Introduction: Deficits in neurocognitive mechanisms such as inhibition control and cognitive flexibility have been suggested to mediate the symptoms in obsessive–compulsive disorder (OCD). These mechanisms are proposedly controlled by the “affective” and “executive” orbitofronto-striato-thalamo-cortical (CSTC) circuits with well-documented morphological and functional alterations in OCD that are associated with OCD symptoms. The precuneus region has been suggested in OCD as another key structure associated with the mechanism of “thought–action fusion.” Our study aimed to elucidate the association of the altered functional coupling of the CSTC nodes (and precuneus), the OCD symptoms, and interference control/cognitive flexibility.Methods: In a group of 36 (17 medicated and 19 drug-free) OCD patients and matched healthy volunteers, we tested functional connectivity (FC) within the constituents of the dorsolateral prefrontal cortex “executive” CSTC, the orbitofrontal cortex/anterior cingulate “affective” CSTC, and precuneus. The functional connections showing the strongest effects were subsequently entered as explanatory variables to multiple regression analyses to identify possible associations between observed alterations of functional coupling and cognitive (Stroop test) and clinical measures .Results: We observed increased FC (FWE p < 0.05 corr.) between CSTC seeds and regions of the parieto-occipital cortex, and between the precuneus and the angular gyrus and dorsolateral prefrontal cortex. Decreased FC was observed within the CSTC loop  and between the anterior cingulate cortex and the limbic lobe. Linear regression identified a relationship between the altered functional coupling of thalamus with the right somatomotor parietal cortex and the Stroop color–word score. Similar association of thalamus FC has been identified also for obsessions severity. No association was observed for compulsions and anxiety.Conclusions: Our findings demonstrate altered FC in OCD patients with a prevailing increase in FC originating in CSTC regions toward other cortical areas, and a decrease in FC within the constituents of CSTC loops. Moreover, our results support the role of precuneus in OCD. The association of the cognitive and clinical symptoms with the FC between the thalamus and somatomotor cortex indicates that cognitive flexibility and inhibitory control are strongly linked and both mechanisms might contribute to the symptomatology of OCD. Obsessive–compulsive disorder (OCD) is a chronic neuropsychiatric disorder characterized by recurrent thoughts (obsessions) and by repetitive behavior (compulsions) that is often reported to “neutralize” obsessions and temporarily reduce anxiety . GeneralNeuroimaging studies have indicated that cognitive flexibility is mainly controlled by the prefrontal cortex (PFC) and frontoparietal attentional network ; currenEven though precuneus (PCU) is not a traditional part of the CSTC loops, it has also been suggested as another key structure in OCD. It is densely projecting to PFC and plays a role in so-called thought–action fusion , 39 undeHowever, the relationship between the fundamental processes of inhibitory control and cognitive flexibility suggested to be altered in OCD and the mechanism of thought–action fusion linked to PCU alterations was not directly examined.Functional alterations within the CSTC loop were repeatedly demonstrated in OCD patients and are still regarded as a central psychopathological mechanism of OCD . HoweverGiven the previous findings, we hypothesized that FC would be increased in all preselected regions of interest (ROIs). We also expected that FC changes in frontal areas including ACC and DLPFC  would correlate with the Stroop test score, while the striatum, thalamus, and precuneus would correlate with clinical symptoms, and none of the FC changes would correlate with the anxiety score.In total, 36 in-patients diagnosed with OCD according to ICD-10  and DSM-The severity of obsessions and compulsions in patients was assessed on the day of fMRI session using the Yale–Brown Obsessive–Compulsive Scale  and curn = 3), 2—moderate (n = 21), or 3—severe (n = 10) clinical symptoms present. In respect to major symptom dimensions of OCD, recruited patients showed most prevalent dimensions of “contamination/washing” (n = 18), “harm/checking” (n = 15), and “symmetry/ordering” (n = 3).The patients were recruited during the initial phases of the cognitive–behavioral therapy program combining both inpatients and day-care patients. All patients were symptomatic at the time of MRI scanning as evaluated by the Y-BOCS scale, with 1—mild  and their medication status was stable for at least 4 weeks prior to the study. The patients did not use antipsychotics  at least 5 days prior to scanning. Only short-acting zolpidem was allowed for insomnia. On the day of fMRI, no psychotropic medication was administered before scanning. While all three patients with mild symptoms were in the subgroup of drug-free patients, 8 of 10 patients with severe symptomatology were recruited as medicated; patients with moderate symptomatology were equally distributed to both patients' subgroups (10/11).red printed in blue ink). The participant is asked to name the color of the word while trying to inhibit the interference of the automatic tendency of reading the word. Response time in the last condition is slowed because of the competing processing of semantic and visual content of the stimuli. Each card has 100 stimuli to read/name. In the case of finishing before the time limit of 45 s, a participant starts naming from the beginning again. During the task, the participant is corrected if making a mistake. The final scores were calculated as the correct answers achieved in 45 s for each of the subtests: word reading score (W), color naming score (C), and color–word naming score (CW).SCWT is a well-established task , 54 for 3 , which was used for spatial normalization and anatomical reference.Imaging data were acquired on a 3T Siemens Prisma MRI scanner  equipped with a standard 64-channel head coil. Resting state fMRI (rs-fMRI) was measured with a gradient echo echo-planar sequence . Whole-brain anatomical scans were also acquired using a 3D T1-weighted magnetization-prepared gradient echo sequence (MP-RAGE), consisting of 240 sagittal slices with a resolution of 0.7 × 0.7 × 0.7 mmwww.nitrc.org/projects/conn/). The fMRI data were corrected for head movement, together with anatomical scans, and were normalized into standard MNI space and segmented into gray matter, white matter, and CSF tissue classes using SPM12 unified segmentation and normalization procedure  of CONN connectivity software , ACC—Anterior cingulate cortex, CN—Caudate nucleus, THA—Thalamus, and PCU—Precuneus. The combined Automated Anatomical Labeling (26 ROIs) and Harvard Oxford Atlas (106 ROIs) provided with the CONN toolbox was used for the FC analysis. For each region of interest (ROI), a representative signal of the ROI was obtained by averaging the residual BOLD time courses across voxels contained in the ROI. The seed-to-voxel connectivity was estimated for the selected seeds by computing Pearson correlation coefficients between the residual BOLD time courses, and further converted to approximately normally distributed Z scores using the Fisher transformation. The group effect was evaluated by between-subject contrast . The same analysis was performed for all six preselected ROIs.The FC analysis proceeded in two steps: first, the relevant functional connections were identified by starting from six key seeds; secondly, the inter-individual differences in strengths of these key connections were analyzed. To establish the overall FC of the network, we used the following bilateral seeds: p level 0.05 to be significant (with cluster-forming threshold p < 0.001). Subsequently, we identified the coordinates of the local maxima of the most significant cluster for each of the six predefined seeds. We identified the anatomical regions  corresponding to these local maxima coordinates. The “slice display” function in the CONN toolbox was used to obtain the presented images of individual brain slices in p level 0.05 with cluster-forming threshold p < 0.001 uncorr.).To avoid a large number of false positives, for the seed-to-voxel FC mapping, we considered only findings at the conservative cluster family-wise error (FWE) corrected Z-values quantifying the raw connectivity strength during rs-fMRI condition for region pairs (seed-cluster) were used as explanatory variables in the regression analysis in order to evaluate their possible effects on inter-subjects variability observed in cognitive interference measured by the Stroop test .The aforementioned significant clusters obtained in the seed-to-voxel analyses were exported from the CONN toolbox in a form of cluster masks representing the spatial maps of individual clusters. Similarly, in case of bilateral seeds, the masks covering both areas from the atlas were created. These masks  were imported to the CONN toolbox and used in the ROI-to-ROI FC analysis. Finally, Fisher-transformed p ≤ 0.05. The Student t-test was used to test between-group differences in age and education level; the Cramer's V test was applied to test between-group differences in sex distribution. The Student t-test was also used to calculate differences in FC of the identified seed-to-target (ROI-to-ROI) pairs in antidepressant-medicated and drug-free patients. The multiple stepwise regression analysis (forward) was used to evaluate whether the functional connections showing the most prominent effects of disease (one for each ROI) explain the variability observed in the cognitive (Stroop CW) and clinical  scores. We used enter/remove thresholds F = 3.84/2.71, approximately corresponding to p = 0.05/0.1 as parameters of the stepwise forward variable selection. Pearson correlation analysis was used to calculate potential associations between the cognitive measure (Stroop performance) and individual scores of applied clinical scales measuring severity of obsessions, compulsions and anxiety.Statistica software v13 was applied in additional data analyses and the significance level was set to p < 0.05) from the matched group of healthy controls in the “color–word naming” subtest score (incongruent word and color). The groups did not differ in the other two subtests that did not involve conflicting information  in the OCD sample compared to the healthy subjects. The preselected seeds showed differences in between-subject contrast  in the seed-to-voxel (whole brain) approach , the DLPFC seed showed only a decrease in functional coupling with the cluster of the left temporo-occipital and posterior divisions of the inferior and middle temporal gyrus. ACC showed only a decrease in FC in the OCD subjects in the hippocampus and the adjacent parahippocampal cortex. In the case of the thalamus, highly significant effects (in the means of increased FC in