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Amyloid self‐assembly of islet amyloid polypeptide (IAPP) is linked to pancreatic inflammation, β‐cell degeneration, and the pathogenesis of type 2 diabetes (T2D). The multifunctional host‐defence peptides (HDPs) cathelicidins play crucial roles in inflammation. Here, we show that the antimicrobial and immunomodulatory polypeptide human cathelicidin LL‐37 binds IAPP with nanomolar affinity and effectively suppresses its amyloid self‐assembly and related pancreatic β‐cell damage in vitro. In addition, we identify key LL‐37 segments that mediate its interaction with IAPP. Our results suggest a possible protective role for LL‐37 in T2D pathogenesis and offer a molecular basis for the design of LL‐37‐derived peptides that combine antimicrobial, immunomodulatory, and T2D‐related anti‐amyloid functions as promising candidates for multifunctional drugs., One for all: A high‐affinity amyloid‐suppressing interaction between the human antimicrobial and immunomodulatory polypeptide cathelicidin LL‐37 and the key type 2 diabetes (T2D) amyloid polypeptide IAPP was identified. The results suggest a protective role for LL‐37 in T2D pathogenesis and offer a molecular basis for the design of novel molecules combining antimicrobial, immunomodulatory, and anti‐amyloid functions as multifunctional drug candidates.

Background Alzheimer’s disease (AD) is the most common neurodegenerative disease ultimately manifesting as clinical dementia. Despite considerable effort and ample experimental data, the role of neuroinflammation related to systemic inflammation is still unsettled. While the implication of microglia is well recognized, the exact contribution of peripheral monocytes/macrophages is still largely unknown, especially concerning their role in the various stages of AD. Objectives AD develops over decades and its clinical manifestation is preceded by subjective memory complaints (SMC) and mild cognitive impairment (MCI); thus, the question arises how the peripheral innate immune response changes with the progression of the disease. Therefore, to further investigate the roles of monocytes/macrophages in the progression of AD we assessed their phenotypes and functions in patients at SMC, MCI and AD stages and compared them with cognitively healthy controls. We also conceptualised an idealised mathematical model to explain the functionality of monocytes/macrophages along the progression of the disease. Results We show that there are distinct phenotypic and functional changes in monocyte and macrophage populations as the disease progresses. Higher free radical production upon stimulation could already be observed for the monocytes of SMC patients. The most striking results show that activation of peripheral monocytes (hyperactivation) is the strongest in the MCI group, at the prodromal stage of the disease. Monocytes exhibit significantly increased chemotaxis, free radical production, and cytokine production in response to TLR2 and TLR4 stimulation. Conclusion Our data suggest that the peripheral innate immune system is activated during the progression from SMC through MCI to AD, with the highest levels of activation being in MCI subjects and the lowest in AD patients. Some of these parameters may be used as biomarkers, but more holistic immune studies are needed to find the best period of the disease for clinical intervention.

Viral infections, such as those caused by Herpes Simplex Virus-1 (HSV-1) and SARS-CoV-2, affect millions of people each year. However, there are few antiviral drugs that can effectively treat these infections. The standard approach in the development of antiviral drugs involves the identification of a unique viral target, followed by the design of an agent that addresses that target. Antimicrobial peptides (AMPs) represent a novel source of potential antiviral drugs. AMPs have been shown to inactivate numerous different enveloped viruses through the disruption of their viral envelopes. However, the clinical development of AMPs as antimicrobial therapeutics has been hampered by a number of factors, especially their enzymatically labile structure as peptides. We have examined the antiviral potential of peptoid mimics of AMPs (sequence-specific N-substituted glycine oligomers). These peptoids have the distinct advantage of being insensitive to proteases, and also exhibit increased bioavailability and stability. Our results demonstrate that several peptoids exhibit potent in vitro antiviral activity against both HSV-1 and SARS-CoV-2 when incubated prior to infection. In other words, they have a direct effect on the viral structure, which appears to render the viral particles non-infective. Visualization by cryo-EM shows viral envelope disruption similar to what has been observed with AMP activity against other viruses. Furthermore, we observed no cytotoxicity against primary cultures of oral epithelial cells. These results suggest a common or biomimetic mechanism, possibly due to the differences between the phospholipid head group makeup of viral envelopes and host cell membranes, thus underscoring the potential of this class of molecules as safe and effective broad-spectrum antiviral agents. We discuss how and why differing molecular features between 10 peptoid candidates may affect both antiviral activity and selectivity.

