Deformable image registration is of essential important for clinical diagnosis, treatment planning, and surgical navigation. However, most existing registration solutions require separate rigid alignment before deformable registration, and may not well handle the large deformation circumstances. We propose a novel edge-aware pyramidal deformable network (referred as EPReg) for unsupervised volumetric registration. Specifically, we propose to fully exploit the useful complementary information from the multi-level feature pyramids to predict multi-scale displacement fields. Such coarse-to-fine estimation facilitates the progressive refinement of the predicted registration field, which enables our network to handle large deformations between volumetric data. In addition, we integrate edge information with the original images as dual-inputs, which enhances the texture structures of image content, to impel the proposed network pay extra attention to the edge-aware information for structure alignment. The efficacy of our EPReg was extensively evaluated on three public brain MRI datasets including Mindboggle101, LPBA40, and IXI30. Experiments demonstrate our EPReg consistently outperformed several cutting-edge methods with respect to the metrics of Dice index (DSC), Hausdorff distance (HD), and average symmetric surface distance (ASSD). The proposed EPReg is a general solution for the problem of deformable volumetric registration. Deformable image registration is to perform spatial transformation between a pair of images and establish a non-linear point-wise correspondence to achieve spatial consistency  for unsupervised volumetric registration. The proposed EPReg is a dual-stream pyramid framework, which utilizes original images and corresponding edge-aware maps to compose dual inputs, and generates multi-scale paired feature maps for recursively transforming the information between images into more representative features to predict more accurate deformation field. Finally, the trained EPReg can perform deformable registration in one forward pass. Extensive experiments on three 3D brain magnetic resonance imaging (MRI) datasets demonstrate that our proposed network achieves satisfactory registration performance.The main contributions of our work are 2-fold.We propose to fully exploit the useful complementary information from the multi-level feature pyramids to predict multi-scale deformation fields. Such coarse-to-fine estimation facilitates the progressive refinement of the predicted registration field, which enables our network to handle large deformations between volumetric images.We integrate edge information with the original images as dual-inputs, which enhances the texture structures of image content, to impel the proposed network pay extra attention to the edge-aware information for structure alignment.The remainder of this paper is organized as follows. Section 2 presents the details of the edge-aware pyramidal deformable network. Section 3 shows the experimental results of the proposed EPReg for the application of brain MRI registration. Section 4 elaborates the discussion of the proposed network, and the conclusion of this study is given in section 5.If) and a moving volume (Im). Their edge maps are denoted as Ef and Em, respectively. The EPReg network leverages deformable pyramid to progressively transform the information between If-Im and Ef-Em into more representative features to predict more accurate deformation field (ϕ4 ~ ϕ1). The trained EPReg can attain deformable registration in one forward pass.The proposed registration network is illustrated in The following subsections first give a brief introduction on the deformable registration and then present the details of our scheme and elaborate the novel edge-aware pyramidal deformable architecture.If. The optimization problem can be defined as:Volumetric registration is to establish the voxel-wise correspondences between different volumes  of the input volumetric pair, which are then used to progressively predict multi-scale deformation field (ϕ4 ~ ϕ1). Such coarse-to-fine estimation based on paired feature pyramids enhances the capability for handing large-scale deformation estimation.The convolutional blocks capture hierarchical paired features :Based on paired feature pyramids, we then progressively carry out the non-rigid registration via the devised deformable block see . The defdi represents the parameter learned by the i-th deformable block fdi, and i = 1, 2, 3. In such a way, based on previous deformation estimation  and paired feature pyramids, each deformable block further estimates extra deformation ϕi, which can integrates with ϕi+1 to attain more accurate non-rigid registration. For the i-th deformable block, the 3D deformable grid Gi is the combination of Gi+1 (with 2×upsampling) and ϕi  volumetric patch at the center of voxel p.where To avoid obtaining an unpractical or discontinuous deformation field, we also add a diffusion regularizer where As the deformation is estimated progressively, we consider similarity loss for each scale of the registration pyramid. Therefore, the total loss is defined as:down2i denotes a down-sample operation with a factor of 2i. Gi is the 3D deformation grid generated to warp the moving volume.where The study protocol was reviewed and approved by the Institution's Ethical Review Board. Experiments were carried on three public brain MRI datasets with manually labeled region of interests (ROIs), including Mindboggle101 : 62 volumes were involved to conduct experiments as described in Kuang and Schmah . SpecifiLPBA40 (40 T1-weighted MRI volumes): 30 volumes were randomly selected for training and the remaining 10 volumes were used for testing.IXI30 (30 T1-weighted MRI volumes): all 30 volumes were used for testing. In order to investigate the generalization ability of the network, we employed the model trained on LPBA40 to register images from IXI30 dataset.All volumes were pre-processed by histogram and intensity normalization, and skull-stripping using FreeSurfer Fischl, .In our experiments, each input volumetric image was resized into the dimension of 192 × 192 × 192. The network was trained on a GPU of NVIDIA Tesla V100. The value of the regularization parameter λ was set empirically as 1000. For the whole registration network, the number of epochs was set to 300. The network was implemented using Pytorch and Adam optimization  Dice, , HausdorRIf and RIm are the segmented ROIs of If and Im, respectively. The HD measures the longest distance over the shortest distances between the segmented ROIs of If and Im. The ASSD is defined as:where BIf and BIm are the segmented surfaces of If and Im, respectively. The operator d is the shortest Euclidean distance operator.where All evaluation metrics were calculated in 3D. A better registration shall have larger DSC, and smaller HD and ASSD.We compared our EPReg network with four cutting-edge brain MRI registration schemes: SyN  are shown in The numerical results on IXI30 are illustrated in t-test was employed to pairwisely compare the registration performance between our method and the other five methods on three different datasets  will be our future work.The problem of registration validation in clinical settings is still an open issue. Most of recent research articles focusing on developing new registration approaches employ DSC as a primary metric to evaluate the registration accuracy (Balakrishnan et al., In this paper, we have presented an edge-aware pyramidal deformable network for unsupervised volumetric registration. The proposed network focuses on the large deformation estimation by progressively predicting the residual deformation. Our key idea is to fully exploit the useful complementary multi-level information from paired features to predict multi-scale deformation fields. We achieve this by developing a deformable pyramid architecture, which can generate multi-scale paired feature maps for progressively transforming the paired information into more representative features to predict more accurate deformation field. In addition, we leverage extra edge information to impel the network pay attention to the edge-aware alignment. Extensive experiments on several 3D MRI datasets demonstrate that our edge-aware pyramidal deformable network achieves satisfactory registration performance. The coarse-to-fine progressive registration procedure is beneficial to compensate for the large-scale deformation, and can be regarded as a general solution for deformable volumetric registration.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.YC, ZZ, YR, CQ, DL, QD, DN, and YW response for study design. YC and CQ implemented the research. YC, ZZ, QD, and YW conceived the experiments. YC, ZZ, and YR conducted the experiments. YC, ZZ, YR, DL, and YW analyzed the results. YC and YW wrote the main manuscript text and prepared the figures. All authors reviewed 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.


