Evaluating the Modulation of the Acoustic Startle Reflex in Children and Adolescents via Vertical EOG and EEG: Sex, Age, and Behavioral Effects.

Acoustic startle reflex (ASR) constitutes a reliable, cross-species indicator of sensorimotor and inhibitory mechanisms, showing distinct signature in cognitive aging, sex, and psychopathological characterization. ASR can be modulated by the prepulse inhibition (PPI) paradigm, which comprises the suppression of reactivity to a startling stimulus (pulse) following a weak prepulse (30- to 500-ms time difference), being widely linked to inhibitory capabilities of the sensorimotor system. If the prepulse-pulse tones are more clearly separated (500-2,000 ms), ASR amplitude is enhanced, termed as prepulse facilitation (PPF), reflecting sustained or selective attention. Our study aimed to investigate early-life sensorimotor sex/age differences using Electroencephalographic recordings to measure muscular and neural ASR in a healthy young population. Sixty-three children and adolescents aged 6.2-16.7 years (31 females) took part in the experiment. Neural ASR was assessed by two different analyses, namely, event-related potentials (ERPs) and first-derivative potentials (FDPs). As expected, PPF showed enhanced responses compared with PPI, as indicated by eyeblink, ERP and FDP measures, confirming the gating effect hypothesis. Sex-related differences were reflected in FDPs, with females showing higher ASR than males, suggesting increased levels of poststartle excitability. Intragroup age effects were evaluated via multipredictor regression models, noticing positive correlation between age versus eyeblink and ERP responses. Attention-related ERPs (N100 and P200) showed distinct patterns in PPI versus PPF, potentially indicative for alternative attentional allocation and block-out of sensory overload. Screening measures of participants' neurodevelopmental (assessed by Wechsler Intelligence Scale for Children) and behavioral (assessed by Child Behavior Checklist) markers were also associated with increased N100/P200 responses, presumably indexing synergy between perceptual consistency, personality profiling, and inhibitory performance. Conclusively, modulation of ASR by PPI and PPF is associated with biological sex and internal/external personality traits in childhood and adolescence, potentially useful to guide symptomatology and prevention of psychopathology.

FLINO: a new method for immunofluorescence bioimage normalization.

MOTIVATION: Multiplexed immunofluorescence bioimaging of single-cells and their spatial organization in tissue holds great promise to the development of future precision diagnostics and therapeutics. Current multiplexing pipelines typically involve multiple rounds of immunofluorescence staining across multiple tissue slides. This introduces experimental batch effects that can hide underlying biological signal. It is important to have robust algorithms that can correct for the batch effects while not introducing biases into the data. Performance of data normalization methods can vary among different assay pipelines. To evaluate differences, it is critical to have a ground truth dataset that is representative of the assay. RESULTS: A new immunoFLuorescence Image NOrmalization method is presented and evaluated against alternative methods and workflows. Multiround immunofluorescence staining of the same tissue with the nuclear dye DAPI was used to represent virtual slides and a ground truth. DAPI was restained on a given tissue slide producing multiple images of the same underlying structure but undergoing multiple representative tissue handling steps. This ground truth dataset was used to evaluate and compare multiple normalization methods including median, quantile, smooth quantile, median ratio normalization and trimmed mean of the M-values. These methods were applied in both an unbiased grid object and segmented cell object workflow to 24 multiplexed biomarkers. An upper quartile normalization of grid objects in log space was found to obtain almost equivalent performance to directly normalizing segmented cell objects by the middle quantile. The developed grid-based technique was then applied with on-slide controls for evaluation. Using five or fewer controls per slide can introduce biases into the data. Ten or more on-slide controls were able to robustly correct for batch effects. AVAILABILITY AND IMPLEMENTATION: The data underlying this article along with the FLINO R-scripts used to perform the evaluation of image normalizations methods and workflows can be downloaded from https://github.com/GE-Bio/FLINO. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An atlas of inter- and intra-tumor heterogeneity of apoptosis competency in colorectal cancer tissue at single-cell resolution.

Cancer cells' ability to inhibit apoptosis is key to malignant transformation and limits response to therapy. Here, we performed multiplexed immunofluorescence analysis on tissue microarrays with 373 cores from 168 patients, segmentation of 2.4 million individual cells, and quantification of 18 cell lineage and apoptosis proteins. We identified an enrichment for BCL2 in immune, and BAK, SMAC, and XIAP in cancer cells. Ordinary differential equation-based modeling of apoptosis sensitivity at single-cell resolution was conducted and an atlas of inter- and intra-tumor heterogeneity in apoptosis susceptibility generated. Systems modeling at single-cell resolution identified an enhanced sensitivity of cancer cells to mitochondrial permeabilization and executioner caspase activation compared to immune and stromal cells, but showed significant inter- and intra-tumor heterogeneity.

