Computed tomography (CT) is the standard imaging test used for the screening and assessment of suspected lung cancer, but distinguishing malignant from benign nodules by CT is an ongoing challenge. Consequently, a large number of avoidable invasive procedures are performed on patients with benign nodules in order to exclude malignancy. Improving cancer discrimination by non-invasive imaging could reduce the need for invasive diagnostics. In this work we focus on developing a gold nanoparticle contrast agent that targets the epidermal growth factor receptor (EGFR), which is expressed on the cell surface of most lung adenocarcinomas. Three different contrast agents were compared for their tumor targeting effectiveness: non-targeted nanoparticles, nanoparticles conjugated with full-sized anti-EGFR antibodies (cetuximab), and nanoparticles conjugated with a single-domain llama-derived anti-EGFR antibody, which is smaller than the cetuximab, but has a lower binding affinity. Nanoparticle targeting effectiveness was evaluated in vitro by EGFR-binding assays and in cell culture with A431 cells, which highly express EGFR. In vivo CT imaging performance was evaluated in both C57BL/6 mice and in nude mice with A431 subcutaneous tumors. The cetuximab nanoparticles had a significantly shorter blood residence time than either the non-targeted or the single-domain antibody nanoparticles. All of the nanoparticle contrast agents demonstrated tumor accumulation; however, the cetuximab-targeted group had significantly higher tumor gold accumulation than the other two groups, which were statistically indistinguishable from one another. In this study we found that the relative binding affinity of the targeting ligands had more of an effect on tumor accumulation than the circulation half life of the nanoparticles. This study provides useful insight into targeted nanoparticle design and demonstrates that nanoparticle contrast agents can be used to detect tumor receptor overexpression. Combining receptor status data with traditional imaging characteristics has the potential for better differentiation of malignant lung tumors from benign lesions. Lung cancer is the leading cause of cancer death worldwide and the number of deaths attributed to lung cancer is expected to continue increasing. Early dIn this work, we focus on developing a nanoparticle contrast agent that targets the epidermal growth factor receptor (EGFR). EGFR belongs to a family of receptor tyrosine kinases that trigger an array of signaling pathways that lead to cell growth, proliferation and survival. Upregulation or oncogenic activation of EGFR can lead to uncontrolled growth and tumor progression. 10–15% of Caucasians and up to 50% of Asians with lung adenocarcinoma have an activating mutation in the EGFR gene, with 40In this study, gold nanoparticles were used as the EGFR-targeted CT contrast agent. The experimental use of nanoparticles as CT contrast agents has been increasing in popularity over the last decade. Gold naSeveral obstacles need to be considered for the successful use of targeted nanoparticle probes as contrast agents. Nanoparticles naturally accumulate in tumors due to the enhanced permeability and retention effect (EPR), which mAnother major challenge for targeted probes is immune recognition. Stealth nanoparticles which are coated with a surface layer of poly(ethylene glycol) (PEG) tend to resist protein adsorption and are therefore not immediately recognized by phagocytic cells. HoweverIn this study we compare the results of targeting using a full-sized anti-EGFR antibody  with targeting using a llama-derived single domain antibody. Llama heavy chain variable region antibody fragments (VHH domains) are the smallest identified antibody fragments that retain target specificity because they are only composed of a single binding domain. EGFR-specific VHH domains have been produced and characterized previously. These d4R (10 mg/mL) was added to 1.5 L of ultrapure water and the solution was brought to boiling. 9 mL of a pre-warmed sodium citrate solution (100 mg/mL) was rapidly injected into the gold solution under vigorous mixing, after which the solution quickly changed color from yellow to deep red. The solution was kept at 100°C and mixed for an additional 15 minutes to complete the reaction, and then the solution was cooled to room temperature. The nanoparticles were then passed through a 0.22 μm filter to remove any aggregates that may have formed during the reaction. The size and distribution of the nanoparticles were determined by transmission electron microscopy (TEM) using an FEI Tecnai G2 Twin TEM  operating at 200 mV.25 nm gold nanoparticles were synthesized via the citrate reduction of gold chloride salt using a modified Frens methodP. 50.55 mAn EGFR-specific VHH domain (VHH-122) was chosen from a llama-VHH domain library, as described previously. VHH-122A full-sized chimeric (human/mouse) clinically-used monoclonal antibody against EGFR  was purchased from the manufacturer (Lilly). C225 has a binding affinity of 0.39 nM for the EGFR extracellular domain and has an approximate molecular weight of 152 kDa.VHH-122 and C225 antibodies were linked to gold nanoparticles via a PEG linker molecule. The PEG molecule contained a reactive NHS ester group  capable of forming covalent bonds with primary amines on the antibodies. The other end of the PEG molecule contained a thiol group  which can bind to the gold nanoparticle surface. The antibodies were PEGylated by reacting the OPSS-PEG-SVA  with the antibodies at a ratio of 20:1 (PEG:antibody) for VHH-122 and 2.5:1 for C225. The reaction was performed in HEPES-buffered saline at pH 8.6 at 4°C overnight. The PEG-antibodies were then mixed with bare gold nanoparticles at a ratio of 50 antibodies/particle for VHH-122 or 10 antibodies/particle for C225. These ratios were chosen based on preliminary testing that showed particle aggregation during the surface conjugation when higher concentrations of protein were added to the surface. These nanoparticles were rocked at 4°C for 2 hours, after which methoxy PEG thiol  was added to the gold nanoparticles in large excess to fill in any gaps remaining on the surface after antibody conjugation. Control (non-targeted) nanoparticles were also produced by adding a large excess of mPEG-SH (5 kDa) to bare gold nanoparticles in the absence of any antibodies. All gold nanoparticles were rocked overnight at 4°C after PEG addition and then rinsed by centrifugation. The nanoparticles were filter-sterilized and concentrated prior to use.The size distribution and surface charge of the gold nanoparticles  were characterized by dynamic light scattering (DLS) and zeta potential using a Zetasizer Nanoseries  at 25°C. Nanoparticles were tested for stability in physiological solutions by incubating them in phosphate-buffered saline (PBS) and culture medium  with 10% fetal bovine serum (FBS). Resistance to aggregation was determined by measuring the peak optical absorbance at the nanoparticle surface plasmon resonance peak wavelength using a Cary 50 UV-Vis spectrophotometer , while size change due to the adsorption of serum proteins was tracked by serial DLS size measurements.Conjugation of the antibodies to the AuNPs was further confirmed and quantified by gel electrophoresis (SDS-PAGE) analysis. After rinsing the targeted AuNPs to remove unbound proteins, the bound antibodies were released from the gold surface by reaction with dithiothreitol (DTT). SDS wasTo verify that the protein conjugation to the nanoparticle surface was via the gold-thiol bond, rather than non-specific protein adsorption on the surface, some bare nanoparticles were conjugated with non-PEGylated VHH-122 and C225 antibodies, followed by the addition of mPEG-SH as described above. Gel electrophoresis analysis was repeated on these samples to quantify the amount of protein attached to the nanoparticle surface non-specifically.2RSOR4R after 15 minutes. The suspensions were then centrifuged to pellet the nanoparticles and the absorbance of each supernatant at 450 nm was measured using a plate reader in order to quantify the amount of EGFR bound to the nanoparticles in the suspension. The ELISA measurements were normalized to both nanoparticle concentration and total antibody concentration in the original sample.Binding of EGFR to the antibody-conjugated AuNPs was assessed by performing an enzyme-linked immunosorbent assay (ELISA) in the nanoparticle suspension. Antibody-conjugated AuNPs were blocked with 10% donkey serum in PBS, then incubated with a soluble recombinant human EGFR extracellular domain-Fc chimera  at 10 nM in blocking buffer for 2 hours. The nanoparticles were then rinsed by repeated rounds of centrifugation and resuspension in PBS. Binding of soluble EGFR to the gold nanoparticles was then detected by adding a horseradish peroxidase-conjugated anti-human IgG antibody, followed by another round of rinsing. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was added and the reaction was stopped by concentrated HR5 copies of EGFR per cell when grown in culture[In vitro cell targeting studies were performed in culture using A431 cells (from ATCC #CRL-1555). The A431 cell line is derived from a human epidermoid carcinoma and is characterized by very high surface expression of the wild type EGFR, estimated at approximately 5x10n culture. A431 ceFixed slides were imaged using a Zeiss Axiovert 135 inverted microscope  and a Cytoviva darkfield condenser . All images were acquired at 200x magnification. Darkfield images were taken of each cell culture sample to detect gold nanoparticles bound to the cells. With darkfield imaging, indirect sample illumination enables image production from light scattered by the samples. Gold nanoparticles are readily detected due to their increased scattering properties relative to the surrounding tissue.For ICP-OES analysis, cells from each sample group were scraped off the slides and collected in a microcentrifuge tube. The cells were then pelleted by centrifugation and the supernatant was removed. Cell pellets were digested in trace metals grade aqua regia for 72 hours. The samples were then filtered through a 0.45-μm filter and diluted with 2% nitric acid to 10 mL. Standard solutions of known gold concentrations (8 concentrations ranging from 0.1–10 ppm) were prepared in 2% aqua regia using certified reference material Gold Standard for ICP . The standards and digested samples were analyzed for gold concentration by ICP-OES .All animals were handled in accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the Institutional Animal Care and Use Committee (IACUC protocol A283-11-11) of Duke University Medical Center. The Duke University Medical Center animal management program is accredited by the American Association for the Accreditation of Laboratory Animal Care and meets National Institute of Health standards as set forth in the ‘‘Guide for the Care and Use of Laboratory Animals” (DHHS Publication No. (NIH) 85–23, Revised 1985). The institution also accepts as mandatory the PHS ‘‘Policy on Humane Care and Use of Laboratory Animals by Awardee Institutions” and ‘‘NIH Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training”. Mice were anesthetized for all procedures and imaging using inhaled 4% isoflurane. Mice were housed in ventilated cages with five single-gender mice per cage. Nude mice additionally had sterilized bedding, food and water. All mice had free access to food and water and regular light/dark cycles. Mouse tumors were monitored by caliper measurement 3 times per week. Mice were euthanized at humane endpoints . At the end of the studies, the mice were euthanized by inhaled isoflurane.For the initial biodistribution imaging study, 3 groups of healthy C57BL/6 mice (n = 3 in each group) were injected by tail vein with C225-AuNPs, VHH-AuNPs or PEG-AuNPs . CT imaging was performed at 0, 2, 4, 12, 24, and 36 hours post-injection to track the dynamic gold nanoparticle concentration. CT enhancement was measured in the blood, liver, spleen and kidney at each time point.6P A431cells in 100 uL Matrigel (Corning). After tumors reached 0.5 cc (10–14 days after injection), mice were injected by tail vein with one of three contrast agents: C225-AuNPs, VHH-AuNPs, or PEG-AuNPs . Dual-energy CT images were acquired immediately after injection and 48 hours post-injection. Gold concentrations were quantified within the tumors at each time point.For the tumor targeting study, 3 groups of 4–6 week old female athymic nude mice (n = 8 in each group) were injected subcutaneously in the flank bilaterally with 1x10PAll CT imaging for this study was performed using a custom-built dual source micro-CT system with twoCT image post-processing was performed as described previously, 29. RawCT images were analyzed using Avizo . Regions of interest including the blood, liver, spleen, and kidneys (for the biodistribution study) and the tumors (for the targeting study) were segmented in Avizo. Because of the high gold concentrations in the blood, liver, and spleen, these organs were readily segmented using the software’s automatic segmentation tools. The tumors and kidneys were segmented by manually drawing rough regions around the region of interest, then thresholding the regions until non-enhancing tissues outside the kidney or tumor were completely excluded. The concentration of gold in each region of interest was measured for each timepoint. For tumor gold quantification, the tumor background signal was subtracted from the measured signal in the enhancing regions before measuring the gold concentration. Nanoparticle blood half-life was calculated for each nanoparticle type by fitting the blood gold concentration data from the biodistribution study to an exponential decay curve.Statistical differences between the PEG, VHH-122 and C225 groups in each study were tested using a one-way ANOVA followed by a post-hoc Tukey test for individual comparisons. Statistical significance was noted at the p<0.05 level for all comparisons . Error bars in all plots represent the standard error of the mean.Gold nanoparticle characterization is shown in Aggregation studies showed that PEG-AuNPs, VHH-AuNPs and C225-AuNPs were stable for > 6 hours (no measurable change in peak absorbance by UV-Vis) in both physiological saline (PBS) and DMEM growth medium + 10% FBS, indicating both stability to particle aggregation and resistance to protein adsorption and resultant particle cross-linking and aggregation in serum. Bare nanoparticles aggregated and sedimented out of suspension within 10 minutes of exposure to PBS. Serial DLS size measurements see  showed tSDS-PAGE quantification of antibodies on the nanoparticle surface following conjugation showed that on average there were ~25 VHH-122 antibodies and ~5 C225 antibodies bound per 25 nm nanoparticle see . To demoBinding of the nanoparticle surface-immobilized C225 and VHH-122 antibodies to soluble EGFR extracellular domain was demonstrated by ELISA in suspension. Results of the ELISA see  were norD R(equilibrium dissociation constant) can be calculated using the following formula:The C225-AuNPs have five times fewer antibodies attached to the surface, so each C225 antibody is much better at binding to EGFR than each VHH-122 antibody. This is consistent with the previously reported binding affinities of these two antibodies. In order to compare the binding affinity seen here on the surface of gold nanoparticles with the published binding affinity, we can calculate the expected fraction of each antibody that should be bound to EGFR using theoretical binding affinities and the known concentrations of EGFR and antibodies in solution. The KR7 nM see , we findFollowing incubation with gold nanoparticles, A431 cells (high EGFR expression) were imaged by darkfield microscopy to visualize gold nanoparticles binding to the cells. Darkfield images are shown in Gold nanoparticle binding to cells was quantified by ICP-OES, which can accurately measure gold concentration in solution after acid digestion of the sample. After collecting cell pellets from each group, the gold concentration within the cell pellet was analyzed. ICP-OES results are shown in Each of the three nanoparticle types  was injected intravenously into healthy C57BL/6 mice (n = 3 for each group) to study the contrast agent effectiveness, biodistribution, and blood half-life. Results are shown in Targeted (C225 and VHH-122) and control (PEG) nanoparticles were injected into nude mice (n = 8 for each group) with bilateral subcutaneous A431 cell line tumors, which demonstrate high levels of EGFR expression. Dual-energy CT was used to visualize and quantify gold within each of the tumors. The mice were imaged 48 hours after contrast agent injection, at which point most of the contrast had been cleared from the bloodstream. In this study we examined the use of three different contrast agents for imaging EGFR-expressing tumors by CT. All of the imaging agents showed effective CT contrast, with high initial enhancement within the blood and high contrast within the tumors and other organs  at later time points. The first contrast agent, PEG-AuNPs, demonstrated a long blood half-life (~20 hours) due to a robust PEG coating which minimized surface protein adsorption and subsequent immune recognition and clearance. The PEG-AuNPs contained no targeting ligand and could only accumulate in tumors due to the leaky vasculature within the tumors (EPR effect). All the tumors in the PEG-AuNP group had a moderate level of gold nanoparticle uptake, showing the inherent effectiveness of EPR in this tumor model.The second contrast agent, VHH-AuNPs, had many (~25) small VHH-122 antibodies on the surface of each nanoparticle. These domains have moderately high binding affinity for EGFR, as demonstrated by the effective binding of soluble EGFR to VHH-AuNPs in solution. The domains are small and lack the Fc portion of the antibody, which limits the ability of phagocytic cells to recognize these nanoparticles and engulf them. The VHH-AuNPs had a long blood half-life (~20 hours), which was indistinguishable from the PEG-AuNP blood half-life, showing that the presence of single-domain antibodies did not interfere with the stealth nature of these nanoparticles. However, these nanoparticles demonstrated less binding affinity for A431 cells in vitro compared to the full-sized C225 antibody. The binding of VHH-AuNPs to A431 cells was successfully demonstrated in vitro, but it was not sufficient to increase the accumulation of these targeted nanoparticles in vivo.The third contrast agent, C225-AuNPs, contained several (~5) full-sized humanized anti-EGFR antibodies conjugated to the nanoparticle surface. These clinically-used antibodies have very high binding affinity for EGFR, as demonstrated by effective in vitro binding both to soluble EGFR in solution and to EGFR-expressing A431 cells in culture. These targeted nanoparticles demonstrated a reduced blood half-life (~11 hours) compared to the other two contrast agents, consistent with increased immune clearance due to the presence of full-sized IgG antibodies on the nanoparticle surface. Despite the reduced blood residence time, the C225-AuNPs had significantly higher tumor uptake than the other two contrast agents. The high EGFR binding affinity of the C225-AuNPs was more than enough the counteract the effects of the reduced blood residence time on total tumor accumulation.Our original hypothesis that VHH-122 antibodies would provide better targeting than full-sized antibodies was not supported by the data. Even though the C225-AuNPs had lower blood half-life , they demonstrated significantly higher tumor accumulation than any of the other nanoparticle types. Surprisingly, the VHH-AuNPs showed no higher accumulation in tumors than the PEG-AuNPs. The VHH-AuNPs showed effective EGFR binding when the EGFR was in solution but showed decreased binding when the EGFR was on the surface of A431 cells. In previous studies with VHH domains[Although one of the primary determinants of tumor accumulation for nanoparticles is the nanoparticle blood residence time, this is clearly not the only important factor in determining total tumor accumulation. In this case, we saw that an antibody-targeted nanoparticle contrast agent with high binding affinity for the target cells can have much higher total tumor accumulation than a non-targeted nanoparticle even if it has a significantly lower blood residence time. The C225 antibody, with high affinity and reduced blood residence time was superior to the VHH-122 antibody, a tumor targeting ligand with lower affinity but a long blood residence time. When making the choice for a tumor targeting ligand, it is important to consider both the effect that the targeting ligand will have on the nanoparticle clearance and the overall targeting efficacy of the targeting ligand. For attachment to nanoparticles, these data suggest that high affinity ligands are preferable for in vivo use, even if they moderately reduce blood residence time. Although these data suggest that ligand affinity has more impact on tumor accumulation than blood half life under these conditions, the effects of these two parameters could be confirmed by expanding this study with additional controls. The ideal controls in this case would be nanoparticles conjugated with non-specific IgG antibodies and nanoparticles with non-specific VHH antibodies (not targeted to EGFR). These control nanoparticles would have similar pharmacokinetics to the corresponding targeted nanoparticles, which would allow for analysis of the effects of affinity and pharmacokinetics isolated from one another.The C225-AuNPs used in this study showed highly effective tumor targeting relative to non-targeted controls. This demonstrates the feasibility of using targeted nanoparticle agents for the detection of surface receptors upregulated on tumor cells, including tumors overexpressing EGFR. The ability to detect tumor EGFR expression (based on the degree of tumor CT enhancement) could be extremely useful in discriminating between benign and malignant lung tumors on CT exam. This can be used to better predict which patients with an identified lung nodule are in need of biopsy and potential surgery and which patients can instead be treated with watchful waiting and periodic follow-up scans. Such methods to improve non-invasive discrimination between benign and malignant nodules could have a large impact on patient well-being by reducing unnecessary invasive procedures.A previous small-scale study initiallWe have compared the effectiveness of three gold nanoparticle contrast agents for the imaging of EGFR-expressing tumors: C225-AuNPs, VHH-AuNPs, and non-targeted control PEG-AuNPs. The C225 and VHH-AuNPs both bind to EGFR in vitro, but the C225-AuNPs show higher binding affinity in cell culture. After in vivo injection, the VHH-AuNPs and PEG-AuNPs show equivalent tumor accumulation, while the C225-AuNPs show much higher accumulation, despite having the lowest half-life of the three groups. All three of the contrast agents could be used effectively for both vascular imaging and tumor imaging by CT, however, the C225-AuNPs showed significantly increased tumor uptake due to specific tumor targeting. This increased enhancement with targeted nanoparticles can be used to better identify receptor status of tumors and improve tumor discrimination and characterization.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.S4 Dataset(XLSX)Click here for additional data file.


