Exploring Top-Down Mass Spectrometric Approaches To Probe Forest Cobra (Naja melanoleuca) Venom Proteoforms.

Snake venoms are comprised of bioactive proteins and peptides that facilitate severe snakebite envenomation symptoms. A comprehensive understanding of venom compositions and the subtle heterogeneity therein is important. While bottom-up proteomics has been the well-established approach to catalogue venom compositions, top-down proteomics has emerged as a complementary strategy to characterize venom heterogeneity at the intact protein level. However, top-down proteomics has not been as widely implemented in the snake venom field as bottom-up proteomics, with various emerging top-down methods yet to be developed for venom systems. Here, we have explored three main top-down mass spectrometry methodologies in a proof-of-concept study to characterize selected three-finger toxin and phospholipase A2 proteoforms from the forest cobra (Naja melanoleuca) venom. We demonstrated the utility of a data-independent acquisition mode "MSE" for untargeted fragmentation on a chromatographic time scale and its improvement in protein sequence coverage compared to conventional targeted tandem mass spectrometry analysis. We also showed that protein identification can be further improved using a hybrid fragmentation approach, combining electron-capture dissociation and collision-induced dissociation. Lastly, we reported the promising application of multifunctional cyclic ion mobility separation and post-ion mobility fragmentation on snake venom proteins for the first time.

Toxic effects of the emerging Alexandrium pseudogonyaulax (Dinophyceae) on multiple trophic levels of the pelagic food web.

The dinoflagellate Alexandrium pseudogonyaulax, a harmful algal bloom species, is currently appearing in increasing frequency and abundance across Northern European waters, displacing other Alexandrium species. This mixotrophic alga produces goniodomins (GDs) and bioactive extracellular substances (BECs) that may pose a threat to coastal ecosystems and other marine resources. This study demonstrated the adverse effects of A. pseudogonyaulax on four marine trophic levels, including microalgae (Rhodomonas salina), microzooplankton (Polykrikos kofoidii) and mesozooplankton (Acartia tonsa), as well as fish gill cells (RTgill-W1, Oncorhynchus mykiss), ultimately leading to enhanced mortality and cell lysis. Furthermore, cell-free supernatants collected from A. pseudogonyaulax cultures caused complete loss of metabolic activity in the RTgill-W1 cell line, indicating ichthyotoxic properties, while all tested GDs were much less toxic. In addition, cell-free supernatants of A. pseudogonyaulax led to cell lysis of R. salina, while all tested GDs were non-lytic. Finally, reduced egg hatching rates of A. tonsa eggs exposed to cell-free supernatants of A. pseudogonyaulax and impaired mobility of P. kofoidii and A. tonsa exposed to A. pseudogonyaulax were also observed. Altogether, bioassay results suggest that the toxicity of A. pseudogonyaulax is mainly driven by BECs and not by GDs, although further research into factors modulating the lytic activity of Alexandrium spp. are needed.

The strength of anticipated distractors shapes EEG alpha and theta oscillations in a Working Memory task.

Working Memory (WM) requires maintenance of task-relevant information and suppression of task-irrelevant/distracting information. Alpha and theta oscillations have been extensively investigated in relation to WM. However, studies that examine both theta and alpha bands in relation to distractors, encompassing not only power modulation but also connectivity modulation, remain scarce. Here, we depicted, at the EEG-source level, the increase in power and connectivity in theta and alpha bands induced by strong relative to weak distractors during a visual Sternberg-like WM task involving the encoding of verbal items. During retention, a strong or weak distractor was presented, predictable in time and nature. Analysis focused on the encoding and retention phases before distractor presentation. Theta and alpha power were computed in cortical regions of interest, and connectivity networks estimated via spectral Granger causality and synthetized using in/out degree indices. The following modulations were observed for strong vs. weak distractors. In theta band during encoding, the power in frontal regions increased, together with frontal-to-frontal and bottom-up occipital-to-temporal-to-frontal connectivity; even during retention, bottom-up theta connectivity increased. In alpha band during retention, but not during encoding, the power in temporal-occipital regions increased, together with top-down frontal-to-occipital and temporal-to-occipital connectivity. From our results, we postulate a proactive cooperation between theta and alpha mechanisms: the first would mediate enhancement of target representation both during encoding and retention, and the second would mediate increased inhibition of sensory areas during retention only, to suppress the processing of imminent distractor without interfering with the processing of ongoing target stimulus during encoding.