Purpose: To report the effects of a single intravitreous injection of bevacizumab for the treatment of secondary choroideal neovascularisation due to large optic disc drusen.Methods: A 14-year-old female patient with painless loss of vision in one eye presented with unusually large optic disc drusen and juxtapapillary choroidal neovascularisation with subretinal hemorrhage. She was treated with a single intravitreous injection of bevacizumab.Results: Visual acuity increased from 20/100 to 20/25 within 4 weeks after injection and remained at this level during the 12-month follow-up period.Conclusions: Bevacizumab is a possible primary treatment option for secondary choroidal neovascularisation due to large optic disc drusen in children as an alternative to other more invasive or complex procedures. Optic disc drusen (ODD) are remnants of mucopolysaccharides from degenerated ganglion cells which calcify and accumulate within the optic nerve head over time . The incWith increasing size, they raise the optic disc, blur out the edges, and lead to abnormal vascular branching and formation of cilioretinal vessels, causing hemorrhages . This deBecause of the rare occurrence of CNVM associated with ODD, different therapeutic approaches have been reported only via small case studies. Next to surgical removal  and photA 14-year-old female patient presented at our clinic with painless loss of vision in her left eye, slowly progressing for over one week. VA had dropped to 20/100 on the affected side, whilst retaining 20/20 vision on the other eye. Intraocular pressure was within physiological limits. Further examination showed a blurry and prominent optic disc with an adjacent decent subretinal hemorrhage Figure 1 . The conAfter discussing the potential treatment options and risks with the child’s parents, a single intravitreous injection of 1.25 mg bevacizumab was given under general anesthesia.VA increased to 20/25 over a period of 4 weeks. The cystoid edema as well as the subretinal hemorrhage resolved, leaving a small subretinal scar and a mild dissociation of the pigment epithelium Figure 2 . The patIn the last decade, intravitreal injections of anti-VEGF agents have proven to be an effective therapy for a variety of retinal and especially macula diseases, for example neovascular age-related degeneration and diabetic macular edema. Disease-adapted guidelines were promoted and published for the initial therapy as well as for the long-term intervention , 12], , . BecauseIn our case, the patient showed a complete remission after only one injection. The options of further injections at a fixed interval, or of close observation and treatment only if required were discussed with the patient and her parents. The decision was made towards short-interval follow-up examinations. Compared to the results of anti-VEGF therapy that have been published in other case series, the juvenile secondary CNVM in our case responded better to this form of treatment. Until today, no further injections were necessary, confirming our choice of treatment plan.The authors declare that they have no competing interests.


Dendritic cells (DCs) are the sentinels of the immune system. They sense, process, and present antigens to T lymphocytes orchestrating the immune response. The discovery of DCs granted the Nobel Prize for Ralph Steinman, who noticed the presence of rare cells in a culture of mice adherent cells with a distinct stellate morphology . Years lSince the discovery of DCs, they have been extensively studied in several contexts, from immunity to pathogens and tumors to the induction of tolerance or an immune response to transplants, to self, and more recently to microbiota and dietary antigens, with new discoveries happening constantly. Recent advances in the field have pointed at new DC function as well as new DC subsets with specific transcriptomes . DCs areThe mechanisms of DC development either from progenitors of bone marrow or from circulating monocytes have also gained much knowledge in the past years, enlightening the signaling pathways activated in the rising of the distinct DC subsets. They are either GM-CSF-mediated STAT5 phosphorylation- or FLT3-L-mediated STAT3 phosphorylation-dependent pathways. Moreover, several transcription factors have been identified as subtype specific for the development of DCs in mouse models , 10, sucThis special issue presents a collection of original research articles that unveil the role of dendritic cells, their cellular interactions, and the molecular mechanisms and signaling pathways involved in clinical and experimental models of infections, inflammatory disease, and cancer. First, Azevedo-Santos et al. investigated the mechanisms of the tolerogenic role of DCs from breast tumor patients. In this patients' cohort, they corroborated previous data by demonstrating that monocyte-derived DCs (mo-DCs) from cancer patients are less mature, have a decreased ability to stimulate T lymphocyte proliferation, and produce the anti-inflammatory cytokine IL-10. In addition, they showed that completely differentiated DCs from patients presented the same phenotype as DCs from healthy donors when cocultured with breast cancer-derived cell lines. Nevertheless, monocytes isolated from cancer patients expressed less GM-CSF and IL-4 receptors than monocytes isolated from healthy donors, which may inhibit the differentiation of inflammatory DCs from circulating monocytes. They correlated the presence of the heat shock protein 27 (Hsp27), which is involved in tumor cell proliferation and invasion, with the tolerogenic profile of DCs. In addition to previous data that showed Hsp27 expression in tumor cells, they also revealed that mo-DCs derived from cancer express higher levels of Hsp70. The direct involvement of DC-expressed Hsp27 with the anti-inflammatory response to the tumor still needs clarification.+ DCs in a mouse model of herpes simplex virus-1- (HSV-1-) induced Behçet's disease, when comparing asymptomatic and symptomatic mice. By performing a set of experiments blocking CD83 in vivo by different techniques, they revealed an improvement on the severity score associated with reduced frequency of CD83+ DCs and downregulation of IL-17 serum levels, which demonstrates the involvement of DCs in the pathology of the disease. Interestingly, ablation of CD83 blockade worsened the symptoms, whereas reestablishment of CD83 inhibition with siRNA treatment, even in the late stage of the disease, restored the beneficial effects and indicates that CD83 is a good candidate for novel therapeutic approaches in Behçet's disease. In the second article, Helmin-Basa et al. detected increased circulating CD83-expressing cDCs in Helicobacter pylori-infected children compared to healthy controls, although the percentages and total number of DCs were similar. The induction of circulating cDC maturation was associated with cDC infiltration in the gastric lamina propria, albeit infiltrating DCs did not express CD83, indicating a more immature profile of DCs in the infected site, which might induce tolerance instead of immunity to local antigens. This set of articles highlighted the importance of DC maturation not only for immunity against HSV-1 and H. pylori infections but also for the immune response-driven pathology.The maturation state of DCs was the aim of investigation of two articles that show its role in different infectious and inflammatory diseases. In the first article, Islam et al. observed a higher frequency of circulating CD83Trypanosoma cruzi strain Ninoa infected mo-DC more effectively than the Brazilian strain CL-Brener or even another Mexican strain INC5, all three classified as the same genotype subgroup. mo-DCs infected with Ninoa produced more TNF-α and IL-10 than mo-DCs infected with the other strains. The response varied also if the mo-DCs were derived from either BALB/c or C56BL/6 mice, evidencing that phylogenetically close pathogens use different pathways for the modulation of DC function, which affects the course of infection and should be taken into consideration for treatment.Another article within this special issue by Barbosa et al. emphasized how DC responses can differ depending on the strain of the pathogen involved, even if the strains are closely related, pointing out that DC functions are responsible for the outcome of the infection. They showed that the Mexican in vitro and in vivo approaches that DCs play the role of intermediary between invariant natural killer T cells (iNKT) and NK cells during Chlamydia pneumoniae infection in mice, which indirectly helps bacterial clearance. They revealed a role for iNKT cells in the regulation of the protective IFN-γ-producing CD27high NK cells by infecting iNKT cell-deficient mice, which displayed changes in NK subsets and an inhibition of NK activation and IFN-γ production. Previous studies demonstrated the role of DCs in NK cell activation and cytokine production; however, in vitro, coculture of NK cells with DCs from iNKT deficient mice resulted in reduced expression of CD69 and CD25 and impaired of IFN-γ production by NK cells compared to coculture with WT DCs. Interestingly, adoptive transfer of DCs from either WT or iNKT-deficient mice to infected mice resulted in the same effect observed in vitro, demonstrating that iNKT interaction with DCs allows them to modulate NK cell responses. Loss et al. also explored the cellular networks important against bacterial invasion. They studied the crosstalk between porcine mo-DCs and intestinal epithelial cells (IEC). Using in vitro assays, they showed that the enteropathogenic bacteria Escherichia coli but not the probiotic bacteria Enterococcus faecium modulated NLRP3-dependent inflammasome signaling. Contact-dependent communication between mo-DCs and IECs increased inflammasome activation in IECs, whereas it weakened IL-1β production by DCs. Altogether, these data illustrate the complexity of cellular interactions necessary to orchestrate the precisely effective immune response by different species to fight against a pathogen. We hope you enjoy the reading of this special issue and that it may give you new insights into the biology of DCs, this unique cell with varied functions in diverse context.The last set of articles explored the interactions of DCs with other cells and their effects on the immune response to infection in different models. Zhao et al. elegantly showed by both


