Homogeneous nutrient distribution throughout three-dimensional (3-D) scaffolds remains a key challenge in tissue engineering. The buildup of cells on scaffold edges and rapid nutrient uptake along the periphery often cause large regions of the centre to be left unoccupied by cells. Microstreaming associated with acoustic cavitation has been exploited to enhance mass transport in oncological drug delivery and transdermal vaccination, making it an attractive mechanism for promoting nutrient and oxygen distribution in tissue engineering scaffolds. In this work, we seek to use protein cavitation nuclei to synthesize ultrasound-responsive collagen scaffolds. Cavitation nuclei were embedded into the scaffold during fabrication and then exposed to 0.5 MHz focused ultrasound at peak negative pressures ranging from 0.5 to 2.7 MPa to induce inertial cavitation. Acoustic data was collected using passive cavitation detection (PCD) and post processed to isolate harmonics and broadband emissions. We compare the benefits and disadvantages of including cavitation nuclei in the scaffold fabrication process versus adding them to surrounding media during ultrasound exposure, discussing potential use of embedded protein nuclei to induce cell migration and differentiation. Additionally, we examine the effects of cavitation, exposure time, and peak negative pressure on the microstructure of collagen scaffolds, namely, pore size, interconnectivity, and percolation diameter.

In the fight against a broad spectrum of human diseases, cavitation techniques show great promise for overcoming physical barriers that lead to suboptimal uptake of passively administered therapeutics. However, small animal testing of candidate therapies remains a poor predictor of clinical success. Here we demonstrate ultrasound-mediated drug delivery in normal and tumour-bearing human livers infused with protein-based cavitation nuclei (PCaN). Whole and partial human livers were obtained immediately from hepatectomy surgeries and were normothermically sustained using a clinically approved perfusion system (OrganOx Metra). Ultrasound was applied using a 0.5 MHz focused source (Sonic Concepts H107) and was monitored with a calibrated linear array (ATS L7-4) for real time structural and cavitational imaging implemented on an array controller (Verasonics Vantage 256). Specifically, the therapy process was monitored using passive acoustic mapping (PAM) of broadband cavitation emissions, employing a non-adaptive beamformer that deconvolves the array point spread function. Levels of fluorescently labelled drugs incorporated in and co-administered with the PCaN were quantified in blood and tissue samples collected during and following treatment, respectively. This presentation highlights PAM observations of broadband cavitation persistence and drug delivery in untargeted and targeted tissues, including the first ever experiments in tumour-bearing human livers.

Objective. Bacterial biofilms represent a major challenge for effective antibiotic therapy as they confer physical and functional changes that protect bacteria from their surrounding environment. In this work, focused ultrasound in combination with cavitation nuclei was used to disrupt biofilms of S. aureus and P. aeruginosa, both of which are on the World Health Organization's priority list for new antimicrobial research. Approach. Single species biofilms were exposed to ultrasound (0.5 MHz center frequency, 0.5-1.5 MPa peak rarefactional pressure, 200 cycle pulses, 5 Hz repetition frequency, 30 s duration), in the presence of two different types of cavitation nuclei. Quantitative passive acoustic mapping (PAM) was used to monitor cavitation emissions during treatment using a calibrated linear array. Main Results. It was observed that the cumulative energy of acoustic emissions during treatment was positively correlated with biofilm disruption, with differences between bacterial species attributed to differences in biofilm morphology. PCaN provided increased biofilm reduction compared to microbubbles due in large part to their persistence over the duration of ultrasound exposure. There was also good correlation between the spatial distribution of cavitation as characterized by PAM and the extent of biofilm disruption observed with microscopy. Significance. Collectively, the results from this work indicate the potential broad applicability of cavitation for eliminating biofilms of priority pathogens and the opportunity presented by PAM for real-time monitoring of antimicrobial processes.

