the COVID-19 pandemic: an education evaluation.

INTRODUCTION: Hospitals had to create new practices and training due to the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) pandemic. An increase in patient acuity and the need for peripherally inserted central catheters (PICC) across the hospital required an urban community hospital to educate and support in-patient nurses to manage PICCs in acute and complex care units. Traditionally, these skills were performed by specialized registered nurses (RNs) from the Vascular Access Team (VAT). This paper highlights the education plan, implementation, and evaluation of a hospital-wide training for RNs and registered practical nurses (RPNs) in in-patient units during the SARS-CoV-2 pandemic. METHODS: Clinical Resource Leaders (CRLs) created a modular approach to upskill existing nurses and train new hires. Various education strategies, such as the use of competency assessments, creating practice supports, and incorporating specialists as a resource, were utilized to ensure knowledge transfer, application, and guidance of evidence-informed clinical practices. Vascular Access Team documentation was utilized to obtain Kirkpatrick's (2021) level 4 evaluation. RESULTS: This training program was implemented after the second wave of the pandemic and was also embedded into nursing orientation. This structured approach ensured that nurses were competent to support the increased acuity and needs of patients. Eighty percent of full-time and part-time nurses were trained to manage PICC lines. CONCLUSION: Education evaluation results show a decrease in PICC-related VAT assistance requests with a baseline of 570 calls down to 149 six months after education was implemented. Leaders are encouraged to ensure teams have role clarity, policies, and practice supports to be successful.

Case of sodium-glucose cotransporter-2 inhibitor-associated euglycaemic diabetic ketoacidosis.

Following non-elective orthopaedic surgery, a 61-year-old man with poorly controlled type 2 diabetes mellitus on empagliflozin developed high anion gap metabolic acidosis in the high-dependency unit. Metabolic acidosis persisted despite intravenous sodium bicarbonate, contributing to tachycardia and a run of non-sustained ventricular tachycardia. He was euglycaemic throughout hospital admission. Investigations revealed elevated urine and capillary ketones, and a diagnosis of sodium-glucose cotransporter-2 inhibitor-associated euglycaemic diabetic ketoacidosis was made. He was treated with an intravenous sliding scale insulin infusion and concurrent dextrose 5% with potassium chloride. Within 24 hours of treatment, his arterial pH, anion gap and serum bicarbonate levels normalised. After a further 12 hours, the intravenous insulin infusion was converted to a basal/bolus regimen of subcutaneous insulin, and he was transferred to the general ward. He was discharged well on subcutaneous insulin 6 days postoperatively.

Impact of Porous Matrices and Concentration by Lyophilization on Cell-Free Expression.

Cell-free expression systems have drawn increasing attention as a tool to achieve complex biological functions outside of the cell. Several applications of the technology involve the delivery of functionality to challenging environments, such as field-forward diagnostics or point-of-need manufacturing of pharmaceuticals. To achieve these goals, cell-free reaction components are preserved using encapsulation or lyophilization methods, both of which often involve an embedding of components in porous matrices like paper or hydrogels. Previous work has shown a range of impacts of porous materials on cell-free expression reactions. Here, we explored a panel of 32 paperlike materials and 5 hydrogel materials for the impact on reaction performance. The screen included a tolerance to lyophilization for reaction systems based on both cell lysates and purified expression components. For paperlike materials, we found that (1) materials based on synthetic polymers were mostly incompatible with cell-free expression, (2) lysate-based reactions were largely insensitive to the matrix for cellulosic and microfiber materials, and (3) purified systems had an improved performance when lyophilized in cellulosic but not microfiber matrices. The impact of hydrogel materials ranged from completely inhibitory to a slight enhancement. The exploration of modulating the rehydration volume of lyophilized reactions yielded reaction speed increases using an enzymatic colorimetric reporter of up to twofold with an optimal ratio of 2:1 lyophilized reaction to rehydration volume for the lysate system and 1.5:1 for the purified system. The effect was independent of the matrices assessed. Testing with a fluorescent nonenzymatic reporter and no matrix showed similar improvements in both yields and reaction speeds for the lysate system and yields but not reaction speeds for the purified system. We finally used these observations to show an improved performance of two sensors that span reaction types, matrix, and reporters. In total, these results should enhance efforts to develop field-forward applications of cell-free expression systems.

