Rewiring yeast sugar transporter preference through modifying a conserved protein motif

Utilization of exogenous sugars found in lignocellulosic biomass hydrolysates, such as xylose, must be improved before yeast can serve as an efficient biofuel and biochemical production platform. In particular, the first step in this process, the molecular transport of xylose into the cell, can serve as a significant flux bottleneck and is highly inhibited by other sugars. Here we demonstrate that sugar transport preference and kinetics can be rewired through the programming of a sequence motif of the general form G-G/F-XXX-G found in the first transmembrane span. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose. Through saturation mutagenesis and subsequent rational mutagenesis, four transporter mutants unable to confer growth on glucose but able to sustain growth on xylose were engineered. Specifically, Candida intermedia gxs1 Phe³⁸Ile³⁹Met⁴⁰, Scheffersomyces stipitis rgt2 Phe³⁸ and Met⁴⁰, and Saccharomyces cerevisiae hxt7 Ile³⁹Met⁴⁰Met³⁴⁰ all exhibit this phenotype. In these cases, primary hexose transporters were rewired into xylose transporters. These xylose transporters nevertheless remained inhibited by glucose. Furthermore, in the course of identifying this motif, novel wild-type transporters with superior monosaccharide growth profiles were discovered, namely S. stipitis RGT2 and Debaryomyces hansenii 2D01474. These findings build toward the engineering of efficient pentose utilization in yeast and provide a blueprint for reprogramming transporter properties.Molecular transporter proteins facilitate monosaccharide uptake and serve as the first step in catabolic metabolism. In this capacity, the preferences, regulation, and kinetics of these transporters ultimately dictate total carbon flux (1–3); and optimization of intracellular catabolic pathways only increases the degree to which transport exerts control over metabolic flux (4, 5). Thus, monosaccharide transport profiles and rates are important design criteria and a driving force to enable metabolic engineering advances, ultimately resulting in a biorefinery concept whereby biomass is converted via microbes into a diverse set of molecules (6–10). Among possible host organisms, Saccharomyces cerevisiae is an emerging industrial organism with well-developed genetic tools and established industrial processes and track record (11–16). However, S. cerevisiae lacks an endogenous xylose catabolic pathway and thus is unable to natively use the second most abundant sugar in lignocellulosic biomass. Decades of research have been focused on improving xylose catabolic pathways in recombinant S. cerevisiae (17–22), but less work has been focused on the first committed step of the process—xylose transport, an outstanding limitation in the efficient conversion of lignocellulosic sugars (23, 24).In S. cerevisiae, monosaccharide uptake is mediated by transporters belonging to the major facilitator superfamily (MFS) (25, 26), a ubiquitous group of proteins found across species (27). The predominant transporters in yeast are members of the HXT family (28) and are marked by efficient hexose transport (29) with lower affinities to xylose thus contributing to diauxic growth and flux limitation when attempting pentose utilization in recombinant S. cerevisiae (30). Previous efforts have attempted to identify heterologous transporters with a higher affinity for xylose over glucose (31–36). However, the vast majority of these transporters are either nonfunctional, not efficient, or not xylose specific (24, 37). Furthermore, nearly all known wild-type transporters that enable growth on xylose in yeast confer higher growth rates on glucose than on xylose (24, 37). As an alternative to bioprospecting, we have previously reported that xylose affinity and exponential growth rates on xylose can be improved via directed evolution of Candida intermedia glucose-xylose symporter 1 (GXS1) and Scheffersomyces stipitis xylose uptake 3 (XUT3) (38). These results demonstrated that mutations at specific residues (e.g., Phe⁴⁰ in C. intermedia GXS1) can have a significant impact on the carbohydrate selectivity of these MFS transporters. The fact that single amino acid substitutions can have such a significant impact on transport phenotype (38–40) indicates how simple homology based searches can be ineffective at identifying efficient xylose transporters (35, 36). However, evidence of natural xylose exclusivity is seen in the Escherichia coli xylE transporter that has recently been crystallized (41). The sequence-function flexibility of MFS transporters potentiates the capability to rewire hexose transporters from being glucose favoring, xylose permissive into being xylose-exclusive transporters.In this work, we report on the discovery of a conserved Gly³⁶-Gly³⁷-Val³⁸-Leu³⁹-Phe⁴⁰-Gly⁴¹ motif surrounding the previously identified Phe⁴⁰ residue of C. intermedia GXS1 that controls transporter efficiency and selectivity. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose, taking the general form G-G/F-XXX-G. We conduct saturation mutagenesis on Val³⁸, Leu³⁹, and Phe⁴⁰ within the variable region of this motif in C. intermedia GXS1 to demonstrate control of sugar selectivity. Next, we combine xylose-favoring mutations to create a unique mutant version of gxs1 that transports xylose, but not glucose. Finally, we demonstrate the importance of this motif in the capacity to rewire the sugar preference of other hexose transporters including S. cerevisiae hexose transporter 7 (HXT7) and S. stipitis glucose transporter/sensor (RGT2, similar to S. cerevisiae RGT2). This work serves to increase our understanding of the structure–function relationships for molecular transporter engineering and demonstrates complete rewiring of hexose transporters into transporters that prefer xylose as a substrate.     

