APOE genotypes are associated with ischemic heart disease (IHD), several other cardiovascular diseases and dementia. Previous studies have not comprehensively considered all genotypes, especially ε2ε2, nor associations by age and sex, although IHD incidence differs by sex. In the UK Biobank, including 391,992 white British participants, we compared effects of APOE genotypes on IHD and its risk factors. Compared to the ε3ε3 genotype, ε2ε2 was not clearly associated with IHD but was associated with lower plasma apolipoprotein B (apoB). The ε2ε3 genotype conferred lower IHD risk, systolic blood pressure (SBP), pulse pressure and plasma apoB than ε3ε3. ε3ε4 and ε4ε4 conferred higher IHD risk, higher pulse pressure and plasma apoB, but lower glycated haemoglobin (HbA1c) than ε3ε3. The associations by age and sex were fairly similar, except ε2ε2 compared to ε3ε3 was marginally positively associated with IHD in the younger age group and nominally inversely associated with SBP in men. ε3ε4 compared to ε3ε3 was nominally positively associated with SBP in women. APOE genotypes affect IHD risk increasingly from ε2ε3, ε3ε3, ε3ε4 to ε4ε4, with similar patterns for pulse pressure and plasma apoB, but not for diabetes. Associations with blood pressure differed by sex. Greater understanding of products of APOE and their effects might generate targets of intervention. Diabetes is an important co-morbidity with a heavy disease burden1. The development of cardiovascular disease (CVD) is complex, with genetic background a contributing factor3. So far, several genes, such as LDLR, PCSK9, HMGCR and SLC12A1 have been recognized for their role in IHD, and correspondingly are therapeutic targets4. Identifying genetic variants that strongly affect CVD can help elucidate targets for intervention6. Notably, sex differences in the incidence of IHD are greater than the differences for diabetes7, raising the possibility of sex-specific targets. The importance of sex-specific investigations is increasingly recognized as a means of investigating disparities and finding more target interventions8. Although prevention and treatment of CVD have improved immensely, declines in CVD mortality have stalled in the United States since 20119, indicating new prevention strategies are needed10, but have become increasingly difficult to identify11. In this situation, re-assessment of “backgrounded” or overlooked targets, such as apolipoprotein E (apoE)12, is increasingly being undertaken14.Ischemic heart disease (IHD) is the leading cause of mortality and morbidity globally16, determined by two single-nucleotide polymorphisms (SNPs), i.e., rs429358 and rs741216. APOE genetic variants are a well-known genetic determinant of longevity17, affecting androgens18 whose relevance to human health in terms of the evolutionary biology trade-off between reproduction and longevity, is increasingly recognized19. ε3  carriage is most common with a frequency of around 78.3%20, although whether ε3 or ε4 is the ancestral form is controversial22. ε2  carriers compared to ε3ε3 carriers have lower risk of IHD26, lower risk of hypertension27, higher risk of type 2 diabetes28, and lower plasma apolipoprotein B (apoB)32. A recent phenome-wide association study (PheWAS) showed ε2 associated with a wide range of age-related outcomes13, while another suggested the ε2ε2 genotype was positively associated with peripheral vascular disease and thromboembolism14. Conversely, ε4  carriers compared to ε3ε3 have a higher risk of IHD33, hypertension27, and higher plasma apoB32 with both positive and inverse associations with type 2 diabetes observed28.The apoE protein has three isoforms  coded by three allelic variants, known as ε2, ε3 and ε4, which form six common genotypes APOE is known to be a key determinant of longevity17 and aging13. However, some of the evidence for effects on CVD concerns older people35. Observational studies in older people can be difficult to interpret because those who have died before recruitment from the exposure, from the outcome or from a competing risk of the outcome are inevitably excluded from the study by prior death, meaning the full effect on the outcome cannot be observed due to selection bias. The small magnitude of genetic associations means genetic studies may be more vulnerable to such bias than traditional observational studies. Studies in younger people are less open to such selection bias36, drawing attention to the importance of age-specific associations which has not been considered in previous studies14. APOE genetic variants may affect androgens18, and androgens affect IHD, suggesting the associations could differ by sex, but previous PheWAS did not consider sex-specific associations14. Nowadays, the importance of investigating sex-specific associations has been recognized and encouraged to provide insights hopefully to reduce disparities and better target interventions8. Here, we made use of a large population-based study of people of mean age 56.9 years, i.e., the UK Biobank, with high-quality indicators of potential population stratification, to assess effects of common APOE genotypes on IHD overall, stratified by age at recruitment to obtain estimates less open to selection bias, and by sex to provide insights about differences by sex. We also similarly assessed associations with traditional IHD risk factors, i.e., blood pressure, type 2 diabetes, glycated hemoglobin (HbA1c), and with plasma apoB given it is increasingly being considered as an important cause of IHD39. We also included low-density lipoprotein (LDL) cholesterol as a positive control outcome, given APOE genotypes are known to affect LDL cholesterol23. Specifically, LDL cholesterol is lower in ε2ε2, ε2ε3 and ε2ε4 carriers, but higher in ε3ε4 and ε4ε4 carriers, than in ε3ε3 carriers23.Most studies of ε2 carriage are based on the ε2ε3 genotype, while effects of ε2ε2 are less well-established, due to the low frequency of ε2. 40. At baseline comprehensive assessments were made, and samples collected. Follow-up via record linkage to hospitalizations and death registration is ongoing40. Genotyping was based on two highly similar genotyping arrays (95% of marker content shared), i.e., the Applied Biosystems UK BiLEVE Axiom Array  and the Applied Biosystems UK Biobank Axiom Array , and was imputed using the Haplotype Reference Consortium (HRC) and the UK10K haplotype resource41. To avoid confounding by population stratification, only participants of white British ancestry were included here. For quality control, we also excluded participants with genetic and reported sex mismatch, sex chromosome aneuploidy, genotyping missing rate > 1.5%, or extensive relatedness (more than 10 putative third degree relatives).The UK Biobank is one of the largest ongoing cohort studies worldwide, which recruited more than 500,000 participants in 2006–2010 intended to be aged 40–69 years from the UK APOE genotypes  with the APOE ε3ε3 genotype. Genotypes were based on combinations of haplotypes derived from rs429358 and rs7412 as shown in Supplementary Table We compared the 42. Taking medication use into account in this fashion may not reflect the effectiveness of medication use, so blood pressure without adjustment for medication use was used in sensitivity analyses. Pulse pressure was the difference between SBP and DBP. HbA1c was measured using HPLC analysis (Bio-Rad VARIANT II Turbo). Plasma apoB was measured using immunoturbidimetric analysis (Beckman Coulter AU5800). LDL cholesterol was measured using enzymatic protective selection analysis (Beckman Coulter AU5800), with 1.1 mmol/L added to LDL cholesterol