Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca2+ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca2+ activation and abolished luminal Ca2+ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [3H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca All of the PCR products were purified using the QIA quick PCR Purification Kit. cDNA fragments containing the desired mutations were removed from the PCR products and used to replace the corresponding wildtype (WT) fragments in the full-length mouse RyR2 cDNA in the expression plasmid pcDNA5 ryanodine binding to cell lysates was performed as described previously [3H]ryanodine at 37°C for 2 h in 300 µl of a binding solution containing 500 mM KCl, 25 mM Tris, 50 mM HEPES (pH 7.4). Free [Ca2+] (0.1 nM to 100 µM) was adjusted by EGTA and CaCl2 solutions using the computer program of Fabiato and Fabiato [3H]ryanodine retained in filters was determined by liquid scintillation counting. Specifically bound [3H]ryanodine was calculated by subtracting nonspecific binding that was determined in the presence of 50 µM unlabeled ryanodine. All binding assays were performed in duplicate. [3H]ryanodine binding data were fitted with the Hill equation using the Prism 8 .Equilibrium ryanodine binding to RyR2. Similarly, we found that the G4935R mutation completely abolished Ca2+ dependent activation of [3H]ryanodine binding to RyR2 when it was expressed alone  and SCD result from GOF defects of the RyR2 channel . However2+ waves under conditions of SR Ca2+ overload such as during physical and emotional stress [2+ waves are well-known cause of DADs, which, in turn, can lead to triggered activity, triggered arrhythmia, and SCD [2+ alternans, EADs, and re-entrant activities [The existence of both GOF and LOF RyR2 mutations has profound implication for the understanding of RyR2-associated cardiac arrhythmias. The GOF RyR2 mutations are believed to enhance the propensity for spontaneous Cal stress . These s and SCD ,31–33. Htivities . TherefoGiven these fundamental differences in their functional impact, the diagnosis and treatment strategies for ventricular arrhythmias associated with GOF or LOF RyR2 mutations would have to be different. EST is widely used for the diagnosis of typical CPVT, which is associated with GOF RyR2 mutations. However, such a test would be ineffective in identifying patients carrying LOF RyR2 mutations because of their lack of response to exercise stress test . In thisIt is of interest to note that although mutations G4935R, S4938F, and R4959Q are all located in the CTD, their impact on RyR2 channel activation is very different. R4959Q does not significantly alter caffeine activation of RyR2, while S4938F markedly suppresses caffeine activation, and G4935R completely abolishes caffeine activation. Thus, it would be challenging to predict whether a mutation will impact channel function solely based on the domain it is located in.2+ oscillations in RyR2-E4146K mutant expressing HEK293 cells. On the other hand, high concentrations of caffeine induced a single peak of Ca2+ release in RyR2-WT expressing HEK293 cells. The exact mechanism underlying this difference is not completely understood. We have shown previously that caffeine induces Ca2+ release/Ca2+ oscillations in RyR2-expressing HEK293 cells by reducing the threshold for SOICR [2+ oscillations in the absence of caffeine. Whereas, in the presence of 10 mM caffeine, the SOICR threshold in RyR2-E4146K expressing HEK293 cells would be reduced to a level at which spontaneous Ca2+ oscillations can now occur. On the other hand, the normal SOICR threshold in RyR2-WT expressing HEK293 cells would be dramatically reduced by 10 mM caffeine, resulting in the depletion of ER Ca2+ stores, which may explain the lack of Ca2+ oscillations in RyR2-WT expressing HEK293 cells in the presence of 10 mM caffeine [Another interesting observation is that the presence of caffeine (10 mM), but not the absence of caffeine, induced Caor SOICR . The obscaffeine .In summary, the present study reveals novel LOF RyR2 mutations associated with IVF and SCD with negative exercise stress test. IVF and SCD associated with loss of RyR2 function represents a new entity of ventricular arrhythmias distinct from CPVT. Different strategies and protocols will be required for the diagnosis and treatment of ventricular arrhythmias associated with enhanced or suppressed RyR2 function.Click here for additional data file.


Ciona robusta Oc protein to show its requirement for ciliary PRC differentiation. Then, transcriptome profiling between the trans-activation and trans-repression Oc phenotypes identified differentially expressed genes that are enriched in exocytosis, calcium homeostasis, and neurotransmission. Finally, comparison of RNA-Seq datasets in Ciona and mouse identifies a set of Oc downstream genes conserved between tunicates and vertebrates. The transcription factor Oc emerges as a key regulator of neurotransmission in retinal cell types.Photoreceptor cells (PRC) are neurons highly specialized for sensing light stimuli and have considerably diversified during evolution. The genetic mechanisms that underlie photoreceptor differentiation and accompanied the progressive increase in complexity and diversification of this sensory cell type are a matter of great interest in the field. A role of the homeodomain transcription factor Onecut (Oc) in photoreceptor cell formation is proposed throughout multicellular organisms. However, knowledge of the identity of the Oc downstream-acting factors that mediate specific tasks in the differentiation of the PRC remains limited. Here, we used transgenic perturbation of the  Onecut (Oc) gene family of transcription factor coding genes plays key roles in the development of the vertebrate eye  for its expression during neurogenesis  have been organized in many different manners in order to enable organisms to perceive light and recognize light direction and, in vertebrates, form high-resolution imaging Lamb, . PRC conOc genes have maintained neurogenic roles described in non-vertebrates, evolving novel functions according to the increased complexity of the nervous system  by miR-9 binding is essential for eye angiogenesis , retinal ganglion cells (RGC), PRC, and amacrine cells (AC) was evidenced by loss-of-function studies in mice . Animals were acclimatized at ~20°C for 2–3 days in open system tanks and fed every day with a solution of marine microalgae concentrates (Shellfish Diet 1800™ Instant Algae®). Subsequently, they were exposed to continuous lighting for a few days in order to accumulate mature gametes and to prevent gamete spawning.Adult specimens of Ciona OC full-length coding sequence fused in frame with the Vp16 activator or WRPW repressor domains . Each electroporation experiment was repeated at least 10 times and GFP-positive embryos (corresponding to 80–90% of the developed