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Carbene-based macromolecules are an emerging new stimuli-sensitive class of biomaterials that avoid the impediments of free radical polymerization but maintain a rapid liquid-to-biorubber transition. Activation of diazirine-grafted polycaprolactone polyol (CaproGlu) is limited to UVA wavelengths that have tissue exposure constraints and limited light intensities. For the first time, UVA is circumvented with visible light-emitting diodes at 445 nm (blue) to rapidly activate diazirine-to-carbene covalent cross-linking. Iridium photocatalysts serve to initiate diazirine, despite having little to no absorption at 445 nm. CaproGlu's liquid organic matrix dissolves the photocatalyst with no solvents required, creating a light transparent matrix. Considerable differences in cross-linking chemistry are observed in UVA vs visible/photocatalyst formulations. Empirical analysis and theoretical calculations reveal a more efficient conversion of diazirine directly to carbene with no diazoalkane intermediate detected. Photorheometry results demonstrate a correlation between shear moduli, joules light dose, and the lower limits of photocatalyst concentration required for the liquid-to-biorubber transition. Adhesion strength on ex vivo hydrated tissues exceeds that of cyanoacrylates, with a fixation strength of up to 20 kg·f·cm2. Preliminary toxicity assessment on leachates and materials directly in contact with mammalian fibroblast cells displays no signs of fibroblast cytotoxicity. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Nanyang Technological University Accepted version T.W.J.S. and I.D. are co-inventors of patent application Hygroscopic, Crosslinking Coatings and Bioadhesives; PCT/ SG2018/050452. The project was supported by the Ministry of Education Tier 1 Grant RG17/18 (S): Novel light activated, diazo protecting groups; Ministry of Education Tier 2 Grant (MOE2018-T2-2-114): CaproGlu, double-sided wet tissue adhesives; NTUitive POC (Gap) Fund NGF/2018/05: Aesthetic Applications of CaproGlu Bioadhesives; and A*Star IAF PP Grant (H19/01/a0/0II9): CathoGlu Bioadhesives-preventing catheter extravasation and skin infections. L.B. acknowledges funding from the Spanish Ministry of Science and Innovation (MCIU), project PID2019- 104654GB-I00.

Bacterial cellulose (BC) based hybrid hydrogels and aerogels have been fabricated by reformatting BC pellicles. BC pellicles are formed by tight fibrillar networks making it challenging to incorporate other materials to form hybrids or composites. In this study, BC pellicles produced from two Acetobacteracea family members are disintegrated into individual nanofibrils via TEMPO-mediated oxidation. The individualized BC nanofibrils about tens of microns in length and 20–50 nm in width are well-dispersed in water with crystallinity and birefringence of cellulose I. These BC nanofibril dispersions are non-Newtonian fluid. Upon complexation with 0.75 mmol/g of Fe3+, the formed BC nanofibril hydrogels display 17-fold increase in storage moduli. Composite aerogels formed by complexation with conductive polymer PEDOT/PSS show characteristics of a non-Hookean foam material with conductivity value of up to 0.86 S/cm for cellulose density of 4.2 mg/cm2.

Enzymes, receptors, and carrier proteins discriminate between enantiomers of natural and synthetic chemicals. Whereas the structural details of this phenomenon have been investigated in enzymes and receptors, much less is known for carrier proteins of hydrophobic ligands, particularly concerning the contribution of asymmetric centers in the side chains of amino acids to chirally selective binding. Working with a pig odorant-binding protein, we have found that the replacement of either one or both isoleucine residues in the binding pocket by leucines abolishes discrimination of menthol and carvone enantiomers. The results indicate that isoleucines are crucial for chiral discrimination of hydrophobic ligands, and that asymmetry in the side chain may be as important as the overall asymmetry of the protein. The results provide suggestions and guidelines for improving chiral selectivity of binding proteins and enzymes, with consequent applications in the production of enantiomerically pure drugs.

The development of protein-polymer complexes using protein cages is garnering attention as a strategy to overcome challenges that relate to immunogenicity and protease degradation. We propose the use of thermoresponsive polymers such as poly(N-isopropylacrylamide) (PNIPAM) to form ferritin-polymer complexes through non-covalent interactions. Ferritin is observed to be released from the complexes after 1–2 days incubation at 37 °C.

Bacterial cellulose (BC), a renewable type of cellulose, has been used in the manufacture of foods, cosmetics, and biomedical products. To produce BC, a high-throughput single-bacterium measurement is necessary to identify the functional bacteria that can produce BC with sufficient amount and desirable morphology. In this study, a continuous-flow intelligent optofluidic device was developed to enable high-throughput single-bacterium profiling of BC. Single bacteria were incubated in agarose hydrogel particles to produce BC with varied densities and structures. An intelligent convolutional neural network (CNN) computational method was developed to analyze the scattering patterns of BC. The BC production and morphology were determined with a throughput of ∼35 bacteria per second. A total of ∼105 single-bacterium BC samples were characterized within 3 hours. The high flexibility of this approach facilitates high-throughput comprehensive single-cell production analysis for a range of applications in engineering biology.

Topical treatments for oral wounds and infections exhibit weak adhesion to wet surfaces which results in short retention duration (6-8 hours), frequent dosing requirement and patient incompatibility. To address these limitations, aqueous composites made of fibrillated bacterial cellulose and photoactive bioadhesives are designed for soft epithelial surfaces. The aqueous composites crosslink upon photocuring within a minute and exhibit a transition from viscous to elastic adhesive hydrogels. The light-cured composites have shear moduli mimicking oral mucosa and other soft tissues. The tunable adhesion strength ranges from 3 to 35 kPa on hydrated tissue-mimicking surfaces (collagen film). The results support the application of bacterial cellulose hydrogel systems for potential treatment of mucosal wounds. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Submitted/Accepted version The project is partially supported by A*STAR IAF PP Grant (H19/01/a0/0II9): CathoGlu Bioadhesives-preventing catheter extravasation and skin infections; Ministry of Education Tier 2 Grant: CaproGlu, Double sided wet-tissue adhesives (MOE2018-T2-2-114).

With a significant proportion of the global population growing older (>60 years), the low success rates of current diagnoses for early neurodegeneration signs are disappointing. Early detection of Alzheimer's disease (AD) can improve acclimatization and quality of life for patients in their later years. Endogenous proteins, such as the most abundant secreted protein in cerebrospinal fluid, lipocalin‐type prostaglandin d synthase (L‐PGDS), can bind the early toxic oligomers of amyloid β (Aβ) peptides implicated in AD and prevent their aggregation. Herein, the utility of L‐PGDS for detection of amyloids is demonstrated. L‐PGDS is conjugated with different iron‐oxide magnetic nanoparticles for contrast‐enhanced visualization using magnetic resonance imaging (MRI). These conjugates inhibit amyloid aggregation in vitro and improve viability in neuronal cells incubated with amyloid fibrils, showing a potential neuroprotective function. L‐PGDS‐ferritin conjugates, when administered intraventricularly, localize to AD‐associated amyloid‐rich regions in mice brain imaged using MRI and histological stains. As a proof‐of‐concept, it is demonstrated that L‐PGDS conjugates could reach the brain regions through non‐invasive intranasal administration. These conjugates are developed as the first entirely protein‐based nanoprobes for early detection of brain amyloids. The results of this study open a wider avenue for study of endogenous proteins as potential theranostics for AD.

