The structure of tyrosine-10 favors ionic conductance of Alzheimer's disease-associated full-length amyloid-ß channels.

Amyloid ß (Aß) ion channels destabilize cellular ionic homeostasis, which contributes to neurotoxicity in Alzheimer's disease. The relative roles of various Aß isoforms are poorly understood. We use bilayer electrophysiology, AFM imaging, circular dichroism, FTIR and fluorescence spectroscopy to characterize channel activities of four most prevalent Aß peptides, Aß1-42, Aß1-40, and their pyroglutamylated forms (AßpE3-42, AßpE3-40) and correlate them with the peptides' structural features. Solvent-induced fluorescence splitting of tyrosine-10 is discovered and used to assess the sequestration from the solvent and membrane insertion. Aß1-42 effectively embeds in lipid membranes, contains large fraction of ß-sheet in a ß-barrel-like structure, forms multi-subunit pores in membranes, and displays well-defined ion channel features. In contrast, the other peptides are partially solvent-exposed, contain minimal ß-sheet structure, form less-ordered assemblies, and produce irregular ionic currents. These findings illuminate the structural basis of Aß neurotoxicity through membrane permeabilization and may help develop therapies that target Aß-membrane interactions.

In pursuit of degenerative brain disease diagnosis: Dementia biomarkers detected by DNA aptamer-attached portable graphene biosensor.

Dementia is a brain disease which results in irreversible and progressive loss of cognition and motor activity. Despite global efforts, there is no simple and reliable diagnosis or treatment option. Current diagnosis involves indirect testing of commonly inaccessible biofluids and low-resolution brain imaging. We have developed a portable, wireless readout-based Graphene field-effect transistor (GFET) biosensor platform that can detect viruses, proteins, and small molecules with single-molecule sensitivity and specificity. We report the detection of three important amyloids, namely, Amyloid beta (Aß), Tau (τ), and α-Synuclein (αS) using DNA aptamer nanoprobes. These amyloids were isolated, purified, and characterized from the autopsied brain tissues of Alzheimer's Disease (AD) and Parkinson's Disease (PD) patients. The limit of detection (LoD) of the sensor is 10 fM, 1-10 pM, 10-100 fM for Aß, τ, and αS, respectively. Synthetic as well as autopsied brain-derived amyloids showed a statistically significant sensor response with respect to derived thresholds, confirming the ability to define diseased vs. nondiseased states. The detection of each amyloid was specific to their aptamers; Aß, τ, and αS peptides when tested, respectively, with aptamers nonspecific to them showed statistically insignificant cross-reactivity. Thus, the aptamer-based GFET biosensor has high sensitivity and precision across a range of epidemiologically significant AD and PD variants. This portable diagnostic system would allow at-home and POC testing for neurodegenerative diseases globally.

Rapid self-test of unprocessed viruses of SARS-CoV-2 and its variants in saliva by portable wireless graphene biosensor.

We have developed a DNA aptamer-conjugated graphene field-effect transistor (GFET) biosensor platform to detect receptor-binding domain (RBD), nucleocapsid (N), and spike (S) proteins, as well as viral particles of original Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus and its variants in saliva samples. The GFET biosensor is a label-free, rapid (≤20 min), ultrasensitive handheld wireless readout device. The limit of detection (LoD) and the limit of quantitation (LoQ) of the sensor are 1.28 and 3.89 plaque-forming units (PFU)/mL for S protein and 1.45 and 4.39 PFU/mL for N protein, respectively. Cognate spike proteins of major variants of concern (N501Y, D614G, Y453F, Omicron-B1.1.529) showed sensor response ≥40 mV from the control (aptamer alone) for fM to nM concentration range. The sensor response was significantly lower for viral particles and cognate proteins of Middle East Respiratory Syndrome (MERS) compared to SARS-CoV-2, indicating the specificity of the diagnostic platform for SARS-CoV-2 vs. MERS viral proteins. During the early phase of the pandemic, the GFET sensor response agreed with RT-PCR data for oral human samples, as determined by the negative percent agreement (NPA) and positive percent agreement (PPA). During the recent Delta/Omicron wave, the GFET sensor also reliably distinguished positive and negative clinical saliva samples. Although the sensitivity is lower during the later pandemic phase, the GFET-defined positivity rate is in statistically close alignment with the epidemiological population-scale data. Thus, the aptamer-based GFET biosensor has a high level of precision in clinically and epidemiologically significant SARS-CoV-2 variant detection. This universal pathogen-sensing platform is amenable for a broad range of public health applications and real-time environmental monitoring.

Scavenging amyloid oligomers from neurons with silica nanobowls: Implications for amyloid diseases.

Amyloid-ß (Aß) oligomers are toxic species implicated in Alzheimer's disease (AD). The prevailing hypothesis implicates a major role of membrane-associated amyloid oligomers in AD pathology. Our silica nanobowls (NB) coated with lipid-polymer have submicromolar affinity for Aß binding. We demonstrate that NB scavenges distinct fractions of Aßs in a time-resolved manner from amyloid precursor protein-null neuronal cells after incubation with Aß. At short incubation times in cell culture, NB-Aß seeds have aggregation kinetics resembling that of extracellular fraction of Aß, whereas at longer incubation times, NB-Aß seeds scavenge membrane-associated Aß. Aß aggregates can be eluted from NB surfaces by mechanical agitation and appear to retain their aggregation driving domains as seen in seeding aggregation experiments. These results demonstrate that the NB system can be used for time-resolved separation of toxic Aß species from biological samples for characterization and in diagnostics. Scavenging membrane-associated amyloids using lipid-functionalized NB without chemical manipulation has wide applications in the diagnosis and therapy of AD and other neurodegenerative diseases, cancer, and cardiovascular conditions.