for people on cholesterol lowering medication43. Similarly, LDL cholesterol without adjustment for medication use was used in sensitivity analyses.The primary outcome was IHD, and the secondary outcomes were systolic blood pressure (SBP) (mmHg), diastolic blood pressure (DBP) (mmHg), pulse pressure (mmHg), type 2 diabetes with or without complications, HbA1c (mmol/mol) and plasma apoB (g/L), with LDL cholesterol (mmol/L) as a positive control outcome. Disease outcomes were based on self-report at baseline, and subsequent record linkage to both primary and secondary diagnoses of hospital episodes, and both primary and secondary causes of death , based on 407,219 unrelated, high quality samples and 147,604 high quality markers, aiming to capture population structure at both sample and marker level. The principal components are associated with self-reported ethnic background and population structure at sub-continental geographic scales2 tests or ANOVA to assess the associations of APOE genotype with baseline characteristics, including age, sex, body mass index (BMI), smoking status, alcohol use, indicators of socio-economic position, including education, average total household income before tax and Townsend deprivation index, physical activity based on the International Physical Activity Questionnaires (IPAQ), and use of medication for lowering cholesterol, blood pressure or for diabetes, and use of exogenous hormones, including hormone replacement therapy and oral contraceptives by women.We used χ44. We also stratified the analyses by age at recruitment using a traditional cut-off of 60 years45 and by sex, with similar adjustments, and compared differences using a z-test46. Given in this study we tested associations of APOE genotypes with seven outcomes including one main outcome IHD and six secondary, possibly correlated, outcomes, we used a Bonferroni correction to account for multiple hypotheses testing, giving a p value threshold of 0.007 (0.05/7).Genetic associations obtained using logistic or linear regression were adjusted for age at recruitment, square of age at recruitment, sex, sex × age at recruitment, sex × square of age at recruitment, genotyping array and the first 40 principal components, as previouslyAll statistical analyses were conducted using R version 3.6.2. This research has been conducted using the UK Biobank Resource under Application number (42468). The UK Biobank has already received ethical approval from the North West Multi-centre Research Ethics Committee (MREC) which covers the UK. It also got the approval from the Patient Information Advisory Group (PIAG) in England and Wales, and from the Community Health Index Advisory Group (CHIAG) in Scotland. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki.This research has been conducted using the UK Biobank Resource under Application number (42468). The UK Biobank has already received ethical approval from the Research Ethics Committee and participants provided written informed consent. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki.Not applicable.After selecting on white British ancestry and quality control criteria, 391,992 participants remained. Among these participants, 33,490 had IHD, and 18,211 had type 2 diabetes.APOE genotype. Mean age was 56.9 years, and 54.1% were women. Mean BMI was 27.4 kg/m2. Just over half had never smoked, 10% were current and 35% were previous smokers. More than 90% were current alcohol users. No differences by APOE genotype were evident for sex, smoking, alcohol use, socio-economic position, use of insulin or hormones (in women). Participants with the ε2ε2 or ε4ε4 genotype were slightly younger than the others. ε4ε4 carriers had lower BMI and were more physically active than others. ε3ε4 and ε4ε4 carriers were more likely to take cholesterol or blood pressure lowering medication than others. As expected ε2ε2, ε2ε3 and ε2ε4 carriers had lower LDL cholesterol than ε3ε3, and ε3ε4 and ε4ε4 had higher LDL cholesterol than ε3ε3 . Findings on blood pressure are somewhat consistent with a previous PheWAS, although they did not include pulse pressure, did not account for effects of anti-hypertensive medications when drug use differs across genotypes  or even reversed, depending on the magnitude of selection bias. The analysis was restricted to the participants of European ancestry, whether the associations apply to other populations are uncertain. However, mechanisms should be consistent across populations although they might be not relevant in all settings64. In the analyses stratified by age at recruitment, 60 years is a slightly arbitrary cut-off. Given increasing IHD prevalence by age we could not create age stratified groups with similar IHD prevalence, but we created groups with similar numbers of events in each group, which gave an age cut-off of 63 years, results were similar using this revised cut-off. Lastly, the number of people with the ε2ε2 genotype may have been inadequate to fully elucidate its role.This study is based on a very large population-based cohort which has the advantage of being relatively young and enabling stratification by age. We controlled for population stratification in several different ways, including restricting the analysis to white British people, excluding people with extensive relatedness and adjusting for principal components. We found little relation of APOE genotypes affect risk of IHD, with approximately lowest to highest risk as ε2ε3, ε3ε3, ε3ε4 and ε4ε4 overall, while the ε2ε2 genotype might be harmful for IHD in younger people. The association of these genotypes with major IHD risk factors, including blood pressure and diabetes however, was not always in the same pattern overall, and some associations differed by sex, highlighting the complexity of IHD etiology, as well as the importance of investigating the role of products of APOE sex-specifically as potential targets of intervention for cardiovascular disease and possibly diabetes prevention.Supplementary Information


Self testing for HIV is a targeted intervention with the potential to increase the access, uptake and frequency of HIV testing and more effectively reach the undiagnosed, especially in priority populations. The objectives of this study were to (1) evaluate the INSTI HIV self-test performance compared with laboratory reference testing, (2) document if intended users can perform the steps to use the HIV self-test device, and (3) document if intended users can successfully interpret contrived positive, negative, and invalid results. Study was intended to be submitted to Health Canada for review for regulatory approval purposes.The study used a cross-sectional design and recruited consenting adults who were representative of intended users of HIV self-testing from four community sites across Ontario, Québec, and Manitoba between August 2019 and March 2020. The results of the observed HIV self-test were compared with results of the Abbott Architect HIV Ag/Ab Combo test. Usability outcomes for critical  and noncritical self-test procedure steps were also determined.n = 522) of participants were between 18 and 45 years of age, 61% (n = 410) were male, 71% (n = 480) had some college or more education, and 45% (n = 307) were employed; identity for race and ethnicity: Caucasian , African, Caribbean or Black , Indigenous  , Asian , Latin American . Primary performance analysis on 678 completed HIV self-tests revealed a positive percent agreement of 100%  and a negative percent agreement of 99.5%  with the comparator method. The overall percent agreement of results interpretation between participant and observer was 93.5% (n = 633). For the 708 participants who took part in the usability study, the average success rate for steps determined to be “critical” for successful completion of the test was 92.4%. 