Hyperpigmentation is a major cosmetic concern that results from the overproduction of melanin from melanocyte cells in the skin. Current formulations of therapeutic drugs and active molecules that are intended to be delivered to the melanocytes are not targeted, leading to inefficient delivery and undesirable side effects. There is a compelling need for delivery vehicles that target melanocytes to effectively regulate melanin production. Self‐assembling protein nanocages (PNCs) have been shown to offer safe and efficient delivery of active molecules. They are versatile protein nanoparticle platforms that can be engineered to display functional ligands to impart specific biological functions. In this work, E2 PNCs are engineered to display alpha‐melanocyte‐stimulating hormone (AlphaMSH) peptides as ligands for targeting and enhancing uptake by melanocytes. At 500 pM, the AlphaMSH‐modified E2 PNCs show 4‐fold increase in melanocyte cell uptake compared to bare E2 PNCs in 2 hours. This increase was less pronounced in keratinocytes. Competitive inhibition assay proves that the MC1R melanocyte cell surface receptor facilitates the uptake of E2 PNCs by mediating interaction with the displayed AlphaMSH peptides. PNCs can thus be used as targeted delivery vehicles of drugs and active molecules to melanocytes for the management of hyperpigmentation.

Accumulation of lipid-laden macrophages (foam cells) is characteristic of atherosclerosis development in the arterial walls. Ferritin nanocages have been found to passively accumulate in the atherosclerotic plaque. Ferritin has been actively investigated as a carrier for contrast agents in atherosclerosis diagnosis. We demonstrate the potential of ferritin as a carrier for therapeutic molecules to mediate cholesterol reduction from foam cells. Cyclodextrin molecules are chemically conjugated to the ferritin nanocages surface or encapsulated within the nanocages using metal co-loading methods. The cyclodextrin-conjugated ferritin has nanomolar affinity to cholesterol molecules. Treatment of foam cells with the conjugates shows decreased levels of intracellular accumulated cholesterol. The preferential localization of ferritin to foam cells is due to transferrin receptor-mediated endocytosis process. These findings show that ferritin nanocages as carriers localize cyclodextrin molecules to foam cells which mediate intracellular cholesterol reduction, thus highlighting its potential use as a therapeutic agent.

Misfolding of Amyloid β (Aβ) peptides leads to the formation of extracellular amyloid plaques. Molecular chaperones can facilitate the refolding or degradation of such misfolded proteins. Here, for the first time, we report the unique ability of Lipocalin-type Prostaglandin D synthase (L-PGDS) protein to act as a disaggregase on the pre-formed fibrils of Aβ(1–40), abbreviated as Aβ40, and Aβ(25–35) peptides, in addition to inhibiting the aggregation of Aβ monomers. Furthermore, our proteomics results indicate that L-PGDS can facilitate extraction of several other proteins from the insoluble aggregates extracted from the brain of an Alzheimer’s disease patient. In this study, we have established the mode of binding of L-PGDS with monomeric and fibrillar Aβ using Nuclear Magnetic Resonance (NMR) Spectroscopy, Small Angle X-ray Scattering (SAXS), and Transmission Electron Microscopy (TEM). Our results confirm a direct interaction between L-PGDS and monomeric Aβ40 and Aβ(25–35), thereby inhibiting their spontaneous aggregation. The monomeric unstructured Aβ40 binds to L-PGDS via its C-terminus, while the N-terminus remains free which is observed as a new domain in the L-PGDS-Aβ40 complex model.

Circulation of cancer cells in the bloodstream is a vital step for distant metastasis, during which cancer cells are exposed to hemodynamic shear stress (SS). The actions of SS on tumor cells are complicated and not fully understood. We previously reported that fluidic SS was able to promote migration of breast cancer cells by elevating the cellular ROS level. In this study, we further investigated the mechanisms regulating SS-promoted cell migration and identified the role of MnSOD in the related pathway. We found that SS could enhance tumor cell adhesion to extracellular matrix and endothelial monolayer, and MnSOD also regulated this process. Briefly, SS stimulates the generation of mitochondrial superoxide in tumor cells. MnSOD then converts superoxide into hydrogen peroxide, which activates ERK1/2 to promote tumor cell migration and activates FAK to promote tumor cell adhesion. Combining our previous and present studies, we present experimental evidence on the pro-metastatic effects of hemodynamic SS and reveal the underlying mechanism. Our findings provide new insights into the nature of cancer metastasis and the understanding of tumor cell responses to external stresses and have valuable implications for cancer therapy development. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Accepted version

Topical approaches to oral diseases require frequent dosing due to limited wound retention times. A mucoadhesive drug delivery platform is proposed for oral wound sites with soft tissue adhesion capability of up to 7 days. Bacterial cellulose (BC) and photoactivated carbene-based bioadhesives are combined to yield flexible film platform for interfacing soft tissues in dynamic, wet environments. Structure-activity relationships evaluate UV dose and hydration state with respect to adhesive strength on soft tissue mimics. The bioadhesive composite has an adhesion strength ranging from 7-17 kPa and duration exceeding 48 hours in wet conditions under sustained shear forces, while other mucoadhesives based on hydrophilic macromolecules exhibit adhesion strength of 0.5-5 kPa and last only a few hours. The work highlights the first evaluation of BC composites for mucoadhesive treatments in the buccal cavity. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Accepted version The project is partially supported by RG17/18 (S); Wound Care Innovation for the Tropics Grant. A*STAR IAF PP Grant (H19/01/a0/ 0II9): CathoGlu Bioadhesives-preventing catheter extravasation and skin infections; Ministry of Education Tier 2 Grant: CaproGlu, Double sided wet-tissue adhesives (MOE2018-T2-2-114); Ministry of Education Tier 1 Grant: Novel light activated, diazo protecting groups.

Tunnel junctions based on Fe storing globular proteins are an interesting class of biomolecular tunnel junctions due to their tunable Fe ion loading, symmetrical structure and thermal stability, and are therefore attractive to study the mechanisms of charge transport (CT) at the molecular level. This paper describes a temperature-induced change in the CT mechanism across junctions with large globular (similar to 25 nm in diameter) E2-proteins bioengineered with Fe-binding peptides from ferritin (E2-LFtn) to mineralise Fe ions in the form of iron oxide nanoparticles (NPs) inside the protein's cavity. The iron oxide NPs provide accessible energy states that support high CT rates and shallow activation barriers. Interestingly, the CT mechanism changes abruptly, but reversibly, from incoherent tunnelling (which is thermally activated) to coherent tunnelling (which is activationless) across the E2-LFtn-based tunnel junctions with the highest Fe ion loading at a temperature of 220-240 K. During this transition the current density across the junctions increases by a factor of 13 at an applied voltage of V = -0.8 V. X-ray absorption spectroscopy indicates that the iron oxide NPs inside the E2-LFtn cages undergo a reversible phase transition; this phase transition opens up new a tunnelling pathway changing the mechanism of CT from thermally activated to activationless tunnelling despite the large size of the E2-LFtn and associated distance for tunnelling. Ministry of Education (MOE) Published version D. Q. acknowledges the support of the Australian Research Council (Grant No. FT160100207). We acknowledge the Ministry of Education (MOE) for supporting this research under award no. MOE2019-T2-1-137. Prime Minister’s Office, Singapore, under its Medium-sized centre program is also acknowledged for supporting this research.

