Dynamics of high-speed electrical tree growth in electron-irradiated polymethyl methacrylate.

Dielectric materials are foundational to our modern-day communications, defense, and commerce needs. Although dielectric breakdown is a primary cause of failure of these systems, we do not fully understand this process. We analyzed the dielectric breakdown channel propagation dynamics of two distinct types of electrical trees. One type of these electrical trees has not been formally classified. We observed the propagation speed of this electrical tree type to exceed 10 million meters per second. These results identify substantial gaps in the understanding of dielectric breakdown, and filling these gaps is paramount to the design and engineering of dielectric materials that are less susceptible to electrostatic discharge failure.

High-throughput screening for cell binding and repulsion peptides on multifunctionalized surfaces.

The adhesion of cells to the extracellular matrix engages cell surface receptors such as integrins, proteoglycans and other types of cell adhesion molecules such as CD44. To closely examine the determinants of cell adhesion, herein we describe the generation of high-density peptide arrays and test the growth of cells on these multifunctionalized surfaces. The peptide library used consists of over 11,000 different sequences, either random or derived from existing proteins. By applying this screen to SW620 mCherry colorectal cancer cells, we select for peptides with both maximum cell adhesion and maximum cell repulsion. All of these extreme properties are based on unique combinations of amino acids. Here, we identify peptides with maximum cell repulsion on secreted frizzled- and Dickkopf-related proteins. Peptides with strong cell repulsion are found at the poles of the TNF-alpha homotrimer. The formation of cellular patterns on alternating highly repulsive and adhesive peptides are examined. Our screen allows the identification of peptides suitable for biomedical and tissue engineering applications.

Designing cytochrome P450 enzymes for use in cancer gene therapy.

Cancer is a significant global socioeconomic burden, as millions of new cases and deaths occur annually. In 2020, almost 10 million cancer deaths were recorded worldwide. Advancements in cancer gene therapy have revolutionized the landscape of cancer treatment. An approach with promising potential for cancer gene therapy is introducing genes to cancer cells that encode for chemotherapy prodrug metabolizing enzymes, such as Cytochrome P450 (CYP) enzymes, which can contribute to the effective elimination of cancer cells. This can be achieved through gene-directed enzyme prodrug therapy (GDEPT). CYP enzymes can be genetically engineered to improve anticancer prodrug conversion to its active metabolites and to minimize chemotherapy side effects by reducing the prodrug dosage. Rational design, directed evolution, and phylogenetic methods are some approaches to developing tailored CYP enzymes for cancer therapy. Here, we provide a compilation of genetic modifications performed on CYP enzymes aiming to build highly efficient therapeutic genes capable of bio-activating different chemotherapeutic prodrugs. Additionally, this review summarizes promising preclinical and clinical trials highlighting engineered CYP enzymes' potential in GDEPT. Finally, the challenges, limitations, and future directions of using CYP enzymes for GDEPT in cancer gene therapy are discussed.

An alcove at the acetyl-CoA synthase nickel active site is required for productive substrate CO binding and anaerobic carbon fixation.

One of the seven natural CO2 fixation pathways, the anaerobic Wood-Ljungdahl pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here, we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO2 fixation and autotrophic growth by the WLP. In vitro spectroscopy, kinetics, binding, and in vivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate that the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Nip site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter "active site". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all "gas handling" metalloenzymes.

Individual Optimization of Reproductive Investment and the Cost of Incubation in a Wild Songbird.

AbstractDespite avid interest in life history trade-offs and the costs of reproduction, evidence that increased parental allocation reduces subsequent breeding productivity is mixed. This uncertainty may be attributable to environmental heterogeneity in space and time, necessitating experiments across a range of ecological contexts. Over three breeding seasons, we cross-fostered clutches between nests to manipulate incubation duration in a wild population of Carolina wrens, a species in which only females incubate, to test for a cost of incubation on current and future reproduction. Prolonged incubation affected maternal productivity in a manner dependent on the current environment and initial investment in eggs, suggesting that incubation is optimized according to other components of reproduction and individual quality. Effects of incubation duration on foster nestling condition varied between years, being costly in one, beneficial in another, and neutral in the third. The proportion of young fledged, females' probability of breeding again within seasons, and subsequent clutch sizes all declined with increasing incubation effort-effects that became more pronounced as seasons progressed. Therefore, costs of incubation were almost entirely dependent on maternal quality and environmental variation, illustrating the importance of conducting experiments across a range of environmental settings for understanding the costs of reproduction and evolution of life histories.