97% (n = 670) of participants found the instructions easy to follow, and 95% (n = 655) of participants indicated that they would use the test again. Of the 404 participants who interpreted the strong positive, weak positive, negative, and invalid contrived results, successful interpretation ranged from 90.6%  to 99.3% .Overall, 77% (The addition of a regulatory-approved self-test into the Canadian HIV testing landscape could significantly increase HIV testing rates. Having a blood-based HIV self-test approved in Canada can offer an accurate, acceptable, and simple alternative to facility-based HIV testing, particularly when impacted by Coronavirus pandemic restrictions.The online version contains supplementary material available at 10.1186/s12889-021-11418-z. Unlike other G7 countries, Canada is not seeing a reduction in the overall number of new people being diagnosed with HIV. Recent data from the Public Health Agency of Canada (PHAC) indicate that an estimated 2242 new HIV infections occurred in Canada in 2018 compared to 2165 in 2016, which is an increase of 3.6%. Approximately 13% of people living with HIV in Canada are still not diagnosed, which represents over 8000 individuals living with HIV who are unaware of their status  × 100, where TP (true positive) is positive self-test in agreement with positive Architect test, and FN  is negative self-test discordant with positive Architect test; and NPA = [TN / (TN + FP)] × 100, where TN (true negative) is negative self-test in agreement with negative Architect test, and FP  is positive self-test discordant with negative Architect test.As a secondary analysis, study participants’ self-interpreted results were compared with the self-test results interpreted by the observer.Due to the impact of COVID-19 on restrictions for facility visits, a total of 767 participants were recruited over the study period instead of the 900 that were targeted for enrollment. Of the 767 participants that started the process, 89 participants were excluded from the Primary Efficacy Analysis for reasons including failure to provide a venous blood sample or failure to provide a self-test result (see Appendix B for full details of excluded participants). Recruited participant numbers varied across the performance, usability and mock results interpretation arms of the study as not all recruited participants completed all elements.n = 522) of the sample was between the ages of 18 and 45 years of age. Over 60% (n = 410) identified as “male” and 37% (n = 247) as “female” with approximately 3% (n = 20) choosing “other”, including transgender, non-binary, queer, and Two Spirit gender identities. In terms of ethno-racial composition, approximately 44% (n = 296) identified as “White”, 17% (n = 113) as “Black”, 14% (n = 95) as “First Nations, Métis or Inuit”, 16% (n = 106) as “Asian”, 7% (n = 46) as “Latin American”, and the remaining 2% (n = 22) as “other”. In terms of educational achievement, approximately 27% (n = 184) of the sample completed High School, 23% (n = 156) completed College, and 48% (n = 322) a University degree or higher. Less than 5% (n = 30) indicated having any kind of reading or writing impairment. Approximately 45% (n = 307) were employed, 23% (n = 153) were unemployed, 13% (n = 87) were students, 2% (n = 16) were retired, and 17% (n = 113) preferred not to answer.Table n = 137) indicated that their status was unknown or had never been tested, while 80% (n = 540) indicated that they were HIV-negative. When asked about HIV testing, 74% (n = 500) indicated having prior experience with testing. When evaluating self-reported risk category for HIV, 600 participants (89%) were considered “high risk” for HIV infection. For specific self-reported sexual behaviours in the year prior to survey, 30% (n = 185) identified as having condomless sex with men while 18% (n = 108) had condomless sex with women. Approximately 30% (n = 181) of participants indicated having multiple sexual partners in the past year, 26 individuals (4%) indicated that their sexual partner was living with HIV, and 51 participants (8%) indicated having ever injected drugs.In terms of self-reported HIV status, 20% (n = 633) (see Table n = 38).After removing the 89 subjects who met the exclusion criteria from the 767 recruited participants, the primary efficacy analysis on the 678 who completed INSTI HIV Self-Test revealed a positive percent agreement of 100%  and a negative percent agreement of 99.5%  when comparing the valid self-tester results to the Abbott Architect (see Table n = 334) of participants were observed to have washed and dried their hands. Just over 92% (n = 650) of participants were able to lance their finger correctly, and 88.7% (n = 627) were able to form a blood droplet, but there was a considerably lower number of participants  who were able to get the blood droplet to fall directly into Bottle 1.From the total of 767 study participants, 59 subjects withdrew after consenting, did not have a venous blood sample collected or did not complete the self-test, leaving 708 available for analysis with the usability assessment. Overall, the average for all expected outcomes was 91.8% and for the 12 critical items, the average was 92.4% (see Table n = 632) of participants performed the procedural steps of the self-test in the correct order, while less than 10% (n = 67) missed any steps and yet continued the process despite this, and only 1.0% (n = 7) quit the process before the test was completed.Following the fingerstick blood collection, there were three types of procedures carried out as part of the test process: (1) shaking bottles (accomplished with range of 88.8 to 90.2% of time), (2) pouring contents into the test device (97.3 to 99.0% of participants did so correctly), and (3) waiting for liquids to disappear before adding next liquid into device (participants did this procedure 98.0 to 98.2% of the time). Over 89% (n = 670) of participants found them easy to follow. In terms of device use, over 90% (n = 626) of participants indicated that they were confident with performing the test on their own, and 96.2% (n = 666) found the device easy to use. 94.7% (n = 655) of participants indicated that they would use the test again and 95.5% (n = 658) would recommend this test to a sexual partner or friend. In terms of where participants would prefer using the self-test, 58.6% (n = 399) indicated that they would do the test at home, while 41.4% (n = 282) indicated that they would prefer to do the test at a clinic.Table n = 395) of participants correctly identified a “strong positive”, 90.6% (n = 366) a “weak positive”, 99.3% (n = 401) a “negative” result, 97.8% (n = 395) an “invalid”  result, and 93.6% (n = 378) an “invalid”  result.A total of 404 participants volunteered to complete the mock test interpretation  Rapid Diagnostic Tests (RDTs) for use at the Point of Care (POC) or for Self-Testing provides performance targets that must be met for clinical sensitivity and specificity: “Evidence that the RDT intended for use at the POC or for Self-Testing has a minimum sensitivity and specificity of ≥99% for HIV antibody detection should be provided” . Primaryn = 633) concordance in the self-test results interpretation between the untrained self-test study participants and the trained observers for the subjects in the primary efficacy analysis. Most discordant results were in the invalid or “do not know/not