Top-down mass spectrometry analysis of capsid proteins of recombinant adeno-associated virus using multiple ion activations and proton transfer charge reduction.

Adeno-associated viruses (AAVs) are common vectors for emerging gene therapies due to their lack of pathogenicity in humans. Here, we present our investigation of the viral proteins (i.e., VP1, VP2, and VP3) of the capsid of AAVs via top-down mass spectrometry (MS). These proteins, ranging from 59 to 81 kDa, were chromatographically separated using hydrophilic interaction liquid chromatography and characterized in the gas-phase by high-resolution Orbitrap Fourier transform MS. Complementary ion dissociation methods were utilized to improve the overall sequence coverage. By reducing the overlap of product ion signals via proton transfer charge reduction on the Orbitrap Ascend BioPharma Tribrid mass spectrometer, the sequence coverage of each VP was significantly increased, reaching up to ∼40% in the case of VP3. These results showcase the improvements in the sequencing of proteins >30 kDa that can be achieved by manipulating product ions via gas-phase reactions to obtain easy-to-interpret fragmentation mass spectra.

Quantum Dots Functionalized Polymeric Nanoparticles as Cancer Theranostics: An Advanced Nanomedicine Strategy.

BACKGROUND: Cancer is a life-threatening disease prevalent worldwide, but its proper treatment has not yet been developed. Conventional therapies, like chemotherapy, sur-gery, and radiation, have shown relapse and drug resistance. Nanomedicine comprising cancer theranostics based on imaging probes functionalized with polymeric nanoconjugates is acquir-ing importance due to its targeting capability, biodegradability, biocompatibility, capacity for drug loading, and long blood circulation time. The application of synthetic polymers contain-ing anti-cancer agents and functionalizing their surface amenities with diagnostic probes offer a nano-combinatorial model in cancer theranostics. OBJECTIVE: This study aimed to highlight the recent advancements in quantum dots-functionalized nanoconjugates and substantial progress in advanced polymeric nanomaterials in cancer theragnostics. METHODS: This review details the synthetic methods for fabricating Quantum Dots (QDs) and QDs-functionalized polymeric nanoparticles, such as the hydrothermal method, solvothermal technique, atomic layer desorption, electrochemical method, microwave, and ultrasonic method. RESULTS: Conjugating nanoparticles with photo-emitting quantum dots has shown efficacy for real-time monitoring and treating multi-drug-resistant cancer. CONCLUSION: Quantum dots are used in phototherapy, bioimaging, and medication delivery for cancer therapy. Real-time monitoring of therapy is possible and multiple models of hybridized quantum dots may be created to treat cancer. This review has discovered that numerous at-tempts have been made to conjugate carbon and graphene-based quantum dots with various biomolecules.

Macrophage membrane coated discoidal polymeric particles for evading phagocytosis.

The purpose of this study was to investigate the potential of discoidal polymeric particles (DPPs) coated with macrophage membranes as a novel drug delivery system. The study aimed to determine whether these coated particles could reduce phagocytosis, and target specific organs, thereby enhancing drug delivery efficacy. In this study, discoidal polymeric particles (DPPs) were synthesized by a top-down fabrication method serving as the core drug delivery platform. The method involved the fusion of macrophage cell membrane vesicles with DPPs, resulting in macrophage membrane coated DPPs. This process aimed to translocate membrane proteins from macrophages onto the DPPs, rendering them structurally and functionally like host cells. The results of this study showed that macrophage membrane coated DPPs exhibited a threefold reduction in phagocytosis compared to bare DPPs. This reduction in phagocytosis indicated the potential of these coated DPPs to evade immune clearance. Time-lapse microscopy further illustrated the distinct interactions of macrophage membrane coated DPPs with immune cells. Biodistribution studies revealed that these coated particles displayed preferential accumulation in the lungs at early time points, followed by sustained accumulation in the liver. In conclusion, this study demonstrated that macrophage membrane coated DPPs represent a unique and promising strategy for drug delivery. These particles can mimic cell surfaces, reduce phagocytosis, and target specific organs. This opens exciting avenues for improving drug delivery efficacy in diverse therapeutic contexts. These findings advance our understanding of nanomedicine's potential in personalized therapies and targeted drug delivery strategies. Supplementary Information: The online version contains supplementary material available at 10.1007/s13534-024-00396-x.

