Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus was discovered in China in late 2019 and subsequently triggered a global pandemic. Dentists, like many other health professionals, are at an increased risk of contracting the virus as they work in close proximity to patients, especially when performing aerosol-generating procedures. Thus, in order for dentists to protect themselves and their patients, it is recommended that practitioners wear filtering facepiece 2 (FFP2) respirators. The prolonged use of these FFP2 respirators has been linked to several side effects. The aim of this paper is to assess the perceived experience associated with N95/FFP2 respirators based on the available literature and data collected through an online survey completed by Italian dental professionals. Articles were included up to May 2020 and literature searches were conducted through The National Library of Medicine, Cochrane Central Register of Controlled Trials, and Embase databases. The search terms included COVID-19, respirators, masks, and discomfort. An online survey was administered to 256 Italian dentists. The results from this survey were in agreement with the available literature. The findings concurred that the prolonged use of respirators was associated with headaches (47.5%), severe exertion and discomfort (50.8%), moderate concentration problems (54.3%), moderate breathing difficulties (63.5%), and consequently, an impaired work ability (85.5%). These findings were not influenced by the number of hours spent wearing the respirator. Despite several side effects, FFP2 respirators are fundamental in protecting dentists and their importance was acknowledged. The virus termed Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (COVID-19—Coronavirus disease 2019) was discovered in Wuhan, Hubei province, China late December 2019 . The mosResearch on COVID-19 focused on the mode of transmission and disease progression in order to prevent contagion spread . It was Dental professionals, anesthetists, and ear-nose-and-throat specialists practice in very close proximity to patients with aerosol-generating procedures (AGPs). The term “aerosol generating procedures” (AGPs) encompass extubation, intubation, positive pressure ventilation (CPAP), tracheostomy, bronchoscopy, and upper gastrointestinal endoscopy. In the dental profession, ultrasonic devices, high-speed drilling, and 3-in-1 air-syringes are considered AGPs. Consequently, the use of a dental dam is highly recommended in order to reduce the spread of microbial loaded droplets . These pAlong with four-handed dentistry with high volume air evacuation, hand hygiene, gowns, gloves, face shields, and goggles are fundamental in order to prevent COVID-19 from infecting dental professionals .Thus, personal protective equipment (PPE) is a fundamental component in healthcare to reduce occupational exposure and risk to their health. A survey among Northern Italian dentists showed how the COVID-19 pandemic affected Italian dental practitioners and their use of PPE. The survey investigated the increasing or modifying use of PPE and their concerns regarding contracting COVID-19 during clinical activity . The word “personal protective equipment” (PPE) encompasses all the protective equipment designed in order to safeguard workers from risks against health and safety. This includes gloves, mop caps, face shields, goggles, gowns, shoe covers, and the appropriate filtering face pieces. Many countries have advocated all of the aforementioned items during the performance of AGPs. COVID-19 is associated with a very small viral particle size and ability to infect the lower respiratory tract. A clear difference exists between examination earloop masks and surgical masks that are produced following the technical standards UNI EN 14638. Surgical masks are divided into three types in accordance with their filtering capacity  and resistance to splashes. Surgical masks under the standard UNI EN 14638 are divided into: Type I, II, and IIR (where R stands for resistance against spraying and humidity). For the technical standard ASTM F2100-11, surgical masks are divided into Levels 1 to 3 and are similar to the aforementioned UNI EN 14638. Type IIR/Level 3 surgical masks filter an average particle size of 2.7 µm and are resistant against droplets and splashes. Hand hygiene is always advised before donning, doffing, or adjusting any mask or respirator.Filtering face pieces (FFP) or respirators, without an exhalation valve, provide two-way protection for the wearer and the patient. These FFP respirators provide a greater facial seal compared to surgical masks. FFP respirators are classified, according to European classification standard EN149:2001 based on their filtering efficiency of <0.3 μm droplets (FFP2 and FFP3). FFP2 can filter up to 94% of particles between 0.1 and 0.3 μm, extending this filtering capacity to 99% for particles of 0.75 μm and are made to the equivalent standard of the N95 respirator, which follows the NIOSH/FDA American standards. FFP3, meanwhile, filter up to 99% of particles with <0.3 μm of diameter and are similar to the American N99. The N99/FFP3 respirators have one-way valves.However, there are conflicting approaches towards the efficacy and implementation of various levels of surgical masks versus N95/FFP2- and even N99/FFP3 respirators among different countries. The Italian “Istituto Superiore di Sanità” (ISS) produced a document  in whichPublic Health England’s guidelines  require an N99/FFP3 respirator in case of AGPs and in some procedures which carry a high risk of infection, such as patients’ intubation and extubation . For dirHealthcare workers experienced masks and respirators differently based on their design features. The design features additionally allow their experience of discomfort and exertion to differ significantly during an 8 h work cycle . The imp2 levels and a decrease in O2 levels due to the important breathing resistance offered by N95 respirators. Hypercapnia was also assessed, in another study, based on a normal working period of one hour [2 and an increase in CO2 levels with the two respirators assessed. Furthermore, several studies assessed the perceived effects of the respirators on the healthcare workers, but no studies have been completed with dental professionals.Moreover, hypercapnia-related problems after many hours of wearing these respirators have been a concern to healthcare workers (HCW) and have been studied under in vitro conditions  and in vone hour . During The objectives of this paper were to evaluate the relevant findings in the literature regarding the rationale for use of an N95/FFP2 respirator related to AGPs particle sizes  that couOur review followed PRISMA guidelines for a scoping review. The studies included in this review matched the predefined criteria according to the PICOS  process as reported in Types of studies and participants: cross sectional and case control studies focusing on healthcare workers.Type of intervention: use of respirators and/or masks in daily practice and their clinical side effects such as discomfort, exertion, breathing difficulties, and headaches.Language: Papers in English, Italian, and Spanish were included.The exclusion criteria were reviews , opinion letters, case reports, case series, congress abstracts, papers not written in English, Italian, and Spanish, and studies not reporting the positive or negative effects on the use of respirators and/or face masks.Three electronic databases were used in the search for studies satisfying the inclusion criteria for studies published until the 31 May 2020: The National Library of Medicine (MEDLINE via Pubmed), Cochrane Central Register of Controlled Trials, and Embase. The search was limited to human subjects. The search strategy employed was the following: ((COVID-19) AND ).Cross-references were also considered together with the personal collections of the authors.All the resulting articles were assessed for eligibility by EDR-MF reviewers independently, who screened the titles and abstracts for possible inclusion in the review, according to the inclusion criteria listed above. The agreement between the reviewers was also calculated (percentage agreement and kappa scores). Reviewers were first trained and calibrated for study screening against another reviewer (CM) with experience in conducting systematic reviews. Abstracts were excluded if they did not fulfil the inclusion criteria. To avoid the exclusion of potentially relevant articles, abstracts not providing the required information were included in the full-text analysis.Full texts of potentially relevant studies were independently assessed by the same reviewers. Any disagreement was resolved by discussion between reviewers. Inter-observer agreement was assessed by means of kappa scores. Papers mentioning SARS-CoV-2 or COVID-19 transmission mode and PPE worn to protect from a possible contagion were included in this review, particularly the ones citing surgical masks and/or N95/FFP2 respirators. Duplicates were removed, articles were sorted by title, abstract, and then, full text. Once a paper was found eligible, its references were screened in order to find new papers for the revision.Moreover, we submitted an anonymous online structured survey to 430 dental professionals belonging to an online community of dentists in order to assess their perceptions while wearing N95/FFP2 respirators. The assurance that the participants were members of the Italian Register of Dentists was given by the fact that it is a closed community and access is granted only if it is provided a registration to the professional order.The 256 questionnaires obtained allow to estimate 47% headaches with an error at 6% and a 95% confidence level (CI95%).The survey was structured in 3 main domains and we asked a total of 16 questions. The first part of the survey regarded demographics , the second focused on their medical respiratory and smoking history and the number of hours spent wearing an N95/FFP2 respirator. The third part was about several possible perceived symptoms related to hypercapnia such as muscular pain, breathing difficulties, headaches, sleepiness, abnormally