Bioindustrial manufacturing readiness levels (BioMRLs) as a shared framework for measuring and communicating the maturity of bioproduct manufacturing processes.

Readiness level (RL) frameworks such as technology readiness levels and manufacturing readiness levels describe the status of a technology/manufacturing process on its journey from initial conception to commercial deployment. More importantly, they provide a roadmap to guide technology development and scale-up from a ''totality of system'' approach. Commercialization risks associated with too narrowly focused R&D efforts are mitigated. RLs are defined abstractly so that they can apply to diverse industries and technology sectors. However, differences between technology sectors make necessary the definition of sector specific RL frameworks. Here, we describe bioindustrial manufacturing readiness levels (BioMRLs), a classification system specific to bioindustrial manufacturing. BioMRLs will give program managers, investors, scientists, and engineers a shared vocabulary for prioritizing goals and assessing risks in the development and commercialization of a bioindustrial manufacturing process.

A wearable patch for continuous monitoring of sweat electrolytes during exertion.

Implementation of wearable sweat sensors for continuous measurement of fluid based biomarkers (including electrolytes, metabolites and proteins) is an attractive alternative to common, yet intrusive and invasive, practices such as urine or blood analysis. Recent years have witnessed several key demonstrations of sweat based electrochemical sensing in wearable formats, however, there are still significant challenges and opportunities in this space for clinical acceptance, and thus mass implementation of these devices. For instance, there are inherent challenges in establishing direct correlations between sweat-based and gold-standard plasma-based biomarker concentrations for clinical decision-making. In addition, the wearable sweat monitoring devices themselves may exacerbate these challenges, as they can significantly alter sweat physiology (example, sweat rate and composition). Reported here is the demonstration of a fully integrated, wireless, wearable and flexible sweat sensing device for non-obtrusive and continuous monitoring of electrolytes during moderate to intense exertion as a metric for hydration status. The focus of this work is twofold: 1- design of a conformable fluidics systems to suit conditions of operation for sweat collection (to minimize sensor lag) with rapid removal of sweat from the sensing site (to minimize effects on sweat physiology). 2- integration of Na+ and K+ ion-selective electrodes (ISEs) with flexible microfluidics and low noise small footprint electronics components to enable wireless, wearable sweat monitoring. While this device is specific to electrolyte analysis during intense perspiration, the lessons in microfluidics and overall system design are likely applicable across a broad range of analytes.

Detection of orexin A neuropeptide in biological fluids using a zinc oxide field effect transistor.

Biomarkers which are indicative of acute physiological and emotional states are studied in a number of different areas in cognitive neuroscience. Currently, many cognitive studies are conducted based on programmed tasks followed by timed biofluid sampling, central laboratory processing, and followed by data analysis. In this work, we present a sensor platform capable of rapid biomarker detection specific for detecting neuropeptide orexin A, found in blood and saliva and known as an indicator of fatigue and cognitive performance. A peptide recognition element that selectively binds to orexin A was designed, characterized, and functionalized onto a zinc oxide field effect transistor to enable rapid detection. The detection limit using the sensor platform was sub-picomolar in water, and picomolar to nanomolar levels in saliva and serum. The transistor and recognition element sensor platform can be easily expanded, allowing for multiple biomarkers to be detected simultaneously, lending itself to complex biomarker analysis applicable to rapid feedback for neuroscience research and physiological monitoring.

Ice restructuring inhibition activities in antifreeze proteins with distinct differences in thermal hysteresis.

Antifreeze proteins (AFPs) share two related properties: the ability to depress the freezing temperature below the melting point of ice (thermal hysteresis; TH); and the ability to inhibit the restructuring of ice into larger crystals. Since the 'hyperactive' AFPs, which have been more recently discovered, show an order of magnitude more TH than previously characterized AFPs, we have now determined their activities in ice restructuring inhibition (IrI) assays. IrI activities of three TH-hyperactive AFPs and three less TH-active AFPs varied over an 8-fold range. There was no obvious correlation between high TH activity and high IrI activity. However, the use of mutant AFPs demonstrated that severe disruption of ice-binding residues diminished both TH and IrI similarly, revealing that that the same ice-binding residues are crucial for both activities. In addition, bicarbonate ions, which are known to enhance the TH activity of AFPs, also enhanced their IrI activity. We suggest that these seemingly contradictory observations can be partially explained by differences in the coverage of ice by TH-hyperactive and non-hyperactive AFPs, and by differences in the stability of AFP-bound ice under supercooled and recrystallization conditions.