Cortical Layer-Dependent Signaling in Cognition: Three Computational Modes of the Canonical Circuit.

The cerebral cortex performs computations via numerous six-layer modules. The operational dynamics of these modules were studied primarily in early sensory cortices using bottom-up computation for response selectivity as a model, which has been recently revolutionized by genetic approaches in mice. However, cognitive processes such as recall and imagery require top-down generative computation. The question of whether the layered module operates similarly in top-down generative processing as in bottom-up sensory processing has become testable by advances in the layer identification of recorded neurons in behaving monkeys. This review examines recent advances in laminar signaling in these two computations, using predictive coding computation as a common reference, and shows that each of these computations recruits distinct laminar circuits, particularly in layer 5, depending on the cognitive demands. These findings highlight many open questions, including how different interareal feedback pathways, originating from and terminating at different layers, convey distinct functional signals.

Heatwave-induced functional shifts in zooplankton communities result in weaker top-down control on phytoplankton.

Freshwater ecosystems are increasingly affected by rising annual mean temperatures and heatwaves. While heatwaves are expected to have more immediate effects than mean temperature increases on local communities, comparative experimental studies are largely lacking. We conducted a 1-month mesocosm experiment to test the effect of different warming treatments, constantly raised temperatures (+3°C) and recurring heatwaves (+6°C), on plankton communities. We specifically tested how shifts in zooplankton trait composition and functional groups are reflected in ecosystem function (top-down control on primary producers). We found that heatwaves had a stronger and more immediate effect on zooplankton trait composition (specifically on body length and body mass) and functional groups. Heatwaves led to the decrease of small-bodied grazers (i.e., Rotifera) and the dominance of larger omnivorous Copepoda, and these shifts resulted in weaker top-down control, leading to elevated phytoplankton biomass. Altogether, our results highlight the importance of the indirect effects of heatwaves via inducing shifts in zooplankton functional groups and trait composition, which may lead to algal blooms.

Impacts of coyote colonization on coastal mammalian predators.

Extreme ecosystem modification by humans has caused drastic reductions in populations and ranges of top mammalian predators, while simultaneously allowing synanthropic mesopredator species to expand. These conditions often result in inflated local densities of highly adaptable mesopredators that disrupt trophic dynamics and place unsustainable predation pressure on native prey populations. Colonization of a dominant predator may lead to top-down control of mesopredators and restore trophic balance. Coyotes are a novel colonizer of some coastal barrier islands of eastern North America, offering an opportunity to test how the addition of an apex predator impacts an established guild of mesopredators. To assess their trophic impact, we conducted 75,576 camera trapping hours over an 18-month study period, capturing > 1.5 million images across 108 coastal camera sites. Using two-species occupancy and habitat use models, we found sizeable effects of coyote habitat use on that of red foxes and free-ranging domestic cats, suggesting that coyotes function as apex predators in barrier island ecosystems. In fact, the only factor that determined the spatial pattern of highly ubiquitous red foxes was the sympatric habitat use of the largest carnivore in the food web-coyotes. That 'novel' apex predators can become established in coastal food webs illustrates the highly dynamic nature of conservation challenges for habitats and species at the edge of the sea.

Rethinking Nanoparticle Synthesis: A Sustainable Approach vs. Traditional Methods.

This review portrays a comparison between green protocols and conventional nanoparticle (NP) synthesis strategies, highlighting each method's advantages and limitations. Various top-down and bottom-up methods in NP synthesis are described in detail. The green chemistry principles are emphasized for designing safe processes for nanomaterial synthesis. Among the green biogenic sources plant extracts, vitamins, enzymes, polysaccharides, fungi (Molds and mushrooms), bacteria, yeast, algae, and lichens are discussed. Limitations in the reproducibility of green protocols in terms of availability of raw material, variation in synthetic protocol, and selection of material due to geographical differences are elaborated. Finally, a conclusion is drawn utilizing green chemical principles, & a circular economy strategy to minimize waste generation, offering a promising framework for the synthesis of NPs emphasizing sustainability.

