Search

Formalin-fixed paraffin-embedded (FFPE) tissue is a ubiquitous and invaluable resource for biomedical research and clinical applications. However, FFPE tissue proteomics is challenging due to protein cross-linking and chemical modification. Laser ablation sampling allows precise removal of material from tissue sections with high spatial control and reproducibility for offline proteomics by liquid chromatography coupled with tandem mass spectrometry. In this work, we used a pulsed mid-infrared laser for microsampling of rat liver tissue for subsequent identification and quantification of proteins. It was found that more proteins were identified by FFPE tissue laser ablation sampling compared to fresh frozen (FF) tissue laser ablation sampling and that more proteins were identified by laser ablation than by manual dissection of FFPE tissue. In contrast to previous studies, no loss of hydrophilic proteins due to residual cross-linking was observed. The efficient capture of proteins by laser ablation microsampling is attributed to efficient laser breakup of the tissue which facilitates downstream processing of the proteins.

This paper presents an innovative approach leveraging Neuronal Manifold Analysis of EEG data to identify specific time intervals for feature extraction, effectively capturing both class-specific and subject-specific characteristics. Different pipelines were constructed and employed to extract distinctive features within these intervals, specifically for motor imagery (MI) tasks. The methodology was validated using the Graz Competition IV datasets 2A (four-class) and 2B (two-class) motor imagery classification, demonstrating an improvement in classification accuracy that surpasses state-of-the-art algorithms designed for MI tasks. A multi-dimensional feature space, constructed using NMA, was built to detect intervals that capture these critical characteristics, which led to significantly enhanced classification accuracy, especially for individuals with initially poor classification performance. These findings highlight the robustness of this method and its potential to improve classification performance in EEG-based MI-BCI systems.

Antioxidant therapies are of interest in the prevention and management of ocular disorders such as cataracts. Although an active area of interest, topical therapy with antioxidants for the treatment of cataracts is complicated by multiple ocular anatomical barriers, product stability, and solubility. Entrapment and delivery of antioxidants with poly(lactic-co-glycolic acid) nanoparticles is a possible solution to these challenges, however, little is known regarding their effects in vitro or in vivo. Our first aim was to investigate the impact of blank and lutein loaded PLGA nanoparticles on viability and development of reactive oxygen species in lens epithelial cells in vitro. Photo-oxidative stress was induced by ultraviolet light exposure with cell viability and reactive oxygen species monitored. Next, an in vivo, selenite model was utilized to induce cataract formation in rodents. Eyes were treated topically with both free lutein and lutein loaded nanoparticles (LNP) at varying concentrations. Eyes were monitored for the development of anterior segment changes and cataract formation. The ability of nanodelivered lutein to reach the anterior segment of the eye was evaluated by liquid chromatography coupled to mass spectrometry of aqueous humor samples and liquid chromatography coupled to tandem mass spectrometry (targeted LC-MS/MS) of lenses. LNP had a minimal impact on the viability of lens epithelial cells during the short exposure timeframe (24 h) and at concentrations < 0.2 μg LNP/μl. A significant reduction in the development of reactive oxygen species was also noted. Animals treated with LNPs at an equivalent lutein concentration of 1,278 μg /mL showed the greatest reduction in cataract scores. Lutein delivery to the anterior segment was confirmed through evaluation of aqueous humor and lens sample evaluation. Topical treatment was not associated with the development of secondary keratitis or anterior uveitis when applied once daily for one week. LNPs may be an effective in the treatment of cataracts., Competing Interests: we wish to confirm that authors Cristina Sabliov, Carlos Astete and Ede Bodoki have intellectual property related to the work presented in the manuscript. The other authors declare no conflict of interest as this does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Carter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In light of recent conflicting reports regarding the hydroformylation catalytic activity derived from cationic Co(II) precatalysts of the form [Co(acac)(bis(phosphine))]BF 4 , the synthetic procedures and characterization of [Co(acac)(dppBz)]BF 4 , 1 , are evaluated. Leveraging calibrated ESI-TOF MS methodologies, substantial quantities of Co(acac) 2 (dppBz), 2 , were observed within samples of 1 . The source of the impurity, 2 , is determined to derive from incomplete protonolysis of the Co(acac) 2 precursor and ligand scrambling occurring during the synthesis of 1 . Revised synthetic procedures using lower temperature conditions and longer reaction times afford analytically pure samples of 1 based on ESI-TOF MS and NMR spectroscopic analysis. Complex 1 is demonstrated to act as a hydroformylation precatalyst for the conversion of 1-hexene to 1-heptanal under relatively mild conditions at 51.7 bar and 140 °C. The presence of impurity 2 is shown to dramatically decrease the catalytic performance derived from 1 .

We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe 2+ utilizing O 2 or H 2 O 2 as oxidant, and the resultant Fe 3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rajapaksha, Yao, Cook, Seibold, Liu, Battaile, Fontenot, Donnarumma, Lovell and Rivera.)

Hematological malignancies are among the top five most frequent forms of cancer in developed countries worldwide. Although the new therapeutic approaches have improved the quality and the life expectancy of patients, the high rate of recurrence and drug resistance are the main issues for counteracting blood disorders. Chemotherapy-resistant leukemic clones activate molecular processes for biological survival, preventing the activation of regulated cell death pathways, leading to cancer progression. In the past decade, leukemia research has predominantly centered around modulating the well-established processes of apoptosis (type I cell death) and autophagy (type II cell death). However, the development of therapy resistance and the adaptive nature of leukemic clones have rendered targeting these cell death pathways ineffective. The identification of novel cell death mechanisms, as categorized by the Nomenclature Committee on Cell Death (NCCD), has provided researchers with new tools to overcome survival mechanisms and activate alternative molecular pathways. This review aims to synthesize information on these recently discovered RCD mechanisms in the major types of leukemia, providing researchers with a comprehensive overview of cell death and its modulation.

Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Sampling thin tissue sections with cellular precision can be accomplished using laser ablation microsampling for mass spectrometry analysis. In this work, the use of a pulsed mid-infrared (IR) laser for selecting small regions of interest (ROI) in tissue sections for offline liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The laser is focused onto the tissue section, which is rastered as the laser is fired. The ablated tissue is captured in a microwell array and processed in situ through reduction, alkylation, and digestion with a low liquid volume workflow. The resulting peptides from areas as small as 0.01 mm 2 containing 5 ng of protein are analyzed for protein identification and quantification using offline LC-MS/MS., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lipid metabolism and glycolysis play crucial roles in the progression and metastasis of cancer, and the use of 3-bromopyruvate (3-BP) as an antiglycolytic agent has shown promise in killing pancreatic cancer cells. However, developing an effective strategy to avoid chemoresistance requires the ability to probe the interaction of cancer drugs with complex tumor-associated microenvironments (TAMs). Unfortunately, no robust and multiplexed molecular imaging technology is currently available to analyze TAMs. In this study, the simultaneous profiling of three protein biomarkers using SERS nanotags and antibody-functionalized nanoparticles in a syngeneic mouse model of pancreatic cancer (PC) is demonstrated. This allows for comprehensive information about biomarkers and TAM alterations before and after treatment. These multimodal imaging techniques include surface-enhanced Raman spectroscopy (SERS), immunohistochemistry (IHC), polarized light microscopy, second harmonic generation (SHG) microscopy, fluorescence lifetime imaging microscopy (FLIM), and untargeted liquid chromatography and mass spectrometry (LC-MS) analysis. The study reveals the efficacy of 3-BP in treating pancreatic cancer and identifies drug treatment-induced lipid species remodeling and associated pathways through bioinformatics analysis., (© 2023 Wiley-VCH GmbH.)

We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe 2+ utilizing H 2 O 2 (not O 2 ) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H 2 O 2 -mediated oxidative stress. In agreement, a P. aeruginosa Δ dps mutant is significantly more susceptible to H 2 O 2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe 2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H 2 O 2 or O 2 but requires divalent cations and 12-mer Pa Dps.

Chromatin structural organization, gene expression and proteostasis are intricately regulated in a wide range of biological processes, both physiological and pathological. Protein acetylation, a major post-translational modification, is tightly involved in interconnected biological networks, modulating the activation of gene transcription and protein action in cells. A very large number of studies describe the pivotal role of the so-called acetylome (accounting for more than 80% of the human proteome) in orchestrating different pathways in response to stimuli and triggering severe diseases, including cancer. NAA60/NatF (N-terminal acetyltransferase F), also named HAT4 (histone acetyltransferase type B protein 4), is a newly discovered acetyltransferase in humans modifying N-termini of transmembrane proteins starting with M-K/M-A/M-V/M-M residues and is also thought to modify lysine residues of histone H4. Because of its enzymatic features and unusual cell localization on the Golgi membrane, NAA60 is an intriguing acetyltransferase that warrants biochemical and clinical investigation. Although it is still poorly studied, this review summarizes current findings concerning the structural hallmarks and biological role of this novel targetable epigenetic enzyme., (© 2022. The Author(s).)

Infrared (IR) laser ablation was used to remove localized tissue regions from which proteins were extracted and processed with a low volume sample preparation workflow for bottom-up proteomics by liquid chromatography tandem mass spectrometry (LC-MS/MS). A polytetrafluoroethylene (PTFE) coated glass slide with 2 mm diameter microwells was used to capture ablated rat brain tissue for in situ protein digestion with submicroliter solution volumes. The resulting peptides were analyzed with LC-MS/MS for protein identification and label-free quantification. The method was used to identify an average of 600, 1350, and 1900 proteins from ablation areas of 0.01, 0.04, and 0.1 mm 2 , respectively, from a 50 μm thick rat brain tissue section. Differential proteomics of 0.01 mm 2 regions captured from cerebral cortex and corpus callosum was accomplished to demonstrate the capabilities of the approach.

Studies of neural population dynamics of cell activity from monkey motor areas during reaching show that it mostly represents the generation and timing of motor behavior. We compared neural dynamics in dorsal premotor cortex (PMd) during the performance of a visuomotor task executed individually or cooperatively and during an observation task. In the visuomotor conditions, monkeys applied isometric forces on a joystick to guide a visual cursor in different directions, either alone or jointly with a conspecific. In the observation condition, they observed the cursor's motion guided by the partner. We found that in PMd neural dynamics were widely shared across action execution and observation, with cursor motion directions more accurately discriminated than task types. This suggests that PMd encodes spatial aspects irrespective of specific behavioral demands. Furthermore, our results suggest that largest components of premotor population dynamics, which have previously been suggested to reflect a transformation from planning to movement execution, may rather reflect higher cognitive-motor processes, such as the covert representation of actions and goals shared across tasks that require movement and those that do not., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Ferritins are iron storage proteins assembled from 24 subunits into a spherical and hollow structure. The genomes of many bacteria harbor genes encoding two types of ferritin-like proteins, the bacterial ferritins (Ftn) and the bacterioferritins (Bfr), which bind heme. The genome of P. aeruginosa PAO1 (like the genomes of many bacteria) contains genes coding for two different types of ferritin-like molecules, ftnA (PA4235) and bfrB (PA3531). The reasons for requiring the presence of two distinct types of iron storage protein in bacterial cells have remained largely unexplained. Attempts to understand this issue in P. aeruginosa through the recombinant expression of the ftnA and bfrB genes in E. coli host cells, coupled to the biochemical and structural characterization of the recombinant 24-mer FtnA and 24-mer BfrB molecules, have shown that each of the recombinant molecules can form an Fe 3+ -mineral core. These observations led to the suggestion that 24-mer FtnA and 24-mer BfrB molecules coexist in P. aeruginosa cells where they share iron storage responsibilities. Herein, we demonstrate that P. aeruginosa utilizes a single heterooligomeric 24-mer Bfr assembled from FtnA and BfrB subunits. The relative content of the FtnA and BfrB subunits in Bfr depends on the O 2 availability during cell culture, such that Bfr isolated from aerobically cultured P. aeruginosa is assembled from a majority of BfrB subunits. In contrast, when the cells are cultured in O 2 -limiting conditions, the proportion of FtnA subunits in the isolated Bfr increases significantly and can become the most abundant subunit. Despite the variability in the subunit composition of Bfr, the 24-mer assembly is consistently arranged from FtnA subunit dimers devoid of heme and BfrB subunit dimers each containing a heme molecule.

