An Engineered Calmodulin-Based Allosteric Switch for Peptide Biosensing

This work describes the development of a new platform for allosteric protein engineering that takes advantage of the ability of calmodulin to change conformation upon binding to peptide and protein ligands. The switch we have developed consists of a fusion protein in which calmodulin is genetically inserted into the sequence of TEM1 β-lactamase. In this approach, calmodulin acts as the input domain, whose ligand-dependent conformational changes control the activity of the β-lactamase output domain. The new allosteric enzyme exhibits up to 120 times higher catalytic activity in the activated (peptide bound) state compared to the inactive (no peptide bound) state in vitro. Activation of the enzyme is ligand-dependent-peptides with higher affinities for wild-type calmodulin exhibit increased switch activity. Calmodulin's ability to "turn on" the activity of β-lactamase makes this a potentially valuable scaffold for the directed evolution of highly specific biosensors for detecting toxins and other clinically relevant biomarkers.Chemistry and Chemical Biolog

Mechanical Reinforcement of Polymeric Fibers through Peptide Nanotube Incorporation

High aspect ratio nanotubular assemblies can be effective fillers in mechanically reinforced composite materials. However, most existing nanotubes used for structural purposes are limited in their range of mechanical, chemical, and biological properties. We demonstrate an alternative approach to mechanical reinforcement of polymeric systems by incorporating synthetic d,l-cyclic peptide nanotube bundles as a structural filler in electrospun poly d-, l-lactic acid fibers. The nanotube bundles self-assemble through dynamic hydrogen bonding from synthetic cyclic peptides to yield structures whose dimensions can be altered based on processing conditions, and can be up to hundreds of micrometers long and several hundred nanometers wide. With 8 wt % peptide loading, the composite fibers are >5-fold stiffer than fibers composed of the polymer alone, according to atomic force microscopy-based indentation experiments. This represents a new use for self-assembling cyclic peptides as a load-bearing component in biodegradable composite materials.Engineering and Applied Science

A Synthetic Circuit for Mercury Bioremediation Using Self-Assembling Functional Amyloids

Synthetic biology approaches to bioremediation are a key sustainable strategy to leverage the self-replicating and programmable aspects of biology for environmental stewardship. The increasing spread of anthropogenic mercury pollution into our habitats and food chains is a pressing concern. Here, we explore the use of programmed bacterial biofilms to aid in the sequestration of mercury. We demonstrate that by integrating a mercury-responsive promoter and an operon encoding a mercury-absorbing self-assembling extracellular protein nanofiber, we can engineer bacteria that can detect and sequester toxic Hg2+ ions from the environment. This work paves the way for the development of on-demand biofilm living materials that can operate autonomously as heavy-metal absorbents.Engineering and Applied Science

The impact of antioxidant diets, nutraceuticals and physical activity interventions in the prevention of cardiometabolic diseases: An overview