the OCD patients) were found in the four main bilateral clusters consisting of the postcentral gyrus and superior parietal lobule, the fusiform and adjacent occipital cortex, and the lateral temporal cortex covering the superior and middle temporal gyri. Only a decrease in thalamic FC in the OCD patients was observed for inward thalamic voxels and the striatum. In the OCD subjects, the caudate nucleus showed an increase in FC in bilateral clusters of post- and precentral gyri, and one cluster located in the left inferior occipital cortex. A decrease in FC was observed in a cluster located in the subcortical gray nuclei  and the left middle frontal gyrus. The precuneus showed a strong increase in FC in the OCD patients in three clusters of the bilateral angular gyrus, and the left middle frontal gyrus and less pronounced decrease in one cluster covering the right limbic lobe . Given the absence of any effect of medication on FC (see below), we analyzed both medicated and unmedicated patients as one group in further analytical steps. See However, not all of the preselected seeds  showed a between-group difference in FC. The following efects were observed in the FC of the selected seeds using the For each selected seed, the functional connection showing the most prominent effect (largest cluster size) (see effects marked by bold in n = 17) and drug-free patients (n = 19) showed no differences in functional seed-to-voxel connectivity (for either FWE corrected p < 0.05 or uncorrected p < 0.001 levels) or in the statistical comparison of FC values extracted for each patient for all of the five seed-target (mask) pairs. The Student t-tests calculated for the individual FCs  did not reveal significant differences between the medicated and unmedicated patients and the results were as follows : DLPFC—Inferior temporal gyrus left ; ACC—Hippocampus left ; THA—Postcentral gyrus right ; CN—Thalamus right ; PCU—Angular gyrus left .Antidepressant-medicated . As no between-group effect was found for the OFC area, only the functional connections for the five remaining seeds were used as explanatory variables for the multiple regression analyses . In order to select specific target regions, Z Fisher values were extracted from the ROI-to-ROI analysis, representing the seed  and the target mask covering the cluster area [labeled using the local maxima for each of the selected effects (the largest cluster size) obtained in the previous step of the between-group analysis]. The forward stepwise multiple regression analysis (with FC of the seed-target pairs used as explanatory variables) revealed a significant effect for the Stroop CW score, showing that score variability is partially explained by the FC between the thalamus and the cluster with local maxima in the right postcentral gyrus  were identified using Pearson correlation in the OCD group between the cognitive measure (Stroop performance in the CW subtest) and the scores of applied clinical scales: Y-BOCS obsessions , Y-BOCS compulsions , and HAM-A . However, the clinical scales were substantially correlated, particularly the severity of compulsions correlated with the severity of obsession  and with evaluated anxiety symptoms .No significant correlations  and executive (DLPFC) CSTC loops, and both increased and decreased connections from subcortical striatal and thalamic regions and PCU. We also confirmed the altered interference control in the OCD sample. Interestingly, while the alterations observed in the FC of the thalamus are associated with altered interference control measured by the Stroop color–word subtest and severity of obsessions evaluated using the Y-BOCS scale, the FC alterations in other regions of interest did not reveal significant associations neither for interference control nor for clinical symptoms.Our findings of impaired performance in the subtest of the color–word list (but not in the two subtests with no conflicting information involved) in the OCD patients are in line with previous studies reporting altered interference control demonstrated by this SCWT subtest , 58. EveCongruently with the majority of the previous studies on FC in OCD –28, 36, post hoc analysis addressing separately medial  and lateral  division of OFC. As we did not identify any between-group differences for these regions, it is unlikely that our negative finding results from parcellation. However, we cannot exclude the role of technical factors in obtaining high-quality images in this specific region near air/tissue interfaces. Hence, due to the occurrence of susceptibility gradients, the fMRI protocol (GRE-EPI sequence) could compromise the detection of neuronal signals in OFC , decreased FC of ACC and DLPFC to temporo-limbic areas, and more opposed regulatory role of PCU to anterior lateral temporal cortex (negative coupling) and more posterior temporo-parietal region such as angular gyrus (positive coupling).Unexpectedly, we did not identify an association between clinical symptoms and cognitive inference and FC originating from striatal and cortical seeds. However, the hyperconnectivity of the thalamus with the somatomotor parietal cortex with local maxima in postcentral gyrus showed association both with obsession severity and with impaired cognitive interference control. This finding is not surprising as these brain areas densely connected by thalamocortical radiations are functionally responsible for speech motor control involving “feedback error detection” in sensory cortices , 75. MorIn line with a recent study , our datWe hypothesize that FC of thalamocortical radiations shows association with OCD symptomatology even during the resting state because it is involved in the preexecution stages of feedback error detection (proactive control). On the other side, the FC of cortical regions (constituents of the CSTCs) might be related more specifically to the clinical symptoms when measured during active state such as symptoms provocation. This assumption should be addressed in future studies comparing the strength of association between expression of clinical symptoms and FC in resting and active (provocation) states.Alternatively, the missing association between FC alterations observed in the other CSTC regions  and clinical symptoms of compulsions  might be related by the complexity and variability of the compulsions. This variability could be highlighted by the fact that our OCD sample comprised an equal proportion of two OCD phenotypes (18/15)—“contamination/washing” and “harm/checking (aggressive obsessions)”—that might not be directly related to the resting-state individual FC values. Moreover, the strong correlation between the compulsion and obsession severities and moderate correlation with the rated anxiety support the concept of compulsions induced by the intrusive thought and performed to reduce the perceived anxiety.p ≤ 0.001). Moreover, the effect of medication on fMRI and FC data is limited, if any , decreased FC of ACC and DLPFC to temporo-limbic areas, and more opposed regulatory role of PCU to anterior lateral temporal cortex (negative coupling) and more posterior temporo-parietal region such as angular gyrus (positive coupling). This could be interpreted as a disconnectivity of the traditional CSTC loops in OCD counterbalanced by hyperconnectivity of CSTC regions manifested outwards. In addition, our results support the role of thalamus and its coupling with the somatomotor area of the parietal cortex in OCD, specifically in interference control and/or cognitive flexibility. Moreover, a similar association was identified also for the severity of obsessions, suggesting that suppression (filtering) of irrelevant information might be linked to the altered FC of the thalamus. We suggest that while the affected cognitive control of the conflicting information in Stroop task and severity of obsessions is linked to altered functional state and individual connections of specific brain networks, the complex behavioral clusters of compulsive symptomatology may be caused by more complex or highly individual alterations of brain dynamics that are not captured by individual connections of the studied key hubs of the CSTC network, or that even do not manifest strongly during resting brain state. Future studies should elucidate if the FC in resting and active (symptoms provocation) states differ in the strength of association with behavioral and neurocognitive expression of OCD. Moreover, these studies should separate variable OCD phenotypes (dimensions) and include a combination of methods aimed at inhibitory control and cognitive flexibility.The datasets generated for this study are available on request to the corresponding author.This study procedure involving human participants and all apllied measures were reviewed and approved by the local Ethics committee of the NIMH in Klecany, Czech Republic in accordance with the Declaration of Helsinki. The patients and healthy participants provided their written informed consent to participate in this study prior to participation.The well-documented alterations of the orbitofronto-striato-thalamo-cortical (CSTC) circuits are considered a central psychopathological mechanism associated with the symptomatology of the obsessive-compulsive disorder (OCD). The impaired mechanisms of inhibitory control and/or cognitive flexibility have been previously suggested to be mediating the compulsive behavior in OCD, with the involvement of affective and/or executive CSTC loops. Our study aimed to investigate the resting state functional coupling of the CSTCs nodes and to elucidate how the altered functional connectivity pattern is linked to OCD symptoms and cognitive interference control mechanism addressed by the Stroop test. Our findings confirmed altered functional connectivity (FC) pattern in OCD patients (both medicated and unmedicated) with a prevailing increase in FC originating in CSTC regions toward other cortical areas, and a decrease in FC mainly within the constituents of CSTC loops. In addition, our findings support the potential role of precuneus in OCD, a structure previously suggested in so-called thought-action fusion mechanism. Importantly, the associations identified in OCD patients between the functional connectivity of the thalamus and both obsessions severity and cognitive performance indicate that the mechanism of interference control might be linked to specific OCD symptoms, potentially mediated by the mutual constituents of affective and executive CSTC circuits.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. Conceptualization: JHo, IF, and PS. Data Acquisition: IF, AF, EN, and PS. Formal Analysis: DG, IF, and JHl. Funding Acquisition: JHo. Methodology: IF, JHo, JK, JHl, PS, DG, and AF. Project Administration: IF, PS, and JHo. Supervision: JHo. Visualization: DG and IF. Writing-Original Draft: IF, JHo, AF, and DG. Writing and Editing: IF, JHo, JHl, DG, AF, EN, PS, and JK.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.


