SENSING AND SEPARATING BIOMOLECULES AT BIOINTERFACES

Ligand-receptor interactions are ubiquitous on cell membranes. Indeed, many important physiological functions primarily involve such interactions. These include cell signaling, pathogen binding, trafficking of lymphocytes, and the immune response.1-4 Therefore, studying ligand-receptor interactions at appropriate model membrane is of importance for both proper understanding of biological functions and applications to biosensors and bioseparations. Supported lipid bilayers are composed of the same lipid molecules found in the plasma cell membranes of living cells and possess the same two-dimensional fluidity as cell membranes, making them capable of mimicking the cell surface. Moreover, supported lipid bilayer-based in vitro assays are appealing because they require only very small sample volumes and they are suitable for multiplexing and high-throughput screening. Recently, our laboratory has combined supported lipid bilayer-coated microfluidic platforms with total internal reflection fluorescence microscopy to obtain equilibrium dissociation constant data for protein-ligand interactions. Using this method, it was found that equilibrium dissociation constants of antibody-ligand interactions at lipid membrane interfaces can be strongly affected by ligand lipophilicity and linker length/structure. These results are described in Chapter III. Monitoring protein-ligand interactions is routinely performed by fluorescently labeling the proteins of interest. Protein labeling can, however, interfere with detection measurements and be highly inconvenient to employ. To solve these problems, a simple and highly sensitive technique for detection of protein-ligand binding at biointerfaces has been developed. The method is based upon modulation of the interfacial pH when the protein binds. This change is detected by pH-sensitive fluorescent dye molecules embedded into the biointerface. The dye fluoresces strongly in the protonated state but becomes inactive upon deprotonation. These results are demonstrated in Chapter IV. Finally, the study of supported lipid bilayer-based electrophoresis is described in Chapter V. Bilayer electrophoresis is an attractive alternative to gel electrophoresis for the separation of membrane components such as lipids and membrane proteins because it is run in native-like environments and avoids exposing the analytes of interest to harsh chemicals. In this study, lipid rafts of varying size were used as separation matrices to separate two similar lipids with different alkyl chains. Lipid rafts of varying size were formed by a process controlled by varying treatment of the solid substrate. Depending on which method was employed, the results showed that lipid raft size could be modulated over five orders of magnitude. Moreover, it was found that the electrophoretic separation of the two lipid components depended on the size of rafts in the bilayer matrix

White-Box AES Implementation Revisited

White-box cryptography is an obfuscation technique for protecting secret keys in software implementations even if an adversary has full access to the implementation of the encryption algorithm and full control over its execution platforms. This concept was presented by Chow et al. with white-box implementations of DES and AES in 2002. The strategy used in the implementations has become a design principle for subsequent white-box implementations. However, despite its practical importance, progress has not been substantial. In fact, it is repeated that as a proposal for a white-box implementation is reported, an attack of lower complexity is soon announced. This is mainly because most cryptanalytic methods target specific implementations, and there is no general attack tool for white-box cryptography. In this paper, we present an analytic toolbox on white-box implementations in this design framework and show how to reveal the secret information obfuscated in the implementation using this. For a substitution-linear transformation cipher on $n$ bits with S-boxes on $m$ bits, if $m_Q$-bit nonlinear encodings are used to obfuscate output values in the implementation, our attack tool can remove the nonlinear encodings with complexity $O(\frac{n}{m_Q}2^{3m_Q})$. We should increase $m_Q$ to obtain higher security, but it yields exponential storage blowing up and so there are limits to increase the security using the nonlinear encoding. If the inverse of the encoded round function $F$ on $n$ bits is given, the affine encoding $A$ can be recovered in $O(\frac{n}{m}\cdot{m_A}^32^{3m})$ time using our specialized affine equivalence algorithm, where $m_A$ is the smallest integer $p$ such that $A$ (or its similar matrix obtained by permuting rows and columns) is a block-diagonal matrix with $p\times p$ matrix blocks. According to our toolbox, a white-box implementation in the Chow et al.\u27s framework has complexity at most $O\left(\min\left\{ \tfrac{2^{2m}}{m}\cdot n^{m+4}, n\log n \cdot 2^{n/2}\right\}\right)$ within reasonable storage, which is much less than $2^n$. To overcome this, we introduce an idea that obfuscates two AES-128 ciphers at once with input/output encoding on 256 bits. To reduce storage, we use a sparse unsplit input encoding. As a result, our white-box AES implementation has up to 110-bit security against our toolbox, close to that of the original cipher. More generally, we may consider a white-box implementation on the concatenation of $t$ ciphertexts to increase security

