A correlative study of the genomic underpinning of virulence traits and drug tolerance of Candida auris.

Candida auris is an opportunistic fungal pathogen with high mortality rates which presents a clear threat to public health. The risk of C. auris infection is high because it can colonize the body, resist antifungal treatment, and evade the immune system. The genetic mechanisms for these traits are not well known. Identifying them could lead to new targets for new treatments. To this end, we present an analysis of the genetics and gene expression patterns of C. auris carbon metabolism, drug resistance, and macrophage interaction. We chose to study two C. auris isolates simultaneously, one drug sensitive (B11220 from Clade II) and one drug resistant (B11221 from Clade III). Comparing the genomes, we confirm the previously reported finding that B11220 was missing a 12.8 kb region on chromosome VI. This region contains a gene cluster encoding proteins related to alternative sugar utilization. We show that B11221, which has the gene cluster, readily assimilates and utilizes D-galactose and L-rhamnose as compared to B11220, which harbors the deletion. B11221 exhibits increased adherence and drug resistance compared to B11220 when grown in these sugars. Transcriptomic analysis of both isolates grown on glucose or galactose showed that the gene cluster was upregulated when grown on D-galactose. These findings reinforce growing evidence of a link between metabolism and drug tolerance. B11221 resists phagocytosis by macrophages and exhibits decreased ß-1,3-glucan exposure, a key determinant that allows Candida to evade the host immune system, as compared to B11220. In a transcriptomic analysis of both isolates co-cultured with macrophages, we find upregulation of genes associated with transport and transcription factors in B11221. Our studies show a positive correlation between membrane composition and immune evasion, alternate sugar utilization, and drug tolerance in C. auris.

Interfacial Assembly of Bacterial Microcompartment Shell Proteins in Aqueous Multiphase Systems.

Compartments are a fundamental feature of life, based variously on lipid membranes, protein shells, or biopolymer phase separation. Here, this combines self-assembling bacterial microcompartment (BMC) shell proteins and liquid-liquid phase separation (LLPS) to develop new forms of compartmentalization. It is found that BMC shell proteins assemble at the liquid-liquid interfaces between either 1) the dextran-rich droplets and PEG-rich continuous phase of a poly(ethyleneglycol)(PEG)/dextran aqueous two-phase system, or 2) the polypeptide-rich coacervate droplets and continuous dilute phase of a polylysine/polyaspartate complex coacervate system. Interfacial protein assemblies in the coacervate system are sensitive to the ratio of cationic to anionic polypeptides, consistent with electrostatically-driven assembly. In both systems, interfacial protein assembly competes with aggregation, with protein concentration and polycation availability impacting coating. These two LLPS systems are then combined to form a three-phase system wherein coacervate droplets are contained within dextran-rich phase droplets. Interfacial localization of BMC hexameric shell proteins is tunable in a three-phase system by changing the polyelectrolyte charge ratio. The tens-of-micron scale BMC shell protein-coated droplets introduced here can accommodate bioactive cargo such as enzymes or RNA and represent a new synthetic cell strategy for organizing biomimetic functionality.

Engaging resident physicians in the design, implementation, and assessment of bedside interdisciplinary rounds.