Hippocrates - Father of Medicine (ca 460-370 BC) - endorsed the cuative effects of foods; he said: "Leave your drugs in the chemist's pot if you can heal the patient with food". This review focuses on the management of cardio-metabolic diseases (CMDs) with nutraceuticals and antioxidant diets such as Allium sativum, turmeric, soybean, peptides, phytosterols, resveratrol, polyphenolic substances etc. CMDs are a cluster of conditions linked to altered fat and carbohydrate metabolism as well as macroand micro-vascular problems. CMDs cause severe pathophysiological and metabolic alterations in the body, resulting in the occurrence of chronic diseases like atherosclerosis, coronary heart disease and stroke, neurodegenerative ailments, fatty liver, kidney malfunction, hypercholesterolaemia, hyperlipidaemia, insulin resi-tance and some cancers, consequently imposing a very high economic burden on the healthcare costs. Currently used pharmacotherapies are not only expensive but also are associated with undesirable adverse events. Thus, there is an urgent need for affordable, cost-effective and alternative safe therapies for the prevention and management of CMDs. Holistic approaches targeted for health promotion and prevention of CMDs include the intake of antioxidant-rich diets, anti-inflammation wholesome foods and moderate physical activity (about 30 min/day). Such strategies will not only prevent obesity-related CMDs, type 2 diabetes mellitus (T2DM), coronary heart disease and stroke, but also will improve the quality of patient's life and consequently reduce healthcare burdens. Nutraceuticals and probiotics exhibit anti-inflammation, anti-aging, anti-obesity and anti-diabetic effects, thereby reducing the adverse health risks associated with CMDs. Antioxidants protect cell membranes and DNA from excessive free radicals, which contribute to CMD related diseases. Physical exercise along with dietary interventions helps to mitigate oxidative stress, improve blood triglyceride levels, increase HDL-cholesterol and reduce LDLcholesterol and reverse the biological markers associated with CMDs. Many studies have provided robust scientific evidence and demonstrated links between dietary interventions, nutraceuticals, probiotics, wholesome foods and physical activity for the prevention of CMDs. The major limitations in promoting nonpharmacological therapies for health and well-being benefits are a lack of public awareness and a paucity of clinical nutrition instruction for medical students on the merits of complementary methods for the prevention and management of CMDs. The goals of this review are to provide up-to-date knowledge about selected nutraceuticals, wholesome foods and physical activity in the prevention of CMDs and the underlying mechanisms associated with each intervention, which will ultimately improve patient's quality of life and assist in reducing healthcare costs globally

Structural, Nanomechanical, and Computational Characterization of d , l -Cyclic Peptide Assemblies

The rigid geometry and tunable chemistry of d,l-cyclic peptides makes them an intriguing building-block for the rational design of nano- and microscale hierarchically structured materials. Herein, we utilize a combination of electron microscopy, nanomechanical characterization including depth sensing-based bending experiments, and molecular modeling methods to obtain the structural and mechanical characteristics of cyclo-[(Gln-d-Leu)4] (QL4) assemblies. QL4 monomers assemble to form large, rod-like structures with diameters up to 2 μm and lengths of tens to hundreds of micrometers. Image analysis suggests that large assemblies are hierarchically organized from individual tubes that undergo bundling to form larger structures. With an elastic modulus of 11.3 ± 3.3 GPa, hardness of 387 ± 136 MPa and strength (bending) of 98 ± 19 MPa the peptide crystals are among the most robust known proteinaceous micro- and nanofibers. The measured bending modulus of micron-scale fibrils (10.5 ± 0.9 GPa) is in the same range as the Young’s modulus measured by nanoindentation indicating that the robust nanoscale network from which the assembly derives its properties is preserved at larger length-scales. Materials selection charts are used to demonstrate the particularly robust properties of QL4 including its specific flexural modulus in which it outperforms a number of biological proteinaceous and nonproteinaceous materials including collagen and enamel. The facile synthesis, high modulus, and low density of QL4 fibers indicate that they may find utility as a filler material in a variety of high efficiency, biocompatible composite materials.Engineering and Applied Science

In Vivo Targeting through Click Chemistry

Targeting small molecules to diseased tissues as therapy or diagnosis is a significant challenge in drug delivery. Drug-eluting devices implanted during invasive surgery allow the controlled presentation of drugs at the disease site, but cannot be modified once the surgery is complete. We demonstrate that bioorthogonal click chemistry can be used to target circulating small molecules to hydrogels resident intramuscularly in diseased tissues. We also demonstrate that small molecules can be repeatedly targeted to the diseased area over the course of at least one month. Finally, two bioorthogonal reactions were used to segregate two small molecules injected as a mixture to two separate locations in a mouse disease model. These results demonstrate that click chemistry can be used for pharmacological drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.Engineering and Applied Science

Bootstrapped Biocatalysis: Biofilm-Derived Materials as Reversibly Functionalizable Multienzyme Surfaces