Stratification of chemotherapy-treated stage III colorectal cancer patients using multiplexed imaging and single-cell analysis of T-cell populations.

Colorectal cancer (CRC) has one of the highest cancer incidences and mortality rates. In stage III, postoperative chemotherapy benefits <20% of patients, while more than 50% will develop distant metastases. Biomarkers for identification of patients at increased risk of disease recurrence following adjuvant chemotherapy are currently lacking. In this study, we assessed immune signatures in the tumor and tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single-cell analysis technology (Cell DIVETM) and evaluated their correlations with patient outcomes. Tissue microarrays (TMAs) with up to three 1 mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin (FOLFOX) chemotherapy. Single sections underwent multiplexed immunofluorescence staining for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and tumor/cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase, and S6). We used annotations and a probabilistic classification algorithm to build statistical models of immune cell types. Images were also qualitatively assessed independently by a Pathologist as 'high', 'moderate' or 'low', for stromal and total immune cell content. Excellent agreement was found between manual assessment and total automated scores (p < 0.0001). Moreover, compared to single markers, a multi-marker classification of regulatory T cells (Tregs: CD3+/CD4+FOXP3+/PD1-) was significantly associated with disease-free survival (DFS) and overall survival (OS) (p = 0.049 and 0.032) of FOLFOX-treated patients. Our results also showed that PD1- Tregs rather than PD1+ Tregs were associated with improved survival. These findings were supported by results from an independent FOLFOX-treated cohort of 191 stage III CRC patients, where higher PD1- Tregs were associated with an increase overall survival (p = 0.015) for CD3+/CD4+/FOXP3+/PD1-. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cell types with stronger correlations with outcomes.

Diverse patterns of vulnerability to visual illusions in children with neurodevelopmental disorders.

Research on how children with neurodevelopmental disorders perceive, process, and interpret visual illusions (VIs) has been extensively focused on children with autism spectrum disorder providing controversial findings. In this study, we investigated the patterns of vulnerability to a wide set of VIs comprising 23 standard text book VIs and their variations in a clinical sample of children with neurodevelopmental disorders compared to typically developing children (TD). A total of 176 children, aged between 4.6 and 13.8 years old, were distributed into four groups: high-functioning autism (HFA; N = 23), attention-deficit/hyperactivity disorder (ADHD; N = 42), specific learning disorder (SLD; N = 70), and TD (N = 41). Regression models, adjusted for sex, age, and non-verbal IQ, showed that HFA was associated with greater responses accuracy than TD children to the full battery of VIs, to the cognitive illusions, to the distortions, and to both geometrical illusions of size/shape (cognitive distortions) and lightness contrast effects (physical distortions). The susceptibility of ADHD children was found attenuated for illusory contours and greater for paradoxical illusions in comparison with TD children. No significant differences were shown between the SLD group and the TD children. Our findings, which were adjusted for the same duration of visual working memory across groups, showed that there is a potential specific tendency of HFA children to failure of processing visual information in context. Contrarily, children with ADHD showed in general normal global processing such as children diagnosed with SLD.

Targeting nucleotide metabolism enhances the efficacy of anthracyclines and anti-metabolites in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) remains the most lethal breast cancer subtype with poor response rates to the current chemotherapies and a lack of additional effective treatment options. We have identified deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) as a critical gatekeeper that protects tumour DNA from the genotoxic misincorporation of uracil during treatment with standard chemotherapeutic agents commonly used in the FEC regimen. dUTPase catalyses the hydrolytic dephosphorylation of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP), providing dUMP for thymidylate synthase as part of the thymidylate biosynthesis pathway and maintaining low intracellular dUTP concentrations. This is crucial as DNA polymerase cannot distinguish between dUTP and deoxythymidylate triphosphate (dTTP), leading to dUTP misincorporation into DNA. Targeting dUTPase and inducing uracil misincorporation during the repair of DNA damage induced by fluoropyrimidines or anthracyclines represents an effective strategy to induce cell lethality. dUTPase inhibition significantly sensitised TNBC cell lines to fluoropyrimidines and anthracyclines through imbalanced nucleotide pools and increased DNA damage leading to decreased proliferation and increased cell death. These results suggest that repair of treatment-mediated DNA damage requires dUTPase to prevent uracil misincorporation and that inhibition of dUTPase is a promising strategy to enhance the efficacy of TNBC chemotherapy.