Bedside interdisciplinary rounds (IDR) improve teamwork, communication, and collaborative culture in inpatient settings. Implementation of bedside IDR in academic settings depends on engagement from resident physicians; however, little is known about their knowledge and preferences related to bedside IDR. The goal of this program was to identify medical resident perceptions about bedside IDR and to engage resident physicians in the design, implementation, and assessment of bedside IDR in an academic setting. This is a pre-post mixed methods survey assessing resident physicians' perceptions surrounding a stakeholder-informed bedside IDR quality improvement project. Resident physicians in the University of Colorado Internal Medicine Residency Program (n = 77 pre-implementation survey responses from 179 eligible participants - response rate 43%) were recruited via e-mail to participate in surveys assessing perceptions surrounding the inclusion of interprofessional team members, timing, and preferred structure of bedside IDR. A bedside IDR structure was created based on input from resident and attending physicians, patients, nurses, care coordinators, pharmacists, social workers, and rehabilitation specialists. This rounding structure was implemented on acute care wards in June 2019 at a large academic regional VA hospital in Aurora, CO. Resident physicians were surveyed post implementation (n = 58 post-implementation responses from 141 eligible participants - response rate 41%) about interprofessional input, timing, and satisfaction with bedside IDR. The pre-implementation survey revealed several important resident needs during bedside IDR. Post-implementation survey results revealed high overall satisfaction with bedside IDR among residents, improved perceived efficiency of rounds, preserved quality of education, and value added by interprofessional input. Results also suggested areas for future improvement including timeliness of rounds and enhanced systems-based teaching. This project successfully engaged residents as stakeholders in system-level interprofessional change by incorporating their values and preferences into a bedside IDR framework.

Hepatitis B e Antigen-Negative Single Hepatocyte Analysis Shows Transcriptional Silencing and Slow Decay of Infected Cells With Treatment.

BACKGROUND: Nucleos(t)ide analogues (NUCs) rarely cure chronic hepatitis B (CHB) because they do not eliminate covalently closed circular deoxyribonucleic acid, the stable replication template. In hepatitis B e antigen (HBeAg)-positive CHB during NUCs, HBV-infected cells decline slowly and are transcriptionally silenced. Whether these occur in HBeAg-negative CHB is unknown. METHODS: Using paired liver biopsies separated by 2.7-3.7 years in 4 males with HIV and HBeAg-negative CHB at both biopsies and 1 male with HIV who underwent HBeAg seroconversion between biopsies, we quantified amounts of viral nucleic acids in hundreds of individual hepatocytes. RESULTS: In the 4 persistently HBeAg-negative participants, HBV-infected hepatocytes ranged from 6.2% to 17.7% (biopsy 1) and significantly declined in 3 of 4 by biopsy 2. In the HBeAg seroconverter, the proportion was 97.4% (biopsy 1) and declined to 81.9% at biopsy 2 (P < .05). We extrapolated that HBV eradication with NUCs would take >100 years. At biopsy 1 in the persistently HBeAg-negative participants, 23%-56.8% of infected hepatocytes were transcriptionally inactive-higher than we observed in HBeAg-positive CHB-and significantly declined in 1 of 4 at biopsy 2. CONCLUSIONS: In HBeAg-negative CHB on NUCs, the negligible decline in infected hepatocytes is similar to HBeAg-positive CHB, supporting the need for more potent therapeutics to achieve functional cure.

Peritoneal Dialysis-Associated Peritonitis Trends Using Medicare Claims Data, 2013-2017.