Cell-free biocatalysis systems offer many benefits for chemical manufacturing, but their widespread applicability is hindered by high costs associated with enzyme purification, modification, and immobilization on solid substrates, in addition to the cost of the material substrates themselves. Here we report a “bootstrapped” biocatalysis substrate material that is produced directly in bacterial culture and is derived from biofilm matrix proteins, which self-assemble into a nanofibrous mesh. We demonstrate that this material can simultaneously purify and immobilize multiple enzymes site-specifically, and directly from crude cell lysates using a panel of genetically programmed, mutually orthogonal conjugation domains. We further demonstrate the utility of the technique in a bi- enzymatic stereoselective reduction coupled with cofactor recycling scheme. The domains allow for several cycles of selective removal and replacement of enzymes under mild conditions to regenerate the catalyst system.Engineering and Applied Science

Transcultural Diabetes Nutrition Therapy Algorithm: The Asian Indian Application

India and other countries in Asia are experiencing rapidly escalating epidemics of type 2 diabetes (T2D) and cardiovascular disease. The dramatic rise in the prevalence of these illnesses has been attributed to rapid changes in demographic, socioeconomic, and nutritional factors. The rapid transition in dietary patterns in India—coupled with a sedentary lifestyle and specific socioeconomic pressures—has led to an increase in obesity and other diet-related noncommunicable diseases. Studies have shown that nutritional interventions significantly enhance metabolic control and weight loss. Current clinical practice guidelines (CPGs) are not portable to diverse cultures, constraining the applicability of this type of practical educational instrument. Therefore, a transcultural Diabetes Nutrition Algorithm (tDNA) was developed and then customized per regional variations in India. The resultant India-specific tDNA reflects differences in epidemiologic, physiologic, and nutritional aspects of disease, anthropometric cutoff points, and lifestyle interventions unique to this region of the world. Specific features of this transculturalization process for India include characteristics of a transitional economy with a persistently high poverty rate in a majority of people; higher percentage of body fat and lower muscle mass for a given body mass index; higher rate of sedentary lifestyle; elements of the thrifty phenotype; impact of festivals and holidays on adherence with clinic appointments; and the role of a systems or holistic approach to the problem that must involve politics, policy, and government. This Asian Indian tDNA promises to help guide physicians in the management of prediabetes and T2D in India in a more structured, systematic, and effective way compared with previous methods and currently available CPGs

Could mitochondrial efficiency explain the susceptibility to adiposity, metabolic syndrome, diabetes and cardiovascular diseases in South Asian populations?

Background South Asians are susceptible to cardiovascular disease (CVD), especially after migration to affluent countries. Contributing factors include high prevalence of diabetes, and possibly insulin resistance. Excess adiposity centrally may underlie such metabolic disturbances. The thrifty genotype, thrifty phenotype, adipose tissue compartment and variable disease selection hypotheses are among the explanations posed. Methods Data from individual studies and review articles known to the authors were examined. A Medline bibliographic database search was also performed. Reference lists were reviewed to identify additional relevant data sources. Key references were examined by both authors. Results We propose, and evaluate, the evidence for a ‘mitochondrial efficiency hypothesis’ i.e. that ancestral changes in mitochondrial coupling efficiency enhanced the successful adaptation of South Asians to environmental stressors by maximizing the conversion of energy to adenosine triphosphate (ATP) rather than heat. This adaptation may be disadvantageous when South Asians are physically inactive and consume high-caloric diets. There is evidence that common mitochondrial mutations vary geographically. Mutations, including those affecting the function of mitochondrial uncoupling proteins (UCPs), may influence the balance of energy and heat production. These may influence basal metabolic rate (BMR), energy efficiency, the tendency to gain weight and hence metabolic disease. UCP gene polymorphisms are related to differences in BMR between African-Americans and Europeans. Similar data for South Asians are lacking but the few studies comparing BMR indicate that South Asians have a lower BMR, which is explained by a lower lean body mass, and higher fat mass. Once adjusted for body composition, BMR is similar. A high fat mass, per se, is a strategy for reducing energy use while conserving body size. Indians in the USA had higher oxidative phosphorylation capacity than Northern European Americans. Conclusion The evidence justifies full exploration of this mitochondrial effeciency hypothesis in South Asians, which may also be relevant to other warm-climate adapted populations