Staphylococcus aureus Subclinical mastitis’ published in International Journal of Molecular Sciences. Milk samples were collected from cows suffering from S. aureus-associated subclinical mastitis and the proteins abundance were identified in comparison with samples collected from the control cows using liquid chromatography-mass spectrometry (LC-MS)-based label free proteomics analysis. Following the MS measurements, the raw spectra were processed using MaxQuant-Andromeda software and the protein identification was carried out through a search against Uniprot FASTA files of the Bos taurus reference proteome. Perseus software analysis was applied for computation of protein abundance. The raw file presented in this DiB was deposition at PRIDE repository in the PRIDE repository with the dataset identifier PXD007516.This dataset is associated with our research article ‘Identification of host-defence related proteins using label-free quantitative proteomic analysis of milk whey from cows with  This mass spectrometry data-in-brief is associated with the research article aimed at identification of protein abundantly present in milk samples of cows suffering from sub-clinical mastitis due to 2S. aureus. Label-free quantitative proteomics analysis was applied for identification of proteins and their comparative abundance among the control and mastitis groups.3Milk samples were collected from dairy cows at the Elora Research Station- Diary Facility at the University of Guelph in accordance to the guidelines set by the Animal Care and Use Committee of the University of Guelph. . The cows were classified as being either healthy or having subclinical mastitis based on history, physical examination and testing of the milk samples by Somatic Cell Counting and bacterial culture.4Whey protein was prepared by removing fat and casein micelles by centrifugation followed by trypsin digestion. Separation of peptides was carried out by liquid chromatography and an Orbitrap-based mass spectrometry system  as described in 5Bos taurus protein reference proteomes (UniProt Proteome ID: UP000009136). Label-free quantification of identified proteins was performed and statistically significant differences in protein abundance in the subclinical group in comparison to that of the control group was determined using Perseus 1.4.1.3 software (http://141.61.102.17/perseus_doku/doku.php?id=start) MaxQuant-Andromeda software suit was utilised to process the raw spectra and to search against the 6PXD007516.The mass spectrometry data was deposited at the ProteomeXchange (PX) Consortium


Background: Adult cancer survivors often experience substantial psychological morbidity following the completion of acute cancer treatment. Unfortunately, current psychological interventions are of limited efficacy. This study explored if metacognitive therapy (MCT); a brief transdiagnostic psychological intervention was potentially efficacious and could be delivered effectively to adult cancer survivors with psychological morbidity.Methods: An open trial with 3- and 6-month follow-up evaluated the treatment effects of MCT in 27 consecutively referred individuals to a clinical psychology health service specializing in psycho-oncology. Each participant received a maximum of six 1-hour sessions of MCT. Levels of anxiety, depression, fear of cancer recurrence, post-traumatic stress symptoms, health related quality of life, and metacognitive beliefs and processes were assessed using self-report questionnaires.Results: MCT was associated with statistically significant reductions across all outcome measures which were maintained through to 6-month follow-up. In the ITT sample on the primary treatment outcome measure, the Hospital Anxiety and Depression Scale-Total, 59% of participants met recovery criteria at post-treatment and 52% at 6-month follow-up, respectively. No participants significantly deteriorated. In the completer sample (N = 20), 80% recovered at post-treatment and 70% at 6-month follow-up. MCT was acceptable to patients with approximately 75% of patients completing all treatment sessions.Conclusion: MCT, a brief transdiagnostic psychological intervention can be delivered effectively to a heterogenous group of cancer survivors with promising treatment effects. Examining the efficacy of brief MCT against the current gold standard psychological intervention would be a valuable advance toward improving the quality of life of cancer survivors. The incidence of cancer in the United Kingdom is projected to increase by 2% over the next 15 years with survival rates also increasing. It is estimated that survival rates have doubled over the past 40 years with a ten-year survival rate of approximately 50%  in 2016,The substantial prevalence and associated problems with psychological morbidity in cancer survivors requires effective interventions. Unfortunately, highly efficacious psychological interventions are unavailable . The mosMetacognitive therapy MCT;  offers aThe S-REF model states that perseveration is guided by positive metacognitive beliefs about the helpfulness of worry and rumination: e.g., “worry will help me be better prepared,” “worry will ensure that I complete my daily tasks.” Unfortunately, worry and rumination achieve the opposite, because the person experiences more negative thoughts and views more situations as potentially dangerous. The individual repeatedly acts as if unwanted negative thoughts are meaningful which leads to the development of an inflexible way of responding to thoughts. A more flexible response style can help to alleviate perseveration. Similarly, the S-REF model specifies that threat monitoring  is determined by positive metacognitive beliefs. More specifically, a person comes to believe that scanning the environment or one’s mind and/or body for symptoms will reduce distress whereas it leads to the persistence of threat and distress. Furthermore, negative metacognitive beliefs about the uncontrollability and danger of worry sustain and increase worry. Modifying negative metacognitive beliefs is fundamentally important in the S-REF model because, if patients believe that worry is uncontrollable, they will not attempt to control it. Therefore, it is possible that through targeting metacognitive beliefs and processes rather than cognitive content, MCT offers a particularly close “fit” with the needs of cancer survivors indicating potential for greater efficacy .The development of MCT for psychological morbidity in cancer is evolving with encouraging evidence for the explanatory and therapeutic utility of MCT. There is increasing evidence for the role of metacognitive beliefs and processes in emotional distress in cancer survivors from cross-sectional and prospective studies  and in aAn open trial with follow-up at 3 and 6 months evaluated the potential efficacy of brief MCT for adult survivors of cancer experiencing emotional distress. Data was also gathered on recruitment and retention rates. All participants gave written informed consent in accordance with the Declaration of Helsinki. Ethical approval was provided by the National Health Service North West Research Ethics Committee (reference 15/NW/0820).Potentially suitable participants were identified from consecutive referrals to an adult clinical heath psychology service which specializes in psychological interventions for cancer patients. Those patients with elevated scores on the Hospital Anxiety and Depression Scale HADS;  and indiTwenty seven cancer survivors participated in the study and met the following inclusion criteria: (i) a score of > 15 on the Hospital Anxiety and Depression Scale-Total (HADS-T); (ii) had been diagnosed with cancer ≥ 6 months previously; (iii) were aged 18 years or over; (iv) had completed acute medical treatment for cancer ; (v) were not receiving concurrent psychological treatment; (vi) were not actively suicidal; (vii) reported no current substance use; (vii) were not experiencing a psychotic or organic illness; (viii) were free from psychotropic medication or has been on a stable dose for at least 8 weeks; and (ix) were able to speak and understand English.Metacognitive therapy was delivered over a maximum of 6 individual face-to face sessions that were 45–60 min in duration. The intervention followed a manualized protocol . As the The HADS is a 14-item self-report questionnaire measuring anxiety and depression (seven items each) over the past week. Each item is rated on a 4-point scale (0–3). Scores for each subscale range from 0 to 21 with higher scores reflecting more sever anxiety or depression. Scores of 11 or more on each of the subscales indicate caseness. Combining the two subscales provides a measure of emotional distress. The HADS-Total is the “gold standard” outcome measure for evaluating the efficacy of interventions on emotional distress in cancer populations, and has excellent psychometric properties .The IES-R is a 22-item self-report questionnaire measuring trauma-related symptoms The total scale score ranges from 0 to -88 with higher scores indicative of more severe trauma symptoms. A total score of ≥ 33 indicates a probable diagnosis of PTSD . The IESThe FCRI is 42-item self-report questionnaire assessing 7 aspects of FCR. Each item is rated on a 5-point scale (0–4). A total score for the FCRI is obtained by summing scores on the 7 subscales, with higher scores indicating greater severity (range 0–168). The FCRI is the most validated measure of FCR across a wide range of cancer types .The FACT-G is a 27 item self-report questionnaire that measures four domains of health-related quality of life (HRQOL). Each item is rated on a 5-point scale from 0  to 4 (very much). The FACT-G total score ranges from 0 to 108 with higher scores indicating a better HRQOL. The FACT-G has been used extensively in mixed cancer populations and has excellent psychometric properties .The MCQ-30 measures 5 domains of metacognition by 30 items. Participants rate the extent to which they “generally agree” with statements presented on a 4-point scale from 1 (do not agree) to 4 (agree very much), providing total scores for each subscale ranging from 6 to24. Higher scores indicate greater conviction in metacognitive beliefs. The MCQ-30 assesses: (1) positive beliefs about worry, (2) negative beliefs uncontrollability and danger of worry, (3) cognitive confidence, (4) beliefs about the need to control thoughts, and (5) cognitive self-consciousness. The MCQ-30 has been validated for use in cancer patients .The CAS-1 is a 10 item self-report questionnaire that assesses metacognitive processes and beliefs. Items 1 to 6 assess the fundamental components of the CAS  Each item is rated on a 10-point scale from 0 (none of the time) to 100 . Items 7 to 10 assess metacognitive beliefs and are not reported in the present study. To provide an overall measure of the CAS, the 6 items were summed and divided by the number of items. The same method has been used previously .d to assess the magnitude of treatment effects from pretreatment to post-treatment and from pre-treatment to both 3- and 6-month follow-ups. To determine the clinical significance of treatment effects the methodology developed by Intention to treat (ITT) analyses were used to determine the potential efficacy of brief MCT for emotional distress in cancer survivors. Missing data for the non-completers in the study were replaced by using the last observation carried forward (LOCF) method. The LOCF has been considered a conservative approach when evaluating treatment outcomes in open trials. Treatment effects across time  were assessed with repeated-measures analysis of variance (ANOVA); the Greenhouse–Geisser correction was applied when the assumption of sphericity was violated. Main effects were followed by Bonferroni-adjusted pairwise comparisons for each outcome measure. Within group effect sizes were calculated using Cohen’s Forty-three consecutive referrals were identified as potentially eligible. There were 16 patients who did not enter the study; 10 did not wish to participate, 3 did not attend the assessment interview 1 patient did not have a have a cancer diagnosis, 1 patient did not meet the threshold for severity of distress with a HADS-T score of less than 16 and 1 patient had a recurrence of cancer.Twenty-seven patients began the trial of whom 20 completed treatment; a completion rate of 74%. Of the seven patients who did not complete the six sessions of MCT; three patients attended only one session, two patients 2 sessions, one patient 3 sessions and the final patient attended 4 sessions but sporadically and decided that it was not feasible to continue therapy. Reasons for non-completion were; one patient was hospitalized for cancer recurrence, one participant stopped therapy to be able to provide full time care for a relative, 2 participants did not wish to undertake psychological therapy and 3 patients dropped out without providing a reason. The demographic and clinical characteristics of the sample shown in There were significant main effects of time on all outcome measures . Follow-Within group effect sizes for the ITT sample are shown in In the ITT