Gender-dependent variations in optical illusions: evidence from N400 waveforms.

OBJECTIVE: The cognitive mechanisms (especially the gender-related ones) underlying optical illusion processing remain elusive. Since the N400 component of event-related potentials (ERPs) is an index of the semantic integration of information processing tasks, the present study focuses on gender-related differences in N400 waveforms elicited during the reasoning process applied to reach a valid conclusion of optical illusions engaging working memory (WM). APPROACH: Fifty-one healthy participants (28 males, age = 34.25 years ± 10.25, years of education = 16.00 years ± 1.78; and 23 females, age = 33.43 ± 7.93, years of education = 15.56 ± 1.82) were measured. The N400 ERP component was evoked by 39 optical illusions adjusted to induce WM. We compared brain activation patterns while participants maintained conclusions of the optical illusions in WM. The N400 of ERPs was recorded during the WM phase, during which participants were required to draw a logical conclusion regarding the correctness of the optical illusions. MAIN RESULTS: Analysis revealed that females compared to males exhibited significantly increased N400 amplitudes located at parietal and occipital sites, whereas males exhibited significantly higher N400 amplitudes located at frontal areas. Furthermore, females compared to males demonstrated significantly prolonged latencies of the N400 component located at right frontotemporal abductions. SIGNIFICANCE: These results suggest that coupling of optical illusions with WM engages distinct gender-related variations of brain semantic processing as reflected by the N400 ERP component. Based on the dual process account, our study gives support to the notion that women tend to employ a more deliberate and slower semantic reasoning than the men who tend to employ an automatic and fast one. Topographically, within the network sub-serving the semantic operation, the posterior brain areas responsible for sensorimotor integration-related processes elicit a greater brain activation among females while the anterior brain areas responsible for control and storage/retrieval operation elicit a greater brain activation among males.

Test-retest reliability of brain oscillations in a prepulse inhibition and facilitation paradigm: effects of gender in healthy humans.

There is a growing interest in assessing the reliability of electroencephalographic (EEG) measures in clinical and research settings. Prepulse inhibition (PPI: representing attentional modulation) and facilitation (PPF: reflecting selective attention) paradigms have been used to study inhibitory function and selective attention, respectively. However, to date, little has been known with regards to the stability of brain oscillatory activity during PPI and PPF. We investigated the stability of event-related EEG oscillations during PPI and PPF in healthy humans over two monthly sessions. Power spectral densities were analysed at traditional frequency bands (delta, alpha, beta sub-bands, and gamma). We assessed test-retest reliability by calculating intraclass correlation coefficients (ICCs, absolute agreement definition) and examined potential effects of gender. The results showed good-to-excellent reproducibility of EEG power (both in PPI and PPF) over all frequency bands (ICCs > 0.75), except for delta (ICCs < 0.75), with alpha exhibiting the highest repeatability performance. In addition, females showed reduced reliability compared to males in both PPI and PPF, possibly attributed to menstrual cycle phase across our female participants. Overall, our findings suggest that brain oscillatory activity can be test-retest reliable, while gender needs to be controlled with caution. Finally, event-related EEG oscillations during both PPI and PPF could provide a complementary tool to study psychopathology in clinical practice.

A novel optical non-intrusive method for measuring acoustic startle reflex in humans.

OBJECTIVE: The development of a new non-intrusive optical system for remotely measuring acoustic startle reflex (ASR) in humans. APPROACH: The eye reflex movement during an acoustic stimulation session is recorded through a high-speed digital camera. The eyes region is isolated by the rest of the face by an advanced pyramid-like feature detection algorithm, which greatly reduces the number of false positives. A separate Lucas-Kanade optical flow routine is designed for the eyeblink movement detection and the startle eyeblink reflex (SEBR) curve extraction. Image masking is implemented for the elimination of unwanted artifacts caused mainly by voluntary eye movement. The proposed system was tested along with a valid EMG system on a sample of 32 healthy randomly selected adults, and the results were compared in order to measure the system's degree of reliability. MAIN RESULTS: To assess the proposed method's validity the EMG data was used as a benchmark. The results showed strong correlation between EMG and Camera acquired results, which proves the validity of the proposed method. Furthermore, by comparing the response probability and the signal to noise ratio (SNR) for the two techniques, we proved that the proposed method can surpass the traditional EMG system in terms of accuracy and reliability. SIGNIFICANCE: The proposed technique presents a simple, robust and reliable non-intrusive means of measuring ASR in humans, with the potential of future implementation on various ASR psychophysiology experiments, such as the study of PPI.