frequent urination, drop in attention levels, and consequently, drop in work ability. Questions about age, hypertension, headaches, muscular pain, and sleepiness were dichotomic (Yes/No questions), whereas questions about drop in attention levels, drop in work ability, breathing difficulties, and exertion were assigned a score ranging from 0 to 10 , categorized in three groups: 1–3 mild symptoms, 4–7 moderate symptoms, and 8–10 severe symptoms. All the questions referred to the experience of healthcare workers in the recent months of the pandemic situation wearing FFP2 respirators.Inclusion criteria: all questionnaires returned, being at least 23 years old, and registered in the Italian National Register of Dentists.Basic information of the trial: trial ID, title, authors, journal information;Eligibility reassessment: all the items in inclusion criteria, final decision;Study design: methods of randomization, allocation concealment, blinding, centers, country, funding;Participants’ information: inclusion and exclusion criteria of the trial, demographic characteristics , number of participants in each group, baseline status;Intervention and comparison: details of the COVID-19 respirator and/or face masks use intervention and control groups, follow-up period, number of participants lost to follow-up and the reasons;Outcome: the name of outcome variables, assessment method, observation time and detailed results;Correspondence: contact address of the original authors.Data were extracted independently by two reviewers using specially designed data extraction forms. Contents of the data extraction included the following:Authors of the primary studies were consulted to obtain any further information not available in the paper. When the study results were published more than once or results were presented in multiple publications, the most complete dataset was identified, and data were included only once.All data collected were analyzed using the software IBM SPSS Statistics 25 . Numbers and percentages of dichotomic variables were reported. Exact Binomial CI95% was reported for headache. Spearman’s rank correlation coefficient test (Spearman’s ρ) was calculated in order to assess whether there were correlations between variables. Chi square test was used to evaluate the relationship between sex and reported variables such as headache and concentration problems. All tests are to be considered with a 0.05 statistical significance level. Following the search criteria in this scoping review, five papers were included, from three nations worldwide. Though the database research identified 72 documents, after having read the abstract and removal of the duplicates, only 35 papers were fully read. As shown by the flow diagram , 6 of thTwo of these five studies ,28 focusShenal et al.  investigRebmann et al.  studied The last article we analyzed, by Chughtai et al. , regardep = 0.007). Headaches were negatively correlated with age  but not correlated with the number of hours spent wearing N95/FFP2 respirators. Moderate difficulties in breathing were reported by 162 participants (63.5% of the sample), with 20.8% of the participants experiencing severe breathing difficulties. Breathing difficulties were not correlated with the hours spent wearing N95/FFP2 respirators.Moderate concentration problems are also reported by the survey with at least 139 participants (54.3%). Concentration problems became more severe for 66 dentists (25.8%) and medical histories indicated the presence of comorbidities  that has an effect on concentration problems among the participating dental professionals. p = 0.012) but not with the number of hours spent wearing respirators.Discomfort and exertion, which are common reports from the study, were a severe consequence of prolonged N95/FFP2 use for 50.8% of the sample (130 participants) and were related to gender, particularly with females , breathing difficulties , concentration problems , and exertion .The presence of concentration problems, exertion, breathing difficulties, and headaches resulted in a moderate impaired working ability for 85.5% of our sample. In fact, an impaired working ability was strongly correlated to headaches  from respirator use. Among these 79 HWC, 62.7% previously presented with no headaches and experienced it when they wore an N95/FFP2 respirator . MoreoveData from our survey, indicating headaches as one of the main outcomes related to FFP2 wear, but not correlated to the hours spent wearing a respirator, is in contrast with previous reports from the literature ,28. MoreAnother significant side effect and relevant outcome in our survey was breathing difficulties, at least moderate for 63.5% of our sample, but not correlated with the hours spent wearing N95/FFP2 respirators. Chughtai et al.  detected2 and CO2 levels, resulting in a statistical increase in CO2 levels compared with baseline measurements, even though this increase was not clinically relevant, and there were no changes in O2 levels. Unfortunately, the results were not assessed in correlation to the headaches, visual challenges, and lightheadedness investigated by the authors. Such substantial differences among our studies are attributable to differences in the respirators worn by participants. Our sample is based on dentists wearing N95/FFP2 respirators without a valve, while the study by Chughtai et al.  was baseDental professionals usually take off masks between patients. We conjecture that the lack of correlation between headaches, breathing difficulties, and other symptoms with the numbers of hours spent wearing a respirator is the result of doffing periods between one patient and the subsequent one.This also applies to Shenal et al. , who anaAn impaired working ability (IWA), not assessed in the examined literature, was one of the strongest outcomes of our sample. It is interesting to note that IWA showed a strong positive correlation with breathing difficulties, concentration problems, and exertion. This could be explained with the perceived hypercapnia phenomena. Despite experiencing IWA and the other side effects, 49.2% of our group confirmed that FFP2 respirators are essential for the wellbeing of patients and healthcare workers, in agreement with Italian and international guidelines ,19. This survey is the first in Italy to assess a problem that, given the actual pandemic situation and consequent rise in the use of respirators, will be a common finding in future months. The results of our survey coincide with the current available literature. However, our study has some limitations, in fact, given the current pandemic situation, we were unable to test a control group of dentists wearing only facial masks. Moreover, it was a self-administered survey, not with an independent operator.N95/FFP2 respirators without valves are currently recommended in order to prevent COVID-19 spread and preserve HCW wellbeing. N95/FFP2is acknowledged by the majority of dental professionals, although the majority experienced several perceived side effects. The practitioners wore the N95/FFP2 respirator for prolonged periods of time and the perceived side effects, such as breathing difficulties, headaches, and concentration problems, lead to an impaired working ability.2 and CO2 to the physical symptoms of healthcare workers. Thus, further studies are required with pulse oximetry to determine the level of hypercapnia in relation to headaches and an impaired working ability. Additionally, the pulse oximetry could assist in the assessment of valve respirators with a surgical mask in an attempt to decrease hypercapnia, since the literature is not conclusive.There are no well-structured studies that correlate the O


Background: Medulloblastoma (MB) is one of the most malignant neuroepithelial tumors in the central nervous system. This study aimed to establish an effective prognostic nomogram and risk grouping system for predicting overall survival (OS) of patients with MB.Materials and Methods: The nomogram was constructed based on data from the database of Surveillance, Epidemiology, and End Results (SEER). This database consisted of 2,824 patients with medulloblastoma and was used as the training cohort. The data of another additional 161 patients treated at the Sun Yat-sen University Cancer Center (SYSUCC) were used as the external validation cohort. Cox regression analysis was used to select independent prognostic factors. Concordance index (C-index) and calibration curve were used to predict the prognostic effect of the nomogram for overall survival.Results: In the training cohort, Cox regression analyses showed that the prognostic factors included histopathology, surgery, radiotherapy, chemotherapy, tumor size, dissemination, and age at diagnosis. The internal and external validated C-indexes were 0.681 and 0.644, respectively. Calibration curves showed that the nomogram was able to predict 1-, 3-, and 5-year OS for patients with MB precisely. Using the training cohort, a risk grouping system was built, which could perfectly classify patients into four risk nomogroups with a 5-year survival rate of 83.9%, 76.5%, 64.5%, and 46.8%, respectively.Conclusion: We built and validated a nomogram and risk grouping system that can provide individual prediction of OS and distinguish MB patients from different risk groups. This nomogram and risk grouping system could help clinicians making better treatment plan and prognostic assessment. Medulloblastoma (MB) is one of the most malignant brain tumors in children . CurrentA nomogram was a graphical mathematical algorithm of a statistical predictive model  to generIn this study, we aimed to establish a nomogram for predicting the prognosis of MB from two different patient populations and to externally validate it.The data of MB patients from the database of the Surveillance, Epidemiology, and End Results (SEER) were extracted using the following specifications: “Incidence—SEER 18 Registries Custom Data , November 2016 Submission (1973–2014 varying).” Inclusion criteria for patient selection included: 1) pathologically proven diagnosis of medulloblastoma; 2) according to the third edition of International