Alzheimer’s disease (AD) is the most prevalent dementia in the world. Its cause(s) are presently largely unknown. The most common explanation for AD, now, is the amyloid cascade hypothesis, which states that the cause of AD is senile plaque forma- tion by the amyloid β peptide, and the formation of neurofibrillary tangles by hyperphosphorylated tau. A second, burgeoning theory by which to explain AD is based on the infection hypothesis. Much experimental and epidemiological data support the involvement of infections in the development of dementia. According to this mechanism, the infection either directly or via microbial virulence factors precedes the formation of amyloid β plaques. The amyloid β peptide, possessing antimicrobial properties, may be beneficial at an early stage of AD, but becomes detrimental with the progression of the disease, concomi- tantly with alterations to the innate immune system at both the peripheral and central levels. Infection results in neuroinflam- mation, leading to, and sustained by, systemic inflammation, causing eventual neurodegeneration, and the senescence of the immune cells. The sources of AD-involved microbes are various body microbiome communities from the gut, mouth, nose, and skin. The infection hypothesis of AD opens a vista to new therapeutic approaches, either by treating the infection itself or modulating the immune system, its senescence, or the body’s metabolism, either separately, in parallel, or in a multi-step way., Basque Government under the grant “Artificial Intelligence in BCAM number EXP. 2019/00432”

Acute lung injury (ALI) leads to progressive loss of breathing capacity and hypoxemia, as well as pulmonary surfactant dysfunction. ALI’s pathogenesis and management are complex, and it is a significant cause of morbidity and mortality worldwide. Exogenous surfactant therapy, even for research purposes, is impractical for adults because of the high cost of current surfactant preparations. Prior in vitro work has shown that poly-N-substituted glycines (peptoids), in a biomimetic lipid mixture, emulate key biophysical activities of lung surfactant proteins B and C at the air-water interface. Here we report good in vivo efficacy of a peptoid-based surfactant, compared with extracted animal surfactant and a synthetic lipid formulation, in a rat model of lavage-induced ALI. Adult rats were subjected to whole-lung lavage followed by administration of surfactant formulations and monitoring of outcomes. Treatment with a surfactant protein C mimic formulation improved blood oxygenation, blood pH, shunt fraction, and peak inspiratory pressure to a greater degree than surfactant protein B mimic or combined formulations. All peptoid-enhanced treatment groups showed improved outcomes compared to synthetic lipids alone, and some formulations improved outcomes to a similar extent as animal-derived surfactant. Robust biophysical mimics of natural surfactant proteins may enable new medical research in ALI treatment.

Alzheimer disease (AD) is one of the most devastating diseases and aging is one of the most important risk factors. For many years huge efforts have been made to better understand the etiopathogenesis of AD. Also, many treatment trials have been performed. At present, we do not what is the exact cause of AD nor how to treat it but we know that neuroinflammation plays an important role, the latter occurring even some 20 years before the clinical appearance of the disease. For the last 25 years, infections have been suspected to play a role in the pathogenesis of AD, and recently, there has been much interesting progress on this aspect. This symposium, with prestigious speakers, would describe recent exciting advances in the field of AD. The main themes would be the general overview on the role of infections in the development of AD; the role of Spirochetes and Chlamicia pneumoniae in the development of AD; the role of amyloid beta (Ab) as an antimicrobial agents and the future for treatment and the interaction between other antimicrobial agents.