Alternatives are needed to remove the pain, injury, cross‐infection, and hazardous waste associated with needle and syringe (N+S)‐based vaccination. Reported here is the use of novel ultrasound‐responsive protein cavitation nuclei (pCaN), formed using the model antigen bovine serum albumin (BSA), to achieve effective transcutaneous delivery. Upon exposure to ultrasound (US), these pCaN instigate cavitation events which propel themselves and co‐located DNA vectors into the skin. US parameters as well as pCaN and DNA concentration are refined to achieve optimal expression of encoded luciferase transgene. Twenty‐four hours post‐treatment, luciferase expression in the skin, by IVIS imaging, was 1.67 × 10 6 ±941943, photons per sec for N+S intradermal injection and 1.49 × 10 6 ±261832 for cavitation‐mediated delivery ( p >0.05). Hence, there is no significant difference in luciferase level achieved, but improved homogeneity and reproducibility of expression are evident in mice treated using US‐mediated cavitation. Despite this equivalence in luciferase levels, a >5× higher level ( p <0.02) of anti‐luciferase antibodies is achieved when cavitation is used versus N+S injection. Antibody levels against BSA, resulting from the use of BSA pCaN, are equivalent for the two groups. PCaN can be formed from a range of antigenic proteins and DNA can encode a range of antigenic proteins, so this approach has wide‐ranging implications for needle‐free vaccination.

Ultrasound-induced cavitation is a promising mechanism for pain-free delivery of vaccines without needles. However, the relationships between cavitation energy and the various bioeffects involved in transdermal vaccination, including skin permeabilization, convective drug transport, reversible and irreversible sonoporation, and immune-stimulation, are not well understood. Previous transdermal ultrasound experiments have demonstrated inhomogeneous delivery across the exposed surface, which remains poorly understood. In this work, we first seek to fully characterize and quantify spatio-temporal cavitation activity across the skin surface, to identify a local cavitation dose that is optimal for all four key bioeffects. We have designed a new in vitro experimental setup that exposes several potential skin models to 265 kHz focused ultrasound, a new generation of protein-based cavitation nuclei (pCaNs), and a fluorescently labelled vaccine analogue, while simultaneously imaging cavitation activity parallel to the skin surface by Passive Acoustic Mapping (PAM). Subsequent staining and multi-photon microscopy of the skin models allows a direct comparison between the PAM-derived spatiotemporal inertial cavitation dose and locations where particular bioeffects were maximized. We will discuss the results of this investigation, how they relate to our recent in vivo study, and whether these findings enable enhancements of the spatial homogeneity and reproducibility of needle-free ultrasound vaccination.

Ultrasound-mediated cavitation shows great promise for improving targeted drug delivery across a range of clinical applications. Cavitation nuclei—sound-sensitive constructs that enhance cavitation activity at lower pressures—have become a powerful adjuvant to ultrasound-based treatments, and more recently emerged as a drug delivery vehicle in their own right. The unique combination of physical, biological, and chemical effects that occur around these structures, as well as their varied compositions and morphologies, make cavitation nuclei an attractive platform for creating delivery systems tuned to particular therapeutics. In this review, we describe the structure and function of cavitation nuclei, approaches to their functionalization and customization, various clinical applications, progress toward real-world translation, and future directions for the field.

Originally developed for diagnostic purposes and already approved for clinical use, lipid and protein-shelled microbubbles were a natural choice as initial nucleation agents for cavitation-based therapies entering the clinic. However, several emerging therapeutic ultrasound applications require nuclei that: (i) are significantly smaller in size, in order to overcome a particular biological barrier such as the leaky vasculature of tumours or the stratum corneum; (ii) offer greatly increased cavitation persistence, both during a single extended ultrasound pulse and in terms of extended circulation following intravenous administration; (iii) have better resilience to sudden ambient pressure changes in order to enable direct injection without nuclei destruction into tissue targets via a needle and syringe; and (iv) are made of materials or have an increased payload or surface area that can interact beneficially with the relevant tissue target during or following cavitation. Gas-stabilizing solid particles will be reviewed in this context, providing an overview of their known characteristics in terms of size distribution, associated acoustic emissions, cavitation thresholds, cavitation persistence and circulation. The relationship between this acoustic characterization and associated bioeffects including drug and vaccine delivery, and immunomodulation will subsequently be explored.