Published studies of famotidine for COVID-19 have focused on acute illness, and none have reported on neuropsychiatric symptoms. This case study reports on an 18-year-old man who sought psychiatric treatment for depression and anxiety, disruptive interpersonal conflicts, and impairments in attention and motivation following mildly symptomatic illness with COVID-19. The neuropsychiatric symptoms, which had been present for 16 weeks at the time of the initial evaluation represented a significant departure from the patient's previous behavioral baseline. The patient had no prior psychiatric history preceding his illness with COVID-19, and no history of any prior treatment with psychopharmacological medications. Famotidine 20 mg twice daily administered orally was begun without any additional medications. At 1-week follow-up the patient was much improved. Improvement was sustained through 12 weeks of follow-up during which the patient continued to take famotidine without apparent side effects. With progression of the COVID-19 pandemic it has become evident that persistent disease-related symptoms may follow acute COVID-19 and may include neuropsychiatric symptoms. Controlled clinical research on famotidine for COVID-19 should follow, as well as the development of valid and reliable research diagnostic criteria to define and operationalize the features of a putative COVID-19 neuropsychiatric residual.Famotidine is of interest as a possible treatment for COVID-19, with effects on disease-related symptoms and survival reported in observational and retrospective studies, as well as  Famotidine, a histamine H2 receptor antagonist with labeled indications for heartburn and gastric reflux, has been suggested as a possible treatment for COVID-19 on the basis of observational and retrospective study evidence. In Wuhan, China, in January of this year, retrospective analysis of data of hospitalized patients indicated increased survival in patients with COVID-19 who had been taking famotidine at the time of admission to the hospital. The difference in mortality rates, 14 vs. 27% favoring those who had taken famotidine, did not reach statistical significance   and 17 oI suspected I just had seasonal allergies/ a cold. When more information about COVID began, including heightened temperature, that is when I began monitoring my temperature,” and at this point, he was afebrile. At about 16 weeks prior to the evaluation, the fatigue recurred, with the onset at that time of the behavioral changes that would eventually lead the patient to seek psychiatric evaluation.Approximately 19 weeks prior to the initial psychiatric evaluation, in the third week of February 2020, the patient experienced the onset of fatigue and cough see . The fatThe week before the onset of cough and fatigue, the patient, who was attending college outside of New York State, went to an indoor conference in New York City that included approximately 100 attendees. He also visited his family at this time, including his father, a public transit worker in New York City with occupational exposure to the subway system. Both the patient and his father experienced the onset of cough and fatigue the week following their contact with one another, with the father experiencing relatively more severe symptoms. Nether the patient nor his father were tested for SARS-CoV-2 antigen, which was not systematically available in New York at that time. Five weeks prior to the initial psychiatric evaluation, the patient tested positive for SARS-CoV-2 antibody, following his father's positive antibody test result.The working DSM-5 psychiatric diagnoses were Other Specified Mental Disorder and Mild Neurocognitive Disorder both due to COVID-19, according to a clinical hypothesis that the patient's presenting neuropsychiatric symptoms were related to prior illness with COVID-19 . “Other …I woke up and got out of bed without feeling awful….” He reported no side effects. At 3-week follow-up, the patient's BDI and BAI scores were 1 and 2, respectively. A friend familiar with the patient's prior behavioral baseline described the patient's behavioral change following presumed COVID-19 illness and prior to treatment with famotidine as “…more irritable or quiet…exhausted.” At 4 weeks following the initiation of famotidine, the friend described, “… he seemed much more conversational as well as productive…very focused….” The patient continues to report he is doing well at the time of this writing, 12 weeks following the initiation of treatment with famotidine, which he continues to take at the initially prescribed dosage of 20 mg twice daily.The patient was begun on oral famotidine 20 mg twice daily as per the drug labeling. No psychopharmacological or other medications were prescribed, melatonin was discontinued, and no behavioral interventions or lifestyle changes were made. On follow-up a week later, the patient said he felt “much better” and noted substantial improvement regarding his symptoms of heightened emotional reactivity and diminished motivation and sustained attention. He estimated the time interval between starting famotidine and symptomatic improvement at 4 days and described his state on the fourth day as “clear-headed,” “This case suggests a possible treatment effect of famotidine for persistent neuropsychiatric symptoms following acute illness with COVID-19. It appears generally consistent with observational and retrospective study evidence for an apparent treatment effect of famotidine on disease-related symptoms and survival in COVID-19 –5.The interval of 16 weeks from the initial onset of neuropsychiatric symptoms following apparent illness with COVID-19 to the initiation of treatment with famotidine is a distinctive aspect of this case. Individuals with persistent symptoms following acute illness with COVID-19, the “long haulers,” are an increasing and arguably a presently relatively underserved population. A CDC study found that 35% of adult outpatients who were symptomatic at the time they tested positive for SARS-CoV-2 antigen had not returned to their usual state of health at 2–3 weeks following testing . ClinicaTo date, the author has treated eight other patients in his general psychiatric practice with famotidine for persistent neuropsychiatric symptoms following acute illness with COVID-19. In contrast to the present case, these other patients were already receiving psychopharmacological treatment at the time of onset of COVID-19 and subsequent treatment with famotidine. Within the limits of uncertainty due to the intermingling of factors including psychiatric baseline, emotional responses to the pandemic, and variability in length of treatment with famotidine, most patients appear to have received some benefit. The most frequent domain of symptomatic improvement appears to be “brain fog,” a term applied to a set of symptomatic features suggestive of problems with executive functions, including sustained attention/working memory and motivation, as well as word-finding and short-term memory. Patients have utilized the term “clearer” in their description of a famotidine effect.Other symptomatic features of a putative COVID-19 neuropsychiatric residual relate to mood, anxiety, and emotional reactivity. Some patients with a prior history of depression describe mood changes following COVID-19 as distinct in quality from their previous depression. The term “despair” has been used, apparently connoting qualities of intensity and hopelessness, which may be of significance regarding suicidal risk. Irritability and highly reactive mood may be evident as interpersonal conflict. The expression of mood and anxiety symptoms may be episodic and paroxysmal. Development of valid and reliable research diagnostic criteria for a putative syndrome of COVID-19 neuropsychiatric residual would provide a basis for defining patient groups for clinical trials and measures of illness severity.Neuroinflammation plays an increasingly appreciated role in psychiatric disorders . SARS-CoSARS-CoV-2 viral persistence may mediate disease-related symptoms following acute COVID-19 illness. Clinical trial data indicate a potential for persistence of SARs-CoV-2; 41.5% of the subjects in a study of lopinavir–ritonavir still had a viral load detectable by oropharyngeal swab at 28 days following randomization , with anIn silico methods, including virtual ligand screening of SARS-CoV-2 proteins against libraries of compounds, predict the binding of famotidine to Mpro , also known as main protease (Mpro), and SARS-CoV-2 papain-like protease (PLpro). Both of these proteases are critical for viral replication, and PLpro additionally has effects on ubiquitination and interferon that may dysregulate host innate immunity.  to Mpro , PLpro  for the publication of any potentially identifiable images or data included in this article.KA evaluated and treated the patient as described in this case report and authored the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aimed to investigate whether personality traits and their facets are associated with a multi-methods assessment of physical activity and walking performance and whether they explain the discrepancy between self-reported and accelerometer-assessed physical activity.n = 239) who were part of a clinical trial. Personality traits and their facets were measured using the 240-item NEO Personality Inventory-3. Physical activity was assessed using questions about frequency, intensity and duration of exercise ) and by tri-axial accelerometers . Walking performance was measured by 6-min walking distance and 10-m walking speed. Linear regression analyses were controlled for age, sex, education, body mass index, disease burden, and intervention group.The participants were community-dwelling, 70–85-year-old men and women from Finland (activity facet of extraversion was positively associated with self-reported MET-minutes, accelerometer-assessed light physical activity and walking performance. The positive emotions facet of extraversion was positively associated with self-reported MET-minutes and walking performance. Openness and its facets and the excitement seeking facet of extraversion were positively associated with walking performance. Conscientiousness and most of its facets were associated with both physical activity and walking performance, but these associations were not statistically significant after accounting for all control variables. The impulsiveness facet of neuroticism was negatively associated with accelerometer-assessed light physical activity and walking performance, but the associations with walking performance attenuated after accounting for all control variables. Accelerometer-assessed moderate-to-vigorous physical activity was not associated with personality traits or facets. Discrepancy analyses suggest that openness and the excitement-seeking facet of extraversion were associated with higher self-reported than accelerometer-assessed physical activity.The excitement-seeking facet of extraversion had better walking performance but also overestimated their self-reported physical activity compared to the accelerometers.Consistently across methods, older adults who scored higher on facets of extraversion and conscientiousness tended to be more active and outperformed peers on walking performance. Older adults who scored higher in the facets of openness and the The online version contains supplementary material available at 10.1186/s11556-020-00251-9. Physical activity has many health benefits throughout the lifespan, but especially in old age it provides protection against common diseases and frailty . Neverthactivity facet of extraversion and physical activity [activity facet is also associated with walking speed [activity tend to be busy, have a lot of energy and live at a rapid tempo [self-consciousness facet of neuroticism has been associated with lower levels of physical activity [self-discipline facet of conscientiousness with higher physical activity [dutifulness facet of conscientiousness with higher physical activity [As Table activity , 22–28, activity . The actng speed . This isid tempo . The eviactivity , 28, 29,activity , 24, 29 activity  and bettactivity . Most ofactivity , 24–27.Most of the research about personality and physical activity is based on self-reported physical activity and the use of multiple measures of physical activity is necessary especially when