Classification of Diseases for Oncology (ICD-O-3) to identify cases of MB, and the following ICD-O-3 codes for histopathology were chosen: 9,470/3 medulloblastoma, NOS; 9,471/3 desmoplastic nodular medulloblastoma; 9,474/3 large cell medulloblastoma; and 3) known survival status and survival time. These data were used as a training cohort.In addition, to validate the nomogram, we searched the Sun Yat-sen University Cancer Center (SYSUCC) database for patients diagnosed with medulloblastoma between 1971 and 2018. The inclusion criteria for case selection were: 1) pathologically confirmed diagnosis of medulloblastoma; 2) no prior anticancer therapy; and 3) complete clinicopathologic characteristics, therapeutic records, and complete follow-up data.https://x-tile.software.informer.com, Yale School of Medicine, New Haven, CT, United States) software for obtaining the best cutoff value. Moreover, dissemination of tumor cells was defined as findings of distant metastasis based on radiographical images or tumor cells in cerebral spinal fluid (CSF).Considering some incomplete data variables from the SEER, we redefine the variables for developing the nomogram. In our study, surgery was categorized as: total resection (TR), subtotal resection (SR), surgery, NOS, and no evidence. Histopathology was categorized as: medulloblastoma, NOS, desmoplastic nodular medulloblastoma, and large cell medulloblastoma. For objectively assessing the prognostic value of age and tumor size for MB patients, the patients were stratified into two categories, namely ≤3 years, >3 years; or ≤6.5 cm, >6.5 cm, and/or unknown, using the X-tile  rates. Based on the contribution of each factor for the outcome in the model, the nomogram can express the relationship between various variables in the model according to drawing a line segment with certain proportion on the same plane, which transformed the complex regression equation into graphic visualization, and made the results of the prediction model more reifiable and convenient for clinician and patients.The performance of the nomogram including its discrimination and calibration was validated using the SYSUCC cohort as external validation. Discrimination was assessed using a concordance index (C-index), and the larger the C-index, the more accurate the nomogram. Calibration was performed by comparing predicted survival rate actual survival rate determined using Kaplan–Meier analysis for 1-, 3-, and 5-year overall survival. In addition, we did bootstrap 1,000 resamples to verify the accuracy of the nomogram in the training cohort and external validation cohort from SYSUCC database.In this study, we defined risk as the harmful factors for prognosis of MB patients. To develop the risk grouping to assess the prognosis of MB patients, we divided patients from the training cohort into four prognostic groups according to the total points of each patient. Furthermore, we used Kaplan–Meier curves to compare the prognosis of patients in the different nomogroups.p-values were two-sided, and p-values <0.05 were considered as statistically significant. The nomogram was constructed by R 3.5.1 (http://www.r-project.org) and all diagrams of this study were drawn by GraphPad Prism 8 (https://www.graphpad.com/scientific-software/prism/).All statistical analyses were performed by the statistical software package of SPSS for Windows, version 22.0 , A total of 2,824 patients were identified from the SEER database as suitable for inclusion in the study and they were grouped as the training cohort . An addiAccording to the cox regression analyses, we identified histopathology, surgery, radiotherapy, chemotherapy, tumor size, dissemination, and age of patients as independent factors correlated with the 1-, 3-, and 5-year OS rates in the training cohort . No signAll patients were classified into four risk nomogroups according to the total score of each factor obtained from the nomogram of the training cohort. The nomogroups of the risk grouping were as follows: nomogroup I, score of 0–3.8 points; nomogroup II, 3.9–6.8 points; nomogroup III, 6.9–11.2 points; and nomogroup IV, ≥11.3 points. As shown in p = 0.018) . As of the last follow-up, the 5-year OS of patients of G3 and G4 subgroups were lower than those of Wnt and SHH subgroups  .As the most common tumor in pediatric central nervous system, MB accounts for 20–25% of all pediatric primary central nervous system tumors, which is classified as WHO grade IV tumor . AlthougChang staging for MB was introduced in 1960s, which classified MB patients into M0, M1, M2, M3, or M4 and T1, T2, T3a, T3b, or T4 according to their clinical features . AlthougIn clinical practice, the most commonly used prognosis system is the four molecular subgroup and the high-risk/average-risk system. However, the inconvenience of molecular detection approaches in daily clinical practice limited the application and prognostic evaluation in the majority of centers. However, the poor-risk/average-risk system is inefficient for stratifying patients into accurate prognosis. Biologic and clinical heterogeneity should be considered to stratify different treatment regimen. However, several clinical trials including SJYC07 presented negative risk-adapted therapy, which only confirmed that “reduced-intensity” therapy benefits a subtype SHH in early childhood medulloblastoma that would improve the progression-free survival. Because the aggressiveness, biological characteristics of individual tumors, and the efficacy of the treatment varied from patient to patient, establishing a better stratified and individualized prognostic model would provide a much more significant experience to both clinicians and patients.However, to the best of our knowledge, there was no nomogram available for the evaluation of MB. The objective of our study is to establish a novel, convenient comprehensive nomogram model to evaluate OS for MB patients. We develop the individual prognostic nomogram from the SEER data set and validate an independent data set from SYSUCC database. Our study shows the value of clinical characteristics predictor of individualized MB prognosis with the nomogram model. The nomograms showed precise and stable prediction when analyzing both the training and the validation cohorts, indicating its good clinical applicability. The traditional prognostic model included the clinical parameters to divide patients into two levels as the poor-risk and average-risk system, which is insufficient for precise prediction. Compared to the previous model, the MB cohort was satisfactorily separated into four distinct prognostic groups with the cutoff point of 3.9, 6.9, and 11.3 in our nomogram model.According to our nomogram, if an MB patient would have histopathology of LC, received radiotherapy, chemotherapy, and total resection, tumor size greater than 6.5 cm, age older than 3 years, and without dissemination, his total score would be 13.9. Therefore, as shown in 2 belong to the high-risk group according to North American stratification. Last but not least, similar high-risk stratification can be applied to younger MB patients younger than 3 years at diagnosis, who were not recommend to receive radiation instead of prolonged and intensive chemotherapy regimen with the aim of reducing the risk of relapse. As a result, prospective study or evidence from multiple databases is needed to confirm the staging system of MB.Seven clinical and pathology factors in our model, including histopathology, surgery, radiotherapy, chemotherapy, tumor size, dissemination, and age, were independently correlated with the OS rates. First, different pathology subtypes may represent distinct biological behaviors of MB, which may affect further treatment strategies. Large-cell and anaplastic MB are regarded as high-risk disease, which demonstrates that patients have a poorer survival and should be received more intensive chemotherapy and higher dose of radiation. However, desmoplastic nodular MB may benefit from a better outcome, because desmoplastic nodular MB shared the best survival in the histopathology in our study, which was consistent with previous study . We stilThere are several limitations in our study. First, this study was a retrospective analysis although we used reliable databases from the SEER system and that of SYSUCC to investigate the applicability of this nomogram in the Caucasian and Asian populations. Second, some details in the SEER database, such as the molecular subgroups, radiotherapy doses, and chemotherapy regimens, were not provided, and other promising prognostic parameters could not be analyzed. Third, information about several important clinicopathological parameters from SEER such as tumor size, tumor location, and histopathology were not complete. To guard against bias, we have to retain these data, which is defined “unknown or NOS.” Fourth, patient cases from the training and validation cohort spanned for nearly half a century, which may have caused certain statistical biases for not accounting advances in techniques of treatment and diagnosis.A nomogram, consisting of seven clinicopathological and treatment-related factors, for predicting the 1-, 3-, and 5-year OS of MB patients was constructed from a Caucasian cohort and was successfully validated in an Eastern database. This proposed survival prognostic model could be useful for differentiating patients of different risk groups to better provide individualized treatment decision and surveillance.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.Our study was approved by the Ethics Committee of Sun-Yat-Sen University Cancer Center Health Authority (identifier: B2020-199-01). Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.All authors participated in this research, including conception and design , data acquisition , data analysis and interpretation , material support , study supervision , as well as drafting the article or critically revising . The final version is ensured and approved by all authors.This research was supported by the Science and Technology Planning Project of Guangdong Province (2017A020215030 and 2014A020212098).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.