Background Tumour-associated macrophages (TAMs) are often implicated in cancer progression but can also exert anti-tumour activities. Selective eradication of cancer-promoting (M2-like) TAM subsets is a highly sought-after goal. Here, we have devised a novel strategy to achieve selective TAM depletion, involving the use of T cell engagers to direct endogenous T cell cytotoxicity towards specific M2-like TAMs. To avoid “on-target off-tumour” toxicities, we have explored localising expression of the T cell engagers to the tumour with enadenotucirev (EnAd), an oncolytic adenovirus in Phase I/II clinical trials. Method A panel of bi- and tri-valent T cell engagers (BiTEs/TriTEs) was constructed, recognising CD3ε on T cells and CD206 or folate receptor β (FRβ) on M2-like macrophages. Initial characterisation of BiTE/TriTE activity and specificity was performed with M1- and M2-polarised monocyte-derived macrophages and autologous lymphocytes from healthy human peripheral blood donors. T cell engagers were inserted into the genome of EnAd, and oncolytic activity and BiTE secretion assessed with DLD-1 tumour cells. Clinically-relevant ex vivo models (whole malignant ascites from cancer patients) were employed to assess the efficacies of the free- and virally-encoded T cell engagers. Results T cells activated by the CD206- and FRβ-targeting BiTEs/TriTEs preferentially killed M2- over M1-polarised autologous macrophages, with EC50 values in the nanomolar range. A TriTE with bivalent CD3ε binding – the first of its kind – demonstrated enhanced potency whilst retaining target cell selectivity, whereas a CD28-containing TriTE elicited non-specific T cell activation. In immunosuppressive malignant ascites, both free and EnAd-encoded T cell engagers triggered endogenous T cell activation and IFN-γ production, leading to increased T cell numbers and depletion of CD11b+CD64+ ascites macrophages. Strikingly, surviving macrophages exhibited a general increase in M1 marker expression, suggesting microenvironmental repolarisation towards a pro-inflammatory state. Conclusions This study is the first to achieve selective depletion of specific M2-like macrophage subsets, opening the possibility of eradicating cancer-supporting TAMs whilst sparing those with anti-tumour potential. Targeted TAM depletion with T cell engager-armed EnAd offers a powerful therapeutic approach combining direct cancer cell cytotoxicity with reversal of immune suppression.

Virus-like particles (VLPs) are stable protein cages derived from virus coats.They have been used extensively as biomolecular platforms, e.g., nanocarriers or vaccines, but a convenient in situ technique is lacking for tracking functional status.Here, we present a simple way to monitor disassembly of 19 F-labeled VLPs derived from bacteriophage Qβ by 19 F NMR. Analysis of resonances, under a range of conditions, allowed determination not only of the particle as fully assembled but also as disassembled, as well as detection of a degraded state upon digestion by cells.This in turn allowed mutational redesign of disassembly and testing in both bacterial and mammalian systems as a strategy for the creation of putative, targeted-VLP delivery systems.

The degradation of long-lived proteins in the body is an important aspect of aging, and much of the breakdown is due to the intrinsic instability of particular amino acids. In this study, peptides were examined to discover if spontaneous nonenzymatic reactions could be responsible for the composition of Alzheimer's (AD) plaque in the human brain. The great majority of AD plaque consists of N-terminally truncated versions of Aβ(1-40/1-42), with the most abundant peptide commencing with Glu (residue 3 in Aβ1-40/1-42) that is present as pyroGlu. Several Asp residues are racemized in Aβ plaque, with residue 1 being predominantly l-isoAsp and peptide bond cleavage next to Ser 8 is also evident. In peptides, loss of the two N-terminal amino acids as a diketopiperazine was demonstrated at pH 7. For the Aβ N-terminal hexapeptide, AspAlaGluPheArgHis, this resulted in the removal of AspAla diketopiperazine and the generation of Glu as the new N-terminal residue. The Glu cyclized readily to pyroGlu. This pathway was altered significantly by zinc, which promoted pyroGlu formation but decreased AspAla diketopiperazine release. Zinc also facilitated cleavage on the N-terminal side of Ser 8. Racemization of the original N-terminal Asp to l-isoAsp was also detected and loss of one amino acid from the N-terminus. These data are therefore entirely consistent with plaque in the human brain forming from deposition of Aβ(1-40/1-42) and, over time, decomposing spontaneously. Since amyloid plaque is present in the human brain for years prior to the onset of AD, gradual spontaneous changes to the polypeptides within it will alter its properties and those of the oligomers that can diffuse from it. Such incremental changes in composition may therefore contribute to the origin of AD-associated cytotoxicity.