Self-assembling Shell Proteins PduA and PduJ have Essential and Redundant Roles in Bacterial Microcompartment Assembly.

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.

BEAMS: a workforce development program to bridge the gap between biologists and material scientists.

To maximize innovation in materials science and synthetic biology, it is critical to master interdisciplinary understanding and communication within an organization. Programming aimed at this juncture has the potential to bring members of the workforce together to frame new networks and spark collaboration. In this article, we recognize the potential synergy between materials and synthetic biology research and describe our approach to this challenge as a case study. A workforce development program was devised consisting of a lecture series, laboratory demonstrations and a hands-on laboratory competition to produce a bacterial cellulose material with the highest tensile strength. This program, combined with support for infrastructure and research, resulted in a significant return on investment with new externally funded synthetic biology for materials programs for our organization. The learning elements described here may be adapted by other institutions for a variety of settings and goals.

Lyophilized Cell-Free Systems Display Tolerance to Organic Solvent Exposure.

Cell-free systems offer a powerful way to deliver biochemical activity to the field without cold chain storage. These systems are capable of sensing as well as biosynthesis of useful molecules at the point of need. So far, cell-free protein synthesis (CFPS) reactions have been studied as aqueous solutions in test tubes or absorbed into paper or cloth. Embedding biological functionality into broadly used materials, such as plastic polymers, represents an attractive goal. Unfortunately, this goal has for the most part remained out of reach, presumably due to the fragility of biological systems outside of aqueous environments. Here, we describe a surprising and useful feature of lyophilized cell-free lysate systems: tolerance to a variety of organic solvents. Screens of individual CFPS reagents and different CFPS methods reveal that solvent tolerance varies by CFPS reagent composition. Tolerance to suspension in organic solvents may facilitate the use of polymers to deliver dry cell-free reactions in the form of coatings or fibers, or allow dosing of analytes or substrates dissolved in nonaqueous solvents, among other processing possibilities.

Silk fibroin as an additive for cell-free protein synthesis.

Cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions. Research into expanding the delivery of these systems by drying or by embedding into other materials is enabling new applications in sensing, point-of-need manufacturing, and responsive materials. Meanwhile, silk fibroin from the silk worm, Bombyx mori, has received attention as a protective additive for dried enzyme formulations and as a material to build biocompatible hydrogels for controlled localization or delivery of biomolecular cargoes. In this work, we explore the effects of silk fibroin as an additive in cell-free protein synthesis (CFPS) reactions. Impacts of silk fibroin on CFPS activity and stability after drying, as well as the potential for incorporation of CFPS into hydrogels of crosslinked silk fibroin are assessed. We find that simple addition of silk fibroin increased productivity of the CFPS reactions by up to 42%, which we attribute to macromolecular crowding effects. However, we did not find evidence that silk fibroin provides a protective effects after drying as previously described for purified enzymes. Further, the enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are inhibited in the presence of the CFPS reaction mixture. Crosslinking attempts did not impact CFPS activity, but did yield localized protein aggregates rather than a hydrogel. We discuss the mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for the design of cell-free devices.

Beta-adrenergic blockade for attenuation of catecholamine surge after traumatic brain injury: a randomized pilot trial.

BACKGROUND: Beta-blockers have been proven in multiple studies to be beneficial in patients with traumatic brain injury. Few prospective studies have verified this and no randomized controlled trials. Additionally, most studies do not titrate the dose of beta-blockers to therapeutic effect. We hypothesize that propranolol titrated to effect will confer a survival benefit in patients with traumatic brain injury. METHODS: A randomized controlled pilot trial was performed during a 24-month period. Patients with traumatic brain injury were randomized to propranolol or control group for a 14-day study period. Variables collected included demographics, injury severity, physiologic parameters, urinary catecholamines, and outcomes. Patients receiving propranolol were compared with the control group. RESULTS: Over the study period, 525 patients were screened, 26 were randomized, and 25 were analyzed. Overall, the mean age was 51.3 years and the majority were male with blunt mechanism. The mean Injury Severity Score was 21.8 and median head Abbreviated Injury Scale score was 4. Overall mortality was 20.0%. Mean arterial pressure was higher in the treatment arm as compared with control (p=0.021), but no other differences were found between the groups in demographics, severity of injury, severity of illness, physiologic parameters, or mortality (7.7% vs. 33%; p=0.109). No difference was detected over time in any variables with respect to treatment, urinary catecholamines, or physiologic parameters. Glasgow Coma Scale (GCS), Sequential Organ Failure Assessment, and Acute Physiology and Chronic Health Evaluation scores all improved over time. GCS at study end was significantly higher in the treatment arm (11.7 vs. 8.9; p=0.044). Finally, no difference was detected with survival analysis over time between groups. CONCLUSIONS: Despite not being powered to show statistical differences between groups, GCS at study end was significantly improved in the treatment arm and mortality was improved although not at a traditional level of significance. The study protocol was safe and feasible to apply to an appropriately powered larger multicenter study. LEVEL OF EVIDENCE: Level 2-therapeutic.