ones) were used partly for statistical analyses and partly were collected and treated for subsequent experiments . For phenotypic analyses of transgenic larvae, about 80 GFP-positive larvae (stage 26), in three experimental replicates, were analyzed under a light microscope and evaluated for their phenotypic alterations of pigmented sensory organs. A total of 233 and 243 transgenic larvae were counted and used for the percentage calculation of the more representative phenotypes observed in the OC::Act and OC::Rep conditions . The mapping of paired-end reads was performed using the bioinformatics tool STAR (version 2.5.0a) . The reference genome version was the assembly Ciona_intestinalis.KH.78 . All the Ensembl gene name codes were shortened for simplicity . Genes were considered differentially expressed (DEGs) when count filtered for padj <0.05 and FC >|1.5|.Cell dissociation and magnetic-activated cell sorting (MACS) were performed as described , transcript sequences were aligned vs. the UniProt Swiss-Prot database (version of November 2016) using BLASTx   were also blasted  vs. the UniProt TrEMBL database (at https://www.uniprot.org/uniprot/?query=reviewed:no version of November 2016).To confirm or update the function associated to https://blast.ncbi.nlm.nih.gov/Blast.cgi) vs. the “non-redundant” (nr) protein database of NCBI, and then we searched for domains using INTERPRO (https://www.ebi.ac.uk/interpro/)  .Ciona and mouse were identified performing (i) a tBLASTx  of Ciona transcripts vs. mouse transcripts, (ii) a tBLASTx  of mouse transcripts vs. Ciona transcripts, and (iii) the Transcriptologs approach : Opsin1 (CiGC28m24), Arrestin (CiGC28m09), Pax3/7 (CiGC42e20), and Synaptotagmin (CiGC26m18). PCR amplification of the other transcription templates was performed with oligos shown in Ciona embryos were performed as previously described by Pezzotti et al.  and WRPW (constitutively repressive) domains under the control of the Gsx promoter, with the aim to investigate the phenotypic consequences on PRC precursor development. Following electroporation of the pGsx>GFP2x construct, control larvae displayed the wild-type head phenotype, in which two pigmented sensory organs are clearly distinguishable, i.e., the otolith, which is a gravity sensory organ located ventrally in the anterior region of the sensory vesicle, and the photosensing ocellus more dorsally, in the wall of the vesicle (Ciona larvae carrying the constitutively repressive form pGsx>OC::WRPW (OC::Rep), 45% of them showed only one apparently normal pigmented cell in the sensory vesicle , which is essential for melanocyte differentiation  and larva stage  and subjected to Illumina HiSeq2500 sequencing (Stranded RNA-Seq). We identified 9,967 Ciona genes whose expression was detected in the two transgenic conditions with at least one match with a protein present in the UniProt Swiss-Prot database. Ciona transgenic transcriptomes revealed about 500 genes with a significant change in expression level (DEGs), 469 of which were found in the OC::Act condition and 36 in the OC::Rep one  showed upregulation in OC::Act and downregulation in OC::Rep, lending themselves as candidate targets in the Oc genetic pathway implicated in PRC differentiation in Ciona. Other five DEGs were downregulated in both conditions  gene encodes a serotonin transporter protein. In vertebrates, Slc7a14 codes for a glycosylated, cationic amino acid transporter protein that has recently been found to underlie the autosomal recessive retinitis pigmentosa  codes for a soluble protein that interacts with the SNARE complex, the molecular machinery of synaptic vesicle exocytosis  belongs to a small GTPase Ras superfamily that regulates many signaling pathways in neuronal functions, including neurotransmitter transmission, cell migration, neurite outgrowth, and neuron proliferation  encodes an endoplasmic reticulum tetratricopeptide repeat-containing adapter protein involved in calcium homeostasis that has been associated with primary open-angle glaucoma  codes for a Ca++-sensor synaptotagmin protein that mediates the exocytotic fusion between vesicle and plasma membranes during synaptic transmission , ENS_05701 (Scgn-like), ENS_02864 (GTPase Rab11a), and ENS_20001 (FKBP25/3) genes encode for factors involved in different steps of neurotransmission as described in Slc6a4b/mouse Slc6a2), whose vertebrate orthologous genes are expressed in the retina . Neither the BLASTx- nor the INTERPRO-based analyses provided any useful information for the annotation of the considered transcripts, except for two genes, ENS_23151 and ENS_21806, downregulated in both transgenic conditions. ENS_23151 encodes a protein with a signal peptide and a non-cytoplasmic domain based on the INTERPRO analysis, indicating a possible translocation across membranes, while ENS_21806 encodes a protein carrying a Slc6sbd_SERT-like domain, the solute-binding domain of serotonin neurotransporters (data not shown)  that were differentially expressed in both species  or downregulated  only under the OC::Act transactivating form. All these genes are expressed in different territories of the vertebrate eye and encode factors involved in synaptogenesis, calcium signaling, or retinal cell specification . The murine ortholog of ENS_04039 Neurogenin gene is involved in rod PRC differentiation (NeuroD4) .To identify conserved fication . In the Ciona. Mab21 (ENS_08580) and Opsin1 (ENS_01146) were significantly downregulated in OC::Rep condition . Mab21 belongs to the male abnormal gene family whose members  play important roles in regulating ocular development in vertebrates  was significantly upregulated (+2.95 log2FC) in the OC::Act condition . This gene belongs to a group of Na+-dependent monoamine transporters encoded by the SLC6 gene family  with two mouse genes that belong to the same gene family of solute carrier 6 (Slc6a1 and Slc6a4) and that are differentially expressed in mouse Oc1/Oc2−/−. Extending this analysis to all Ciona and mouse DEGs, we greatly increased the number of Oc targets shared across chordates. We found that 45 mouse DEGs are paralogs or orthologs of DEGs in Ciona (−50), we can assume that even if ENS_07757 and ENS_09380 are not the computationally defined orthologs of Slc6a1 and Slc6a4, they share a high similarity level, which may be the reason for preserving the same functionality between paralogous.Considering the significant phylogenetic distance between nditions , while iin Ciona , 4. SincOc and DEG expression patterns (double WISH)  showed no detectable mRNA signal in WISH, while some other riboprobes did not work in double WISH experiments. However, we show that four DEGs  are specifically co-expressed with Oc, while one DEG (ENS_23151) is expressed in tissues adjacent to OC-expressing cells  were overexpressed in the progenitor cells of the sensory vesicle of OC::Act embryos and downregulated