Old proteins are widely distributed in the body. Over time, they deteriorate and many spontaneous reactions, for example isomerisation of Asp and Asn, can be replicated by incubation of peptides under physiological conditions. One of the signatures of long-lived proteins that has proven to be difficult to replicate in vitro is cleavage on the N-terminal side of Ser residues, and this is important since cleavage at Ser, and also Thr, has been observed in a number of human proteins. In this study, the autolysis of Ser- and Thr-containing peptides was investigated with particular reference to discovering factors that promote cleavage adjacent to Ser/Thr at neutral pH. It was found that zinc catalyses cleavage of the peptide bond on the N-terminal side of Ser residues and further that this process is markedly accelerated if a His residue is adjacent to the Ser. NMR analysis indicated that the imidazole group co-ordinates zinc and that once zinc is co-ordinated, it can polarize the carbonyl group of the peptide bond in a manner analogous to that observed in the active site of the metalloexopeptidase, carboxypeptidase A. The hydroxyl side chain of Ser/Thr is then able to cleave the adjacent peptide bond. These observations enable an understanding of the origin of common truncations observed in long-lived proteins, for example truncation on the N-terminal side of Ser 8 in Abeta, Ser 19 in alpha B crystallin and Ser 66 in alpha A crystallin. The presence of zinc may therefore significantly affect the long-term stability of cellular proteins.

BACKGROUND:Diabetic macular oedema (DMO) is the commonest cause of vision loss in people with diabetes. Laser photocoagulation can be effective in the treatment of DMO; however, its mechanism of action is still poorly understood. A better understanding of these mechanisms may allow the development of therapeutic approaches that could avoid the deleterious adverse events associated with photocoagulation.METHODS:We have used proteomic techniques to identify the protein changes induced by threshold intensity retinal laser treatment in a rodent model of diabetic retinopathy. Retinae were obtained from diabetic Dark Agouti rats 8 weeks following laser treatment. Extracted proteins from lasered and non-lasered diabetic retinae were separated and compared using two-dimensional gel electrophoresis.RESULTS:Image analysis revealed 24 protein spots with decreased expression after laser treatment and 9 spots with increased expression. On lasered retinal gels, four spots were uniquely expressed, with eight unique spots on non-lasered gels. Twenty-two protein spots of interest were identified using matrix-assisted desorption ionization-mass spectrometry with database matching. Following laser, Wnt-5 beta, LEK-1, GADPH, claudin-12 and calretinin were significantly down-regulated in expression.CONCLUSIONS:The present study provides a proteomic insight into the underlying biological basis for the therapeutic effects of laser for DMO. We provide further evidence of the involvement of Wnt pathway signalling in the neural retina in DMO, and for up to 2 months following laser treatment. Changes in LEK-1 and claudin-12 may have effector roles, and changes in glyceraldehyde-3-phosphate dehydrogenase and calretinin may reflect the altered retinal microenvironment resulting from laser treatment.

Reduced expression of a similar to 150kDa protein was unexpectedly observed while investigating Norrin protein in a transgenic murine model in which Muller cells can be selectively and inducibly disrupted. Isolation of this unknown protein via ion exchange and hydrophobic interaction chromatography followed by Tandem mass spectrometry identified it as Inter-photoreceptor retinoid-binding protein (IRBP). Significantly reduced IRBP mRNA expression was observed at the early and late stages after Muller cell disruption. IRBP protein expression was also consistently reduced to 5.7% of the control level as early as 1week after Muller cell disruption. This down-regulation of IRBP was accompanied by focal hyperfluorescent dots and cytotoxic N-retinylidene-N-retinylethanolamine (A2E) accumulation. In vitro treatment of cone photoreceptor cell lines with conditioned medium collected from stressed Muller cells suggested that Muller cells regulated photoreceptors expression of IRBP via secreted factor(s). In vivo studies suggested that one of these secreted factors was tumour necrosis factor alpha (TNF alpha). These findings suggest that dysregulation of IRBP expression caused by Muller cell dysfunction may be an important early event in photoreceptor degeneration in some retinal diseases.

