Polymeric microfluidic platform combined with Fourier Transform infrared imaging to explore biomolecular reactions

Diseases such as Alzheimer’s and Parkinson’s are classified as B-amyloid diseases due to the presence of plaques composed of B-amyloid fibrils, aggregations of misfolded proteins, in the affected tissues. Poorly functioning bioenergetics reactions such as those in cytochrome oxidase are linked to cardiomyopathies. Very early reaction intermediates such as misfolding in proteins that arise in less than a millisecond can lead to a cascade that results in diseases. Reaction mechanisms and kinetics of such sub-millisecond events are especially difficult to investigate experimentally due to (i) the lack of suitable methods to rapidly mix reactants, and/or (ii) lack of facile detection methods that are sensitive to molecular structure. Current techniques for such investigations are impractical in many cases or have serious limitations. The most promising method to investigate fast reactions integrates microfluidic continuous-flow reactors (MCFMs) with Fourier Transform infrared (FTIR) imaging to obtain sub-millisecond temporal resolution and molecular-bond structural resolution. My thesis primarily focuses on developing polymeric MCFMs compatible with FTIR imaging and developing robust methods for high-fidelity FTIR imaging and data analysis of sub-millisecond biomolecular reactions. We developed polymeric MCFMs using a low-cost cyclic olefin copolymer (COC) that is physically and spectrally biocompatible, and well suited for microfabrication. We used strong covalent bonding between device layers to enable the high flow rates needed to probe sub-millisecond reactions and developed robust FTIR imaging and analysis algorithms to extract high-quality FTIR spectral data. After validating the ability of the platform to provide both change in structural details of biomolecules and associated kinetics, we applied the platform and showed the ability of dodine as a chemical denaturant to enable FTIR protein dynamic studies by tracing the conformational change of apomyoglobin, and its unfolding kinetics. We successfully showed that the secondary structures of apomyoglobin behave differently during unfolding, and the unfolding kinetics changed depending on the dodine concentration

Viral dissemination and immune activation modulate antiretroviral drug levels in lymph nodes of SIV-infected rhesus macaques

Introduction and methodsTo understand the relationship between immunovirological factors and antiretroviral (ARV) drug levels in lymph nodes (LN) in HIV therapy, we analyzed drug levels in twenty-one SIV-infected rhesus macaques subcutaneously treated with daily tenofovir (TFV) and emtricitabine (FTC) for three months.ResultsThe intracellular active drug-metabolite (IADM) levels (TFV-dp and FTC-tp) in lymph node mononuclear cells (LNMC) were significantly lower than in peripheral blood mononuclear cells (PBMC) (P≤0.005). Between Month 1 and Month 3, IADM levels increased in both LNMC (P≤0.001) and PBMC (P≤0.01), with a steeper increase in LNMC (P≤0.01). The viral dissemination in plasma, LN, and rectal tissue at ART initiation correlated negatively with IADM levels at Month 1. Physiologically-based pharmacokinetic model simulations suggest that, following subcutaneous ARV administration, ART-induced reduction of immune activation improves the formation of active drug-metabolites through modulation of kinase activity and/or through improved parent drug accessibility to LN cellular compartments.ConclusionThese observations have broad implications for drugs that need to phosphorylate to exert their pharmacological activity, especially in the settings of the pre-/post-exposure prophylaxis and efficacy of antiviral therapies targeting pathogenic viruses such as HIV or SARS-CoV-2 replicating in highly inflammatory anatomic compartments

Pre-coder design over two-symbol extension for K-user cyclic interference channels

The authors consider K-user cyclic single-input–single-output (SISO) interference channel (IC) where each receiver is interfered with from only one neighbouring transmitter. In the K-user cyclic SISO IC, K/2 sum degrees of freedom can be achieved when two-symbol extension is applied even without channel state information at the transmitter. They derive an approximation of the ergodic sum rate as a function of pre-coders and propose designs of pre-coders to maximise the ergodic sum rate

Plasmon-Triggered Upconversion Emissions and Hot Carrier Injection for Combinatorial Photothermal and Photodynamic Cancer Therapy

Despite the unique ability of lanthanide-doped upconversion nanoparticles (UCNPs) to convert near-infrared (NIR) light to high-energy UV–vis radiation, low quantum efficiency has rendered their application unpractical in biomedical fields. Here, we report anatase titania-coated plasmonic gold nanorods decorated with UCNPs (Au NR@aTiO2@UCNPs) for combinational photothermal and photodynamic therapy to treat cancer. Our novel architecture employs the incorporation of an anatase titanium dioxide (aTiO2) photosensitizer as a spacer and exploits the localized surface plasmon resonance (LSPR) properties of the Au core. The LSPR-derived near-field enhancement induces a threefold boost of upconversion emissions, which are re-absorbed by neighboring aTiO2 and Au nanocomponents. Photocatalytic experiments strongly infer that LSPR-induced hot electrons are injected into the conduction band of aTiO2, generating reactive oxygen species. As phototherapeutic agents, our hybrid nanostructures show remarkable in vitro anticancer effect under NIR light [28.0% cancer cell viability against Au NR@aTiO2 (77.3%) and UCNP@aTiO2 (98.8%)] ascribed to the efficient radical formation and LSPR-induced heat generation, with cancer cell death primarily following an apoptotic pathway. In vivo animal studies further confirm the tumor suppression ability of Au NR@aTiO2@UCNPs through combinatorial photothermal and photodynamic effect. Our hybrid nanomaterials emerge as excellent multifunctional phototherapy agents, providing a valuable addition to light-triggered cancer treatments in deep tissue

Enzymatic Synthesis of Self-assembled Dicer Substrate RNA Nanostructures for Programmable Gene Silencing

Enzymatic synthesis of RNA nanostructures is achieved by isothermal rolling circle transcription (RCT). Each arm of RNA nanostructures provides a functional role of Dicer substrate RNA inducing sequence specific RNA interference (RNAi). Three different RNAi sequences (GFP, RFP, and BFP) are incorporated within the three-arm junction RNA nanostructures (Y-RNA). The template and helper DNA strands are designed for the large-scale in vitro synthesis of RNA strands to prepare self-assembled Y-RNA. Interestingly, Dicer processing of Y-RNA is highly influenced by its physical structure and different gene silencing activity is achieved depending on its arm length and overhang. In addition, enzymatic synthesis allows the preparation of various Y-RNA structures using a single DNA template offering on demand regulation of multiple target genes