Therapeutic hypothermia for acute myocardial infarction and cardiac arrest

This report focuses on cardioprotection and describes the advantages and disadvantages of various methods of inducing therapeutic hypothermia (TH) with regard to neuroprotection and cardioprotection for patients with cardiac arrest and ST-segment elevation myocardial infarction (STEMI). TH is recommended in cardiac arrest guidelines. For patients resuscitated after out-of-hospital cardiac arrest, improvements in survival and neurologic outcomes were observed with relatively slow induction of TH. More rapid induction of TH in patients with cardiac arrest might have a mild to modest incremental impact on neurologic outcomes. TH drastically reduces infarct size in animal models, but achievement of target temperature before reperfusion is essential. Rapid initiation of TH in patients with STEMI is challenging but attainable, and marked infarct size reductions are possible. To induce TH, a variety of devices have recently been developed that require additional study. Of particular interest is transcoronary induction of TH using a catheter or wire lumen, which enables hypothermic reperfusion in the absence of total-body hypothermia. At present, the main methods of inducing and maintaining TH are surface cooling, endovascular heat-exchange catheters, and intravenous infusion of cold fluids. Surface cooling or endovascular catheters may be sufficient for induction of TH in patients resuscitated after out-of-hospital cardiac arrest. For patients with STEMI, intravenous infusion of cold fluids achieves target temperature very rapidly but might worsen left ventricular function. More widespread use of TH would improve survival and quality of life for patients with out-of-hospital cardiac arrest; larger studies with more rapid induction of TH are needed in the STEMI population.     

Reconstruction of complex aplasia cutis congenita

Aplasia cutis congenita is a rare scalp defect with occasional absence of underlying bone and dura. Patients with small defects respond well to conservative management. However, larger defects often can pose management and technical challenge. Successful management of a large, composite aplasia cutis congenita defect using Integra dermal regeneration matrix is presented. At 16 months, the patient showed a stable and supple scalp with evidence of calvarial regeneration.     

Liver disease in Viet Nam: screening, surveillance, management and education: a 5-year plan and call to action

Despite a high prevalence of liver disease in Viet Nam, there has been no nationwide approach to the disease and no systematic screening of at-risk individuals. Risk factors include chronic hepatitis B (estimated prevalence of 12%), chronic hepatitis C (at least 2% prevalence), and heavy consumption of alcohol among men. This combination of factors has resulted in liver cancer being the most common cause of cancer death in Viet Nam. There is a general lack of understanding by both the general public and health-care providers about the major risk to health that liver disease represents. We report here the initial steps taken as part of a comprehensive approach to liver disease that will ultimately include nationwide education for health-care providers, health educators, and the public; expansion of nationwide screening for hepatitis B and C followed by hepatitis B virus vaccination or treatment of chronic hepatitis B and/or hepatitis C; education about alcoholic liver disease; long-term surveillance for liver cancer; reduction of infection transmission related to medical, commercial, and personal re-use of contaminated needles, syringes, sharp instruments, razors, and inadequately sterilized medical equipment; and ongoing collection and analysis of data about the prevalence of all forms of liver disease and the results of the expanded screening, vaccination, and treatment programs. We report the beginning results of our pilot hepatitis B screening program. We believe that this comprehensive nationwide approach could substantially reduce the morbidity and mortality from liver disease and greatly lessen the burden in terms of both lives lost and health-care costs.     

Qualitative comparison of coronary angiograms between 4 french catheters with an Advanced Cardiovascular Injection System and 6 french catheters with manual injection

Objectives: This study aimed to test whether advanced cardiovascular injection (ACI) via 4 French (Fr) catheters is more advantageous than manual injection with 6 Fr catheters. Background: For coronary angiography, proponents of contrast media ACI believe it utilizes smaller‐diameter catheters when compared with manual injection, without compromising the quality of the angiogram, and has the potential to reduce the amount of contrast used and lessen the use of closure devices. Methods: In a prospective study, 200 consecutive patients referred for elective coronary angiography were randomized to standard 6 Fr catheters with manual injection versus 4 Fr catheters with ACI. The study's primary endpoint is the quality of the coronary angiogram. Secondary endpoints include groin complications, utilization of closure devices, and volume of contrast media. Thirty‐six patients who underwent ad hoc percutaneous coronary intervention were excluded from the analysis. Results: The final analysis included 80 patients treated with 4 Fr catheters with ACI and 84 patients treated with 6 Fr catheters with manual injection. The groups had similar demographic and clinical characteristics. The quality of the angiogram was similar between groups with the exception of more left coronary sinus flush with 4 Fr catheters (3.57 ± 1.1 vs. 2.98 ± 0.9, P < 0.001) and less closure device use with 4 Fr (25 vs. 71.4%, P < 0.01) compared to 6 Fr catheters. The procedure duration, volume of contrast used, vascular complications, and time to ambulation were similar between groups (all P > 0.05). Conclusions: 4 Fr diagnostic catheters with ACI offer similar quality coronary angiograms, similar rates of vascular complications, procedure duration, contrast used, and time to ambulation, but have a significantly lesser need for closure devices compared to the 6 Fr system with manual injection. © 2011 Wiley‐Liss, Inc.     

Electrodeposited nickel–graphene nanocomposite coating: effect of graphene nanoplatelet size on its microstructure and hardness

In this study, the effect of graphene nanoplatelet (GNP) size on the microstructure and hardness of the electrodeposited nickel–graphene nanocomposite coatings were investigated. GNPs with different sizes were prepared by using a high energy ball milling technique. The experimental result revealed the high energy ball milling technique could reduce the size, increase the surface area, and improve the dispersion ability of GNPs. The microstructure, hardness, and components of the nanocomposite coatings were greatly affected by GNP sizes. The highest microhardness was measured to be 273 HV for the nanocomposite coatings containing 5 h-milled GNPs, which is increased up to ∼47% compared to pristine Ni coating. The enhancement in the hardness is attributed to the uniform dispersion of the small GNP sizes inside the Ni matrix and the Ni grain size reduction when using milled GNPs.

The effect of graphene nanoplatelet size on the microstructure and hardness of electrodeposited nickel–graphene nanocomposite coatings was investigated.