in OC::Rep, in agreement with log2FC in RNA-Seq datasets  and for spatial correlation between le WISH) . We geneng cells . Importadatasets . Expressteration .Prox1 and Lhx5, which are dysregulated in our RNA-Seq dataset and in that of mouse Oc1/Oc2−/− double mutant retinas  and reduced (Lhx5) transcript staining in the sensory vesicle of OC::Act transgenic tailbud stage 22 embryos in comparison with controls (Lhx5 upregulation in OC::Rep embryos that is not supported by RNA-Seq data (Ciona Pax3/7 (ENS_13561) and Mab21 (ENS_08580), orthologous genes of two important factors for PRC formation , which shows a negative log2FC in both OC::Act and OC::Rep RNA-Seq datasets, is downregulated only in the sensory vesicle of OC::Rep embryos and is upregulated in OC::Act embryos , neurotransmission , opsin transport , and calcium homeostasis . Given the activating or inhibitory role of Oc on gene expression, we propose that Ciona Oc serves as a regulator of proteins that mediate synaptic activities. Furthermore, Oc itself and some of these DEG genes with neurosynaptic activity are not restricted to PRC, suggesting that Oc could use the same target genes to promote neural cell type differentiation in various regions of the nervous system , PRC differentiation , inhibition of Müller cell differentiation and positive regulation of optic nerve projections (Ebf1), ciliogenesis (Mak), synapsis formation, and GABA release  , and of the rhodopsin-transporter Rab11a (ENS_02864) decreased under a predominantly repressive Oc regulation (OC::Rep) , the gene coding for the delta subunit of rod-specific photoreceptor phosphodiesterase (Pde6d), a key enzyme in the phototransduction cascade, dropped under the transactivating condition  can be found at: QV, AF, GF, VN, and FB did the molecular experiments. CC did the bioinformatics analyses. QV, CC, AL, and PS drafted the manuscript and prepared the figures. RK, AL, and PS supervised the experiments. MC supervised the bioinformatics analyses. PS and AL edited and revised the manuscript. All authors approved the manuscript for publication.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.


Neglecting oral hygiene in adolescents negatively affects dental caries and periodontal diseases, in addition to social and emotional well-being. Thus, the TikTok platform as a social media could be a powerful channel to provide health-related information and educational content. This study aims to assess the quality of the TikTok videos corresponding to #oralhealtheducation. Sixty-nine videos were identified. Three oral health professionals (OHP), three health education professionals (HEP), and ten of TikTok’s target audience watched and evaluated the videos from a qualitative questionnaire. OHP detected false or incorrect information in 11.6% (8/69) of the videos. At least two HEPs reported being unable to detect this type of content or whether the video met dental ethics standards in both the videos. Disagreement was observed among the professionals themselves. The evaluation indicated that TikTok’s target audience was satisfied with the products viewed with an average score of >2.5, unlike the professionals, whose average score was <2.5 on a scale of 0 to 5. Users are advised to think critically and to consider the content of TikTok oral health videos with caution. The involvement of health professionals in the writing and validation of the videos could be an added value to positively respond to the needs of the adolescents. Oral diseases represent a significant health burden for individuals and populations . The advOral health care education represents a public health care priority since maintaining a good level of oral hygiene is an essential part of overall health . Oral diThe change in health behaviors has undergone a revolution with the growth of the digital environment . Mobile Health-related audiovisual content can impact society. Regardless of the content, dramatic means are used in social network videos to capture the short attention span of the audience and to compensate for the almost endless supply of other videos. Different categories of videos may attract different users and thus have different characteristics . The conTikTok, formerly known as musical.ly was created in September 2016 in China . The govAlthough less popular than female reproductive health, and acne treatment, information on oral hygiene is present on the platform. Also, it is generally accepted that nutrition and diet should be included for a holistic health program since they are common risk factors for oral diseases . The queThe aim of this study is to assess the quality of the content related to oral health education on TikTok platform, as well as to evaluate its possible impact on the young population.This study was designed as an observational retrospective study based on the content of TikTok videos. As a social media platform, TikTok provides access to short videos, from 3 to 60 s, shared by TikTok users. The videos are accessible from a mobile application or accessible online on a website without a necessary user account. The TikTok version was 20.2.5 (3 July 2021). This study did not require any regulatory approval. All respondents were informed of the process and purpose of the study. This research was performed in accordance with the STROBE guidelines .A total of 203 TikTok videos were extracted with the hashtag #oralhealtheducation, which was associated with 6.8 million views on 28 September 2021 . Next, tTwo academic researchers  specifically created an online questionnaire for the purpose of this study. This questionnaire was elaborated using the DISCERN instrument developed for judging the quality of written consumer health information on treatment choices , and froThe version was tested with a random selection of 10 videos, adjusted and finally approved by an oral health professional (DB) and a public health professional (DT). The final version of the questionnaire is available in After watching the 69 selected videos, two academic researchers  completed The online questionnaire was constructed to be sure that all the raters provided an answer to each question; no missing data were allowed. For the categorical item, we provided the number of agreements  and the number of “yes” per agreement for categorical variables (yes/no). For the ordinal item, we provided the number of agreements on the five-item responses  and the number of raters who agree (score: 4) or strongly agree (score: 5).p-value indicates whether the calculated kappa is significantly different from zero and thus different from a random agreement; if the p-value is less than or equal to 5%, we conclude that the agreement between raters is significantly different from the value that would be obtained by chance.To measure the inter- and intra-rater reliability between OHP and HEP, the Kappa of Fleiss (Kf) between categorical variables was used. A poor agreement corresponds to Kf < 0, a slight agreement to 0 <Kf < 0.2, a weak agreement to 0.2 < Kf < 0.4, an average agreement to 0.4 < Kf < 0.6, a good agreement to 0.6 < Kf < 0.8 and an excellent agreement to Kf > 0.8. The For the ordinal variables, the intra-class coefficient (ICC) and its 95% confidence interval were calculated. An ICC close to 1 indicates high similarity between responses in the same group, while an ICC close to zero means that values in the same group are not similar.Statistical analysis was performed using the package “irr” of the R Project for Statistical Computing (version 4.1.1. 