Although integrated traditional Chinese medicine (TCM) has long been indicated to be effective in the treatment of sciatica and is widely used in the management of this condition, the mechanism by which integrated TCM alleviates sciatica has not yet been fully defined, and the effect of integrated TCM on gene expression in the peripheral blood of patients with sciatica is still unknown. We performed this study to investigate the effect of integrated TCM on peripheral blood gene expression in patients with sciatica and to explore new clues for studying the mechanism of integrated TCM in alleviating sciatica.We used a microarray to identify differentially expressed genes (DEGs) in the peripheral blood of patients with sciatica and healthy controls (DEGs-baseline), bioinformatic analysis to reveal the characteristics of DEGs-baseline, and the key genes that contribute to the gene dysregulation. A microarray was also used to identify DEGs in the peripheral blood of patients with sciatica after integrated TCM treatment compared with those at baseline, and the expression levels of DEGs were validated by qRT-PCR.We identified 153 DEGs-baseline, which included 131 upregulated genes and 22 downregulated genes. Bioinformatic analysis revealed that most of the DEGs-baseline were related to immunity and the inflammatory response and that TLR4, MMP9, MPO, CAMP, RETN, TLR5, and IL1RN were key genes involved in the dysregulation of genes in the peripheral blood of patients with sciatica. The expression levels of TLR5, IL1RN, SLC8A1, RBM20, GPER1, IL27, SOCS1, and GRTP1-AS1 were decreased in the peripheral blood of patients after integrated TCM treatment compared with that at baseline, which was accompanied by relief of pain.Integrated TCM treatment relieved pain while regulating the gene expression of TLR5, IL1RN, SLC8A1, RBM20, GPER1, IL27, SOCS1, and GRTP1-AS1 in the peripheral blood of patients with sciatica. Our study provides new clues for studying the mechanism of TCM in treating sciatica.The online version contains supplementary material available at 10.1186/s13018-021-02280-1. Sciatica is characterized by neuropathic pain in the sciatic nerve, and the lifetime incidence is up to 40% . Lumbar Many methods have been reported to improve sciatica, and 90% of acute sciatica can be effectively relieved by nonsurgical treatment . IntegraThis study was approved by the Ethics Committee of Sichuan Provincial Orthopedics Hospital, and written informed consent was obtained. From April 2018 to December 2019, 25 patients and 25 healthy volunteers were enrolled in this observational study. Patients were enrolled from those who chose to receive nonsurgical treatment with integrated TCM in our hospital. Diagnostic criteria of sciatica was set according to the guidelines of the North American Spine Society : (1) a hPeripheral blood was collected and frozen in PAXgene tubes, and then, total RNA was extracted and purified using the PAXgeneTM Blood RNA Kit . An Agilent SurePrint G3 Microarray (8 × 60 K) was used for the microarray experiment, which was performed by Shanghai Biotech Co. . These procedures were described in our previous study .Two groups of DEGs were identified: DEGs in the peripheral blood of patients with sciatica and healthy controls (DEGs-baseline) and DEGs in the peripheral blood of patients with sciatica after integrated TCM treatment compared with those at baseline (DEGs-TCM).t tests (P < 0.05). Gene expression data sets are accessible on the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo) under the number GSE150408 and GSE124272.Data from the chip scan were log2 normalized for comparative analyses. Unrecognized probes were discarded. We deleted unrecognized probes. The average value of the data from the probes corresponding to the same gene was used for analysis. Genes with an absolute fold change (FC) of ≥ 1.5 were identified as DEGs, and the FC data were filtered by Enrichment analysis was performed with the web-based portal Metascape , which iTo identify key genes in the dysregulation of gene expression in the peripheral blood of patients at baseline and in healthy controls, we used the STRING database to construct the PPI network of DEGs-baseline with a combined score > 0.4. Cytoscape software (V3.6.1) was used to visualize the PPI network. Disconnected nodes were excluded, the degree of centrality of the DEGs-baseline was calculated, and a submodule was clustered, as we previously described .-ΔΔCq method. β-Actin was used as the normalization control for mRNA. The design and synthesis of the primers and the qRT-PCR procedure were described previously [t test with a P value < 0.05. Statistical analysis was performed and visualized as we previously described [qRT-PCR was performed to measure the expression levels of DEGs using the 2eviously . The priescribed .P < 0.000), from an average of 7.4 ± 0.81 at baseline to 2.1 ± 0.97 14 days after integrated TCM treatment, indicating that all patients achieved clinical remission.We enrolled 25 patients with sciatica aged between 19 and 54 years (mean age 40 years) who were seen in our department for integrated TCM treatment and 25 healthy volunteers aged between 19 and 30 years (mean age 23 years) as healthy controls. All patients underwent standard integrated TCM treatment for sciatica for at least 14 days. The VAS score for pain in patients decreased  in the peripheral blood of patients at baseline and in the peripheral blood of healthy controls, as well as expression levels of DEGs-TCM in the peripheral blood of patients at baseline and in the peripheral blood of patients after integrated TCM treatment.qRT-PCR showed that the expression levels of TLR4, MMP9, MPO, CAMP, RETN, TLR5, and IL1RN were increased in the peripheral blood of patients at baseline compared with the healthy controls Fig. ; the expWith microarray analysis, we identified 153 DEGs in the peripheral blood of sciatica patients and healthy controls. Bioinformatic analysis revealed that these DEGs were mainly related to immunity and the inflammatory response and that TLR4, MMP9, MPO, CAMP, RETN, TLR5, and IL1R were the most important genes contributing to the dysregulation of these DEGs.In the PPI network of DEGs-baseline, TLR4 had the highest centrality degree, which was consistent with our previous study . TLR4 isAfter integrated TCM treatment, the pain of the patients was relieved, and the expression levels of TLR5, IL1RN, SLC8A1, RBM20, GPER1, IL27, SOCS1, and GRTP1-AS1 in peripheral blood were downregulated compared with baseline. We also found that TLR5, IL1RN, and SLC8A1 were upregulated in sciatica patients at baseline compared with healthy controls and downregulated after integrated TCM treatment compared with baseline, indicating that these genes played an important role in the pathogenesis and remission of sciatica. In the future, the expression of these genes can be considered as an objective indicator for evaluating the efficacy of integrated TCM in treating sciatica in the clinical setting.Animal experiments have shown that the knockout or blockade of TLR5 can reduce pain , 36, whiOur study had limitations. Some other factors, such as miRNAs and lncRNAs, are involved in the process of protein translation from mRNA, and protein expression is not only determined by mRNA. We did not perform western blotting to measure the protein levels of these genes, and the protein levels and mRNA levels of these genes may not be consistent. Changes in gene expression and alleviation of sciatica were simultaneously observed after integrated TCM treatment in the present study; whether integrated TCM treatment relieved sciatica by regulating the expression of these genes still needs further investigation.In conclusion, we found that integrated TCM treatment alleviated sciatica while regulating the gene expression of TLR5, IL1RN, SLC8A1, RBM20, GPER1, IL27, SOCS1, and GRTP1-AS1 in the peripheral blood of patients. Our study provides new clues for studying the mechanism of TCM in treating sciatica.Additional file 1. Sequences of primers used for qRT-PCR


Microscopic examination is essential in urine analysis. This is a simple way to collect informative data but it is also labor-intensive, time-consuming, and requires experienced staff for accurate results and interpretation. Several automated urine analyzers have been introduced for urine analysis in medical laboratories. The aim of this study was to assess and compare the performance of the most common three automated urine analyzers, Cobas 6500, UN3000-111b and iRICELL 3000.and Methods: A total of 100 routine urine samples were used in the study. Results from the three machines were compared with the routine procedure results including physical, chemical and sediment analysis.There was good correlation of urine physical and chemical analyses between the three analyzers with an overall concordance level of more than 80%. For sediment analysis, the degree of concordance between manual analysis and the three instruments was very good to good for white blood cells, red blood cells and epithelial cells, and moderate for bacteria. There were fair to good agreements between manual microscopy and the three instruments, Cobas 6500, UN3000-111b and iRICELL 3000, for casts .The three automated urine analyzers showed similar performances and good correlation with manual microscopy. The results of this study indicate that automated urine analyzers could be used for initial urine testing to reduce high workloads and to save time, but manual microscopic analysis by experienced staff is still necessary to classify urine sediments for confirmation, especially in pathologic specimens. Urinalysis includes visual examination, evaluation of chemical parameters and microscopic examination. The traditional procedure for urinalysis recommended by the Clinical and Laboratory Standards Institute (CLSI) and the European Urinalysis Guidelines consists of two steps ,2. The fIn recent years, a new generation of more sophisticated automated urinalysis machines has been introduced. Each platform has different advantages and disadvantages . Automat22.1One hundred freshly-collected urine specimens from admitted in- and out-patients which were submitted to the Microscopy Laboratory of the Department of Pathology, Faculty of Medicine, Prince of Songkla University, Thailand for routine diagnostic urinalysis were randomly selected and included in the study. The samples were collected in preservative-free containers and transferred to four different test tubes for three automated urine analyzers without centrifuging and manual microscopy by 3 experienced medical technologists, independently using the same microscope slide. All samples were analyzed within 2 h of their arrival at the laboratory.This study was approved by the Institutional Ethics Committee of the Faculty of Medicine, Prince of Songkla University (REC.62-470-5-7).2.22.2.1The Cobas 6500 is a combination of 2 analyzers including the Cobas u601 which analyzes physical and chemical components, and the Cobas u701 which performs microscopic examinations. The Cobas u601 can evaluate leukocytes, erythrocytes, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, pH, colour and turbidity with reflectance photometry and specific gravity with refractometry. For the Cobas u701, a urine specimen is pipetted into a special cuvette and the machine centrifuges the cuvette at 2000 rpm for 10 s. A microscopic camera takes 15 real images of each centrifuged specimen that are then analyzed by particle recognition software for erythrocytes, leukocytes, squamous epithelial cells, non-squamous epithelial cells, bacteria, hyaline casts, pathological casts, crystals, yeasts, mucus and sperm.2.2.2The