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At least 1,063 records · Page 59

Dosimetric and biological impact of activity extravasation of radiopharmaceuticals in PET imaging

The increasing use of nuclear medicine and PET imaging has intensified scrutiny of radiotracer extravasation. To our knowledge, this topic is understudied but holds great potential for enhancing our understanding of extravasation in clinical PET imaging. This work aims to (1) quantify the absorbed doses from radiotracer extravasation in PET imaging, both locally at the site of extravasation and with the extravasation location as a source of exposure to bodily organs and (2) assess the biological ramifications within the injection site at the cellular level. A radiation dosimetry simulation was performed using a whole-body 4D Extended Cardiac-Torso (XCAT) phantom embedded in the GATE Monte Carlo platform. A 10-mCi dose of 18 F-FDG was chosen to simulate a typical clinical PET scan scenario, with 10% of the activity extravasated in the antecubital fossa of the right arm of the phantom. The extravasation volume was modeled as a 5.5 mL rectangle in the hypodermal layer of skin. Absorbed dose contributions were calculated for the first two half-lives, assuming biological clearance thereafter. Dose calculations were performed as absorbed doses at the organ and skin levels. Energy deposition was simulated both at the local extravasation site and in multiple organs of interest and converted to absorbed doses based on their respective masses. Each simulation was repeated ten times to estimate Monte Carlo uncertainties. Biological impacts on cells within the extravasated volume were evaluated by randomizing cells and exposing them to a uniform radiation source of 18 F and 68 Ga. Particle types, their energies, and direction cosines were recorded in phase space files using a separate Geant4 simulation to characterize their entry into the nucleus of the cellular volume. Subsequently, the phase space files were imported into the TOPAS-nBio simulation to assess the extent of DNA damage, including double-strand breaks (DSBs) and single-strand breaks (SSBs). Organ-level dosimetric estimations are presented for 18 F and 68 Ga radionuclides in various organs of interest. With 10% extravasation, the hypodermal layer of the skin received the highest absorbed dose of 1.32 ± 0.01 Gy for 18 F and 0.99 ± 0.01 Gy for 68 Ga. The epidermal and dermal layers received absorbed doses of 0.07 ± 0.01 Gy and 0.13 ± 0.01 Gy for 18 F, and 0.14 ± 0.01 Gy and 0.29 ± 0.01 Gy for 68 Ga, respectively. In the extravasated volume, 18 F caused an average absorbed dose per nucleus of 0.17 ± 0.01 Gy, estimated to result in 10.58 ± 0.50 DSBs and 268.11 ± 12.43 SSBs per nucleus. For 68 Ga, the absorbed dose per nucleus was 0.11 ± 0.01 Gy, leading to an estimated 6.49 ± 0.34 DSBs and 161.24 ± 8.12 SSBs per nucleus. Absorbed doses in other organs were on the order of micro-gray (µGy). The likelihood of epidermal erythema resulting from extravasation during PET imaging is low, as the simulated absorbed doses to the epidermis remain below the thresholds that trigger such effects. Moreover, the organ-level absorbed doses were found to be clinically insignificant across various simulated organs. The minimal DNA damage at the extravasation site suggests that long-term harm, such as radiation-induced carcinogenesis, is highly unlikely.

DNA strand breaks↗

Advances in targeted proteomics and applications to biomedical research

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity (Shi et al., Proteomics , 12, 1074–1092, 2012) herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications in human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data‐independent acquisition (DIA) with targeted data extraction on fast scanning high‐resolution accurate‐mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large‐scale quantification of hundreds of target proteins are discussed.

Shi, Tujin↗

Structural characterization and computational analysis of PDZ domains in Monosiga brevicollis