Scent-driven Selective Attention on Gambling Outcome: Implications for Responsible Gambling.

Many casinos diffuse a pleasant ambient scent into their facilities as a customer experience management practice, but the ethics of this scenting process is questionable. Although the effect of a pleasant scent on cognitive, emotional, and behavioral responses has been well-documented, its effect on attention during gambling has yet to be explored. Grounded in the tenets of the top-down control of attention and cross-modal correspondence between vision and olfaction, we conduct two eye-tracking experiments that involve different electronic casino games including video slots and live Cussec. The findings consistently show that pleasant ambient scent prolongs attention and induces more frequent attention to the win/loss areas on the video screen. The findings add to the implications related to responsible gambling by inspiring the stakeholders to consider the use of ambient scent in the gambling environment. Theoretically, the findings offer insights into scent as the catalyst that directs attention to goal-related information, while scent and goal do not need to be congruent in traits.

Impact of COVID-19 pandemic on microplastic occurrence in aquatic environments: A three-year study in Taihu Lake Basin, China.

The impact of the Coronavirus Disease 2019 (COVID-19) pandemic on microplastic (MP) occurrence in aquatic environments deserves an in-depth study. In this study, the occurrence of MPs and environmental flux of plastics before (2019) and during (2020 and 2021) the pandemic were comparatively investigated in various aquatic compartments in the Taihu Lake Basin in China. The field-based investigations from 2019 to 2021 for Taihu Lake have shown that, at the onset of the outbreak, the MP abundance declined at a rate of 62.3 %, but gradually recovered to the pre-pandemic level. However, the amount of plastics being released into aquatic environments showed a declining trend in 2020 and 2021 compared to those in 2019, with decrease rates of 13.7 % and 15.8 %, respectively. Characterization analysis of MP particles and source apportionment framework implied that while the contributions of tire abrasion and domestic waste to MP occurrence were depleted owing to the reduction in human activity during the pandemic, weathering and fragmentation of retained plastics contributed to the recovery of stored MPs. This study provides insights into the anthropogenic influences on MP occurrence, and supports policymakers in managing and controlling plastic contamination in large freshwater systems in the "new normal" phase.

Characterization of a Monoclonal Antibody by Native and Denaturing Top-Down Mass Spectrometry.

Established in recent years as an important approach to unraveling the heterogeneity of intact monoclonal antibodies, native mass spectrometry has been rarely utilized for sequencing these complex biomolecules via tandem mass spectrometry. Typically, top-down mass spectrometry has been performed starting from highly charged precursor ions obtained via electrospray ionization under denaturing conditions (i.e., in the presence of organic solvents and acidic pH). Here we systematically benchmark four distinct ion dissociation methods─namely, higher-energy collisional dissociation, electron transfer dissociation, electron transfer dissociation/higher-energy collisional dissociation, and 213 nm ultraviolet photodissociation─in their capability to characterize a therapeutic monoclonal antibody, trastuzumab, starting from denatured and native-like precursor ions. Interestingly, native top-down mass spectrometry results in higher sequence coverage than the experiments carried out under denaturing conditions, with the exception of ultraviolet photodissociation. Globally, electron transfer dissociation followed by collision-based activation of product ions generates the largest number of backbone cleavages in disulfide protected regions, including the complementarity determining regions, regardless of electrospray ionization conditions. Overall, these findings suggest that native mass spectrometry can certainly be used for the gas-phase sequencing of whole monoclonal antibodies, although the dissociation of denatured precursor ions still returns a few backbone cleavages not identified in native experiments. Finally, a comparison of the fragmentation maps obtained under denaturing and native conditions strongly points toward disulfide bonds as the primary reason behind the largely overlapping dissociation patterns.

Bioinspired Molecular Scalpel for Two-Dimensional Metal-Organic Nanosheet: Design Strategies and Recent Progress.

