Ion and Molecular Sieving With Ultrathin Polydopamine Nanomembranes.

In contrast to biological cell membranes, it is still a major challenge for synthetic membranes to efficiently separate ions and small molecules due to their similar sizes in the sub-nanometer range. Inspired by biological ion channels with their unique channel wall chemistry that facilitates ion sieving by ion-channel interactions, the first free-standing, ultrathin (10-17 nm) nanomembranes composed entirely of polydopamine (PDA) are reported here as ion and molecular sieves. These nanomembranes are obtained via an easily scalable electropolymerization strategy and provide nanochannels with various amine and phenolic hydroxyl groups that offer a favorable chemical environment for ion-channel electrostatic and hydrogen bond interactions. They exhibit remarkable selectivity for monovalent ions over multivalent ions and larger species with K+/Mg2+ of ≈4.2, K+/[Fe(CN)6]3- of ≈10.3, and K+/Rhodamine B of ≈273.0 in a pressure-driven process, as well as cyclic reversible pH-responsive gating properties. Infrared spectra reveal hydrogen bond formation between hydrated multivalent ions and PDA, which prevents the transport of multivalent ions and facilitates high selectivity. Chemically rich, free-standing, and pH-responsive PDA nanomembranes with specific interaction sites are proposed as customizable high-performance sieves for a wide range of challenging separation requirements.

A Rare Case of Hepatic Schwannoma in the Setting of Schwannomatosis.

Schwannomas are benign nerve sheath tumors commonly found in the head, neck, vestibular system, and extremities. Primary hepatic schwannomas are exceptionally rare, with 34 cases reported to date according to our review of the literature. This case report describes a 79-year-old man with a medical history of skin and thyroid cancer, who presented with no clinical symptoms and underwent a follow-up MRI due to an initial scan indicating a suspicious hepatic mass resembling an atypical hemangioma. The MRI revealed a 3.6 cm left hepatic mass concerning for an intrahepatic cholangiocarcinoma. Histopathological and immunohistochemical studies of a biopsy of the liver mass confirmed the presence of a benign hepatic schwannoma. Further evaluation revealed multiple spinal schwannomas, leading to the diagnosis of schwannomatosis. The diagnosis of hepatic schwannomas poses challenges through imaging alone. This case underscores the importance of microscopic evaluation in accurately diagnosing hepatic masses. Additionally, the presence of concurrent schwannomas should be considered in patients initially diagnosed with isolated schwannomas.

Risks of the ketogenic diet in CKD - the con part.

The ketogenic diet is a very low carbohydrate diet that has received a lot of attention for its role in the treatment of type 2 diabetes and obesity. For patients with chronic kidney disease, there is limited evidence on the risks and/or benefits of this diet. However, from the limited evidence that does exist, there are several inferences that can be drawn regarding this diet for patients with kidney disease. The ketogenic diet may not be better than comparator higher carbohydrate diets over the long term. The diet also has low adherence levels in studies lasting ≥12 months. The diet's emphasis on fat, which often comes from animal fat, increases the consumption of saturated fat, which may increase the risk of heart disease. It has the potential to worsen metabolic acidosis by increasing dietary acid load and endogenous acid production through the oxidation of fatty acids. In addition, the diet has been associated with an increased risk of kidney stones in patients using it for the treatment of refractory epilepsy. For these reasons, and for the lack of safety data on it, it is reasonable for patients with kidney disease to avoid utilizing the ketogenic diet as a first-line option given alternative dietary patterns (like the plant-dominant diet) with less theoretical risk for harm. For those adopting the ketogenic diet in kidney disease, a plant-based version of the ketogenic diet may mitigate some of the concerns with animal-based versions of the ketogenic diet.

High-Throughput Analyses of Therapeutic Antibodies Using High-Field Asymmetric Waveform Ion Mobility Spectrometry Combined with SampleStream and Intact Protein Mass Spectrometry.

Intact protein mass spectrometry (MS) coupled with liquid chromatography was applied to characterize the pharmacokinetics and stability profiles of therapeutic proteins. However, limitations from chromatography, including throughput and carryover, result in challenges with handling large sample numbers. Here, we combined intact protein MS with multiple front-end separations, including affinity capture, SampleStream, and high-field asymmetric waveform ion mobility spectrometry (FAIMS), to perform high-throughput and specific mass measurements of a multivalent antibody with one antigen-binding fragment (Fab) fused to an immunoglobulin G1 (IgG1) antibody. Generic affinity capture ensures the retention of both intact species 1Fab-IgG1 and the tentative degradation product IgG1. Subsequently, the analytes were directly loaded into SampleStream, where each injection occurs within ∼30 s. By separating ions prior to MS detection, FAIMS further offered improvement in signal-overnoise by ∼30% for denatured protein MS via employing compensation voltages that were optimized for different antibody species. When enhanced FAIMS transmission of 1Fab-IgG1 was employed, a qualified assay was established for spiked-in serum samples between 0.1 and 25 µg/mL, resulting in ∼10% accuracy bias and precision coefficient of variation. Selective FAIMS transmission of IgG1 as the degradation surrogate product enabled more sensitive detection of clipped species for intact 1Fab-IgG1 at 5 µg/mL in serum, generating an assay to measure 1Fab-IgG1 truncation between 2.5 and 50% with accuracy and precision below 20% bias and coefficient of variation. Our results revealed that the SampleStream-FAIMS-MS platform affords high throughput, selectivity, and sensitivity for characterizing therapeutic antibodies from complex biomatrices qualitatively and quantitatively.

