Nonspecific Targets Research Articles

Nanoparticles to Target Asthma.

Asthma is a heterogenous chronic lung disease that affects nearly 340 million people globally. Airway hyperresponsiveness, remodeling (thickening, fibrosis), and mucus hypersecretion are some hallmarks of asthma. With several current treatments having serious side effects from long-term use, and a proportion of patients with uncontrolled asthma, there is urgent need for new therapies. With increasing understanding of asthma pathophysiology, there is a recognized need to target therapies to specific cell types of the airway which necessitates identification of delivery systems that can overcome increased mucus and thickened airways. Nanoparticles (NPs) that are highly customizable (material, size, charge, surface modification) are a potential solution for delivery systems of a wide variety of cargoes (nucleic acids, proteins, and/or small molecules), as well as sole therapeutics for asthma. However, there is need to consider the safety of the NPs in terms of potential for inflammation, toxicity, non-specific targets, and accumulation in organs. Ongoing clinical trials using NPs, some FDA-approved for therapeutics in other diseases, provide confidence regarding potential safety and efficacy of NPs in asthma treatment. This review highlights the current state of the use of NPs in asthma, identifying opportunities for further improvements in NP design and utilization for targeting this chronic lung disease.

Incorporating Single-Copper Sites and Host Defense Peptides into a Nanoreactor for Antibacterial Therapy by Bioinspired Design

A sustainable solution to the dramatic spread of antibiotic resistance threatening public health security is the development of antibiotic-free antimicrobial substances. Inspired by natural host defense mechanisms involving amino-terminal copper−nickel binding motif (ATCUN) antimicrobial peptides (AMPs), we have designed and prepared an artificial complex (Cu@G-AMPs) incorporating single-atom Cu catalysts for antibacterial therapy. The substrate of the complex, formed from guanine doped with abundant heteroatoms, anchored single Cu atoms with a coordination number of 2 and an average bond length of 1.91Å. Interestingly, Cu@G-AMPs, exhibiting Fenton-like catalytic activity, caused the inactivation of methicillin-resistant Staphylococcus aureus (MRSA) by generating and delivering reactive oxygen species (ROS) cargo. Mechanistically, the intrinsic stress response system of MRSA underwent an irreversible collapse when Cu@G-AMPs initiated its offensive program associated with non-specific targets. Furthermore, Cu@G-AMPs, which inherited the immunomodulatory properties of AMPs, sequentially carried out the functions of pulling edge closure, stabilizing granulation tissue, promoting collagen fiber proliferation, alleviating inflammation, and promoting neovascularization in wound areas infected by MRSA. Our results show that Cu@G-AMPs will provide a new perspective on untangling the complex regulatory networks that resistant bacteria have cultivated to deactivate commercial antibiotics.

Strategies for overcoming the lung surfactant barrier and achieving success in antimicrobial photodynamic therapy

The impressive increase in antimicrobial resistance has required the development of alternative treatments that act on multiple non-specific molecular targets and are effective against a broad range of microorganisms. Antimicrobial Photodynamic Therapy (aPDT) is based on microbial inactivation from oxidative stress and represents an important tool for inactivating microorganisms with low risk of resistance selection. Therefore, our research group has been devoted to demonstrating its effectiveness against pathogens that cause pneumonia, one of the most lethal infections worldwide. Previous studies reported the efficiency and safety of an in vitro photoinactivation protocol for Streptococcus pneumoniae and the delivery of infrared light (external illumination) and photosensitizer (PS) in an animal model. However, the in vivo inactivation of microorganisms still poses challenges due to the presence of lung surfactant (LS), which traps PSs, preventing them from reaching the microbial target. This study investigated different approaches such as use of emulsifiers, perfluorocarbon, oxygen nanobubbles, and copolymer towards overcoming LS and optimizing aPDT response. The most promising strategy consisted in combining indocyanine green (ICG) with GantrezTM AN-139 - a Polyvinyl Methyl Ether/Maleic Anhydride copolymer (PVM/MA) – showing high microbial inactivation and safety for human lung epithelial (A549) and fibroblast (MRC-9) cell lines. The in vitro experiments provided an alternative to overcome the limited PS distribution through LS and will serve as the basis for in vivo studies.