Pancreatic islet encapsulation within biosynthetic materials has had limited clinical success due to loss of islet function and cell death. As an alternative encapsulation material, a silk-based scaffold was developed to reestablish the islet microenvironment lost during cell isolation. Islets were encapsulated with ECM proteins (laminin and collagen IV) and mesenchymal stromal cells (MSCs), known to have immunomodulatory properties or to enhance islet cell graft survival and function. After a 7 day in vitro encapsulation, islets remained viable and maintained insulin secretion in response to glucose stimulation. Islets encapsulated with collagen IV, or laminin had increased insulin secretion at day 2 and day 7, respectively. A 3.2-fold synergistic improvement in islet insulin secretion was observed when islets were co-encapsulated with MSCs and ECM proteins. Furthermore, encapsulated islets had increased gene expression of functional genes; insulin I, insulin II, glucagon, somatostatin, and PDX-1, and lower expression of the de-differentiation genes cytokeratin 19 and vimentin compared to non-encapsulated cells. This work demonstrates that encapsulation in silk with both MSCs and ECM proteins enhances islet function and with further development may have potential as a suitable platform for islet delivery in vivo.

Peptoids are a rapidly developing class of biomimetic polymers based on oligo-N-substituted glycine backbones, designed to mimic peptides and proteins. Inspired by natural antimicrobial peptides, a group of cationic amphipathic peptoids has been successfully discovered with a potent and broad-spectrum activity against pathogenic bacteria; however, there are limited studies to address the in vivo pharmacokinetics of the peptoids. Herein, 64Cu labeled DOTA conjugates of three different peptoids and two control peptides were synthesized and assayed in vivo by both biodistribution studies and small animal positron emission tomography (PET). The study was designed in a way to assess how structural differences of the peptidomimetics affect in vivo pharmacokinetics. As amphipathic molecules, major uptake of the peptoids occurred at the liver. Increased kidney uptake was observed by deleting one hydrophobic residue in the peptoid, and 64Cu-3 achieved the highest kidney uptake of all the conjugates tested in this study. In comparison to peptides, our data indicated that peptoids had general in vivo properties of higher tissue accumulation, slower elimination, and higher in vivo stability. Different administration routes (intravenous, intraperitoneal, and oral) were investigated with peptoids. When administered orally, the peptoids showed poor bioavailability, reminiscent to that of peptide. But, remarkably longer passage through the gastrointestinal (GI) tract without rapid digestion was observed for peptoids. These unique in vivo properties of peptoids were rationalized by efficient cellular membrane permeability and protease resistance of peptoids. The results observed in the biodistribution studies could be confirmed by the PET imaging, which provides a reliable way to evaluate in vivo pharmacokinetic properties of peptoids noninvasively and in real time. The pharmacokinetic data presented here can provide an insight for further development of the antimicrobial peptoids as pharmaceuticals.

Free-solution conjugate electrophoresis (FSCE) is a method of DNA sequencing that eliminates the need for viscous polymer solutions by tethering a carefully designed, mobility modifying "drag-tag" to each DNA molecule to achieve size-based separations of DNA. The most successful drag-tags to date are genetically engineered, highly repetitive polypeptides ("protein polymers") that are designed to be large, water-soluble, and completely monodisperse. Positively charged arginines were deliberately introduced at regular intervals into the amino acid sequence to increase the hydrodynamic drag without increasing drag-tag length. Additionally, a one-step purification method that combines affinity chromatography and on-column tag cleavage was devised to achieve the required drag-tag monodispersity. Sequencing with a read length of approximately 180 bases was successfully achieved with a known sequence in free-solution electrophoresis using one of these positively charged drag-tags. This preliminary result is expected to lead to further progress in FSCE sequencing with ~400 bases read length possible when more "highly" positively charged protein polymers of larger size are generated with the intein system.

Protein-based polymers are increasingly being used in biomaterial applications because of their ease of customization and potential monodispersity. These advantages make protein polymers excellent candidates for bioanalytical applications. Here we describe improved methods for producing drag-tags for free-solution conjugate electrophoresis (FSCE). FSCE utilizes a pure, monodisperse recombinant protein, tethered end-on to a ssDNA molecule, to enable DNA size separation in aqueous buffer. FSCE also provides a highly sensitive method to evaluate the polydispersity of a protein drag-tag and thus its suitability for bioanalytical uses. This method is able to detect slight differences in drag-tag charge or mass. We have devised an improved cloning, expression, and purification strategy that enables us to generate, for the first time, a truly monodisperse 20 kDa protein polymer and a nearly monodisperse 38 kDa protein. These newly produced proteins can be used as drag-tags to enable longer read DNA sequencing by free-solution microchannel electrophoresis.

