Interfacial Interactions between Nanoplastics and Biological Systems: toward an Atomic and Molecular Understanding of Plastics-Driven Biological Dyshomeostasis.

Micro- and nano-plastics (NPs) are found in human milk, blood, tissues, and organs and associate with aberrant health outcomes including inflammation, genotoxicity, developmental disorders, onset of chronic diseases, and autoimmune disorders. Yet, interfacial interactions between plastics and biomolecular systems remain underexplored. Here, we have examined experimentally, in vitro, in vivo, and by computation, the impact of polystyrene (PS) NPs on a host of biomolecular systems and assemblies. Our results reveal that PS NPs essentially abolished the helix-content of the milk protein ß-lactoglobulin (BLG) in a dose-dependent manner. Helix loss is corelated with the near stoichiometric formation of ß-sheet elements in the protein. Structural alterations in BLG are also likely responsible for the nanoparticle-dependent attrition in binding affinity and weaker on-rate constant of retinol, its physiological ligand (compromising its nutritional role). PS NP-driven helix-to-sheet conversion was also observed in the amyloid-forming trajectory of hen egg-white lysozyme (accelerated fibril formation and reduced helical content in fibrils). Caenorhabditis elegans exposed to PS NPs exhibited a decrease in the fluorescence of green fluorescent protein-tagged dopaminergic neurons and locomotory deficits (akin to the neurotoxin paraquat exposure). Finally, in silico analyses revealed that the most favorable PS/BLG docking score and binding energies corresponded to a pose near the hydrophobic ligand binding pocket (calyx) of the protein where the NP fragment was found to make nonpolar contacts with side-chain residues via the hydrophobic effect and van der Waals forces, compromising side chain/retinol contacts. Binding energetics indicate that PS/BLG interactions destabilize the binding of retinol to the protein and can potentially displace retinol from the calyx region of BLG, thereby impairing its biological function. Collectively, the experimental and high-resolution in silico data provide new insights into the mechanism(s) by which PS NPs corrupt the bimolecular structure and function, induce amyloidosis and onset neuronal injury, and drive aberrant physiological and behavioral outcomes.

Genetically encoded discovery of perfluoroaryl macrocycles that bind to albumin and exhibit extended circulation in vivo.

Peptide-based therapeutics have gained attention as promising therapeutic modalities, however, their prevalent drawback is poor circulation half-life in vivo. In this paper, we report the selection of albumin-binding macrocyclic peptides from genetically encoded libraries of peptides modified by perfluoroaryl-cysteine SNAr chemistry, with decafluoro-diphenylsulfone (DFS). Testing of the binding of the selected peptides to albumin identified SICRFFC as the lead sequence. We replaced DFS with isosteric pentafluorophenyl sulfide (PFS) and the PFS-SICRFFCGG exhibited KD = 4-6 µM towards human serum albumin. When injected in mice, the concentration of the PFS-SICRFFCGG in plasma was indistinguishable from the reference peptide, SA-21. More importantly, a conjugate of PFS-SICRFFCGG and peptide apelin-17 analogue (N3-PEG6-NMe17A2) showed retention in circulation similar to SA-21; in contrast, apelin-17 analogue was cleared from the circulation after 2 min. The PFS-SICRFFC is the smallest known peptide macrocycle with a significant affinity for human albumin and substantial in vivo circulation half-life. It is a productive starting point for future development of compact macrocycles with extended half-life in vivo.

BinderSpace: A package for sequence space analyses for datasets of affinity-selected oligonucleotides and peptide-based molecules.

Discovery of target-binding molecules, such as aptamers and peptides, is usually performed with the use of high-throughput experimental screening methods. These methods typically generate large datasets of sequences of target-binding molecules, which can be enriched with high affinity binders. However, the identification of the highest affinity binders from these large datasets often requires additional low-throughput experiments or other approaches. Bioinformatics-based analyses could be helpful to better understand these large datasets and identify the parts of the sequence space enriched with high affinity binders. BinderSpace is an open-source Python package that performs motif analysis, sequence space visualization, clustering analyses, and sequence extraction from clusters of interest. The motif analysis, resulting in text-based and visual output of motifs, can also provide heat maps of previously measured user-defined functional properties for all the motif-containing molecules. Users can also run principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE) analyses on whole datasets and on motif-related subsets of the data. Functionally important sequences can also be highlighted in the resulting PCA and t-SNE maps. If points (sequences) in two-dimensional maps in PCA or t-SNE space form clusters, users can perform clustering analyses on their data, and extract sequences from clusters of interest. We demonstrate the use of BinderSpace on a dataset of oligonucleotides binding to single-wall carbon nanotubes in the presence and absence of a bioanalyte, and on a dataset of cyclic peptidomimetics binding to bovine carbonic anhydrase protein. BinderSpace is openly accessible to the public via the GitHub website: https://github.com/vukoviclab/BinderSpace.