Molecularly Imprinted Viral Protein Integrated Zn-Cu-In-Se-P Quantum Dots Superlattice for Quantitative Ratiometric Electrochemical Detection of SARS-CoV-2 Spike Protein in Saliva.

Solution-processable colloidal quantum dots (QDs) are promising materials for the development of rapid and low-cost, next-generation quantum-sensing diagnostic systems. In this study, we report on the synthesis of multinary Zn-Cu-In-Se-P (ZCISeP) QDs and the application of the QDs-modified electrode (QDs/SPCE) as a solid superlattice transducer interface for the ratiometric electrochemical detection of the SARS-CoV-2-S1 protein in saliva. The ZCISeP QDs were synthesized through the formation of In(Zn)PSe QDs from InP QDs, followed by the incorporation of Cu cations into the crystal lattice via cation exchange processes. A viral-protein-imprinted polymer film was deposited onto the QDs/SPCE for the specific binding of SARS-CoV-2. Molecular imprinting of the virus protein was achieved using a surface imprinting electropolymerization strategy to create the MIP@QDs/SPCE nanosensor. Characterization through spectroscopic, microscopic, and electrochemical techniques confirmed the structural properties and electronic-band state of the ZCISeP QDs. Cyclic voltammetry studies of the QDs/SPCE superlattice confirmed efficient electron transport properties and revealed an intraband gap energy state with redox peaks attributed to the Cu1+/2+ defects. Binding of SARS-CoV-2-S1 to the MIP@QDs/SPCE cavities induced a gating effect that modulated the Fe(CN)6 3-/4- and Cu1+/2+ redox processes at the nanosensor interface, producing dual off/on ratiometric electrical current signals. Under optimal assay conditions, the nanosensor exhibited a wide linear detection range (0.001-100 pg/mL) and a low detection limit (0.34 pg/mL, 4.6 fM) for quantitative detection of SARS-CoV-2-S1 in saliva. The MIP@QDs/SPCE nanosensor demonstrated excellent selectivity against nonspecific protein targets, and the integration with a smartphone-based potentiostat confirmed the potential for point-of-care applications.

Accuracy of molecular diagnostic assays for detection of Mycobacterium bovis: A systematic review and meta-analysis

Bovine tuberculosis (bovine TB) is a chronic wasting disease of cattle caused primarily by Mycobacterium bovis. Controlling bovine TB requires highly sensitive, specific, quick, and reliable diagnostic methods. This systematic review and meta-analysis evaluated molecular diagnostic tests for M. bovis detection to inform the selection of the most viable assay. On a per-test basis, loop-mediated isothermal amplification (LAMP) showed the highest overall sensitivity of 99.0% [95% CI: 86.2%-99.9%] and specificity of 99.8% [95% CI: 96.2%-100.00%]. Quantitative real-time polymerase chain reaction (qPCR) outperformed conventional PCR and nested PCR (nPCR) with a diagnostic specificity of 96.6% [95% CI: 88.9%-99.0%], while the diagnostic sensitivity of 70.8% [95% CI: 58.6–80.5%] was comparable to that of nPCR at 71.4% [95% CI: 60.7–80.2%]. Test sensitivity was higher with the input of milk samples (90.9% [95% CI: 56.0%-98.7%]), while specificity improved with tests based on major M. bovis antigens (97.8% [95% CI: 92.3%-99.4%]), the IS6110 insertion sequence (95.4% [95% CI: 87.6%-98.4%]), and the RD4 gene (90.7% [95% CI: 52.2%-98.9%]). The design of the currently available molecular diagnostic assays, while mostly based on nonspecific gene targets, prevents them from being accurate enough to diagnose M. bovis infections in cattle, despite their promise. Future assay development should focus on the RD4 region since it is the only target identified by genome sequence data as being distinctive for detecting M. bovis. The availability of a sufficiently accurate diagnostic test combined with the routine screening of milk samples can decrease the risk of zoonotic transmissions of M. bovis.

Understanding the rapid spread of antimicrobial resistance genes mediated by IS26.