Thermo-responsive polymer “nanogels” (crosslinked hydrogel particles with sub-100 nm diameters) are intriguing for many potential applications in biotechnology and medicine. There have been relatively few reports of electrostatically neutral, thermosensitive nanogels comprising a high fraction of hydrophilic co-monomer. Here we demonstrate the syntheses and characterization of novel, nonionic nanogels based on random N,N-diethylacrylamide (DEA) / N,N-dimethylacrylamide (DMA) copolymers, made by free-radical, surfactant-free dispersion polymerization. The volume phase transition temperatures of these DEA/DMA nanogels are strongly affected by co-monomer composition, providing a way to “tune” the phase transition temperature of these non-ionic nanogels. While DEA nanogels (comprising no DMA) can be obtained at 70°C by standard emulsion precipitation, DEA/DMA random copolymer nanogels can be obtained only in a particular range of temperatures, above the initial phase transition temperature and below the critical precipitation temperature of the DEA/DMA copolymer, controlled by co-monomer composition. Increasing percentages of DMA in the nanogels raises the phase transition temperature, and attenuates and broadens it as well. We find that concentrated DEA/DMA nanogel dispersions are optically clear at room temperature. This good optical clarity was exploited for their use in a novel DNA sieving matrix for microfluidic chip electrophoresis. An ultrafast, high-efficiency dsDNA separation was achieved in less than 120 seconds for dsDNA ranging from 75 bp-15000 bp.

In this report, we demonstrate the purification of DNA and RNA from a 10% serum sample using an oligonucleotide capture matrix. This approach provides a one-stage, completely aqueous system capable of purifying both RNA and DNA for downstream PCR amplification. The advantages of utilizing the polymer capture matrix method in place of the solid-phase extraction method is that the capture matrix eliminates both guanidine and the 2-propanol wash that can inhibit downstream PCR and competition with proteins for the binding sites that can limit the capacity of the device. This method electrophoreses a biological sample (e.g., serum) containing the nucleic acid target through a polymer matrix with covalently bound oligonucleotides. These capture oligonucleotides selectively hybridize and retain the target nucleic acid, while the other biomolecules and reagents (e.g., SDS) pass through the matrix to waste. Following this purification step, the solution can be heated above the melting temperature of the capture sequence to release the target molecule, which is then electrophoresed to a recovery chamber for subsequent PCR amplification. We demonstrate that the device can be applied to purify both DNA and RNA from serum. The gag region of HIV at a starting concentration of 37.5 copies per microliter was successfully purified from a 10% serum sample demonstrating the applicability of this method to detect viruses present in low copy numbers.

Surfactant protein C (SP-C) is a hydrophobic lipopeptide that is critical for lung function, in part because it physically catalyzes the formation of surface-associated surfactant reservoirs. Many of SP-C's key biophysical properties derive from its highly stable and hydrophobic α-helix. However, SP-C's posttranslational modification with N-terminal palmitoyl chains also seems to be quite important. We created a new (to our knowledge) class of variants of a synthetic, biomimetic family of peptide mimics (peptoids) that allow us to study the functional effects of biomimetic N-terminal alkylation in vitro. Mimics were designed to emulate the amphipathic patterning, helicity, and hydrophobicity of SP-C, and to include no, one, or two vicinal amide-linked, N-terminal octadecyl chains (providing a reach equivalent to that of natural palmitoyl chains). Pulsating bubble surfactometry and Langmuir-Wilhelmy surface balance studies showed that alkylation improved biomimetic surface activities, yielding lower film compressibility and lower maximum dynamic surface tensions. Atomic force microscopy studies indicated that alkyl chains bind to and retain segregated interfacial surfactant phases at low surface tensions by inducing 3D structural transitions in the monolayer's fluid-like phase, forming surfactant-associated reservoirs. Peptoid-based SP-C mimics are easily produced and purified, and offer much higher chemical and secondary structure stability than polypeptide-based mimics. In surfactant replacements intended for medical use, synthetic SP mimics reduce the odds of pathogen contamination, which may facilitate the wider use of surfactant treatment of respiratory disorders and diseases.