sure” (uncertainty) response categories.There was a 93.5%  interpreted their self-test results as invalid, including one participant whose venous blood tested positive with the Abbott Architect. Of these, 22 were also interpreted as invalid by the observers, including the one participant living with HIV; however, 15 were interpreted as negative by the observers indicating there was a visible control dot present in the self-test which was either not observed by the self-tester, or not interpreted correctly. In some of these cases, the control dot intensity was indicated to be faint. A faint, but visible control dot may also have led to “do not know/not sure” interpretations recorded by both the self-testers and observers. Mitigations may be needed to improve the fingerstick blood collection process since usability results indicated that nearly 1 in 5 participants had difficulties in getting a free-flowing blood drop to fall into the INSTI sample diluent (Bottle 1). These mitigations could include revisions to the package insert to provide more clear instructions on the proper use of the lancet and subsequent blood drop collection, as well alerting the self-tester that even a faint dot intensity is considered valid. Consequently, self-test users should be instructed to conduct self-testing in a well-lit area.n = 22/37) of invalid self-test results and for several self-test results that were still valid. A further mitigation to address the critical factor for the success in getting the blood drop into Bottle 1 could be to revise the self-test package insert to indicate, with illustration, that one should not scrape or wipe the blood into Bottle 1.There was an observed tendency for some self-testers to wipe their fingertip on the rim of the INSTI Bottle 1 (sample diluent) instead of allowing the blood drop to fall freely into the sample diluent. This was indicated for 60% (n = 366) for the weak positive result to 99.3% (n = 401) for the negative result, indicating that untrained self-testers can successfully interpret a range of self-test results including strong positive, weak positive, negative, and invalid test results. However, mitigations to the package insert to call attention to the possibility of faint dot intensity could improve the lower success rate in interpreting weak positive results.The mock results interpretations for the 404 participants who volunteered to participate in this portion of the study show a high concordance with the expected results. The percent agreement with expected results ranged from 90.6% (n = 670) of participants found the instructions for use easy to follow and 96.2% (n = 666) found the self-test device easy to use. 94.7% (n = 655) of participants would use the INSTI HIV Self-Test again, and 95.5% (n = 658) would recommend its use to a sexual partner or friend. Of interest and for consideration – approximately 60% (n = 399) of participants indicated a preference for using the self-test in a home environment while 40% (n = 282) would prefer to use the test at a clinic or health care facility, suggesting that both assisted (supervised) and unassisted self-testing strategies will likely be needed for “real world” access and uptake.In general, participants expressed a high level of satisfaction with the HIV-ST experience as shown in Table Overall, acceptance of the INSTI HIV Self-Test by intended users in this study was high, and similar to studies that reported that oral fluid-based HIV self-testing is highly acceptable among a variety of populations , 16, 17.The impact of the COVID-19 pandemic and its related public health control measures on HIV treatment and prevention, including access to facility-based testing, has had profound effects on individuals, communities, and societies across the world . ImplemeAdditional file 1. Appendix A – Inclusion and Exclusion Criteria; Appendix B – Exclusion List; Appendix C – Survey Instrument.


Protein-based nanofibres are commonly used in the biomedical field to support cell growth. For this study, the cell viability of wool keratin-based nanofibres was tested. Membranes were obtained by electrospinning using formic acid, hexafluoroisopropanol, and water as solvents. For aqueous solutions, polyethylene oxide blended with keratin was employed, and their use to support in vitro cell interactions was also validated. Morphological characterization and secondary structure quantification were carried out by SEM and FTIR analyses. Although formic acid produced the best nanofibres from a morphological point of view, the results showed a better response to cell proliferation after 14 days in the case of fibres from hexafluoroisopropanol solution. Polyethylene oxide in keratin nanofibres was demonstrated, over time, to influence in vitro cell interactions, modifying membranes-wettability and reducing the contact between keratin chains and water molecules, respectively. Keratin is a natural protein extensively used to produce scaffolds thanks to its biocompatibility and biodegradability . In the Recent studies have demonstrated that the peculiar structure of proteins may influence their interaction with cells, thus addressing their fate in vitro . In the case of keratin, previous works have reported that extraction and manufacturing methods play a relevant role in the protein chemical structure at the sub-micro scale , signifiAs reported in previous studies, the use of different solvents and the integration of additional polymers in solution can affect the micro and nanostructural properties of NFs, thus altering the local interface with cells . As a fuBesides, several studies have broadly demonstrated that some solvents can be undesirable during the production of protein-based NFs, due to the presence of low volatile residual solvent with aggressive moieties that tend to be hardly removed from fibres , and, inIn this context, the integration of synthetic polymers with recognized properties in the biomedical field in terms of non-toxic response , hexafluoroisopropanol (HFIP), and water. Moreover, a synthetic water-soluble polymer such as PEO will also be considered to validate its use to support in vitro cell interactions.Wool fibres were used to obtain keratin powder (KS) by sulfitolysis, as reported in previous works . Unless Polymeric solutions made of KS  were employed to produce NFs-membranes by a single jet electrospinning prototype. It consists of a flat plate collector, a KDS 200 high-precision syringe pump , a stainless steel needle, and an SL50 high-voltage generator . The morphology of NFs was observed using a XEVO series scanning electron microscope (Carl Zeiss Microscopy GmbH) with an acceleration voltage of 15 kV at about 30 mm working distance. The samples were sputter-coated with a 20 nm-thick gold layer in rarefied argon (20 Pa), using a Quorum SC7620 sputter coater, with a current of 20 mA for 120 s. The software Fiji-ImageJ 1.51 was used on SEM images to estimate the diameter distribution of NFs-membranes.