UN3000-111b has 2 component analyzers in a single platform including a chemical component  for analyzing the physical and chemical parts of urine and a fluorescence flow cytometry component  for microscopic examination of sediments. The UC-3500 analyzer can evaluate leukocytes, erythrocytes, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, pH, colour and turbidity using reflectance photometry and specific gravity using refractometry. Particle identification and characterization are achieved by detection of forward scatter and fluorescence, and adaptive cluster analysis is performed with the UF-5000 analyzer. Additionally, the UN3000-111b includes the UD-10 which is a urine particle digital imaging device, capturing images of urine and classifying the particles into 8 classes on the basis of their size, and giving a detailed view of urine particles to the user. The UD-10 device confirms abnormal results after particle analysis.2.2.3The iRICELL 3000 is a combination of 2 component analyzers including a chemical component (iChemVELOCITY) and a microscopic examination component (iQ200). The Iris iQ200 uses Digital Flow Morphology technology with Auto-Particle Recognition (APR) software to classify and quantify the cells and particles in uncentrifuged urine. In the system, the particles in a urine specimen are imaged in a planar plane in flow cells by hydrodynamic focusing sheath fluid, then the hundreds of captured images from its digital camera are identified and counted by APR software, and each particle is auto-classified by size, shape, contrast and texture. The results are classified into 12 particle categories, red blood cells (RBCs), white blood cells (WBCs), WBC clumps, hyaline casts, pathological casts (or unclassified casts), squamous epithelial cells, non-squamous epithelial cells, bacteria, yeasts, crystals, mucus and sperm.The characteristics of the Cobas 6500, UN 3000-111b and iRICELL 3000 are provided in 2.3g) for 5 min. After centrifugation, the sediment contents were resuspended and then slides were prepared and examined by light microscope at magniﬁcations of 100x  for casts and 400x  for RBCs, WBCs, epithelial cells, and bacteria. The particles were counted per field, and the results were classified into 3 categories for evaluation . WBCs, RBCs and epithelial cells were classified semi-quantitatively . Bacteria were classified as negative, few, moderate, or numerous. Casts were classified as not found and found. Manual microscopy was used as the reference standard method for urine sediment evaluation in all reports.Manual microscopic sediment examinations were performed following the CLSI approved guidelines for urinalysis . 10 mL All samples were independently examined by 3 experienced medical technologists using the same microscope slide. The results were accepted when two or three evaluators reported the same category of cells or particles. If all 3 assessors reached notably different results for any particular slide, the analysis was repeated with a new urine sample in order to resolve the discrepancy.2.42.4.1In order to evaluate the analytical performances of the workstations, between- and within-run variations and carry-over measurements of the workstations were evaluated against control materials. Liquichek, urinalysis control level 1 and 2  was used to provide analytical quality control, including for WBCs and RBCs. We used 20 repetitions for both within-run (20 times within a day) and between-run (once per day on 20 separate days) precision. The precision of each automated urine analyzer was assessed by mean ± SD and percentage coefficient of variation (CV%). CV values less than 30% are considered to be acceptable.2.4.2r). For the chemical components, the analytical detection of pH, glucose, protein, bilirubin, urobilinogen, blood, ketones, nitrite, and leukocyte esterase was compared between the analyzers. The analytical assessment of glucose and protein between the three machines was compared with the results of a Cobas c501 analyzer, a fully automated analyzer, which has been wildly used for clinical chemistry testing and has been used in our laboratory for a long time. The Cobas c501 performs urinalysis based on standard methods, notably colorimetry for protein analysis and an enzymatic method for glucose analysis. The results of the analysis of the chemical components of the three machines were considered to be concordant if they were within one grading difference result from the Cobas c501. The pairwise concordance rates between the three instruments were defined as the percentage of results within ±1 grade from the best-fit line.We compared the specific gravity results of the three automated urine analyzers with refractometry and compared the results statistically using regression analysis and coefficient correlation  and negative predictive value (NPV) were calculated for these data. Concordance rates of urine sediment with the same grade and within ±1 grade difference between the machines were calculated.33.1The within-run and between-run coefficients of variations of the RBCs and WBCs for the three automated urine analyzers, Cobas 6500, UN3000-111b, and iRICELL 3000, are shown in 3.2r) and linear regression analysis of specific gravity for comparison of the machine results with the refractometry method results as the gold standard method are presented in Pearson’s coefficient correlation . Although, within-run and between-run precision of the three automated urine analyzers for RBCs and WBCs were acceptable at all levels, only the Cobas 6500 showed a high degree of precision of both RBCs and WBCs with CV < 10%. The precision of the data which was produced by the three analyzers in this study was similar to previous studies . The conSeveral microscopy comparison studies have shown that automated analyzers mostly have similar performances and with results comparable with manual microscopy. One of these studies, conducted by Baken et al. , compareThe limitations of the present study were the small sample size and the low number of pathological samples, thus further studies are needed with larger sample sizes to validate our findings. Despite these limitations, this study has the benefit that the measurements were obtained from real patient samples and therefore the results can be applied directly to the clinical situation.5The Cobas 6500, UN3000-111b and iRICELL 3000 analyzers showed similar performances in physical, chemical and sediment examinations in most of the parameters analyzed. Therefore, we can conclude that the these three fully automated urine analyzers can be safely used in urine analysis. The automated urine analyzers can reduce the workload involved in analysis of a large number of urine samples, save time, and decrease human error in preparations for microscopic analysis, such as centrifugation. However, manual urinalysis is still recommended for confirmation of findings, particularly in urine samples flagged as some type of pathology.10.13039/100016545Roche Diagnostics , Sysmex Corporation , and Iris Diagnostics .This work was supported by three companies – Piraya Tantisaranon: Conceptualization, Methodology, Validation, Data curation, Formal analysis, Writing - original draft. Kanyarat Dumkengkhachornwonga: Validation, Investigation. Peechana Aiadsakunb: Validation, Investigation. Areerat Hnoonual: Conceptualization, Writing - review & editing, Supervision.None.


Menthol is often used as a cold-mimicking substance to allegedly enhance performance during physical activity, however menthol-induced activation of cold-defence responses during exercise can intensify heat accumulation in the body. This meta-analysis aimed at studying the effects of menthol on thermal perception and thermophysiological homeostasis during exercise. PubMed, EMBASE, Cochrane Library, and Google Scholar databases were searched until May 2020. Menthol caused cooler thermal sensation by weighted mean difference (WMD) of − 1.65  and tended to improve thermal comfort  during physical exercise. However, there was no meaningful difference in sweat production , deep body temperature , and heart rate  between the treatment groups. Menthol improved the performance time in certain subgroups, which are discussed. Our findings suggest that different factors, viz., external application, warmer environment, and higher body mass index can improve menthol’s effects on endurance performance, however menthol does not compromise warmth-defence responses during exercise, thus it can be safely applied by athletes from the thermoregulation point of view. The most common naturally occurring form of menthol is the l-isomer, which is used in various products, e.g., candies, beverages, cigarettes, and toothpastes, mainly because of its cooling, analgesic, and anti-inflammatory effects3. It has long been assumed that menthol might improve different aspects of physical performance such as endurance, speed, strength, and joint range of motion, consequently it is often used by athletes in the form of sprays, creams, tapes, beverages, etc.5.Menthol (2-isopropyl-5-methylcyclohexanol) is a lipophilic, organic compound which can be extracted from essential oils of aromatic plants or produced syntheticallya), it increases the thermal and circulatory strain6. Endurance exercise capacity at a high Ta is impaired by heat stress prior to exercise7, and hyperthermia induces fatigue during short intense activities and prolonged exercise in the heat8. On the contrary, physical cooling of the body before and during exercise in the warmth improves exercise endurance and reduces cardiovascular strain9. A recent meta-analysis of 45 studies also concluded that physical cooling improves aerobic and anaerobic exercise performance in hot conditions10. From animal experiments it is known that the transient receptor potential (TRP) melastatin-8 (M8) channel, formerly called as menthol receptor, is a universal cold sensor in the thermoregulation system. The pharmacological modulation of TRPM8 with systemic  administration of an antagonist changes the activity of the cold-activated neural pathway11, which raises the possibility that activation of TRPM8 with ligand agonists like menthol can have similar effects to physical cooling before or during physical exercise. Indeed, in exercising humans menthol administration resulted in increased thermogenesis12, decreased sweating14, and more pronounced skin vasoconstriction16, consequently in elevated deep body temperature (Tb)18; that is the same pattern of thermoregulatory effector recruitment which can be observed as part of the cold-defence responses19. Importantly, the menthol-induced decrease in heat loss and elevation in deep Tb can increase the risk for heat exhaustion and adverse cardiovascular events in the warmth17, therefore, the safety of menthol application in physical exercise, especially at high Ta, remains questionable. In contrast with the aforementioned studies showing an increased risk for the onset of heat-related illnesses in association with menthol application, several human studies showed beneficial effects of menthol on physiological, psychological, and performance parameters during physical exercise27, while a decent number of studies found no effect31.Warming-up before an exercise is often used to optimize muscle temperature and, thereby, maximal muscle power production, however, at high ambient temperatures (Ta). Menthol-containing products can be administered externally  or internally . External application has been shown to be more beneficial than the internal in sports physiology and on endurance performance25, whereas other authors found that internally applied menthol is more effective27, and yet others