The quest to develop ideal nanoparticles capable of molecular, cellular, and tissue level imaging is ongoing. Since certain imaging probes and nanoparticles face drawbacks such as low aqueous solubility, increased ROS generation leading to DNA damage, apoptosis, and high cellular/organ toxicities, the development of versatile and biocompatible nanocarriers becomes necessary. Protein nanoparticles (PNPs) are one such promising class of nanocarriers that possess most of the desirable properties of an ideal nanocarrier for bioimaging applications. PNPs demonstrate high aqueous solubility, minimal cytotoxicity, and multi-cargo loading capacity. They are also amenable to surface-functionalization, as well as modulation of their hydrophobicity and hydrophilicity. The use of PNPs for bioimaging applications has made rapid advancements in the past two decades. Being comparatively less explored, the field opens up a plethora of opportunities and focus areas to engineer ideal bioimaging protein nanocarriers. The use of PNPs as carriers of their natural ligands as well as other heavy metals and fluorescent probes, along with drug molecules for combined theranostic applications has been reported. In addition, surface functionalization to impart specificity of targeting the PNPs has been shown to reduce nonspecific cellular interactions, thus reducing systemic toxicity. PNPs have been explored for their application in imaging of numerous cancers, cardiovascular diseases as well as imaging of the brain using near infrared fluorescence (NIRF) imaging, magnetic resonance imaging (MRI), X-ray computed tomography (CT), positron emission tomography (PET), single-photon emission computed tomography (SPECT), ultrasound (US), and photoacoustic (PA) imaging. Nanyang Technological University This work is partially supported by the School of Chemical and Biomedical Engineering at Nanyang TechnologicalUniversity, Singapore.

The utilization of surface-active engineered protein nanocages as stabilizers for emulsions provides avenues for the design of new tailor-made functional materials in various fields including food, pharmaceutical, and biotechnology. They can be used to codeliver bioactive molecules of different polarities in a tailored manner to the body, act as a platform for screening cells or enzymes, or function as targeted drug delivery systems. Knowledge on the mechanisms that underlie the protein nanocage-driven stabilization of emulsions and their colloidal structure can have direct implications for the rational design of the new advanced functional colloids. This contribution summarizes the recent progress in protein nanocage-stabilized emulsions. It discusses the advances in the precision bioengineering of protein nanocages for emulsion design, highlights challenges in the characterization of structure and dynamics in these materials, and demonstrates selected applications in the field of functional food materials. Published version The work was supported by NTUitive (project no. NGF-2018-04-022) to S.L. and the Swiss National Science Foundation (project no. 200021_192051) to S.S.

Investigating plant structure is fundamental in botanical science and provides crucial knowledge for the theories of plant evolution, ecophysiology and for the biotechnological practices. Modern plant anatomy often targets the formation, localization and characterization of cellulosic, lignified or suberized cell walls. While classical methods developed in the 1960s are still popular, recent innovations in tissue preparation, fluorescence staining and microscopy equipment offer advantages to the traditional practices for investigation of the complex lignocellulosic walls. Our goal is to enhance the productivity and quality of microscopy work by focusing on quick and cost-effective preparation of thick sections or plant specimen surfaces and efficient use of direct fluorescent stains. We discuss popular histochemical microscopy techniques for visualization of cell walls, such as autofluorescence or staining with calcofluor, Congo red (CR), fluorol yellow (FY) and safranin, and provide detailed descriptions of our own approaches and protocols. Autofluorescence of lignin in combination with CR and FY staining can clearly differentiate between lignified, suberized and unlignified cell walls in root and stem tissues. Glycerol can serve as an effective clearing medium as well as the carrier of FY for staining of suberin and lipids allowing for observation of thick histological preparations. Three-dimensional (3D) imaging of all cell types together with chemical information by wide-field fluorescence or confocal laser scanning microscopy (CLSM) was achieved., Investigating plant structure is fundamental in botanical science. Across disciplines, such as plant development, ecophysiology and biotechnology, particular interest is focused on the complex structure of lignocellulosic walls. We review the recent innovations in tissue preparation and fluorescence staining for lignin and suberin. We demonstrate simple and cost-effective protocols for preparation of plant samples for microscopy using hand-cut sections and clearing with glycerol. Autofluorescence of lignin in combination with direct stains such as Congo red and fluorol yellow can clearly differentiate between lignified, suberized and unlignified cell wall domains.

Endogenous brain proteins can recognize the toxic oligomers of amyloid-β (Aβ) peptides implicated in Alzheimer’s disease (AD) and interact with them to prevent their aggregation. Lipocalin-type Prostaglandin D Synthase (L-PGDS) is a major Aβ-chaperone protein in the human cerebrospinal fluid. Here we demonstrate that L-PGDS detects amyloids in diseased mouse brain. Conjugation of L-PGDS with magnetic nanoparticles enhanced the contrast for magnetic resonance imaging. We conjugated the L-PGDS protein with ferritin nanocages to detect amyloids in the AD mouse model brain. We show here that the conjugates administered through intraventricular injections co-localize with amyloids in the mouse brain. These conjugates can target the brain regions through non-invasive intranasal administration, as shown in healthy mice. These conjugates can inhibit the aggregation of amyloids in vitro and show potential neuroprotective function by breaking down the mature amyloid fibrils.

Nanoformulation of therapeutic and diagnostic agents is beneficial as theyaccumulate at tumor sites due to enhanced permeability and retention effects.However, many in vitro studies performed on 2D cultures lack the realisticdimension of in vivo systems. In this work, a photosensitizer, acridine orange(AO), used in photodynamic therapy of cancer, is nanoformulated byentrapment in thermophilic ferritin. The efficacy is tested on 2D and 3D invitro models and complemented with studies on the cellular uptake routes.Ferritin from the archaeonArchaeoglobus fulgidus(AfFtn) exhibits the uniqueproperty of divalent metal ion or ionic strength mediated assembly, making itan interesting nanocarrier for the entrapment of small molecules. Thephotosensitization and toxicity studies on 2D monolayers and 3D spheroidmodels of colorectal cancer cells show that AO loaded in AfFtn is functional.The killing efficiency in 2D monolayers reaches 50% while the growth of 3Dspheroids is arrested after the first day with a subsequent 10% reduction oftumor diameter over the next 2 days. Results show that AO loaded AfFtn hasbetter penetration ability than free AO alone in 3D spheroid models indicatingthat nanocage formulation provides for an efficient delivery vehicle into thetumor tissue. Nanyang Technological University The authors acknowledge funding from the NTU-Northwestern Institutefor Nanomedicine (Grant Number: M4081504.F40). The authors thank Dr.Mridul Sarker for his help with transmission electron microscopy.

Graphene quantum dots (GQDs) have been emerging as next‐generation bioimaging agents because of their intrinsic strong fluorescence, photostability, aqueous stability, biocompatibility, and facile synthesis. In this work, GQDs are encapsulated in ferritin protein nanocages to develop multi‐functional nanoplatforms toward multi‐modal imaging and cancer therapy. Encapsulation of ultra‐small GQDs is expected to reduce their quick excretion from the body and increase their bioimaging efficiency. To expand the functionality of protein nanocages as multi‐modal imaging nanoprobes capable of both fluorescence and magnetic resonance imaging (MRI), GQDs and iron are encapsulated inside the core of AfFtn‐AA (an engineered ferritin nanocage derived from the archaeon Archaeoglobus fulgidus ). The co‐encapsulation is achieved through an iron‐mediated, self‐assembly of ferritin dimers resulting in the formation of GQD–iron complex in the ferritin nanocages ((GQDs/Fe)AA). The (GQDs/Fe)AA shows high relaxivities in MRI and pH‐sensitive fluorescence with strong fluorescence at low pH values and on MDA‐MB‐231 cells. As an imaging agent and a drug nanocarrier, (GQDs/Fe)AA exhibits negligible cytotoxicity on the cells and a high loading capacity (35%) of doxorubicin. Taken together, the (GQDs/Fe)AA shows promising applications in cancer diagnosis and therapy as a pH‐responsive fluorophore, MRI agent, and drug nanocarrier. Accepted version