−1 band resolution and 50 scans) between 4000 and 650 cm−1.A thermo nicolet nexus spectrometer with a smart endurance accessory was employed to record all spectra in ATR (4 cm−1 to intermolecular β–sheets [−1 to intramolecular β–sheets [−1 can be assigned to inter and/or intramolecular β–sheets [−1 to β–turns [−1 to α–helix [−1 to random coil [−1) and the bands of carboxylic groups  were also taken into account [−1, fixing the band position and allowing any positive value for the height of Gaussians [The peak of Amide I was fitted with Gaussians  to quantify the content of secondary structures in KS. A baseline between Amide I and Amide II peaks was also used. Furthermore, the second derivate method with a quadratic Savitzky-Golay smoothing  was employed to determine the wavenumbers. The frequencies were assigned as follow: 1611, 1618, 1625, and 1695 cmβ–sheets ,20,21; 1β–sheets ; the banβ–sheets ,23, and  α–helix ,25,26; 1dom coil ,25. More account ,27,28; haussians .2 cell culture flask in Eagle’s alpha minimum essential medium (α–MEM) supplemented with 10% fetal bovine serum (Sigma-Aldrich), antibiotic solution  and 2 mM of L-glutamine, incubated at 37 °C in a humidified atmosphere with 5% CO2 and 95% air. hMSCs from 4–6 passages were used for cell proliferation assays. Before in vitro studies, membranes were cut to put in 96-well cell culture, washed and sterilized in 70% ethanol for 30 min before being washed three times with phosphate-buffered saline (PBS). hMSCs were seeded at 6 × 103 onto KSFA, KSPEO and KSHFIP fibres for proliferation assays assessed via cell counting kit-8 reagent  up to 14 days. Briefly, cell culture media was removed and changed at each time point by 100 µL of fresh medium with 10 µL of CCK-8 reagent per well and incubated for 4 h in standard conditions. The supernatant was collected, and absorbance was measured at 450 nm using a microplate reader. Results are presented as mean ± standard deviation (n = 3). Analysis of variance (ANOVA) with Tukey’s post hoc test was used to detect differences between groups. A value of p < 0.05 was considered to determine statistically significant differences.For in vitro assays, human mesenchymal stem cells  were used. hMSCs were cultured in a 75 cmOver the last few years, the use of natural proteins has been variously applied to the fabrication of fibrous scaffolds with improved surface properties in terms of bio-recognition and adhesiveness that identify key features for the fabrication of platforms suitable for in vitro cell interaction studies ,29. In p−1 is assigned to Amide A; the peak of Amide I at 1650 cm−1 and the peak of Amide II at 1540 cm−1 can be assigned to the stretching vibrations of C=O bonds and the in-plane bending modes of N–H bonds, with some contributions of C–N stretching vibrations. The peak at 1200 cm−1 corresponds to Amide III  [−1 is related to the stretching vibration of Bunte salts [FTIR spectra of all samples are reported in rations) . It alsorations) . The peate salts .2 rocking, C–C stretching, C–O stretching at 841 cm−1; CH2 twisting, CH2 rocking at 960 cm−1; CH2 rocking, C–C, C–O stretching at 1061 cm−1; C–C, C–O stretching at 1096 cm−1; CH2 rocking, C–O stretching at 1145 cm−1; CH2 twisting at 1242 and 1279 cm−1, CH2 wagging at 1341 cm−1; C–C stretching, CH2 wagging at 1358 cm−1 and CH2 scissoring at 1464 cm−1 [The spectrum of PEO is characterized by bands identified as follows: CH464 cm−1 ,34,35.It is important to remember that the solvents can destabilize the hydrogen bonds, the hydrophobic interactions and the solvation shell of proteins, as well as preferentially stabilize α–helix, β–sheet, β–turn or random coil conformation ,37,38. SThe CCK-8 assay evaluated the biological response of hMSCs seeded onto KSFA, KSHFIP and KSPEO. In this work, the role of morphology, i.e., fibre diameter distribution and chemical properties on in vitro hMSC response, was investigated. The most homogeneous fibres with the smallest diameter distribution  were obtained using FA as solvent. Flat ribbons with a size  considerably greater than the former case were produced when HFIP was used. In the literature, similar results were reported in the case of silk fibroin electrospun fibres from equivalent solvents in solution . Therefo2 [The fabrication of KSPEO-NFs by electrospinning has been extensively studied in the literature ,18. In t2 ,34.−1, as-spun KSFA-NFs showed a peak at 1715–1722 cm−1 while KSHFIP-NFs had a peak at 1723–1738 cm−1 and another at 1760 cm−1. The bands above 1700 cm−1 are associated with the COOH modes of protonated glutamic and aspartic acid side chains [N- or O-formylations in lysine and serine/threonine residues, whose aldehyde group generates a peak above 1710 cm−1 [Compared with pure KS (lyophilized powder), new peaks were found in the spectra of as-spun samples using solvents, e chains ,42. Howe710 cm−1 ,44.−1, which might be assigned to the vibration of secondary alcohols (C–O stretch at 1100 cm−1) or ester groups (C–O–C stretch at 1100–1030 cm−1) [−1 and, consequently, solvent traces could be the source of this signal [−1 and 1760 cm−1 gives evidence of the formation of ester groups in KSHFIP-NFs [−1 and 1388 cm−1 increased when FA and HFIP were used to KS-NFs electrospin in comparison with pure KS (lyophilized powder), 3 groups in amino acid side chains  [In the KSHFIP-NFs case, one additional peak was found at 1090–1100 cm30 cm−1) ,45,46. Is signal . NeverthHFIP-NFs ,46. ConsHFIP-NFs , it is pleucine) . This re−1, 1464 cm−1 and the triplet peak between 1000 and 1200 cm−1) [−1 and the peak at 1040 cm−1) is verified [KSPEO spectra show that the electrospinning process does not vary the bands associated with PEO crystallinity (the peaks at 841 cm00 cm−1) ,49 in coverified ,49.When PEO was used to electrospin KS in an aqueous solution, a significant reduction in intramolecular β–sheet and intermolecular β–sheet was observed. Intramolecular β–sheets are formed within individual proteins, and intermolecular β–sheets are formed among separate proteins (causing protein aggregation) . This reAs far as the biological response of hMSCs is concerned, it is worth mentioning that the poor initial response of KSHFIP may be ascribable to the effect of fibre diameters at the sub micrometric scale, which provide topological signals in vitro that are less often recognized by cells rather than fibres at the nanoscale . Additionally, it is suggested that KSFA and KSHFIP samples provide a major exposition of aliphatic side chains that probably reduced the wettability of KS-NFs, see c, limitiThe presence of PEO into KSPEO-NFs supports the initial cell growth, thus confirming its favourable contribution on in vitro tests. However, its mechanism of cell proliferation slowed down as a function of culture time. As reported in the literature , the macIn other terms, the presence of PEO-chains promotes a better hMSC recognition onto KS-NFs-by increasing fibre hydrophilicity at the microscopic scale, but reducing cell proliferation over time by inhibiting keratin interactions on a molecular scale into the culture. Hence, KSHFIP-NFs are the best choice in terms of cell proliferation, in agreement with evidence related to the modification of secondary structures of the protein .The results demonstrated that, even though solvents (HFIP and FA) and synthetic water-soluble polymers (PEO) increase the processability of proteins in electrospinning, those affect to some extent the bioactivity of biomacromolecules, like wool keratin. In the HFIP case, the hydrogen bonds that enclose polypeptide chains of KS stabilize α–helix conformations and enhance the viability of wool keratin-based NFs during their interaction with hMSC. In terms of nanostructural properties, the best NFs (the most homogeneous fibres with the narrowest diameter distribution) were obtained using FA; however, hMSC proliferation was worse than the cell response observed in HFIPKS-NFs. PEO demonstrated a dual effect on in vitro cell interactions when used to produce electrospun-NFs for scaffold materials by modifying the wettability of membranes and by reducing the accessibility of solvent molecules into keratin chains. Finally, the increase of α–helix conformations in KS-NFs seems to enhance the cell viability of hMSC over time.