An estimate is worth about a thousand experiments: using order-of-magnitude estimates to identify cellular engineering targets.

Biotechnological processes use microbes to convert abundant molecules, such as glucose, into high-value products, such as pharmaceuticals, commodity and fine chemicals, and energy. However, from the outset of the development of a new bioprocess, it is difficult to determine the feasibility, expected yields, and targets for engineering. In this review, we describe a methodology that uses rough estimates to assess the feasibility of a process, approximate the expected product titer of a biological system, and identify variables to manipulate in order to achieve the desired performance. This methodology uses estimates from literature and biological intuition, and can be applied in the early stages of a project to help plan future engineering. We highlight recent literature examples, as well as two case studies from our own work, to demonstrate the use and power of rough estimates. Describing and predicting biological function using estimates guides the research and development phase of new bioprocesses and is a useful first step to understand and build a new microbial factory.

Evidence for Improved Encapsulated Pathway Behavior in a Bacterial Microcompartment through Shell Protein Engineering.

Bacterial microcompartments are a class of proteinaceous organelles comprising a characteristic protein shell enclosing a set of enzymes. Compartmentalization can prevent escape of volatile or toxic intermediates, prevent off-pathway reactions, and create private cofactor pools. Encapsulation in synthetic microcompartment organelles will enhance the function of heterologous pathways, but to do so, it is critical to understand how to control diffusion in and out of the microcompartment organelle. To this end, we explored how small differences in the shell protein structure result in changes in the diffusion of metabolites through the shell. We found that the ethanolamine utilization (Eut) protein EutM properly incorporates into the 1,2-propanediol utilization (Pdu) microcompartment, altering native metabolite accumulation and the resulting growth on 1,2-propanediol as the sole carbon source. Further, we identified a single pore-lining residue mutation that confers the same phenotype as substitution of the full EutM protein, indicating that small molecule diffusion through the shell is the cause of growth enhancement. Finally, we show that the hydropathy index and charge of pore amino acids are important indicators to predict how pore mutations will affect growth on 1,2-propanediol, likely by controlling diffusion of one or more metabolites. This study highlights the use of two strategies to engineer microcompartments to control metabolite transport: altering the existing shell protein pore via mutation of the pore-lining residues, and generating chimeras using shell proteins with the desired pores.

De novo design of signal sequences to localize cargo to the 1,2-propanediol utilization microcompartment.

Organizing heterologous biosyntheses inside bacterial cells can alleviate common problems owing to toxicity, poor kinetic performance, and cofactor imbalances. A subcellular organelle known as a bacterial microcompartment, such as the 1,2-propanediol utilization microcompartment of Salmonella, is a promising chassis for this strategy. Here we demonstrate de novo design of the N-terminal signal sequences used to direct cargo to these microcompartment organelles. We expand the native repertoire of signal sequences using rational and library-based approaches and show that a canonical leucine-zipper motif can function as a signal sequence for microcompartment localization. Our strategy can be applied to generate new signal sequences localizing arbitrary cargo proteins to the 1,2-propanediol utilization microcompartments.

Experience in optimizing fertility outcomes in men with congenital adrenal hyperplasia due to 21 hydroxylase deficiency.