A cyclic product that forms spontaneously from peptides that contain a penultimate Asp, Asn or isoAsp residue at the N-terminus has been characterized. This 2,5-diketopiperazine derivative forms under physiological conditions and is stable, showing little degradation even following heating at 60 °C. A mechanism for its formation from Asn and Asp peptides is proposed that involves a succinimide or isoaspartate intermediate. A diketopiperazine-modified peptide was also detected in human lens extracts. Since peptides that contain the diketopiperazine moiety are not readily hydrolysed by leucine aminopeptidase, it is hypothesized that proteins and peptides modified in this way in the body may not readily be digested by the normal proteolytic machinery of cells.

Some amino acids are particularly susceptible to degradation in long-lived proteins. Foremost among these are asparagine, aspartic acid and serine. In the case of serine residues, cleavage of the peptide bond on the N-terminal side, as well as racemisation, has been observed. To investigate the role of the hydroxyl group, and whether cleavage and racemisation are linked by a common mechanism, serine peptides with a free hydroxyl group were compared to analogous peptides where the serine hydroxyl group was methylated. Peptide bond cleavage adjacent to serine was increased when the hydroxyl group was present, and this was particularly noticeable when it was present as the hydroxide ion. Adjacent amino acid residues also had a pronounced affect on cleavage at basic pH, with the SerPro motif being especially susceptible to scission. Methylation of the serine hydroxyl group abolished truncation, as did insertion of a bulky amino acid on the N-terminal side of serine. By contrast, racemisation of serine occurred to a similar extent in both O-methylated and unmodified peptides. On the basis of these data, it appears that racemisation of Ser, and cleavage adjacent to serine, occur via separate mechanisms. Addition of water across the double bond of dehydroalanine was not detected, suggesting that this mechanism was unlikely to be responsible for conversion of l-serine to d-serine. Abstraction of the alpha proton may account for the majority of racemisation of serine in proteins.

Structural analysis of a novel UV filter present in the lens of the thirteen-lined ground squirrel has shown that it is related in structure to N-acetyl-3-hydroxykynurenine. This finding is consistent with the fact that the squirrel lenses also contain high levels of this tryptophan metabolite. Analysis of both NMR and mass spectrometric data suggested that the novel UV filter compound forms by condensation of proline with N-acetyl-3-hydroxykynurenine. Its absorption maximum at 340 nm is more than 20 nm lower than that of the kynurenines and it may therefore assist in filtering the more damaging shorter wavelengths of UVA.

Summary Nonenzymatic post‐translational modification ( PTM ) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC –tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of S er and T hr residues by glutathione ( GSH ) and its metabolites were unambiguously identified. Thirty‐four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine ( DHA ) and dehydrobutyrine ( DHB ) formation through β‐elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH . In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein–protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between β B 2‐ & β B 2‐, β A 4‐ & β A 3‐, γ S ‐ & β B 1‐, and β A 4‐ & β A 4‐crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long‐lived proteins and that DHA ‐ and DHB ‐mediated protein crosslinking may be the source of the long‐sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long‐lived proteins of other tissues leading to protein aggregation diseases.

Scientific workflows are gaining popularity, and repositories of workflows are starting to emerge. In this paper we describe TopicsExplorer , a data exploration approach for myExperiment.org , a collaborative platform for the exchange of scientific workflows and experimental plans. Our approach uses a variant of topic modeling with tags as features, and generates a browsable view of the repository. TopicsExplorer has been fully integrated into the open-source platform of myExperiment.org , and is available to users at www.myexperiment.org/topics. We also present our recently developed personalization component that customizes topics based on user feedback. Finally, we discuss our ongoing performance optimization efforts that make computing and managing personalized topic views of the myExperiment.org repository feasible.