RATIONALE & OBJECTIVE: The occurrence and consequences of peritoneal dialysis (PD)-associated peritonitis limit its use in populations with kidney failure. Studies of large clinical populations may enhance our understanding of peritonitis. To facilitate these studies we developed an approach to measuring peritonitis rates using Medicare claims data to characterize peritonitis trends and identify its clinical risk factors. STUDY DESIGN: Retrospective cohort study of PD-associated peritonitis. SETTING & PARTICIPANTS: US Renal Data System standard analysis files were used for claims, eligibility, modality, and demographic information. The sample consisted of patients receiving PD treated at some time between 2013 and 2017 who were covered by Medicare fee-for-service (FFS) insurance with paid claims for dialysis or hospital services. EXPOSURES/PREDICTORS: Peritonitis risk was characterized by year, age, sex, race, ethnicity, vintage of kidney replacement therapy, cause of kidney failure, and prior peritonitis episodes. OUTCOME: The major outcome was peritonitis, identified using ICD-9 and ICD-10 diagnosis codes. Closely spaced peritonitis claims (30 days) were aggregated into 1 peritonitis episode. ANALYTICAL APPROACH: Patient-level risk factors for peritonitis were modeled using Poisson regression. RESULTS: We identified 70,271 peritonitis episodes from 396,289 peritonitis claims. Although various codes were used to record an episode of peritonitis, none was used predominantly. Peritonitis episodes were often identified by multiple aggregated claims, with the mean and median claims per episode being 5.6 and 2, respectively. We found 40% of episodes were exclusively outpatient, 9% exclusively inpatient, and 16% were exclusively based on codes that do not clearly distinguish peritonitis from catheter infections/inflammation ("catheter codes"). The overall peritonitis rate was 0.54 episodes per patient-year (EPPY). The rate was 0.45 EPPY after excluding catheter codes and 0.35 EPPY when limited to episodes that only included claims from nephrologists or dialysis providers. The peritonitis rate declined by 5%/year and varied by patient factors including age (lower rates at higher ages), race (Black > White>Asian), and prior peritonitis episodes (higher rate with each prior episode). LIMITATIONS: Coding heterogeneity indicates a lack of standardization. Episodes based exclusively on catheter codes could represent false positives. Peritonitis episodes were not validated against symptoms or microbiologic data. CONCLUSIONS: PD-associated peritonitis rates decline over time and were lower among older patients. A claims-based approach offers a promising framework for the study of PD-associated peritonitis.

The Return on Investment of a Province-Wide Quality Improvement Initiative for Reducing In-Hospital Sepsis Rates and Mortality in British Columbia, Canada.

OBJECTIVES: Sepsis is a life-threatening medical emergency. There is a paucity of information on whether quality improvement approaches reduce the in-hospital sepsis caseload or save lives and decrease the healthcare system and society's cost at the provincial/national levels. This study aimed to assess the outcomes and economic impact of a province-wide quality improvement initiative in Canada. DESIGN: Retrospective population-based study with interrupted time series and return on investment analyses. SETTING: The sepsis cases and deaths averted over time for British Columbia were calculated and compared with the rest of Canada (excluding Quebec and three territories). PATIENTS: Aggregate data were obtained from the Canadian Institute for Health Information on risk-adjusted in-hospital sepsis rates and sepsis mortality in acute care sites across Canada. INTERVENTIONS: In 2012, the British Columbia Sepsis Network was formed to reduce sepsis occurrence and mortality through education, knowledge translation, and quality improvement. MEASUREMENTS AND MAIN RESULTS: A return on investment analysis compared the financial investment for the British Columbia Sepsis Network with the savings from averted sepsis occurrence and mortality. An estimated 981 sepsis cases and 172 deaths were averted in the post-British Columbia Sepsis Network period (2014-2018). The total cost, including the development and implementation of British Columbia Sepsis Network, was $449,962. Net savings due to cases averted after program costs were considered were $50.6 million in 2018. This translates into a return of $112.5 for every dollar invested. CONCLUSIONS: British Columbia Sepsis Network appears to have averted a greater number of sepsis cases and deaths in British Columbia than the national average and yielded a positive return on investment. Our findings strengthen the policy argument for targeted quality improvement initiatives for sepsis care and provide a model of care for other provinces in Canada and elsewhere globally.

Functional subgroups of cochlear inner hair cell ribbon synapses differently modulate their EPSC properties in response to stimulation.