Cutting mechanics of wood by beetle larval mandibles.

Wood boring is a feature of several insect species and is a major cause of severe and irreparable damage to trees. Adult females typically deposit their eggs on the stem surface under bark scales. The emerging hatchlings live within wood during their larval phase, avoiding possible predation, whilst continually boring and tunneling through wood until pupation. A study of wood boring by insects offers unique insights into the bioengineering principles that drive evolutionary adaptations. We show that larval mandibles of the coffee wood stem borer beetle (Xylotrechus quadripes: Cerambycidae) have a highly sharp cusp edge to initiate fractures in Arabica wood and a suitable shape to generate small wood chips that are suitable for digestion. Cuticle hardness at the tip is significantly enhanced through zinc-enrichment. A hollow architecture significantly reduces bending stresses at the mandibular base without compromising the structural integrity. Finite element model of the mandible showed highest stresses in the tip region; these decreased to significantly lower values at the start of the hollow section. A scaling model based on a fracture mechanics framework shows the importance of the mandible shape in generating optimal chip sizes. These findings contain general principles in tool design and put in focus interactions of insects and their woody hosts.

Ion channel formation by N-terminally truncated Aß (4-42): relevance for the pathogenesis of Alzheimer's disease.

Aß deposition is a pathological hallmark of Alzheimer's disease (AD). Besides the full-length amyloid forming peptides (Aß1-40 and Aß1-42), biochemical analyses of brain deposits have identified a variety of N- and C-terminally truncated Aß variants in sporadic and familial AD patients. However, their relevance for AD pathogenesis remains largely understudied. We demonstrate that Aß4-42 exhibits a high tendency to form ß-sheet structures leading to fast self-aggregation and formation of oligomeric assemblies. Atomic force microscopy and electrophysiological studies reveal that Aß4-42 forms highly stable ion channels in lipid membranes. These channels that are blocked by monoclonal antibodies specifically recognizing the N-terminus of Aß4-42. An Aß variant with a double truncation at phenylalanine-4 and leucine 34, (Aß4-34), exhibits unstable channel formation capability. Taken together the results presented herein highlight the potential benefit of C-terminal proteolytic cleavage and further support an important pathogenic role for N-truncated Aß species in AD pathophysiology.

Direct DNA Methylation Profiling with an Electric Biosensor.

DNA methylation is one of the principal epigenetic mechanisms that control gene expression in humans, and its profiling provides critical information about health and disease. Current profiling methods require chemical modification of bases followed by sequencing, which is expensive and time-consuming. Here, we report a direct and rapid determination of DNA methylation using an electric biosensor. The device consists of a DNA-tweezer probe integrated on a graphene field-effect transistor for label-free, highly sensitive, and specific methylation profiling. The device performance was evaluated with a target DNA that harbors a sequence of the methylguanine-DNA methyltransferase, a promoter of glioblastoma multiforme, a lethal brain tumor. The results show that we successfully profiled the methylated and nonmethylated forms at picomolar concentrations. Further, fluorescence kinetics and molecular dynamics simulations revealed that the position of the methylation site(s), their proximity, and accessibility to the toe-hold region of the tweezer probe are the primary determinants of the device performance.

DNA Nanotweezers and Graphene Transistor Enable Label-Free Genotyping.

Electronic DNA-biosensor with a single nucleotide resolution capability is highly desirable for personalized medicine. However, existing DNA-biosensors, especially single nucleotide polymorphism (SNP) detection systems, have poor sensitivity and specificity and lack real-time wireless data transmission. DNA-tweezers with graphene field effect transistor (FET) are used for SNP detection and data are transmitted wirelessly for analysis. Picomolar sensitivity of quantitative SNP detection is achieved by observing changes in Dirac point shift and resistance change. The use of DNA-tweezers probe with high-quality graphene FET significantly improves analytical characteristics of SNP detection by enhancing the sensitivity more than 1000-fold in comparison to previous work. The electrical signal resulting from resistance changes triggered by DNA strand-displacement and related changes in the DNA geometry is recorded and transmitted remotely to personal electronics. Practical implementation of this enabling technology will provide cheaper, faster, and portable point-of-care molecular health status monitoring and diagnostic devices.

Gelatin Methacrylate Hydrogels as Biomimetic Three-Dimensional Matrixes for Modeling Breast Cancer Invasion and Chemoresponse in Vitro.

Recent studies have shown that three-dimensional (3D) culture environments allow the study of cellular responses in a setting that more closely resembles the in vivo milieu. In this context, hydrogels have become popular scaffold options for the 3D cell culture. Because the mechanical and biochemical properties of culture matrixes influence crucial cell behavior, selecting a suitable matrix for replicating in vivo cellular phenotype in vitro is essential for understanding disease progression. Gelatin methacrylate (GelMA) hydrogels have been the focus of much attention because of their inherent bioactivity, favorable hydration and diffusion properties, and ease-of-tailoring of their physicochemical characteristics. Therefore, in this study we examined the efficacy of GelMA hydrogels as a suitable platform to model specific attributes of breast cancer. We observed increased invasiveness in vitro and increased tumorigenic ability in vivo in breast cancer cells cultured on GelMA hydrogels. Further, cells cultured on GelMA matrixes were more resistant to paclitaxel treatment, as shown by the results of cell-cycle analysis and gene expression. This study, therefore, validates GelMA hydrogels as inexpensive, cell-responsive 3D platforms for modeling key characteristics associated with breast cancer metastasis, in vitro.