studying older adults. Among older adults, most of the daily activity comes from light intensity activities that are easily under-reported in questionnaires  and, theThe correlation between self-reports and accelerometers vary between studies but is usually low or at most moderate . Even thactivity facet of extraversion and physical functioning. For other facets the approach was exploratory, because to our knowledge, there is only one previous study about associations of all facet-level personality traits with walking performance [The purpose of this study was to investigate whether personality traits and their facets 1) are associated with accelerometer-assessed and self-reported physical activity and walking performance, and 2) explain the discrepancy between accelerometer-assessed and self-reported physical activity. We expected that extraversion, openness and conscientiousness have positive associations and neuroticism a negative association with physical functioning. In the facet level, the strongest associations was expected to found between the formance  and one formance . By addrwww.isrctn.com/ISRCTN52388040.This study examined cross-sectional data collected in a post-intervention assessment of a randomized controlled trial “Promoting safe walking among older people: the effects of a physical and cognitive training intervention vs. physical training alone on mobility and falls among older community-dwelling men and women” (the PASSWORD study) . The stuThe participants were randomly selected from the Finnish National Registry. Recruitment started with an information letter sent to a random sample of 70- to 85-year-old community-dwelling older adults living in the city of Jyväskylä, Finland, followed up by a phone call. During the phone interview, willingness to participate in the study was confirmed and participants were screened for inclusion and exclusion criteria. The inclusion criteria at baseline were being at most moderately active , able to walk 500 m without assistance and to score ≥ 24 points on the Mini Mental State Examination. Exclusion criteria included severe chronic condition, medication, or other factors that may affect study participation, excessive alcohol use, difficulties in communication due to severe hearing or vision problems and another family member participating in the study . The samn = 239) who completed the NEO-PI-3. All of these participants had participated in physical training alone or combined physical and cognitive training during the last 12 months. Compared to the rest of the original sample (n = 75), the participants included in this study (n = 239) were younger  and had a higher level of education .We have previously reported the baseline associations of extraversion and neuroticism  with physical activity in the same sample . This stn = 5) were excluded from this study. Of those 239 participants who had filled the NEO-PI-3 questionnaire at least partly, 193 (81%) had filled it completely without any missing values and the rest of the participants (n = 46) had seven missing values at the most. These were imputed with the neutral option 2 [The NEO-PI-3 was used to assess personality traits and facets , 40. It Physical activity was assessed by both accelerometers and self-reports. Participants were asked to wear a tri-axial accelerometer, model UKK RM42 , in an elastic band on their right hip during waking hours for seven consecutive days except during water-based activities . The accFor discrepancy analysis, average daily metabolic equivalent (MET)-minutes from accelerometer data were calculated with the common thresholds for light, moderate and vigorous activity: 1.6* light + 3*moderate + 6*vigorous physical activity . BecauseTwo walking performance tests were used in this study: the 6-min walking test evaluates community walking  and the The background variables included sex, age, body mass index (BMI), education and chronic diseases. Sex and date of birth were drawn from the Finnish Population Registry. BMI was calculated from staff-measured height and weight. Education was categorized to low (at most primary school), medium  and high (college or university degree). Self-report chronic diseases diagnosed by physicians were reported at baseline and were updated with new verified diagnoses that occurred during the intervention. Answers to questions on chronic diseases were categorized into metabolic, cardiovascular, pulmonary, musculoskeletal, neurological and mental diseases. Number of disease categories was calculated and used in analyses with a categorization of 0, 1, 2 and 3 or more disease categories.All statistical analyses were carried out using SPSS, version 25 . Demographic characteristics of the study sample were described using proportions, means and standard deviations and minimum and maximum values.Associations of personality traits and their facets with physical activity and walking speed were analyzed by linear regression. Every trait and facet were analyzed in separate models to avoid multicollinearity. The models with all five traits in the same model are presented in supplementary material  included a trait or facet and sex and age as covariates while the rest of the covariates  were included in the second model (M2). M2 models for light physical activity also included MVPA as a covariate and M2 models for MVPA also included light physical activity as a covariate. Because the variable had a right-skewed distribution in self-reported MET-minutes, the logarithm transformation was used in linear regression analysis.The Benjamini-Hochberg procedure was used to correct the high number of comparisons with a false discovery rate of 0.10 , 48. TheThe descriptive statistics for main study variables are presented in Table At the broad five trait level, extraversion was positively associated with self-reported MET-minutes and conscientiousness with both light physical activity and self-reported MET-minutes Table , 4 and 5competence was associated with both self-reported MET-minutes and light physical activity, C4 achievement-striving and C6 deliberation with light physical activity and C5 self-discipline with self-reported MET-minutes. The inclusion of all control variables in M2 models reduced the associations of conscientiousness facets with both self-reported MET-minutes and light physical activity. After the Benjamini-Hochberg corrections, only C4 achievement-striving remained statistically significant predictor of light physical activity and none of the facets for self-reported MET-minutes. The E4 activity facet of extraversion was consistently associated with both light physical activity and self-reported MET-minutes and E6 positive emotions facet with self-reported MET-minutes in all models  and Benjamini-Hochberg corrections.At the facet level, higher scores in most facets of conscientiousness were associated with higher physical activity Table , Fig. 5:activity, E5 excitement seeking and E6 positive emotions had positive associations with walking performance tests, but E5 excitement seeking was associated only with walking speed and the association of E4 activity with walking speed was not statistically significant in M2  reported more physical activity than what was assessed by the accelerometers  and discrepancy.The results for openness in this study were in line with previous studies showing a weak positive association between openness and self-reported physical activity , 9 and nFacets of conscientiousness were consistently related to physical activity and none of the facets stood out from the rest, supporting the previous studies about the positive association between conscientiousness and physical activity –9. One eThis study has some limitations. While this was one of the largest studies to report on the associations between personality traits and accelerometer-measures, the sample size provided limited power to detect the typical small effects. In our relatively small sample, only the largest effects remained significant after accounting for multiple-testing, increasing the risk of false negative. Hence, these exploratory findings should be replicated with other samples and also with corresponding samples that have not participated in a training intervention. In addition, owing to the cross-sectional nature of this study, we cannot draw any conclusions about the causal relationships between the study variables.Accelerometers have some limitations related to the cut-off points for older adults and their capability to capture different types of physical activities , 31. ThiAccelerometers record body acceleration in all daily activities (when a participant wears the accelerometer)  whereas Despite these limitations, this study was one of the first to investigate the role of personality in the discrepancy between physical activity measures and contributes to our understanding of how personality is linked to different measures of physical activity and walking performance. The major strengths of this study were the use of multiple measures of physical activity and walking performance and an in-depth assessment of personality at both trait- and facet-levels. Another relatively unique feature of this study was the use of a post-intervention sample. Unfortunately, we did not have access to the NEO-PI-3 at baseline data collection and therefore data collection occurred one-year after a physical activity intervention was used. As such, the findings illustrate the role of individual differences in personality for physical functioning even after older adults engaged in a standardized intervention to increase mobility.This study has provided a deeper insight into how facet-level personality tendencies explain part of the variability in physical activity and walking performance. Older adults with higher impulsiveness and lower self-control, who are less energetic and have less optimistic tendencies are at a higher risk for physical inactivity and walking limitations. Testing for personality characteristics in health care settings could help to identify individuals with higher risk for limitations. Interventions to promote physical functioning could also benefit from a more individually tailored approach. However, more research is needed on effective ways to promote physical functioning among people with personality characteristics that increase the risk for low physical functioning. In addition, a greater focus on personality characteristics could produce interesting findings that help us understand the discrepancy between self-reports and activity monitors.Additional file 1: Table S1. Associations of personality traits and facets with physical activity, discrepancy between physical activity measurements and walking performance. Description: Results of the regression analyses were five personality traits were tested in the same regression models.Additional file 2: Table S2. Descriptive statistics for facets. Description: Descriptive statistics for facets.Additional file 3: Table S3. Pearson bi-variate correlations between personality facets and physical functioning. Description: Pearson bi-variate correlations between personality facets and physical functioning.Additional file 4: Table S4. Associations of personality traits and facets with accelerometer-assessed moderate-to-vigorous physical activity. Description: Results of the regression analyses for accelerometer-assessed moderate-to-vigorous physical activity.