Nondestructive in situ identification of crystal orientation of anisotropic ZnO nanostructures.

We present a novel method for direct, fast, nonambiguous, and nondestructive identification of the growth direction and orientation of individual ZnO nanostructures in the device-ready environment by exploiting high-resolution confocal Raman mapping. Various features of the Raman spectrum of ZnO nanostructures, vapor deposition grown nanobelts and peptide-assisted vertical nanorods, were found to be sensitive to the relative orientation of the crystal plane. Furthermore, we discovered that the waveguiding property of the ZnO nanobelt is also orientation dependent and results in either apparent enhancement or suppression of Raman scattering from the underlying substrate. We demonstrate that various features of Raman spectrum of ZnO and the modulation of the substrate signal can be employed for the rapid and nondestructive identification of the crystal growth direction and orientation of these nanostructures even after integration into devices, which is impossible with current electron microscopy and diffraction techniques. We believe that the general features observed here are equally applicable to other wurtzite nanostructures (ZnS, GaN) which are critical in optoelectronics, lasing, and piezotronic applications.

Morphological control and assembly of zinc oxide using a biotemplate.

Zinc oxide is a wide band gap material that has significant applications in photovoltaics, piezoelectrics and optoelectronics. Traditionally, ZnO has been synthesized using high temperatures and harsh reaction conditions. Recently, benign reaction conditions have been used to synthesize ZnO using amine and citrate additives. In this study, peptide phage display was performed to identify a peptide, termed Z1, that binds to and directs the growth of ZnO hexagonal nanocrystals. By altering the concentration of Z1 peptide, the ZnO nanocrystal morphology can be tailored. Additionally, Z1 peptide was used to direct the growth of ZnO structures on free-standing silk films. The results presented here demonstrate the utility of peptides in controlling the structure and deposition of ZnO.

Metal ion-dependent, reversible, protein filament formation by designed beta-roll polypeptides.

BACKGROUND: A right-handed, calcium-dependent beta-roll structure found in secreted proteases and repeat-in-toxin proteins was used as a template for the design of minimal, soluble, monomeric polypeptides that would fold in the presence of Ca2+. Two polypeptides were synthesised to contain two and four metal-binding sites, respectively, and exploit stacked tryptophan pairs to stabilise the fold and report on the conformational state of the polypeptide. RESULTS: Initial analysis of the two polypeptides in the presence of calcium suggested the polypeptides were disordered. The addition of lanthanum to these peptides caused aggregation. Upon further study by right angle light scattering and electron microscopy, the aggregates were identified as ordered protein filaments that required lanthanum to polymerize. These filaments could be disassembled by the addition of a chelating agent. A simple head-to-tail model is proposed for filament formation that explains the metal ion-dependency. The model is supported by the capping of one of the polypeptides with biotin, which disrupts filament formation and provides the ability to control the average length of the filaments. CONCLUSION: Metal ion-dependent, reversible protein filament formation is demonstrated for two designed polypeptides. The polypeptides form filaments that are approximately 3 nm in diameter and several hundred nm in length. They are not amyloid-like in nature as demonstrated by their behaviour in the presence of congo red and thioflavin T. A capping strategy allows for the control of filament length and for potential applications including the "decoration" of a protein filament with various functional moieties.

Thermodynamic stability of a cold-adapted protein, type III antifreeze protein, and energetic contribution of salt bridges.

A thermodynamic analysis of a cold-adapted protein, type III anti-freeze protein (AFP), was carried out. The results indicate that the folding equilibrium of type III AFP is a reversible, unimolecular, two-state process with no populated intermediates. Compared to most mesophilic proteins whose folding is two-state, the psychrophilic type III AFP has a much lower thermodynamic stability at 25 degrees C, approximately 3 kcal/mol, and presents a remarkably downshifted stability-temperature curve, reaching a maximum of 5 kcal/mol around 0 degrees C. Type III AFPs contain few and non-optimally distributed surface charges relative to their mesophilic homologs, the C-terminal domains of sialic acid synthases. We used thermodynamic double mutant cycles to evaluate the energetic role of every surface salt bridge in type III AFP. Two isolated salt bridges provided no contribution to stability, while the Asp36-Arg39 salt bridge, involved in a salt bridge network with the C-terminal carboxylate, had a substantial contribution (approximately 1 kcal/mol). However, this contribution was more than counteracted by the destabilizing effect of the Asp36 carboxylate itself, whose removal led to a net 30% increase in stability at 25 degrees C. This study suggests that type III AFPs may have evolved for a minimally acceptable stability at the restricted, low temperature range (around 0 degrees C) at which AFPs must function. In addition, it indicates that salt bridge networks are used in nature also for the stability of psychrophilic proteins, and has led to a type III AFP variant of increased stability that could be used for biotechnological purposes.