sample, most participants were recovered on the HADS-Total at post-treatment and across the follow-up period. In terms of the proportion of patients that responded to treatment, 81% were improved at post-treatment and 74% at 6-month follow-up. Examination of the recovery rates for those patients that completed treatment shows recovery rates of 80% at post-treatment and 70% at 6-month follow-up. A summary of the clinical significance of treatment outcomes is shown in This study provides further support for the potential of brief MCT to alleviate psychological morbidity in cancer survivors. Following six 1-hour sessions of MCT, there were significant reductions in anxiety depression, post-traumatic stress symptoms, FCR and improvements in quality of life. There were also significant reductions in metacognitive beliefs and the CAS as predicted by the metacognitive model , 1996. TThe within group effect sizes on FCR provide the opportunity to benchmark the effects of brief MCT with those reported in recent randomized controlled trial evaluating an integrative approach for FCR. The psychological treatment in the trials conducted by The present open trial is a valuable step in the translation of MCT from adult mental health populations to cancer survivors and is following the recommended framework for translating psychological interventions to a new population . The limA comparatively small sample was used, but the sample appeared representative of cancer survivors referred to the clinical health psychology service. Other limitations include the lack of ethnic diversity and that most of the sample were female, thereby compromising external validity. Treatment outcome was assessed exclusively by self-report questionnaires in the present study. Although exclusive reliance on self-report questionnaires could be considered a methodological weakness, the study was not focused on changes psychiatric diagnosis, rather the study was designed to measure general distress for which the “gold standard” outcome measure for evaluating the efficacy of interventions on emotional distress in cancer was used .Overcoming other limitations of open trials can be achieved through conducting randomized controlled evaluation. It would be valuable to assess the hypothesized mechanisms of change in the context of an RCT against the current recommended treatment approaches, it may be that the treated patients who recover change to most on metacognitive variables regardless of the treatment received. There were statistically significant reductions in all metacognitive beliefs and the CAS over treatment, which were maintained through to the 6-month follow up assessment. This study adds to the extant literature that MCT has the potential to be an efficacious psychological intervention for adult cancer survivors. Given the limited outcomes of currently available interventions, there is an obvious need to conduct a controlled evaluation of the potential of brief MCT to alleviate psychological morbidity in cancer survivors.PF and PS designed the study. AB, LF, and PF were the therapists. PF drafted the manuscript. GA and HU recruited and assessed participants at intake and following treatment. All authors have contributed to drafting and revising the manuscript and approved its submission.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.


Nanoscale surface morphology of plant fibers has important implications for the interfacial bonding in fiber-polymer composites. In this study, we investigated and quantified the effect of plasma-surface modification on ramie plant fibers as a potential tool for simple and efficient surface modification. The extensive investigation of the effects of plasma treatment of the fiber surface nano-morphology and its effect on the fiber-polymer interface was performed by Low-Voltages Scanning Electron Microscopy (LV-SEM), infrared spectroscopy (FT-IR) analysis, fiber-resin angle measurements and mechanical (tensile) testing. The LV-SEM imaging of uncoated plasma treated fibers reveals nanostructures such as microfibrils and elementary fibrils and their importance for fiber mechanical properties, fiber wettability, and fiber-polymer matrix interlocking which all peak at short plasma treatment times. Thus, such treatment can be an effective in modifying the fiber surface characteristics and fiber-polymer matrix interlocking favorably for composite applications. The u/g cm−3)  and they/g cm−3) ,6.Among lignocellulosic fibers, ramie fiber is one of the most widely used fibers in the field of polymer composites due to its high crystallinity and commercial availability ,8,9 as wPlasma treatment presents an attractive method for the surface modification of various materials. It offers many advantages compared with chemical treatments such as simplicity, low energy consumption, short treatment times, low cost, and it does not require water or any potentially hazardous chemicals ,19,20. DTherefore, in the present study we use plasma-surface treatment with the goal of maximizing the exposure of crystalline cellulose microfibrils by removing the amorphous constituents such as hemicellulose and lignin from the fiber surface, without adversely affecting the fiber mechanical properties. To identify and image the crystalline cellulose microfibrils within elementary fiber we present a detailed investigation of the surface topography and single fiber mechanical testing of ramie fibers under different durations of plasma treatment. We use Low Voltage Scanning Electron Microscopy (LV-SEM) to observe the presence and orientation of nanoscale structures (cellulose microfibrils) on the fiber surface after plasma treatment without any sample coating and subsequently investigate the relationship between exposed nanostructures, surface wettability, fiber-phenolic matrix interlocking, and single fiber mechanical properties, to define what constitutes optimal plasma treatment conditions for ramie fiber – phenolic polymer composites. Ramie fibers (Boehmeria nivea) were purchased from Wild Fibers store, Birmingham, UK and used as received for the experiments a. The moIn this investigation, a Zepto plasma-surface cleaner  with a borosilicate glass cylindrical chamber (2.6 Liter) and power supply frequency of 40 kHz  was usedA Nova Nano SEM 450 Scanning Electron Microscopy  was used to observe the fiber surface topography of untreated and plasma treated fibers. Moreover, single ramie fibers were cryo-fractured in the liquid nitrogen to observe the fiber cell wall structure by LV-SEM. The fracture surfaces of tensile test samples were also observed by LV-SEM. Natural plant fibers are not conductive materials; therefore, the observations were performed using a low accelerating voltage (1 kV) to avoid fiber charging. The images were collected using a through lens detector (TLD) at around 4.5 mm working distance with a beam deceleration of 2000 V. Fast Fourier Transform (FFT) image analysis method in ImageJ was used to observe the nanoscale structure orientation on the fiber surface.−1 to 600 cm−1 and the average of scan repetitions was 8 scans for each sample (untreated and plasma treated fibers) at 2 cm−1 of resolution.A PerkinElmer FTIR spectrometer  was used to investigate the surface chemical compositions of untreated and plasma treated ramie fibers in single reflection diamond ATR accessory with an angle of incidence of 16° from the perpendicular. Typically, the depth of light penetration in this technique is a few microns, depending on the wavelength, the angle of incidence, the refractive indices, and the sample . In thisTo evaluate the effect of plasma treatment on the fiber surface wettability, the single-fiber drop technique was used using drop-shape analyzer instrument . Phenolic resin and its catalyst (phencat 382) were mixed in the ratio of 100:5 w/w and then used as a test liquid. This kind of resin is usually used as a matrix in polymer-fiber composites . A 5 μL A tensile testing machine  with a 500 N load cell was used to test the mechanical tensile properties of single ramie fibers. Thirty isolated single fibers were prepared for untreated and plasma treated fibers at different times . Each single fiber with a gauge length of 5 mm was mounted on a cardboard frame by using cyanoacrylate glue. The fibers were carefully glued in the exact center of the cardboard. Thereafter, the sample was clamped onto the testing machine and just before the beginning of each test, the supporting side of the card was carefully cut off. The samples were tested at a constant crosshead displacement rate of 40 %/min and according to the ASTM D 3822-01 standard. The tests were carried out at room temperature (22 ± 3 °C). During the test, the force-strain values were recorded, and these values were used to measure the fiber tensile properties (ultimate strength and Young’s modulus). Only samples that broke in the middle of their gauge length were used to calculate the tensile strength and Young’s modulus, whereas the fibers that broke near to the glue clamp or card frame were not used in the calculations. Before testing the fiber, images at different locations along the fiber gauge length were taken using an optical microscope . The cross-section of each single fiber was assumed to be circular. The fiber diameter was directly measured from the images and hence the cross-sectional area of each single fiber was calculated from the average of three apparent fiber diameter measurements (30 samples for each treatment time).As the single fiber failure is elastic without any signs of plastic deformation, it can be assumed that the cross-sectional area of the fractured fibers has not changed significantly after the test. Therefore, after testing, LV-SEM was used to observe the cross-section area of the fractured fibers. Fractured fibers with a flat and clear cross-section end a were seThe cross-sectional area measurements highly affect the final tensile properties of single fibers . TherefoThe as received fibers were cryo-fractured to investigate the fiber cross-section structure by LV-SEM a. The LVThe surface morphology of untreated and plasma treated ramie fibers are shown in When increasing the plasma treatment time to 2 min, those bright nanostructures (microfibrils) are seen more clearly on the fiber surface with their increased height indicating a large amount of amorphous material has been removed, leading to an increased surface roughness as presented in However, longer plasma treatment durations (3 and 4 min) leads to fewer, more isolated microfibrils as well as increases in the interfibrillar distance, especially after 4 min of plasma treatment as shown in According to our LV-SEM observations, the plasma treatment is not creating holes or cracks on the fiber surface even after 4 min treatment duration. However, it is also important to mention that the LV-SEM observations in this work indicated that any pre-existing holes or cracks became wider and deeper as the treatment duration increases as shown in The ATR-FTIR absorption spectra of untreated and plasma treated ramie fibers are shown in −1 and 1024 cm−1), particularly after 4 min of plasma treatment. They are characteristic bands of the hydroxyl groups (O–H) present in the cellulose structures [−1 after 2 min of plasma treatment  which might be due to the remaining of the crystalline cellulose [−1 is assigned to the absorbed water in crystalline cellulose [−1 for the plasma treated fiber is probably due to the temperature effect and bond cleavage with plasma to form free radicals. The band near 1235 cm−1 is possibly due to the (C–O) vibration of esters, ethers, and phenolic groups related to the presence of waxes on the fiber surface and the decreasing of this band after plasma treatment  are considered to be wettable surfaces and hydrophilic properties (high surface energy) , and a lThe surface tension forces between fiber and phenolic resin enable the resin to spread across the fiber surface, as shown in The fractured surface morphology of the tensile test of single ramie fibers is shown in To determine to what extent plasma treatment durations affect the mechanical properties of ramie fibers, tensile properties (strength and Young’s modulus) were measured. Using the two different methods to measure the cross-sectional area of single fibers described in the experimental section, the strength and Young’s modulus with plasma treatment time were evaluated. The non-circular cross-section of single ramie fiber is clearly illustrated in Plasma-surface treatment of ramie fibers has created surface nanoscale structures after only 1 min of treatment. We identified these nanostructures to be crystalline cellulose microfibrils and report for the first time direct observation of the elementary fibrils. Exposing cellulose microfibrils resulted in a nanoscale surface roughness which increases the surface area and led to better wettability and fiber-matrix interaction. The use of plasma-surface modification could have immense benefits in the production of fiber-polymer composites with good interface integrity and could pose a big step forward in replacing fossil-fuel-based fibers.