Tissue Adequacy and Safety of Percutaneous Transthoracic Needle Biopsy for Molecular Analysis in Non-Small Cell Lung Cancer: A Systematic Review and Meta-analysis

OBJECTIVE: We conducted a systematic review and meta-analysis of the tissue adequacy and complication rates of percutaneous transthoracic needle biopsy (PTNB) for molecular analysis in patients with non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: We performed a literature search of the OVID-MEDLINE and Embase databases to identify original studies on the tissue adequacy and complication rates of PTNB for molecular analysis in patients with NSCLC published between January 2005 and January 2020. Inverse variance and random-effects models were used to evaluate and acquire meta-analytic estimates of the outcomes. To explore heterogeneity across the studies, univariable and multivariable meta-regression analyses were performed. RESULTS: A total of 21 studies with 2232 biopsies (initial biopsy, 8 studies; rebiopsy after therapy, 13 studies) were included. The pooled rates of tissue adequacy and complications were 89.3% (95% confidence interval [CI]: 85.6%-92.6%; I(2) = 0.81) and 17.3% (95% CI: 12.1%-23.1%; I(2) = 0.89), respectively. These rates were 93.5% and 22.2% for the initial biopsies and 86.2% and 16.8% for the rebiopsies, respectively. Severe complications, including pneumothorax requiring chest tube placement and massive hemoptysis, occurred in 0.7% of the cases (95% CI: 0%-2.2%; I(2) = 0.67). Multivariable meta-regression analysis showed that the tissue adequacy rate was not significantly lower in studies on rebiopsies (p = 0.058). The complication rate was significantly higher in studies that preferentially included older adults (p = 0.001). CONCLUSION: PTNB demonstrated an average tissue adequacy rate of 89.3% for molecular analysis in patients with NSCLC, with a complication rate of 17.3%. PTNB is a generally safe and effective diagnostic procedure for obtaining tissue samples for molecular analysis in NSCLC. Rebiopsy may be performed actively with an acceptable risk of complications if clinically required

Impact of Hapten Presentation on Antibody Binding at Lipid Membrane Interfaces

AbstractWe report the effects of ligand presentation on the binding of aqueous proteins to solid supported lipid bilayers. Specifically, we show that the equilibrium dissociation constant can be strongly affected by ligand lipophilicity and linker length/structure. The apparent equilibrium dissociation constants (KD) were compared for two model systems, biotin/anti-biotin and 2,4-dinitrophenyl (DNP)/anti-DNP, in bulk solution and at model membrane surfaces. The binding constants in solution were obtained from fluorescence anisotropy measurements. The surface binding constants were determined by microfluidic techniques in conjunction with total internal reflection fluorescence microscopy. The results showed that the bulk solution equilibrium dissociation constants for anti-biotin and anti-DNP were almost identical, KD(bulk)=1.7±0.2 nM vs. 2.9±0.1 nM. By contrast, the dissociation constant for anti-biotin antibody was three orders of magnitude tighter than for anti-DNP at a lipid membrane interface, KD=3.6±1.1 nM vs. 2.0±0.2 μM. We postulate that the pronounced difference in surface binding constants for these two similar antibodies is due to differences in the ligands’ relative lipophilicity, i.e., the more hydrophobic DNP molecules had a stronger interaction with the lipid bilayers, rendering them less available to incoming anti-DNP antibodies compared with the biotin/anti-biotin system. However, when membrane-bound biotin ligands were well screened by a poly(ethylene glycol) (PEG) polymer brush, the KD value for the anti-biotin antibody could also be weakened by three orders of magnitude, 2.4±1.1μM. On the other hand, the dissociation constant for anti-DNP antibodies at a lipid interface could be significantly enhanced when DNP haptens were tethered to the end of very long hydrophilic PEG lipopolymers (KD=21±10nM) rather than presented on short lipid-conjugated tethers. These results demonstrate that ligand presentation strongly influences protein interactions with membrane-bound ligands