Abstract Identification of the molecular networks that facilitated the evolution of multicellular animals from their unicellular ancestors is a fundamental problem in evolutionary cellular biology. Choanoflagellates are recognized as the closest extant nonmetazoan ancestors to animals. These unicellular eukaryotes can adopt a multicellular‐like “rosette” state. Therefore, they are compelling models for the study of early multicellularity. Comparative studies revealed that a number of putative human orthologs are present in choanoflagellate genomes, suggesting that a subset of these genes were necessary for the emergence of multicellularity. However, previous work is largely based on sequence alignments alone, which does not confirm structural nor functional similarity. Here, we focus on the PDZ domain, a peptide‐binding domain which plays critical roles in myriad cellular signaling networks and which underwent a gene family expansion in metazoan lineages. Using a customized sequence similarity search algorithm, we identified 178 PDZ domains in the Monosiga brevicollis proteome. This includes 11 previously unidentified sequences, which we analyzed using Rosetta and homology modeling. To assess conservation of protein structure, we solved high‐resolution crystal structures of representative M. brevicollis PDZ domains that are homologous to human Dlg1 PDZ2, Dlg1 PDZ3, GIPC, and SHANK1 PDZ domains. To assess functional conservation, we calculated binding affinities for mbGIPC, mbSHANK1, mbSNX27, and mbDLG‐3 PDZ domains from M. brevicollis . Overall, we find that peptide selectivity is generally conserved between these two disparate organisms, with one possible exception, mbDLG‐3. Overall, our results provide novel insight into signaling pathways in a choanoflagellate model of primitive multicellularity.

Gao, Melody↗

Structural and functional validation of a highly specific Smurf2 inhibitor

Smurf1 and Smurf2 are two closely related member of the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase family and play important roles in the regulation of various cellular processes. Both were initially identified to regulate transforming growth factor‐β and bone morphogenetic protein signaling pathways through regulating Smad protein stability and are now implicated in various pathological processes. Generally, E3 ligases, of which over 800 exist in humans, are ideal targets for inhibition as they determine substrate specificity; however, there are few inhibitors with the ability to precisely target a particular E3 ligase of interest. In this work, we explored a panel of ubiquitin variants (UbVs) that were previously identified to bind Smurf1 or Smurf2. In vitro binding and ubiquitination assays identified a highly specific Smurf2 inhibitor, UbV S2.4, which was able to inhibit ligase activity with high potency in the low nanomolar range. Orthologous cellular assays further demonstrated high specificity of UbV S2.4 toward Smurf2 and no cross‐reactivity toward Smurf1. Structural analysis of UbV S2.4 in complex with Smurf2 revealed its mechanism of inhibition was through targeting the E2 binding site. In summary, we investigated several protein‐based inhibitors of Smurf1 and Smurf2 and identified a highly specific Smurf2 inhibitor that disrupts the E2–E3 protein interaction interface.

59 BASIC BIOLOGICAL SCIENCES↗

Cisplatin-Induced Alteration on Membrane Composition of A549 Cells Revealed by ToF-SIMS

Cisplatin has been clinically used for treatment of solid tumors such as non-small-cell lung cancer for decades. However, tumor resistance may be acquired with losing the antitumor activity of cisplatin. As cellular membrane is the first barrier that cisplatin has to overcome before its further action inside the cells, the membrane composition must play a vital role in the cisplatin uptake and excretion which further influences cisplatin sensitivity. In this work, we applied ToF-SIMS surface analysis combined with principle component analysis to distinguish the differences of cell membrane composition between non-small-cell lung cancer cells (A549) and its cisplatin resistant counterpart A549/DDP cells. The decreased phosphatidylcholine content and more abundant cholesterol were observed in the drug resistant cell surfaces, indicating the decreased membrane fluidity of A549/DDP cells. Moreover, we further compared membrane composition of A549 and A549/DDP cells after being treated with different concentrations of cisplatin. A higher composition level of proteins was discovered on all groups of A549/DDP cell membranes. The altered surface chemistry of cellular membranes induced by cisplatin indicate the significance of membrane structures in the drug resistance, which deserves further investigations to this regard.

Zhang, Yanyan↗

Cell-Free Protein Synthesis for High-Throughput Biosynthetic Pathway Prototyping

Biological systems provide a sustainable and complimentary approach to synthesizing useful chemical products. Metabolic engineers seeking to establish economically viable biosynthesis platforms strive to increase product titers, rates, and yields. Despite continued advances in genetic tools and metabolic engineering techniques, cellular workflows remain limited in throughput. It may take months to test dozens of unique pathway designs even in a robust model organism, such as Escherichia coli. In contrast, cell-free protein synthesis enables the rapid generation of enzyme libraries that can be combined to reconstitute metabolic pathways in vitro for biochemical synthesis in days rather than weeks. Cell-free reactions thereby enable comparison of hundreds to thousands of unique combinations of enzyme homologs and concentrations, which can quickly identify the most productive pathway variants to test in vivo or further characterize in vitro. Furthermore, this cell-free pathway prototyping strategy provides a complementary approach to accelerate cellular metabolic engineering efforts toward highly productive strains for metabolite production.