What to look for in case of polysplenia and/or unusual disposition of several intra-abdominal organs. Anysmay, polysplenia syndrome is an unusual disposition of intra-abdominal organs and unlike situs inversus it’s a spectrum of abnormalities and not a single set.An asymptomatic 62-year-old woman underwent abdominopelvic computed tomography (CT) for inaugural diabetes.CT showed dorsal pancreatic (P) agenesis (A) Figure .Incidental abnormalities were found, including:– Four spleens (S), one adjacent to the stomach Figure .– Right renal hypotrophy Figure .– Midline falciform ligament Figure .– Duplicated inferior vena cava system (IVC) with dilated azygos (A) and hemiazygos (H) continuation and no communication with hepatic veins (N) Figure .– Intestinal nonrotation: the small bowel was right-sided (S), the colon was left-sided (C), the superior mesenteric artery (A) was to the right of the vena (V), and there was no midline crossing by the duodenum (D) under the aorto-mesenteric junction (P) Figure .The diagnosis of type II diabetes was retained and after four months of metformin and insulin therapy, the rate of HbA1c was almost normalized. The remaining of the follow-up was unremarkable.Heterotaxy syndrome (HS) (or situs ambiguus) is the result of an early embryological developmental failure in which there is an abnormal arrangement of thoraco-abdominal organs. In contrast to situs inversus, HS is not characterized by a single set of abnormalities but rather a spectrum.Polysplenia syndrome (PS) (or left isomerism) is the subtype of HS with features of bilateral left-sidedness. No single anomaly is pathognomonic but the association of a sufficient number allows the diagnosis. The commonest is the presence of multiple spleens, right- or left-sided, with a consistent relationship to the stomach.As in the present case, the other intra-abdominal abnormalities include:– midline liver with or without biliary abnormality,– truncated pancreas with presence of the head and a variable portion of the body,– azygos continuation of the IVC,– midline or right-sided aorta,– right-sided stomach and/or abnormalities of the mesentery rotation.Compared to the other HS  PS is often detected incidentally in adults. Indeed, it is associated with less severe or no congenital heart disease and no immune system deficiency .


GGCX gene of infertile men with asthenozoospermia. Overall, we revealed that the GGCX-MGP system is integrated with the intercellular calcium signaling to promote sperm maturation.A low-calcium microenvironment is essential for spermatozoa to mature in the epididymis; however, it remains unclear how dysregulation of epididymal luminal calcium is associated with male infertility. Using a warfarin-induced vitamin K2 deficiency rat model, we found that vitamin-K-dependent γ-glutamyl carboxylase (GGCX) and matrix Gla protein (MGP) were essential in extracellular calcium signaling of the intercellular communication required for epididymal sperm maturation. We found that GGCX and MGP co-localized in the vesicular structures of epididymal cells and spermatozoa. Calcium-regulated MGP binds to proteins in a biphasic manner; sub-millimolar calcium enhances, whereas excessive calcium inhibits, the binding. Bioinformatic analysis of the calcium-dependent MGP-bound proteome revealed that vesicle-mediated transport and membrane trafficking underlie the intercellular communication networks. We also identified an SNP mutation, rs699664, in the   •Epididymal sperm maturation requires VK2-dependent GGCX-mediated MGP carboxylation•A GGCX SNP mutation is found in infertile men suffering from asthenozoospermia•Carboxylated-MGP regulates intercellular calcium signaling in the epididymal lumen•Calcium-regulated MGP binds to proteins in a biphasic-manner and favors low levels Cell Biology; Developmental Biology; Specialized Functions of Cells Proper intercellular communication is essential for multicellular organisms to function and survive normally, and aberrant exchange of information may result in pathological conditions and diseases. Extracellular vesicles  are the information carriers for cell-to-cell communication in most cell types . One cel2+ level in the lumen can result in defective spermatozoa and impaired fertility, without affecting spermatogenesis  and continues the carboxylation reaction . WarfariGGCX rs699664, which has been reported to cause a change from 325Arg to Gln, in association with idiopathic asthenozoospermia in infertile patients. Our study suggests that mutations of GGCX or dysfunction of the GGCX-MGP system in calcium-regulated epididymal sperm maturation is the pathological cause in some infertile men.Overall, our data reveal a role of VK2-dependent GGCX-MGP pathway in extracellular calcium-dependent vesicle-mediated cell-to-cell communication during sperm maturation. In extending the biological relevance of VKD-MGP signaling from rats to humans, we also identified an SNP mutation of As a first step to determine the expression level of the VK-cycle-associated proteins in the epididymis, real-time PCR method was used, and results showed that the mRNAs of GGCX, MGP, and VKORC1 were all expressed in the wild-type (WT) rat epididymis, whereas VKORC1L1 was detected at a negligible level A. The mRTo determine the cellular distribution and subcellular localization of the proteins, we performed confocal imaging and immunofluorescence labeling experiments. The results showed that both GGCX and MGP proteins were absent when the spermatozoa first entered the initial segment, but they appeared strongly in the epithelial cells of the caput and corpus epididymidis, then slightly decreased in the cauda epididymidis of WT males D and 1E.in situ MGP carboxylation by GGCX occurs not only in epididymal epithelial cells but also in the epididymal luminal microenvironment.To determine the cellular colocalization of MGP and GGCX, double-immunofluorescence labeling of rat epididymal cryosections were performed . Colocalin vivo, we established a warfarin-induced VK2-deficient rat model by using a modified protocol supplemented with VK1 to avoid VK1-deficiency-induced hemorrhage, as described previously  was analyzed by inductively coupled plasma mass spectrometry (ICP-MS)  to reveal the relative Cat groups A. Resultfication B. The re(ICP-MS) C. The CaTo examine the MGP carboxylation status in the epididymis after warfarin treatment, double-immunofluorescence labeling was performed on WT rat epididymal cryosections with an anti-full length MGP antibody and the conformation-specific anti-Gla residue antibody that recognizes proteins containing active γ-carboxyglutamyl (Gla) residues A. Most o2+, and Mg2+ . The analysis yielded over 1,000 proteins  in both the 32 kDa bands of epididymis and kidney with molecular sizes ranging from 10–530 kDa (data not shown). Of these proteins, 102 and 112 proteins were identified with molecular size between 20 and 25 kDa in the 32 kDa bands of the epididymis and kidney, respectively F. BioinfMGP and GGCX in a cohort of 199 patients with idiopathic asthenozoospermia and 110 fertile controls. The sperm samples collected from fertile healthy donors exhibited normal motility, morphology and sperm counts. We observed a significant association of an SNP mutation rs699664 in GGCX with male infertility under the recessive model  is a protein with an isoelectric point of 8.8, so that at the acidic pH of ∼6.8 in the epididymal lumen , the ucMConsistently, our result of titrating calcium concentrations versus the density of the 32 kDa MGP band in cultured DC2 cell lysates see E revealeGGCX SNP mutation rs699664 in the asthenozoospermic samples compared with controls, but we found no differences in MGP. It is known that the GGCX rs699664 SNP mutation results in the amino acid change from 325Arg to Gln in GGCX (To extend our rat study on VKD proteins to human reproductive health, we identified a significant difference in the  in GGCX  and is a in GGCX . Astheno in GGCX . Althoug in GGCX . Biochem in GGCX  and it min situ in the lumen. Therefore, both GGCX carboxylation and the prevention of MGP mineralization may be regulated by the epididymal luminal microenvironment. We speculate that dysregulation of such luminal GGCX-MGP system is the underlying cause of sperm maturation defects from warfarin administration and from the GGCX rs699664 mutation. Since the amino acid in position 325 of GGCX is near the MGP's binding site, which is located outside the ER lumen (GGCX rs699664 SNP mutation might thereby result in an unfavorable condition for MGP binding during carboxylation, leading to unsuccessful MGP carboxylation and eventually male infertility.Previous investigations indicate that MGP's carboxylation status does not affect its secretion into the extracellular environment . ConsistER lumen , it is bER lumen . Since uER lumen , the funIn summary, our study demonstrates that the VK2-dependent protein MGP and its carboxylation enzyme GGCX play an essential role in epididymal sperm maturation and male fertility in rats and that the mutation of GGCX is associated with one form of human male infertility. Our study also suggests that the VKD signaling axis is a promising target for the development of diagnostic products and therapies for male infertility. We also showed that the GGCX-MGP system participates in vesicle-mediated transport, presumably between epithelial cells and spermatozoa in the epididymis and that it is subject to the regulation by luminal calcium in a biphasic manner. In view of the versatile function of EVs in many physiological processes and the ubiquitous expression of MGP, our findings also provide insight into the physiology of VKD GGCX-MGP-dependent calcium signaling in other organs, such as kidney.Although this study provided insights into the VKD regulation of epididymal sperm maturation, subsequent studies are necessary to elucidate clearly the molecular mechanism of the GGCX-MGP system for sperm maturation in men. Such studies include providing the direct evidence for the exact roles and contributions of the GGCX mutations, defining the calcium-dependent MGP-mediated intercellular interaction networks, determining the physiological roles of biphasic calcium dependence of MGP-protein complexation, as well as identifying the potential transepithelial transportation pathways for these complexes across epididymis.All methods can be found in the accompanying