Mental processing comparison between past and future employing a novel EEG analysis based on radiated EM field estimation.

BACKGROUND: The inverse problem solution in the field of ElectroEncephaloGraphy (EEG) analysis has been addressed in the scientific literature for many decades, utilizing either mathematical techniques for measurement fitting or pure ElectroMagnetic (EM) methods involving complex head models for the prediction of the near field. NEW METHOD: A novel radiated EM field estimation analysis scheme is proposed for EEG analysis, based on the determination of a grid of equivalent distributed EM sources with equal magnetic moments, in order to compute the extrapolated far field. A Pattern Search approach is adopted to minimize the Mean Absolute Relative Error between the EM near field created by the source grid and the EM field extracted by the measurements. RESULTS: The application of the method on a subject's brain activity recordings in the context of "Protagoras" mental-auditory experiment demonstrates the capability of the proposed scheme to compare the subject's concentration differences between the limit of present and past versus the limit of present and future. COMPARISON WITH EXISTING METHODS: The proposed method combines features from different existing methods, both in terms of mathematical and EM theory techniques, in order to extend their capabilities and transform the conventional analysis of EEG recordings to a far field radiation basis. CONCLUSIONS: The treatment of the brain as an equivalent far field radiator can be a useful and promising new perspective to the established analysis of EEG recordings arising from brain activity during mental processing.

Dendritic Cell Migration to Skin-Draining Lymph Nodes Is Controlled by Dermatan Sulfate and Determines Adaptive Immunity Magnitude.

For full activation of naïve adaptive lymphocytes in skin-draining lymph nodes (LNs), presentation of peptide:MHC complexes by LN-resident and skin-derived dendritic cells (DCs) that encountered antigens (Ags) is an absolute prerequisite. To get to the nearest draining LN upon intradermal immunization, DCs need to migrate from the infection site to the afferent lymphatics, which can only be reached by traversing a collagen-dense network located in the dermis of the skin through the activity of proteolytic enzymes. Here, we show that mice with altered collagen fibrillogenesis resulting in thicker collagen fibers in the skin display a reduced DC migration to the draining LN upon immune challenge. Consequently, the initiation of the cellular and humoral immune response was diminished. Ag-specific CD8+ and CD4+ T cells as well as Ag-specific germinal center B cells and serum immunoglobulin levels were significantly decreased. Hence, we postulate that alterations to the production of extracellular matrix, as seen in various connective tissue disorders, may in the end affect the qualitative outcome of adaptive immunity.

Aristotle Meets Zeno: Psychophysiological Evidence.

This study, a tribute to Aristotle's 2400 years, used a juxtaposition of valid Aristotelian arguments to the paradoxes formulated by Zeno the Eleatic, in order to investigate the electrophysiological correlates of attentional and /or memory processing effects in the course of deductive reasoning. Participants undertook reasoning tasks based on visually presented arguments which were either (a) valid (Aristotelian) statements or (b) paradoxes. We compared brain activation patterns while participants maintained the premises / conclusions of either the valid statements or the paradoxes in working memory (WM). Event-related brain potentials (ERPs), specifically the P300 component of ERPs, were recorded during the WM phase, during which participants were required to draw a logical conclusion regarding the correctness of the valid syllogisms or the paradoxes. During the processing of paradoxes, results demonstrated a more positive event-related potential deflection (P300) across frontal regions, whereas processing of valid statements was associated with noticeable P300 amplitudes across parieto-occipital regions. These findings suggest that paradoxes mobilize frontal attention mechanisms, while valid deduction promotes parieto-occipital activity associated with attention and/or subsequent memory processing.

Dermatan Sulfate-Free Mice Display Embryological Defects and Are Neonatal Lethal Despite Normal Lymphoid and Non-Lymphoid Organogenesis.

The epimerization of glucuronic acid into iduronic acid adds structural variability to chondroitin/dermatan sulfate polysaccharides. Iduronic acid-containing domains play essential roles in processes such as coagulation, chemokine and morphogen modulation, collagen maturation, and neurite sprouting. Therefore, we generated and characterized, for the first time, mice deficient in dermatan sulfate epimerase 1 and 2, two enzymes uniquely involved in dermatan sulfate biosynthesis. The resulting mice, termed DKO mice, were completely devoid of iduronic acid, and the resulting chondroitin sulfate chains were structurally different from the wild type chains, from which a different protein binding specificity can be expected. As a consequence, a vast majority of the DKO mice died perinatally, with greatly variable phenotypes at birth or late embryological stages such as umbilical hernia, exencephaly and a kinked tail. However, a minority of embryos were histologically unaffected, with apparently normal lung and bone/cartilage features. Interestingly, the binding of the chemokine CXCL13, an important modulator of lymphoid organogenesis, to mouse DKO embryonic fibroblasts was impaired. Nevertheless, the development of the secondary lymphoid organs, including the lymph nodes and spleen, was normal. Altogether, our results indicate an important role of dermatan sulfate in embryological development and perinatal survival.