59 BASIC BIOLOGICAL SCIENCES↗

Anchorage of curcumin onto PVP enhances anti-tumor effect of curcumin

Curcumin, a natural polyphenol compound, exhibits wide range of pharmacological activities. However, its poor aqueous solubility, weak stability, and rapid metabolism limit its potential development. In this study, solid dispersion of curcumin with PVP was developed. This study aimed to evaluate the loading capacity, stability, solubility, and anti-tumor activity of PVP-Cur solid dispersion. Scanning electron microscope (SEM), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), and fourier transform infrared (FTIR) spectroscopy were used to investigate physical characterization of PVP-Cur. Loading capacity, stability, and solubility of PVP-Cur were evaluated by spectrophotometer. The biological effects of PVP-Cur on tumor cell proliferation and apoptosis were analyzed using CCK-8 assay and 7-AAD/AnnexinV-APC assay, respectively. PVP-cur-induced activation of caspase-3 and caspase-9 were evaluated by in vitro caspase activity assay and western blotting. It was interesting to note that PVP: curcumin in the ratios of 6:1 or 8:1 exhibited the highest loading capacity. Anchorage of curcumin onto PVP increased its solubility and stability. Compared with curcumin alone, PVP-Cur displayed an increased ability in abrogating tumor cell proliferation and inducing tumor cell apoptosis via mitochondria signaling, which was attributed by the enhanced cellular uptake of PVP-Cur. Anchorage of curcumin onto PVP was an effective way to increase the solubility and stability of curcumin. PVP-Cur increased anti-tumor ability and cellular uptake relative to curcumin, indicating that PVP-Cur is a promising candidate for anti-cancer drug development.

60 APPLIED LIFE SCIENCES↗

Synthesis and in silico and in vitro evaluation of trimethoxy-benzamides designed as anti-prion derivatives

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are neurodegenerative disorders which affect mammals, including the human species, and arise after the conversion of the monomeric cellular prion protein (PrP{sup C}) into the aggregated scrapie form (PrP{sup Sc}). There is no therapy to treat TSEs and the identification of compounds that bind PrP{sup C}, preventing its conversion into PrP{sup Sc}, is a viable therapeutic strategy. We designed and synthesized six novel trimethoxy-benzamide compounds as anti-prion drug candidates. Molecular docking analyses predicted that all the derivatives bind to a hotspot region located in the PrP globular domain with very similar spatial orientation and interaction mode. Although none of the analogs inhibited in vitro-aggregation of recombinant PrP (rPrP) in a cell-free conversion assay, the RT-QuIC, compound 8a accelerated rPrP conversion into PrP{sup Sc}-like species. STD-NMR and ITC analyses indicated that both 8a and 8b bind to rPrP{sup 90–231}. These analogs were toxic to PrP{sup Sc}-infected cell lines, hence we could not assess their anti-prion activity by using this cellular approach, although this toxicity was cell line-dependent. These results point out that the 4-amino-quinoline trimethoxy-benzamide scaffold described herein represents a novel chemical pattern useful as a starting point for future structural optimization in the design of PrP ligands with improved affinity and safety profiles.

60 APPLIED LIFE SCIENCES↗

Enhancing glycan isomer separations with metal ions and positive and negative polarity ion mobility spectrometry-mass spectrometry analyses

Glycomics has become an increasingly important field of research since glycans play critical roles in biology processes ranging from molecular recognition and signaling to cellular communication. Glycans often conjugate with other biomolecules such as proteins and lipids, and alter their properties and functions, so understanding the effect glycans have on cellular systems is essential. However the analysis of glycans is extremely difficult due to their complexity and structural diversity (i.e., the number and identity of monomer units, and configuration of their glycosidic linkages and connectivities). In this work, we coupled ion mobility spectrometry with mass spectrometry (IMS-MS) to characterize glycan standards and biologically important isomers of synthetic αGal-containing O-glycans including glycotopes of the protozoan parasite Trypanosoma cruzi, which is the causative agent of Chagas disease. IMS-MS results showed significant differences for the glycan structural isomers when analyzed in positive and negative polarity and complexed with different metal cations. These results suggest specific metal ions or ion polarities could be used to target and baseline separate glycan isomers of interest with IMS-MS.