Characterizing the Interactions of Cell-Membrane-Disrupting Peptides with Lipid-Functionalized Single-Walled Carbon Nanotubes.

Lipid-functionalized single-walled carbon nanotubes (SWNTs) have garnered significant interest for their potential use in a wide range of biomedical applications. In this work, we used molecular dynamics simulations to study the equilibrium properties of SWNTs surrounded by the phosphatidylcholine (POPC) corona phase and their interactions with three cell membrane disruptor peptides: colistin, TAT peptide, and crotamine-derived peptide. Our results show that SWNTs favor asymmetrical positioning within the POPC corona, so that one side of the SWNT, covered by the thinnest part of the corona, comes in contact with charged and polar functional groups of POPC and water. We also observed that colistin and TAT insert deeply into the POPC corona, while crotamine-derived peptide only adsorbs to the corona surface. In separate simulations, we show that three examined peptides exhibit similar insertion and adsorption behaviors when interacting with POPC bilayers, confirming that peptide-induced perturbations to POPC in conjugates and bilayers are similar in nature and magnitude. Furthermore, we observed correlations between the peptide-induced structural perturbations and the near-infrared emission of the lipid-functionalized SWNTs, which suggest that the optical signal of the conjugates transduces the morphological changes in the lipid corona. Overall, our findings indicate that lipid-functionalized SWNTs could serve as simplified cell membrane model systems for prescreening of new antimicrobial compounds that disrupt cell membranes.

Computational Modeling of the Virucidal Inhibition Mechanism for Broad-Spectrum Antiviral Nanoparticles and HPV16 Capsid Segments.

Solid core nanoparticles (NPs) coated with sulfonated ligands that mimic heparan sulfate proteoglycans (HSPGs) can exhibit virucidal activity against many viruses that utilize HSPG interactions with host cells for the initial stages of infection. How the interactions of these NPs with large capsid segments of HSPG-interacting viruses lead to their virucidal activity has been unclear. Here, we describe the interactions between sulfonated NPs and segments of the human papilloma virus type 16 (HPV16) capsids using atomistic molecular dynamics simulations. The simulations demonstrate that the NPs primarily bind at the interfaces of two HPV16 capsid proteins. After equilibration, the distances and angles between capsid proteins in the capsid segments are larger for the systems in which the NPs bind at the interfaces of capsid proteins. Over time, NP binding can lead to breaking of contacts between two neighboring proteins. The revealed mechanism of NPs targeting the interfaces between pairs of capsid proteins can be utilized for designing new generations of virucidal materials and contribute to the development of new broad-spectrum non-toxic virucidal materials.

Surfactant-Controlled Mobility of Oil Droplets in Mineral Nanopores.

Polymer flooding is one of the widely used enhanced oil recovery (EOR) methods. However, tuning polymer properties to achieve improved performance in porous mineral rocks of diverse oil reservoirs remains one of the challenges of EOR processes. Here, we use molecular dynamics (MD) simulations to examine decane/water mixtures with surfactant additives in calcite and kaolinite mineral nanopores and characterize surfactant properties associated with increased fluid mobility and improved wettability in planar and constricted nanopore geometries. Cetyltrimethylammonium chloride (CTAC) and sodium dodecyl sulfate (SDS) surfactants are found to modulate the contact angles of decane droplets and reduce the decane density on mineral surfaces. CTAC can enhance and unblock the flow of decane droplets through narrowing nanopores with constricted geometries while aiding in decane droplet shape deformation, whereas SDS leads to decane droplets stalling in front of constrictions in nanopores. We hypothesize that the inability of the cationic CTAC headgroup to form hydrogen bonds is one of the key factors leading to enhanced CTAC-coated decane flow through constricted nanopores. The obtained molecular view of equilibrium and dynamic properties of complex fluids typical of oil reservoirs can provide a basis for the future design of new molecules for EOR processes.

Collecting duct carcinoma and endemic nephropathy - case reportS and literature review.