The COVID-19 pandemic has created an unprecedented health crisis and a substantial socioeconomic impact. It also affects tuberculosis (TB) control severely worldwide. Interruptions of many TB control programs because of the COVID-19 pandemic could result in significant setbacks. One of the targets that can be affected is the WHO’s End TB Strategy goal to eliminate catastrophic costs of TB-affected households by 2030. Disruptions to TB programs and healthcare services due to COVID-19 could potentially prolong diagnostic delays and worsen TB treatment adherence and outcomes. The economic recession caused by the pandemic could significantly impact household financial capacity because of the reduction of income and the rise in unemployment rates. All of these factors increase the risk of TB incidence and the gravity of economic impact on TB-affected households, and hamper efforts to eliminate catastrophic costs and control TB. Therefore, efforts to eliminate the incidence of TB-affected households facing catastrophic costs will be very challenging. Because financial constraint plays a significant role in TB control, the improvement of health and social protection systems is critical. Even before the pandemic, many TB–high-burden countries (HBCs) lacked robust health and social protection systems. These challenges highlight the substantial need for a more robust engagement of patients and civil society organizations and international support in addressing the consequences of COVID-19 on the control of TB. It has a huge social impact: retracting economic growth and increasing rates of unemployment, poverty, and food insecurity.4 The pandemic also raises a particular concern for regular healthcare provision as healthcare resources are being shifted to COVID-19–related services6 and as patients are starting to avoid healthcare services for non-COVID-19–related health problems.7The COVID-19 pandemic has created an unprecedented health crisis, with 15.5 million confirmed cases and over 633,000 deaths worldwide in the first 7 months of the pandemic.8 Before the onset of the COVID-19 pandemic, TB was the single biggest infectious disease killer.9 Although the incidence of TB worldwide remains high, that number has remained relatively stable and many countries have even shown significant progress in recent years.9 However, because millions of people with TB depend on large-scale TB control programs,9 interruptions due to the COVID-19 pandemic could result in significant setbacks.8Tuberculosis (TB) control is one of the health domains that are affected by this pandemic.10 The disruptions to TB programs and healthcare services due to COVID-19 could potentially prolong diagnostic delays, increase the rate of undetected TB cases in the community, and leave TB patients untreated, which all would lead to more transmission and TB cases in the upcoming years. If no further action is taken, TB mortality could also increase.11In 2015, the WHO End TB Strategy set three main milestones for 2020: 20% reduction of TB incidence and 35% reduction of TB mortality compared with 2015, and zero percent of TB-affected households facing catastrophic costs due to TB.13 These costs may further reduce household financial capacity and cast them into a poverty trap because catastrophic costs may hamper further access to health care. The COVID-19 pandemic has set an exceptional crisis in a short period and it is expected to affect most people living in poor households. Income loss, rising prices, and limited social protection are expected to push about 71 million people into extreme poverty in 2020 alone.14 These figures have been much more severe in low- and middle-income countries (LMICs) that lack strong social protection programs.15 It is plausible that accessing TB-related services, therefore, will aggravate this financial burden, and eliminating the incidence of catastrophic costs due to TB will be very challenging.Potential setbacks are also expected in the achievement of the target of eliminating the incidence of catastrophic costs due to TB. Accessing TB-related services has substantial economic consequences, and such consequences can be catastrophic, particularly for poor households.There has been little discussion on how the COVID-19 pandemic will impact progress toward the elimination of catastrophic costs due to TB. In this article, we describe progress toward the target of eliminating the incidence of catastrophic costs due to TB, the potential impact of COVID-19 on future target achievement, and potential measures that can be implemented to cushion this effect of the pandemic.18 Whereas catastrophic expenditure focuses on direct medical costs only, TB-related catastrophic costs also include indirect costs because of accessing TB-related services. For the WHO’s End TB Strategy framework, if the total costs incurred by a TB-affected household exceed 20% of household annual income, the costs are defined as catastrophic. Most studies concerning TB-related catastrophic costs have used this definition, using various thresholds in the sensitivity analyses.19In 2015, the WHO End TB Strategy set a new concept of “catastrophic costs,” which is different from “catastrophic expenditures,” a similar-sounding indicator commonly used to measure progress toward universal health coverage (UHC).23 Thirty-one other countries are preparing survey and expected to collect data in 2020 or 2021. Until now, the national surveys revealed that incidence of TB-related catastrophic costs remains high in many countries, ranging from 19% (95% CI: 15–25) in Lesotho to 83% (95% CI: 80–86) in Timor Leste.25 Among households affected by multidrug-resistant (MDR) TB, the incidence was consistently higher than among TB-affected households, ranging from 67% (95% CI: 62–72) in the Philippines and 67% (95% CI: 42–85) in Benin to 100% (95% CI: 92–100) in Uganda.25 In addition to the national costs surveys, many studies reported the incidence of catastrophic costs at the local level, such as district, province, or several provinces.28 Despite the various methods applied, the studies at the local level also suggest that the incidence of TB-related catastrophic costs remains high.The WHO has encouraged many countries to implement a national TB patient costs survey. This is crucial to obtain a clear picture of the financial burden faced by TB-affected households. By July 2019, 17 countries had accomplished the national costs survey, whereas nine countries were underway.Looking at the current picture, eliminating the incidence of catastrophic costs remains very challenging. In the current context of the COVID-19 pandemic and global economic recession, the proximate WHO End TB Strategy milestone of zero TB-affected families incurring catastrophic costs by 2025 seems unlikely to be achieved.29 This could also reduce healthcare service availability for TB, as many TB-related resources are designated to COVID-19 services.31The COVID-19 pandemic has affected TB control programs and the incidence of catastrophic costs by distorting the health system at different levels. 8 In many LMICs, where healthcare resources are scarce, the severe reduction of TB-related healthcare services is likely to increase diagnostic delays and decrease the TB notifications.In the primary care level, the pandemic could affect most health promotion and disease prevention programs. When physical distancing measures are promoted, primary healthcare facilities should reduce TB-related health promotion programs, which are delivered in groups. Contact tracing has also been affected because of a much more limited number of healthcare staff to do such program including TB preventive therapy that could increase the likelihood of secondary cases in households. Most TB-related services are also closed.In some countries, TB-related services are provided in secondary care–level facilities. MDR TB services, including the diagnostic procedure, are also often delivered in hospital. With an increasing volume of COVID-19 cases in hospitals, TB-related services and its healthcare staff are reduced—all eventually interrupting TB services in hospitals and likely to increase patients’ diagnostic delays.One of modalities to reduce TB diagnostic delay is the rapid GeneXpert device. However, because of the pandemic and the vast need to identify SARS-CoV-2 virus, some countries encourage the use of GeneXpert to test suspected COVID-19 patients. For COVID-19 control, this initiative boosts the COVID-19 testing scale but also reduces the TB test capacity, particularly for countries that rely heavily on the GeneXpert device . It would, therefore, hinder the identification of TB cases and increase its diagnostic delay.32 It may lead people to seek care with private healthcare providers that are not designated as COVID-19–related services and are not linked to the NTP. This preference could increase and, together with potential diagnostic delay, eventually may result in much higher pre-diagnostic costs incurred by suspected TB patients and their households.33The diagnostic delay and not notified cases are also aggravated by patient avoidance of healthcare services, in particular public healthcare services that are linked to the National Tuberculosis Program (NTP) and now commonly designated for COVID-19 services. Avoidance comes from a real concern that visiting such healthcare services may increase the risk of being infected by COVID-19.8 Their avoidance of the already reduced TB-related services may lead to a higher number of interrupted TB treatments or even to patients being lost to follow-up. Patients who have interrupted TB treatment usually face prolonged TB treatment. Patients who are lost to follow-up may face more severe symptoms, fail to complete their treatment, undergo category 2 TB treatment , or even encounter (multi)drug-resistant TB. It would increase the risk of hospitalization and more expensive treatment; two