Spiral ganglion neurons (SGNs) form single synapses on inner hair cells (IHCs), transforming sound-induced IHC receptor potentials into trains of action potentials. SGN neurons are classified by spontaneous firing rates as well as their threshold response to sound intensity levels. We investigated the hypothesis that synaptic specializations underlie mouse SGN response properties and vary with pillar versus modiloar synapse location around the hair cell. Depolarizing hair cells with 40 mM K+ increased the rate of postsynaptic responses. Pillar synapses matured later than modiolar synapses. Excitatory postsynaptic current (EPSC) amplitude, area, and number of underlying events per EPSC were similar between synapse locations at steady state. However, modiolar synapses produced larger monophasic EPSCs when EPSC rates were low and EPSCs became more multiphasic and smaller in amplitude when rates were higher, while pillar synapses produced more monophasic and larger EPSCs when the release rates were higher. We propose that pillar and modiolar synapses have different operating points. Our data provide insight into underlying mechanisms regulating EPSC generation.NEW & NOTEWORTHY Data presented here provide the first direct functional evidence of late synaptic maturation of the hair cell- spiral ganglion neuron synapse, where pillar synapses mature after postnatal day 20. Data identify a presynaptic difference in release during stimulation. This difference may in part drive afferent firing properties.

Resolution of subcomponents of synaptic release from postsynaptic currents in rat hair-cell/auditory-nerve fiber synapses.

The synapse between inner hair cells and auditory nerve fiber dendrites shows large excitatory postsynaptic currents (EPSCs), which are either monophasic or multiphasic. Multiquantal or uniquantal (flickering) release of neurotransmitter has been proposed to underlie the unusual multiphasic waveforms. Here the nature of multiphasic waveforms is analyzed using EPSCs recorded in vitro in rat afferent dendrites. Spontaneous EPSCs were deconvolved into a sum of presumed release events having monophasic EPSC waveforms. Results include, first, the charge of EPSCs is about the same for multiphasic versus monophasic EPSCs. Second, EPSC amplitudes decline with the number of release events per EPSC. Third, there is no evidence of a mini-EPSC. Most results can be accounted for by versions of either uniquantal or multiquantal release. However, serial neurotransmitter release in multiphasic EPSCs shows properties that are not fully explained by either model, especially that the amplitudes of individual release events are established at the beginning of a multiphasic EPSC, constraining possible models of vesicle release.NEW & NOTEWORTHY How do monophasic and multiphasic waveshapes arise in auditory-nerve dendrites; mainly are they uniquantal, arising from release of a single vesicle, or multiquantal, requiring several vesicles? The charge injected by excitatory postsynaptic currents (EPSCs) is the same for monophasic or multiphasic EPSCs, supporting uniquantal release. Serial adaptation of responses to sequential EPSCs favors a multiquantal model. Finally, neurotransmitter partitioning into similar sized release boluses occurs at the first bolus in the EPSC, not easily explained with either model.

Suppression of pancreatic cancer liver metastasis by secretion-deficient ITIH5.

BACKGROUND: Previously, we identified ITIH5 as a suppressor of pancreatic ductal adenocarcinoma (PDAC) metastasis in experimental models. Expression of ITIH5 correlated with decreased cell motility, invasion and metastasis without significant inhibition of primary tumour growth. Here, we tested whether secretion of ITIH5 is required to suppress liver metastasis and sought to understand the role of ITIH5 in human PDAC. METHODS: We expressed mutant ITIH5 with deletion of the N-terminal secretion sequence (ITIH5Δs) in highly metastatic human PDAC cell lines. We used a human tissue microarray (TMA) to compare ITIH5 levels in uninvolved pancreas, primary and metastatic PDAC. RESULTS: Secretion-deficient ITIH5Δs was sufficient to suppress liver metastasis. Similar to secreted ITIH5, expression of ITIH5Δs was associated with rounded cell morphology, reduced cell motility and reduction of liver metastasis. Expression of ITIH5 is low in both human primary PDAC and matched metastases. CONCLUSIONS: Metastasis suppression by ITIH5 may be mediated by an intracellular mechanism. In human PDAC, loss of ITIH5 may be an early event and ITIH5-low PDAC cells in primary tumours may be selected for liver metastasis. Further defining the ITIH5-mediated pathway in PDAC could establish future therapeutic exploitation of this biology and reduce morbidity and mortality associated with PDAC metastasis.

Burden and Cost of Caring for US Veterans With CKD: Initial Findings From the VA Renal Information System (VA-REINS).