Peptide toxins that target vertebrate voltage-gated sodium channels underly the painful stings of harvester ants.

Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain, and other neurotoxic symptoms in vertebrates. Here, we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.

Imaging high jitter, very fast phenomena: A remedy for shutter lag.

Dielectric breakdown is an example of a natural phenomenon that occurs on very short time scales, making it incredibly difficult to capture optical images of the process. Event initiation jitter is one of the primary challenges, as even a microsecond of jitter time can cause the imaging attempt to fail. Initial attempts to capture images of dielectric breakdown using a gigahertz frame rate camera and an exploding bridge wire initiation were stymied by high initiation jitter. Subsequently, a novel optical delay line apparatus was developed in order to effectively circumvent the jitter and reliably image dielectric breakdown. The design and performance of the optical delay line apparatus are presented. The optical delay line increased the image capture success rate from 25% to 94% while also permitting enhanced temporal resolution and has application in imaging other high-jitter, extremely fast phenomena.

Label-Free Imaging of Solid-Phase Peptide Synthesis Products and Their Modifications Tethered in Microspots Using Time-of-Flight Secondary Ion Mass Spectrometry.

Time-of-flight secondary ion mass spectrometry is used to analyze solid-phase synthesis products in 60 µm spots of high-density peptide arrays. As a result, a table of specific fragments for the individual detection of amino acids and their side chain protecting groups within peptides is compiled. The specific signal of an amino acid increases linearly as its number increases in the immobilized peptide. Mass-to-charge ratio values are identified that can distinguish between isomers such as leucine and isoleucine. The accessibility of the N-terminus of polyalanine will be studied depending on the number of its residues. The examples provided in the study demonstrate the significant potential of time-of-flight secondary ion mass spectrometry for high-throughput screening of functional groups and their accessibility to chemical reactions occurring simultaneously in hundreds of thousands of microreactors on a single microscope slide.

Iron Oxide Nanoparticles: Green Synthesis and Their Antimicrobial Activity.

The rise of antimicrobial resistance caused by inappropriate use of these agents in various settings has become a global health threat. Nanotechnology offers the potential for the synthesis of nanoparticles (NPs) with antimicrobial activity, such as iron oxide nanoparticles (IONPs). The use of IONPs is a promising way to overcome antimicrobial resistance or pathogenicity because of their ability to interact with several biological molecules and to inhibit microbial growth. In this review, we outline the pivotal findings over the past decade concerning methods for the green synthesis of IONPs using bacteria, fungi, plants, and organic waste. Subsequently, we delve into the primary challenges encountered in green synthesis utilizing diverse organisms and organic materials. Furthermore, we compile the most common methods employed for the characterization of these IONPs. To conclude, we highlight the applications of these IONPs as promising antibacterial, antifungal, antiparasitic, and antiviral agents.

Ant venoms contain vertebrate-selective pain-causing sodium channel toxins.

Stings of certain ant species (Hymenoptera: Formicidae) can cause intense, long-lasting nociception. Here we show that the major contributors to these symptoms are venom peptides that modulate the activity of voltage-gated sodium (NaV) channels, reducing their voltage threshold for activation and inhibiting channel inactivation. These peptide toxins are likely vertebrate-selective, consistent with a primarily defensive function. They emerged early in the Formicidae lineage and may have been a pivotal factor in the expansion of ants.

The association of spirometric small airways obstruction with respiratory symptoms, cardiometabolic diseases, and quality of life: results from the Burden of Obstructive Lung Disease (BOLD) study.