showed no effect of menthol independently from the application method31.The observed discrepancies among the studies may originate from differences in study designs, application methods , and experimental conditions  of the overall endurance performance during physical exercise in healthy humans.32  protocolsA search of the PubMed, EMBASE, and Cochrane Controlled Trials Registry databases was performed until May 2020 using the following search key: “(menthol OR mint OR peppermint OR mentha OR spearmint) AND ”. We restricted our search to randomized controlled human trials published in English without time period limitations. A manual search of the reference lists of identified full-text articles was also performed in Google Scholar for eligible studies. The search was conducted separately by two authors , who also assessed study eligibility and extracted data from the selected studies independently. Disagreements were resolved, if needed, by a third party (ZR).b, sweat production, heart rate, performance time, and power output in menthol-treated and control healthy subjects before and during physical exercise. For all parameters, the maximal change from baseline after menthol treatment  was extracted to assess the acute effect of menthol. In each study we calculated the difference between the menthol-treated and control groups, which was then included in the analyses. This approach allowed for taking into considerations differences in experimental protocols between studies. From all included articles, we extracted the group size, the reported mean values and standard deviations (SD) of the parameters of interest, and the level of statistical significance . To analyse the effects of menthol under different conditions, we also divided the studies into subgroups, which were determined on the basis of known influencing factors34, and data availability. The main influencing factors were grouped in three categories: characteristics of the subject (body mass index [BMI] and heat acclimation), study protocol , and environmental circumstances (airflow and Ta).After screening on the titles and abstracts of the identified publications, the full texts of eligible articles were obtained. We included studies which reported at least one of the following values: thermal sensation (TS), thermal comfort (TC), T35.In each study, we calculated the maximum change in the outcome parameter from baseline after menthol application and the change from baseline until the same time point in the control group. Then, we calculated the weighted mean difference (WMD) with 95% confidence interval (CI) in the change of the parameter between the menthol-treated and the control groups. The statistical analysis was performed according to the standard methods of meta-analysis by using a random effects model. The effects were considered significant when p < 0.05. Using both p value and CI allowed us to detect physiologically relevant differences between the groups even in the case of overlapping CIs36). In the analysed studies, TS scales were used with ranges of 7-point25, 9-point22, and 20-point30, while in case of TC, the authors used 4-point25, 7-point23, and 20-point ranges30. Since TS and TC were determined by different visual analogue scales in the studies, in order to make the reported TS and TC values comparable for our meta-analysis, while also minimizing the need for conversion of the originally reported data in the studies, if required, the reported values were extrapolated into a unified scale, ranging from 0 to 20. The scales were bilateral, i.e., 0 corresponded to neutral, in several studies. For example, TS was assessed with a 9-point scale ranging from very cold (− 4) to neutral (0) to very hot (4). Thus, after extrapolation of the endpoints of the original scales to 0 and 20, in case of TS the middle of the unified scale (10) represented the neutral situation and an increase in TS between 0 (very cold) and 20 (very hot) indicated a weaker cold or stronger warmth sensation. To assess TC, three types of scales were used in the analysed studies: 20-cm visual analogue scale 30, 7-point scale 23, and 4-point scale 25. In the studies using 7-point scale, we did not find any value smaller than 0, thus we considered the scale from 0 (comfortable) to 3 (uncomfortable) and these endpoints were extrapolated to 20 and 0, respectively. In the 4-point scale, the 1 (comfortable) and 4 (uncomfortable) endpoints were extrapolated to 20 and 0, respectively. As result, the unified 20-point scale, ranging from very uncomfortable to very comfortable, resembled the one used in the pioneer study by Gagge et al.37. In this scale, a higher TC value between 0 (very uncomfortable) and 20 (very comfortable) corresponded to more pleasant comfort feeling.To study perceptual responses, data on TS and TC were collected. By definition, TS identifies the relative intensity of the temperature being sensed, and, as such, provides the body with information about the thermal environment, while TC means subjective indifference with the thermal environment, so that thermal pleasure is perceived when a stimulus aims to restore TC  protocols, a longer performance time indicates improved endurance as the subjects are able to perform the exercise for a longer time period, whereas in time-trial (TT) protocols the subjects aim at finishing a predefined exercise task as fast as they can, thus longer performance time indicates reduced endurance in these tasks. Therefore, in our analyses, we always separated the TTE and TT protocols in different groups when the investigated outcome was performance time, similarly as in previous studies2 statistical test, where I2 is the proportion of total variation attributable to between-study variability . Publication bias was assessed by Egger’s test and visual inspection of funnel plots .41. All included studies had randomized, crossover design and included data from a total of 177 athletes. The participant characteristics in the studies are presented in Supplementary Table 31 and possibly another one which did not report the sex ratio in the sample26. In 12 trials, the participants were refrained from strenuous exercise, alcohol and caffeine intake before the experiments41, two studies included unspecified training limitations24, and three articles did not report any limitations31.The flowchart of study selection is presented in Fig. 26 to 71 min28, while in TTE protocols, it ranged between 1 min41 and 61 min20, with an average of 27 min. In two studies the subjects exercised for a fixed time duration of 45 min17 or 2 × 20 min18, while the exercise duration was calculated from distance and speed in one of the studies26. In all studies we considered the beginning of the exercise as the baseline, which was before the menthol treatment, except for two studies, in which menthol administration was before the start of the trial25. It should be noted that the TTE test was preceded by 45-min exercise without interruption in the study by Barwood et al.14, during which the subjects were repeatedly treated with menthol, thus we considered the beginning of the 45-min preliminary fatiguing task as the baseline for TS, TC, and deep Tb. The TTE test was also preceded by physical exertion and repeated menthol administration in the study by Saldaris et al.41, but in that study the 3 × 30 min trials were interrupted by breaks and resting between the trials, as well as, before the TTE test. Therefore, we considered the beginning of the TTE test as the baseline in that study.The mean exercise duration was 30 min in TT tests, ranging from 1 minThe used doses of menthol varied due to differences in administration method , concentration (0.01–8%), volume (25–500 ml), and surface of the treated body area (4–91%). The most commonly used concentrations and volumes were, respectively, 0.01% and 25 ml in internal, while 0.2% and 100 ml in external administration routes. The details of the used administration routes, doses, and experimental procedures are summarised in Supplementary Table 1, the TS score decreased in the menthol-treated groups as compared to controls in seven studies41, while two studies reported a slight increase in TS25. Accordingly, the overall WMD between the menthol-treated and control groups was − 1.65    Fig. . The TS-b . We found that the volume of sweat production did not differ significantly between the menthol-treated and control groups during exercise   , heart rate (a nonspecific indicator of metabolic rate), and deep T°C) Fig. . Further°C) Fig. .Figure 530, whole-body creaming13 or immersion18, and gel on the face25  with menthol has bigger impact on endurance performance than other areas in TTE protocols, we performed a sensitivity analysis , which showed no difference in the final pooled results (Supplementary Table as (above 31 °C) performance time was significantly increased in response to menthol compared with Tas ranging from ~ 20 to 30 °C , while there was no significant difference between the subgroups in TT protocols   of menthol’s effect were not investigated. In our study, we aimed at filling those gaps by studying the effects of menthol also on thermophysiological parameters, i.e., sweat production, heart rate, and deep Tb, and by identifying different phenotypes and environmental factors which can augment or attenuate menthol’s effects. We show that the use of menthol does not lead to compromised warmth-defence responses during physical exercise, since it does not affect sweat production, heart rate, and deep Tb. We also identify bodily , methodological , and environmental factors, such air movement (fan use) and higher Ta, which enhance the beneficial effects of menthol on performance time.In the present study, we show that the application of menthol improves TS, TC, and power output during physical exercise. Our results about thermal perception are in harmony with the findings of a previous meta-analysis45. The active muscle generates heat during physical exercise, thereby constituting an internal heat stress for the body, which is further augmented when physical activity is performed in the heat46. The heat load leads to worsening of TS and TC44, while behavioural and autonomic warmth-defence mechanisms are recruited to prevent an excessive increase in deep Tb46. When the defence mechanisms are compromised or exhausted, bodily homeostasis cannot be maintained, and heat-related illnesses, such as exertional heat stroke in the most severe forms, develop44. Efforts should be made to prevent the simultaneous presence of severe external and internal heat load to the individuals. There are, however, certain scenarios, when prevention of these conditions is not possible. The most obvious examples include the strenuous physical activity of firefighters, soldiers, and professional athletes in hot environments.Thermoregulatory changes, particularly in TS and TC, during physical exercise are of high importance, as they are considered among the limiting factors of endurance performance, and, as such, play a role in the development of fatigue47, France48, the US49, Australia50, and Japan50. These countries can be actual or potential hosts of upcoming worldwide, summertime sport events, e.g., Summer Olympic Games, thus pre-cautions should be implemented in order to prevent heat-related illnesses of the athletes during the games. In addition to physical methods of cooling, menthol may be also used as a pharmacological cooling intervention prior to and during exercise in hot conditions50. The improved TC in response to menthol can increase the thermal tolerance in athletes51, which can lead to better performance. It should be noted, however, that menthol may not be safely used to