conditions that are risk factors for catastrophic costs due to TB.34More than 50% of TB patients in Kenya and India are in fear of contracting COVID-19 at a health facility.For drug-resistant TB patients, the pandemic causes a much more complicated situation. They still need to visit a hospital, where such care is often delivered, with a much higher caution from either themselves or healthcare staff to prevent the COVID-19 infection. If a patients’ concern of contracting COVID-19 leads to dismissal from accessing MDR-TB treatment, the risk increases of a more severe condition, longer treatment period, hospitalization, or extensively drug-resistant TB. All will result in a higher cost and risk of experiencing catastrophic costs or impoverishment.36 The fear of visiting healthcare facilities because of COVID-19 may reduce such unnecessary costs. The pandemic may also raise awareness of respiratory disease symptoms and encourage people to seek care for respiratory complaints. In some cases, this may reduce diagnostic delay of TB and prevent unnecessary costs in the pre-diagnostic phase.Despite the risk of increasing TB-related costs, the pandemic may have positive effects as well concerning certain costs. Tuberculosis patients and their families are known to spend unnecessary costs on diagnostic tests, consultation, and hospitalization.37 The job loss, income reduction, and poverty are risk factors for catastrophic costs due to TB.13The COVID-19 pandemic also caused an economic recession that significantly impacts household financial capacity because of the rise in unemployment rates and the decrease of income.38 This could lead to poor nutrition in the long run. Poor nutrition is a risk factor for the development of TB and, if a patient receives TB treatment, it may negatively affect TB treatment.39 The treatment may be unsuccessful and need to be continued to a longer period. When the TB treatment is prolonged, it can increase the risk of catastrophic costs due to TB.Poor households that face unemployment and substantial income loss may be forced to buy cheaper, less nutritious meals.40 Setting up comprehensive social protection programs is critical.The impact of COVID-19 on populations and TB-related health systems, particularly in LMICs, requires a robust and comprehensive response that incorporates a long-term strategy. Achieving the WHO End TB Strategy targets require measures beyond providing high-quality healthcare services. These ambitious targets, which are supposed to be achieved in the next 15 years, require political commitment to provide adequate financial protection for TB patients and their affected households. Several studies reported that providing national health insurance within the UHC framework is not enough to achieve the target of eliminating the incidence of catastrophic costs.42 A global economic recession will retract economic growth and result in scarce financial resources. Governments may shift national budget priorities to economic development and may limit their budgets for health-related programs. While many high-income countries may start to provide economic stimulus packages, most TB HBCs often depend on the external funds from donors.7 Many TB HBCs and LMICs need help from higher income countries to mount a sustained response. This would involve a substantial increase of investments in the provision of UHC, strengthening the national healthcare system with a particular focus on primary-level health care, and the provision of social support to vulnerable population groups.However, social protection systems in many TB HBCs are suboptimal, and are at risk of further weakening due to the pandemic. It is very difficult for countries to build institutional capacity during the pandemic, which would result in a limited and delayed response.44 Many social protection programs are detached from each other.45Many countries provide TB-related services in a vertical system, detached from their national healthcare system and health financing system. This is a common approach applied, particularly when the major funding comes from international donors. However, it also may result in a fragmented, ineffective system to combat TB. Tuberculosis-related social protection is also often provided in a TB-specific approach that targets people diagnosed with TB and their affected household in the form of cash transfers or food parcels.46 This approach should improve equitable access to TB-related services, from health promotion to rehabilitation. It should also reduce the high costs incurred for accessing TB services, and therefore improve TB treatment outcomes. The social protection strategy also requires a clear understanding of patients’ needs and the government’s resources. In some setting, the NTP and the national health insurance are fragmented, and the integration of both could help control TB and avoid unnecessary costs. Any financial support should also engage patients and civil society organizations with the effort of preventing much more severe impact of COVID-19 on TB control programs.Nevertheless, this pandemic, which reduces fiscal capacity of countries, may remind them to develop innovative social protection interventions for TB-affected households that are integrated into other social protection schemes instead of having many programs which are not linked to each other.It is clear that the COVID-19 pandemic will affect the WHO targets to reduce TB incidence and death rate and to eliminate the incidence of catastrophic costs. It is plausible that the risk of facing catastrophic costs increases in several ways. In response, it is important that health and social protection systems should be strengthened, particularly in TB HBCs, to reduce the devastating impact of the pandemic for TB-affected households. It is also important as part of strategies to attain the target of eliminating the TB burden worldwide. International support is required for countries that lack robust health and social protection systems.


Measuring optically detected magnetic resonance (ODMR) of diamond nitrogen vacancy centers significantly depends on the photon detectors used. We study camera-based wide-field ODMR measurements to examine the performance in thermometry by comparing the results to those of the confocal-based ODMR detection. We show that the temperature sensitivity of the camera-based measurements can be as high as that of the confocal detection and that possible artifacts of the ODMR shift are produced owing to the complexity of the camera-based measurements. Although measurements from wide-field ODMR of nanodiamonds in living cells can provide temperature precisions consistent with those of confocal detection, the technique requires the integration of rapid ODMR measurement protocols for better precisions. Our results can aid the development of camera-based real-time large-area spin-based thermometry of living cells. Nanodiamond (ND) spin-based thermometry is a novel opto-microwave hybrid technique that can probe subcellular temperatures with distinct photostability18, various functionalized surfaces21, and low cytotoxicity26. The technique is based on optically detected magnetic resonance (ODMR) of electron spins in diamond nitrogen vacancy (NV) color defect centers, where the fluorescence of NDs is measured under microwave irradiation to detect the fluorescence decrease at an electron spin resonance of about 2.87 GHz30. The ODMR frequency is temperature dependent, and the temperature of NDs can be probed by measuring the ODMR frequency shift42. Biological applications of this method have been demonstrated for various cultured cells, including fibroblasts31, neurons23, and stem cells43. It has been recently applied to in vivo nematode worms for studying physiological thermogenesis44 and the temperature dependence of embryogenesis45. These demonstrations have proven the usefulness of ND spin-based thermometry.Subcellular thermometry has great potential for studying the molecular mechanisms of temperature-related biological phenomena, such as the variation of cell-death types in photothermal cancer therapy28. An example for biological thermometry is the multi-point ODMR method36 that was used for real-time biological thermometry studies45. Camera-based wide-field detection offers parallel probing of multiple NDs simultaneously that enables spin-based ND thermometry at multiple points in a large field of view47. Combining wide-field ODMR detection with multi-point ODMR methods will enable real-time, large-area monitoring of temperatures under the biological microscope. However, it has been recently reported that multi-point ODMR methods introduce several measurement artifacts in confocal ODMR detection48. It is important to analyze the operational process and associated artifacts of wide-field ODMR detection prior to the integration of multi-point ODMR methods, which can add complex artifacts.To further integrate this technique into biological thermometry, real-time operations and large-area probing are required. ND spin-based thermometry is mainly classified into two types depending on the fluorescence detectors used, that is, photon-counter-based confocal detection and camera-based wide-field detection. Confocal detection is a point-by-point ND probing technique that is widely used for NV-spin manipulation and detection combined with rapid timing control of photon detection and microwave pulsingThis study clarifies factors that can substantially affect temperature measurements in wide-field ODMR detection, including sensitivity degradation from background fluorescence and artifacts from selection of binning regions near target NDs. By properly adjusting these factors, we show that, in living cells, wide-field ODMR detection can provide thermometric performance that are comparable to those provided by confocal detection. Furthermore, we technically estimate multi-point ODMR methods can be integrated