Although collecting duct carcinoma is a subtype of renal cell carcinoma, several studies implicate association with urothelial carcinoma. The coexistence of collecting duct carcinoma and another renal neoplasm is rare. Endemic nephropathy is a renal disease causing chronic renal failure. It is highly associated with urothelial neoplasm and occurs in endemic villages in Bosnia, Croatia, Bulgaria, Romania and Serbia. Recent studies have confirmed the important role of exposure to aristolochic acid as an etiologic factor. We present three cases of collecting duct carcinoma with literature overview. In one case, we describe collecting duct carcinoma with metachronous urothelial carcinoma of the pyelon and urinary bladder in an endemic nephropathy patient. To our knowledge, this is the first case report describing this coexistence. Certain similarities between collecting duct carcinoma and urothelial carcinoma were found, e.g., higher incidence in female compared to male, higher mean age, and multifocal and multicentric occurrence of the tumor. Our observations support the hypothesis that collecting duct carcinoma and urothelial carcinoma could be connected.

Transition of Metastable Cross-α Crystals into Cross-ß Fibrils by ß-Turn Flipping.

The ensemble of native, folded state was once considered to represent the global energy minimum of a given protein sequence. More recently, the discovery of the cross-ß amyloid state revealed that deeper energy minima exist, often associated with pathogenic, fibrillar deposits, when the concentration of proteins reaches a critical value. Fortunately, a sizable energy barrier impedes the conversion from native to pathogenic states. However, little is known about the structure of the related transition state. In addition, there are indications of polymorphism in the amyloidogenic process. Here, we report the first evidence of the conversion of metastable cross-α-helical crystals to thermodynamically stable cross-ß-sheet-like fibrils by a de novo designed heptapeptide. Furthermore, for the first time, we demonstrate at atomic resolution that the flip of a peptide plane from a type I to a type II' turn facilitates transformation to cross-ß structure and assembly of a dry steric zipper. This study establishes the potential of a peptide turn, a common protein secondary structure, to serve as a principal gatekeeper between a native metastable folded state and the amyloid state.

High-resolution imaging of cellular dopamine efflux using a fluorescent nanosensor array.

Intercellular communication via chemical signaling proceeds with both spatial and temporal components, but analytical tools, such as microfabricated electrodes, have been limited to just a few probes per cell. In this work, we use a nonphotobleaching fluorescent nanosensor array based on single-walled carbon nanotubes (SWCNTs) rendered selective to dopamine to study its release from PC12 neuroprogenitor cells at a resolution exceeding 20,000 sensors per cell. This allows the spatial and temporal dynamics of dopamine release, following K+ stimulation, to be measured at exceedingly high resolution. We observe localized, unlabeled release sites of dopamine spanning 100 ms to seconds that correlate with protrusions but not predominately the positive curvature associated with the tips of cellular protrusions as intuitively expected. The results illustrate how directionality of chemical signaling is shaped by membrane morphology, and highlight the advantages of nanosensor arrays that can provide high spatial and temporal resolution of chemical signaling.

Chronic dietary exposure to aristolochic acid and kidney function in native farmers from a Croatian endemic area and Bosnian immigrants.

BACKGROUND AND OBJECTIVES: Improvements in agricultural practices in Croatia have reduced exposure to consumption of aristolochic acid-contaminated flour and development of endemic (Balkan) nephropathy. Therefore, it was hypothesized that Bosnian immigrants who settled in an endemic area in Croatia 15-30 years ago would be at lower risk of developing endemic nephropathy because of reduced exposure to aristolochic acid. To test this hypothesis, past and present exposure to aristolochic acid, proximal tubule damage as a hallmark of endemic nephropathy, and prevalence of CKD in Bosnian immigrants were analyzed. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In this cross-sectional observational study from 2005 to 2010, 2161 farmers were divided into groups: indigenous inhabitants from endemic nephropathy and nonendemic nephropathy villages and Bosnian immigrants; α-1 microglobulin-to-creatinine ratio >31.5 mg/g and eGFR<60 ml/min per 1.73 m(2) were considered to be abnormal. RESULTS: CKD and proximal tubule damage prevalence was significantly lower in Bosnian immigrants than inhabitants of endemic nephropathy villages (6.9% versus 16.6%; P<0.001; 1.3% versus 7.3%; P=0.003, respectively); 20 years ago, Bosnian immigrants observed fewer Aristolochia clematitis in cultivated fields (41.9% versus 67.8%) and fewer seeds among wheat seeds (6.1% versus 35.6%) and ate more purchased than homemade bread compared with Croatian farmers from endemic nephropathy villages (38.5% versus 14.8%, P<0.001). Both Croatian farmers and Bosnian immigrants observe significantly fewer Aristolochia plants growing in their fields compared with 15-30 years ago. Prior aristolochic acid exposure was associated with proximal tubule damage (odds ratio, 1.64; 95% confidence interval, 1.04 to 2.58; P=0.02), whereas present exposure was not (odds ratio, 1.31; 95% confidence interval, 0.75 to 2.30; P=0.33). Furthermore, immigrant status was an independent negative predictor of proximal tubule damage (odds ratio, 0.40; 95% confidence interval, 0.19 to 0.86; P=0.02). CONCLUSIONS: Bosnian immigrants and autochthonous Croats residing in endemic areas are exposed significantly less to ingestion of aristolochic acid than in the past. The prevalence of endemic nephropathy and its associated urothelial cancers is predicted to decrease over time.