TNF and TACE are significantly decreased in lung adenocarcinoma patients, while the TNFR1/TNFR2 balance are increased. We conclude that the biomarker potential of TNFα alone will most likely not provide conclusive findings, but that TACE could have a key role along with the delicate balance of sTNFα/tmTNFα as well as TNFR1/TNFR2, hence stressing the importance of more research into the potential of rationalized treatments that combine TNFα pathway modulators with immunotherapy for lung cancer patients.Tumor necrosis factor-alpha (TNFα) can bind two distinct receptors (TNFR1/2). The transmembrane form (tmTNFα) preferentially binds to TNFR2. Upon tmTNFα cleavage by the TNF-alpha-converting enzyme (TACE), its soluble (sTNFα) form is released with higher affinity for TNFR1. This assortment empowers TNFα with a plethora of opposing roles in the processes of tumor cell survival (and apoptosis) and anti-tumor immune stimulation (and suppression), in addition to angiogenesis and metastases. Its functions and biomarker potential to predict cancer progression and response to immunotherapy are reviewed here, with a focus on lung cancer. By mining existing sequencing data, we further demonstrate that the expression levels of  About 60 years ago, it was reported that bacterial endotoxin administration to mice resulted in the release of a serological protein with necrotic anti-tumor activity at high concentrations. Due to the latter characteristic, this protein was termed tumor necrosis factor (TNF)  and consSoluble TNFα is mainly secreted by activated macrophages  and to aIn general, sTNFα is rapidly released upon trauma or infection, as it is bestowed with a determining role in immunoregulatory processes such as immune ontogeny, inflammation, and apoptosis . As a soIn comparison, tmTNFα is an important mediator of immune-cell crosstalk. To illustrate, when DCs express tmTNFα, the latter can interact with TNFR2 on NK cells, resulting in increased NK cell proliferation and cytotoxic activity . MoreoveTNFR1, also known as tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) or CD120a, is expressed on almost all host cells including various tumor cell types ,24,25,26+ T cells [In terms of biological effects, high affinity binding to TNFR1 (and not TNFR2) has been demonstrated to result in DC activation and subsequent stimulation of antigen specific CD8 T cells . That TN T cells  further  T cells , and NK  T cells . Yet, it T cells ,34,36. T T cells ,38,39. M T cells ,41,42. T T cells ,44.+ T cells [Next to their roles in immune cell proliferation and activation, triggered TNFR1 and TNFR2 can also induce apoptotic and necrotic cell death. In the case of TNFR1, this is the result of the interaction between the receptors’ death domain (DD) and the adapter protein TNF receptor-associated death domain (TRADD). Upon interaction of DD-TRADD, the activation of cysteine-aspartyl-specific proteases (caspases) is stimulated ,46. Alth T cells . As outl T cells . Althoug T cells , the “cl T cells , while t T cells ,49,50. A T cells ,52 and J T cells , and thi T cells ,55. In tIn conclusion, the presence of two functional isoforms (soluble and transmembrane) next to two receptors with specific expression, affinity, and downstream signaling avenues collectively serves the pleiotropic job description of TNFα. As regulator of cytotoxic, pro-, and anti-inflammatory functions, it is evident that TNFα can play a critical role in the development of chronic inflammatory diseases as well as cancer, as outlined below.Despite TNFα’s denomination, in vitro reported tumor necrosis after high TNFα concentrations appeared a phenomenon that is not so straightforwardly translated to successful cancer treatments in vivo. The latter is partly explained by TNFα’s multifunctionality as a cytotoxic but also immune modulating cytokine. As the immune system plays a complex role on the tumor microenvironmental (TME) battlefield, TNFα is used as a weapon to modulate and/or kill tumor cells, immune cells, and/or endothelial cells .Previously observed direct anti-tumoral properties of TNFα in vivo are among others based on its capacity to hinder tumor-associated blood vessel formation (angiogenesis) via selective endothelial cytotoxicity and necrotic hemorrhage . This seWhile a high concentration of TNFα has been linked to hemorrhagic necrosis, it is now widely accepted that chronic exposure to TNFα is more likely to promote tumor progression. First, it has been demonstrated repeatedly that chronic inflammation, in which the innate immune system plays a leading role, can promote cancer onset as well as progression and metastasis, typifying the “never-healing-wound” character of solid cancers ,70. IndeEven if chronic inflammation is not involved in the onset of tumor cell transformation, the immune system often becomes a co-worker during cancer progression. Today it is generally accepted that the immune system can identify and control nascent malignancies in a process called cancer immunosurveillance. In contrast, the latter can also promote tumor progression through the selection of poorly immunogenic variants and suppression of anti-tumor immunity. Together, the dual host-protective and tumor-promoting actions of immunity are referred to as cancer immunoediting and comprise three distinct phases: the elimination, equilibrium, and escape phase ,79,80.high (M1) TAMs and granulocytes can secrete, among others, TNFα, IL-1, and IL-12 to further ameliorate a Th1-polarized anti-tumor immune profile [The elimination phase is characterized by an imbalance towards more anti-tumor immunity, installed by adaptive as well as innate immune cells. Characteristic for a potent Th1-oriented tumor-associated antigen (TAA) specific adaptive immune response is the presence of immunogenic TAAs, presented via MHC-I on the surface of tumor cells, together with Fas, TRAIL, and IFN-γ receptors and the presence of perforin, granzymes, IFN-α/β/γ, IL-1, IL-12, and TNFα within the TME. Hence, TNFα can ameliorate this phase through its involvement in activation of T cells, macrophages, and NK cells. For example, it was shown that TNFR1 signaling promotes the accumulation of anti-tumor M1 polarized tumor-associated macrophages (TAMs) by suppressing the M2-polarizing release of IL-13 from eosinophils co-recruited with inflammatory monocytes . On thei profile .During the equilibrium phase, anti- and pro-tumor immunity are in balance and/or immune-mediated tumor dormancy is installed . It was + T-cells, TNFα has been shown to induce myelopoiesis in tumor-bearing mice. Furthermore, cells of the myeloid lineage can be recruited to become immune-suppressive regulatory myeloid cells, which decrease TAA-specific CD8+ T cell mediated tumor cytotoxicity [In the escape phase, TNFα has several effects capable of lowering the antitumor immune response by facilitating the accumulation and/or activation of a wide range of immunosuppressive cells such as Tregs , regulattoxicity . Moreovetoxicity . TNFα altoxicity . As IDO1toxicity .Although both TNFα and its receptors’ precise role within the complex and ever-evolving TME are far from fully understood, substantial evidence