Paramedic-Performed Prehospital Tele-Ultrasound: A Powerful Technology or an Impractical Endeavor? A Scoping Review.

Ultrasound with remote assistance (tele-ultrasound) may have potential to improve accessibility of ultrasound for prehospital patients. A review of recent literature on this topic has not been done before, and the feasibility of prehospital tele-ultrasound performed by non-physician personnel is unclear. In an effort to address this, the literature was qualitatively analyzed from January 1, 2010 - December 31, 2021 in the MEDLINE, EMBASE, and Cochrane online databases on prehospital, paramedic-acquired tele-ultrasound, and ten articles were found. There was considerable heterogeneity in the study design, technologies used, and the amount of ultrasound training for the paramedics, preventing cross-comparisons of different studies. Tele-ultrasound has potential to improve ultrasound accessibility by leveraging skills of a remote ultrasound expert, but there are still technological barriers to overcome before determinations on feasibility can be made.

ZHX2 promotes HIF1α oncogenic signaling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease, which warrants the critical need to identify new therapeutic targets. We show that Zinc Fingers and Homeoboxes 2 (ZHX2) is amplified or overexpressed in TNBC cell lines and patients. Functionally, depletion of ZHX2 inhibited TNBC cell growth and invasion in vitro, orthotopic tumor growth, and spontaneous lung metastasis in vivo. Mechanistically, ZHX2 bound with hypoxia-inducible factor (HIF) family members and positively regulated HIF1α activity in TNBC. Integrated ChIP-seq and gene expression profiling demonstrated that ZHX2 co-occupied with HIF1α on transcriptionally active promoters marked by H3K4me3 and H3K27ac, thereby promoting gene expression. Among the identified ZHX2 and HIF1α coregulated genes, overexpression of AP2B1, COX20, KDM3A, or PTGES3L could partially rescue TNBC cell growth defect by ZHX2 depletion, suggested that these downstream targets contribute to the oncogenic role of ZHX2 in an accumulative fashion. Furthermore, multiple residues (R491, R581, and R674) on ZHX2 are important in regulating its phenotype, which correspond with their roles on controlling ZHX2 transcriptional activity in TNBC cells. These studies establish that ZHX2 activates oncogenic HIF1α signaling, therefore serving as a potential therapeutic target for TNBC.

Von Hippel-Lindau tumor suppressor pathways & corresponding therapeutics in kidney cancer.

The identification and application of the Von Hippel-Lindau (VHL) gene is a seminal breakthrough in kidney cancer research. VHL and its protein pVHL are the root cause of most kidney cancers, and the cascading pathway below them is crucial for understanding hypoxia, in addition to the aforementioned tumorigenesis routes and treatments. We reviewed the history and functions of VHL/pVHL and Hypoxia-inducible factor (HIF), their well-known activities under low-oxygen environments as an E3 ubiquitin ligase and as a transcription factor, respectively, as well as their non-canonical functions revealed recently. Additionally, we discussed how their dysregulation promotes tumorigenesis: beginning with chromosome 3 p-arm (3p) loss/epigenetic methylation, followed by two-allele knockout, before the loss of complimentary tumor suppressor genes leads cells down predictable oncological paths. These different pathways can ultimately determine the grade, outcome, and severity of the deadliest genitourinary cancer. We finished by investigating current and proposed schemes to therapeutically treat clear cell renal cell carcinoma (ccRCC) by manipulating the hypoxic pathway utilizing Vascular Endothelial Growth Factor (VEGF) inhibitors, mammalian target of rapamycin complex 1 (mTORC1) inhibitors, small molecule HIF inhibitors, immune checkpoint blockade therapy, and synthetic lethality.

Hypoxia-Driven Effects in Cancer: Characterization, Mechanisms, and Therapeutic Implications.

Hypoxia, a common feature of solid tumors, greatly hinders the efficacy of conventional cancer treatments such as chemo-, radio-, and immunotherapy. The depletion of oxygen in proliferating and advanced tumors causes an array of genetic, transcriptional, and metabolic adaptations that promote survival, metastasis, and a clinically malignant phenotype. At the nexus of these interconnected pathways are hypoxia-inducible factors (HIFs) which orchestrate transcriptional responses under hypoxia. The following review summarizes current literature regarding effects of hypoxia on DNA repair, metastasis, epithelial-to-mesenchymal transition, the cancer stem cell phenotype, and therapy resistance. We also discuss mechanisms and pathways, such as HIF signaling, mitochondrial dynamics, exosomes, and the unfolded protein response, that contribute to hypoxia-induced phenotypic changes. Finally, novel therapeutics that target the hypoxic tumor microenvironment or interfere with hypoxia-induced pathways are reviewed.