Soil-transmitted helminthiasis affects almost 2 billion people worldwide in tropical climates. Preventive chemotherapy, using the benzimidazoles  is the current main recommended control strategy. Nevertheless, there is limited efficacy of these drugs against hookworm infection and, to a greater extent, against trichuriasis. We describe a protocol for a trial investigating the efficacy and safety of the co-administration of ivermectin and albendazole against trichuriasis.Trichuris trichiura infection as assessed by Kato-Katz 14–21 days after treatment. Secondary outcomes include CRs against concomitant soil-transmitted helminth (STH) infections  and egg reduction rates (ERRs) against STH at 14–21 days, 180 days and 360 days. Tolerability of treatment, infection status assessed by polymerase chain reaction (PCR), and potential benefits of deworming on nutritional and morbidity indicators will be assessed. The primary analysis will include an available-case set and use logistic regression models adjusted for age, sex and weight.A double-blind, placebo-controlled randomized controlled trial will be conducted in three countries  with the aim to determine the efficacy, safety and extended effects of co-administered ivermectin and albendazole compared to standard albendazole monotherapy. We will enroll 600 participants aged 6–60 years in each setting. The primary outcome is cure rate (CR) against This trial will provide robust results on the efficacy and safety of co-administration of ivermectin and albendazole with the aim to better inform WHO recommendations on control of STHs. Furthermore, secondary and explanatory outcomes will provide direct evidence on the extended effects of combination therapy and insight on the relationship between nutrition and morbidity parameters and infection status and intensity.NCT03527732 (date assigned: 17 May 2018).The online version of this article (10.1186/s12879-019-3882-x) contains supplementary material, which is available to authorized users. Ascaris lumbricoides (roundworm), Trichuris trichiura (whipworm) and Necator americanus or Ancylostoma duodenale (hookworms), the most common STHs, account for an annual burden of 1.9 million disability-adjusted life-years (DALYs) related to infection *100 .Nutritional and morbidity indicators will be analyzed using logistic and linear regression as appropriate. To compare individual’s changes in nutrition/morbidity categories as an effect from treatment, McNemar’s test will be applied. The analysis after 6 and 12 months of follow-up will be complemented by generalized estimating equation models with independent correlation structure and empirical estimators to account for missing data.AEs will be evaluated descriptively as the difference of proportion reporting AEs before and after treatment.Soil-transmitted helminthiasis remains a public health burden in many low- and middle-income countries . Since tWith the scaling up of PC programs, mounting drug pressure increases the risk for drug resistance against the benzimidazoles in populations infected by STHs. Nonetheless, combination treatment of two or more drugs can provide heightened efficacy and protection against drug resistance . Our datWe will report not only on efficacy and safety outcomes of combination ivermectin and albendazole therapy, but also the effects of drug administration on morbidity in a broad age range. In the midst of controversy on the impact of PC through mass deworming campaigns, this trial provides direct evidence in determining the relationship between deworming campaigns and clinical morbidity and nutritional indicators , 40–44. We will also report on the differences in CRs and sensitivity between the current diagnostic method recommended by the WHO (Kato-Katz smear) and DNA-based methods (qPCR). A major drawback of the Kato-Katz method is the low sensitivity for infections of low intensity . As counIn conclusion, this trial will aim to generate evidence to inform future WHO guidelines of STH therapy and its impact on morbidity. Currently, the combination of ivermectin and albendazole is a safe and effective treatment that is currently given to more than 500 million individuals yearly as part of the Global Programme to Eliminate Lymphatic Filariasis; and there may be opportunities to transition the program to control and elimination of soil-transmitted helminthiasis , 49. HowAdditional file 1:World Health Organization Trial Registration Data Set for the efficacy and safety of IVM-ALB co-administration trial summarizing the most important trial information. (DOCX 20 kb)Additional file 2:SPIRIT checklist for the efficacy and safety of IVM-ALB co-administration trial referring to study protocol elements within manuscript sections. (DOC 122 kb)


Local dynamic FC was examined by calculating the dynamic regional homogeneity (dReHo), and large-scale dynamic FC (dFC) was investigated between regions with significant dReHo group differences. For the PTSD patients, we also investigated the relationship between symptom severity and dFC/dReHo. Our results showed that PTSD patients were characterized by I) increased dynamic (more variable) dReHo in left precuneus (PCu); II) increased dynamic (more variable) dFC between the left PCu and left insula; and III) decreased dFC between left PCu and left inferior parietal lobe (IPL), and decreased dFC between left PCu and right PCu. However, there is no significant correlation between the clinical indicators and dReHo/dFC after the family-wise-error (FWE) correction. These findings provided the initial evidence that PTSD is characterized by aberrant patterns of fluctuating communication within brain system such as the default mode network (DMN) and among different brain systems such as the salience network and the DMN.Posttraumatic stress disorder (PTSD) is a psychiatric condition that can emerge after exposure to an exceedingly traumatic event. Previous neuroimaging studies have indicated that PTSD is characterized by aberrant resting-state functional connectivity (FC). However, few existing studies on PTSD have examined dynamic changes in resting-state FC related to network formation, interaction, and dissolution over time. In this study, we compared the dynamic resting-state local and large-scale FC between PTSD patients ( Posttraumatic stress disorder (PTSD) is a psychiatric condition that can emerge after exposure to an exceedingly traumatic event . In the One effective approach for exploring brain communication is through the analysis of resting-state fMRI studies . Recent Regional homogeneity (ReHo) is one of the commonly used algorithms in measuring local FC –23. ReHoThe investigation of dFC and dReHo in the resting state may provide new insight into the aberrant brain connectivity in PTSD. Previous studies investigating major depression , schizopAlthough there are few studies available that have focused on the dynamic brain activity in PTSD, prior static studies have suggested that the symptoms of PTSD are associated with the DMN. Mounting evidence has indicated that PTSD is associated with aberrant DMN connectivity –35. A stIn order to explore the characteristic resting-state temporal variability of PTSD, we decided to measure both large-scale and local dynamic FC. Based on previous findings, we hypothesized that PTSD patients would exhibit altered dReHo in regions within the DMN. We also expected regions with dReHo alterations to show aberrant dFC and the connectivity measures to be associated with subjects’ symptomatology.Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV); IV) no psychiatric medications or substance abuse; V) no MR imaging contraindications; VI) no head trauma or neurologic disorders; VII) fulfills the criteria of DSM-IV and has a Clinical-Administered PTSD Scale (CAPS) score >40; and VIII) not pregnant or nursing. After considering the strict requirements, eight subjects were excluded, five of them for failing to obtain the CAPS score >40. Twenty-two demographically matched healthy controls (HCs) were recruited for this study. The inclusion criteria for HCs were as follows: I) age >18 years; II) right-hand dominance; III) no preexisting psychiatric disorders or physical conditions as determined by a structural clinical interview using the DSM-IV; IV) no psychiatric medications or substance abuse; V) no MR imaging contraindications; and VI) not pregnant or nursing. Each participant provided written informed consent, which was obtained prior to the MRI scanning.Permission to undertake this study was granted by the ethics committee of Guangdong Second Provincial General Hospital. In January and February 2017, we recruited 30 trauma-exposed subjects from a serious highway traffic accident in Guangdong province. Prior to the examination, none of the patients had undergone any psychotherapy. The inclusion criteria for the PTSD patients were as follows: I) age >18 years; II) right-hand dominance; III) no preexisting psychiatric disorders or physical conditions as determined by a structural clinical interview using the PTSD diagnosis was determined following the DSM-IV diagnostic criteria. Before undergoing resting-state MRI, all PTSD patients were screened with CAPS  in orderEach of the participants underwent a resting-state MRI in a 3.0-T MR imager  equipped with a 32-channel head coil at the Department of Medical Imaging in Guangdong Second Provincial General Hospital. A diagnostic T1-weighted image and a T2 fluid attenuated inversion recovery (T2-FLAIR) image were taken to exclude participants with brain lesions. The resting-state fMRI data were acquired using gradient echo-planar imaging (EPI) with the following parameters: repetition time (TR)/echo time (TE) = 2,000 ms/30 ms; matrix = 64 × 64; field-of-view = 230 mm × 230 mm; flip angle = 90; slice thickness = 3.6 mm, 0.6-mm gap; interleaved scanning; 38 transverse slices covering the whole brain at all 240 volumes were acquired for each participant within 480 s; each volume was aligned along the anterior–posterior commissure. Each participant was instructed to lie still and to avoid falling asleep or thinking of anything in particular during MR scanning.http://rfmri.org/DPARSF) and the SPM12 package (www.fil.ion.ucl.ac.uk/spm) based on MATLAB . The first 10 volumes of each dataset were discarded for signal equilibration. The remaining data were performed using slice timing correction and realignment and co-registered with the anatomical scan. The co-registered T1-weighted images were segmented into gray matter, white matter, and cerebrospinal fluid. And then the functional images were normalized into the Montreal Neurological Institute (MNI) space with a voxel size of 3 × 3 × 3 mm3. The head movement