Many anti-cancer drugs are used in chemotherapy; however, little is known about their efficacy against circulating tumor cells (CTCs). In this study, we investigated whether the pulsatile fluidic shear stress (SS) in human arteries can affect the efficacy of anti-cancer drugs. Cancer cells were circulated in our microfluidic circulatory system, and their responses to drug and SS treatments were determined using various assays. Breast and cervical cancer cells that stably expressed apoptotic sensor proteins were used to determine apoptosis in real-time by fluorescence resonance energy transfer (FRET)-based imaging microscopy. The occurrence of cell death in non-sensor cells were revealed by annexin V and propidium iodide staining. Cell viability was determined by MTT assay. Intracellular reactive oxygen species (ROS) levels were determined by staining cells with two ROS-detecting dyes: 2′,7′-dichlorofluorescin diacetate and dihydroethidium. Fluidic SS significantly increased the potency of the ROS-generating drugs doxorubicin (DOX) and cisplatin but had little effect on the non-ROS-generating drugs Taxol and etoposide. Co-treatment with SS and ROS-generating drugs dramatically elevated ROS levels in CTCs, while the addition of antioxidants abolished the pro-apoptotic effects of DOX and cisplatin. More importantly, the synergistic killing effects of SS and DOX or cisplatin were confirmed in circulated lung, breast, and cervical cancer cells, some of which have a strong metastatic ability. These findings suggest that ROS-generating drugs are more potent than non-ROS-generating drugs for destroying CTCs under pulsatile fluidic conditions present in the bloodstream. This new information is highly valuable for developing novel therapies to eradicate CTCs in the circulation and prevent metastasis.

Imaging of immune cells has wide implications in understanding disease progression and staging. While optical imaging is limited in penetration depth due to light properties, magnetic resonance (MR) imaging provides a more powerful tool for the imaging of deep tissues where immune cells reside. Due to poor MR signal to noise ratio, tracking of such cells typically requires contrast agents. This report presents an in-depth physical characterization and application of archaeal magnetoferritin for MR imaging of macrophages – an important component of the innate immune system that is the first line of defense and first responder in acute inflammation. Magnetoferritin is synthesized by loading iron in apoferritin in anaerobic condition at 65 °C. The loading method results in one order of magnitude enhancement of r1 and r2 relaxivities compared to standard ferritin synthesized by aerobic loading of iron at room temperature. Detailed characterizations of the magnetoferritin revealed a crystalline core structure that is distinct from previously reported ones indicating magnetite form. The magnetite core is more stable in the presence of reducing agents and has higher peroxidase-like activities compared to the core in standard loading. Co-incubation of macrophage cells with magnetoferritin in-vitro shows significantly higher enhancement in T2-MRI contrast of the immune cells compared to standard ferritin.

Stimuli-sensitive biomaterials that are activated by light are in need of formulations that are stable under indoor lighting yet can be activated under direct sunlight. Carbene-based bioadhesives are a new generation of film-forming polymers that are stable under indoor lighting yet are rapidly activated with low-energy UVA light, but have never been evaluated under sunlight exposure. Previous investigations have evolved two flexible carbene-based platforms, where aryl-diazirine is grafted on to polyamidoamine dendrimers (PAMAM-NH2; generation-5) or hydrophobic liquid polycaprolactone tetrol to yield G5-Dzx and CaproGlu, respectively. For the first time the activation of G5-Dzx and CaproGlu is investigated by natural sunlight with intensities up to 10 mW.cm-2. Structure-property relationships of bioadhesion are investigated by: (1) joules dose of sunlight (2) bioadhesive polymer structure; and (3) optical concentrators of magnifying glass and Fresnel lens. Using only natural sunlight, adhesion strength could be tuned from 20-150 kPa with crosslinking achieved in under 1 min. The results show that carbene-based polymers are a class of stimuli-sensitive biomaterials that are stable to indoor lighting, yet can be rapidly activated under direct sunlight, which may be useful for topical film forming polymers or as active ingredients in sunscreen formulation. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Accepted version The authors would like to acknowledge the funding support that was provided by the Agency for Science, Technology and Research (A*Star, Singapore) IRG17283008 ‘Microprocessor based methods of composite curing’ & IAF PP Grant (H19/01/a0/0II9 ‘CathoGlu Bioadhesivespreventing catheter extravasation and skin infections’; Tier 1 Grant RG17/18 (S): Novel light activated, diazo protecting groups, Ministry of Education (Singapore) Tier 2 Grant (MOE2018-T2-2-114): CaproGlu, Double sided wet-tissue adhesives; and Nanyang Technological University President's Graduate Scholarship provided to Nigel C.S. Tan. This material is based upon work supported by the National Science Foundation (USA) under grant no. 1460110.

We report the development of a self-assembling protein nanocage as a contrast agent for magnetic resonance imaging (MRI). The protein nanocage is derived from genetically engineered ferritin from Archaeoglobus fulgidus (AfFtnAA). Iron (Fe) was loaded in a controlled manner within the core of the ferritin nanocage, resulting in the formation of iron oxide magnetic nanostructures (MNS). Using a variety of structural and magnetic characterization methods, we have demonstrated the magnetic and domain structures of the MNS formed within the protein nanocage and their contribution on water proton relaxation in MRI. With the primary focus on cardiac imaging for identification of atherosclerotic lesion, macrophage cell line was chosen for in vitro studies. The cytocompatibility of Fe-loaded engineered ferritin nanocages ((Fe)AfFtnAA) was confirmed by cell viability and oxidative stress measurements. The ferritin nanocages were successfully internalized by macrophage cells in a dose dependent manner and visualized under MRI. The drop in relaxation time with increasing concentration validated their potential as a contrast agent in MRI. Enhanced uptake and diagnostic capability in MRI of Fe-loaded ferritin nanocages imply their use as a “natural” probe for targeting and imaging plaque macrophages.

Ligand binding experiments between small chemicals and proteins and the evaluation of dissociation constants of their complexes in competitive binding assays often rely on displacement of reporter probes by the tested ligand. The most widely adopted protocol uses a fluorescent ligand which changes its emission spectrum when bound to a protein. A decrease of fluorescence, caused by the addition of a second ligand to the complex is generally interpreted as displacement of the fluorescent probe by the ligand, and therefore as a measure of the affinity of the ligand for the protein. Working with an odorant-binding protein (OBP), we found drastic differences in the calculated affinities when using 1-aminoanthracene or N-phenyl-1-naphthylamine as the fluorescent reporter. This fact was quite unexpected, as OBPs are small compact proteins with a single binding pocket without allosteric sites. Such observation raises doubts on the reliability of the fluorescent binding assay, perhaps the most widely used approach to evaluate affinities of small organic compounds to OBPs and other binding proteins. We recommend that the results of fluorescent binding experiments with OBPs should be confirmed by using two different probes or alternative methods. The reliability of current protocols for ligand binding assays is rather limited, while we still wait for a label-free approach that could be simple, fast and free from the use of radioactive tracers.