Environmental Molecular Sciences Laboratory↗

The origin and evolution of viruses inferred from fold family structure

The canonical frameworks of viral evolution describe viruses as cellular predecessors, reduced forms of cells, or entities that escaped cellular control. The discovery of giant viruses has changed these standard paradigms. Their genetic, proteomic and structural complexities resemble those of cells, prompting a redefinition and reclassification of viruses. In a previous genome-wide analysis of the evolution of structural domains in proteomes, with domains defined at the fold superfamily level, we found the origins of viruses intertwined with those of ancient cells. In this review, we extend these data-driven analyses to the study of fold families confirming the co-evolution of viruses and ancient cells and the genetic ability of viruses to foster molecular innovation. The results support our suggestion that viruses arose by genomic reduction from ancient cells and validate a co-evolutionary ‘symbiogenic’ model of viral origins.

59 BASIC BIOLOGICAL SCIENCES↗

Concurrent metabolism of pentose and hexose sugars by the polyextremophile Alicyclobacillus acidocaldarius

Abstract Alicyclobacillus acidocaldarius is a thermoacidophilic bacterium capable of growth on sugars from plant biomass. Carbon catabolite repression (CCR) allows bacteria to focus cellular resources on a sugar that provides efficient growth, but also allows sequential, rather than simultaneous use when more than one sugar is present. The A. acidocaldarius genome encodes all components of CCR, but transporters encoded are multifacilitator superfamily and ATP-binding cassette-type transporters, uncommon for CCR. Therefore, global transcriptome analysis of A. acidocaldarius grown on xylose or fructose was performed in chemostats, followed by attempted induction of CCR with glucose or arabinose. Alicyclobacillus acidocaldarius grew while simultaneously metabolizing xylose and glucose, xylose and arabinose, and fructose and glucose, indicating that CCR did not control carbon metabolism. Microarrays showed down-regulation of genes during growth on one sugar compared to two, and occurred primarily in genes encoding: (1) regulators; (2) enzymes for cell wall synthesis; and (3) sugar transporters.

59 BASIC BIOLOGICAL SCIENCES↗

In vivo biosensors: mechanisms, development, and applications

Abstract In vivo biosensors can recognize and respond to specific cellular stimuli. In recent years, biosensors have been increasingly used in metabolic engineering and synthetic biology, because they can be implemented in synthetic circuits to control the expression of reporter genes in response to specific cellular stimuli, such as a certain metabolite or a change in pH. There are many types of natural sensing devices, which can be generally divided into two main categories: protein-based and nucleic acid-based. Both can be obtained either by directly mining from natural genetic components or by engineering the existing genetic components for novel specificity or improved characteristics. A wide range of new technologies have enabled rapid engineering and discovery of new biosensors, which are paving the way for a new era of biotechnological progress. Here, we review recent advances in the design, optimization, and applications of in vivo biosensors in the field of metabolic engineering and synthetic biology.

Shi, Shuobo↗

A Reconfigurable In Vitro Model for Studying the Blood–Brain Barrier

Much of what is currently known about the role of the blood–brain barrier (BBB) in regulating the passage of chemicals from the blood stream to the central nervous system (CNS) comes from animal in vivo models (requiring extrapolation to human relevance) and 2D static in vitro systems, which fail to capture the rich cell–cell and cell–matrix interactions of the dynamic 3D in vivo tissue microenvironment. In this work we have developed a BBB platform that allows for a high degree of customization in cellular composition, cellular orientation, and physiologically-relevant fluid dynamics. The system characterized and presented in this study reproduces key characteristics of a BBB model (e.g. tight junctions, efflux pumps) allowing for the formation of a selective and functional barrier. We demonstrate that our in vitro BBB is responsive to both biochemical and mechanical cues. This model further allows for culture of a CNS-like space around the BBB. As a result, the design of this platform is a valuable tool for studying BBB function as well as for screening of novel therapeutics.