Deep-ultraviolet laser ablation with a pulsed 193 nm ArF excimer laser was used to remove localized regions from tissue sections from which proteins were extracted for spatially resolved proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). The ability to capture intact proteins by ablation at 193 nm wavelength was verified by matrix-assisted laser desorption ionization (MALDI) of the protein standard bovine serum albumin (BSA), which showed that BSA was ablated and captured without fragmentation. A Bradford assay of the ablated and captured proteins indicated 90% efficiency for transfer of the intact protein at a laser fluence of 3 kJ/m 2 . Rat brain tissue sections mounted on quartz microscope slides and ablated in transmission mode yielded 2 μg protein per mm 2 as quantified by the Bradford assay. Tissue areas ranging from 0.06 mm 2 to 1 mm 2 were ablated and the ejected material was collected for proteomic analysis. Extracted proteins were digested and the resulting peptides were analyzed by LC-MS/MS. The proteins extracted from the ablated areas were identified and the average number of identified proteins ranged from 85 in the 0.06 mm 2 area to 2400 in the 1 mm 2 area of a 50 μm thick tissue. In comparison to infrared laser ablation of equivalent sampled areas, both the protein mass and number of proteins identified using DUV laser ablation sampling were approximately four times larger., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Stem cell-based therapies carry significant promise for treating human diseases. However, clinical translation of stem cell transplants for effective treatment requires precise non-destructive evaluation of the purity of stem cells with high sensitivity (<0.001% of the number of cells). Here, a novel methodology using hyperspectral imaging (HSI) combined with spectral angle mapping-based machine learning analysis is reported to distinguish differentiating human adipose-derived stem cells (hASCs) from control stem cells. The spectral signature of adipogenesis generated by the HSI method enables identifying differentiated cells at single-cell resolution. The label-free HSI method is compared with the standard techniques such as Oil Red O staining, fluorescence microscopy, and qPCR that are routinely used to evaluate adipogenic differentiation of hASCs. HSI is successfully used to assess the abundance of adipocytes derived from transplanted cells in a transgenic mice model. Further, Raman microscopy and multiphoton-based metabolic imaging is performed to provide complementary information for the functional imaging of the hASCs. Finally, the HSI method is validated using matrix-assisted laser desorption/ionization-mass spectrometry imaging of the stem cells. The study presented here demonstrates that multimodal imaging methods enable label-free identification of stem cell differentiation with high spatial and chemical resolution., Competing Interests: Conflict of Interest The authors declare no conflict of interest.

A full-fledged neural network modeling, based on a Multi-layered Nonlinear Autoregressive Exogenous Neural Network (NARX) architecture, is proposed for quasi-static and dynamic hysteresis loops, one of the most challenging topics for computational magnetism. This modeling approach overcomes drawbacks in attaining better than percent-level accuracy of classical and recent approaches for accelerator magnets, that combine hybridization of standard hysteretic models and neural network architectures. By means of an incremental procedure, different Deep Neural Network Architectures are selected, fine-tuned and tested in order to predict magnetic hysteresis in the context of electromagnets. Tests and results show that the proposed NARX architecture best fits the measured magnetic field behavior of a reference quadrupole at CERN. In particular, the proposed modeling framework leads to a percent error below 0.02% for the magnetic field prediction, thus outperforming state of the art approaches and paving a very promising way for future real time applications.

Heterogeneity in the distribution of nutrients and oxygen gradients during biofilm growth gives rise to changes in phenotype. There has been long term interest in identifying spatial differences during biofilm development including clues that identify chemical heterogeneity. Laser ablation sample transfer (LAST) allows site-specific sampling combined with label free proteomics to distinguish radially and axially resolved proteomes for Pseudomonas aeruginosa biofilms. Specifically, differential protein abundances on oxic vs. anoxic regions of a biofilm were observed by combining LAST with bottom up proteomics. This study reveals a more active metabolism in the anoxic region of the biofilm with respect to the oxic region for this clinical strain of P. aeruginosa, despite this organism being considered an aerobe by nature. Protein abundance data related to cellular acclimations to chemical gradients include identification of glucose catabolizing proteins, high abundance of proteins from arginine and polyamine metabolism, and proteins that could also support virulence and environmental stress mediation in the anoxic region. Finally, the LAST methodology requires only a few mm2 of biofilm area to identify hundreds of proteins., Competing Interests: The authors have declared that no competing interests exist.

In neural networks literature, there is a strong interest in identifying and defining activation functions which can improve neural network performance. In recent years there has been a renovated interest in the scientific community in investigating activation functions which can be trained during the learning process, usually referred to as trainable, learnable or adaptable activation functions. They appear to lead to better network performance. Diverse and heterogeneous models of trainable activation function have been proposed in the literature. In this paper, we present a survey of these models. Starting from a discussion on the use of the term "activation function" in literature, we propose a taxonomy of trainable activation functions, highlight common and distinctive proprieties of recent and past models, and discuss main advantages and limitations of this type of approach. We show that many of the proposed approaches are equivalent to adding neuron layers which use fixed (non-trainable) activation functions and some simple local rule that constrains the corresponding weight layers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Aim: Prostate cancer represents the most common cancer afflicting men. It may be asymptomatic at the early stage. In this paper, we propose a methodology aimed to detect the prostate cancer grade by computing non-invasive shape-based radiomic features directly from magnetic resonance images., Materials and Methods: We use a freely available dataset composed by coronal magnetic resonance images belonging to 112 patients. We represent magnetic resonance slices in terms of formal model, and we exploit model checking to check whether a set of properties (formulated with the support of pathologists and radiologists) is verified on the formal model. Each property is related to a different cancer grade with the aim to cover all the cancer grade groups., Results: An average specificity equal to 0.97 and an average sensitivity equal to 1 have been obtained with our methodology., Conclusion: The experimental analysis demonstrates the effectiveness of radiomics and formal verification for Gleason grade group detection from magnetic resonance.