Insertion sequences (ISs) promote the transmission of antimicrobial resistance genes (ARGs) across bacterial populations. However, their contributions and dynamics during the transmission of resistance remain unclear. In this study, we selected IS26 as a representative transposable element to decipher the relationship between ISs and ARGsand to investigate their transfer features and transmission trends. We retrieved 2656 translocatable IS 26 -bounded units with ARGs (tIS26-bUs-ARGs) in complete bacterial genomes from theNCBI RefSeq database. In total, 124 ARGs spanning 12 classes of antibiotics were detected, and the average contribution rate of IS26 to these genes was 41.2%. We found that IS 26 -bounded units (IS26-bUs) mediated extensive ARG dissemination within the bacteria of theGammaproteobacteria class, showing strong transfer potential between strains, species, and even phyla. The IS26-bUs expanded in bacterial populations over time, and their temporal expansion trend was significantly correlated with antibiotic usage. This wide dissemination could be due to the nonspecific target site preference of IS26. Finally, we experimentally confirmed that the introduction of a single copy of IS26 could lead to the formation of a composite transposon mediating the transmission of "passenger" genes. These observations extend our knowledge of the IS26and provide new insights into the mediating role of ISs in the dissemination of antibiotic resistance.

Detection of SARS-CoV-2 Spike Protein Using Micropatterned 3D Poly(3,4-Ethylenedioxythiophene) Nanorods Decorated with Gold Nanoparticles.

The sensitivity and fabrication process of the detection platform are important for developing viral disease diagnosis. Recently, the outbreak of SARS-CoV-2 compelled us to develop a new detection platform to control such diseases in the future. We present an electrochemical-based assay that employs the unique properties of gold nanoparticles (AuNPs) deposited on 3D carboxyl-functionalized poly(3,4-ethylenedioxythiophene) (PEDOTAc) nanorods for specific and sensitive detection of SARS-CoV-2 spike protein (S1). The 3D-shaped PEDOTAc nanorods offer an ample surface area for receptor immobilization grown on indium-tin oxide surfaces through transfer-printing technology. Characterization via electrochemical, fluorescence, X-ray photoelectron spectroscopy, and scanning electron microscopy techniques confirmed the structural and morphological properties of the AuNPs-decorated PEDOTAc. In contrast to antibody-based assays, our platform employs ACE2 receptors for spike protein binding. Differential pulse voltammetry records current responses, showing linear sensitivity from 100 ng to 10 pg/mL of S1. In addition, the SARS-CoV-2 assay (CoVPNs) also exhibited excellent selectivity against nonspecific target proteins (H9N2, IL-6, and Escherichia coli). Furthermore, the developed surface maintained good stability for up to 7 consecutive days without losing performance. The results provide new insight into effective 3D conductive nanostructure formation, which is promising in the development of versatile sensory devices.

Abstract C025: Colli-Pee™ UAS™ 40mL: A first-void urine collection device with potential to improve access to cancer screening when paired with an appropriate clinical assay