This is the pre-peer reviewed version of the following article: Easy Formation of Functional Liposomes in Water Using a pH‐Responsive Microbial Glycolipid: Encapsulation of Magnetic and Upconverting Nanoparticles, which has been published in final form at https://doi.org/10.1002/cnma.201900318. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. The compartmentalization of colloids into topologically closed, vesicular, microphases offers an attractive mean to concentrate a functional cargo in aqueous solutions for a range of biomedical, cosmetic, and biotechnological applications. In this paper, we develop a simple, phospholipid‐free, phase change method employing a pH‐responsive glycolipid. The method is applied to the encapsulation of a sonicated, metastable, aqueous dispersion of functional colloids in the lumen of lipid vesicles: uncoated magnetic maghemite γ‐Fe2O3 and oleic‐acid coated upconverting NaYF4 : Yb/Ln (Ln=Er or Tm) nanoparticles (NPs). We find a stable liposomal dispersion containing a sub‐population of crowded liposomes with high concentrations of NPs. The encapsulated NPs, formed at nearly neutral pH and room temperature, are stable over time and towards extrusion. The vesicular microphase is entirely composed of pH‐responsive glycolipids, which undergo a pH‐mediated mesoscopic structural transition from an open lamellar (24). We also show that encapsulation successfully works with a stable colloidal aqueous dispersion of iron clusters stabilized in ferritin cages. This compartmentalization approach combining self‐assembly with an orthogonal nonequilibrium dispersion of nanoparticles shows untapped potential for synthesizing unusual classes of mixed matter.

The mechanism of long-range charge transport across tunneling junctions with monolayers of ferritin is investigated. It is shown that the mechanism can be switched between coherent tunneling, sequential tunneling, and hopping by changing the iron content inside the ferritin. This study shows that ferritins are an interesting class of biomolecules to control charge transport.

Bacterial cellulose (BC) has been gaining importance over the past decades as a versatile material that finds applications in diverse industries. However, a secured supply is hindered by the slow production rate and batch-to-batch variability of the yield. Here, we report a rational approach for characterising the BC production process using Design of Experiment (DoE) methodology to study the impact of different parameters on desired process attributes. Notably, we found that the carbon source used for bacterial growth significantly impacts the interplay between the process variables and affects the desired outcomes. We therefore, propose that the highest priority process outcome in this study, the yield, is a function of the carbon source and optimal reactor design. Our systematic approach has achieved projected BC yields as high as ∼40 g/L for Gluconacetobacter hansenii 53582 grown on sucrose as the carbon source compared to the widely reported yields of ∼10 g/L. National Research Foundation (NRF) This research was funded by the Singapore National Research Foundation (NRF) Biological Design, Tools, and Applications (BDTA) Grant (NRF2013-THE001-046).

Bacterial cellulose (BC) is a biocompatible material with versatile applications. However, its large-scale production is challenged by the limited biological knowledge of the bacteria. The advent of synthetic biology has lead the way to the development of BC producing microbes as a novel chassis. Hence, investigation on optimal growth conditions for BC production and understanding of the fundamental biological processes are imperative. In this study, we report a novel analytical platform that can be used for studying the biology and optimizing growth conditions of cellulose producing bacteria. The platform is based on surface growth pattern of the organism and allows us to confirm that cellulose fibrils produced by the bacteria play a pivotal role towards their chemotaxis. The platform efficiently determines the impacts of different growth conditions on cellulose production and is translatable to static culture conditions. The analytical platform provides a means for fundamental biological studies of bacteria chemotaxis as well as systematic approach towards rational design and development of scalable bioprocessing strategies for industrial production of bacterial cellulose. NRF (Natl Research Foundation, S’pore) Published version

Using particles with different functionalities for treating cancer has many advantages over other methods (for example, better access to remote parts of the body); however, current chemical (for example, chemotherapy) and biological (for example, immunotherapy) methods still face many challenges. Here, we describe a fundamentally different approach: using the physical force of an expanding stimuli-responsive hydrogel to rupture cancer cells attached on its surface. Specifically, we coated temperature-responsive hydrogels with a layer of cell-adherent arginine-glycine-aspartate (RGD) peptides. The approach involved first allowing cancer cells to attach onto the surface of the hydrogels, and then applying a change in temperature. As the hydrogel underwent a chemical transformation and expanded due to the stimulus, the cancer cells attached to it ruptured. The results from staining the cells with trypan blue, observing them using SEM, and analyzing them using the MTT assay showed that both breast and lung cancer cells died after the hydrogel expanded; hence, we showed that this physical force from the expanding hydrogel is strong enough to rupture the cancer cells. In addition, the force derived from the expanding hydrogel was determined separately to be larger than that needed to rupture typical cells. This physical approach is conceptually simple, technically easy to implement, and potentially generalizable for rupturing a wide range of cells. A hydrogel that uses physical force rather than drugs or radiation to kill breast and lung cancer cells has been developed. The approach by Siowling Soh at the National University of Singapore and colleagues exploits the temperature responsiveness of soft, watery polymers known as poly(N-isopropylacrylamides). The researchers coated the hydrogel with cell-adhering arginine-glycine-aspartate peptides, using a dopamine and lysine co-polymer to anchor the biomolecules. After seeding cancer cells onto the peptide-coated hydrogel, they dropped sample temperatures from 37°C to 22°C. The hydrogel expanded due to a phase transition, providing a force large enough to rupture the cancer cells attached to the surface — an easy-to-implement strategy effective for particles made of these coated hydrogels with sizes ranging from micrometers to millimeters. Measurements revealed the hydrogels can apply enough force to rupture a variety of cell types. Using functionalized particles for treatment of cancer is advantageous because they can access remote parts of the body and is minimally invasive. However, current chemical and biological methods still face challenges. A novel approach that uses the physical force of stimuli-responsive hydrogels is introduced. Temperature-responsive hydrogels were coated with cell-adherent molecules. After attaching cancer cells on its surface and changing the temperature, the force of the expanding stimuli-responsive hydrogel ruptures the cells. Comparing to other chemical and biological methods, this physical approach may be conceptually simpler, technically easier to implement, and more general for different types of cancer cells.

Raman spectroscopy (RS) for selective quantification of protein species in mixed solutions holds enormous potential for advancing protein detection technology to significantly faster, cheaper, and less technically demanding platforms. However, even with powerful computational methods such as nonlinear least squares regression, protein quantification in such complex systems suffers from relatively poor accuracy, especially in comparison with established methods. In this work, a combination of the expanded set of spectral information provided by polarized Raman spectroscopy (PRS) that is otherwise unavailable in conventional RS was, to our knowledge, explored to enhance the quantitative accuracy and robustness of protein quantification for the first time. A mixture containing two proteins, lysozyme and α-amylase, was used as a model system to demonstrate enhanced quantitative accuracy and robustness of selective protein quantification using PRS. The concentrations of lysozyme and α-amylase in mixtures were estimated using data obtained from both traditional RS and PRS. A new method was developed to select highly sensitive peaks for accurate concentration estimation to take advantage of additional spectra offered by PRS. The root-mean squared errors (RMSE) of estimation using traditional RS and PRS were compared. A drastic improvement in RMSE was observed from traditional RS to PRS, where the RMSEs of α-amylase and lysozyme concentrations decreased by 11 and 7 times, respectively. Therefore, this technique is a successful demonstration in achieving greater accuracy and reproducibility in the estimation of protein concentration in a mixture, and it could play a significant role in future multiplexed protein quantification platforms. Copyright © 2015 John Wiley & Sons, Ltd.