Flexible navigation relies on a cognitive map of space, thought to be implemented by hippocampal place cells: neurons that exhibit location-specific firing. In connected environments, optimal navigation requires keeping track of one's location and of the available connections between subspaces. We examined whether the dorsal CA1 place cells of rats encode environmental connectivity in four geometrically identical boxes arranged in a square. Rats moved between boxes by pushing saloon-type doors that could be locked in one or both directions. Although rats demonstrated knowledge of environmental connectivity, their place cells did not respond to connectivity changes, nor did they represent doorways differently from other locations. Place cells coded location in a global reference frame, with a different map for each box and minimal repetitive fields despite the repetitive geometry. These results suggest that CA1 place cells provide a spatial map that does not explicitly include connectivity., Competing Interests: Declaration of interests K.J.J. is a non-shareholding director of Axona, Ltd., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

A method for selecting electroencephalographic (EEG) signals in motor imagery-based brain-computer interfaces (MI-BCI) is proposed for enhancing the online interoperability and portability of BCI systems, as well as user comfort. The attempt is also to reduce variability and noise of MI-BCI, which could be affected by a large number of EEG channels. The relation between selected channels and MI-BCI performance is therefore analyzed. The proposed method is able to select acquisition channels common to all subjects, while achieving a performance compatible with the use of all the channels. Results are reported with reference to a standard benchmark dataset, the BCI competition IV dataset 2a. They prove that a performance compatible with the best state-of-the-art approaches can be achieved, while adopting a significantly smaller number of channels, both in two and in four tasks classification. In particular, classification accuracy is about 77-83% in binary classification with down to 6 EEG channels, and above 60% for the four-classes case when 10 channels are employed. This gives a contribution in optimizing the EEG measurement while developing non-invasive and wearable MI-based brain-computer interfaces.

Animal behavior is highly structured. Yet, structured behavioral patterns-or "statistical ethograms"-are not immediately apparent from the full spatiotemporal data that behavioral scientists usually collect. Here, we introduce a framework to quantitatively characterize rodent behavior during spatial (e.g., maze) navigation, in terms of movement building blocks or motor primitives. The hypothesis that we pursue is that rodent behavior is characterized by a small number of motor primitives, which are combined over time to produce open-ended movements. We assume motor primitives to be organized in terms of two sparsity principles: each movement is controlled using a limited subset of motor primitives (sparse superposition) and each primitive is active only for time-limited, time-contiguous portions of movements (sparse activity). We formalize this hypothesis using a sparse dictionary learning method, which we use to extract motor primitives from rodent position and velocity data collected during spatial navigation, and successively to reconstruct past trajectories and predict novel ones. Three main results validate our approach. First, rodent behavioral trajectories are robustly reconstructed from incomplete data, performing better than approaches based on standard dimensionality reduction methods, such as principal component analysis, or single sparsity. Second, the motor primitives extracted during one experimental session generalize and afford the accurate reconstruction of rodent behavior across successive experimental sessions in the same or in modified mazes. Third, in our approach the number of motor primitives associated with each maze correlates with independent measures of maze complexity, hence showing that our formalism is sensitive to essential aspects of task structure. The framework introduced here can be used by behavioral scientists and neuroscientists as an aid for behavioral and neural data analysis. Indeed, the extracted motor primitives enable the quantitative characterization of the complexity and similarity between different mazes and behavioral patterns across multiple trials (i.e., habit formation). We provide example uses of this computational framework, showing how it can be used to identify behavioural effects of maze complexity, analyze stereotyped behavior, classify behavioral choices and predict place and grid cell displacement in novel environments.

Post-traumatic stress disorder (PTSD) is a widespread psychiatric injury that develops serious life-threatening symptoms like substance abuse, severe depression, cognitive impairments, and persistent anxiety. However, the mechanisms of post-traumatic stress injury in brain are poorly understood due to the lack of practical methods to reveal biochemical alterations in various brain regions affected by this type of injury. Here, we introduce a novel method that provides quantitative results from Raman maps in the paraventricular nucleus of the thalamus (PVT) region. By means of this approach, we have shown a lipidome comparison in PVT regions of control and PTSD rat brains. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was also employed for validation of the Raman results. Lipid alterations can reveal invaluable information regarding the PTSD mechanisms in affected regions of brain. We have showed that the concentration of cholesterol, cholesteryl palmitate, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, ganglioside, glyceryl tripalmitate and sulfatide changes in the PVT region of PTSD compared to control rats. A higher concentration of cholesterol suggests a higher level of corticosterone in the brain. Moreover, concentration changes of phospholipids and sphingolipids suggest the alteration of phospholipase A2 (PLA2) which is associated with inflammatory processes in the brain. Our results have broadened the understanding of biomolecular mechanisms for PTSD in the PVT region of the brain. This is the first report regarding the application of Raman spectroscopy for PTSD studies. This method has a wide spectrum of applications and can be applied to various other brain related disorders or other regions of the brain.

The moral decisions we make during this period, such as deciding whether to comply with quarantine rules, have unprecedented societal effects. We simulate the "escape from Milan" that occurred on March 7th-8th 2020, when many travelers moved from a high-risk zone (Milan) to southern regions of Italy (Campania and Lazio) immediately after an imminent lockdown was announced. Our simulations show that fewer than 50 active cases might have caused the sudden spread of the virus observed afterwards in these regions. The surprising influence of the actions of few individuals on societal dynamics challenges our cognitive expectations - as in normal conditions, collective dynamics are rather robust to the decisions of few "cheaters". This situation therefore requires novel educational strategies that increase our awareness and understanding of the unprecedented effects of our individual moral decisions., Competing Interests: The authors declare that they have no conflict of interest., (© 2021 The Authors.)

Iron homeostasis offers a significant bacterial vulnerability because pathogens obtain essential iron from their mammalian hosts, but host-defenses maintain vanishingly low levels of free iron. Although pathogens have evolved mechanisms to procure host-iron, these depend on well-regulated iron homeostasis. To disrupt iron homeostasis, our work has targeted iron mobilization from the iron storage protein bacterioferritin (BfrB) by blocking a required interaction with its cognate ferredoxin partner (Bfd). The blockade of the BfrB-Bfd complex by deletion of the bfd gene (Δ bfd ) causes iron to irreversibly accumulate in BfrB. In this study we used mass spectrometry and NMR spectroscopy to compare the proteomic response and the levels of key intracellular metabolites between wild type (wt) and isogenic Δ bfd P. aeruginosa strains. We find that the irreversible accumulation of unusable iron in BfrB leads to acute intracellular iron limitation, even if the culture media is iron-sufficient. Importantly, the iron limitation and concomitant iron metabolism dysregulation trigger a cascade of events that lead to broader metabolic homeostasis disruption, which includes sulfur limitation, phenazine-mediated oxidative stress, suboptimal amino acid synthesis and altered carbon metabolism.