Kidney disease is a common, complex, costly, and life-limiting condition. Most kidney disease registries or information systems have been limited to single institutions or regions. A national US Department of Veterans Affairs (VA) Renal Information System (VA-REINS) was recently developed. We describe its creation and present key initial findings related to chronic kidney disease (CKD) without kidney replacement therapy (KRT). Data from the VA's Corporate Data Warehouse were processed and linked with national Medicare data for patients with CKD receiving KRT. Operational definitions for VA user, CKD, acute kidney injury, and kidney failure were developed. Among 7 million VA users in fiscal year 2014, CKD was identified using either a strict or liberal operational definition in 1.1 million (16.4%) and 2.5 million (36.3%) veterans, respectively. Most were identified using an estimated glomerular filtration rate laboratory phenotype, some through proteinuria assessment, and very few through International Classification of Diseases, Ninth Revision coding. The VA spent ∼$18 billion for the care of patients with CKD without KRT, most of which was for CKD stage 3, with higher per-patient costs by CKD stage. VA-REINS can be leveraged for disease surveillance, population health management, and improving the quality and value of care, thereby enhancing VA's capacity as a patient-centered learning health system for US veterans.

The risk of medically uncontrolled secondary hyperparathyroidism depends on parathyroid hormone levels at haemodialysis initiation.

BACKGROUND: Optimal parathyroid hormone (PTH) control during non-dialysis chronic kidney disease (ND-CKD) might decrease the subsequent risk of parathyroid hyperplasia and uncontrolled secondary hyperparathyroidism (SHPT) on dialysis. However, the evidence for recommending PTH targets and therapeutic strategies is weak for ND-CKD. We evaluated the patient characteristics, treatment patterns and PTH control over the first year of haemodialysis (HD) by PTH prior to HD initiation. METHODS: We studied 5683 incident HD patients from 21 countries in Dialysis Outcomes and Practice Patterns Study Phases 4-6 (2009-18). We stratified by PTH measured immediately prior to HD initiation and reported the monthly prescription prevalence of active vitamin D and calcimimetics over the first year of HD and risk of PTH >600 pg/mL after 9-12 months on HD. RESULTS: The 16% of patients with PTH >600 pg/mL prior to HD initiation were more likely to be prescribed active vitamin D and calcimimetics during the first year of HD. The prevalence of PTH >600 pg/mL 9-12 months after start of HD was greater for patients who initiated HD with PTH >600 (29%) versus 150-300 (7%) pg/mL (adjusted risk difference: 19%; 95% confidence interval : 15%, 23%). The patients with sustained PTH >600 pg/mL after 9-12 months on HD were younger, more likely to be black, and had higher serum phosphorus and estimated glomerular filtration rates at HD initiation. CONCLUSIONS: Increased PTH before HD start predicted a higher PTH level 9-12 months later, despite greater use of active vitamin D and calcimimetics. More targeted PTH control during ND-CKD may influence outcomes during HD, raising the need for PTH target guidelines in these patients.

Association of erythropoietin resistance and fibroblast growth factor 23 in dialysis patients: Results from the Japanese Dialysis Outcomes and Practice Patterns Study.