parameters were obtained from the realignment steps in the DPARSF. We took the mean FD Jenkinson . By sliding the onset of each window by 2 dynamics (4 s), for a total of 101 overlapping windows in the first level analysis, the dReHo was estimated by using the calculated SD of the ReHo through the windows at each voxel, yielding a set of ReHo maps for each participant.t-test with head motion parameters , age, and sex as covariates was performed to test the difference in dReHo maps between the PTSD patients and HC at each voxel. Multiple comparisons correction was performed with Gaussian random field (GRF) theory at the cluster level .Two-sample p < 0.05 (uncorrected).To determine whether the dReHo metrics were associated with clinical indicators, we performed general linear models with the clinical indicators  and mean dReHo values from clusters with significant group differences as independent variables, and head motion parameters, age, and sex as covariates. The correlation analysis was accomplished with the SPSS software with a significance threshold of z transformed Pearson correlation coefficient between the averaged time course of all voxels in the seed and the time course of all other voxels in the whole brain, yielding a set of sliding window zFC maps for each participant. The dFC was estimated by calculating the standard deviation in zFC values through windows at each voxel.Voxel-wise seed-based FC analyses were performed using the DPARSF 4.3. We employed the aberrant dReHo region, which we calculated above, as a seed region. Then we used the sliding-window approach as we have used in the dReHo calculation; the time course was segmented into 60-s Hamming windows by sliding the onset of each window by 2 dynamics, for a total of 101 overlapping windows in the first level analysis. Within each sliding window, the whole brain FC maps for the seed region were computed as the Fisher t-test with head motion parameters , age, and sex as covariates was performed to investigate the difference of dFC values between the PTSD group and the HC group at each voxel. Multiple comparisons were performed with GRF correction at the cluster level .Two-sample p < 0.05 (uncorrected).To explore the relationship between dFC metrics and clinical indicators, we performed general linear models with the clinical indicators and mean dFC values from clusters with significant group differences as independent variables, and head motion parameters, age, and sex as covariates. The correlation analysis was accomplished with SPSS software with significance threshold of The demographic and clinical data are summarized in Next, we conducted analyses to test the association between the clinical indicators  of PTSD and dReHo within the PTSD group. There is no significant correlation between the clinical indicators and dReHo/dFC after FWE correction  in left PCu, suggesting that the neurofluctuation of the left PCu is unstable in the PTSD patients in the resting state. The PCu is a key hub of the pDMN –49 and iSeveral studies have identified aberrant activities in the DMN of PTSD patients , 34, 35.In the present study, the left insula was the only region in the brain that exhibited significant positive dynamic correlation with the left PCu in the PTSD group. The insula is a key hub in the SN and is thought to be involved in the detection of personally salient internal and external stimuli that guide behaviors in order to maintain equilibrium . In addiThere are some limitations in the present study that should be highlighted. Firstly, little information is available on the meaning of the resting-state dFC in neurocognitive functioning. For example, it remains unknown whether the abnormal dynamic activities in the resting state are intrinsic properties or are affected by the present-moment cognitive activities , 60. As In conclusion, this resting-state dFC (combine the dReHo and dFC) study provided evidence that the PTSD patients exhibited aberrant dReHo and dFC in comparison with the HC. Decreased variability within the DMN may suggest dysfunction of self-referential processing in PTSD patients, while increased variability between the insula and PCu may suggest dysregulation between the DMN and the SN.Permission to undertake this study was granted by the ethics committee of Guangdong Second Provincial General Hospital.SF designed the experiment. SF and XM carried out the experiment. ZB collected and sorted out the data. YW, MLiu, YY, ZL, SH, MLi, and KH helped on data management and processing. SF and GJ wrote the manuscript.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.


Low socioeconomic status (SES) is a barrier for cardiovascular disease (CVD) risk screening and a determinant of poor CVD outcomes. This study examined the associations between access to health-promoting facilities and participation in a CVD risk screening program among populations with low SES residing in public rental flats in Singapore.Data from Health Mapping Exercises conducted from 2013 to 2015 were obtained, and screening participation rates of 66 blocks were calculated. Negative binomial regression was used to test for associations between distances to four nearest facilities  and block participation rate in CVD screening. We also investigated potential heterogeneity in the association across regions with an interaction term between distance to each facility and region.The analysis consisted of 2069 participants. The associations were only evident in the North/North-East region for subsidized private clinic and park. Specifically, increasing distance to the nearest subsidized private clinic and park was significantly associated with lower  and higher  screening participation rates respectively.Our findings could potentially inform the planning of future door-to-door screenings in urban settings for optimal prioritization of resources. To increase participation rates in low SES populations, accessibility to subsidized private clinics and parks in a high population density region should be considered. We further tested potential heterogeneity in the association by considering an interaction between the distance from the nearest health-promoting facility and region. Singapore is a city state in South-East Asia, comprising three major ethnic groups . Public rental flats  database in Singapore Heart Foundation. HME  had a single encounter.©   or with a difference of greater than or equal to two years between calculated and self-reported ages were excluded from the analyses. Hence, our analyses comprised of 2069 HME participants. To account for demographic factors in the variability of block screening participant rate, participants were aggregated by block to obtain the number of participants stratified by: age (<40 or ≥40 years old), ethnicity (Chinese or non-Chinese), and gender . We used 40 years old as the age cut-off as Singapore’s MOH recommends CVD risk screening for adults ≥40 years old  was calculated by dividing the number of participants in a block  by three times the total number of one- or two-room apartments in a block, multiplied by 100. We first performed a descriptive analysis on the block characteristics and reported their medians, interquartile ranges (IQR), and minimum and maximum values.et al., P-value exceeding 0.05.Due to the presence of over-dispersion in the data, we used negative binomial regressions  with their respective 95% confidence intervals (CI) and A total of 66 blocks were in our study, with the majority (53.0%) located in the Central/West regions, and the remaining blocks almost equally distributed between the East (24.2%) and North/North-East (22.7%) regions. These blocks housed a median of 216 one- or two-room apartments  than overall and those aged <40 (0.7 per 100 apartment-years). Meanwhile the East region had the highest rate (6.2 per 100 apartment-years), followed by the Central/West (4.5 per 100 apartment-years) and North/North-East (4.0 per 100 apartment-years) regions.For the unadjusted analyses  and parks in the North/North-East regions  were significantly associated with block screening participation rate  and to the nearest subsidized private clinic (every 100 m increase) was significantly associated with a 93% increase and 12% decrease in block screening participation rates respectively. Interestingly, these were only observed in the North/North-East regions where all blocks except one were in the North-East, and this region has the highest population density  in our sample had to be excluded due to missing data and discrepancies in self-reported and calculated ages. We performed two sensitivity analyses that did not exclude participants with discrepancies between self-reported and calculated ages by using age calculated from date of birth and self-reported age which reduced the loss of participation count to 5% (n = 2488) and 11% (n = 2330) respectively. Sensitivity analyses yielded similar findings (see Appendices 1 and 2).Our study has limitations. First, the non-participants were unavailable and hence blocks were used as the unit of analysis instead of individuals. To estimate participation rate per 100 apartment-years, we assumed that the composition of residents were similar across each apartment over the three consecutive years. There is likely small changes in the composition of residents given the short period of three years as the Ethnic Integration Policy (EIP) imposes ethnicity quota to maintain good ethnic mix in public housing estates  not only face barriers to cardiovascular disease (CVD) risk screening, but have also been shown to have higher incidence and mortality of CVDs.A wide range of individual factors have been shown to be associated with CVD risk screening participation, but little attention has been given to physical environment factors.Associations between physical environment and health behavior such as diet and physical activity have been well studied, but not with health screening behavior.What this study adds:This is the first study to examine the associations between access to health-promoting facilities and health screening participation among populations with low SES by integrating data from different sources.This study demonstrated that these associations varied based on the type of health-promoting facilities and the regions the blocks were located in.Our analyses illustrate how access to health-promoting facilities could influence participation in CVD risk screening and how it could advise the planning of future door-to-door screening initiatives in an urban setting for low SES population.