Purpose: A ferritin-containing nanoparticle conjugated with a target-specific antibody was investigated as a MRI contrast agent for tumor detection. A genetically modified ferritin to markedly improve Fe (III) payload (up to 7,000 Fe ions), was chemically tethered to a monoclonal antibody against rat Nectin-like molecule 5 (Necl-5). Necl-5 is a cell surface glycoprotein that is highly expressed on the cell surface of many common epithelial cancers, including prostate cancer. It was previously demonstrated that this novel nanoconjugate agent exhibited effective in vitro targeting of Necl- 5 expressing tumor cells and exhibited strong MRI contrast characteristics via shortening of T 2 . Here, we demonstrate that the nanoconjugate-Necl-5 interaction can be exploited to target and detect tumor in vivo by MRI. Procedure: Using an in vivo tumor model (i.e., tumor size 0.5 -1 cm, immunodeficient beige/nude/xid mouse, xenograft injection with transformed rat prostate cells), efficacy of the conjugate targeting the tumor was examined. We used two injection strategies, a direct and a tail vein injection (0.8 mg, 300 μL per subject). Pre-injection baseline and postinjection scans were performed with the following spin-echo sequence parameters: Field of view = 90x53mm, reconstruction matrix size = 192x114, slice thickness = 1mm (10 slices), repetition time (TR) = 2070 ms, echo times (TE) = 11-198 ms in 11ms steps (18 echoes), number of averages = 2, acquisition time per scan = 7min 56s. Results: All T 2 data obtained were converted to R 2 for demonstration purposes (R 2 = 1/T 2 ). The tail vein injected conjugate significantly increased R 2 response (22.9 ± 5.2 s -1 ) as compared to control (13.5 ±1.7 s -1 ) at 4 h. The weaker R 2 increase was noted (15.2 ± 2.0 s -1 ) at 24 h. No notable changes in R 2 were observed in surrounding tissues regardless the stages of the measurement. We also measured the initial conjugate kinetics for both injection methods with respect to the ability of targeting the tumor. Direct injection of the nanoconjugate in to the center of the tumor showed a stronger and more rapid increase in R2 than the tail vein injection. Conclusion: The nanoconjugate interacts strongly and selectively in situ with Necl-5 overexpressing tumor cells. Direct injection of the nanoconjugate into the body of the tumor caused a more significant in situ R 2 increase in MRI than the tail vein injection. Varying degrees of R 2 increase within the tumor mass is likely to represent different distribution patterns of the conjugate, reflective of tumor heterogeneity.

Vaults are naturally occurring ovoid nanoparticles constructed from a protein shell that is composed of multiple copies of major vault protein (MVP). The vault-interacting domain of vault poly(ADP-ribose)-polymerase (INT) has been used as a shuttle to pack biomolecular cargo in the vault lumen. However, the interaction between INT and MVP is poorly understood. It is hypothesized that the release rate of biomolecular cargo from the vault lumen is related to the interaction between MVP and INT. To tune the release of molecular cargos from the vault nanoparticles, we determined the interactions between the isolated INT-interacting MVP domains (iMVP) and wild-type INT and compared them to two structurally modified INT: 15-amino acid deletion at the C terminus (INTΔC15) and histidine substituted at the interaction surface (INT/DSA/3 H) to impart a pH-sensitive response. The apparent affinity constants determined using surface plasmon resonance (SPR) biosensor technology are 262 ± 4 nM for iMVP/INT, 1800 ± 160 nM for iMVP/INTΔC15 at pH 7.4. The INT/DSA/3 H exhibits stronger affinity to iMVP (KDapp = 24 nM) and dissociates at a slower rate than wild-type INT at pH 6.0.

In the present study, we present a room temperature-, one-pot method for the synthesis of functional poly (tannic acid) (PTA)-based core@shell nanocomposites (core@PTA). The addition of tannic acid (TA) to gold (Au) salt solutions at mildly alkaline pH induced reduction of the metal salts to Au nanoparticles (NPs) while TA was oxidized and self-polymerized before encapsulating the AuNPs to form Au@PTA. Through the use of coordination chemistry, the synthesized Au@PTA was utilized as a naked eye sensor for iron (III) ions and H2O2. In the presence of Fe3+, Au@PTA aggregated and resulted in visible color change, showing high selectivity towards Fe3+ with visual detection limit of 20 μM. Extension of the approach is used to detect H2O2 with visual detection limit of 0.4 μM. Observation of the UV–vis spectra after the addition of analytes for the two detection systems revealed an additional peak in the H2O2 detection system at ∼ 650 nm. This is due to the presence of “wire-like” oligomer structures in the H2O2 detection system as opposed to nanocomposite aggregation observed in the former system. Accepted version

The electrons generated by photosynthetic water splitting have been studied for direct electron transfer under light irradiation. Isolated thylakoids are incorporated as biocatalysts with indium tin oxide (ITO) as the electrode in a two-chamber photosynthetic electrochemical cell (PEC). The generated photocurrent is compared between deposited and dispersed thylakoids. The physical properties of deposited thylakoids are observed using field emission scanning electron microscopy (FESEM) and absorbance spectroscopy techniques. The results show the presence of thylakoids with bound photosystems including light harvesting antennas and other protein complexes. Further investigations reveal that the direction of electron transfer can be tuned by varying the applied potentials to promote bi-directional photocurrent. The maximum cathodic photocurrent is obtained at 50 mV vs. standard calomel electrode (SCE), while the maximum anodic photocurrent is enhanced with increasing positive potential applied. Our observation indicates the possibility of either reduction of O2 or H2O2 as the prominent cathodic photocurrent source, while electron transfer from thylakoids to ITO yields the anodic photocurrent.

Ceramic-polymer hybrid particles, intended for osteomyelitis treatment, were fabricated by preparing poly(lactic-co-glycolic acid) particles through an emulsion solvent evaporation technique, followed by calcium phosphate (CaP) coating via a surface adsorption-nucleation method. The presence of CaP coating on the surface of the particles was confirmed by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy. Subsequently, two antibiotics for treating bone infection, nafcillin (hydrophilic) and levofloxacin (amphiphilic), were loaded into these hybrid particles and their in vitro drug release studies were investigated. The CaP coating was shown to reduce burst release, while providing sustained release of the antibiotics for up to 4 weeks. In vitro bacterial study against Staphylococcus aureus demonstrated the capability of these antibiotic-loaded hybrid particles to inhibit biofilm formation as well as deteriorate established biofilm, making this hybrid system a potential candidate for further investigation for osteomyelitis treatment.

Bacterial cellulose (BC) is a biocompatible material with high purity and robust mechanical strength used to fabricate desirable scaffolds for 3D cell culture and wound healing. However, the chemical resistance of BC and its insolubility in the majority of solutions make it difficult to manipulate using standard chemical methods. In this study, a microfluidic process is developed to produce hollow BC microspheres with desirable internal structures and morphology. Microfluidics is used to generate a core-shell structured microparticle with an alginate core and agarose shell as a template to encapsulate Gluconacetobacter xylinus for long-term static culture. G. xylinus then secretes BC, which becomes entangled within the shell of the structured hydrogel microparticles and forms BC microspheres. The removal of the hydrogel template via thermal-chemical treatments yields robust BC microspheres exhibiting a hollow morphology. These hollow microspheres spontaneously assemble as functional units to form a novel injectable scaffold. In vitro, a highly porous scaffold is created to enable effective 3D cell culture with a high cell proliferation rate and better depth distribution. In vivo, this injectable scaffold facilitates tissue regeneration, resulting in rapid wound-healing in a Sprague Dawley rat skin model.