2021-08-10).The oldest video was posted 515 days ago, on 1 May 2020. We estimated that one video with the hashtag #oralhealtheducation was shared every two weeks. The median length of the included videos was 20 s , with 44.9% of videos (31/69) that mentioned at least one commercial brand. The median number of likes was seven . Only 6 videos received more than 1000 likes , including 4 videos posted in the last 6 months.Among the included videos, 88.4% featured at least one person (61/69) and 11.6% did not feature anyone (8/69). Only one video featured more than five people. The main character presented himself as a health professional  due to his pseudonym in 24.6% of the included videos (15/61) or due to his professional outfit when at least one person is featured in 54.1% (33/61). The scene took place in a dental office in 15.9% of the videos (11/69) and 27.5% of the videos presented dummy material  (19/69).Concerning the staging of videos, 73.9% had a musical background (51/69), 17.4% were entirely subtitled (12/69) and 84.0% were composed of key text-messages (58/69). The humor tone was used in less than 6% of the videos (4/69). The video setting and staging characteristics are described in Oral care products presented or used in the videos are described in p-values when comparing the whole responses, regardeless of OHP and HEP groups.The analysis of the objectives of the video in relation to oral health education are presented in The OHP and HEP agreed to concerning the three main objectives of the TikTok videos corresponding to #oralhealtheducation . The firThe target audience for the TikTok videos corresponding to #oralhealtheducation is summarized in The public targeted by Tiktok videos was clearly identified in only 13% of videos (9/69) for OHP and in 4% of videos (3/69) for HEP.According to OHP, the oral health subject was mastered in 28,9% (20/69) of videos and the vocabulary used was appropriated in 36.2% (25/69) of videos. They agreed that 42.0% (29/69) of videos complied with dental ethics or dental professionals and 28.9% (20/69) of videos applied official recommendations. They detected false or erroneous information in 11.6% (8/69) of videos. HEP evaluated that the oral health subject was mastered in 4.3% (3/69) of videos but at least one HEP answered “Do not know” in 84.1% (58/69) of videos. They judged that vocabulary used was appropriate in 8.7% (6/69) of videos but at least one HEP didn’t know in 68.1% (47/69) of cases. For all the videos, at least two of the three HEP declared to be unable to detect false or erroneous information or to know if the video respected dental ethics or dental professionals.The subjective evaluation of the quality was realized by OHP, HEP and TikTok’s target audience. The results are presented in The average score given by TikTok’s target audience was always higher than OHP which was always higher than HEP. The average score given by the TikTok’s target audience was always higher than 2.5 whereas for the OHP end the EHP, the average score was always lower than 3. For the TikTok’s target audience, the videos were able to increase awareness of the importance of oral health and to provide relevant information related to oral health.Social media use among teens is nearly ubiquitous . TikTok,The choice of the hashtag #oralhealtheducation allowed us to focus our research on videos that focused on oral health promotion and were targeted to the general population. The #oralhealth that reached a larger audience was not specific enough to the oral health promotion that was the focus of this article. It referred to numerous videos dealing with the curative aspect  or the aesthetic aspect and was very often addressed to professionals and not to the general population.Oral health education will not be a major target of the TikTok Health platform in 2021. The hashtag #oralhealtheducation corresponded to 203 TikTok videos and only 69 were in English or French and related to human oral health education. These videos received 141,720 “likes”, were commented 1358 times and shared 3295 times. Compared with other health-related topics, oral health education doesn’t appear to be of high interest for TikTokers and viewers. For example, the keyword “diabetes” referred to 199 TikTok videos which received 2.75 million “likes” and were commented on and shared thousands of times .Our research is relevant to analyze oral health education-related videos on TikTok as well as their potential effect on behavior. TikTok users are often adolescents and therefore belong to a potentially vulnerable and influenceable group of individuals . MoreoveIn some cases, such as childhood caries, general population interventions  have been shown to be more successful than population-specific and individual interventions . TikTok The TikTok videos were mainly focused on oral hygiene tips which is a one key element of the prevention of oral diseases ,44,45. TFifty percent of the videos included images of toothbrushes, with manual toothbrushes being overrepresented (74.3%). However, basic messages such as the indication of type of toothbrushes including the hardness of the filaments ,52 and oAmong the oral hygiene components, the interdental thread is presented (30.4%). As previously described, there is a lack of criteria on the indications, choice and methods of use of the product. The floss is the interdental device the more present in the videos . Cochrane Review on flossing underlined there is some evidence that flossing added to toothbrushing in adults may reduce gum disease and plaque compared to toothbrushing alone. But, overall, the evidence was low to very low- certainty . MoreoveConcerning tongue cleaning, the description of tongue scraper in the videos corresponding to hashtag #oralhealtheducation is interesting due to the fact that its efficiency was proven to fight against halitosis and gingivitis ,65. The use of chemical agents such as toothpaste and mouthwash was also presented in the videos (47.8%). Broadly speaking, there are two types of mouthwash: cosmetic and therapeutic. Cosmetic mouthwash may temporarily control bad breath and leave behind a pleasant taste, but have no chemical or biological application beyond their temporary benefit. Therapeutic