has emerged that TNFα signaling has a paradoxical and dual role in cancer progression and the dynamic immunoediting process. While it has been held responsible for key tumor promoting features such as the maintenance of an antigen-ignorant chronic inflammatory state and immunosuppressive effects that hinder TAA-specific effector functions, its documented anti-tumor assets in vitro and in vivo hamper a clear-cut conclusion.Lung cancer remains the leading cause of cancer-related deaths worldwide, responsible for almost 1.8 million deaths in 2020 alone . Lung caTNF is one of the co-occurring frequently altered immune genes found within the TCGA pan-cancer lung adenocarcinoma (LUAD) dataset (n = 507) [TNF gene region impact lung cancer progression differently. While the 238 G>A polymorphism, found in the promoter region of TNFα, has a favorable prognostic association for NSCLC [As the human respiratory tract is continuously exposed to air that can potentially contain airborne pathogens, lungs necessitate a unique, fine-tuned, and rapidly acting pulmonary immune system to maintain homeostasis . When thn = 507) . Howeveror NSCLC , the 308or NSCLC . Another opposing effect is found for sTNFα and tmTNFα on lung cancer growth . When muTNF, TACE, TNFR1, and TNFR2 expression profiles within a healthy tissue and TCGA pan-cancer LUAD transcriptomic data set. We found a significant reduction of TNF, TACE, and TNFR2 transcripts within the LUAD cohort  has been linked to a better prognosis for lung cancer patients. This implies that the biomarker potential of tmTNFα and TNFR2 for lung cancer progression most likely pivots on the delicate balances of sTNFα over tmTNFα as well as TNFR1 over TNFR2. + TAA-specific cytotoxic effector cells [The goal of antitumor immunotherapy is to completely and specifically eradicate both primary and metastatic tumor lesions by mobilized cytotoxic effector cells. Hence, immunotherapy can achieve actual cures of advanced lung cancer patients, representing an unprecedented reality ,113. Theor cells ,115. Sinor cells . Notablyor cells  yet haveor cells . Togetheor cells , these pIn search for clues, the relationship between TNFα and immune checkpoint signaling in the TME is being explored, hinting towards a lead role for TAMs. While IFN-γ is the main regulator of PD-L1 expression in tumor cells, PD-L1 expression in TAMs seems to be regulated via TNFα . In 2017+ T cell transfer, TNFα appeared to be a crucial factor in the incitement of melanoma dedifferentiation, which resulted in immune escape and melanoma relapse [+ T cells in melanoma, with subsequent lack of response to anti-PD-1 therapy [+ T cells [As melanoma remains the textbook example for immunotherapy responsiveness, TNFα targeting studies are most numerous for this cancer type. Overall, preclinical TNFα blockade has been shown to reduce the induction of irAEs upon ICI combinations and even improve therapeutic effectiveness of ICIs ,124. Upo relapse . Bertran therapy . Hence,  T cells . A few y T cells  that is + T cells sorted out of NSCLC patients’ peripheral blood mononuclear cells before and during anti-PD-1 mAb therapy [TACE expression within the responder group specifically, linked to more release of sTNFα from its tmTNFα form , although we were unable to draw any conclusions for TNF due to the lack of representative data, underscoring the current lack of human data on gene expression profiles of the different TNFα family members before, during, and after antitumor immunotherapy.In contrast to melanoma, only a handful of preclinical studies have explored the potential of TNFα pathway modulating strategies to treat lung cancer with rather contradictory findings. For example, anti-TNFα antibody treatment has been tested together with an intrapulmonary IFN-beta immuno-gene therapy (Ad.IFNβ) in an orthotopic mouse model of lung cancer. The rationale for this was that blocking the pro-inflammatory actions of TNFα could reduce the induction of dose-limiting pulmonary inflammation upon Ad.IFNβ delivery. However, upon administration, the anti-TNFα antibody not only significantly reduced the pulmonary inflammation but also the therapeutic effect of Ad.IFNβ delivery. Hence, TNFα proved to be both a dose-limiting factor as well as crucial for the anti-tumor immune stimulatory capacity of Ad.IFNβ . More st therapy , we wereNFα form E. While r groups F. Finall+ neovasculature in vivo, the rapid destruction of the tumor neovasculature and complete regression of large, established tumors was demonstrated. In contrast, selective blockage of sTNFα via INB03 led to a reduced carcinogen-induced tumor incidence and growth rate [+ T cells as well as the onset of irAEs, rationalizing combined TNFα-blockade with immunotherapy to treat melanoma. The multitude of contradictory findings currently poses a stalemate for TNFα pathway-affecting strategies in combination with immunotherapy to treat lung cancer and suggests the need for additional research into biomarkers to guide rationalized therapy combinations. This conundrum is reflected by the range of preclinical studies that report on the therapeutic efficacy of TNFα upon its administration as well as its inhibition . To illuwth rate . MoreoveTNF, and because of the significant reduction in TACE, sTNFα protein is likely to be most decreased. Together with the notion that tmTNFα, and not sTNFα, has been shown to play a key role in Th1-polarized antitumor immunity and improved lung cancer patient survival [+ Tregs have been found in the TME of human advanced lung cancers, and TNFR2 has recently been identified as a tumor-promoting oncogene with new biomarker potential for cancer [TNFR2 is markedly decreased in the lung TME. Moreover, pre-clinically, TNFR2 agonists as well as antagonists have been linked to antitumoral effects, arguing against the effectiveness of TNFR2 modulation for lung cancer therapy [By mining existing next-generation sequencing data from LUAD patients, the latter were shown to contain less survival ,134, thir cancer ,136. How therapy ,139,140.To conclude, numerous studies point out that TNFα signaling is extensively involved in lung tumor progression and response to (immuno-)therapy. However, the underlying mechanisms of the different TNFα family members that modulate tumor prognosis and response to treatment remain to be revealed. We summarized our main findings in