BACKGROUND: Fibroblast growth factor 23 (FGF23) plays an important role in chronic kidney disease (CKD)-related mineral and bone disorders. High FGF23 levels are associated with increased risk of anaemia in non-haemodialysis CKD patients. FGF23 also negatively regulates erythropoiesis in mice. We hypothesized that higher FGF23 levels are associated with increased erythropoietin hyporesponsiveness among haemodialysis patients. METHODS: The study included 1044 patients from the Japanese Dialysis Outcomes and Practice Patterns Study (J-DOPPS) phase 5 (2012-2015). The outcome was erythropoiesis-stimulating agent hyporesponsiveness (ESA-hypo), defined as mean Hgb <10 g/dL and standardized mean ESA dose >6000 u/week over 4 months following FGF23 measurement. The association between ESA-hypo and FGF23 was estimated using multivariable-adjusted logistic generalized estimating equation regression models. RESULTS: Patients with higher levels of FGF23 were younger and had higher levels of serum albumin, creatinine, albumin-corrected calcium, phosphorus, PTH, 25(OH)-vitamin D, and had higher percentages of intravenous (IV) iron, IV vitamin D and cinacalcet use. ESA-hypo was present in 144 patients (13.8%). Compared with the third quintile of FGF23 levels, the odds ratio (95% CI) of ESA-hypo was 2.14 (0.99, 4.62) and 1.74 (0.74, 4.11) for the first and fifth quintiles, respectively. CONCLUSION: The lowest and highest levels of FGF23 were associated with higher odds of ESA-hypo in patients on maintenance haemodialysis, although the associations were not statistically significant. The relationship between FGF23 and anaemia, and particularly the increased risks of ESA-hypo at low FGF23 levels which might be the result of energy saving, must be confirmed in larger clinical studies.

Visualizing in Vivo Dynamics of Designer Nanoscaffolds.

Enzymes of natural biochemical pathways are routinely subcellularly organized in space and time in order to improve pathway efficacy and control. Designer scaffolding platforms are under development to confer similar benefits upon engineered pathways. Herein, we evaluate bacterial microcompartment shell (pfam0936-domain) proteins as modules for constructing well-defined nanometer scale scaffolds in vivo. We use a suite of visualization techniques to evaluate scaffold assembly and dynamics. We demonstrate recruitment of target cargo molecules onto assembled scaffolds by appending reciprocally interacting adaptor domains. These interactions can be refined by fine-tuning the scaffold expression level. Real-time observation of this system reveals a nucleation-limited step where multiple scaffolds initially form within a cell. Over time, nucleated scaffolds reorganize into a single intracellular assembly, likely due to interscaffold competition for protein subunits. Our results suggest design considerations for using self-assembling proteins as building blocks to construct nanoscaffolds, while also providing a platform to visualize scaffold-cargo dynamics in vivo.

Preparation and Characterization of Biobased Lignin-Co-Polyester/Amide Thermoplastics.

More than 23 million tonnes of lignin are produced annually in the US from wood pulping and 98% of this lignin is burnt. Therefore, creating products from lignin, such as plastics, offers an approach for obtaining sustainable materials in a circular economy. Lignin-based copolymers were synthesized using a single pot, solvent free, melt condensation reaction. The synthesis occurred in two stages. In the first stage, a biobased prepolymer consisting of butanediol (BD, 0.8-1 molar content) and a diacid (succinic (SA), adipic (AA) and suberic acids (SuA), with varying amounts of diaminobutane (DAB, 0-0.2 molar content) was heated under vacuum and monitored by Fourier transform infra-red (FTIR) spectroscopy and electrospray ionization-mass spectrometry (ESI-MS). In the second stage, prepolymer was mixed with a softwood kraft lignin (0-50 wt.%) and further reacted under vacuum at elevated temperature. Progression of the polymerization reaction was monitored using FTIR spectroscopy. The lignin-copolyester/amide properties were characterized using tensile testing, X-ray diffraction (XRD), dynamic mechanical analysis (DMA), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) techniques. Lignin co-polymer tensile (strength 0.1-2.1 MPa and modulus 2 to 338 MPa) properties were found to be influenced by the diacid chain length, lignin, and DAB contents. The lignin-copolymers were shown to be semi-crystalline polymer and have thermoplastic behavior. The SA based copolyesters/amides were relatively stiff and brittle materials while the AA based copolyesters/amides were flexible and the SuA based copolyesters/amides fell in-between. Additionally, > 30 wt.% lignin the lignin- copolyesters/amides did not exhibit melt behavior. Lignin-co-polyester/amides can be generated using green synthesis methods from biobased building blocks. The lignin- copolyesters/amides properties could be tuned based on the lignin content, DAB content and diacid chain length. This approach shows that undervalued lignin can be used in as a macromonomer in producing thermoplastic materials.