Monodisperse protein polymers engineered by biosynthetic techniques are well suited to serve as a basis for creating comb-like polymer architectures for biomaterial applications. We have developed a new class of linear, cationic, random-coil protein polymers designed to act as scaffolds for multivalent display. These polymers contain evenly spaced lysine residues that allow for chemical or enzymatic conjugation of pendant functional groups. Circular dichroism (CD) spectroscopy and turbidity experiments have confirmed that these proteins have a random coil structure and are soluble up to at least 65 °C. Cell viability assays suggest these constructs are non-toxic in solution up to a concentration of 100 μM. We have successfully attached a small bioactive peptide, a peptoid-peptide hybrid, a poly(ethylene glycol) (PEG) polymer, and a fluorophore to the protein polymers by chemical or enzymatic coupling, demonstrating their suitability to serve as multivalent scaffolds in solutions or as gels.

Since the widespread use of exogenous lung surfactant to treat neonatal respiratory distress syndrome, premature infant survival and respiratory morbidity have dramatically improved. Despite the effectiveness of the animal-derived surfactant preparations, there still remain some concerns and difficulties associated with their use. This has prompted investigation into the creation of synthetic surfactant preparations. However, to date, no clinically used synthetic formulation is as effective as the natural material. This is largely because the previous synthetic formulations lacked analogues of the hydrophobic proteins of the lung surfactant system, SP-B and SP-C, which are critical functional constituents. As a result, recent investigation has turned toward the development of a new generation of synthetic, biomimetic surfactants that contain synthetic phospholipids along with a mimic of the hydrophobic protein portion of lung surfactant. In this Account, we detail our efforts in creating accurate mimics of SP-C for use in a synthetic surfactant replacement therapy. Despite SP-C's seemingly simple structure, the predominantly helical protein is extraordinarily challenging to work with given its extreme hydrophobicity and structural instability, which greatly complicates the creation of an effective SP-C analogue. Drawing inspiration from Nature, two promising biomimetic approaches have led to the creation of rationally designed biopolymers that recapitulate many of SP-C's molecular features. The first approach utilizes detailed SP-C structure-activity relationships and amino acid folding propensities to create a peptide-based analogue, SP-C33. In SP-C33, the problematic and metastable polyvaline helix is replaced with a structurally stable polyleucine helix and includes a well-placed positive charge to prevent aggregation. SP-C33 is structurally stable and eliminates the association propensity of the native protein. The second approach follows the same design considerations but makes use of a non-natural, poly-N-substituted glycine or "peptoid" scaffold to circumvent the difficulties associated with SP-C. By incorporating unique biomimetic side chains in a non-natural backbone, the peptoid mimic captures both SP-C's hydrophobic patterning and its helical secondary structure. Despite the differences in structure, both SP-C33 and the SP-C peptoid mimic capture many requisite features of SP-C. In a surfactant environment, these analogues also replicate many of the key surface activities necessary for a functional biomimetic surfactant therapy while overcoming the difficulties associated with the natural protein. With improved stability, greater production potential, and elimination of possible pathogenic contamination, these biomimetic surfactant formulations offer not only the potential to improve the treatment of respiratory distress syndrome but also the opportunity to treat other respiratory-related disorders.

Antimicrobial peptides (AMPs) and their mimics are emerging as promising antibiotic agents. We present a library of “ampetoids” (antimicrobial peptoid oligomers) with helical structures and biomimetic sequences, several members of which have low-micromolar antimicrobial activities, similar to cationic AMPs like pexiganan. Broad-spectrum activity against six clinically relevant BSL2 pathogens is also shown. This comprehensive structure–activity relationship study, including circular dichroism spectroscopy, minimum inhibitory concentration assays, hemolysis and mammalian cell toxicity studies, and specular x-ray reflectivity measurements shows that the in vitro activities of ampetoids are strikingly similar to those of AMPs themselves, suggesting a strong mechanistic analogy. The ampetoids' antibacterial activity, coupled with their low cytotoxicity against mammalian cells, make them a promising class of antimicrobials for biomedical applications. Peptoids are biostable, with a protease-resistant N -substituted glycine backbone, and their sequences are highly tunable, because an extensive diversity of side chains can be incorporated via facile solid-phase synthesis. Our findings add to the growing evidence that nonnatural foldamers will emerge as an important class of therapeutics.