mouthwash, by contrast, has active ingredients intended to help control or reduce conditions like bad breath, gingivitis, plaque, and tooth decay. This product contains active molecules that can help to fight against oral diseases such as caries or periodontal diseases but can help to fight against systemic diseases such as COVID-19 ,67,68,69In terms of oral health education, nutrition and diet should be included in a global health program as they constitute common risk factors for non-communicable diseases ,72. ReviFrom our analysis of the dental hygiene products contained in TikTok, it appears that even professionals have difficulty distinguishing whether the information contained in the videos comes from reliable sources. To stay safe from false or harmful content, creators on TikTok should clearly indicate where their information comes from, and users should remain vigilant . LikewisEvaluation criteria are needed regarding the quality of the video that is uploaded by each video uploader about oral health education on TikTok, with the hope that viewers can receive useful information, good quality content. The videos analyzed were mainly made by a TikToker that was presented as a health professional (65.5%) due to his pseudonym or his professional outfit. Moreover, the scene took place in a dental office in 15.9% of the videos. Although TikTok has no means of monitoring to confirm that contributors are accredited health professionals, it was found that the number of health professionals using TikTok for health communication and awareness campaigns was increasing . Even if the content of the TikTok videos related to oral health education is interesting, it is important to analyze how the message was delivered. The OHP evaluated that the subject was mastered in less than one third of the videos and the vocabulary appropriated in a little more than a third of the videos. The HEP evaluated that the subject and the vocabulary was correct in respectively 4.3% and 1.4% of the videos. This difference between the professionals demonstrated the difficulty to evaluate the TikTok videos contain. OHP did not agree concerning the fact that TikTok videos applied or not official recommendation (49.3% of agreement) and the fact that false or erroneous information was presented or not (46.4% of agreement). The evaluation of other TikTok videos in other health areas, such as “diabetes” or “acne”, has already shown serious shortcomings as well as the risk of spreading misinformation ,78.In addition, the subjective evaluation demonstrated that TikTok’s target audience and professionals have totally different demands and expectations in terms of content and staging. The TikTok’s target audience were satisfactory with the TikTok videos corresponding to #oralhealtheducation whereas the professionals were not. The TikTok’s target audience quoted the videos as (i) attractive, (ii) able to increase awareness of the importance of oral health, (iii) to provide relevant information related to oral health, and (iv) able to change oral hygiene habits. So, TikTok’s target audience that have no formation in the oral health domain cannot differentiate between reliable content and potentially biased content and should be more careful with the content of the videos related to oral health education.Future audiovisual productions should be oriented to professional and domestic environments. In the oral healthcare sector, several fundamental trends are driving the adoption of advanced digital solutions to continue in the process of excellence. Advances in technology, prevailing patient preferences and dental school curriculums, as well as emerging treatment guidelines, are accelerating the adoption of future audiovisual productions with a simplified digital format. Access to quality continuing education, targeted to the specific needs of practitioners, is fully compatible with the principles that guide social media. They will also reduce inequalities in access to training for dentists. Social media must continue to be developed in the domestic environment with tools, such as TikTok, targeting teens and the nomad population. The framework is to empower, promote oral health and knowledge in the detection and self-management of the most common oral symptoms or diseases with a universal language, both of which are currently lacking. The content will have to evolve towards more rigor and scientific quality and evidence-based information, with full transparency regarding conflicts of interest.There are several strengths to our analysis. This study is the first to analyze the TikTok videos corresponding to the hashtag #oralhealtheducation. Moreover, the point of view of three different groups of viewers  was compared. However, this study also has limitations. First, the videos were only identified using #oralhealtheducation, and additional hashtags such as #toothbrushing, #mouthwash, #teethcleaning, among other related hashtags, often co-occurring with #oralhealtheducation, might yield subsets of videos with differing content, sentiment, and levels of incorrect content and misinformation. Secondly, the videos were included only if they were in French or in English, which probably excluded some interesting videos. Thirdly, due to the fact that the health-related audiovisual can impact the society, it could have been interesting to add a group of audiovisual communication professionals and some more specific questions. Fourthly, it is not possible to know if the TikToker is an oral health professional since only his pseudonym or outfit could give some indication but without real evidence. Fifthly, teenagers are not probably the only targeted audience. Although the videos are intended for teenagers, the video metrics  do not determine whether the audience figures are teenagers or other target audiences. Sixthly, unlike other content platforms, such as Youtube, that have their own campaigns, the TikTok application does permit the evaluation of the impact of the content. Finally, this study included only 10 young adults and the questionnaire had few questions regarding subjective evaluation. It might be interesting to conduct another study with a larger panel of users and targeted questions about their perception and knowledge of oral hygiene before and after viewing TikTok videos.Our results indicate that the content of TikTok videos corresponding to the hashtag #oralhealtheducation is not always reliable, as has already been demonstrated in other studies of health-related videos. Moreover, our study showed the difference of opinion between the professionals themselves but also with the users. Thus, we recommend that users be careful and critical about the content of videos related to oral health. The involvement of health professionals in the writing and validation of the videos could be an added value to positively respond to the needs of adolescents.