improve TC in athletes competing at cold environments . Our analysis, to our knowledge for the first time, also shows that the application of menthol did not result in compromised warmth defences.As part of the global climate change, the incidence of heat waves has increased in different countries, including, for example, the UK43. It is also known that menthol evokes its thermoregulatory effects through the TRPM8 channel, which, at least in rodents, serves as a universal cold receptor for the body19. The activation of TRPM8  leads to the recruitment of cold-defence responses, which aim at elevating (but at least preventing the drop in) deep Tb11. These thermophysiological effects of menthol, which were mostly discovered in animal experiments, imply a risk of menthol application in humans during physical exercise, since an adverse thermoregulatory effect, viz., an overt increase in deep Tb, can not be ruled out. However, in humans thermal signals from the skin are less important for autonomic thermoregulation because the greater thermal inertia makes transient thermal exposures less threatening, thus decreases the importance of signals from the skin42. Hence, activating peripheral cold receptors, such as TRPM8, with menthol in humans can have smaller effects on Tb than in rodents. It should be also noted that signals used for behavioural thermoregulation, which can be triggered through altered TS or TC, can differ from signals for autonomic thermoregulation 52. For example, antagonists of the TRP vanilloid-1 channel readily affect autonomic thermoeffectors in rats53, but fail to affect the behavioural thermoeffectors in the same species, at least as concluded from one study54. Moreover, the mode of action for the thermal effect of TRP vanilloid-1 channel antagonists differs between rodents and humans55. Therefore, activation of peripheral thermosensation with menthol in humans can have smaller effects on deep Tb than in rodents.Menthol has been identified earlier as a cold-mimicking substance, and its beneficial effects on sport performance have been also reported in a recent reviewb. It should be noted that sweat rate could be also an important indicator of thermoregulatory warmth defence. We found only three studies25, which reported sweat rate, but in all of them only the averaged sweat rate was reported for the treatment groups. In two studies25, there was no significant difference in sweat rate between menthol-treated and control groups, whereas in the third study the average sweat rate was significantly reduced after menthol treatment14. However, sweat rate is not steady, but rather a dynamic parameter during exercise. It was shown that during exercise sweating rate increased abruptly for 8 min after the onset of sweating and then continued increasing at a much lower rate56, therefore the average sweat rate for the entire duration of the exercise should be interpreted with caution. As an alternative, we compared the exercise durations between the menthol-treated and control groups of the studies that reported sweat production and found that the difference between the treatment groups was less than 3 min in all studies30. We believe that such minimal difference in exercise duration between treatment groups of the same study did not have a significant influence on sweat production. Our results suggest that with regards to thermoregulation homeostasis, menthol can be safely applied during physical exercise in humans. Nevertheless, it is also possible that the administered doses of menthol and the treated surface area were not sufficient in the most of the analysed studies to trigger cold-defence responses, thereby leading to a change in thermophysiological parameters, including deep Tb. Furthermore, we pointed out different influencing factors, which can help to augment the performance-improving effects of menthol. Among environmental factors, we found that the use of a fan  and higher Ta increased the efficacy of menthol on endurance performance. The beneficial effects of menthol were more pronounced in subjects with higher BMI, while acclimation to heat did not influence the effects.In the present study, we collected the available information about the thermoregulation homeostasis in menthol-treated athletes performing exercise, and conducted meta-analysis of the obtained data. We showed that at the used doses, menthol exerted beneficial effects on endurance performance, but it had no significant effect on any of the thermoregulation-related parameters, which included sweating production, heart rate, and deep T30. The study with the biggest effect size showed a significant improvement in power output18, whereas power output did not differ statistically between the menthol-treated and control groups in either of the studies with smaller effect size58. Based on the risk of bias assessment, we found that blinding was not feasible in many studies, because of the characteristic odour of menthol. Furthermore, the allocation concealment was not indicated in some articles31, which could have also influenced the effectiveness of menthol application. These methodological and medical differences in study design can explain the considerably high between-study heterogeneity (indicated by an I2 of more than 50%), as observed in our analysis (Figs. Some limitations of our study should be also mentioned. There were inter-study differences in the design of the analysed studies regarding, for example, the sample population, the menthol administration route and dose, the exercise protocol, and the measurement of the outcome parameters. For example, the assessment of power output differed in the three analysed studiesis Figs. -S6. To aOur findings suggest that menthol can be safely used during physical exercise to improve thermal perception. Due to its beneficial effects on TS and TC, it can be used as an alternative to mitigate the impact of heat exposure on the individuals. External application of menthol in a warmer environment with air movement is more efficient, especially in subjects with higher BMI than 23. The validation of our results in targeted human trials is subject for future research.Supplementary Information.


Internet addiction has become a major global concern and a burden on mental health. However, there is a lack of consensus on its link to mental health outcomes.The aim of this study was to investigate the associations between internet addiction severity and adverse mental health outcomes.First-year undergraduates enrolled at Sichuan University during September 2015, 2016, 2017, and 2018 were invited to participate in the current study survey, 85.13%  of whom fully responded. Young’s 20-item Internet Addiction Test, Patient Health Questionnaire-15, Patient Health Questionnaire-9, Symptom Checklist 90, Six-Item Kessler Psychological Distress Scale, and Suicidal Behaviors Questionnaire-Revised were used to evaluate internet addiction, four psychopathologies , serious mental illness, and lifetime suicidality.The prevalence of students with mild, moderate, and severe internet addiction was 37.93% , 6.33% , and 0.20% , respectively. The prevalence rates of high somatic symptom severity, clinically significant depression, psychoticism, paranoid ideation, and serious mental illness were 6.54% , 4.09% , 0.51% , 0.52% , and 1.88% , respectively, and the lifetime prevalence rates of suicidal ideation, suicidal plan, and suicidal attempt were 36.31% , 5.13% , and 1.00% , respectively. The prevalence rates and odds ratios (ORs) of the four psychopathologies and their comorbidities, screened serious mental illness, and suicidalities in the group without internet addiction were much lower than the average levels of the surveyed population. Most of these metrics in the group with mild internet addiction were similar to or slightly higher than the average rates; however, these rates sharply increased in the moderate and severe internet addiction groups. Among the four psychopathologies, clinically significant depression was most strongly associated with internet addiction after adjusting for the confounding effects of demographics and other psychopathologies, and its prevalence increased from 1.01%  in the students with no addiction to 4.85% , 24.81% , and 58.73% (37/63) in the students with mild, moderate, and severe internet addiction, respectively. The proportions of those with any of the four psychopathologies increased from 4.05%  to 11.72% , 36.89% (739/2003), and 68.25% (43/63); those with lifetime suicidal ideation increased from 24.92%  to 47.56% , 67.70% (1356/2003), and 73.02% (46/63); those with a suicidal plan increased from 2.59%  to 6.77% , 16.72% (335/2003), and 31.75% (20/63); and those with a suicidal attempt increased from 0.50%  to 1.23% , 3.54% (71/2003), and 12.70% (8/63), respectively.Moderate and severe internet addiction were strongly associated with a broad group of adverse mental health outcomes, including somatic symptoms that are the core features of many medical illnesses, although clinically significant depression showed the strongest association. This finding supports the illness validity of moderate and severe internet addiction in contrast to mild internet addiction. These results are important for informing health policymakers and service suppliers from the perspective of resolving the overall human health burden in the current era of “Internet Plus” and artificial intelligence. The internet has conceptually transformed the Earth into a high-dimensional information network village, and human experience has benefited from the unprecedented availability and exchange of information. However, the potential adverse effects of internet exposure on human health have emerged as a major global concern .In June 2017, the English addiction therapist Mandy Saligari warned parents that giving a smartphone to their child may be as damaging as giving them “a gram of cocaine” due to its potential addictive features and association with adverse mental consequences . In ChinSince Dr. Kimberly Young published a case report in 1996, internet addiction, sometimes called internet dependence or problematic internet use (PIU), has been increasingly conceptualized as “a kind of psychopathological disorder” . StudiesAfter a wide-ranging debate on whether internet addiction primarily reflects the adverse effects of internet contents  or technologies -20, someTo overcome these limitations, in the present study, we investigated the prevalence of four representative psychopathologies , which could reflect a broad range of symptoms and different levels of severity of clinical manifestation of mental disorders, as well as serious mental illness and suicidality among groups with different levels of internet addiction severity. The study was conducted using a large Chinese undergraduate sample comprising the largest sample evaluated in internet addiction research to date.The present study, which was approved by the Ethics Committee of West China Hospital, Sichuan University (no. 2016-171), is based on the cross-sectional data from the Online Psychosomatic Health Survey (OPHS) system of Sichuan University. Sichuan University is a national comprehensive university located in southwestern China whose undergraduates come from all of the provincial administrative regions of China. Every respondent was informed of the aim of this investigation prior to the formal survey, and internet-based informed consent was obtained from each participant.The questions and scales in the OPHS system were designed and selected by a group of researchers from the Mental Health Center, West China Hospital of Sichuan University, and the Centre for Educational and Health Psychology of Sichuan