Protein self-assembly is a ubiquitous phenomenon, traditionally studied for its links to amyloid pathologies, which has also gained attention as its physiological roles and possible biotechnological applications emerged over time. It is also known that varying the conditions to which proteins are exposed can lead to aggregate polymorphism. To understand the factors that trigger aggregation and/or direct it toward specific outcomes, we performed a multifaceted structural characterization of the thermally induced self-assembly process of MNEI, a model protein able to form amyloid aggregates under nondenaturing conditions. MNEI is also known for its extreme sweetness which, combined with a considerable thermal stability, makes the protein a promising alternative sweetener. Fourier-transformed infrared spectroscopy and electron microscopy data showed that the presence of NaCl accelerates the kinetics of fibrillar aggregation, while disfavoring the population of off-pathway states that are instead detected by native gel electrophoresis at low ionic strength. NMR studies revealed how NaCl modulates the self-assembling mechanism of MNEI, switching the process from soluble oligomeric forms to fibrils. Comparative analysis demonstrated that the presence of NaCl induces local differences in the protein dynamics and surface accessibility, without altering the native fold. We identified the regions most affected by the presence of NaCl, which control the aggregation process, and represent hot spots on the protein surface for the rational design of new mutants with controlled aggregation propensity., (© 2019 Federation of European Biochemical Societies.)

We consider the ways humans engage in social epistemic actions, to guide each other's attention, prediction, and learning processes towards salient information, at the timescale of online social interaction and joint action. This parallels the active guidance of other's attention, prediction, and learning processes at the longer timescale of niche construction and cultural practices, as discussed in the target article.

A multimodal workflow for mass spectrometry imaging was developed that combines MALDI imaging with protein identification and quantification by liquid chromatography tandem mass spectrometry (LC-MS/MS). Thin tissue sections were analyzed by MALDI imaging, and the regions of interest (ROI) were identified using a smoothing and edge detection procedure. A midinfrared laser at 3-μm wavelength was used to remove the ROI from the brain tissue section after MALDI mass spectrometry imaging (MALDI MSI). The captured material was processed using a single-pot solid-phase-enhanced sample preparation (SP3) method and analyzed by LC-MS/MS using ion mobility (IM) enhanced data independent acquisition (DIA) to identify and quantify proteins; more than 600 proteins were identified. Using a modified database that included isoform and the post-translational modifications chain, loss of the initial methionine, and acetylation, 14 MALDI MSI peaks were identified. Comparison of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the identified proteins was achieved through an evolutionary relationships classification system., (© 2019 John Wiley & Sons, Ltd.)

Context: In the last 10 years, assisted reproductive technologies (ARTs) have offered infertile couples an opportunity to complete their reproductive project. However, the high failure rate could be explained with the complex human reproduction system. In ART, the decrease of the success is due to the conditions far from the natural ones., Aims: The aim of this study is to evaluate deoxyribonucleic acid (DNA) damage of spermatozoa before and after selection procedures, using a new technique able to quantize sperm DNA damage., Settings and Design: They were involved 43 males domiciled permanently in two areas with different Environmental Impact, HEI (high environmental impact) and LEI (Low environmental impact), they are aged between 24 and 31 years with various degrees of dyspermia., Subjects and Methods: The 43 males were divided into two groups: 21 in Group A (EIL) and 22 in Group B (EIH). The samples must be aliquoted into parts of 0.5 mL: Group (a) Control, no processing; Group (b) Swim-up (SUP) from semen; Group (c) classic SUP; Group (d) density gradient centrifugation (DGC). All samples were subjected to a quantitative dosage of p53 protein, before and after processing., Statistical Analysis Used: For the development of the probability and significance of the data, the Student's t -test was used., Results: From our data, it emerges that Groups D and B provide a superior quality about motility, vitality, and apoptosis indexes compared to other conventional techniques. In Group B, apoptosis is comparable to Group D, but they have slightly lower about motility and vitality. Group C is the one that has lower parameters than the other techniques. Regarding the evaluation of p53 protein, the results are conflicting with the evaluation of apoptosis; in fact, in Group D, the values are significantly higher than the other techniques., Conclusions: Sperm separation is an important moment in ART techniques. From our data, it emerges a greater fragility of DNA in the male spermatozoa who reside permanently in areas with high environmental impact., Competing Interests: The authors declared that they have no conflict of interests. They have not received financial rewards for the execution of the procedure or any sponsorship. The author declares that this study was made in accordance with the Italian principles governing research.[4041]., (Copyright: © 2020 Journal of Human Reproductive Sciences.)

Photooxidation of peptides and proteins by pulsed ultraviolet laser irradiation of an electrospray in the ion source of a mass spectrometer was demonstrated. A 193-nm excimer laser at 1.5-mJ pulse energy was focused with a cylindrical lens at the exit of a nanoelectrospray capillary and ions were sampled into a quadrupole time-of-flight mass spectrometer. A solution containing a peptide or protein and hydrogen peroxide was infused into the spray at a flow rate of 1 μL/min using a syringe pump. The laser creates OH radicals directly in the spray which modify biomolecules within the spray droplet. These results indicate that photochemical oxidation of proteins can be initiated directly within electrospray droplets and detected by mass spectrometry.

Premotor neurons play a fundamental role in transforming physical properties of observed objects, such as size and shape, into motor plans for grasping them, hence contributing to "pragmatic" affordance processing. Premotor neurons can also contribute to "semantic" affordance processing, as they can discharge differently even to pragmatically identical objects depending on their behavioural relevance for the observer (i.e. edible or inedible objects). Here, we compared the response of monkey ventral premotor area F5 neurons tested during pragmatic (PT) or semantic (ST) visuomotor tasks. Object presentation responses in ST showed shorter latency and lower object selectivity than in PT. Furthermore, we found a difference between a transient representation of semantic affordances and a sustained representation of pragmatic affordances at both the single neuron and population level. Indeed, responses in ST returned to baseline within 0.5 s whereas in PT they showed the typical sustained visual-to-motor activity during Go trials. In contrast, during No-go trials, the time course of pragmatic and semantic information processing was similar. These findings suggest that premotor cortex generates different dynamics depending on pragmatic and semantic information provided by the context in which the to-be-grasped object is presented.