Time series demonstrate that live cell imaging of lung lobes can be performed for at least 4 h post mortem. Time-lapse movies illustrate the dynamics of the nanoparticles within the pulmonary circulation and their uptake by immune cells. Moreover, the exchange of nanoparticle material between cancer cells was observed over time. Fluorescent monocytes in lungs of CD68-EGFP transgenic mice could be visualized within blood vessels in the process of interaction with tumor cells and nanoparticles. This high resolution ex vivo live cell imaging approach provides an excellent 4D tool to obtain valuable information on the behavior of tumor and immune cells at first encounter with nanoparticles and may contribute to the understanding of how nanoparticles interact with cells supporting the development of therapeutic strategies based on nanoparticulate drug delivery systems.A successful clinical translation of novel nanoparticle-based cancer therapeutics requires a thorough preclinical investigation of their interaction with immune, tumor and endothelial cells as well as components of the tumor-microenvironment. Although high-resolution microscopy images of fixed tumor tissue specimens can provide valuable information in this regard, they are only static snapshots of a momentary event. Here we describe a superior alternative fluorescence microscopy approach to assess the feasibility of investigating nanoparticle-cell interactions in the mouse lung live and over time at nanometer resolution. We applied fluorescent lung tumor cells and Barium-based fluorescently labeled nanoparticles to nude mice or to CD68-EGFP transgenic mice for visualization of the monocyte-macrophage lineage. Shortly before imaging, fluorescently labeled lectin was intravenously injected for staining of the blood vessels. The lung was filled  Routinely used chemotherapy has a number of weaknesses, including poor pharmacokinetics, low specificity and off target effects, leading to substantial side-effects. Nanoparticle (NP)-based cancer therapeutics have therefore been increasingly explored, because NPs have a number of advantages compared to “naked” therapies: (i) improved drug solubility and stability, (ii) prolonged drug half-live in plasma, (iii) minimized off-target effects, (iv) improved accumulation of drugs at a target site due to the possibility of functionalization and (v) better controlled concentration of drugs by packaging in NPs. Furthermore, recent efforts allowed the incorporation of therapeutic agents into biocompatible NPs, therefore, reducing undesirable local or systemic effects  and T cells as promising targets for pulmonary NP therapies , magnetic resonance imaging (MRI), and positron emission tomography (PET) or ultrasound do not provide the resolution to image at the cellular or nanomaterial resolution.Imaging of the numerous processes and interactions of NPs with lung immune cells, microenvironment and/or tumor cells and the elimination of NPs from the lung has traditionally been done by histology and immunohistochemistry. While these microscopic methods provide cellular nanoscale resolution, they are only static snapshots of momentary events and cannot provide the same information as live cell imaging. Other live cell or ex vivo live cell confocal microscopy of entire mouse lung lobes provides an excellent 4D tool for imaging of several dynamic processes in tumor tissue, such as the traffic of cells, shedding of extracellular vesicles (EVs) and the accumulation of NPs in tumor tissue.Here we demonstrate an alternative technique for monitoring the live interaction of monocytes/macrophages and tumor cells with NPs in the lung. We show that in vitro/in vivo use. The hydrodynamic diameter of the particles was around 120 nm. Fluorescent labeling of Ba-NPs was performed via EDC/NHS coupling chemistry of amino-functionalized Atto488  or Cy3 fluorescent dyes to COOH groups of the stabilizing polymer. Subsequently, the labeled particles were dialyzed against water in order to remove non-bounded dye. The Ba-NPs were tested in vitro and were found to be non-toxic and had no effect on the vitality of mammalian cells.Barium sulfate (BaSO4)-based nanoparticles (Ba-NPs) were produced by chemical precipitation at ambient conditions. After a purification step the particles were sterically stabilized using a biocompatible polymer. The so obtained highly stable colloidal suspension was sterilized and formulated for Lectin-Alexa647 (Alexa647 labeled Isolectin B4 from Bandeiraea simplicifolia) was a kind gift from Roche Pharma, Penzberg.n = 5 per test series) were conducted at 4 h and 24 h incubation time and with a final Ba-NP concentration of 15–45 mg/ml. The results were compared against a positive (100% death cells) and negative control . The same experiments were performed with A549 cells with a Ba-NPs concentration of 30 mg/mL and 24 h incubation time.A cell proliferation assay (MTT-assay) was performed using the MCF-7 in order to assess the cytotoxicity of the Ba-NPs. The experiments (2 atmosphere. A549-mCherry cells were grown in DMEM high glucose (Gibco) supplemented with 10% fetal calf serum (FCS) (Gibco). LL/2-RFP cells were grown in DMEM high glucose supplemented with 1% penicillin/streptomycin, 1% non-essential amino acids (Gibco) and 10% FCS. When indicated, 1 × 106 A549-mCherry cells were incubated with 13 mg/ml Ba-Atto488 NPs overnight, washed of excess NPs the next morning and run over a 40 μm cell strainer (Corning) before application to the mice.Human A549-mCherry lung tumor cells  and murine LL/2-red fluorescence protein (RFP) (Lewis Lung) cancer cells  were kept at 37°C and 5% COin-vivo procedures were performed in compliance with the guidelines of the European Directive (2010/63/EU) and the German ethical laws and were approved by the administration of Lower Saxony, Germany.All animal ad libitum.Pathogen-free male NMRI-Fox1nu/nu mice, 6–8 weeks of age were purchased from Charles River Laboratories Inc. C57BL/6-Tg(CD68-EGFP)1Drg/J were originally purchased from The Jackson Laboratory and then bred in house. All animals were housed in a controlled environment with a regular 12 h dark:light cycle, at 22°C and were fed laboratory chow and tap water 6 LL/2-RFP cells or 1 × 106 Ba-Atto488-NP-loaded A549-mCherry cells or with 6 mg Ba-Cy3 NPs, 5 min before sacrifice.Healthy mice were injected intravenously (i.v.) either with 1 × 106 A549-mCherry cells into male NMRI-Fox1nu/nu mice. The development of lung metastasis was assessed by microCT  every other week. Once lung metastasis were visible (about 8 weeks after induction), 6 mg Ba-Atto488 NPs were injected i.v. 5 min before sacrifice.Experimental lung metastases were obtained by i.v. injection of 1 × 10All mice were i.v. injected with 100 μg Lectin-Alexa647 shortly before sacrifice.The lungs were prepared as described previously with some modifications  and the whole equipment was turned on the evening before imaging to minimize tissue movement due to temperature changes.A Zeiss LSM880 confocal laser scanning microscope  was used, equipped with an Airyscan detection unit and GaAsP-PMT/Spectral detectors, a 20 × air objective lens , a motorized stage, incubator for live cell conditions and a tuneable laser (470–670 nm). mCherry and Red Fluorescence Protein (RFP) were excited at 561 nm, Ba-Atto488 NPs and CD68-EGFP were excited at 488 nm and Lectin-Alexa647 was excited at 633 nm. The experiments were performed under live cell environmental conditions  overnight and then cleared using the ethyl-3-phenylprop-2-enoate  protocol described before  and imaged using an Axiovert 200 M inverted microscope (Carl Zeiss Microscopy GmbH).All images, including volumetric 3D images of microscopy z-stacks, were processed and analyzed with the software Imaris 9.1.2 (Bitplane), Imaris Stitcher, FIJI (an image-processing package based on ImageJ) and Graph Pad Prism 7.05 . Maximum 3D projections of z-stacks are presented. Videos were produced from time series using Imaris 9.1.2.ex vivo live cell imaging we chose three biological scenarios in the mouse lung:To demonstrate the feasibility of (i) To evaluate the immediate interaction of NPs and tumor cells with the immune cells and the surrounding lung tissue we either i.v. injected LL/2-RFP cells  To evaluate the inter-cellular behavior of tumor cells, we incubated A549-mCherry cells with Ba-Atto488 NPs overnight and injected them i.v. into Nu/Nu mice.