Abstract In recent years, liquid biopsy has become increasingly important for cancer screening, detection and monitoring as cancer-specific biomarkers can be captured non-invasively in biological fluids. First-void urine (FVU) offers distinct advantages, such as ease of collection and large available volumes. Colli-Pee™ UAS™ 40 mL is a FVU self-collection device pre-filled with UAS, a non-lytic liquid preservative. Usability questionnaires from 98 healthy and diseased users showed that Colli-Pee™ was clear (90%) and comfortable (68%) to use, underscoring its potential to improve access to cancer screening for underserved communities. Here, we evaluated the performance of the device for FVU collection and urinary analyte preservation. FVU samples were collected from healthy donors with the Colli-Pee™ UAS 40 mL device to evaluate analyte preservation (n=14). Samples were stored at room temperature (RT), with aliquots processed at baseline and 8 days, or after freeze/thaw cycling (-20°C/-4°F to 40°C/104°F) to simulate ambient temperature transport. Cell-free DNA (cfDNA, Qiagen Circulating Nucleic Acid Kit) and extracellular vesicle RNA (EV RNA, Qiagen exoRNeasy Maxi Kit) were extracted from sample supernatants and evaluated for analyte preservation via qPCR assays targeting the β-globin gene (cfDNA) or the GAPDH mRNA (EV RNA). Cellular pellets were obtained at each time point by centrifugation and genomic DNA was extracted (Qiagen PowerFecal Pro DNA Kit) to evaluate both host cell integrity and microbial growth. Both were inferred based on the amount of change in qPCR signal for a human DNA target (TS143) and a non-specific bacterial target (16S). Data are presented as mean ± SD. Analysis of RT-stored samples confirmed that both urinary cfDNA and EV RNA were well preserved (ΔCt: 0.01 ± 1.04 and 0.58 ± 1.15, respectively). Similarly, qPCR of host and microbial DNA recovered from the cellular pellet showed no change after storage (ΔCt: 0.00 ± 0.50 and 0.06 ± 0.51, respectively), demonstrating that host cells remained intact and that UAS™ preservative prevented microbial growth. Under simulated ambient temperature transportation conditions, cfDNA (ΔCt: 0.57 ± 1.58), EV RNA (1.05 ± 0.94), and host cell integrity (0.63 ± 0.73) were successfully maintained. Urinary cfDNA, EV RNA, and host cell integrity from FVU samples were effectively preserved up to 7 days post-collection at RT, and under simulated ambient temperature transportation. By enabling standardized volumetric FVU collection and analyte preservation, the Colli-Pee™ UAS™ 40 mL device demonstrates its viability as a non-invasive FVU self-sampling device for oncology applications, simplifying serial and at-home collection. In addition to the advantages of FVU, Colli-Pee™ collection devices can improve access to testing by allowing for samples to be collected at home and transported at ambient temperature, which can improve the chances of early detection and outcomes for diverse patients in at-risk populations. Citation Format: Kyle MacDonald, Melissa Richer, Thanh Dang, Alejandra Rioscortes, Cameron Wood, Nette Meers, Savannah Colameco, Koen Beyers, Graham Morse, Tara Crawford Parks. Colli-Pee™ UAS™ 40mL: A first-void urine collection device with potential to improve access to cancer screening when paired with an appropriate clinical assay [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C025.

Fluorescent bacteria detection in water using cell imprinted polymer (CIP) coated microparticles in a magnetophoretic microfluidic device

Molecular imprinting has advanced towards synthesizing whole-cell imprints of microorganisms such as bacteria on various sensor surfaces including wire electrodes, quartz crystal microbalances, and microparticles (MPs). We recently introduced cell-imprinted polymers (CIPs) coated on MPs, called CIP-MPs, for bacteria recovery from water. In this paper, we have advanced towards rapid fluorometric Escherichia coli (E. coli) detection by applying fluorescent magnetic CIP-MPs, which were captured by soft ferromagnetic microstructures integrated into a microfluidic channel. The shape of ferromagnetic microstructures was optimized numerically to enhance the magnetophoretic accumulation of CIP-MPs in the microchannel. The device was then fabricated and the flow rate required to enhance bacteria capturing efficiency by CIP-MPs was determined. Decreasing the flow rate reduced the flow-induced drag and increased the interactions between the bacteria and imprinted CIP cavities. Fluorescent imaging of the accumulated CIP-MPs was done before and after bacteria capturing to quantify the changes in the fluorescence intensity as E. coli cells were captured by CIP-MPs in the microchannel. The dose-response curve of the sensor at 0–109 CFU/mL bacterial counts was obtained. Using the 3- and 10-sigma methods on the dose-response curve, the limits of detection (LOD) and quantification (LOQ) of the sensor were determined to be 4 × 102 and 3 × 103 CFU/mL, respectively, within a dynamic range of 102–107 CFU/mL. Lastly, the specificity of the CIP-MPbased sensor towards E. coli was tested and confirmed using Sarcina as a non-specific target bacterium. In summary, our developed sensor offers a promising approach for rapid and low-cost detection of bacteria in water and is suitable for development of portable and durable all-polymeric sensors for point-of-need detection.

The developing role of extracellular vesicles in autoimmune diseases: special attention to mesenchymal stem cell-derived extracellular vesicles.