into wide-field detection with realistic camera acquisition parameters. We discuss the possibility of realizing large-area spin-based thermometry operable in real-time, which should be an important technological milestone of quantum sensor applications in biology.43  was set to 10. The obtained 16-bit To compare the ODMR spectra of the confocal and wide-field detections, we used a home-built microscope that can perform ODMR measurements in both detection modes Fig. a. The mimentclass2pt{minimmentclass2pt{minimm is the photon–pixel conversion factor of the camera, given by R and The SNR of the ODMR spectra is dependent on the total photon count measured by the detectors, that is, the EMCCD camera for the wide-field and APD for the confocal detection, and we determined the measurement parameters of wide-field detection that provide a comparable SNR in the confocal method. In wide-field detection, the full-well capacity (FWC) and digital resolution of the analog-to-digital converter (ADC) of the camera strongly limit the measurement strategy. The FWC of our EMCDD is 185,000 e49C, dD/dT indicate the ODMR contrast, ODMR linewidth, total detected photon count corresponding to either of the detectors  via Eq.  the mechanical distortion of the microscope system and intra-cellular transportation move the NDs in the z axis, and (2) there are a number of blurred ND spots  in a single focal plane owing to the cellular height. Figure z-positional variation of z-position is shifted, the ND is defocused, exhibiting the 3D shape of the point spread function. Figure z-position determined by Gaussian fitting and the corresponding temperature sensitivity estimated based on the spectral shape and the photon count rate. The center frequency exhibits a fluctuation of 370 kHz (s.e.), except at 52, whereas the deviation at Next, the effect of the The present results regarding the dependence of the wide-field ODMR on the positional variation of the NDs can be summarized as: (1) The measurement noise (or temperature precision) is dependent on the ND fluorescence flux summed up in the ROI. (2) The blurred fluorescence spots of individual NDs at a single focal plane do not create artifacts of ODMR shift as long as the ROI is properly set. (3) The pixel saturation of the camera requires particularly careful treatment, as it may cause measurement artifacts. Increasing the fluorescence intensity and number of measurable NDs in a single image is an important factor in achieving high SNR, but it inevitably saturates the fluorescence of the brighter NDs. This is because the excitation power is usually adjusted such that the majority of the NDs gain 40,000–50,000 pixel values. There should be a tradeoff between the efficient optical excitation power and the number of available NDs depending on the real experiments on biological samples. Parameter optimization, such as the adjustment of optical magnification and exposure time, is not always possible in a dynamic biological environment; this necessitates the present characterization of pixel saturation such that it can be effectively addressed. Note also that the splitting of ODMR dip varies by particles because of the differences in transverse component of zero-field splitting parameters and effective microwave power relative to the saturated intensity. These spectral variations can affect the curve-fitting results, leading to a variation in the sensitivity and precision.z-positional distribution of NDs in HeLa cells . We performed super-resolution imaging of ND-labeled HeLa cells that were fixed on a coverslip, as shown in Fig. xy plane and z)43, and more NDs can be focused in the wide-field fluorescence image in contrast to HeLa cells.Before working on cells, we quantify the z-direction because of the substantial height of cells in the z axis compared to the focal depth. In the subsequent experiments involving wide-field ODMR measurements in living ND-labeled HeLa cells, 10–30 NDs were predominantly available for measurements. Note that the number of NDs inside cells can be increased by employing a higher ND concentration. The selection of the ND concentration is dependent on the purpose. In our experiment, we used a relatively low concentration to focus on individual NDs so that the principles of the system, with regard to the wide-field ODMR detection relative to the confocal one, could be investigated.Although there are approximately 200 NDs in the cells, not all NDs can be used for thermometry because (1) some NDs (approximately 20–30%) show broad spectral lines that cannot provide high sensitivity  and (2) practically, only 10–20% of the NDs can be focused in the tolerable focusing range in the 1–5 in Fig. 53. Indeed, the fluorescence spots of the NDs are sometimes merged or overlapped during the measurements because of the intra-cellular transportation and merger of endosomes. NDs 1 and 2 are insignificantly fluorescence-saturated and doughnut shaped owing to the electron overflow in the pixel arrays. This saturation effect appears as the relatively small ODMR contrasts of NDs 1 and 2, compared to those of NDs 3–5. Accordingly, the estimated sensitivities for NDs 1, 2 are 2.1 and 2.2 K/3–5, which show 1.9, 1.8, and 1.7 K/54 and relatively worse than those on the coverslip in this study because (1) the background fluorescence reduces the ODMR contrast and (2) the ODMR spectra are broad in cells.Having understood the system principles of wide-field detection in detail and its connection to confocal detection, we apply it to multiple NDs in living HeLa cells. Figure We then performed intracellular temperature sensing of living HeLa cells in the present wide-field detection. Figure 43. The ODMR linewidth , Dulbecco’s phosphate-buffered saline (D-PBS(-)), and 4% paraformaldehyde phosphate buffer solution were purchased from Fujifilm Wako Pure Chemical Corporation . D-MEM and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific . Collagenase type I was purchased from Koken Co. . A cell counting plate was purchased from Fukae Kasei Co. . Acti-stain 488 phalloidin was purchased from Cytoskeleton . Hoechst 33342 was purchased from Thermo Fisher Scientific . Fluoromount was purchased from Diagnostic BioSystems .The NDs (NDNV100nmHi10ml) were purchased from Adámas Nanotechnologies , and they contained approximately 500 NV centers per particle. E-MEM with HeLa cells were incubated with the NDs  to enable fine translation and positional scanning. Note that an external magnetic field was not applied in this study. Microwaves were generated from a microwave source  and amplified by a maximum factor of 45 dB . The microwaves were fed to the linear antenna . The fiber-coupled fluorescence was detected using APD . For wide-field ODMR detection, the excitation laser was focused on the back focal plane of the objective to illuminate the entire area of the field of view, and the fluorescence was imaged with an EMCCD camera  via a N image sets for the microwave ON and OFF states. To obtain the ODMR contrast, pixel values of the microwave ON images  were divided by those of the microwave OFF images (reference images). This image data processing was performed using Fiji/ImageJ software64. The obtained ODMR spectra were fitted with Gaussian in a practical manner because there are no simple analytic expressions for the ODMR spectral shape of ensemble NV centers65.The obtained 48.In the home-built stage-top incubator, we heated the antenna-integrated dishes in two directions, that is, from the direction of the oil-immersion objective and metal cap Fig. a. The tementclass2pt{minim


The ability of the immune system to kill tumour cells is a natural and extremely effective defence mechanism for fighting cancer. Cytotoxic-T-cells are a critical component of our immune system which function is to eliminate cancer cells. In some cancers, especially those with a rich tumour stroma, these cytotoxic-T-cells are unable to reach and kill the tumour cells. Cancer-associated fibroblasts are the most abundant cells in the tumour stroma and play a key role of the recruitment, infiltration and function of cytotoxic T-cells in the tumour, via several molecular mechanisms which we describe in this review.The tumour microenvironment (TME) is the complex environment in which various non-cancerous stromal cell populations co-exist, co-evolve and interact with tumour cells, having a profound impact on the progression of solid tumours. The TME is comprised of various extracellular matrix (ECM) proteins in addition to a variety of immune and stromal cells. These include tumour-associated macrophages, regulatory T cells (Tregs), myeloid-derived suppressor cells, as well as endothelial cells, pericytes and cancer-associated fibroblasts (CAFs). CAFs are the most abundant stromal cell population in many tumours and support cancer progression, metastasis and resistance to therapies through bidirectional signalling with both tumour cells and other cells within the TME. More recently, CAFs have been shown to also affect the anti-tumour immune response through direct and indirect interactions with immune cells. In this review, we specifically focus on the interactions between CAFs and cytotoxic CD8+ T cells, and on how these interactions affect T cell recruitment, infiltration and function in the tumour. We additionally provide insight into the therapeutic implications of targeting these interactions, particularly in the context of cancer immunotherapy. Our understanding of the so-called tumour microenvironment (TME) has seen significant advancement through a large number of studies conducted over the last decade ,4,5,6,7.Cancer-associated fibroblasts (CAFs) are the dominant stromal cell population in many solid tumours ,9,10 andHowever, despite the variety of CAF-mediated tumour-promoting functions, CAF ablation strategies have largely been deemed as deleterious. This was exemplified by the landmark studies of Özdemir et al.  and RhimWhat