The exceptional chemical and physical properties of graphene oxide (GO) make it an attractive nanomaterial for biomedical applications, particularly in drug delivery. In this work we synthesized a novel, GO-based nanocarrier for the delivery of docetaxel (DTX), a potent hydrophobic chemotherapy drug. The GO was functionalized with transferrin (Tf)-poly(allylamine hydrochloride) (PAH), which provided targeted and specific accumulation to extracellular Tf receptors and stabilized GO in physiological solutions. Tf was conjugated to PAH via amide covalent linkages, and Tf-PAH coated the surface of DTX-loaded GO through electrostatic interactions. The morphology and structure of the resulting nanostructure, along with its surface modifications, were verified by use of Fourier transform infrared (FT-IR) and UV-vis spectroscopy, atomic force microscopy (AFM), and scanning electron microscopy (SEM). DTX was loaded at a relatively high loading capacity of 37% and released in a pH-dependent and sustained manner under physiological conditions. The targeting efficiency and cytotoxicity of this drug delivery system were evaluated on MCF-7 breast cancer cells. Improved efficacy of targeted DTX-loaded nanocarrier was observed compared to nontargeted carrier and free DTX, especially at high drug concentrations. The Tf-PAH-functionalized GO nanocarrier is a promising candidate for targeted delivery and controlled release of DTX.

In conventional in vitro encapsulation of molecular cargo, the multi-subunit ferritin protein cages are disassembled in extremely acidic pH and re-assembled in the presence of highly concentrated cargo materials, which results in poor yields due to the low-pH treatment. In contrast, Archaeoglobus fulgidus open-pore ferritin (AfFtn) and its closed-pore mutant (AfFtn-AA) are present as dimeric species in neutral buffers that self-assemble into cage-like structure upon addition of metal ions.To understand the iron-mediated self-assembly and ascorbate-mediated disassembly properties, we studied the iron binding and release profile of the AfFtn and AfFtn-AA, and the corresponding oligomerization of their subunits.Fe(2+) binding and conversion to Fe(3+) triggered the self-assembly of cage-like structures from dimeric species of AfFtn and AfFtn-AA subunits, while disassembly was induced by dissolving the iron core with reducing agents. The closed-pore AfFtn-AA has identical iron binding kinetics but lower iron release rates when compared to AfFtn. While the iron binding rate is proportional to Fe(2+) concentration, the iron release rate can be controlled by varying ascorbate concentrations.The AfFtn and AfFtn-AA cages formed by iron mineralization could be disassembled by dissolving the iron core. The open-pores of AfFtn contribute to enhanced reductive iron release while the small channels located at the 3-fold symmetry axis (3-fold channels) are used for iron uptake.The iron-mediated self-assembly/disassembly property of AfFtn offers a new set of molecular trigger for formation and dissociation of the protein cage, which can potentially regulate uptake and release of molecular cargo from protein cages.

E2 protein cages, which consist of the assembled 60 subunits, were simulated at two levels of protonation to mimic their electrostatic properties at pH 4 and 7 using all-atom models. Starting with the initial configuration of the assembled 60-mer, either the truncation of C-terminus or the protonation state of pH 4 induces the disassembly of 60-mer, leading to the formation of trimers. Hydrodynamic radii (R h) of E2 monomer, trimer, and 60-mer are calculated from diffusivities, which agree well with experimental values. R h become smaller for the disassembled E2, confirming the formation of clusters smaller than 60-mer. Anionic Asp419 and Glu421 at the C-terminus interact with Lys240 of a neighboring trimer, while hydrophobic Leu424 and Met425 at the C-terminus interact with Pro313∼Ala318 of a neighboring trimer. When anionic residues of E2 60-mer are protonated (neutralized), the assembled 60-mer are stabilized by hydrophobic interactions of C-terminus. These indicate that the stability of E2 60-mer is attributed to both electrostatic and hydrophobic inter-trimer interactions of the neighboring C-termini. These findings support the experimental interpretation regarding the formation of trimer as an intermediate between E2 monomer and 60-mer, and help explain the key role of C-terminus for electrostatic and hydrophobic inter-trimer interactions. MOE (Min. of Education, S’pore) Accepted version

In the aromatic amino acid biosynthesis pathway, chorismate presents a branch point intermediate that is converted to tryptophan, phenylalanine (Phe), and tyrosine (Tyr). In bacteria, three enzymes catalyze the conversion of chorismate to hydroxyphenylpyruvate or pyruvate. The enzymes, chorismate mutase (CM), prephenate dehydratase (PDT), and prephenate dehydrogenase (PDHG) are either present as distinct proteins or fusions combining two activities. Gene locus AF0227 of Archaeoglobus fulgidus is predicted to encode a fusion protein, AroQ, containing all three enzymatic domains. This work describes the first characterization of a trifunctional AroQ. The A. fulgidus aroQ gene was cloned and overexpressed in Escherichia coli. The recombinant protein purified as a homohexamer with specific activities of 10, 0.51, and 50 U/mg for CM, PDT, and PDHG, respectively. Tyr at 0.5 mM concentration inhibited PDHG activity by 50%, while at 1 mM PDT was activated by 70%. Phe at 5 μM inhibited PDT activity by 66% without affecting the activity of PDHG. A fusion of CM, PDT, and PDHG domains is evident in the genome of only one other organism sequenced to date, that of the hyperthermophilic archaeon, Nanoarchaeum equitans. Such fusions of contiguous activities in a biosynthetic pathway may constitute a primitive strategy for the efficient processing of labile metabolites.

Plasma-assisted reactive rf magnetron sputtering deposition is used to fabricate vanadium oxide films on glass, silica and silicon substrates. The process conditions are optimized to synthesize phase-pure vanadium pentoxide (V2O5) featuring a nanocrystalline structure with the predominant (0?0?1) crystallographic orientation, surface morphology with rod-like nanosized grains and very uniform (the non-uniformity does not exceed 4%) coating thickness over large surface areas. The V2O5 films also show excellent and temperature-independent optical transmittance in a broad temperature range (20?95??C). The results are relevant to the development of smart functional coatings with temperature-tunable properties.

Controlling the self-assembly behavior of caged proteins expands their potential applications in nanotechnology. While the structure of a caged E2 protein from pyruvate dehydrogenase is inert to any pH change, the incorporation of switchable GALA peptide that undergoes a coil-to-helix transition at acidic pH modulates its self-assembly property. By substituting the native α-helix at the C-terminus of the E2 protein with the GALA peptide, we report the first engineered caged protein with reversible inversed pH-responsive behavior. The redesigned caged E2 protein assumes an assembly profile that is distinct from the native state; it disassembles at pH 7.0 and self-assembles at pH 4.0 in a reversible manner. This unique reversible pH trigger suggests the applicability of the self-assembly control on other multi-subunit macromolecules.

The feasibility of forming porous metallic materials by the plasma spraying process was studied. The process involved the co-spraying of two materials: aluminum (Al) and calcium oxide (CaO), the former being the metallic phase that eventually was retained as the porous structure, while the latter being the sacrificial phase was removed giving rise to pores in the Al matrix. For this the Al and the CaO powders of four different compositions were blended and plasma sprayed on a mold cavity. The CaO was dissolved from the sprayed structure by treating in either water or glycerol. The surface and cross sectional microstructure of the specimens after treatment in water and glycerol showed a more or less uniform pore distribution. The dissolution of CaO, leading to the porous microstructure was confirmed by the X-ray diffractometry. In the XRD spectra, the specimens in the as-sprayed untreated condition exhibited peaks corresponding to both CaO and Al, while, the peaks corresponding to CaO were absent in specimens after water and glycerol treatments confirming CaO dissolution. The study clearly showed that porous aluminum material could be manufactured by plasma spraying.