CT analysis of thoracolumbar body composition for estimating whole-body composition

Background To evaluate the correlation between single- and multi-slice cross-sectional thoracolumbar and whole-body compositions. Methods We retrospectively included patients who underwent whole-body PET–CT scans from January 2016 to December 2019 at multiple institutions. A priori-developed, deep learning-based commercially available 3D U-Net segmentation provided whole-body 3D reference volumes and 2D areas of muscle, visceral fat, and subcutaneous fat at the upper, middle, and lower endplate of the individual T1–L5 vertebrae. In the derivation set, we analyzed the Pearson correlation coefficients of single-slice and multi-slice averaged 2D areas (waist and T12–L1) with the reference values. We then built prediction models using the top three correlated levels and tested the models in the validation set. Results The derivation and validation datasets included 203 (mean age 58.2years; 101 men) and 239 patients (mean age 57.8years; 80 men). The coefficients were distributed bimodally, with the first peak at T4 (coefficient, 0.78) and the second peak at L2-3 (coefficient 0.90). The top three correlations in the abdominal scan range were found for multi-slice waist averaging (0.92) and single-slice L3 and L2 (0.90, each), while those in the chest scan range were multi-slice T12–L1 averaging (0.89), single-slice L1 (0.89), and T12 (0.86). The model performance at the top three levels for estimating whole-body composition was similar in the derivation and validation datasets. Conclusions Single-slice L2–3 (abdominal CT range) and L1 (chest CT range) analysis best correlated with whole-body composition around 0.90 (coefficient). Multi-slice waist averaging provided a slightly higher correlation of 0.92.Key points In single-slice analysis, the L2–3 and L1 levels had the closest correlations with whole-body composition. Multi-slice waist averaging (0.92; correlation) showed a better correlation than the L2–3 single-slice analysis (0.90) in the abdomen. Multi-slice T12–L1 averaging (0.89) provided a comparable correlation to the L1 level in the chest (0.89).This work was supported by the Korea Medical Device Development Fund grant funded by the Korean government (the Ministry of Science and ICT, the Ministry of Trade Industry and Energy, the Ministry of Health & Welfare, Republic of Korea, the Ministry of Food and Drug Safety) (Project Number: 202011A03). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Genetic assessment of pathogenic germline alterations in lysosomal genes among Asian patients with pancreatic ductal adenocarcinoma

Background Lysosomes are closely linked to autophagic activity, which plays a vital role in pancreatic ductal adenocarcinoma (PDAC) biology. The survival of PDAC patients is still poor, and the identification of novel genetic factors for prognosis and treatment is highly required to prevent PDAC-related deaths. This study investigated the germline variants related to lysosomal dysfunction in patients with PDAC and to analyze whether they contribute to the development of PDAC. Methods The germline putative pathogenic variants (PPV) in genes involved in lysosomal storage disease (LSD) was compared between patients with PDAC (n = 418) and healthy controls (n = 845) using targeted panel and whole-exome sequencing. Furthermore, pancreatic organoids from wild-type and KrasG12D mice were used to evaluate the effect of lysosomal dysfunction on PDAC development. RNA sequencing (RNA-seq) analysis was performed with established PDAC patient-derived organoids (PDOs) according to the PPV status. Results The PPV in LSD-related genes was higher in patients with PDAC than in healthy controls (8.13 vs. 4.26%, Log2 OR = 1.65, P = 3.08 × 10–3). The PPV carriers of LSD-related genes with PDAC were significantly younger than the non-carriers (mean age 61.5 vs. 65.3 years, P = 0.031). We further studied a variant of the lysosomal enzyme, galactosylceramidase (GALC), which was the most frequently detected LSD variant in our cohort. Autophagolysosomal activity was hampered when GALC was downregulated, which was accompanied by paradoxically elevated autophagic flux. Furthermore, the number of proliferating Ki-67+ cells increased significantly in pancreatic organoids derived from Galc knockout KrasG12D mice. Moreover, GALC PPV carriers tended to show drug resistance in both PDAC cell line and PDAC PDO, and RNA-seq analysis revealed that various metabolism and gene repair pathways were upregulated in PDAC PDOs harboring a GALC variant. Conclusions Genetically defined lysosomal dysfunction is frequently observed in patients with young-onset PDAC. This might contribute to PDAC development by altering metabolism and impairing autophagolysosomal activity, which could be potentially implicated in therapeutic applications for PDAC.This work was supported by the National Research Foundation of Korea funded by the Korean Government (MSIT) (Grant No. NRF-2021R1A2C3005360) (YK) and the Ministry of Health & Welfare, Republic of Korea (Grant No. HI18C1876) (SSY). This study was supported by the Future Medicine 20 × 30 Project of the Samsung Medical Center (Grant No. SMX1230041, SMO1230021) and a Samsung Medical Center Research and Development Grant (Grant No. SMO1230661) (JKP)