(iii) To visualize the tumor cell dynamics in the tumor mass and the interactions of NPs with tumor tissue we used the model of experimental lung metastasis.The use of the fluorescent protein-expressing-cancer cells (A549-mCherry and LL/2-RFP), transgenic fluorescent protein expressing animals (CD68-EGFP), and fluorescent tags (Lectin-A647 and Ba-NPs) enabled the easy discrimination of tumor cells, tumor masses, blood vessels and NPs and their specific interactions Hoffman, . The humin vitro (data not shown).As an illustrative example for NPs we chose Ba-based NPs of about 120 nm in size that are non-toxic. Cell proliferation tests (MTT assay) at 4 h incubation showed no cytotoxic effects over the tested concentration range while incubation at 24 h time showed a concentration dependent trend, but still no relevant cytotoxic effects  . MTT assin vivo with the widely used protein Lectin, which is known to bind to glycoproteins located in the glycocalyx and in the basal membrane of endothelial cells. The visualization of the blood vessels provided a good “counterstain” of the lung tissue structure and excellent means to monitor the stability of the tissue over the time measurements.Blood vessels were stained We chose to sacrifice the animals shortly after injection of NPs because we were interested in visualizing the immediate reaction of tumor and immune cells to NPs in the lung. The filling of the lungs with agarose following the sacrifice of the mice prevented the collapse of the lungs and maintained the structural integrity of the lung. Environmental control settings during the image acquisition process provided physiological conditions to maximize the length of cell viability within the explanted lung. The total process from i.v. injection of NPs or tumor cells to acquisition of the first image took on average 40–60 min.ex vivo imaging approach allowed us to assess several live cell processes in the lung tissue at cellular resolution and up to a time period of at least 4 h. These findings are in accordance with results previously described by van den Bijgaart et al.  which can be generated by primary tumors and can lead to metastasis. From the circulation, the cells reach the lung within minutes. We therefore i.v. injected LL/2-RFP cells into CD68-EGFP transgenic mice to examine the behavior of tumor cells at the immediate contact with immune cells.In the first hour of acquisition we could observe the recruitment of macrophages/monocytes to the tumor cell-rich regions and their surveillance . SupplemThe dynamic movement of monocytes/macrophages and tumor cells were tracked over time in lungs of CD68-EGFP mice which received an i.v. injection of LL/2-RFP tumor cells as shown in No other cell is more appropriate than the macrophage to show the need that cells have to be examined in their natural setting. Only recently, Hussel and Bell stressed in a review that alveolar macrophages exist in a unique microenvironment of the airway lumen which can have a considerable influence on many aspects of their phenotype, function and turnover . The described technique using agarose to fill the lung is thus mostly suited for visualization of processes that occur within the blood and lymphatic vessels and interaction of NPs with blood residing immune cells as well as endothelial cells. However, some aspects such as the induction of cell apoptosis by drugs or the internalization of NPs by alveolar macrophages may well be studied by including the drugs or NPs in the agarose. We included Ba-Cy3 NPs in the agarose and could indeed detect the phagocytosis of NPs by macrophages (data not shown).A limitation of the technique is the necessity to use agarose to fill the lung in order to prevent collapsing of the lung. This evidently implies a somewhat unphysiological environment in the bronchial and alveolar spaces and most certainly represents a barrier for alveolar macrophages and their mobility. Processes that occur in these spaces can therefore only be observed in limits. Especially the phagocytosis of NPs and tumor cells by alveolar macrophages are likely to be hampered. We have attempted to image ex vivo live cell imaging may thus be used to test whether nanovaccines can be efficiently drained into the lymphatic system, enabling accumulation in lymph nodes which contain a high number of immune cells and which coordinate diverse immunomodulation events. This method may be also used to test the size dependency of NPs for lymphatic drainage of NPs. Reddy et al. reported for instance that after intradermal injection, interstitial flow transported 25 nm small nanoparticles highly efficiently into lymphatic capillaries and their draining lymph nodes, targeting half of the lymph node–residing dendritic cells, whereas 100 nm NPs were only 10% as efficient . Furthermore, dark areas were visible in the center of the nodules, suggestive of necrosis . This led to a very limited distribution of Ba-Atto488 NPs in the tumor nodules when compared to the healthy lung tissue, where the NPs were generally well-dispersed throughout. Some lectin-stained vessels are present at the border of the tumor and these are partly leaky, which is demonstrated by an accumulation of NPs in the surrounding tumor tissue  (Sanna and Sechi, ex vivo visualization procedures, including light sheet microscopy and histology, as shown in ex vivo live cell confocal imaging of entire lung lobes is thus a good preclinical method of pre-assessing the accumulation of NP-based drugs at the desired site of action, in particular in the case of tumor masses.Following confocal live cell visualization, the tissues can be prepared for all other common Also the behavior of individual or clusters of cells within a tumor mass can be studied. We have for instance observed astonishingly strong movement of tumor cells in already established tumor masses that were induced several weeks before imaging . This suex vivo lung live cell imaging, an example of which can be seen in The process of extra- and intravasation of tumor cells into and from blood vessels are also processes that could be studied in detail by Another process that we observed with the presented technique is the exchange of material between different cells. In the following example we have incubated A549-mCherry cells with Ba-Atto488 overnight and then injected them i.v. in a NMRI-Fox1nu/nu mouse. Intercellular organelle and EV exchange has been described many times, but most have only been shown in cell culture (Rogers and Bhattacharya, ex vivo live cell microscopy approach. As shown in The process of EV production by tumor cells was also captured by our ex vivo live cell imaging are oncosomes. Thus, this method may support the better understanding of EVs and their distinctive roles and may also contribute to the clarification of the controversy that exists to date on the nomenclature of EVs (Meehan et al., A second example of EVs shedding of similar size can be seen in Ex vivo live cell confocal microscopy provides a new way of studying the interactions of nanoparticles with immune and tumor cells and the vessel endothelium. As we show by several examples in the mouse lung, the method presents a notable alternative to more demanding techniques such as intravital lung microscopy using windows and to in vivo optical imaging that provide substantially lower resolution. It is mostly suitable for the visualization of processes that occur within the vessel/tumor interfaces, such as delivery of NPs to the tumor, interaction of NPs with blood monocytes, intra- and extravasation. Thus, the method may support the characterization of nanoparticulate therapies, studying the dependence of NP uptake on the heterogeneity and microenvironmental features of tumors as well as the optimization of NPs size and functionalization for specific targeting. By engineering specific NP surfaces it should in theory be possible to modulate and control host responses. Such processes may also be studied by this technique. While we are using fluorescently tagged cell lines and transgenic mice to visualize specific cells, an alternative or additional approach could be the injection of fluorescent antibodies before sacrifice and imaging of the lung. Moreover, the approach is able to supply new information on cell processes that are not directly and instantaneously affected by a loss of air flow or breathing mechanics, such as the behavior of tumor masses in the lung, including the production of extracellular vesicles and their fate, intercellular exchange of material/organelles and the processing of tumor material by immune cells. While we focus on interactions with nanoparticles, this method may provide novel information on a number of other cellular events, such as the phagocytosis of tumor cells by macrophages and other cells; the immediate cellular effects of treatments such as chemotherapies, including the live visualization of apoptotic events; the immediate reaction of immune cells at encounter of allergens and real-time detection of intracellular and metabolic processes in live cells.All datasets generated for this study are included in the article/The animal study was reviewed and approved by Nds. Landesamt für Verbraucherschutz und Lebensmittelsicherheit.FR-G conceived and designed the experiments, acquired and analyzed the data, and wrote the paper. NF acquired and analyzed the light sheet microscopy data. AK designed and prepared the nanoparticles. FA conceived the experiments and contributed to writing the paper. MM conceived, designed, prepared the experiments, and wrote the paper. All authors contributed to the article and approved the submitted version.AK was employed by nanoPET Pharma GmbH. The remaining 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.