Endemic (Balkan) nephropathy is aristolochic acid nephropathy.

Endemic nephropathy is a syndrome that comprises two entities: chronic interstitial nephropathy and urothelial cell cancers predominantly of the upper urinary tract. The etiological agent for the disease is aristolochic acid, a compound found in the plants of Aristolochia spp. The development of urothelial cancers is characterized by the formation of aristolactam DNA adducts leading to mutations, predominantly A: T->T: A transversions. In order to comprehensively understand the gene regulation programs in upper urothelial cancers we performed integrated miRNA and mRNA expression profiling of paired tumours and unaffected urothelium samples. The obtained data will help us to understand the carcinogenesis caused by aristolochic acid and might be the source for the design of a diagnostic biomarker.

Consensus statement on screening, diagnosis, classification and treatment of endemic (Balkan) nephropathy.

Currently used diagnostic criteria in different endemic (Balkan) nephropathy (EN) centers involve different combinations of parameters, various cut-off values and many of them are not in agreement with proposed international guidelines. Leaders of EN centers began to address these problems at scientific meetings, and this paper is the outgrowth of those discussions. The main aim is to provide recommendations for clinical work on current knowledge and expertise. This document is developed for use by general physicians, nephrologists, urologist, public health experts and epidemiologist, and it is hoped that it will be adopted by responsible institutions in countries harboring EN. National medical providers should cover costs of screening and diagnostic procedures and treatment of EN patients with or without upper urothelial cancers.

Solubilization of therapeutic agents in micellar nanomedicines.

We use atomistic molecular dynamics simulations to reveal the binding mechanisms of therapeutic agents in PEG-ylated micellar nanocarriers (SSM). In our experiments, SSM in buffer solutions can solubilize either ≈11 small bexarotene molecules or ≈6 (2 in low ionic strength buffer) human vasoactive intestinal peptide (VIP) molecules. Free energy calculations reveal that molecules of the poorly water-soluble drug bexarotene can reside at the micellar ionic interface of the PEG corona, with their polar ends pointing out. Alternatively, they can reside in the alkane core center, where several bexarotene molecules can self-stabilize by forming a cluster held together by a network of hydrogen bonds. We also show that highly charged molecules, such as VIP, can be stabilized at the SSM ionic interface by Coulombic coupling between their positively charged residues and the negatively charged phosphate headgroups of the lipids. The obtained results illustrate that atomistic simulations can reveal drug solubilization character in nanocarriers and be used in efficient optimization of novel nanomedicines.

Aristolactam-DNA adducts are a biomarker of environmental exposure to aristolochic acid.

Endemic (Balkan) nephropathy is a chronic tubulointerstitial disease frequently accompanied by urothelial cell carcinomas of the upper urinary tract. This disorder has recently been linked to exposure to aristolochic acid, a powerful nephrotoxin and human carcinogen. Following metabolic activation, aristolochic acid reacts with genomic DNA to form aristolactam-DNA adducts that generate a unique TP53 mutational spectrum in the urothelium. The aristolactam-DNA adducts are concentrated in the renal cortex, thus serving as biomarkers of internal exposure to aristolochic acid. Here, we present molecular epidemiologic evidence relating carcinomas of the upper urinary tract to dietary exposure to aristolochic acid. DNA was extracted from the renal cortex and urothelial tumor tissue of 67 patients that underwent nephroureterectomy for carcinomas of the upper urinary tract and resided in regions of known endemic nephropathy. Ten patients from nonendemic regions with carcinomas of the upper urinary tract served as controls. Aristolactam-DNA adducts were quantified by (32)P-postlabeling, the adduct was confirmed by mass spectrometry, and TP53 mutations in tumor tissues were identified by chip sequencing. Adducts were present in 70% of the endemic cohort and in 94% of patients with specific A:T to T:A mutations in TP53. In contrast, neither aristolactam-DNA adducts nor specific mutations were detected in tissues of patients residing in nonendemic regions. Thus, in genetically susceptible individuals, dietary exposure to aristolochic acid is causally related to endemic nephropathy and carcinomas of the upper urinary tract.