IntroductionIn this study, we evaluated the scope of acute non-specific back pain (ANSBP) content available on TikTok  in 2021. It is plausible that TikTok’s popularity among teenagers, adolescents, and young adults may influence decision-making about what constitutes appropriate ANSBP self-care among a younger age cohort.MethodsWe examined 157 of the most viewed videos available through the hashtag #backpain available on TikTok in September 2021. We examined the following research questions: (1) What are the metadata characteristics of the videos in the final data set?, (2) What are the creator identities reflected in the final data set in this study?, (3) What are the ANSBP self-care content themes in the final data set?, and (4) What are the characteristics of the data set based on a low back pain reference checklist based on consensus guidelines?.ResultsWe identified clear differences based on TikTok creator identity in our data set of most popular videos. We examined videos created by chiropractors, fitness professionals, influencers, physicians, physiotherapists, and other creator identities. We found that the TikTok videos created by chiropractors were consistently among the most viewed, most commented, and most shared. Conversely, chiropractic TikTok videos consistently had the lowest self-care reference checklist scores relative to all other disciplines. That is, TikTok videos created by chiropractors were least likely to reflect the scientific consensus on treating ANSBP.DiscussionTikTok is an increasingly popular medium for disseminating short health messages. The main cohort using TikTok is young and at risk of ANSBP. However, we postulate that the messages reaching young TikTok users overall do not generally reflect the self-care advice described in consensus guidelines. ConclusionTikTok is a popular social media channel among young people. However, the most viewed TikTok videos about ANSBP are not produced by mainstream health professionals and the videos featuring the #backpain hashtag do not generally reflect contemporary evidence-based practice. There is considerable scope for mainstream health professionals to provide evidence-informed self-management and self-care content for ANSBP on TikTok. This exploratory study aims to increase the understanding of acute non-specific back pain (ANSBP) content available on TikTok . This paper will also examine whether approaches used to analyse YouTube  health videos can be applied to TikTok health content. Low back pain imposes a high social and financial burden in all countries across the globe . In the Epidemiology of ANSBP in younger populationsAcute low back pain is generally regarded as lasting less than six weeks . In mostSelf-management and self-care of low back painMost people manage their low back pain with minimal assistance from health care providers. Independent management without assistance from health care providers is generally referred to as self-care . By contAbout TikTokTikTok is a video-sharing focused social networking service launched in 2018 ,13. TikTHealth information on TikTokGoogle Scholar suggests scientific engagement with TikTok began in 2018. In 2021, health content on TikTok had still not been widely examined in scientific literature. In 2021, there was nascent literature on analysing TikTok videos for health self-care. Among the health care conditions researched were COVID-19 , diabeteTikTok has been associated with misinformation in the mass media. In 2021, media monitoring organisation NewsGuard reported COVID-19 vaccine misinformation directed at children aged under 13 on TikTok . The creApproaches to TikTok Health Video AnalysisTikTok in 2021 was a relatively novel medium for health content. The limited peer-reviewed health literature on TikTok in 2021 revealed heterogeneous approaches to research into this social media platform. However, by 2021, there was a larger body of research describing the use of YouTube videos to reach specific groups with condition-specific health messages. In 2021, YouTube was the second most popular social medium and the most popular video social medium . The appSeveral investigators have examined the methods used to analyse YouTube health content. Drozd and colleagues noted that most researchers develop independent scoring systems and that no commonly agreed on methods exist . SystemaResearchers have adopted the methods used for analysing YouTube videos for analysing TikTok health content. These approaches include analysis of information quality using the DISCERN instrument, by creator professional background, by expert clinical review, or by metadata  ,21,33. TThis exploratory study aimed to evaluate the scope of ANSBP content available on TikTok in September 2021. In order to evaluate the scope of ANSBP TikTok content, we examined the following research questions (RQs). RQ1: What are the metadata characteristics of the videos in the final data set?, RQ2: What are the creator identities reflected in the final data set in this study?, RQ3: What are the ANSBP self-care content themes in the final data set?, and RQ4: What are the characteristics of the data set based on a low back pain reference checklist?.We modified the methods developed by Zheluk and Maddock for their analysis of YouTube videos about acute non-specific low back pain (ALBP) . First, Data collection and cleansing to obtain the final data setWe first identified ANSBP TikTok videos for analysis through three steps.Step 1: Selection of Search Terms for Content DiscoveryUsers can access TikTok videos through the TikTok algorithm or hashtags (#). The TikTok algorithm will serve individualised content to each TikTok user . HashtagStep 2: Raw Data SetWe initially identified a raw data set of the 200 most viewed TikTok videos by searching for #backpain in the TikTok app on September 30, 2021. These 200 TikTok videos were our raw data set, which represented approximately 47% of all TikTok views for #backpain as of September 2021 . Second,Step 3: Cleansing the Raw Data to Produce the Final Data SetOf the 200 TikTok videos identified, 43 non-English, duplicate, and not relevant TikTok videos unrelated to back or spinal pain were excluded. The final data set consisted of 157 TikTok videos in English that were relevant to ANSBP. Relevant videos were those that featured spinal pain as the primary theme. The final data set represented approximately 46% of all TikTok videos tagged with #backpain as of September 2021.Research QuestionsWe used the final data set in order to answer four RQs.RQ1: What are the Metadata Characteristics of Videos in the Final Data Set?We used the metadata obtained via the TSDT to answer this question. Zheluk and Maddock used descriptive statistics to examine length of YouTube videos, the number of views, and channel names . In thisRQ2: What are the TikTok Creator Identities in the Final Data Set?We coded the 157 unique TikTok videos in the final data set according to the creator’s identity. We used the same author identities as Zheluk and Maddock did in their analysis of back pain YouTube videos . We usedRQ3: What are the Intervention Themes in the Final Data Set?We first coded each TikTok video according to one of three intervention themes: education, real-time exercise, or real-time treatment. See Table RQ4: What are the Characteristics of the Final Data Set Based on a Reference Checklist?We analysed the final data set based on the ALBP reference checklist originally developed for the analysis of YouTube videos by Zheluk and Maddock . See TabIntercoder reliabilityCoding was conducted by the three authors of this study. Intercoder reliability was achieved through intercoder consensus ,42. FollWe identified clear differences based on the creator’s identity in the TikTok final data set. We found that the TikTok videos created by chiropractors were consistently among the most viewed, most commented and most shared. Conversely, chiropractic TikTok videos generally had the lowest self-care reference checklist scores relative to all other disciplines. That is, TikTok videos