has been made clear through these studies is that there is significant phenotypic heterogeneity within the CAF population with certain subtypes acting to restrain, rather than promote tumour progression . There iHowever, whilst our understanding of CAF heterogeneity has improved over the last half decade, CAF subtyping is still very much in its infancy and identifying unique CAF markers of CAF subtypes has proven to be a significant challenge in the field of cancer biology. At this early stage, it is currently unknown whether this subtyping of CAF populations will confer therapeutic benefit. We believe therefore, that it is important to also further characterise the functional interactions of CAFs within the TME, alongside approaches to delineate CAF heterogeneity, for the rational design of therapeutic strategies that target the pro-tumourigenic functions of CAFs.Studies conducted in recent years have developed a greater appreciation for the role of the immune system in regulating/deregulating cancer progression. This has also led to a surge of interest in the field of immuno-oncology, with immunotherapeutic strategies such as adoptive transfer of chimeric antigen receptor (CAR) T cells and immune checkpoint inhibitors (ICIs) being the subject of a large number of clinical trials over the last decade. Critically, in the context of solid tumours, the efficacy of these immunotherapeutic strategies is dependent on a functional cytotoxic CD8+ T cell response within the TME ,26. GiveThe cancer immunity cycle proposed by Chen and Mellman describes a series of stepwise events implicit to an effective anti-tumour T cell response . CriticaGiven the fact that CD8+ T cells directly mediate tumour cell killing and their importance in the field of immuno-oncology, the literature detailed herein is specifically focused on the effects of CAFs on cytotoxic CD8+ T cells. Nonetheless, it should be mentioned that the pleiotropic functions of CAFs extend also to regulating the activity of other T lymphocytes such as regulatory T cells (Tregs) and CD4+ T helper cells and other immune cell populations present in the tumour microenvironment .The ability of the immune system to mount a successful response relies, in part, on the recruitment of various immune cells to the site of inflammation. This recruitment is dependent on a highly sophisticated system involving chemokines and their corresponding chemokine receptors, enabling a highly specific and localised immune response. Accordingly, upon their activation within the TME, CAFs can modulate the recruitment of peripheral CD8+ T cells towards the tumour through secretion of numerous cytokines and chemokines. For instance, activated CAFs are able to promote the trafficking of CD8+ T cells away from the juxta-tumoral compartment and towards the pan-stromal compartment of PDAC tumours in a chemokine-dependent manner . The juxA key hallmark of activated CAFs is their ability to aberrantly deposit extracellular matrix (ECM) proteins such as collagen, fibronectin, hyaluronan, in addition to matrix degradation enzymes . This deConversely, several lines of evidence from a series of independent studies may call into question the importance of CAF-mediated ECM deregulation as a means of reducing T cell infiltration. Utilising novel computational imaging techniques, one study showed that while spatial distribution of CD8+ T cells in close proximity with tumour cells correlated with an increased overall patient survival in pancreatic cancer, the density of collagen 1 deposition and number of αSMA+ CAFs do not correlate with a reduced CD8+ T cell infiltrate . In contTherefore, at least in the context of PDAC and colorectal carcinoma, it still remains unclear whether CAF-mediated ECM deposition impedes or favours infiltration of CD8+ T cells into the tumour. CAFs have also been shown to negatively regulate the infiltration of CD8+ T cells into solid tumours through regulation of their secretome. Increased secretion of CAF-derived TGF-β has been shown to correlate with a reduced accumulation of CD8+ T cells within both metastatic urothelial cancer and metastatic colorectal carcinoma and a reduced response to immune checkpoint inhibitors ,44. In bIt is important to also note the role of tumour hypoxia as another factor responsible for the reduced infiltration of CD8+ T cells in many solid tumours. CAF deposition of ECM contributes to the generation of a hypoxic TME, and in turn, the CAFs upregulate the expression of angiogenic factors such as VEGF ,46. Endo+ T cell function [+ T cells through simultaneous upregulation of PD-L2 and Fas ligand, leading to increased tumour survival [The immune system is well-orchestrated and in a healthy organism exists in a state of homeostasis. The immune system can be rapidly mobilised to act against invading pathogens or neoplastic cells whilst simultaneously, self-tolerance is maintained by negative regulation of cell activation—preventing the indiscriminate killing of host cells, as seen in autoimmune disorders . Immune function . More refunction . This safunction . Howeverfunction . This sufunction . Interessurvival . Strikinsurvival .In addition to upregulation of immune checkpoint molecules on their own cell surface, CAFs can also act indirectly through paracrine signalling interactions to upregulate the expression of immune checkpoint molecules on both tumour cells and other cells of the TME. For instance, CAF-derived CXCL5 was shown to upregulate the expression of PD-L1 in murine melanoma and colorectal carcinoma cell lines upon activation the CXCR4 in a PI3K-AKT-dependent manner . SimilarThe role of CAF metabolic reprogramming in promoting tumour progression and contributing to therapeutic resistance has now been well established. For instance, paracrine transfer of CAF metabolites such as deoxycytidine to tumour cells contributes to gemcitabine resistance in the context of PDAC . A more Another example of T cell suppression caused by CAF metabolic reprogramming has recently been observed using CAFs derived from prostate cancer patients . This stCross-presentation of tumour-specific antigens by antigen-presenting cells (APCs) such as dendritic cells (DCs) is critical for the generation of antigen-specific T cells and a sustained anti-tumour immune response. The release and cross-presentation of tumour antigens to prime CD8+ T cells is the integral step in the initiation of the “Cancer Immunity cycle” described by Chen and Mellman . TherefoThere is also evidence that CAFs may act directly on CD8+ T cells to interfere with the process of antigen recognition and activation, through inhibition of the T cell receptor (TCR). Utilising both in vitro and syngeneic orthotopic murine models of PDAC, a study showed a suppressive effect of CAF-derived transforming growth factor beta-induced protein (TGF-βi) on the antigen-specific activation of ovalbumin specific CD8+ T cells . MechaniFinally, while the topic of CAF heterogeneity will be covered in greater detail in another review in this journal issue, recent evidence has revealed a novel subpopulation of CAFs with antigen-presenting capabilities. These so-called “apCAFs” were found to express MHC class II at their cell surface, whilst also possessing the capacity to present the ovalbumin peptide to CD4+ T cells ex vivo . Co-stimSince the breakthrough approval of the immune checkpoint CTLA-4 inhibitor ipilimumab for the treatment of metastatic melanoma in 2011, the field of clinical immuno-oncology has seen rapid development and a surge of interest. The success of ipilimumab represents an important proof of concept for therapeutic modulation of the immune system, with CTLA-4 being the first target to bear translational relevance. This has led to the emergence of new and improved checkpoint inhibitors over the last decade, the use of which has been extended to an increasing number of indications for widespread clinical benefit . Immune Currently, the use of these immunotherapeutic strategies has seen the most widespread clinical benefit in the context of non-solid cancers and cancers with a non-prominent tumour stroma ,92. In sSeveral strategies to overcome the inhibitory effects of CAFs on T-cells are beginning to emerge; however, the field is still very much in its infancy. As previously discussed, IL-6 and CXCL12 are two CAF-derived factors that impair T-cell function and recruitment to the TME ,79. AccoOverall, however, there is currently a rather limited body of work focused on therapeutically exploiting CAF-mediated deregulation of CD8+ T cell function with this area of research needing further attention in future studies.There is now a growing body of evidence detailing the mechanisms through which CAFs can negatively regulate the activity of cytotoxic CD8+ T cells within the TME of solid malignancies. As presented herein, this CAF mediated regulation of T cell function is mediated through interference with T cell-recruitment, infiltration, and cytotoxic activity. Future studies must seek to further characterise these molecular interactions between CAFs and CD8+ T cells. In particular, greater emphasis needs to be made into delineating the precise role of CAF production of ECM components in regulating T cell infiltration in the TME. As described, the jury is still out on whether deposition of ECM serves to facilitate T cell infiltration or whether it forms a barrier to biophysically impede the movement of T cells, as canonically believed to be the case.Furthermore, future efforts must be made to build upon the basic science of CAF-T cell biology presented within this review to facilitate the rational development of therapeutic strategies which seek to target these interactions. Additionally, previous studies already showed that large-scale genetic ablation of the CAF population as a whole is deleterious and translates to increased tumour aggressiveness and reduced survival in preclinical studies ,20, owin