On the other hand, we showed that the pGRS is not associated with cardio-metabolic comorbidities in children and that certain environmental factors interact with the genetic predisposition to the disease. Finally, according to the results derived from a weight-reduction metformin intervention in children with obesity, we discarded the utility of the pGRS as a pharmacogenetics marker of metformin response.Polygenetic risk scores (pGRSs) consisting of adult body mass index (BMI) genetic variants have been widely associated with obesity in children populations. The implication of such obesity pGRSs in the development of cardio-metabolic alterations during childhood as well as their utility for the clinical prediction of pubertal obesity outcomes has been barely investigated otherwise. In the present study, we evaluated the utility of an adult BMI predisposing pGRS for the prediction and pharmacological management of obesity in Spanish children, further investigating its implication in the appearance of cardio-metabolic alterations. For that purpose, we counted on genetics data from three well-characterized children populations , following both cross-sectional and longitudinal designs, expanding childhood and puberty. As a result, we demonstrated that the pGRS is strongly associated with childhood BMI Z-Score (B = 1.56, SE = 0.27 and  Among noncommunicable common diseases, overweight and obesity in children are a public health problem that has raised concern worldwide . CharactTwin studies have proven a strong heritable component of body mass index (BMI), and genome-wide association studies (GWAS) have shown that adult BMI is influenced by hundreds of common genetic variants . EvidencAlthough initial expectations for obesity GWASs were high, the results derived after two decades of research have not met the previsions  of the BMI heritability ). ConseqThe inclusion of adult-BMI SNPs under a pGRS could serve therefore as an excellent predictive, and preventive, tool for facing the obesity-associated morbidity and mortality from the early periods of life. Although some previous studies have already investigated the utility of adult-BMI pGRSs for the management of obesity in children ,20,21,22(1)To demonstrate how a pGRS can quantify inherited susceptibility to obesity and its cardio-metabolic comorbidities in children.(2)To evaluate the effects of genetic predisposition for obesity during childhood and how they evolve when entering puberty.(3)To describe the plausible modulatory role of environmental factors over inherited genetic susceptibility in children.(4)To investigate the utility of the pGRS for the pharmacological management of obesity in children.The present study design consisted of three independent children populations following cross-sectional and longitudinal approaches. A general description for each study population as well as each study design are presented in N = 1699), current genetic analyses were performed in a subset population of 574 children (293 girls) who had good quality DNA samples. Children were recruited at three Spanish health institutions: Lozano Blesa University Clinical Hospital in Zaragoza, Santiago de Compostela University Clinical Hospital in Santiago de Compostela and Reina Sofia University Clinical Hospital in Córdoba. Obesity status was defined according to BMI by using the age- and sex-specific cut-off points proposed by Cole et al. (2000) ,2726,27]. (2000) . For theWith the aim of studying the effects of the pGRS on BMI changes during the course of childhood and puberty, we also performed a longitudinal analysis using data from 96 boys and girls undergoing sexual maturation B recruithttps://www.clinicaltrialsregister.eu/ctr-search/trial/2010-023061-21/ES). Among the 160 subjects participating in the original RCT, 124 (59 placebo (29 boys) and 65 treated children (32 boys)) had an appropriate DNA sample quality for the present genetic analyses. General characteristics of the selected study population at baseline and post-treatment stages are summarized in the In order to test whether the constructed pGRS presents utility for the pharmacological management of obesity in children, a third obesity cohort was submitted to genetic analyses in the present work. This cohort corresponded to a previous multicentre and double blind randomized controlled trial (RCT) conducted in 124 children with obesity C. A compAll described projects were conducted in accordance with the Declaration of Helsinki (Edinburgh 2000 revised) and followed the recommendations of the Good Clinical Practice of the CEE (Document 111/3976/88 July 1990) and the legally enforced Spanish regulation, which regulates the clinical investigation of human beings . The Ethics Committee on Human Research of the University of Granada (ID code: 01/2017), the Ethics Committee of the Reina Sofía University Clinical Hospital of Cordoba (ID code: 260/3408), the Bioethics Committee of the University of Santiago de Compostela (ID codes: 2011/198 and 2016/522), the Ethics Committee in Clinical Research of Aragon (ID codes: 12/2010 and 22/2016) and the Ethics Committee for Biomedical Research of Andalusia on 15 January 2012 (acta 1/12) (ID code: 2010-2739) have approved all experiments and procedures. All parents or guardians provided written informed consent, and the children gave their assent.® DNA Investigator Kit for coagulated samples and the Qiamp® DNA Mini & Blood Mini Kit for noncoagulated samples . All extractions were purified using a DNA Clean and Concentrator kit from Zymo Research . Genotyping was performed by TaqMan allelic discrimination assay using the QuantStudio 12K Flex Real-Time PCR System .In all participants of the present study genomic DNA was extracted from peripheral white blood cells using two kits, the QiampA total of 56 previously BMI-associated SNPs from the largest and most comprehensive GWAS performed to date in obesity research  were conIn all described cohorts, body weight (kg), height (cm) and waist circumference (cm) were measured using standardized procedures, and the BMI Z-score was calculated based on the Spanish standards reference . Blood pFor study population 1, environmental exposures were further assessed through an interview that focused on PA, sedentary behaviors, disease family history and familial educational level (at mother’s and father’s levels separately). Among all available environmental data, only quantitative or ordinal variables were selected for interaction analyses.This resulted in 47 lifestyle questions described in the t-tests, pairwise Mann–Whitney U-tests and Dunn tests were applied conveniently as post-hoc analyses to determine which experimental groups differ from each other. Values in descriptive tables are expressed as mean and standard deviation, or median and range if not normally distributed. In the descriptive statistics of the longitudinal cohort, within-group changes from baseline to puberty in all continuous measurements were assessed by means of a paired design; employing either a paired t-test or a Wilcoxon signed-rank test. Between-group differences were instead assessed by conveniently applying One-way Anova, Kruskal–Wallis or Welch tests to the computed delta values (T1–T0) for each continuous measurement.All continuous variables were tested for normality using the Shapiro–Wilk test and transformed when necessary by means of the natural log or the rank-based inverse normal transformation. Heteroscedasticity between experimental groups was explored by means of the Levene test. One-way Anova, Kruskal-Wallis and the Welch test were employed to assess group differences in measurements according to standard statistical assumptions. Pairwise-stats R package. This function uses the Akaike information criterion (AIC) to select variables for a linear model. Adjusted R2 and model deviance (D2) were calculated to assess the amount of outcome variability explained by each model. In all analyses, age, gender, pubertal stage, origin, height and BMI Z-score where adjusted for as confounders when necessary. Linear regression models were evaluated by model control (investigating linearity of effects on outcome(s), consistency with a normal distribution and variance homogeneity). All residuals- vs.-fitted, normal Q-Q, scale-location and residuals- vs.-leverage plots are available upon request. A p-value < 0.05 was considered as statistically significant. Given the number of analyzed markers, we also considered the false discovery rate (FDR) as in Benjamini and Hochberg to correct for multiple hypothesis testing in all analyses.In the study population 1, logistic regression models were applied in order to test whether higher genetic risk scores were observed for subjects with obesity than for normal weight controls. A logistic regression model was further applied for comparing obesity prevalence among participants presenting a high-risk genetic profile  versus those belonging to the reference quartile (Q1). Multiple linear regressions were employed instead to investigate the relationship between continuous measurements (including BMI Z-score) and the pGRS. For these analyses, the pGRS was treated both as a continuous and discrete variable (quartiles). To determine which SNPs within the pGRS had an independent contribution in the association with BMI Z-Score, we further performed stepwise linear regression using the “step” function included in the PredictABEL and the pROC R packages.The ability of the pGRS to comprehensively discriminate between normal weight and subjects with obesity was quantified  using the area under the curve (AUC) of the receiver operating characteristic curve. This plot represents the true positive rate (sensitivity) versus the false-positive rate (specificity) and is equivalent to the overall probability that the predicted risk of an individual with disease is higher than the predicted risk of an individual without disease ,39. Mode1–T0).In order to study the ability of the pGRS as a predictor of future obesity outcomes following puberty entrance (study population 2), we performed logistic regression models with the dichotomized pGRS as an independent predictor variable (1st and 2nd tertiles vs. 3rd tertil), including the longitudinal experimental group classifications from All statistical analyses were performed in R environment, version 3.6.0 .E) for each collected lifestyle factor (E) individually. In addition to the pGRS × E interaction term, each tested model also included covariates such as origin and puberty, in accordance with previously published recommendations ), which could be sufficient for clinical discrimination. All these results are in concordance with previous insights from Butler et al., (2019) , who demIn previous studies performed in adults, obesity pGRSs have also yielded secondary findings for multiple cardio-metabolic outcomes, including a heightened risk of all-cause mortality, diabetes, coronary artery disease, hypertension, stroke, and venous thromboembolism, all of them after correcting for BMI. While we knew the clinical association of obesity with these outcomes and conditions, the pGRS correlation now adds the genomic underpinning. To date, no studies have demonstrated such associations in children populations otherwise. Here, we only found slightly significant associations (BMI-adjusted) between the pGRS and certain lipid metabolism outcomes , none ofFrom our gene-environment cross-sectional approach, we found that only the educational level of the parents demonstrated a significant interaction with the pGRS. Compared with other socioeconomic indicators, the educational level of the mother is the one that had presented the strongest association with unhealthy factors in literature, such as adiposity, in both children and adolescents ,48. PartAlthough we have previously shown that certain individual obesity-SNPs could act as pharmacogenetics regulators of metformin response in children with obesity , we hereAmong the limitations of our current approach, we can highlight the inclusion of only European ancestry individuals, limiting extrapolation for other ancestries and the lack of objectively measured physical activity and diet assessments. These important questions remain unanswered and will define the potential benefit derived from using this obesity pGRS.Prevention of key medical conditions such as obesity has been a long-standing dream that largely remains unfulfilled. If we are to take advantage of the opportunity, we need to know as much as possible for prediction, acknowledging it will never be deterministic. The obesity pGRS reported in the present study provides an extremely powerful tool for the early risk detection. While there remains uncertainties and practical limitations for making such pGRS results widely available, such as the requirement for considerable education for the medical community and the general population, we are moving in the right direction for someday pre-empting important conditions that would have otherwise been manifest.