Ultrathin two-dimensional (2D) metal-organic nanosheets (MONs) have attracted continued attention in the field of advanced functional materials. Their nanoscale thickness, high surface-to-volume ratio, and abundant accessible active sites, are superior advantages compared with their 3D bulk counterparts. Bioinspired molecular scalpel strategy is a promising method for the creation of 2D MONs, and may solve the current shortcomings of MONs synthesis. This review aims to provide a state-of-the-art overview of molecular scalpel strategies and share the results of current development to provide a better solution for MONs synthesis. Different types of molecular scalpel strategies have been systematically summarized. Both mechanisms, advantages and limitations of multiform molecular scalpel strategies have been discussed. Besides, the challenges to be overcome and the question to be solved are also introduced.

An OpenSim thoracolumbar spine model applying a bottom-up modelling approach is similar to a top-down approach.

The kinetic demands of the spine can be assessed using a top-down (TD) or bottom-up (BU) approach, which start calculations from the either the hands or from the feet, respectively. Biomechanists have traditionally favored a BU approach, though existing modeling approaches encourage a TD approach. Regardless of the approach the demands should be similar, provided the external forces and linked segment parameters are equivalently measured and modeled. Demonstrating a level of agreement between the two approaches can help evaluate a model. Further, having both approaches can be advantageous when data is inaccurate or unavailable for one. The purpose of this study was to compare the internal moments and forces at multiple lumbar and thoracic intervertebral joint (IVJ) levels during lifting tasks from an established OpenSim thoracolumbar spine model that applies a TD approach and a similar model modified to adopt a BU approach. Kinematics and external forces were recorded from twelve participants during sagittal and lateral lifts of different lifting speeds and crate masses. For both approaches IVJ kinetics were estimated using a standard OpenSim modeling pipeline. The BU and TD approach IVJ joint moments generally agreed both temporally (R2 = .94 ± .17) and in magnitude (RMSE=6.2 ± 3.5 Nm) of the primary planes of movement. There were however some temporal fit exceptions for off axes moments with low magnitudes (i.e., < 10 Nm). Bland-Altman plots also indicated acceptable agreement for IVJ peak forces (BU-TD difference of 12 ± 111 and 8 ± 31 N in compression and resultant shear, respectfully). These results support the application of the BU approach and the assigned linked segment parameters of the model. The new BU model is available on the SimTK site (https://simtk.org/projects/spine_ribcage).

Shape Dependent Therapeutic Potential of Nanoparticulate System: Advance Approach for Drug Delivery.

Drug delivery systems rely heavily on nanoparticles because they provide a targeted and monitored release of pharmaceuticals that maximize therapeutic efficacy and minimize side effects. To maximize drug internalization, this review focuses on comprehending the interactions between biological systems and nanoparticles. The way that nanoparticles behave during cellular uptake, distribution, and retention in the body is determined by their shape. Different forms, such as mesoporous silica nanoparticles, micelles, and nanorods, each have special properties that influence how well drugs are delivered to cells and internalized. To achieve the desired particle morphology, shape-controlled nanoparticle synthesis strategies take into account variables like pH, temperatures, and reaction time. Top-down techniques entail dissolving bulk materials to produce nanoparticles, whereas bottom-up techniques enable nanostructures to self-assemble. Comprehending the interactions at the bio-nano interface is essential to surmounting biological barriers and enhancing the therapeutic efficacy of nanotechnology in drug delivery systems. In general, drug internalization and distribution are greatly influenced by the shape of nanoparticles, which presents an opportunity for tailored and efficient treatment plans in a range of medical applications.

The interaction of top-down and bottom-up attention in visual working memory.

Understanding the interplay between top-down and bottom-up attention in visual working memory (VWM) is crucial, although the specific challenges arising from this interaction remain ambiguous. In this study, we address this complexity by examining how cue informativeness and probe status of the salient items influence this interaction. Through three experiments, we manipulated top-down attention by varying probe frequencies using pre-cues and bottom-up attention by varying the visual salience of memory items. Experiment 1 explored cue informativeness at 100% and 50%, while Experiments 2 and 3 maintained cue informativeness at 80% and 50%. Additionally, Experiment 1 tested a few of the salient items, Experiment 2 excluded them, and Experiment 3 tested half of them in each cue condition. Across all experiments, we consistently observed cueing benefits for cue-directed items, albeit with costs to non-cued items. Furthermore, cue informativeness and the probe status of salient items emerged as critical factors influencing the interaction between top-down and bottom-up attention in VWM. These findings underscore the pivotal roles of cue informativeness and salient item relevance in shaping the dynamics of top-down and bottom-up attention within VWM.

Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins.

Within the intricate landscape of the proteome, approximately 30% of all proteins bind metal ions. This repertoire is even larger when considering all the different forms of a protein, known as proteoforms. Here, we propose the term "metalloforms" to refer to different structural or functional variations of a protein resulting from the binding of various hetero- or homogeneous metal ions. Using human Cu(I)/Zn(II)-metallothionein-3 as a representative model, we developed a chemical proteomics strategy to simultaneously differentiate and map Zn(II) and Cu(I) metal binding sites. In the first labeling step, N-ethylmaleimide reacts with Cysteine (Cys), resulting in the dissociation of all Zn(II) ions while Cu(I) remains bound to the protein. In the second labeling step, iodoacetamide is utilized to label Cu(I)-bound Cys residues. Native mass spectrometry (MS) was used to determine the metal/labeling protein stoichiometries, while bottom-up/top-down MS was used to map the Cys-labeled residues. Next, we used a developed methodology to interrogate an isolated rabbit liver metallothionein fraction containing three metallothionein-2 isoforms and multiple Cd(II)/Zn(II) metalloforms. The approach detailed in this study thus holds the potential to decode the metalloproteoform diversity within other proteins.

Enhanced Declustering Enables Native Top-Down Analysis of Membrane Protein Complexes using Ion-Mobility Time-Aligned Fragmentation.

Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary for understanding the functional roles of these biomolecules. Recent research is expanding the application of native MS towards membrane proteins directly from isolated membrane preparations or from purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; the latter enables therapeutic protein targets to be screened against multiplexed preparations of compound libraries. In both cases, the resulting spectra are increasingly complex to assign/interpret, and the key to these new directions of native MS research is the ability to perform native top-down analysis, which allows unambiguous peak assignment. To achieve this, detergent removal is necessary prior to MS analyzers, which allow selection of specific m/z values, representing the parent ion for downstream activation. Here, we describe a novel, enhanced declustering (ED) device installed into the first pumping region of a cyclic IMS-enabled mass spectrometry platform. The device enables declustering of ions prior to the quadrupole by imparting collisional activation through an oscillating electric field applied between two parallel plates. The positioning of the device enables liberation of membrane protein ions from detergent micelles. Quadrupole selection can now be utilized to isolate protein-ligand complexes, and downstream collision cells enable the dissociation and identification of binding partners. We demonstrate that ion mobility (IM) significantly aids in the assignment of top-down spectra, aligning fragments to their corresponding parent ions by means of IM drift time. Using this approach, we were able to confidently assign and identify a novel hit compound against PfMATE, obtained from multiplexed ligand libraries.

Orbitofrontal cortex modulates auditory cortical sensitivity and sound perception in Mongolian gerbils.

Sensory perception is dynamic, quickly adapting to sudden shifts in environmental or behavioral context. Although decades of work have established that these dynamics are mediated by rapid fluctuations in sensory cortical activity, we have a limited understanding of the brain regions and pathways that orchestrate these changes. Neurons in the orbitofrontal cortex (OFC) encode contextual information, and recent data suggest that some of these signals are transmitted to sensory cortices. Whether and how these signals shape sensory encoding and perceptual sensitivity remain uncertain. Here, we asked whether the OFC mediates context-dependent changes in auditory cortical sensitivity and sound perception by monitoring and manipulating OFC activity in freely moving Mongolian gerbils of both sexes under two behavioral contexts: passive sound exposure and engagement in an amplitude modulation (AM) detection task. We found that the majority of OFC neurons, including the specific subset that innervates the auditory cortex, were strongly modulated by task engagement. Pharmacological inactivation of the OFC prevented rapid context-dependent changes in auditory cortical firing and significantly impaired behavioral AM detection. Our findings suggest that contextual information from the OFC mediates rapid plasticity in the auditory cortex and facilitates the perception of behaviorally relevant sounds.