The majority of clinically used contrast agents (CAs) for magnetic resonance imaging have low relaxivities and thus require high concentrations for signal enhancement. Research has turned to multivalent, macromolecular CAs to increase CA efficiency. However, previously developed macromolecular CAs do not provide high relaxivities, have limited biocompatibility, and/or do not have a structure that is readily modifiable to tailor to particular applications. We report a new family of multivalent, biomacromolecular, genetically engineered protein polymer-based CAs; the protein backbone contains evenly spaced lysines that are derivatized with gadolinium (Gd(III)) chelators. The protein's length and repeating amino acid sequence are genetically specified. We reproducibly obtained conjugates with an average of 8-9 Gd(III) chelators per protein. These multivalent CAs reproducibly provide a high relaxivity of 7.3 mM (-1) s (-1) per Gd(III) and 62.6 mM (-1) s (-1) per molecule. Furthermore, they can be incorporated into biomaterial hydrogels via chemical cross-linking of the remaining free lysines, and provide a dramatic contrast enhancement. Thus, these protein polymer CAs could be a useful tool for following the evolution of tissue engineering scaffolds.

Microfluidic laboratory-on-a-chip (LOC) systems based on a modular architecture are presented. The architecture is conceptualized on two levels: a single-chip level and a multiple-chip module (MCM) system level. At the individual chip level, a multilayer approach segregates components belonging to two fundamental categories: passive fluidic components (channels and reaction chambers) and active electromechanical control structures (sensors and actuators). This distinction is explicitly made to simplify the development process and minimize cost. Components belonging to these two categories are built separately on different physical layers and can communicate fluidically via cross-layer interconnects. The chip that hosts the electromechanical control structures is called the microfluidic breadboard (FBB). A single LOC module is constructed by attaching a chip comprised of a custom arrangement of fluid routing channels and reactors (passive chip) to the FBB. Many different LOC functions can be achieved by using different passive chips on an FBB with a standard resource configuration. Multiple modules can be interconnected to form a larger LOC system (MCM level). We demonstrated the utility of this architecture by developing systems for two separate biochemical applications: one for detection of protein markers of cancer and another for detection of metal ions. In the first case, free prostate-specific antigen was detected at 500 aM concentration by using a nanoparticle-based bio-bar-code protocol on a parallel MCM system. In the second case, we used a DNAzyme-based biosensor to identify the presence of Pb 2+ (lead) at a sensitivity of 500 nM in

Polymers of N-substituted glycines (“peptoids”) containing chiral centers at the α position of their side chains can form stable structures in solution. We studied a prototypical peptoid, consisting of five para -substituted ( S )- N -(1-phenylethyl)glycine residues, by NMR spectroscopy. Multiple configurational isomers were observed, but because of extensive signal overlap, only the major isomer containing all cis -amide bonds was examined in detail. The NMR data for this molecule, in conjunction with previous CD spectroscopic results, indicate that the major species in methanol is a right-handed helix with cis -amide bonds. The periodicity of the helix is three residues per turn, with a pitch of ≈6 Å. This conformation is similar to that anticipated by computational studies of a chiral peptoid octamer. The helical repeat orients the amide bond chromophores in a manner consistent with the intensity of the CD signal exhibited by this molecule. Many other chiral polypeptoids have similar CD spectra, suggesting that a whole family of peptoids containing chiral side chains is capable of adopting this secondary structure motif. Taken together, our experimental and theoretical studies of the structural properties of chiral peptoids lay the groundwork for the rational design of more complex polypeptoid molecules, with a variety of applications, ranging from nanostructures to nonviral gene delivery systems.