The degree to which elevated mortality associated with weekend or night-time hospital admissions reflects poorer quality of care (‘off-hours effect’) is a contentious issue. We examined if off-hours admissions for primary percutaneous coronary intervention (PPCI) were associated with higher adjusted mortality and estimated the extent to which potential differences in door-to-balloon (DTB) times—a key indicator of care quality for ST elevation myocardial infarction (STEMI) patients—could explain this association.Nationwide registry-based prospective observational study using Myocardial Ischemia National Audit Project data in England. We examined how off-hours admissions and DTB times were associated with our primary outcome measure, 30-day mortality, using hierarchical logistic regression models that adjusted for STEMI patient risk factors. In-hospital mortality was assessed as a secondary outcome.From 76 648 records of patients undergoing PPCI between January 2007 and December 2012, we included 42 677 admissions in our analysis. Fifty-six per cent of admissions for PPCI occurred during off-hours. PPCI admissions during off-hours were associated with a higher likelihood of adjusted 30-day mortality . The median DTB time was longer for off-hours admissions  than regular hours . After adjusting for DTB time, the difference in adjusted 30-day mortality between regular and off-hours admissions for PPCI was attenuated and no longer statistically significant .Higher adjusted mortality associated with off-hours admissions for PPCI could be partly explained by differences in DTB times. Further investigations to understand the off-hours effect should focus on conditions likely to be sensitive to the rapid availability of services, where timeliness of care is a significant determinant of outcomes. Internationally, hospital admission during the weekend has been associated with higher mortality rates than during weekdays, a phenomenon that is popularly referred to as the ‘weekend effect’.The issue of the weekend effect has prompted much debate due to questions on the extent to which it exists in the first place, and if so, the range of possible explanations on what causes it.An important consideration is the possibility that an off-hours effect may not exist uniformly across all conditions. In fact, when Bell and Redelmeier demonstrated higher mortality associated with weekend hospital admissions in their seminal study, they posited that a weekend effect would only exist for certain conditions that are more sensitive to the rapid availability of specialised services and personnel.Current guidelines for the treatment of ST elevation myocardial infarction (STEMI) requires a rapid and coordinated response at time of patient admission.In this study, we made use of data from a nationally mandated population-based acute coronary syndromes (ACS) registry in England to (1) assess if off-hours admissions for PPCI in England were associated with higher adjusted mortality and (2) examine the extent to which potential differences in DTB times between regular and off-hours PPCIs could explain this association.We carried out a registry-based prospective observational cohort study using the Myocardial Ischaemia National Audit Project (MINAP) data, a clinical registry of patients hospitalised with ACS in England and Wales  risk score to adjust for patient case-mix. The AMG risk score was derived from the Global Registry of Acute Coronary Events (GRACE) risk score, modified and adopted for MINAP data by the National Institute for Health and Care Excellence.We included hospital annual PPCI volume to account for potential hospital volume effects on outcomes. To account for seasonality effects and aggregate time trends, we included controls for month and year. We also controlled for deprivation using the Index of Multiple Deprivation (included as deciles), which ranks small areas in England (Lower-layer Super Output areas) from one (most deprived area) to 32 844 (least deprived area).Patient DTB times were subsequently added to our logistic regression model (as a continuous variable) to determine if accounting for DTB times changed our previous adjusted point estimates for patient mortality following PPCIs performed during off-hours relative to regular hours. A significant change in the point estimates would be indicative of DTB times being a mediating factor of any association that may exist between admission time for PPCI and mortality.Overall model performance was assessed using the area under the receiver operating characteristics curve (C statistic).Statistical analyses was performed using STATA MP V.14.Our analytical cohort consisted of 42 677 patients directly admitted for PPCI to PPCI-capable hospitals in England between 2007 and 2012. Overall, 56% of these admissions occurred during off-hours. The regular hours cohort had a lower proportion of current smokers but were on average, older, and had a larger proportion of patients with a history of stroke and chronic renal failure. They were also generally sicker based on clinical risk factors, as reflected by the higher average AMG risk score compared with the off-hours group . There were no statistically significant differences between the two groups with respect to the other baseline patient characteristics included in the study.Overall, there was no statistically significant difference in unadjusted 30-day or in-hospital mortality rate between the two groups. The unadjusted 30-day mortality rate was 3.91% for PPCI admissions during regular hours and 3.86% for admissions during off-hours (p=0.80). The unadjusted in-hospital mortality rates for regular and off-hours admissions were 2.81% and 2.84% (p=0.82), respectively.After adjusting for the patient risk factors described in The model had good discriminative value, strongly predictive of mortality, with a C-statistic of 0.86 for 30-day mortality and 0.91 for in-hospital mortality.The median DTB time for PPCI was longer during off-hours  than regular hours , a statistically significant difference of 7 min (p<0.001). Weekday night-time admissions for PPCI had higher median DTB times than the corresponding weekday daytime admissions . During Overall, there was a modest increase in the gap in DTB times between regular and off-hours PPCIs at higher percentiles—for example, at the 95th percentile, the DTB times were 118 and 131 min, respectively .When we accounted for PPCI DTB time in the hierarchical logistic regression models, the difference in adjusted 30-day mortality and in-hospital mortality between regular and off-hours PPCI admissions was attenuated and no longer statistically significant . Online Our results were robust to using an alternative definition of off-hours—patients admitted for PPCI during off-hours had a significantly higher likelihood of adjusted 30-day and in-hospital mortality compared with regular hours, and after controlling for DTB time, the difference in adjusted 30-day and in-hospital mortality was attenuated and no longer statistically significant . In addiThe relationship between DTB times and mortality is detailed in In this study, we assessed if off-hours admissions of STEMI patients undergoing PPCIs in England were associated with higher adjusted mortality and evaluated the extent to which potential differences in DTB times between regular and off-hours PPCIs could explain this association. First, we found admissions for PPCI during off-hours were significantly associated with higher adjusted 30-day and in-hospital mortality compared with admissions during regular hours. Second, we confirmed small but significant differences in DTB times between regular and off-hours PPCIs—DTB times were, on average, longer for patients admitted during off-hours. After accounting for DTB time, the difference in adjusted 30-day and in-hospital mortality between regular and off-hours PPCIs was attenuated and no longer statistically significant, suggesting that higher adjusted mortality associated with off-hours admissions could be partly explained by differences in DTB times.While previous studies have found elevated mortality associated with weekend or night-time admissions, the reason why this phenomenon generates debate is the implication of poorer quality of care during off-hours admissions. If, for instance, elevated mortality during off-hours entirely reflected higher severity of illness, this would not necessarily be an ‘off-hours effect’ in the sense that the term usually generates debate. Potential selection bias may explain some of the excess mortality associated with off-hours admissions, where routinely collected in-patient administrative data may not be able to fully account for severity of illness.Previous studies that assessed STEMI mortality outcomes based on admission times found discrepant findings but differed widely on study design. Some were limited to a single high-volume PCI centre,We made use of a national clinical registry of ACS patients in England to better adjust for illness severity by accounting for clinical risk factors specific to STEMI. We focused on PPCI, which is superior to fibrinolytic therapy if performed rapidly by a team of expertsOur findings should be interpreted with several limitations in mind. First, even though we have taken measures to carefully adjust for patient risk factors, potential biases may have been introduced by unobserved patient factors that might vary based on time of presentation. In addition, we had to exclude patients with missing AMG risk score data. Fifty-four per cent of admissions in this excluded group were during off-hours, similar to the proportion of off-hours admissions in our analytical cohort (56%). The DTB times and unadjusted mortality rates for this excluded group are reported in Second, we lacked information on hospital-level factors—we did not have access to the name or location of the hospitals. Further, MINAP does not have information on hospital staffing levels. Therefore, we could not assess how these factors may contribute to variations in DTB times and PPCI outcomes. Third, we did not have access to potentially important clinical variables on procedural characteristics .In assessing the reperfusion times for STEMI patients in England, it should be noted that the DTB times reported in this study show good overall performance, both in terms of the proportion of PPCIs conducted within the 90 min benchmark recommended by clinical practice guidelinesBell and Redelmeier considered acute myocardial infarction (AMI) a ‘control’ condition, where a weekend effect was not anticipated and not found in their study.The logical next step would be to understand the stages in the diagnostic, decision-making and treatment pathways of STEMI patients where potential delays occur that may lead to longer DTB times for off-hours admissions. Policy measures to address the off-hours effect should be based on a detailed understanding of how and why care quality varies in the care pathway for acute conditions that are more sensitive to the rapid availability of key resources and personnel. Therefore, it would be beneficial for future research to focus on understanding the temporal variation in care quality for these conditions, where the timely delivery of care can significantly improve outcomes—for example, Bray and colleagues found several patterns of variation in different aspects of quality in acute stroke care.