42 ENGINEERING↗

Machine learning models for segmentation and classification of cyanobacterial cells

Abstract Timelapse microscopy has recently been employed to study the metabolism and physiology of cyanobacteria at the single-cell level. However, the identification of individual cells in brightfield images remains a significant challenge. Traditional intensity-based segmentation algorithms perform poorly when identifying individual cells in dense colonies due to a lack of contrast between neighboring cells. Here, we describe a newly developed software package called Cypose which uses machine learning (ML) models to solve two specific tasks: segmentation of individual cyanobacterial cells, and classification of cellular phenotypes. The segmentation models are based on the Cellpose framework, while classification is performed using a convolutional neural network named Cyclass. To our knowledge, these are the first developed ML-based models for cyanobacteria segmentation and classification. When compared to other methods, our segmentation models showed improved performance and were able to segment cells with varied morphological phenotypes, as well as differentiate between live and lysed cells. We also found that our models were robust to imaging artifacts, such as dust and cell debris. Additionally, the classification model was able to identify different cellular phenotypes using only images as input. Together, these models improve cell segmentation accuracy and enable high-throughput analysis of dense cyanobacterial colonies and filamentous cyanobacteria.

Huffine, Clair A.↗

Functional Performances of CuZnAl Shape Memory Alloy Open-Cell Foams

Shape memory alloys (SMAs) with cellular structure offer a unique mixture of thermo-physical–mechanical properties. These characteristics can be tuned by changing the pore size and make the shape memory metallic foams very attractive for developing new devices for structural and functional applications. In this work, CuZnAl SMA foams were produced through the liquid infiltration of space holder method. In comparison, a conventional CuZn brass alloy was foamed trough the same method. Functional performances were studied on both bulk and foamed SMA specimens. Calorimetric response shows similar martensitic transformation (MT) below 0 °C. Compressive response of CuZnAl revealed that mechanical behavior is strongly affected by sample morphology and that damping capacity of metallic foam is increased above the MT temperatures. The shape memory effect was detected in the CuZnAl foams. The conventional brass shows a compressive response similar to that of the martensitic CuZnAl, in which plastic deformation accumulation occurs up to the cellular structure densification after few thermal cycles.

36 MATERIALS SCIENCE↗

Laser 3D Printing of Fe-Based Bulk Metallic Glass: Microstructure Evolution and Crack Propagation

This work presents a comprehensive study of microstructure evolution, crack development and hardness performance of Fe-based bulk metallic glass parts processed by laser 3D printing. The combination of a low scan speed and high energy density generates a low temperature gradient, leading to supercooling and the formation of coarse dendrite and cellular crystal microstructure. Columnar dendrites grow in a single direction with large stress, and cracks are easily formed between the dendrites. The stress intensity factor (SIF) caused by the crack surface tension is small, the crack healing effect is weak, and it is difficult to prevent crack propagation. Cellular grain growth is uniform and more easily accommodates the strain, preventing crack initiation and propagation. In contrast, use of a high scan speed with low energy input produces a fine and uniform dendrite or even nanocrystalline, microstructure with microcracks. The SIF caused by the crack surface tension is large, and the crack healing effect is strong and prevents crack initiation and propagation. For low energy density, the hardness of the sample is similar to the hardness of the cast amorphous. This result indicates that the printing sample retained some amorphous characteristics.

36 MATERIALS SCIENCE↗

High Temperature Corrosion of a Pt-30 wt.% Rh Alloy in a Phosphorizing Gas

High temperature corrosion of a Pt-30 wt.% Rh alloy in a phosphorizing gas was isothermally investigated at 1285 K using a gas switching technique. Diffusion of P into the alloy created an outer layer of Pt-rich liquid and blocky (Pt, Rh) 2 P precipitates along with an inner layer of fcc and (Pt, Rh) 2 P plates in a cellular microstructure. Concentration profiles measured by SEM-WDS and EPMA across the layers at room temperature showed that there were three fcc phases: first was a 12 at.% Rh phase in the outer layer; second was a 37 at.% Rh phase in the cellular microstructure; and third was the initial 43 at.% Rh alloy. Also, the EPMA data registered approximately 0.1 at.% P in fcc of these layers. Based on the surrounding binary phase diagrams and the experimental data obtained in this study, a partial Pt-Rh-P phase diagram was constructed. A diffusion path for the corrosion microstructure was drawn on the partial phase diagram to help develop a step by step model for how the microstructure evolved. In conclusion, growth kinetics of the inner layer were used to calculate a P diffusivity of about 10 –12 m 2 /s in the Pt-Rh alloy at 1285 K, suggesting rapid diffusion by either an interstitial or interstitialcy mechanism.

36 MATERIALS SCIENCE↗