The PCR for SARS-CoV-2 was positive for two patients , whose “before” temperatures were 36.4 °C and 36.5 °C, respectively. Conclusions: Forehead temperatures increase during dental procedures and are conditioned by the patient’s behavior. An isolated temperature reading does not identify children infected by SARS-CoV-2.Background: To date, the efficacy of temperature readings of children in the dental setting for COVID-19 screening has not been evaluated. The aim of this pilot study was to assess the usefulness of forehead temperature measurements in a dental clinic for COVID-19 screening in healthy children (without systemic disease) and in children with neurodevelopmental disorders. Methods: Using an infrared thermometer, we recorded the forehead temperature of 200 pediatric patients . We performed temperature measurements “before”, “during”, and “after” the dental procedure. Oropharyngeal swabs were taken of all participants to detect SARS-CoV-2. Results: Sex, age, administration of local anesthesia, and use of rotary instrumentation did not affect the temperature values. In the children with neurodevelopmental disorders with a value of 1 on the Frankl behavior scale, the temperatures were significantly higher than in those with values of 2, 3, and 4 ( In February 2020, the World Health Organization (WHO) officially reported the Coronavirus disease 2019 (COVID-19), and, only a month later, declared the COVID-19 pandemic  and coulA recently published review that compiled information on 10,251 children from 31 countries concluded that the most prevalent manifestations of COVID-19 were fever 63.3%) and cough 33.7%) [3.3% and % 3.3% a. This prTo date, the efficacy of temperature readings of children in the dental setting for COVID-19 screening has not been evaluated. Their applicability is still less predictable in children with neurodevelopmental disorders (ND) because their thermoregulation might be abnormal due to autonomous dysregulation  and/or dWe selected a study group of convenience consisting of 200 pediatric patients  who visited the Odontology Unit for Patients with Special Needs and Pediatric Dentistry of the University of Santiago de Compostela (Spain) between December 2020 and February 2021. During the participant recruitment period there were no vaccines available for children. The applied exclusion criteria were as follows: symptoms suggestive of SARS-CoV-2 infection, being within an isolation period, having direct contact with a patient with COVID-19 in the past 10 days, having received nonsteroidal anti-inflammatory drugs or other type of antipyretic in the last 24 h, and refusing to participate in the study.The following information was collected from all participants: sex, age and presence of systemic disease . In terms of dental treatment, we recorded whether local infiltration anesthesia was administered, whether rotary instrumentation was employed, and the degree of the child’s collaboration according to the Frankl scale, which includes the following options: 1, “definitely negative”; 2, “negative”; 3, “positive”; and 4, “definitely positive” .® thermometer (Food and Drug Administration-certified JXB-178). To record the temperature, the thermometer was placed 5 cm from the central growth point of the hair . After remaining in the waiting room for 5–10 min, the companion’s temperature was recorded, and an initial (“before”) reading was performed on the child. A second reading was performed on the patient halfway through the dental consultation (“during”), and the last reading was taken approximately 3 min after completing the procedure (“after”).All temperature readings were made with a non-contact infrared BerrcomThe patients passed through a second office in which an oropharyngeal swab was taken from them; this was sent to the laboratory for processing. We performed nucleic acid extraction in a MicrolabStarlet IVD platform using the STARMag 96 × 4 Universal Cartridge Kit . To detect SARS-CoV-2, we applied the Allplex™ 2019-nCoV Assay , a multiplex one-step real-time reverse transcription (rRT)-PCR assay targeting a conserved region in the structural protein envelope E-gene for pan-Sarbecovirus detection, RNA-dependent RNA polymerase (RdRP) and nucleocapsid (N) genes specific for SARS-CoV-2. For the rRT-PCR, we employed the CFX96™ system . We analyzed the results using Seegene Viewer-specific 2019-nCoV software (Seegene).Before the dental intervention, we informed the patients’ guardians regarding the purpose of this study and obtained their consent. Approval by the ethics committee was not necessary, given that the measure was included in the current anti-COVID-19 protocol of the University of Santiago de Compostela.We used the Shapiro–Wilk test to compare the normality of the study variables. If the distribution was normal, we applied the T-test and the ANOVA test; when it was not normal, we used the Mann–Whitney U test and the Wilcoxon test. To compare the temperature values over time, we used a mixed linear model.Fifty-nine percent of the participants were boys, and 41% were girls. The patients’ mean age was 8.5 years . We administered local infiltration anesthesia to 38% of the patients. For 39% of the patients, we used rotary instrumentation. The degree of cooperation according to the Frankl scale was one in 11 participants , two in 38 cases (19%), three in 59 cases (29.5%) and four in the remaining 92 cases (46%).The companions’ mean temperature was 36.411 ± 0.230 °C . There were no significant differences between the companions’ recorded temperatures and the children’s “before” temperatures.The mean “before”, “during”, and “after” temperatures were 36.401 ± 0.256 °C , 36.892 ± 0.438 °C , and 36.600 ± 0.349 °C , respectively. The “during” temperature values increased by a mean of 0.371 °C compared with the “before” readings. After completing the procedure, the temperature decreased, but the “after” temperature values were still a mean of 0.144 °C higher than those of the “before” readings. To detect differences between the “before”, “during”, and “after” temperature values, we proposed the following model: Temperature ~ Time of measurement + (1|Patient). By applying this model, we showed that there were significant differences in the temperatures depending on when they were measured .p = 0.047), but there were no statistically significant differences in the “during” and “after” measurements  , the temperature readings were significantly higher than in the patients with values of 2, 3, and 4 (ctively) . All of The SARS-CoV-2 PCR was positive for two patients , whose “before” temperatures were 36.4 °C and 36.5 °C, respectively. In all of the remaining participants, the PCR was negative, including a healthy individual with a “before” temperature of 37.9 °C and another with an ND who reached 38.0 °C in the “after” measurement.This pilot study confirmed that forehead temperatures increase during dental procedures and that, in children with ND, the temperatures are determined by the patient’s level of behavioral control. To our knowledge, this is the first study to demonstrate that forehead temperature readings are not useful for COVID-19 triaging, either in children with ND or healthy children.It has been confirmed that non-contact infrared thermometers are useful instruments for determining the body temperatures of children older than 1 month both in the outpatient and hospital settings . HoweverThe reorganization of oral healthcare in the pandemic scenario  has not had a profound effect on the dental anxiety of children . TherefoPsychological stress can trigger physiological responses, including cutaneous vasoconstriction, tachycardia, glucocorticoid release, and increased body temperature . AlthougThe elderly was one of the most affected groups during the pivotal phase of the pandemic, and it is considered highly unlikely that a single temperature reading could detect nursing home residents infected with SARS-CoV-2 . It is e