Autoimmune diseases are complex, chronic inflammatory conditions initiated by the loss of immunological tolerance to self-antigens. Nowadays, there is no effective and useful therapy for autoimmune diseases, and the existing medications have some limitations due to their nonspecific targets and side effects. During the last few decades, it has been established that mesenchymal stem cells (MSCs) have immunomodulatory functions. It is proposed that MSCs can exert an important therapeutic effect on autoimmune disorders. In parallel with these findings, several investigations have shown that MSCs alleviate autoimmune diseases. Intriguingly, the results of studies have demonstrated that the effective roles of MSCs in autoimmune diseases do not depend on direct intercellular communication but on their ability to release a wide spectrum of paracrine mediators such as growth factors, cytokines and extracellular vehicles (EVs). EVs that range from 50 to 5,000nm were produced by almost any cell type, and these nanoparticles participate in homeostasis and intercellular communication via the transfer of a broad range of biomolecules such as modulatory proteins, nucleic acids (DNA and RNA), lipids, cytokines, and metabolites. EVs derived from MSCs display the exact properties of MSCs and can be safer and more beneficial than their parent cells. In this review, we will discuss the features of MSCs and their EVs, EVs biogenesis, and their cargos, and then we will highlight the existing discoveries on the impacts of EVs from MSCs on autoimmune diseases such as multiple sclerosis, arthritis rheumatic, inflammatory bowel disease, Type 1 diabetes mellitus, systemic lupus erythematosus, autoimmune liver diseases, Sjögren syndrome, and osteoarthritis, suggesting a potential alternative for autoimmune conditions therapy.

2D vanadium disulfide nanosheets assisted ultrasensitive, rapid, and label-free electrochemical quantification of cancer biomarker (MMP-2)

Cancer is one of the most tormenting global health burdens reporting high mortality and morbidity worldwide. Matrix metalloproteinase 2 (MMP-2) protein has elevated expression for most types of cancers, including prostate and breast cancer. Therefore, accurate and specific detection of MMP-2 biomarker is crucial for screening, treatment, and prognosis of related cancer. In this work, we have proposed a label-free electrochemical biosensor for the detection of MMP-2 protein. This biosensor was fabricated using hydrothermally synthesized vanadium disulfide (VS2) nanosheets with monoclonal anti-MMP2 antibodies biofunctionalized using a suitable linker. The VS2 nanomaterials were synthesized hydrothermally at different reaction temperatures (140 °C, 160 °C, 180 °C and 200 °C) generating different morphologies from a 3D bulk cubic structure at 140 °C to 2D nanosheets at 200 °C. Owing to the advantages of 2D VS2 nanosheets with high surface-to-volume ratio, excellent electrochemical response and high antibody loading possibility, it was selected for fabricating an MMP-2 specific biosensor. The antibody–antigen binding event is analyzed by recording electrochemical impedance spectroscopy signals for different target MMP-2 protein concentrations. The sensitivity and lower limit of detection were 7.272 (ΔR/R)(ng ml)−1 cm−2 and 0.138 fg ml−1, respectively in 10 mM phosphate buffer saline for this proposed sensor. Further, interference studies were also performed which demonstrates the sensor to be highly selective against non-specific target proteins. This 2D VS2 nanosheet-based electrochemical biosensor is a sensitive, cost-effective, accurate, and selective solution for cancer diagnosis.

The COVERED FACE: FaceNet-based partially occluded person recognition - on attention mechanism

This paper investigates the problem of improving the recognition accuracy of partially occluded faces. We aim to insert the attention mechanism blocks into the FaceNet to improve its recognition capability. FaceNet has excellent performance as a general-purpose face recognition system. However, recognizing faces with incomplete facial features is a challenge for feature extraction networks. To overcome this weakness and improve the recognition range of FaceNet, this paper proposes to incorporate the attention mechanism in the feature extraction network so that the attention of the model will be focused on the face features that are not occluded. The addition of the attention mechanism makes the application scenario of FaceNet more flexible and improves the anti-interference ability and robustness of the model. The results show that FaceNet trained by non-specific target dataset can improve the performance after adding the attention mechanism. In detail, the improvement in recognition for regular datasets is small. Nevertheless, the performance of the model is significantly improved for certain datasets with extreme conditions, such as face images with occlusions. In general, inserting the attention mechanism blocks in FaceNet can improve its value and expand its usage environment.

Benefits from prospective memory offloading depend on memory load and reminder type.