Polypeptide-templated synthesis of hexagonal silica platelets.

Several studies have demonstrated the use of biomimetic approaches in the synthesis of a variety of inorganic materials. Poly-L-lysine (PLL) promotes the precipitation of silica from a silicic acid solution within minutes. The molecular weight of PLL was found to affect the morphology of the resulting silica precipitate. Larger-molecular weight PLL produced hexagonal silica platelets, whereas spherical silica particles were obtained using low-molecular weight PLL. Here we report on the polypeptide secondary-structure transition that occurs during the silicification reaction. The formation of the hexagonal silica platelets is attributed to the PLL helical chains that are formed in the presence of monosilicic acid and phosphate ions. Hexagonal PLL crystals can also serve as templates in directing the growth of the silica in a manner that generates a largely mesoporous silica phase that is oriented with respect to the protein crystal template.

Entrapment of enzymes and nanoparticles using biomimetically synthesized silica.

Entrapment of enzymes and nanoparticles using biosilicification reactions.

Partitioning of fish and insect antifreeze proteins into ice suggests they bind with comparable affinity.

Antifreeze proteins (AFPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. AFPs from insects are much more effective at depressing the freezing point than fish AFPs. Here, we have investigated the possibility that insect AFPs bind more avidly to ice than fish AFPs. Because it is not possible to directly measure the affinity of an AFP for ice, we have assessed binding indirectly by examining the partitioning of proteins into a slowly growing ice hemisphere. AFP molecules adsorbed to the surface and became incorporated into the ice as they were overgrown. Solutes, including non-AFPs, were very efficiently excluded from ice, whereas AFPs became incorporated into ice at a concentration roughly equal to that of the original solution, and this was independent of the AFP concentration in the range (submillimolar) tested. Despite their >10-fold difference in antifreeze activity, fish and insect AFPs partitioned into ice to a similar degree, suggesting that insect AFPs do not bind to ice with appreciably higher affinity. Additionally, we have demonstrated that steric mutations on the ice binding surface that decrease the antifreeze activity of an AFP also reduce its inclusion into ice, supporting the validity of using partitioning measurements to assess a protein's affinity for ice.

A facile method for determining ice recrystallization inhibition by antifreeze proteins.

Ice recrystallization, the growth of large ice crystals at the expense of small ones, stresses freeze tolerant organisms and causes spoilage of frozen foods. This process is inhibited by antifreeze proteins (AFPs). Here, we present a simple method for determining the ice recrystallization inhibition (RI) activity of an AFP under physiological conditions using 10microl glass capillaries. Serial dilutions were prepared to determine the concentration below which RI activity was no longer detected, termed the RI endpoint. For type III AFP this was 200nM. The capillary method allows samples to be aligned and viewed simultaneously, which facilitates RI endpoint determination. Once prepared, the samples can be used reproducibly in subsequent RI assays and can be archived in a freezer for future reference. This method was used to detect the elution of type III AFP from a Sephadex G-75 size-exclusion column. RI activity was found at the expected V(e) for a 7kDa protein and also unexpectedly in the void volume.

The effect of hydrophobic analogues of the type I winter flounder antifreeze protein on lipid bilayers.