Identification of critical chemical modifications and paratope mapping by size exclusion chromatography of stressed antibody-target complexes.

Therapeutic proteins including antibodies and Fc-fusion proteins undergo a large number of chemical modifications during cell culture, purification, storage and in human circulation. They are also exposed to harsh conditions during stress studies, including elevated temperature, extremes of pH, forced oxidation, physiological pH, UV light to assess the possible degradation pathways and suitability of methods for detecting them. Some of these modifications are located on residues in binding regions, leading to loss of binding and potency and classified as critical quality attributes. Currently, criticality of modifications is assessed by a laborious process of collecting antibody fractions from the soft chromatography techniques ion exchange and hydrophobic interaction chromatography and characterizing the fractions one-by-one for potency and chemical modifications. Here, we describe a method for large-scale, parallel identification of all critical chemical modifications in one experiment. In the first step, the antibody is stressed by one or several stress methods. It is then mixed with target protein and separated by size-exclusion chromatography (SEC) on bound antibody-target complex and unbound antibody. Peptide mapping of fractions and statistical analysis are performed to identify modifications on amino acid residues that affect binding. To identify the modifications leading to slight decreases in binding, competitive SEC of antibody and antigen mixtures was developed and described in a companion study by Shi et al, where target protein is provided at lower level, below the stoichiometry. The newly described method was successfully correlated to crystallography for assessing criticality of chemical modifications and paratope mapping. It is more sensitive to low-level modifications, better streamlined and platform ready.

Identification of critical chemical modifications by size exclusion chromatography of stressed antibody-target complexes with competitive binding.

Chemical modifications (attributes) in the binding regions of stressed therapeutic proteins may affect binding to target and efficacy of therapeutic proteins. The method presented here describes the criticality assessment of therapeutic antibody modifications by size-exclusion chromatography (SEC) of competitive binding between a stressed antibody and its target, human epidermal growth factor receptor-2 (HER2), followed by SEC fractionation and peptide mapping characterization of bound and unbound antibodies. When stressed antibody and its target were mixed at a stoichiometric molar ratio of 1:2, only antibody-receptor complex eluted from SEC, indicating that binding was not decreased to break the complex. When a smaller amount of the receptor was provided (1:1), the antibody species with modifications reducing binding eluted as unbound from SEC, while the antibody-receptor complex eluted as the bound fraction. Peptide mapping revealed ratios of modifications between unbound and bound fractions. Statistical analysis after triplicate measurements (n = 3) indicated that heavy chain (HC) D102 isomerization and light chain (LC) N30 deamidation were four-fold higher in unbound fraction with high statistical significance. Although HC N55 deamidation and M107 oxidation were also abundant, they were not statistically different between unbound and bound. Our findings agree with previously published potency measurements of collected CEX fractions and the crystal structure of antibody and HER2. Overall, competitive SEC of stressed antibody-receptor mixture followed by peptide mapping is a useful tool in revealing critical residues and modifications involved in the antibody-target binding, even if they elute as a complex from SEC when mixed at 1:2 stoichiometric ratio.

Hypoxia and Oxygen-Sensing Signaling in Gene Regulation and Cancer Progression.

Oxygen homeostasis regulation is the most fundamental cellular process for adjusting physiological oxygen variations, and its irregularity leads to various human diseases, including cancer. Hypoxia is closely associated with cancer development, and hypoxia/oxygen-sensing signaling plays critical roles in the modulation of cancer progression. The key molecules of the hypoxia/oxygen-sensing signaling include the transcriptional regulator hypoxia-inducible factor (HIF) which widely controls oxygen responsive genes, the central members of the 2-oxoglutarate (2-OG)-dependent dioxygenases, such as prolyl hydroxylase (PHD or EglN), and an E3 ubiquitin ligase component for HIF degeneration called von Hippel-Lindau (encoding protein pVHL). In this review, we summarize the current knowledge about the canonical hypoxia signaling, HIF transcription factors, and pVHL. In addition, the role of 2-OG-dependent enzymes, such as DNA/RNA-modifying enzymes, JmjC domain-containing enzymes, and prolyl hydroxylases, in gene regulation of cancer progression, is specifically reviewed. We also discuss the therapeutic advancement of targeting hypoxia and oxygen sensing pathways in cancer.

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis.

Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatography - top-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping.

Cooperative adhesion and friction of compliant nanohairs.

The adhesion and friction behavior of soft materials, including compliant brushes and hairs, depends on the temporal and spatial evolution of the interfaces in contact. For compliant nanofibrous materials, the actual contact area individual fibers make with surfaces depends on the preload applied upon contact. Using in situ microscopy observations of preloaded nanotube hairs, we show how nanotubes make cooperative contact with a surface by buckling and conforming to the surface topography. The overall adhesion of compliant nanohairs increases with increasing preload as nanotubes deform and continuously add new side-wall contacts with the surface. Electrical resistance measurements indicate significant hysteresis in the relative contact area. Contact area increases with preload (or stress) and decreases suddenly during unloading, consistent with strong adhesion observed for these complaint nanohairs.