BACKGROUND: Spirometric small airways obstruction (SAO) is common in the general population. Whether spirometric SAO is associated with respiratory symptoms, cardiometabolic diseases, and quality of life (QoL) is unknown. METHODS: Using data from the Burden of Obstructive Lung Disease study (N = 21,594), we defined spirometric SAO as the mean forced expiratory flow rate between 25 and 75% of the FVC (FEF25-75) less than the lower limit of normal (LLN) or the forced expiratory volume in 3 s to FVC ratio (FEV3/FVC) less than the LLN. We analysed data on respiratory symptoms, cardiometabolic diseases, and QoL collected using standardised questionnaires. We assessed the associations with spirometric SAO using multivariable regression models, and pooled site estimates using random effects meta-analysis. We conducted identical analyses for isolated spirometric SAO (i.e. with FEV1/FVC ≥ LLN). RESULTS: Almost a fifth of the participants had spirometric SAO (19% for FEF25-75; 17% for FEV3/FVC). Using FEF25-75, spirometric SAO was associated with dyspnoea (OR = 2.16, 95% CI 1.77-2.70), chronic cough (OR = 2.56, 95% CI 2.08-3.15), chronic phlegm (OR = 2.29, 95% CI 1.77-4.05), wheeze (OR = 2.87, 95% CI 2.50-3.40) and cardiovascular disease (OR = 1.30, 95% CI 1.11-1.52), but not hypertension or diabetes. Spirometric SAO was associated with worse physical and mental QoL. These associations were similar for FEV3/FVC. Isolated spirometric SAO (10% for FEF25-75; 6% for FEV3/FVC), was also associated with respiratory symptoms and cardiovascular disease. CONCLUSION: Spirometric SAO is associated with respiratory symptoms, cardiovascular disease, and QoL. Consideration should be given to the measurement of FEF25-75 and FEV3/FVC, in addition to traditional spirometry parameters.

Screening for Primordial RNA-Peptide Interactions Using High-Density Peptide Arrays.

RNA-peptide interactions are an important factor in the origin of the modern mechanism of translation and the genetic code. Despite great progress in the bioinformatics of RNA-peptide interactions due to the rapid growth in the number of known RNA-protein complexes, there is no comprehensive experimental method to take into account the influence of individual amino acids on non-covalent RNA-peptide bonds. First, we designed the combinatorial libraries of primordial peptides according to the combinatorial fusion rules based on Watson-Crick mutations. Next, we used high-density peptide arrays to investigate the interaction of primordial peptides with their cognate homo-oligonucleotides. We calculated the interaction scores of individual peptide fragments and evaluated the influence of the peptide length and its composition on the strength of RNA binding. The analysis shows that the amino acids phenylalanine, tyrosine, and proline contribute significantly to the strong binding between peptides and homo-oligonucleotides, while the sum charge of the peptide does not have a significant effect. We discuss the physicochemical implications of the combinatorial fusion cascade, a hypothesis that follows from the amino acid partition used in the work.

Switchable Explosives: Performance Tuning of Fluid-Activated High Explosive Architectures.

We present our discovery of switchable high explosives (HEs) as a new class of energetic material that cannot detonate unless filled with a fluid. The performance of fluid-filled additive-manufactured HE lattices is herein evaluated by analysis of detonation velocity and Gurney energy. The Gurney energy of the unfilled lattice was 98% lower than that of the equivalent water-filled lattice and changing the fluid mechanical properties allowed tuning of the Gurney energy and detonation velocity by 8.5% and 13.4%, respectively. These results provide, for the first time since the development of HEs, a method to completely remove the hazard of unplanned detonations during storage and transport.

Intra-colony venom diversity contributes to maintaining eusociality in a cooperatively breeding ant.

BACKGROUND: Eusociality is widely considered to evolve through kin selection, where the reproductive success of an individual's close relative is favored at the expense of its own. High genetic relatedness is thus considered a prerequisite for eusociality. While ants are textbook examples of eusocial animals, not all ants form colonies of closely related individuals. One such example is the ectatommine ant Rhytidoponera metallica, which predominantly forms queen-less colonies that have such a low intra-colony relatedness that they have been proposed to represent a transient, unstable form of eusociality. However, R. metallica is among the most abundant and widespread ants on the Australian continent. This apparent contradiction provides an example of how inclusive fitness may not by itself explain the maintenance of eusociality and raises the question of what other selective advantages maintain the eusocial lifestyle of this species. RESULTS: We provide a comprehensive portrait of the venom of R. metallica and show that the colony-wide venom consists of an exceptionally high diversity of functionally distinct toxins for an ant. These toxins have evolved under strong positive selection, which is normally expected to reduce genetic variance. Yet, R. metallica exhibits remarkable intra-colony variation, with workers sharing only a relatively small proportion of toxins in their venoms. This variation is not due to the presence of chemical castes, but has a genetic foundation that is at least in part explained by toxin allelic diversity. CONCLUSIONS: Taken together, our results suggest that the toxin diversity contained in R. metallica colonies may be maintained by a form of group selection that selects for colonies that can exploit more resources and defend against a wider range of predators. We propose that increased intra-colony genetic variance resulting from low kinship may itself provide a selective advantage in the form of an expanded pharmacological venom repertoire. These findings provide an example of how group selection on adaptive phenotypes may contribute to maintaining eusociality where a prerequisite for kin selection is diminished.