Prospective memory (PM) refers to the ability to remember to complete a task at the appropriate moment in the future. Past research has found reminders can improve PM performance in both laboratory and naturalistic settings, but few projects have examined the circumstances when and what types of reminders are most beneficial. Three experiments in the present study tested the influence of reminders in an event-based PM task under different cognitive loads. An additional study examined how effective reminders of different types were. In Experiments 1 (specific targets) and 2 (nonspecific targets), load was manipulated by having participants respond to a single (low load) or multiple (high load) targets. In Experiment 3, the association between target-action word pairs was manipulated by presenting strongly associated pairs (low load) or weakly associated pairs (high load). Experiment 4 used target-action word pairs and varied the type of reminder. Participants in the reminder conditions had target (Experiments 1 and 2), target and action (Experiment 3) or target and/or action (Experiment 4) reminders listed at the top of the screen throughout the PM task. Across the first 3 experiments, it was found that the benefit of reminders was greater under high load than low load conditions. Experiment 4 found that target-action reminders improved PM, while target-only or action-only reminders did not. Importantly, the improvements in PM from reminders occurred without cost to ongoing task performance. Together these results suggest that reminders can be beneficial for reducing PM failures, particularly under high load, without the potential downside of increased effort expenditure. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Effects of 10 Hz individualized repetitive transcranial magnetic stimulation on patients with disorders of consciousness: a study protocol for an exploratory double-blind crossover randomized sham-controlled trial

BackgroundRepetitive transcranial magnetic stimulation (rTMS), as a non-invasive brain stimulation technique, has shown potentials for consciousness recovery of patients with disorders of consciousness (DoC), as, to a certain extent, it is effective in regulating the excitability of central nervous system. However, it is difficult to achieve satisfactory effect with “one size fits all” rTMS treatment due to different clinical conditions of patients. There is an urgent need to develop individualized strategy to improve the effectiveness of rTMS on patients with DoC.MethodsOur protocol is a randomized double-blind sham-controlled crossover trial that includes 30 DoC patients. Each patient will received 20 sessions, in which 10 sessions will be rTMS-active stimulus, and the other 10 sessions will be sham stimulus, separated by no less than 10 days’ washout period. The rTMS-active will include 10 Hz rTMS over the individualized-targeted selection area for each patient according to the different insult regions of the brain. Coma Recovery Scale-Revised (CRS-R) will be used as primary outcome at baseline, after the first stage of stimulation, at the end of the washout period, and after the second stage of stimulation. Secondary outcomes will be measured at the same time, including efficiency, relative spectral power, and functional connectivity of high-density electroencephalograph (EEG). Adverse events will be recorded during the study.DiscussionrTMS has obtained grade A evidence in treating patients with several central nervous system diseases, and there has been some evidence showing partial improvement on level of consciousness in DoC patients. However, the effectiveness of rTMS in DoC is only 30~36%, mostly due to the non-specific target selection. In this protocol, we present a double-blind crossover randomized sham-controlled trial based on the individualized-targeted selection strategy that aims to study the effectiveness of rTMS therapy for DoC, and the result may provide new insights to non-invasive brain stimulation.Trial registrationClinicalTrials.gov: NCT05187000. Registered on January 10, 2022.

Exploring membrane binding targets of tau oligomers on phase-separated lipid bilayers using multiscale MD simulations.

Self-aggregation of tau has been linked to the onset of Alzheimer's. Recent studies indicate that the disordered tau oligomers are more neurotoxic than the ordered tau fibrils in the intracellular neurofibrillary tangle. Details of tau oligomer interactions with the lipid nanodomains in phase-separated lipid bilayers (raft membranes), a model of neuronal membranes, are unknown. Using coarse-grained (CG) and all-atom (AA) MD simulations, a tau-K18 monomer containing the microtubule-binding domain (R1-R2-R3-R4) and its disordered oligomers were simulated. By mixing phosphatidylcholine, cholesterol, ganglioside (GM1), and phosphatidylserine (PS) lipids in water, raft membranes containing lipid nanodomains, liquid-ordered (Lo), liquid-disordered (Ld), and Lo/Ld boundary (Lod) on both lipid leaflets, as well as PS- or GM1-cluster on one lipid leaflet, were simulated. Lipid-binding kinetics and energetics of tau-K18 oligomers to these nanodomains were investigated. Within 20 μs, tau-K18 oligomers bound to the Lod domains in membranes without PS or GM1. In contrast, exclusive binding to PS- or GM1-clusters in membranes containing PS or GM1 occurred in less than 6 μs. After a CG to AA transformation and a 200 ns-AA simulation, disruptions of lipid chain order and beta-sheet formation on raft membranes containing membrane-bound tau-K18 oligomers were found. By mutating three hydrophobic residues to anionic residues at the R2 and R3 domain regions of tau-K18, the mutated tau-K18 oligomers bound much weaker to the PS- or GM1-cluster domains than the wild type did, suggesting the importance of electrostatic interactions in the tau-lipid interactions. Since PS and GM1 are found in the inner and outer leaflets of neuronal membranes, respectively, our results suggest that the PS- and GM1-clusters are leaflet-specific targets while the Lod domains are non-specific targets in membrane-associated tauopathies in Alzheimer's. (Supported by NSF [OAC-153159] and Welch Foundation [W-2057-2021-327].)