The effect of four synthetic analogues of the 37-residue winter flounder type I antifreeze protein (AFP), which contain four Val, Ala or Ile residues in place of Thr residues at positions 2, 13, 24 and 37 and two additional salt bridges, on the binary lipid system prepared from a 1:1 mixture of the highly unsaturated DGDG and saturated DMPC has been determined using FTIR spectroscopy. In contrast to the natural protein, which increases the thermotropic phase transition, the Thr, Val and Ala analogues decreased the thermotropic phase transitions of the liposomes by 2.2 degrees Celsius, 3.4 degrees Celsius and 2.4 degrees Celsius, while the Ile analogue had no effect on the transition. Experiments performed using perdeuterated DMPC showed that the Ala and Thr peptides interacted preferentially with the DGDG in the lipid mixture, while the Val peptide showed no preference for either lipid. The results are consistent with interactions involving the hydrophobic face of type I AFPs and model bilayers, i.e. the same face of the protein that is responsible for antifreeze properties. The different effects correlate with the helicity of the peptides and suggest that the solution conformation of the peptides has a significant role in determining the effects of the peptides on thermotropic membrane phase transitions.

Structural characterization of the ADAM 16 disintegrin loop active site.

ADAM's have various roles in intercellular adhesion and are thought to function by binding integrins through a 13 amino acid motif called the disintegrin loop. Xenopus laevis sperm express the protein ADAM 16, and peptides with the sequence of its disintegrin loop cause downstream events in eggs that require a rise in intracellular calcium similar to that occurring at fertilization. We characterized the portion of the ADAM 16 disintegrin loop responsible for causing egg activation. A peptide based on the C-terminal half of the motif, which includes a known integrin-binding sequence, is a partial agonist of calcium release. A peptide with the N-terminal sequence of the motif activates eggs in a manner virtually identical to the full-length peptide but lacks a recognized integrin-binding sequence. None of these peptides alter the permeability or fluidity of liposomes made from membrane lipids of X. laevis eggs. This result reflects the fact that the peptides do not cause calcium to leak across the egg membrane and indirectly provides evidence that they act through a receptor on the egg surface. The infrared spectrum of the full-length peptide has a strong absorption peak corresponding to a beta-turn. We predict this structure occurs at the N-terminal sequence MPKT. A rearranged peptide lacking any turns fails to activate eggs. These results provide the first structural information about the active site of an ADAM disintegrin loop. We interpret these results in terms of active site sequences from other ADAM's and the role of integrins during fertilization.

Lipid unsaturation determines the interaction of AFP type I with model membranes during thermotropic phase transitions.

We have previously shown that antifreeze protein (AFP) type I from winter flounder interacts with the acyl chains of lipids in model membranes containing a mixture of dimyristoylphosphatidylcholine (DMPC) and the plant thylakoid lipid digalactosyldiacylglycerol (DGDG), most likely through hydrophobic interactions. By contrast, in studies with pure phospholipid membranes, no such interaction was seen. DGDG is a highly unsaturated lipid, which renders these studies quite different from the previous studies of AFP-membrane interaction where the lipids were saturated or trans-unsaturated. Therefore, it seemed possible that either the digalactose headgroups or the unsaturated DGDG acyl chains, or both, may be important for interactions of membranes with AFP type I. To distinguish between these possibilities, we catalytically hydrogenated the DGDG to obtain a galactolipid with completely saturated fatty acyl chains. The results with the hydrogenated DGDG were strikingly different from those obtained previously with the unsaturated DGDG; the clear binding of AFPs to the bilayer appeared to be lost. Nevertheless, the temperature-dependent folding of AFP type I was inhibited in the presence of liposomes containing either the unsaturated or the hydrogenated DGDG. The results indicate that the liposomes and protein still interact, even following hydrogenation of the acyl chains, perhaps at the membrane-solution interface.

A mechanism for stabilization of membranes at low temperatures by an antifreeze protein.

Polar fish, cold hardy plants, and overwintering insects produce antifreeze proteins (AFPs), which lower the freezing point of solutions noncolligatively and inhibit ice crystal growth. Fish AFPs have been shown to stabilize membranes and cells in vitro during hypothermic storage, probably by interacting with the plasma membrane, but the mechanism of this stabilization has not been clear. We show here that during chilling to nonfreezing temperatures the alpha-helical AFP type I from polar fish inhibits leakage across model membranes containing an unsaturated chloroplast galactolipid. The mechanism involves binding of the AFP to the bilayer, which increases the phase transition temperature of the membranes and alters the molecular packing of the acyl chains. We suggest that this change in acyl chain packing results in the reduced membrane permeability. The data suggest a hydrophobic interaction between the peptide and the bilayer. Further, we suggest that the expression of AFP type I in transgenic plants may be significant for thermal adaptation of chilling-sensitive plants.