Structures of the R-type human Cav2.3 channel reveal conformational crosstalk of the intracellular segments.

The R-type voltage-gated Ca2+ (Cav) channels Cav2.3, widely expressed in neuronal and neuroendocrine cells, represent potential drug targets for pain, seizures, epilepsy, and Parkinson's disease. Despite their physiological importance, there have lacked selective small-molecule inhibitors targeting these channels. High-resolution structures may aid rational drug design. Here, we report the cryo-EM structure of human Cav2.3 in complex with α2δ-1 and ß3 subunits at an overall resolution of 3.1 Å. The structure is nearly identical to that of Cav2.2, with VSDII in the down state and the other three VSDs up. A phosphatidylinositol 4,5-bisphosphate (PIP2) molecule binds to the interface of VSDII and the tightly closed pore domain. We also determined the cryo-EM structure of a Cav2.3 mutant in which a Cav2-unique cytosolic helix in repeat II (designated the CH2II helix) is deleted. This mutant, named ΔCH2, still reserves a down VSDII, but PIP2 is invisible and the juxtamembrane region on the cytosolic side is barely discernible. Our structural and electrophysiological characterizations of the wild type and ΔCH2 Cav2.3 show that the CH2II helix stabilizes the inactivated conformation of the channel by tightening the cytosolic juxtamembrane segments, while CH2II helix is not necessary for locking the down state of VSDII.

Development and Validation of a Novel Skills Training Model for PCNL, an ESUT project.

BACKGROUND AND AIM: The aim of this study is to validate a totally non biologic training model that combines the use of ultrasound and X ray to train Urologists and Residents in Urology in PerCutaneous NephroLithotripsy (PCNL). METHODS: The training pathway was divided into three modules: Module 1, related to the acquisition of basic UltraSound (US) skill on the kidney; Module 2, consisting of correct Nephrostomy placement; and Module 3, in which a complete PCNL was performed on the model. Trainees practiced on the model first on Module 1, than in 2 and in 3. The pathway was repeated at least three times. Afterward, they rated the performance of the model and the improvement gained using a global rating score questionnaire. RESULTS: A total of 150 Urologists took part in this study. Questionnaire outcomes on this training model showed a mean 4.21 (range 1-5) of positive outcome overall. Individual constructive validity showed statistical significance between the first and the last time that trainees practiced on the PCNL model among the three different modules. Statistical significance was also found between residents, fellows and experts scores. Trainees increased their skills during the training modules. CONCLUSION: This PCNL training model allows for the acquisition of technical knowledge and skills as US basic skill, Nephrostomy placement and entire PCNL procedure. Its structured use could allow a better and safer training pathway to increase the skill in performing a PCNL.

Resemblance-Ranking Peptide Library to Screen for Binders to Antibodies on a Peptidomic Scale.

A novel resemblance-ranking peptide library with 160,000 10-meric peptides was designed to search for selective binders to antibodies. The resemblance-ranking principle enabled the selection of sequences that are most similar to the human peptidome. The library was synthesized with ultra-high-density peptide arrays. As proof of principle, screens for selective binders were performed for the therapeutic anti-CD20 antibody rituximab. Several features in the amino acid composition of antibody-binding peptides were identified. The selective affinity of rituximab increased with an increase in the number of hydrophobic amino acids in a peptide, mainly tryptophan and phenylalanine, while a total charge of the peptide remained relatively small. Peptides with a higher affinity exhibited a lower sum helix propensity. For the 30 strongest peptide binders, a substitutional analysis was performed to determine dissociation constants and the invariant amino acids for binding to rituximab. The strongest selective peptides had a dissociation constant in the hundreds of the nano-molar range. The substitutional analysis revealed a specific hydrophobic epitope for rituximab. To show that conformational binders can, in principle, be detected in array format, cyclic peptide substitutions that are similar to the target of rituximab were investigated. Since the specific binders selected via the resemblance-ranking peptide library were based on the hydrophobic interactions that are widespread in the world of biomolecules, the library can be used to screen for potential linear epitopes that may provide information about the cross-reactivity of antibodies.