Dermatan sulfate epimerase 1 deficient mice as a model for human abdominal wall defects.

BACKGROUND: Dermatan sulfate (DS) is a highly sulfated polysaccharide with a variety of biological functions in extracellular matrix organization and processes such as tumorigenesis and wound healing. A distinct feature of DS is the presence of iduronic acid, produced by the two enzymes, DS-epimerase 1 and 2, which are encoded by Dse and Dsel, respectively. METHODS: We have previously shown that Dse knockout (KO) mice in a mixed C57BL/6-129/SvJ background have an altered collagen matrix structure in skin. In the current work we studied Dse KO mice in a pure NFR genetic background. RESULTS: Dse KO embryos and newborns had kinked tails and histological staining revealed significantly thicker epidermal layers in Dse KO mice when compared with heterozygote (Het) or wild-type (WT) littermates. Immunochemical analysis of the epidermal layers in newborn pups showed increased expression of keratin 5 in the basal layer and keratin 1 in the spinous layer. In addition, we observed an abdominal wall defect with herniated intestines in 16% of the Dse KO embryos. Other, less frequent, developmental defects were exencephaly and spina bifida. CONCLUSION: We conclude that the combination of defective collagen structure in the dermis and imbalanced keratinocyte maturation could be responsible for the observed developmental defects in Dse KO mice. In addition, we propose that Dse KO mice could be used as a model in pathogenetic studies of human fetal abdominal wall defects.

New insights into the pathobiology of Down syndrome--hyaluronan synthase-2 overexpression is regulated by collagen VI α2 chain.

Down syndrome (DS) is a common birth defect characterized by the trisomy of chromosome 21. DS-affected umbilical cords (UCs) of fetuses show altered architecture of the extracellular matrix. Overexpression of the chromosome 21 genes encoding the collagen type VI (COLVI) chains α1(VI) and α2(VI), COL6A1 and COL6A2, respectively, has also reported to occur in the nuchal skin of DS fetuses. The aim of this study was therefore to evaluate the COLVI content in euploid and DS-affected UCs and human skin fibroblasts, and to investigate the relationships between COLVI and hyaluronan (HA) and HA synthase-2 (HAS2). We found that the UCs of DS fetuses showed denser staining of COLVI and increased COL6A2 expression at both early and term gestational ages. In vitro expression studies in DS-derived fibroblasts showed similarly increased amounts of α1(VI) and α2(VI) chains at the protein and transcriptional level, supporting the hypothesis of the gene dosage effect. Furthermore, increased levels of HA and HAS2 were also found in DS-derived skin fibroblast cultures. Notably, silencing of COL6A2 in DS-derived cells resulted in downregulation of HAS2, with a simultaneous decrease in secreted HA. Exogenous addition of COLVI to normal fibroblasts did not have any effect on HAS2 expression. In conclusion, UCs and skin fibroblasts in DS show significant increases in COLVI and HA; the overexpression of COL6A2 in DS tissue and cells is closely related to the increased expression of HAS2. These data may explain the DS phenotypes and their effects in organ tissue maturation.

Enhanced antitumour activity of cyclopendadienyl-substituted metallocene dihalides in human breast and colon cancer cells.

Metallocene dihalides, which are cyclopentadienyl complexes with the general formula R2MX2 (where R=eta(5)-C5H5, eta(5)-CH3C5H4, eta(5)-SiMe3C5H4 etc.; M=Ti, Zr, Hf, V or Nb; and X=halogen), are highly effective agents against Ehrlich ascites tumour cells and lymphocytic leukaemia. The aim of this study was to evaluate the antitumor activity of the various metallocene dihalides and particularly their effects on cell proliferation of human breast and colon cancer cells. The growth inhibition of the antitumour metallocenes (eta(5)-C5H5)2TiCl2 and (eta(5)-C5H5)2VCl2 and four ring-substituted derivatives in HT-29 (colon cancer) and MCF-7 (breast cancer) cell lines is reported. The results showed that ring-substitution of metallocenes gave similar or even better activity in cell proliferation reduction, in both cell lines, especially in HT-29 and suggested that ring-substitution may enhance the inhibitory activity of the metallocene compound family.