Evolutionary development of vision has provided us with the capacity to detect moving objects. Concordant shifts of visual features suggest movements of the observer, whereas discordant changes are more likely to be indicating independently moving objects, such as predators or prey. Such distinction helps us to focus attention, adapt our behavior, and adjust our motor patterns to meet behavioral challenges. However, the neural basis of distinguishing self-induced and self-independent visual motions is not clarified in unrestrained animals yet. In this study, we investigated the presence and origin of motion-related visual information in the striatum of rats, a hub of action selection and procedural memory. We found that while almost half of the neurons in the dorsomedial striatum are sensitive to visual motion congruent with locomotion , only a small subset of them are composed of fast-firing interneurons that could also perceive self-independent visual stimuli. These latter cells receive their visual input at least partially from the secondary visual cortex (V2). This differential visual sensitivity may be an important support in adjusting behavior to salient environmental events. It emphasizes the importance of investigating visual motion perception in unrestrained animals. Evolution of vision has provided us with the capacity to detect moving objects. However, visual motion perception may occur both because of the movement of an observer in an otherwise stationary environment and because of independently moving observed objects. Distinguishing these two sources or simply recognizing truly moving objects when the observer is also moving is essential for survival; it allows the reliable detection of predators or prey in real-world situations. In this study, we investigated the presence and origin of motion-related visual information in the striatum of rats, a hub of action selection and procedural memory. We found that while many of the striatal neurons responded to the apparent visual motion while the rat was running in an environment with striped walls, only a subset of them could also respond to moving stripes with similar properties if watched passively. This differential visual sensitivity may be an important mechanism of adjusting behavior to salient environmental events. The need for congruent locomotion to evoke neuronal responses in many of the recorded neurons emphasizes the importance of investigating visual motion perception in unrestrained animals. Besides the well-known motor functions of the basal ganglia , there iPrevious experiments that studied the motion sensitivity of the CPu at the single-neuron level employed anesthetized or restrained animals. Thus, they were not designed for deciphering if the CPu is equally sensitive for the self-motion-induced  and the self-motion-independent object motion. More profoundly, there is no existing evidence that the CPu neurons can distinguish between these two conditions at all nor whether they can integrate the visual motion percepts with spatial representations or other aspects of striatal information processing.To answer this question, we investigated the response profiles of the dorsal CPu neurons to self-independent and self-motion-induced visual motion stimuli in freely moving rats. We did this by conducting large-scale, high-density extracellular recordings. We also made efforts to show the role of the corticostriatal pathway in shaping these responses, as there is no consensus regarding the origin of the striatal visual information either ,20–22.N = 32, 285–655 g; 3–12 mo old). The intermediate part of the dorsomedial striatum has been reported to display visual stimulus—induced electrophysiological responsivity in many species  / [FRpeak + FRpeak-180°]).Space-resolved neuronal activity patterns were also analyzed with respect to the local luminance at the instantaneous positions of the rats: each location was assigned with the local luminance value of the “striped” pattern . Circular histogram of the “stripe phase”–resolved activity pattern was generated by averaging the activity patterns across all 14 cycles and all trials of the given condition. For the “uniform” condition with nonmodulated gray pattern, the same cycle phase values were used in the analysis as determined in the “striped” condition. Activity patterns were tested for circular uniformity using the Rayleigh test, and only those neurons were included in the population analysis that showed a significant deviation from the von Mises distribution either in the “striped” or in the “uniform” condition. This deviation was quantified as the MI of circular nonuniformity (MI = [FRmax − FRmin] / [FRmax + FRmin]).Power spectrum and time-resolved spectral analysis were performed on the low-pass-filtered and downsampled  LFP signals using multitaper spectral analysis. Spectra were whitened by multiplying with the corresponding frequencies and expressed as decibels (dB). For all local spike—LFP coupling analyses, the median of the local LFP signals recorded on the same shank has been used. For phase coupling of the CPu neurons to V2 LFP, the median LFP of all V2 recording sites was used instead. Subjectively noisy LFP channels due to recording sites with too-high impedance were discarded. LFP signals were filtered in 5 frequency bands  with an eighth-order zero-phase-lag Butterworth filter, and phase values were obtained using Hilbert transform. No 50 Hz notch filtering was used in the data acquisition or during the data analysis. LFP phase histograms were constructed by taking the corresponding instantaneous LFP phase values corresponding to the isolated single-unit spikes, and their deviation from the circular uniform distribution was tested using the Rayleigh test. The bin size of the histograms was 18° and was smoothed by calculating a 5 bin wide moving average of the histograms. The phase values corresponding to the peaks and troughs of the smoothed distributions were taken as preferred and unpreferred phases, respectively. Phase MIs were calculated from smoothed occurrence frequencies of spikes at the preferred and unpreferred phases to account for possible asymmetric distributions  the following way: Both signals were iteratively filtered between 1 and 100 Hz in 1 Hz steps, with an eighth-order, zero-phase-lag passband filters, and their cross-correlations were calculated for  ms lags, in 2 ms steps. The magnitudes of the cross-correlograms were taken as the absolute values of its Hilbert transforms for each frequency. To remove the additive distortion caused by the symmetric correlation of the synchronous events , the minima of each symmetric time bin pairs have been subtracted from the same bins. The principle of this approach is demonstrated by analyzing 2 artificial LFP signals and explained on Median LFP signals were calculated for each recording shank . Granger causality was estimated for each V2 and CPu signal pairs. Only time lags in the range of  ms were S1 Data(XLSX)Click here for additional data file.S1 Fig(A) Original tile-scan image of the BDA-labeled axons on a representative example slice (left). Middle panel shows the intensity-thresholded image, while right panel denotes the calculated binned axon density. (B) Original tile-scan of the cFos+ neuronal somata on the same slice (left) and its inverted version (middle). Inset of the middle panel shows a magnified image of a cFos+ neuron. Note the red dot placed at the center of the soma by the automated detection algorithm. Right panel shows the binned density of the automatically detected cFos+ neurons. (C) Manually generated image mask to avoid artefact detection. Black mask covers the non-CPu structures, while red masks cover the intra-CPu artefacts . (D) Distribution of intra-CPU masks at all slices along the vertical (blue) and mediolateral (red) axes. Note that the masks were distributed uniformly and covered only approximately 1% of the total CPu area. (E) Correlation of the binned BDA and cFos density values of the representative slice shown on panels A—C. The correlation coefficient of the value pairs and the significance level of the correlation are displayed above the plot.  Small (left) and large (right) magnification photomicrographs of the V2 injection site of the BDA tracer (F) and the V1 (G) of a stimulated animal. Color channels are identical to those on (TIF)Click here for additional data file.S2 Fig(A—C) Waveform and spike train characteristics of a representative PFN (A), FFN (B) and TFN (C). Top left panels: mean extracellular spike waveforms on the 8 recording sites of the corresponding electrode shank. Top-right panels: distribution of ISIs, ISI2s are aggregated. Note the high proportion of long ISIs for PFNs (red arrow). Bottom-left and right panels display the postspike suppression histograms and autocorrelograms, respectively. (D) Classification of the recorded neurons based on the features of their spike waveforms (left panel) and spike trains (right panel). (E) Proportion of the recorded PFNs (64%), FFNs (22%), TFNs (8%), and unclassified neurons (6%) among all recorded single units. CPu, caudate putamen; FFN, fast-firing neuron; ISI, interspike interval; ISI2s, interspike interval exceeding 2 s; PFN, phasically firing neuron; TFN, tonically firing neuron.(TIF)Click here for additional data file.S3 FigThe left panels of each example show the spatially resolved firing patterns in the uniform and striped environments . Right panels show the details of statistical testing of the space-resolved visual modulation. The thick black lines show the real FR differences in the two environments, while the gray curves denote the label-shuffled surrogate firing pattern differences. Purple and green lines depict the “local” and “global” significance thresholds, respectively. Spatial locations breaking the global significance limits are marked with thick red lines in both the left and right panels. aVis, active visual; FR, firing rate.(TIF)Click here for additional data file.S4 FigThe plots are showing the comparison of the normalized FRs of each pVis+ neurons in the uniform (abscissa) and in the striped maze (ordinate). Identity and regression lines are shown in black and red, respectively. Correlation coefficients and significance levels are shown above each plot. To form the FR, data points of only those consecutive trials were included in which the condition was changing from “uniform” to “striped” or vice versa. FR values were normalized by using the maximum rate during the “uniform” trials. FR, firing rate; pVis, passive visual.(TIF)Click here for additional data file.S5 FigThe plots show the comparison of the normalized FRs of each place cell—like neurons in the uniform (abscissa) and in the striped maze (ordinate). Identity and regression lines are shown in black and red, respectively. Correlation coefficients, significance levels, and the coefficients of the regression lines  are shown above each plot. To form the FR, data points of only those consecutive trials were included in which the condition was changing from “uniform” to “striped” or vice versa. FR values were normalized by using the maximum rate during the “uniform” trials. FR, firing rate.(TIF)Click here for additional data file.S6 Fig(A) Modulation indices of the rate remapping depend on the temporal velocity of the perceived visual motion, determined by the running velocity. Slower speeds elicited weaker decrease  of the FRs. (B) Comparison of the mean running speeds in the striped and uniform environments. (C-E) Analysis of aVis+ neurons’ activity patterns in the reward zones, where the rats were not running—thus, no visual motion was perceived—but they still observed the stationary patterns of the walls. (C) The average time spent at the reward zones in trials when uniform or striped pattern were presented was similar. (D) The FRs of the aVis+ neurons were almost identical at the reward zones, with no respect to the pattern or context. (E) The distribution of the significance levels of the cell-by-cell comparison of the firing patterns depict that only a small minority of the aVis+ neurons perceived the stationary patterns as different. (F) Two representative neurons excited (left) or inhibited (right) by the global brightness increase. (G) Percent of neurons responsive to global luminance change. Note that in contrast to the aVis+ neurons, more than half of the pVis+ neurons were sensitive to brightness. (H) Response profile of a representative neuron triggered by the individual sinusoidal cycles of the grated pattern. Local luminance levels are shown above the plot; 2 full stripe cycles are shown for better visibility. Note that the stripe phase—resolved firing pattern (red) is similarly uniform as the pattern in the uniform trials (blue). (I) Mean stripe phase—resolved circular modulation indices of the aVis+ neurons were similar in the two conditions. aVis, active visual; FR, firing rate.(TIF)Click here for additional data file.S7 FigPreferred LFP phases are shown as histograms for pVis−/aVis− (top row), pVis+ (second row), and aVis+ (third row) neurons separately. Bottom row shows the comparison of phase coupling strength expressed as the distribution of the phase modulation indices for the three groups of neurons Click here for additional data file.S8 Fig(A) A demonstrative example of 2 artificial LFP signals with a phase-delayed continuous theta oscillation, 2 time-delayed discrete gamma bursts in the low- and high-gamma band, and a coincident square wave resembling a recording artefact. (B) Raw time and frequency—resolved cross-correlograms constructed from individual cross-correlograms calculated after iterative filtering for each frequency band. (C) The same time and frequency—resolved cross-correlogram after the suppression of the zero-lag correlation components. Note the emergence of 3 domains (red arrows), representing the phase-shifted theta oscillations and the gamma bursts with the appropriate time lags. Note also the lack of the artefact’s spectral profile, which dominated the raw spectrogram on panel B. LFP, local field potential.(TIF)Click here for additional data file.