OBJECTIVE: Men with congenital adrenal hyperplasia (CAH) have impaired fertility. We aimed to assess fertility outcomes and the importance of hypogonadotropic hypogonadism, testicular failure and the presence of testicular adrenal rest tumours (TART). DESIGN: Retrospective analysis of men attending an adult CAH clinic in a tertiary centre. PATIENTS: Fifty men with CAH due to 21 hydroxylase deficiency were identified of whom 35 were salt wasting and 15 were non-salt-wasting. MEASUREMENTS: Review of fertility history and parameters including luteinizing hormone (LH), follicle-stimulating hormone (FSH), androstenedione, 17-hydroxyprogesterone (17-OHP), semen analysis and the presence of testicular adrenal rest tissue (TART) on ultrasound. RESULTS: TART were detected by ultrasound in 21 (47%), and their presence was associated with an elevated FSH (P = 0·01). Severe oligospermia was present in 11 of 23 (48%), and this was associated with an elevated FSH (P = 0·02), suppressed LH (P < 0·01) and TART (P = 0·03) when compared to those with a sperm count >5 × 10(6) per ml. Of those that desired fertility, 10 of 17 (59%) required treatment intensification and four underwent in vitro fertilization. Intensification resulted in a rise in median LH (0·6-4·3 IU/l; P = 0·01). Live birth rate was 15 of 17 (88%) with a median (range) time to conception of 8 (0-38) months. CONCLUSIONS: Suppressed LH is a marker for subfertility and is often reversible. Testicular failure is closely associated with TART formation. If TART are detected, sperm cryopreservation should be offered given the risk of progression to irreversible testicular failure. Male fertility in CAH can be improved by intensified treatment and assisted reproductive technology.

Screening for mutations in 17ß-hydroxysteroid dehydrogenase and androgen receptor in women presenting with partially virilised 46,XY disorders of sex development.

CONTEXT AND OBJECTIVE: The precise diagnosis of partially virilised women with 46,XY disorders of sex development (DSD) is often obscure. In practice, this group often comes under the poorly defined, clinically based label of partial androgen insensitivity syndrome (PAIS). In a previous study, we found that 5α-reductase 2 (SRD5A2) mutations occurred in 43% of women in this subgroup. We expand this work to include biochemical and genetic screening for 17ß-hydroxysteroid dehydrogenase (HSD17B3) and androgen receptor (AR) mutations. METHODS: Analysis of serum androgens (androstenedione and testosterone) and genetic analyses for HSD17B3 and AR were performed in 42 women from 36 pedigrees with partially virilised 46,XY DSD in whom SRD5A2 deficiency had been excluded by urine steroid profiling. RESULTS: Out of 36 unrelated women, 14 (38%) were found to have HSD17B3 mutations and one (2.7%) to have an AR defect. Six novel pathogenic HSD17B3 mutations were identified: three splice site mutations and three missense changes. Seven patients with HSD17B3 deficiency tested before gonadectomy had basal testosterone/androstenedione (T/A) ratio <0.8 (sensitivity 100% and specificity 91%). CONCLUSIONS: HSD17B3 deficiency is prevalent in the adolescent and adult 46,XY female DSD population and is often associated with lesser degrees of virilisation compared with those with 5α-reductase deficiency. This diagnosis should be considered for individuals labelled as PAIS, particularly, but not exclusively, those who present with virilisation at puberty or primary amenorrhoea. Before gondadectomy, T/A ratio is useful to aid diagnosis, but after gonadectomy sequencing of HSD17B3 must be performed to confirm the diagnosis.

Turner's syndrome: challenges of late diagnosis.

Girls and women with Turner's syndrome who come to medical attention older than 12 years present a challenge of medical management. Puberty is already delayed and some compromises have to be made in adjusting the timing of artificially induced puberty to optimise overall outcome with respect to stature, secondary sex characteristics, and psychosocial endpoints. Additionally, individuals who present with primary amenorrhoea to adult services might miss the opportunity for effective growth hormone treatment. Further, induction of puberty regimens lack an evidence base or even clear guidelines for the timing and dose of oestrogen replacement. We have searched the scientific literature to inform management of Turner's syndrome.

Propofol infusion syndrome: a lethal condition in critically injured patients eliminated by a simple screening protocol.