RNA was obtained from discrete locations of frozen rat brain tissue sections through infrared (IR) laser ablation using a 3-μm wavelength in transmission geometry. The ablated plume was captured in a microcentrifuge tube containing RNAse-free buffer and processed using a commercial RNA purification kit. RNA transfer efficiency and integrity were evaluated based on automated electrophoresis in microfluidic chips. Reproducible IR-laser ablation of intact RNA was demonstrated with purified RNA at laser fluences of 3-5 kJ/m 2 (72 ± 12% transfer efficiency) and with tissue sections at a laser fluence of 13 kJ/m 2 (79 ± 14% transfer efficiency); laser energies were attenuated ∼20% by the soda-lime glass slides used to support the samples. RNA integrity from tissue ablation was >90% of its original RIN value (∼7) and the purified RNA was sufficiently intact for conversion to cDNA and subsequent qPCR assay., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Structural roles of loop regions are frequently overlooked in proteins. Nevertheless, they may be key players in the definition of protein topology and in the self-assembly processes occurring through domain swapping. We here investigate the effects on structure and stability of replacing the loop connecting the last two β-strands of RNase A with the corresponding region of the more thermostable Onconase. The crystal structure of this chimeric variant (RNaseA-ONC) shows that its terminal loop size better adheres to the topological rules for the design of stabilized proteins, proposed by Baker and coworkers [43]. Indeed, RNaseA-ONC displays a thermal stability close to that of RNase A, despite the lack of Pro at position 114, which, due to its propensity to favor a cis peptide bond, has been identified as an important stabilizing factor of the native protein. Accordingly, RNaseA-ONC is significantly more stable than RNase A variants lacking Pro114; RNaseA-ONC also displays a higher propensity to form oligomers in native conditions when compared to either RNase A or Onconase. This finding demonstrates that modifications of terminal loops should to be carefully controlled in terms of size and sequence to avoid unwanted and/or potentially harmful aggregation processes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this study, we asked whether the event-related potentials associated to cue and target stimuli of a Central Cue Posner Paradigm (CCPP) may encode key parameters of Bayesian inference - prior expectation and surprise - on a trial-by-trial basis. Thirty-two EEG channel were recorded in a sample of 19 young adult subjects while performing a CCPP, in which a cue indicated (validly or invalidly) the position of an incoming auditory target. Three different types of blocks with validities of 50%, 64%, and 88%, respectively, were presented. Estimates of prior expectation and surprise were obtained on a trial-by-trial basis from participants' responses, using a computational model implementing Bayesian learning. These two values were correlated on a trial-by-trial basis with the EEG values in all the electrodes and time bins. Therefore, a Spearman correlation metrics of the relationship between Bayesian parameters and the EEG was obtained. We report that the surprise parameter was able to classify the different validity blocks. Furthermore, the prior expectation parameter showed a significant correlation with the EEG in the cue-target period, in which the Contingent Negative Variation develops. Finally, in the post-target period the surprise parameter showed a significant correlation in the latencies and electrodes in which different event-related potentials are induced. Our results suggest that Bayesian parameters are coded in the EEG signals; and namely, the CNV would be related to prior expectation, while the post-target components P2a, P2, P3a, P3b, and SW would be related to surprise. This study thus provides novel support to the idea that human electrophysiological neural activity may implement a (Bayesian) predictive processing scheme.

A combination of matrix-assisted laser desorption ionization (MALDI) imaging and infrared (IR) laser ablation sampling with offline electrospray ionization mass spectrometry (ESI-MS) was used to determine the distribution of the fungicide imazalil in apples. MALDI images were used to determine the penetration depth of imazalil up to 7 days after its application. IR laser ablation sampling and ESI-MS were used to quantify the rate of penetration of the fungicide, which was determined to be approximately 1 mm per day. Imazalil concentration decreased in the apple skin over the course of the experiment, and after 7 days the fungicide was detected at 0.015 ppm 6 mm inside the apple. Approximately 60% of the pesticide remained in the skin after 7 days. This work demonstrates the utility of MALDI imaging for spatial localization of fungicide in fruit in combination with IR laser ablation and ESI-MS for quantitative analysis.

Introduction and Objectives: Protein p53 role in the spermatogenesis is demonstrated, it guarantees both the appropriate quality and quantity of mature spermatozoa. In this observational study we evaluate the eventual correlation between "corrected" p53 concentration on human spermatozoa DNA and male fertility potential., Materials and Methods: Our work is based on an observational study made of 169 male in a period between March 2012 and February 2017. The entire study group is composed by 208 male partners aged between 26-38 years with ejaculate volume from 0.6 to 5.8 mL and heterogeneous seminal valuation: 86/208 (41,3%) normospermic; 19/208 (9,1%) mild oligospermic; 51/208 (24,5%) moderate oligospermic to; 52/208 (25,1%) with severe oligospermic. The "control" group A includes 39 male partners considered "fertile", because we did the p53 "corrected" test on their spermatozoa after 28 ± 3,5 days from the positives of their partners pregnancy test (betaHCG> 400 m U/m L). The group B, subdivided in B1, B2 and B3, includes 169 male partners for a observational period of 60 months. This partners don't report previous conceptions, they aren't smokers, don't make use neither of alcohol nor drugs and don't present pathologic varicocele studied with ecoColorDoppler. They are all married or stable cohabitants from a period of 27-39 months and report to have frequent sex without protection with their partners. Determination of p53 procedure: To separate the spermatozoa from seminal fluid we utilized the Differex™ kit System and the DNA IQ™ kit (Promega). For the p53 test we used the direct DuoSet IC kit and quantitative (R&D System). The p53 values were corrected in respect to the spermatozoa concentration expressed in ng/millions of spermatozoa., Results: Group A (39 male) presents "correct" p53 values that vary from 0.35 to 3.20 ng/millions of spermatozoa and group B presents values that vary from 0.68 to 14.53. From group B (48 male) in the observational period we have recorded 21 pregnancies with initial "correct" p53 values that vary from a minimum of 0.84 to a maximum of 3.29. In the subgroup B1 we obtained 8 pregnancies from male partners with a "correct" p53 concentration included between 0.84 to 1.34. In the subgroup B2 we obtained 13 pregnancies from male partners with a "correct" p53 concentration included between 1.66 and 3.29. In the subgroup B3 (121 male) there weren't neither pregnancies nor miscarriages and "correct" p53 values were included between 3.58 and 14.53., Conclusion: The results show that the member of the group A with values of 'corrected' p53 between 0.35 and 3.20 were considered "Fertile", although in the observational period 3 miscarriages happened for 3 partners. 36 partners on 39 (92,3%) had a p53 concentration inferior to 1.65, this value were considered as the extreme to identify this group. The member of the group B1 had "corrected" p53 concentration that were included in the group. In the group B2 were observe 13 pregnancies, so its member were considered "potentially fertile" In the group B3 (121 male) weren't observe neither pregnancies nor miscarriages, so its member were considered "potentially infertile". If further studies confirm these data, we will consider the p53 test ELISA inspected in "correct" p53 as a new and accurate marker of the potential of male fertility., Competing Interests: There are no conflicts of interest.