Lung adenocarcinoma (LUAD), a major and fatal subtype of lung cancer, caused lots of mortalities and showed different outcomes in prognosis. This study was to assess key genes and to develop a prognostic signature for the patient therapy with LUAD.  RNA expression profile and clinical data from 522 LUAD patients were accessed and downloaded from the Cancer Genome Atlas (TCGA) database. Differentially expressed genes (DEGs) were extracted and analyzed between normal tissues and LUAD samples. Then, a 14-DEG signature was developed and identified for the survival prediction in LUAD patients by means of univariate and multivariate Cox regression analyses. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to predict the potential biological functions and pathways of these DEGs.  Twenty-two out of 5924 DEGs in the TCGA dataset were screened and associated with the overall survival (OS) of LUAD patients. 14CID="C008" value=" "DEGs were finally selected and included in our development and validation model by risk score analysis. The ROC analysis indicated that the specificity and sensitivity of this profile signature were high. Further functional enrichment analyses indicated that these DEGs might regulate genes that affect the function of release of sequestered calcium ion into cytosol and pathways that associated with vibrio cholerae infection.  Our study developed a novel 14-DEG signature providing more efficient and persuasive prognostic information beyond conventional clinicopathological factors for survival prediction of LUAD patients. Lung cancer continues to be the leading cause of cancer-related mortality around the world , in whicDifferentially expressed genes (DEGs) that regulated by gene transcription are implicated in diverse biological processes. Gene-expression profiling analysis made some progresses in predicting overall survival (OS) in NSCLC , 6, 7. MAlthough several genes or long noncoding RNA expression signatures, including programmed death-ligand 1(PD-L1), have been recently proposed for predicting the OS in NSCLC , 8–10, tIn this study, we identified a 14-DEG signature as a predictor of survival risk of LUAD patients using a cohort of 522 cases from The Cancer Genome Atlas (TCGA) database. We employed a survival-associated risk score formula to identify a novel 14-DEG prognostic signature from the TCGA dataset of 522 LUAD patient samples. To show the conscientiousness of this signature, the specificity and sensitivity of our model were examined by the area under ROC curve (AUROC) analysis. A 14-DEG signature which could distinguish patients between good and poor survival was developed by means of Cox regression analysis and risk score model method. A higher area under curve (AUC) of the receiver operating characteristic (ROC) curve confirmed good sensitivity and specificity of the prognostic model, while multivariate Cox regression analysis and stratified analysis indicated the independence of predictive capacity of the 14-DEG prognostic signature. Besides, the functional enrichment analysis demonstrated that the 14-DEG may be probably involved in the progression of LUAD through exerting their roles in LUAD-related function of release of sequestered calcium ion into cytosol and pathways that associated with vibrio cholerae infection. Therefore, our finding may provide insights into the predictive capacity of DEG signature elaborating LUAD.https://cancergenome.nih.gov/), including clinical features. The patients with the following criteria were filtered: patients with complete information of RNA expression profiles and clinical factors .The RNA-Seq data set of patients with LUAD was downloaded from the TCGA database  from the univariate analysis were considered as potential candidate DEGs associated with OS. To determine the independent predictive capacity of the 14-DEG signature for LUAD patients, a stepwise multivariate Cox regression analysis was executed to identify the predictive model with the best explanatory and informative efficacy.The RNA-seq expression values were transformed in log2 format to normalize the data. Univariate Cox regression analysis using the “Survival” R package was performed to clarify the association between DEG expression and patient survival. The DEGs (i=1N(Exp(i) · coe(i)) ) was developed to predict the risk score for each patient based on the multivariate Cox regression analysis. In accordance with our risk scoring system, patients were classified into high-risk and low-risk groups according to the median risk score. A Kaplan-Meier overall survival curve of the different stages was plotted, and the hazard ratio was calculated. Subsequently, the log-rank test was utilized to determine the survival differences between high-risk and low-risk groups. The sensitivity and specificity of the DEG prognostic model to predict clinical outcome were evaluated by calculating the area under curve (AUC) of the receiver operating characteristic (ROC) curve in the R package of “survival ROC” [A mathematical formula  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was carried out for DEGs with the aid of clusterProfiler R package. Only terms with The whole construction process in our research was showed in According to the defined criteria, a total of 5924 DEGs (including 5147 upregulated and 777 downregulated) were extracted between LUAD and normal samples . The resE-06, a set of 22 DEGs was selected. These DEGs were utilized in stepwise multivariate Cox regression analysis, and finally, 14 DEGs  were identified . ROC curves indicated that the AUC of the 14-gene signature was 0.769  by calculating the expression levels of the 14 DEGs in each patient. The log-rank test was used to determine the survival differences. As depicted in as 0.769 , which pp = 7e − 16, shown in p < 0.001) (n = 26), stratification analysis was carried out only in stage I, II, and III patients. Log-rank test for patients in stage I demonstrated that the 14-DEG signature could distinguish patients with significantly different survival time  and III (p = 9e − 05) patients  . Therefo Figures . BesidesThe biological functions and pathway analyses were conducted using R package clusterProfiler. The results showed that DEGs were enriched in 181 GO biological process (BP), 10 cellular component (CC), and 14 molecular function (MF) terms. The GO categories of GO: 0051209~release of sequestered calcium ion into cytosol (BP), GO: 0005788~endoplasmic reticulum lumen (CC) and GO: 0008191~metalloendopeptidase inhibitor activity (MF) were mainly clustered, respectively . The topNSCLC is a global health threat with high morbidity and mortality, up to 0.6 and 0.1 percent, respectively . LUAD acIncreasing evidences suggest that DEGs play indispensable and important roles in the tumorigenesis, TNM staging, and progression of lung cancer. Although several researches have identified a number of DEGs with prognostic value in NSCLC, especially in LUSC , 11, fewα (ERO1A) is a poor prognostic marker for non-small-cell lung cancer [In this study, 14-DEG signature related to overall survival of LUAD patients was identified. By means of univariate Cox regression analysis and stepwise multivariate Cox regression analysis, a novel 14-gene  prognostic signature was established and validated to demonstrate high specificity and sensitivity in predicting the overall survival time of LUAD patients. We calculated the RiskScore of each patient through the formula and the expression of selected DEGs. The patients were divided into high- and low-risk group by the median RiskScore ; then, we obtained the survival curve according to the survival rate of all LUAD patients. To our knowledge, C1QTNF6 has been recently identified as a novel biomarker exacerbating the outcome of lung adenocarcinoma patients . Combineg cancer . Level og cancer . Regulatg cancer , 20. FetThe carcinogenesis of LUAD is a multistep process hallmarked by a series of genetic alterations. In order to gain a further insight into the functional roles of the 14 DEGs, the correlation between their expression levels and the coexpressed protein-coding genes were analyzed. In the present study, we performed GO and KEGG enrichment analysis to explore the functions of the predictive DEGs. The results indicated that the prognostic 14-DEGs were involved in significant functional process, such as release of sequestered calcium ion into cytosol (BP), endoplasmic reticulum lumen (CC), and metalloendopeptidase inhibitor activity (MF) and enriched in KEGG pathways including vibrio cholerae infection, protein processing in endoplasmic reticulum, and calcium signaling pathway. Therefore, it is convincing to infer that the fourteen prognostic DEGs participate in the progression of LUAD in these LUAD-related biological pathways. However, further experimental studies are needed to confirm the functions of these DEGs. Our findings provide insights into prognosis-related genes of LUAD and may have a positive clinical capacity for prognosis prediction and target therapy in LUAD management.In summary, this study identified a novel 14-DEG prognostic signature which could predict the survival risk of LUAD patients. The signature exhibited independent prognostic capacity of clinicopathological factors and could predict survival outcomes of LUAD patients within the same TNM stage. This signature could be utilized to identify patients with high-risk scores who may be further desperate for more effective and individualized therapy. It could not only serve as a novel potential biomarker for the survival risk stratification of LUAD patient but also provide us a better understanding of molecular mechanisms involved in the development of LUAD. However, further molecular investigations, such as exploring the underlying mechanisms of these DEGs in LUAD development and performing independent cohorts of large sample sizes from institutions across the country or world, are necessary to confirm accuracy and stability for the prediction signature.