Chronic kidney disease progression and mortality risk profiles in Germany: results from the Chronic Kidney Disease Outcomes and Practice Patterns Study.

BACKGROUND: Chronic kidney disease (CKD) progression among German patients in a representative setting has not been described previously. The Verband Deutsche Nierenzentren and Chronic Kidney Disease Outcomes and Practice Patterns Study established a longitudinal observational cohort among German CKD patients to research variations in patient care and outcomes in real-world nephrology practices. METHODS: A cohort of CKD Stages 3 (25%) and 4 (75%) patients was established from German nephrologist-run CKD clinics in 2013-16. Linear models were used to determine the estimated glomerular filtration rate (eGFR) slope during follow-up and Cox models were used to assess outcomes of end-stage kidney disease (ESKD) and death. RESULTS: A total of 1834 patients (median age 75 years, 58% male, 42% diabetics, median baseline eGFR 25 mL/min/1.73 m2) were followed for a median of 29 months. More than 50% had slow or no decline and 17% declined ≥5 mL/min/1.73 m2/year. After 4.5 years, the incidence of ESKD was 8% and of deaths without ESKD 16% among patients with eGFR ≥30 mL/min/1.73 m2 and 37% and 19% for eGFR <30 mL/min/1.73 m2. Adjusted models showed higher risks of ESKD or death for patients with worse kidney function at baseline, male sex, diabetes and higher blood pressure; a higher risk of ESKD with higher albuminuria; and a higher risk of death with older age or cardiovascular comorbidity. CONCLUSIONS: Routine nephrology care of patients in Germany comprises mostly elderly patients, many with slow CKD progression. Identification of risk factors for CKD progression and mortality may help guide resources by closer follow-up of high-risk patients.

Can oral health and oral-derived biospecimens predict progression of dementia?

Growing evidence indicates that oral health and brain health are interconnected. Declining cognition and dementia coincide with lack of self-preservation, including oral hygiene. The oral microbiota plays an important role in maintaining oral health. Emerging evidence suggests a link between oral dysbiosis and cognitive decline in patients with Alzheimer's disease. This review showcases the recent advances connecting oral health and cognitive function during aging and the potential utility of oral-derived biospecimens to inform on brain health. Collectively, experimental findings indicate that the connection between oral health and cognition cannot be underestimated; moreover, oral biospecimens are abundant and readily obtainable without invasive procedures, which may help inform on cognitive health.

Development of a Provincial initiative to improve glucose control in critically ill patients.

OBJECTIVE: To describe the development, implementation and initial evaluation of an initiative to improve glucose control in critically ill patients. DESIGN: Glucose control in critically ill patients was chosen by critical care leaders as a target for improvement. This was an observational study to document changes in processes and measures of glucose control in each intensive care unit (ICU). ICU nurse educators were interviewed to document relevant changes between April 2012 and April 2016. SETTING: 16 ICUs in British Columbia, Canada. PARTICIPANTS: ICU leaders. INTERVENTION(S): A community of practice (CoP) was formed, guidelines were adopted, two learning sessions were held, and an electronic system to collect data was created. Then, each ICU introduced their own educational and process interventions. MAIN OUTCOME MEASURE(S): Average hyperglycemic index (area under the curve of serum glucose concentration versus time above the upper limit (10 mmol/l) divided by time on insulin infusion), number of hypoglycemic events (<3.5 mmol/l) divided by time on insulin infusion and standardized mortality rate (actual/predicted hospital mortality) for each 3-month period. RESULTS: Although there were some isolated points and short trends that indicated special cause variation, there were no major trends over time and no obvious association with any of the process changes for each hospital. However, the average hyperglycemic index was higher in some of the smaller hospitals than in the larger hospitals. CONCLUSIONS: In this, 4-year observation of glucose control in ICUs within a CoP, the lack of sustained improvement suggests the need for more active and durable interventions.