UNLABELLED: Propofol infusion syndrome (PIS) is defined by arrhythmia, rhabdomyolysis, lactic acidosis, and unrecognized leads to death. We sought to determine the incidence of PIS in trauma patients and evaluate the efficacy of a prospective screening protocol in this patient population. MATERIALS AND METHODS: In Phase I of the before-and-after study (1st January, 2005-31st December, 2005), trauma patients who received propofol were evaluated. Records were reviewed for demographics, injury severity, propofol time, dose, and rates, laboratory values, and adverse events. Patients were identified with PIS based on two of the following criteria: (1) cardiac arrhythmia/collapse, (2) metabolic acidosis, (3) rhabdomyolysis, and (4) acute kidney injury. Phase II (1st January, 2006-31st December, 2011) consisted of a prospective screening protocol (elevated lactate or creatine phosphokinase (CPK)) to identify patients at risk for PIS. RESULTS: 207 patients were identified in Phase I. 6 (2.9%) developed PIS with a 50% mortality. No differences were seen in age, gender, or mechanism. PIS patients were more injured (median ISS 44 vs 26, p=0.04; median head AIS 5 vs 4, p=0.003) and received more propofol (median 50,350 vs 9770 mg, p=0.001) with longer infusion times (413 vs 65 h, p=0.001). Sodium, creatinine, and CPK levels were higher in those that developed PIS (160 vs 145 mmol/L, p=0.001; 4.3 vs 1.1mg/dL, p=0.005; 59,871 vs 520 U/L; p=0.002). Pre-screening PIS incidence was 2.9% (6/207), but after screening (January 2006) the incidence dropped to 0.19% (2/1038, p<0.001). CONCLUSIONS: PIS is a morbid and lethal entity associated with sedation of critically injured patients. A simple screening procedure utilizing serum CPK (<5000 U/L) can essentially eliminate the development of PIS.

Sustainability of Improved Glycemic Control After Diabetes Self-Management Education.

Objective. The purpose of this study was to evaluate glycemic control as measured by A1C during a 2-year period after patients received diabetes self-management education (DSME). Methods. Patients who completed DSME in 2009 and received medical follow-up with A1C measurements for at least 2 years after DSME were included in the evaluation. Primary endpoints were changes in A1C from before to immediately after, 1 year after, and 2 years after DSME. Secondary outcomes included the effects of the following factors on change in A1C: sex, duration of diabetes, uncontrolled diabetes (A1C ≥ 9%), health insurance coverage, and self-reported education level. Results. Forty-three patients were included in the evaluation. Mean A1C before DSME was 10.2 ± 3.7%. Mean A1C after DSME was 7.8 ± 2.2% (P < 0.0001), a 23.5% reduction. Mean A1C at 1 and 2 years after DSME was 7.8 ± 2.1% for each year and remained unchanged from just after DSME to 1 and 2 years after DSME (P > 0.05). Patients with a duration of diabetes of < 1 year had a significantly greater reduction in mean A1C than those with a duration of diabetes ≥ 1 year (28.7 and 20.2%, respectively, P = 0.001). Conclusion. DSME improved glycemic control to a substantial degree, and the effect was sustained for up to 2 years. Although the reduction in A1C was significant for all patients receiving DSME, there was a significantly greater reduction for patients who had a duration of diabetes of < 1 year than for those with a duration of diabetes > 1 year.

Liver dysfunction in Turner syndrome and its relationship to exogenous oestrogen.

Liver dysfunction is commonly seen in women with Turner syndrome and can manifest in a variety of pathologies. In this review, we discuss the spectrum of liver anomalies associated with this syndrome, and discuss some possible aetiological factors and relationships with exogenous oestrogen.

Predicting baccalaureate nursing students' first-semester outcomes: HESI Admission Assessment.

As a means of promoting evidence-based admission selection decisions in a baccalaureate school of nursing, the faculty at a college of nursing in the southeastern part of the United States investigated the value of including preadmission exam scores as one criterion in the admission protocol. The purpose of this study was to evaluate the use of Elsevier's HESI Admission Assessment (A(2)) exam as a predictor of student success. Four A(2) exams were administered to baccalaureate nursing students: reading comprehension, vocabulary & general knowledge, math, and anatomy & physiology. The mean of reading comprehension scores and vocabulary & general knowledge scores constituted the students' English scores, and the mean of all 4 exam scores constituted the students' A(2) composite scores. A(2) scores were correlated with final course grades in the 3 first-semester nursing courses (N = 184). There was a significantly positive (P ≤ .01) relationship between A(2) scores and final course grades for the 3 first-semester nursing courses-as A(2) scores increased, so did final course grades. Faculty concluded that A(2) scores provided a valuable measure of students' ability to succeed within the nursing program and, as such, they enabled faculty to make evidence-based decisions regarding applicant selection.