A novel alanine dehydrogenase (AlaDH) showing no significant amino acid sequence homology with previously known bacterial AlaDHs was purified to homogeneity from the soluble fraction of the hyperthermophilic archaeon Archaeoglobus fulgidus . AlaDH catalyzed the reversible, NAD + -dependent deamination of l -alanine to pyruvate and NH 4 + . NADP(H) did not serve as a coenzyme. The enzyme is a homodimer of 35 kDa per subunit. The K m values for l -alanine, NAD + , pyruvate, NADH, and NH 4 + were estimated at 0.71, 0.60, 0.16, 0.02, and 17.3 mM, respectively. The A. fulgidus enzyme exhibited its highest activity at about 82°C (203 U/mg for reductive amination of pyruvate) yet still retained 30% of its maximum activity at 25°C. The thermostability of A. fulgidus AlaDH was increased by more than 10-fold by 1.5 M KCl to a half-life of 55 h at 90°C. At 25°C in the presence of this salt solution, the enzyme was ∼100% stable for more than 3 months. Closely related A. fulgidus AlaDH homologues were found in other archaea. On the basis of its amino acid sequence, A. fulgidus AlaDH is a member of the ornithine cyclodeaminase-μ-crystallin family of enzymes. Similar to the μ-crystallins, A. fulgidus AlaDH did not exhibit any ornithine cyclodeaminase activity. The recombinant human μ-crystallin was assayed for AlaDH activity, but no activity was detected. The novel A. fulgidus gene encoding AlaDH, AF1665, is designated ala .

Facile synthesis of metal@polymer nanocomposites were achieved using self‐assembled tannic acid (TA) shells without crosslinkers. The TA shell was assembled under mildly alkaline conditions in reaction time of 20 min under constant vortexing. Universal synthesis method was demonstrated by the synthesis of Ag@TA and Au@TA nanocomposites. We propose that the shell formation is due to TA undergoing oxidative self‐polymerization to poly(tannic acid) or a supramolecular aggregate of oxidized TA held together by charge transfer, hydrogen bond, and π–π interactions, similar to dopamine polymerization. Gibbs free energy calculations suggest that polymerization is energetically favorable. Synthesized Ag@TA exhibited antibacterial functionality with Escherichia coli minimum inhibitory concentration of 100 µg mL−1 up to 48 h. The population of E. coli was also reduced by 99% within 5 h when incubated with 100 µg mL−1 of Ag@TA nanocomposite. Au@TA also functions as a reduction catalyst. It reduces 4‐nitrophenol to 4‐aminophenol in the presence of NaBH4 with a rate constant of k = 0.63 min−1 μmol−1. For comparison, using Au nanoparticles yields a rate constant of 0.14 min−1 μmol−1. The ease of synthesis renders the nanocomposites superior to others, with potential for large‐scale application. Accepted version

Circulating tumor cells (CTCs) are mainly responsible for the cause of cancer metastasis. Although most CTCs can be destroyed by the bloodstream, some of them can still manage to withstand hemodynamic shear stress in blood stream. To study the effects of fluidic micro-environment on CTCs, we designed a microfluidic system that can produce various levels of shear stress which can be generated in human artery under resting or exercise condition. We also generated three human breast cancer cell lines with increased ability to form lung metastases in nude mice (i.e. 213-M1A>231-M1>231-C3). All three cell lines can produce a fluorescence resonance energy transfer (FRET)-based sensor which can reveal apoptosis in real-time by changing its color from green to blue. Besides these, we also investigated the effects of shear stress on multiple types of cancer cells including lung cancer (A549), ovarian cancer (2008), breast cancer (UACC-893) and leukemia (K562). For all the cell lines, except the non-attached K562 cells, cells were cultured on petri-dishes, detached by trypsinization and re-suspended in a normal culture medium to a cell density of 2x105 cells/ml. One milliliter of this cell suspensions was circulated in our microfluidic system under the shear stresses of 15, 30, 45 and 60 dyne/cm2 for 2-18 h. The effects of shear stress on morphology and viability of CTCs were determined by FRET imaging microscopy and MTT assay. And the impact of shear stress on causing necrosis in CTCs were examined by propidium iodide (PI) staining and LDH assay. Our findings are summarized below: 1) High shear stress (SS60, 4 h) is more potent to destroy CTCs than low shear stress (SS15, 4 h). 2) High shear stress can destroy most of the CTCs via necrosis. 3) This high level of shear stress is theoretically achievable via physical exercise. 4) Highly metastatic 231-M1A cells are more resistant to high shear stress compared to less metastatic 231-C3 cells. 5) Leukemia K562 cells representing the white blood cells showed stronger resistance to high shear stress compared to the cancer cells tested in this study. We think these findings can help people to understand how metastatic CTCs resist shear force-induced cell death which are useful for designing more effective therapies against metastatic CTCs. Additionally, the finding that high shear stress is more effective to kill CTCs may inspire cancer patients to consider exercise as a way to increase their hemodynamic shear stress, consequently destroy CTCs and prevent cancer metastasis. Citation Format: Sagar Regmi, Afu Fu, Sierin Lim, Kathy Qian Luo. Destruction of circulating tumor cells by fluid shear stresses generated in a microfluidic system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2325. doi:10.1158/1538-7445.AM2017-2325

In biomineralization processes, a supramolecular organic structure is often used as a template for inorganic nanomaterial synthesis. The E2 protein cage derived from Geobacillus stearothermophilus pyruvate dehydrogenase and formed by the self-assembly of 60 subunits, has been functionalized with non-native iron-mineralization capability by incorporating two types of iron-binding peptides. The non-native peptides introduced at the interior surface do not affect the self-assembly of E2 protein subunits. In contrast to the wild-type, the engineered E2 protein cages can serve as size- and shape-constrained reactors for the synthesis of iron nanoparticles. Electrostatic interactions between anionic amino acids and cationic iron molecules drive the formation of iron oxide nanoparticles within the engineered E2 protein cages. The work expands the investigations on nanomaterial biosynthesis using engineered host-guest encapsulation properties of protein cages.

Archaeoglobus fulgidus ferritin (AfFtn) is the only tetracosameric ferritin known to form a tetrahedral cage, a structure that remains unique in structural biology. As a result of the tetrahedral (2-3) symmetry, four openings (∼45 Å in diameter) are formed in the cage. This open tetrahedral assembly contradicts the paradigm of a typical ferritin cage: a closed assembly having octahedral (4-3-2) symmetry. To investigate the molecular mechanism affecting this atypical assembly, amino acid residues Lys-150 and Arg-151 were replaced by alanine. The data presented here shed light on the role that these residues play in shaping the unique structural features and biophysical properties of the AfFtn. The x-ray crystal structure of the K150A/R151A mutant, solved at 2.1 Å resolution, indicates that replacement of these key residues flips a "symmetry switch." The engineered molecule no longer assembles with tetrahedral symmetry but forms a typical closed octahedral ferritin cage. Small angle x-ray scattering reveals that the overall shape and size of AfFtn and AfFtn-AA in solution are consistent with those observed in their respective crystal structures. Iron binding and release kinetics of the AfFtn and AfFtn-AA were investigated to assess the contribution of cage openings to the kinetics of iron oxidation, mineralization, or reductive iron release. Identical iron binding kinetics for AfFtn and AfFtn-AA suggest that Fe(2+) ions do not utilize the triangular pores for access to the catalytic site. In contrast, relatively slow reductive iron release was observed for the closed AfFtn-AA, demonstrating involvement of the large pores in the pathway for iron release.