Absolute Proteome Quantification in the Gas-Fermenting Acetogen Clostridium autoethanogenum.

Microbes that can recycle one-carbon (C1) greenhouse gases into fuels and chemicals are vital for the biosustainability of future industries. Acetogens are the most efficient known microbes for fixing carbon oxides CO2 and CO. Understanding proteome allocation is important for metabolic engineering as it dictates metabolic fitness. Here, we use absolute proteomics to quantify intracellular concentrations for >1,000 proteins in the model acetogen Clostridium autoethanogenum grown autotrophically on three gas mixtures (CO, CO+H2, or CO+CO2+H2). We detect the prioritization of proteome allocation for C1 fixation and the significant expression of proteins involved in the production of acetate and ethanol as well as proteins with unclear functions. The data also revealed which isoenzymes are likely relevant in vivo for CO oxidation, H2 metabolism, and ethanol production. The integration of proteomic and metabolic flux data demonstrated that enzymes catalyze high fluxes with high concentrations and high in vivo catalytic rates. We show that flux adjustments were dominantly accompanied by changing enzyme catalytic rates rather than concentrations. IMPORTANCE Acetogen bacteria are important for maintaining biosustainability as they can recycle gaseous C1 waste feedstocks (e.g., industrial waste gases and syngas from gasified biomass or municipal solid waste) into fuels and chemicals. Notably, the acetogen Clostridium autoethanogenum is being used as a cell factory in industrial-scale gas fermentation. Here, we perform reliable absolute proteome quantification for the first time in an acetogen. This is important as our work advances both rational metabolic engineering of acetogen cell factories and accurate in silico reconstruction of their phenotypes. Furthermore, this absolute proteomics data set serves as a reference toward a better systems-level understanding of the ancient metabolism of acetogens.

A peptide toxin in ant venom mimics vertebrate EGF-like hormones to cause long-lasting hypersensitivity in mammals.

Venoms are excellent model systems for studying evolutionary processes associated with predator-prey interactions. Here, we present the discovery of a peptide toxin, MIITX2-Mg1a, which is a major component of the venom of the Australian giant red bull ant Myrmecia gulosa and has evolved to mimic, both structurally and functionally, vertebrate epidermal growth factor (EGF) peptide hormones. We show that Mg1a is a potent agonist of the mammalian EGF receptor ErbB1, and that intraplantar injection in mice causes long-lasting hypersensitivity of the injected paw. These data reveal a previously undescribed venom mode of action, highlight a role for ErbB receptors in mammalian pain signaling, and provide an example of molecular mimicry driven by defensive selection pressure.

Design Strategy for Nanostructured Arrays of Metallodielectric Cuboids to Systematically Tune the Optical Response and Eliminate Spurious Bulk Effects in Plasmonic Biosensors.

Plasmonic biosensors are a powerful tool for studying molecule adsorption label-free and with high sensitivity. Here, we present a systematic study on the optical properties of strictly regular nanostructures composed of metallodielectric cuboids with the aim to deliberately tune their optical response and improve their biosensing performance. In addition, the patterns were tested for their potential to eliminate spurious effects from sensor response, caused by refractive index changes in the bulk solution. Shifts in the plasmonic spectrum are exclusively caused by the adsorbing molecules. For this purpose, nanopatterns of interconnected and separated cubes with dimensions ranging from 150 to 600 nm have been fabricated from poly(methyl methacrylate) using electron-beam lithography followed by metallization with gold. It is shown that a small lateral pattern size, a high aspect ratio, and short connection lengths are favorable to generate extinction spectra with well-separated and pronounced peaks. Furthermore, for selected nanostructures, we have been able to identify reflection angles for which the influence of the bulk refractive index on the position of the plasmonic peaks is negligible. It is shown that sensor operation under these angles enables monitoring of in situ biomolecule adsorption with high sensitivity providing a promising tool for high-throughput applications.