created by chiropractors were least concordant with the reference checklist and, thus, these were least likely to reflect scientific consensus on treating ANSBP.Metadata characteristics of the videos in the final data setWe examined six different metadata characteristics Table . We founTikTok creator identities in the final data setWe coded the 157 TikTok videos in the final data set into six creator identities Table . ChiroprIntervention themes in the final data setWe examined TikTok videos in the final data from the perspective of three types of intervention: education, exercises, or treatment Table . The salCharacteristics of the final data set based on a reference checklistWe analysed the final data set based on the reference checklist. We found differences in the self-care advice contained in TikTok videos based on creator identity Table . Most noTikTok is an increasingly popular medium for disseminating short health messages. This exploratory study aimed to evaluate the scope of ANSBP content available on TikTok as of September 2021. The main cohort using TikTok is young and at risk of ANSBP. However, we suggest that the messages reaching young TikTok users overall do not generally reflect the self-care advice described by Foster et al. ,40. We fMetadataThe metadata for the final data set revealed that chiropractic TikTok videos were consistently the most viewed, commented, and shared in our data set. Zheluk and Maddock  similarly found chiropractic videos were consistently the most commonly viewed and most commented videos on YouTube . We did Creator identitiesChiropractors were the main creator identity identified in our study. Chiropractic TikTok videos in our data set were the oldest of all identities, with an average (mean) of 316 days since publication. This suggests chiropractors have been early adopters of TikTok as a medium. Chiropractic TikTok videos were also the most popular. The higher average age of chiropractic TikTok videos may partially account for their higher view counts. Zheluk and Maddock similarly found a high number of ANSBP videos on YouTube were created by chiropractors [Intervention themesWe examined education, exercise, and treatment as potential intervention themes. The most common intervention theme in our data set was exercise. The limited information about duration, frequency, progression of exercises, and precautions suggest that individuals using this information may be more likely to gain little benefit or risk further pain and injury from conducting these exercises. The short mean duration of TikTok videos in our dataset  compared to YouTube ALBP videos may influence content considerations. TikTok videos in our final dataset frequently featured a single activity. Activities included an exercise, a discussion of pain, or chiropractic manipulation. By contrast, YouTube videos generally offer a slower-paced and more comprehensive examination of low back pain topics. Influencers describing pain and activities of daily living were another notable finding. We coded these influencer TikTok videos as education.Reference checklistOverall, we found limited concordance between popular chiropractic and fitness TikTok videos and the ANSBP reference checklist. Most notably, few videos carried any information about when to visit a medical professional or any other precautionary information. This suggests that by excluding disclaimer information, the short duration of TikTok videos may expose creators to some medicolegal risk. Most identities similarly demonstrated limited concordance with the reference checklist. However, TikTok offers creators the option of creating three and five-minute videos. We found some of the longer videos offered greater scope for presenting content similar to YouTube. This suggests that TikTok has the potential to offer guideline-concordant content and more complex advice. Conversely, these longer videos may be less appealing to TikTok audiences .Translation of methods from YouTube to TikTokWe believe that methods translated from YouTube, as described in this paper, may also be appropriate for the analysis of ANSBP video content on TikTok. Specifically, the methods that may be applied to TikTok videos include: (1) the analysis by creator identity to analyse TikTok metadata. The constantly changing popularity of individual TikTok videos suggests individual videos may not offer the optimal unit of analysis. We suggest an analysis of data sets aggregated by creator identity may provide a more consistent approach to the analysis of broader trends in TikTok health information; (2) The analysis of TikTok videos by intervention themes such as education, treatment, and exercise, in combination with creator identity and metadata appears to offer insights into the scope of health information available beyond simple description. These themes should be grounded themes, i.e., they should be based on analysis of the specific data set; and (3) the use of a reference checklist to assess TikTok videos against consensus guidelines as an approach to evaluating ANSBP content across social media.LimitationsThis paper had several limitations. First, we did not examine the literature on digital health interventions. This study examined the scope of ANSBP TikTok videos available in 2021. By conducting this study we also aimed to extend the methods used for the analysis of TikTok videos. Second, we did not examine TikTok videos from the perspective of misinformation. Suarez and colleagues define health misinformation as a “health-related claim that is based on anecdotal evidence, false, or misleading owing to the lack of existing scientific knowledge” . In thisFurther researchIn this paper, we made limited comparisons between YouTube and TikTok from the perspective of translating methods for analysing videos across social media platforms. We believe further research that examines the optimal use of the unique features of each popular social media platform by various health disciplines is merited.Second, there appear to be several opportunities for health professionals seeking to use TikTok for the dissemination of ANSBP information. We identified a potential demand for information about ADL based on TikTok videos produced by influencers. We suggest that the limited presence of mainstream health professionals such as physicians and physiotherapists offer opportunities to reach younger age cohorts with health messages about ANSBP and other health conditions. The use of TikTok by younger age cohorts to self-care and self-manage ANSBP thus also merits further research.Third, we believe chiropractic TikTok videos merit further research. Chiropractic TikTok videos featuring young women and ASMR represent a novel health marketing phenomenon. We found TikTok videos created by chiropractors to be consistently rated poorly by coders against the ANSBP reference checklist. This marketing phenomenon may be influencing future expectations of what constitutes appropriate ANSBP care among younger TikTok users. Fourth, content analysis cannot examine the actual impact of TikTok videos on individual decision making. Future research may establish this causal relationship. In summary, TikTok is a popular social medium that is under-researched. This paper contributes to the TikTok methods literature and to the understanding of the scope of ANSBP information available on TikTok.TikTok is a popular social media channel among young people and it is plausible that it may influence decision making about what constitutes appropriate ANSBP self-care in young people. However, we found that most messages reaching younger people about ANSBP self-care do not reflect evidence-informed guidelines. The most viewed TikTok videos about ANSBP are not produced by mainstream health professionals. There is scope for mainstream health professionals to provide evidence-informed self-management and self-care content for ANSBP. This requires that mainstream health professionals adopt creative approaches to transmit their ideas effectively via the novel TikTok social media platform.