A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

Iterative algorithm-guided design of massive strain libraries, applied to itaconic acid production in yeast.

Metabolic engineering requires multiple rounds of strain construction to evaluate alternative pathways and enzyme concentrations. Optimizing multigene pathways stepwise or by randomly selecting enzymes and expression levels is inefficient. Here, we apply methods from design of experiments (DOE) to guide the construction of strain libraries from which the maximum information can be extracted without sampling every possible combination. We use Saccharomyces cerevisiae as a host for a novel six-gene pathway to itaconic acid, selected by comparing alternative shunt pathways that bypass the mitochondrial TCA cycle. The pathway is distinctive for the use of acetylating acetaldehyde dehydrogenase to increase cytosolic acetyl-CoA pools, a bacterial enzyme to synthesize citrate in the cytosol, and an itaconic acid exporter. Precise control over the expression of each gene is enabled by a set of promoter-terminator pairs that span a 174-fold range. Two large combinatorial libraries (160 variants, 2.4 Mb and 32 variants, 0.6 Mb) are designed where the expression levels are selected by statistical methods (I-optimal response surface methodology, full factorial, or Plackett-Burman) with the intent of extracting different types of guiding information after the screen. This is applied to the design of a third library (24 variants, 0.5 Mb) intended to alleviate a bottleneck in cis-aconitate decarboxylase (CAD) expression. The top strain produces 815 mg/l itaconic acid, a 4-fold improvement over the initial strain achieved by iteratively balancing pathway expression. Including a methylated product in the total, the strain produces 1.3 g/l combined itaconic acids. Further, a regression analysis of the libraries reveals the optimal expression level of CAD as well as pairwise interdependencies between genes that result in increased titer and purity of itaconic acid. This work demonstrates adapting algorithmic design strategies to guide automated yeast strain construction and learn information after each iteration.

Maturation of Spontaneous Firing Properties after Hearing Onset in Rat Auditory Nerve Fibers: Spontaneous Rates, Refractoriness, and Interfiber Correlations.

Auditory nerve fibers (ANFs) exhibit a range of spontaneous firing rates (SRs) that are inversely correlated with threshold for sounds. To probe the underlying mechanisms and time course of SR differentiation during cochlear maturation, loose-patch extracellular recordings were made from ANF dendrites using acutely excised rat cochlear preparations of different ages after hearing onset. Diversification of SRs occurred mostly between the second and the third postnatal week. Statistical properties of ANF spike trains showed developmental changes that approach adult-like features in older preparations. Comparison with intracellularly recorded EPSCs revealed that most properties of ANF spike trains derive from the characteristics of presynaptic transmitter release. Pharmacological tests and waveform analysis showed that endogenous firing produces some fraction of ANF spikes, accounting for their unusual properties; the endogenous firing diminishes gradually during maturation. Paired recordings showed that ANFs contacting the same inner hair cell could have different SRs, with no correlation in their spike timing. SIGNIFICANCE STATEMENT: The inner hair cell (IHC)/auditory nerve fiber (ANF) synapse is the first synapse of the auditory pathway. Remarkably, each IHC is the sole partner of 10-30 ANFs with a range of spontaneous firing rates (SRs). Low and high SR ANFs respond to sound differently, and both are important for encoding sound information across varying acoustical environments. Here we demonstrate SR diversification after hearing onset by afferent recordings in acutely excised rat cochlear preparations. We describe developmental changes in spike train statistics and endogenous firing in immature ANFs. Dual afferent recordings provide the first direct evidence that fibers with different SRs contact the same IHCs and do not show correlated spike timing at rest. These results lay the groundwork for understanding the differential sensitivity of ANFs to acoustic trauma.