Applying machine learning to investigate lipid domain formation behaviors in the presence of amyloidogenic proteins.

Cell membranes are not homogeneous but contain lipid nanodomains distributed on both leaflets of the lipid bilayers. Using microsecond molecular dynamics (MD) simulations, we have simulated phase-separated lipid bilayers (raft membranes) containing lipid nanodomains that mimic the heterogeneous structures of neuronal plasma membranes. Our recent studies indicate that these lipid nanodomains, e.g., the mixed liquid-ordered (Lo) and liquid-disordered (Ld) boundaries or Lod and ganglioside-cluster, represent non-specific and specific membrane damage targets of amyloidogenic protein, e.g., tau, aggregates (oligomers), in raft membranes. However, a robust computational tool to classify lipids into these lipid nanodomains and quantitate the spatial and temporal evolutions of the domain structures in the presence of amyloidogenic oligomers is not available. In this study, we have modified two unsupervised Machine Learning (ML) algorithms, Non-negative Matrix Factorization (NMFk) and K-Mean Clustering, to investigate lipid domain formation behaviors. These algorithms are currently used in mutation analysis of cancer genomes and feature extractions. In this work, NMFk was used to decompose our time-resolved lipid-lipid proximity data matrix into two latent “signature” and “activity” matrices, which contain a set of probability configurations and the time-dependent activity of those configurations, respectively, of the lipids participating in each nanodomain, e.g., Lo and Lod. On the other hand, by implementing additional spatial information to the dataset used in NMFk, the K-Mean Clustering sorts lipids into nanodomains and determines the latent temporal and spatial evolutions of each domain. Hence, by combining both ML algorithms, this study provides new insights into understanding protein-induced membrane damage mechanisms of amyloidogenic oligomers associated with lipid domain disruption behaviors that may contribute to the pathogenesis of Alzheimer's. (Supported by NSF [OAC 153159] and Welch Foundation [W-2057-2021-327].)

Surface-induced protein folding of human islet amyloid polypeptide on neuronal membrane domains.

Aggregated human islet amyloid polypeptide (hIAPP) has been associated with the development of type II diabetes. However, recent evidence suggests that disordered hIAPP aggregates, or hIAPP oligomers, may also bind to the neurons in the brain causing membrane damage and thereby contributing to the cognitive decline of people with Alzheimer's Disease (AD). At present, the detailed membrane damage mechanisms of disordered hIAPP oligomers on neuronal membranes are unknown. Using both coarse-grained (CG) and all-atom (AA) molecular dynamics (MD) simulations, binding of disordered hIAPP oligomers (monomer, dimer, and tetramer) to phase-separated lipid bilayers (raft membranes), a model of neuronal plasma membrane domains, was investigated. We observed membrane binding of hIAPP oligomers to raft membranes containing mixed liquid-ordered (Lo) and liquid-disordered (Ld) boundary or Lod domains on both lipid leaflets of the lipid bilayer. However, much stronger binding to ganglioside (GM1) clusters embedded in the Lo domains and phosphatidylserine (PS) clusters embedded in the Lo and Lod domains, located in only one leaflet of the lipid bilayer, was evident. All oligomers binding events occurred within 10 μs. Our results suggest that the Lod domains represent the non-specific targets in both lipid leaflets, while the PS-clusters and GM1-clusters represent the specific targets in the inner lipid leaflet and outer lipid leaflet, respectively, of the plasma membranes of neurons. After a CG-to-AA resolution transformation and a 200 ns-long AA MD simulation, surface-induced protein folding, particularly the formation of localized inter-chain and intra-chain beta-sheets, in several hIAPP-raft complexes, especially those involving Lod domains, was evident. We propose that these localized beta-sheets may act as nucleation seeds to attract nearby hIAPP or non-hIAPP oligomers to form larger surface-bound amyloid aggregates and create membrane damage to the neurons that leads to the pathogenesis of AD.