Human communication is a traditional topic of research in many disciplines such as psychology, linguistics and philosophy, all of which mainly focused on language, gestures and deictics. However, these do not constitute the sole channels of communication, especially during online social interaction, where instead an additional critical role may be played by sensorimotor communication (SMC). SMC refers here to (often subtle) communicative signals embedded within pragmatic actions - for example, a soccer player carving his body movements in ways that inform a partner about his intention, or to feint an adversary; or the many ways we offer a glass of wine, rudely or politely. SMC is a natural form of communication that does not require any prior convention or any specific code. It amounts to the continuous and flexible exchange of bodily signals, with or without awareness, to enhance coordination success; and it is versatile, as sensorimotor signals can be embedded within every action. SMC is at the center of recent interest in neuroscience, cognitive psychology, human-robot interaction and experimental semiotics; yet, we still lack a coherent and comprehensive synthesis to account for its multifaceted nature. Some fundamental questions remain open, such as which interactive scenarios promote or do not promote SMC, what aspects of social interaction can be properly called communicative and which ones entail a mere transfer of information, and how many forms of SMC exist and what we know (or still don't know) about them from an empirical viewpoint. The present work brings together all these separate strands of research within a unified overarching, multidisciplinary framework for SMC, which combines evidence from kinematic studies of human-human interaction and computational modeling of social exchanges., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A 193-nm wavelength deep ultraviolet laser was used for ambient laser ablation electrospray ionization mass spectrometry of biological samples. A pulsed ArF excimer laser was used to ablate solid samples, and the resulting plume of the desorbed material merged with charged electrospray droplets to form ions that were detected with a quadrupole time-of-flight mass spectrometer. Solutions containing peptide and protein standards up to 66-kDa molecular weight were deposited on a metal target, dried, and analyzed. No fragmentation was observed from peptides and proteins as well as from the more easily fragmented vitamin B 12 molecule. The mass spectra contained peaks from multiply charged ions that were identical to conventional electrospray. Deep UV laser ablation of tissue allowed detection of lipids from untreated tissue. The mechanism of ionization is postulated to involve absorption of laser energy by a fraction of the analyte molecules that act as a sacrificial matrix or by residual water in the sample., (© 2019 John Wiley & Sons, Ltd.)

Infrared laser ablation microsampling was used with data-dependent acquisition (DDA) and ion mobility-enhanced data-independent acquisition (HDMS E ) for mass spectrometry based bottom-up proteomics analysis of rat brain tissue. Results from HDMS E and DDA analyses of the 12 laser ablation sampled tissue sections showed that HDMS E consistently identified approximately seven times more peptides and four times more proteins than DDA. To evaluate the impact of ultra-performance liquid chromatography (UPLC) peak congestion on HDMS E and DDA analysis, whole tissue digests from rat brain were analyzed at six different UPLC separation times. Analogous to results from laser ablated samples, HDMS E analyses of whole tissue digests yielded about four times more proteins identified than DDA for all six UPLC separation times., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Infrared (IR) laser ablation at 3 μm wavelength was used to extract enzymes from tissue and quantitatively determine their activity. Experiments were conducted with trypsin, which was ablated, captured and then used to digest bovine serum albumin (BSA). BSA digests were evaluated using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) and sequence coverage of 59% was achieved. Quantification was performed using trypsin and catalase standards and rat brain tissue by fluorescence spectroscopy. Both enzymes were reproducibly transferred with an efficiency of 75 ± 8% at laser fluences between 10 and 30 kJ/m 2 . Trypsin retained 37 ± 2% of its activity and catalase retained 50 ± 7%. The activity of catalase from tissue was tested using three consecutive 50 μm thick rat brain sections. Two 4 mm 2 regions were ablated and captured from the cortex and cerebellum regions. The absolute catalase concentration in the two regions was consistent with previously published data, demonstrating transfer of intact enzymes from tissue., (Copyright © 2018 Elsevier B.V. All rights reserved.)

High resolving power ion mobility (IM) allows for accurate characterization of complex mixtures in high-throughput IM mass spectrometry (IM-MS) experiments. We previously demonstrated that pure component IM-MS data can be extracted from IM unresolved post-IM/collision-induced dissociation (CID) MS data using automated ion mobility deconvolution (AIMD) software [Matthew Brantley, Behrooz Zekavat, Brett Harper, Rachel Mason, and Touradj Solouki, J. Am. Soc. Mass Spectrom., 2014, 25, 1810-1819]. In our previous reports, we utilized a quadrupole ion filter for m/z-isolation of IM unresolved monoisotopic species prior to post-IM/CID MS. Here, we utilize a broadband IM-MS deconvolution strategy to remove the m/z-isolation requirement for successful deconvolution of IM unresolved peaks. Broadband data collection has throughput and multiplexing advantages; hence, elimination of the ion isolation step reduces experimental run times and thus expands the applicability of AIMD to high-throughput bottom-up proteomics. We demonstrate broadband IM-MS deconvolution of two separate and unrelated pairs of IM unresolved isomers (viz., a pair of isomeric hexapeptides and a pair of isomeric trisaccharides) in a simulated complex mixture. Moreover, we show that broadband IM-MS deconvolution improves high-throughput bottom-up characterization of a proteolytic digest of rat brain tissue. To our knowledge, this manuscript is the first to report successful deconvolution of pure component IM and MS data from an IM-assisted data-independent analysis (DIA) or HDMSE dataset.