Antiretroviral therapy (ART) demonstrates high efficacy in reducing the risk of HIV transmission to sexual partners. However, it is not clear if the use of pre-exposure prophylaxis (PrEP) in HIV-1-serodiscordant couples is necessary during natural conception when the HIV-positive partner exhibits a suppressed viral load. The purpose of this study was to assess the role of PrEP during natural conception in this population.A retrospective, multicenter study was conducted in a cohort of HIV-1-serodiscordant couples  with childbearing desires. HIV-positive male partners were treated with ART and achieved viral suppression for more than half a year. The HIV-negative female partners were either treated with PrEP or not treated with PrEP, and outcomes were compared between the two treatment groups.Of 246 HIV-1-serodiscordant couples in whom the HIV-positive partner achieved viral suppression, 104 seronegative women were treated with PrEP during natural conception and 142 seronegative women were not treated with PrEP. There were 410 condom-less sexual acts in couples treated with PrEP and 615 condom-less sexual acts in couples not treated with PrEP. We observed no instances of HIV transmission in HIV-1-serodiscordant couples with or without the use of PrEP during the process of natural conception.Our results show that PrEP had minimal influence in reducing the risk of HIV transmission during natural conception in HIV-1-serodiscordant couples with a stably suppressed viral load. Thus, it may be an acceptable option for HIV-negative partners to not use PrEP during the process of natural conception if the HIV-positive partner has achieved viral suppression for more than half a year. Antiretroviral therapy (ART) has not only significantly reduced the risk of sexual transmission of the human immunodeficiency virus (HIV), but has also dramatically improved the life expectancy in people living with HIV , 2. A stIn developing countries such as China, reproductive options for HIV-serodiscordant couples are limited in that intrauterine insemination and in vitro fertilization can be expensive or are not accessible to HIV-infected people. With the effectiveness of ART in reducing the risk of sexual transmission, natural conception has become a relatively safe and exciting reproductive option for HIV-1-serodiscordant couples. In addition to timed intercourse and sustained viral suppression in HIV-positive partners, PrEP is used in HIV-negative partners to reduce the risk of HIV infection during the process of natural conception , 8.Recent studies have indicated that linked HIV infections were not observed in serodiscordant couples having condom-less sex when the HIV-positive partner exhibited a stably suppressed plasma viral load , 10. As This retrospective study examined the medical records of HIV-1-serodiscordant couples who were treated from January 2008 to January 2018 at sexually transmitted disease/acquired immunodeficiency syndrome clinics of Beijing Youan Hospital, Capital Medical University and the Third People’s Hospital of Shenzhen. All couples expressed a desire to conceive and were given general HIV information as well as information about the risk of transmitting HIV during natural conception. They were also told that it was unclear whether PrEP provided any additional benefit in reducing HIV transmission between partners in couples with a virally suppressed HIV-positive partner. The choice to use or not use PrEP was voluntary. The couples gave informed consent to have data extracted from their medical records. Ethical approval was obtained from the Ethics Committee of Beijing Youan Hospital, Capital Medical University.Preparation for fertilization was done as previously described . BrieflyThe HIV-positive male partner in the HIV-1-serodiscordant couples was treated with ART. Natural conception was advised in couples whose positive-partner exhibited sustained undetectable plasma viral load (< 50 copies/ml) for at least 6 months. Unprotected sexual relations were limited to the periovulatory period (timed intercourse).The HIV-1-discordant couples were divided into those who seronegative female partners were treated with PrEP and those whose seronegative female partners were not treated with PrEP. In the PrEP-treated group, zidovudine (AZT) + lamivudine (3TC), tenofovir/emtricitabine (TDF/FTC), TDF/FTC + raltigravir (RAL) or lopinavir/ritonavir (LPV/r)-based regimens  was administrated to the seronegative female partner starting the morning of luteinizing hormone-peak until 28 days after the last instance of condom-less intercourse. The antiretrovirals used for PrEP were chosen based on availability. The seronegative female partners were tested for HIV infection at 6, 12, and 24 weeks after the last occurrence of condom-less intercourse. After successful conception, the couples resumed condom use during sexual activities, and PrEP antiretroviral use ceased.p-value < 0.05 indicated statistical significance. All data were analyzed with SPSS version 17.0 .A descriptive analysis of the sociodemographic and clinical characteristics of the HIV-1-serodiscordant couples was performed. We analyzed the epidemiological characteristics, clinical profile of the HIV-positive partners, total number of unprotected acts of vaginal intercourse, and risk of horizontal transmission. We compared characteristics and outcomes between PrEP use and non-PrEP use groups using the chi-square or Mann-Whitney U test and stratified by sex. The number of vertical transmissions was recorded. A A total of 246 HIV-1-serodiscordant couples  were included in this study. Of the 246 couples, 104 seronegative women were treated with PrEP. The remaining 142 seronegative women were not treated with PrEP. The characteristics of the couples are shown in Table A total of 410 condom-less sexual acts were reported for the couples that received PrEP whereas 615 condom-less sexual acts were reported for the couples that did not receive PrEP. Seroconversion did not occur in either group of HIV-1-serodiscordant couples during natural conception. The number of successful pregnancies was 104 in the PrEP group and 142 in the non-PrEP group (Table PrEP has been shown to be an effective option for preventing the transmission of HIV. A previously published study reported a 99% reduction in the risk of HIV infection when PrEP was taken 7 days a week . A sub-sNatural conception is becoming an acceptable option for some HIV-serodiscordant couples in resource-limited settings, since reproductive technologies such as in vitro fertilization and intracytoplasmic sperm injection with prepared sperm are either too expensive or not available to HIV-infected people . With coOur study analyzed the use of PrEP during natural conception in HIV-1-serodiscordant couples in which the viral load in the HIV-infected partner was suppressed. Our results showed that there were no cases of HIV seroconversion in uninfected sexual partners in both the PrEP use and non-PrEP use groups. This suggests that natural conception is relatively safe without PrEP use when timed intercourse is employed in HIV-1-serodiscordant couples with a virally suppressed HIV-positive partner. Natural conception without PrEP has also been analyzed in other studies , 15.In 1994, a study showed that HIV-infected men treated with zidovudine monotherapy were half as likely to transmit infection to their female partners compared to untreated men . In 2009The effectiveness of ART in preventing HIV transmission is dependent upon an HIV-positive individual maintaining full virological suppression in plasma. Once an individual on ART is virally suppressed and maintains good drug adherence, the risk of viral load rebound is very low. The PARTNER 2 study  which inOur findings provide new evidence for the “U = U” message and support the concept that HIV-positive people who are adhering to treatment and exhibit a suppressed viral load do not transmit HIV to HIV-negative sexual partners. The message has allowed HIV-serodiscordant couples to engage in condom-less sexual acts without fear. Furthermore, it can reduce the burden of drug use during pregnancy without PrEP and reduce the potential risk of the drugs. However, in some cases, couples are reluctant to disclose their HIV or serodiscordant relationship status to families or communities and feel constrained to rely on each other for social support in coping with HIV. PrEP offers potential relief from stress and may increases trust within the relationship. Recognizing the issues these couples face enables physicians to offer honest and nonjudgmental preconception counseling .There were several limitations to our study. First, this study was conducted retrospectively by case selection and was not randomized, so there was the potential of selection bias. Second, the number of HIV-serodiscordant couples was small. A larger prospective study should be conducted to evaluate the efficacy of PrEP use during natural conception among HIV-serodiscordant couples.In summary, our study examined whether PrEP use during natural conception was beneficial in reducing the risk of HIV transmission among HIV-1-serodiscordant couples that have an HIV-positive partner who has achieved long-term viral suppression. Our study found no cases of HIV seroconversion in the PrEP group and the non-PrEP group. Our results indicate that PrEP use during natural conception may not be necessary in HIV-1-serodiscordant couples if the HIV-positive partner has stably suppressed plasma viral loads. Furthermore, our findings provide new evidence for the message of “U = U.” The decision to undergo PrEP during the process of natural conception in HIV-serodiscordant couples should be based on sustained viral suppression in the HIV-positive partner and acceptance of “U = U” in the HIV-negative partner.