Multiple protein-patterned surface plasmon resonance biochip for the detection of human immunoglobulin-G.

A portable surface plasmon resonance (SPR) measurement prototype integrated with a multiple protein-patterned SPR biochip is introduced for label-free and selective detection of human immunoglobulin-G (H-IgG). The polyclonal anti-H-IgG antibodies derived from goat, rabbit, and mouse were immobilized through polydimethylsiloxane (PDMS) microchannels to fabricate the patterned SPR biochip. The PDMS surface was functionalized using 3-aminopropyltrimethoxysilane and bonded to carbodiimide-activated gold substrates to construct irreversibly bonded hydrophilic microfluidic chip at room temperature. For SPR measurement, a custom-made system is developed with a high angular scanning accuracy of 0.005° and a wide scanning range of 30°-80° that avoids the conventional requirement of expensive goniometric stages and detector arrays. The SPR biochip immobilized with 750μg/mL goat anti-H-IgG demonstrated detection of H-IgG with a detection limits of 15 μg/mL, and linear response through a wide concentration range (15-225 μg/mL) of high coefficient of determination (R2 =0.99661). The selectivity of the sensor was investigated by exposing them to two different non-specific targets (bovine serum albumin and polyvalent antivenom). The results indicate negligible sensor response towards nonspecific targets (0.25° for 30 μg/mL bovine serum albumin (BSA) and 0.25° for 30 μg/mL polyvalent antivenom) in comparison to H-IgG (1.5° for 30 μg/mL).

Enhancement in Site-Specific Delivery of Chloramphenicol Using Bacterially Sensitive Microparticle Loaded Into Dissolving Microneedle: Potential For Enhanced Effectiveness Treatment of Cellulitis.

One of the biggest challenges in infectious disease treatment is the existence of bacterial infections in underskin wound tissue, such as cellulitis. Compared to other treatments, it is harder for antibacterial drugs to penetrate the physical barrier on the affected skin with a nonspecific target, making conventional therapy for cellulitis infection more difficult and considered. In this novel research, we pioneer a combined strategy of dissolving microneedles (MNs) and bacteria-sensitive microparticles (MPs) for enhanced penetration and targeted delivery of chloramphenicol (CHL) to the infection site specifically. The polycaprolactone polymer was used to make MPs because of its sensitivity to bacterial enzyme stimuli. The best microparticle formulation was discovered and optimized using the Design-Expert application. Furthermore, this study evaluated the antibacterial activity of MPs in vitro and in vivo on the mutant Drosophila larval infection model. This strategy shows improvement in the antibacterial activity of MPs and higher retention duration compared to conventional cream formulation, and the inclusion of these MPs into dissolving MNs was able to greatly improve the dermatokinetic characteristics of CHL in ex vivo evaluation. Importantly, the antimicrobial efficacy in an ex vivo infection model demonstrated that, following the use of this strategy, bacterial bioburdens decreased by up to 99.99% after 24 h. The findings offered a proof of concept for the enhancement of CHL dermatokinetic profiles and antimicrobial activities after its preparation into bacteria-sensitive MPs and distribution by MNs. Future research should investigate in vivo effectiveness in an appropriate animal model.

Non-Specific Targets for Correction of Pneumonia Caused by Aerosols Containing Damaging Factors of Various Nature

This review article provides data on the current state of the pathogenesis peculiarities of body and lung inflammation (pneumonia) under the influence of damaging factors of various nature: infectious agents, chemical toxicants, as well as incorporated radionuclides, etc. The peculiarities of inflammation itself, as a typical pathological process, are considered. Information on mediators that induce the so-called pro-resolving phase of inflammation manifestations is given. Approaches to the neuroimmune correction of non-specific inflammation are substantiated. Data on the following alternative approaches to the correction of nonspecific inflammation are summarized: factors of the coagulation system, modulators of the integrated stress response, and modulators of sigma-1 receptors. Based on the data presented, general directions for the treatment of nonspecific pneumonia are formulated, including reflexogenic and anti-inflammatory therapy in combination with multimodal drugs, as well as pro-resolving therapy in combination with drugs that prevent fibrosis.