Micro/Nanostructured Coating for Cotton Textiles That Repel Oil, Water, and Chemical Warfare Agents

Using a lotus leaf as our model, we fabricated an extremely low surface energy micro/nanostructured coating for textiles that repel oil, water, and chemical warfare agents (CWAs) using a simple process that is suitable for large scale production. This coating, called “OmniBlock”, consisted of approximately 200-nm silica nanoparticles, tetraethylorthosilicate, 3-glycidoxypropyl trimethoxysilane, and a perfluorooctanoic acid-free fluoropolymer (Fluorolink S10) that was cross-linked between Si-O-Si groups via a sol-gel process. The perfluorooctanoic acid-free fluoropolymer-coated silica nanoparticles were simply applied to the surface of a cotton fabric by a dip–dry–cure process, forming dense, continuous, and uniform layers of OmniBlock coating. OmniBlock modified the surface of the cotton fibers, creating a rough, high surface area uniform coating with many micro-crevasses. As a result, n-dodecane, water, and CWAs beaded up without wetting the surface, exhibiting large contact angles of 154° for water and 121° for n-dodecane, with a small shedding angle of 5° and contact angle hysteresis of 3.2° for water. The designed coating showed excellent liquid repellence properties against three types of CWAs: 129°, 72°, and 87° for sulfur mustard (HD), soman (GD), and VX nerve agents, respectively. Furthermore, OmniBlock coating shows good mechanical properties under tensile strength and wash tests. This remarkable ability to repel CWAs is likely to have potential military applications in personal protective equipment systems requiring self-cleaning functions

The role of nutritional status in the relationship between diabetes and health-related quality of life

BACKGROUND/OBJECTIVES: The association between nutritional status and health-related quality of life (HRQoL) among patients with type 2 diabetes mellitus (T2DM) is not fully understood. This study was conducted to understand the role of nutritional status on HRQoL among people with and without T2DM. SUBJECTS/METHODS: Structured survey and direct measurement of anthropometric data were conducted among people with and without T2DM. Nutritional status was measured with Mini Nutritional Assessment tool and HRQoL was measured with a 36-item Short Form Healthy Survey. Data collection was conducted in Chuncheon, South Korea with 756 participants who are older than 40 yrs of age. RESULTS: This study found that overall HRQoL were significantly lower in people with T2DM than people without T2DM after controlling for key covariates. When stratified by nutritional status, a greater degree of negative impact of T2DM on overall physical HRQoL was observed among well-nourished or at risk of malnutrition, whereas significant and more evident negative impact of diabetes on overall psychological HRQoL was observed only among malnourished. CONCLUSIONS: The study results suggest the role of nutritional status among people with T2DM on overall, especially psychological aspects of HRQoL. Future longitudinal or intervention studies are warranted to test the impact of nutritional status on HRQoL among people with T2DM

PH modulation method to detect ligand-receptor binding

The present disclosure relates to detecting receptor-ligand binding by measuring local pH modulation using a pH-sensitive fluorophore.U

PH modulation method to detect ligand-receptor binding

The present disclosure relates to detecting receptor-ligand binding by measuring local pH modulation using a pH-sensitive fluorophore.U