Background: MNEI and its variant Y65R-MNEI are sweet proteins with potential applications as sweeteners in food industry. Also, they are often used as model systems for folding and aggregation studies., Methods: X-ray crystallography was used to structurally characterize Y65R-MNEI at five different pHs, while circular dichroism and fluorescence spectroscopy were used to study their thermal and chemical stability. ThT assay and AFM were used for studying the kinetics of aggregation and morphology of the aggregates., Results: Crystal structures of Y65R-MNEI revealed the existence of a dimer in the asymmetric unit, which, depending on the pH, assumes either an open or a closed conformation. The pH dramatically affects kinetics of formation and morphology of the aggregates: both MNEI and Y65R-MNEI form fibrils at acidic pH while amorphous aggregates are observed at neutral pH., Conclusions: The mutation Y65R induces structural modifications at the C-terminal region of the protein, which account for the decreased stability of the mutant when compared to MNEI. Furthermore, the pH-dependent conformation of the Y65R-MNEI dimer may explain the different type of aggregates formed as a function of pH., General Significance: The investigation of the structural bases of aggregation gets us closer to the possibility of controlling such process, either by tuning the physicochemical environmental parameters or by site directed mutagenesis. This knowledge is helpful to expand the range of stability of proteins with potential industrial applications, such as MNEI and its mutant Y65R-MNEI, which should ideally preserve their structure and soluble state through a wide array of conditions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Converging evidence shows that hand-actions are controlled at the level of synergies and not single muscles. One intriguing aspect of synergy-based action-representation is that it may be intrinsically sparse and the same synergies can be shared across several distinct types of hand-actions. Here, adopting a normative angle, we consider three hypotheses for hand-action optimal-control: sparse-combination hypothesis (SC) - sparsity in the mapping between synergies and actions - i.e., actions implemented using a sparse combination of synergies; sparse-elements hypothesis (SE) - sparsity in synergy representation - i.e., the mapping between degrees-of-freedom (DoF) and synergies is sparse; double-sparsity hypothesis (DS) - a novel view combining both SC and SE - i.e., both the mapping between DoF and synergies and between synergies and actions are sparse, each action implementing a sparse combination of synergies (as in SC), each using a limited set of DoFs (as in SE). We evaluate these hypotheses using hand kinematic data from six human subjects performing nine different types of reach-to-grasp actions. Our results support DS, suggesting that the best action representation is based on a relatively large set of synergies, each involving a reduced number of degrees-of-freedom, and that distinct sets of synergies may be involved in distinct tasks.

Infrared laser ablation coupled to vacuum capture was employed to collect material from fingermarks deposited on surfaces of different porosity and roughness. Laser ablation at 3 μm was performed in reflection mode with subsequent capture of the ejecta with a filter connected to vacuum. Ablation and capture of standards from fingermarks was demonstrated on glass, plastic, aluminum, and cardboard surfaces. Using matrix assisted laser desorption ionization (MALDI), it was possible to detect caffeine after spiking with amounts as low as 1 ng. MALDI detection of condom lubricants and detection of antibacterial peptides from an antiseptic cream was demonstrated. Detection of explosives from fingermarks left on plastic surfaces as well as from direct deposition on the same surface using gas chromatography mass spectrometry (GC-MS) was shown. Graphical Abstract ᅟ.

Cisplatin (CDDP) can form interprotein cross-links, leading to the formation of platinated oligomers. A dimer, a trimer and higher oligomers of bovine pancreatic ribonuclease (RNase A) obtained upon reaction with CDDP in 1:10 protein to metal ratio at 37°C have been previously characterized. Here, we verify the ability of carboplatin and oxaliplatin to induce RNase A oligomerization under the same experimental conditions. The amount of formed RNase A oligomers was compared with that obtained in the reaction of the protein with CDDP. Among the three anticancer agents, CDDP is the most reactive and the most effective in inhibiting the ribonucleolytic activity of the protein. Oxaliplatin is the least potent oligomerization agent. Biophysical characterizations of structure and stability of platinated dimers formed in the presence of carboplatin and oxaliplatin suggest that they have a similar thermal stability and are more prone to dissociation than the corresponding dimer obtained with CDDP. Oligomers obtained in the presence of carboplatin are the most active. X-ray structures of the monomeric adducts that RNase A forms with the three drugs provide a rational basis to explain the different effects of the three anticancer agents on enzymatic activity and protein aggregation. Although platinated oligomers of RNase A formed upon reaction with CDDP, carboplatin and oxaliplatin retain a residual ribonuclease activity, they do not show cytotoxic action, suggesting that protein aggregation processes induced by Pt-based drugs can represent a collateral drawback, which affects the functional state of protein targets and reduces the efficacy of Pt-based drug treatment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Infrared (IR) laser ablation was used to remove material from tissue sections mounted on microscope slides, with subsequent capture in a solvent-containing microcentrifuge tube. Experiments conducted with a 3200-bp double-stranded plasmid DNA template demonstrated IR-laser ablation transfer of intact DNA. The transfer efficiency and the molecular integrity of the captured DNA were evaluated using Sanger sequencing, gel electrophoresis, and fluorimetric analysis. The plasmid DNA was reproducibly transferred with an efficiency of 59 ± 3% at laser fluences of between 10 and 20 kJ/m 2 at a wavelength of 3 μm. IR laser ablation sample transfer was then used to ablate and capture DNA from 50-μm-thick rat brain and kidney tissue sections. DNA was extracted from the captured material using five commercial DNA extraction kits that employed significantly divergent methodologies, with all kits recovering sufficient DNA for successful amplification by polymerase chain reaction (PCR). Four sets of primers were employed, targeting one region of the CYP 11b2 gene (376 bp) and three different regions of the Snn1g gene (298, 168, and 281 bp). The PCR results were not consistently reliable when using unpurified ablation samples; however, after extraction, all samples produced PCR products of the expected size. This work expands the sampling capabilities of IR laser ablation, demonstrating that DNA can be isolated from tissue samples for genomic assays. Due to the small size of the ablation regions (1 mm 2 ), this technique will be useful for sampling discrete cell populations from tissue sections. Graphical abstract Infrared laser ablation transfer of intact DNA from a tissue section.