University. This survey was conducted through internet messages with a website link. Students voluntarily logged in with their student ID and were able to quit at any time without penalty. The final survey domains included in the OPHS system were: sociodemographics, childhood adversity, Adolescent Self-Rating Life Events Check List, psychopathologies , psychological distress, suicidality, and internet usage. In addition to demographics of biological sex and age , data were collected using the Young 20-item Internet Addiction Test (IAT), Patient Health Questionnaire-15 (PHQ-15), Patient Health Questionnaire-9 (PHQ-9), psychoticism and paranoid ideation subscales of the Symptom Checklist 90 (SCL-90), six-item Kessler psychological distress scale (K6), and Suicidal Behaviors Questionnaire-Revised (SBQ-R) for the specified research aims. The raw data were saved and stored in a manner only accessible by the research administrators.All undergraduate freshmen enrolled in university during September 2015, September 2016, September 2017, and September 2018 were invited to participate. For inclusion in the study, participants were required to finish the entire self-administered questionnaire on the internet by October 31 of their year of enrollment. Altogether, 37,187 freshmen were invited to participate, 34,140 (91.80%) of whom agreed to participate and logged on to the online survey system. Participants in this study were excluded for the following reasons: failure to complete all of the survey questions, taking less than 10.0 minutes to finish the survey (the median time to finish the survey was 30.6 minutes), did not turn in the questionnaire by November, were not between 15 and 23 years old, or provided obvious dishonest information . The final sample comprised 31,659 respondents, yielding an overall effective response rate of 85.13%.Young IAT has been internationally validated to assess internet addiction ,30. The The PHQ-15, based on the diagnostic criteria of depression from DSM-IV, was used to measure high somatic symptom severity of participants in this study. PHQ-15 is a self-administered, internationally validated, and widely used questionnaire for screening somatization and monitoring somatic symptom severity in clinical practice and research. Its test-retest reliability coefficient was found to be 0.75 in a Chinese population ,32. SubjThe nine questions of PHQ-9 ask about the participant’s experience in the last 2 weeks, with the possible responses ranging from 0  to 3 (“nearly every day”) and a score ranging from 0 to 27. A cutoff of ≥10, reported as the optimal cutoff to detect major depressive episodes in the Chinese population, was used to define clinically significant depression in the present study . CronbacSubscales of the SCL-90 were used to assess psychoticism and paranoid ideation in this study. High validity and reliability of SCL-90 has been demonstrated both in the Chinese general population and with university students ,35. The The K6 is a self-report 6-item scale that asks respondents to rate how frequently they have felt “nervous,” “hopeless,” “restless or fidgety,” “so depressed that nothing could cheer you up,” “that everything was an effort,” and “worthless” during the past 30 days. The 5-point response options for each question ranged from 0 (none) to 4  . For theIn this study, the first SBQ-R question was used to survey lifetime suicidality in participants. This self-report questionnaire consists of 4 questions used to identify suicide risk and has been validated for use with university students. Both single SBQ-R Item 1 and SBQ-R total scores have been recommended for use in clinical and nonclinical settings . QuestioData analyses were conducted using SPSS 22.0 software. The mean (95% CI) and median age, number of respondents in each demographic and internet addiction severity group, and prevalence rates (95% CIs) across levels of internet addiction severity, psychopathology, serious mental illness, and suicidality were calculated. The Chi-square test was used to compare prevalence rates among demographic or internet addiction severity groups, and the adjusted ORs and their 95% CIs of other mental health outcomes among the internet addiction severity groups were estimated and compared using binary logistic regression, adjusting for confounding effects of demographics  and the interactions among psychopathologies. A two-tailed alpha level of .05 was used to evaluate statistical significance.The 31,659 respondents included 16,109 (50.88%) men and 15,550 (49.12%) women. The groups aged 15-17 years, 18 years, and 19-23 years included 4821 (15.23%), 19,034 (60.12%), and 7804 (24.65%) respondents, respectively.Overall, the rates of those without internet addiction and those with mild, moderate, and severe internet addiction were 55.5%, 37.9%, 6.3%, and 0.2%, respectively. Internet addiction severity rates differed between sexes, among the age groups, and among the year-of-survey groups. The rate of moderate internet addiction was higher in women than in men. The oldest group (aged 19-23 years) included more respondents without internet addiction and fewer individuals with mild internet addiction than the other two age groups. Among the year-of-survey groups, the rates of all levels of internet addiction severity increased with recency .Overall, the prevalence rates of high somatic symptom severity, depression, psychoticism, paranoid ideation, and serious mental illness in the current sample were 6.5%, 4.1%, 0.5%, 0.5%, and 1.9%; lifetime prevalence rates of suicidal ideation, suicidal plan, and suicidal attempt were 36.3%, 5.1%, and 1.0%, respectively. All of these rates significantly increased from the group without internet addiction to the groups with mild, moderate, and severe internet addiction. Compared with the average levels of the surveyed population, these rates in the group without internet addiction were much lower, whereas those in the group with mild internet addiction were similar  or mildly higher ; however, these rates sharply increased in the moderate and severe internet addiction groups . AccordiAmong all respondents, the rates of those who had at least one, two, and three of the four psychopathologies were 9.2%, 1.9%, and 0.4%, respectively. These rates increased significantly from the group without internet addiction to those with mild, moderate, and severe internet addiction . AccordiThe IAT, which is the most widely used internet addiction survey instrument in the world, allows researchers to differentiate the severity of internet addiction, as in the present study . HoweverFirst, by comparing the prevalence rates and ORs of mental health outcomes among the differentiated internet addiction severity groups, the present study found a surprising incremental pattern of these conditions from the group without internet addiction through the group with severe internet addiction. That is, the prevalence rates of the four psychopathologies, serious mental illness, and suicidality in the group without internet addiction were much lower than the average levels of the surveyed population, and those in the group with mild internet addiction were similar to  or mildly higher than  the average levels of the surveyed population; however, the prevalence rates and their OR increments from mild to moderate and severe internet addiction were surprisingly large for most conditions. Previous surveys typically compared prevalence rates and ORs of individual syndromes between the groups without and with internet addiction. Based on this approach in the original studies, Ho et al  performeSecond, by investigating the largest number (four) of representative psychopathologies in the context of an internet addiction survey to date, the present study enabled an analysis of the overlap between these psychopathologies and their comorbidity, demonstrating a clear relationship with internet addiction severity. Previous surveys have typically reported an association of internet addiction with individual psychopathology because of a focus on the investigation of a single or very small number of psychopathologies. This may explain a possible significant underestimation of the prevalence of psychopathologies in groups with varying internet addiction severities, in addition to a lack of prior understanding of the pattern of overlap and comorbidity of the mental health outcomes linked to internet addiction. For example, the meta-analysis by Ho et al  showed tThird, the present study is, to our knowledge, the first to investigate the association of internet addiction with high somatic symptom severity or serious mental illness. Consequently, it was found that internet addiction was strongly associated not only with mental symptoms, serious mental illness, and suicidality but also with somatic symptoms, which are the core features of many medical illnesses . Given tThe overall prevalence rates of internet addiction , psychopathologies , and serious mental illness (1.9%) in the present sample of university freshmen were lower than those reported in other recent surveys  would be only partially helpful in view of an expected limitation of correspondence of the data of this study and the university clinic data. Disparities between prevalence rates based on mental health clinic presentation–based estimates and community survey–based estimates are expected. Epidemiological studies have clearly documented that health care–seeking rates of individuals with mental disorders in general communities is low, especially in low and middle income countries such as China, which provides partial support for our current survey approach rather than a medical records–based analysis. It has been argued that cultural issues leading to increased stigmatization and alternate support–seeking behavior may underly this pattern of reporting ,46.This study investigated internet addiction but we were not able to identify IGD  nor did we identify the proportion of the student sample using the internet predominantly to access online games as opposed to other content such as social media and information. Further information on the proportion of different internet activities that participants engaged in will be necessary to achieve any detailed understanding of the relationship of internet use with addiction and other psychopathologies that are a significant concern for the healthy development of our youth in the information age. To gain insight into the etiology and possible therapeutic interventions for internet addiction and IGD, further research must include data on the proportional access of participants to online gaming, social media, and social messaging as well as access to other risk-related content such as pornography.We investigated a broader group of mental health outcomes as possibly being associated with internet addiction than previous studies, but did not include all mental health factors. Our participants were first-year students at a single university, although they were from all of the provincial administrative regions of China. The gross enrollment rate in higher education in China from 2015 to 2018 ranged from 40% to 48.1% . HealthyThe present study showed that internet addiction is strongly associated with a broad group of mental health problems , which strongly supports the illness validity of moderate and severe, but not mild, internet addiction. This study is also meaningful for health policy makers and service suppliers to manage the so-called internet addiction or other mental health outcomes relevant to internet usage, especially from the perspective of resolving the overall human health burden in the current era of “Internet Plus” and artificial intelligence.