The treatment of dentin before the use of self-adhesive cements is still a crucial point to achieve the best bond strength values. The objective of this study was to evaluate the bond strength between dentin and composite resin using different adhesion strategies with self-adhesive resin cement. Forty healthy third human molars were randomly divided into 4 groups (n = 10): CA (control); application of self-adhesive cement , AD + CA: only application of conventional adhesive  + self-adhesive cement, AC + AD + CA; conditioning with 37% phosphoric acid for 15 seconds + application of conventional adhesive + self-adhesive cement and AC + CA; conditioning with 37% phosphoric acid for 15s + self-adhesive cement. Blocks made of composite resin  were cemented over dentin. The samples were stored for 24h in distilled water at 37ºC and then were sectioned on a metallographic cutter to obtain tooth picks with approximately 1.0 mm2 in cross section. A universal testing machine was used with a speed of 0.5 mm/min to test the microtensile bond strength,. The fracture patterns were classified as adhesive, cohesive and mixed failures. The data (MPa) were analyzed statistically by One-way ANOVA and Holm-Sidak test (α=5%). The AC + AD + CA and AC + CA groups had the highest averages, while the CA and AD + CA groups had the lowest bond strength values. Adhesive failure was prevalent in all groups. Conditioning with 37% phosphoric acid for 15s increases the adhesion of the self-adhesive resin cement to the dentin, regardless of the use of dental adhesive system. Key words:Resin cement, microtensile bond strength, acid conditioning. Self-adhesive cements, by simplifying the number of steps in the technique, make the cementation procedure less sensitive to errors, unlike what happens with conventional cements, with more clinical stages -5. AnothThe manufacturer’s recommendation does not require the prior stage of conditioning the dental substrates, and washing with application of primer and adhesive, as self-adhesive cements are capable of modifying the smear layer and incorporating it into the hybrid layer -10. Thiset al. , extracted and preserved in 0.1% Timol at 4ºC, were sectioned in the occlusal third of the crown, in the mesio-distal direction, perpendicularly along the long axis of the tooth and another cut parallel to this was made in the third of the crown exposing the dentin fully. Pumice stone prophylaxis was performed on the surfaces and the cut teeth were washed and kept in distilled water at 37°C in an oven.They were randomly divided into 4 groups (n = 10);Group CA (control): application of self-adhesive cement Rely-X U200 only following the manufacturer’s recommendations;Group AD + CA: application of Adper Single Bond 2 adhesive  on the dentin surface with microbrush, photopolymerized for 30 seconds, prior to the application of the self-adhesive cement Rely-X U200;Group AC + AD + CA: conditioning with 37% phosphoric acid for 15s flushing with running water from the triple syringe for 30s, lightly drying the dentin surface with a cotton ball, in order to maintain a slight shine on the surface, applying the Adper Single Bond 2 adhesive prior to the application of the Rely-X U200 self-adhesive cement;Group AC + CA: conditioning with 37% phosphoric acid for 15s and application of self-adhesive cement Rely-X U200.After the application of the resin cement, blocks made of light-cured resin  were cemented over the dentin. The photoactivation of the resin cement was carried out by a Radii photopolymerizer , with LED application for 60s, with 1200 mW/cm². Then, the samples were stored for 24 hours in distilled water at 37°C.After that time, the samples were sectioned in a metallographic cutter (Isomet) to obtain toothpicks with approximately 1.0 mm² of cross section. For the microtensile test, a universal testing machine (EMIC - DL2000) with a speed of 0.5 mm/min was used. The failure pattern was analyzed under an optical microscope and classified into adhesive, cohesive and mixed failures. Prior to the analyzes, fracture resistance data were assessed for normality using the Kolmogorov-Smirnov test. The data (MPa) were analyzed statistically by One-way ANOVA and Holm-Sidak test (5%).Analysis of variance at one criterion showed that there was a statistically significant difference in the strength values of the Bonding between the protocols used. According to the Holm Sidak test, AC + AD + CA and AC + CA demonstrated the highest bond strength values, which did not differ. While AD + CA and CA showed the lowest values of bond strength with no difference between them.The predominant failure pattern among all evaluated techniques was the adhesive type failure. AD + CA obtained the highest number of adhesive failures 100%) and AC + CA the lowest (86%)  positively influences the adhesion of the cementation process of self-adhesive materials. However, because there are still contradictions in the literature on the subject, self-adhesive cementation and its protocols need more research, both in the laboratory and, especially, in clinical trials.Etching with 37% phosphoric acid for 15s increases the adhesion of the self-adhesive resin cement to the dentin substrate, regardless of the use of an intermediate adhesive system.


Escherichia coli bioparticles, and (c) mitogen-induced leukocyte proliferation and cytokine release. In men, lower childhood socioeconomic status predicted decrements in immunological performance across functional assays, along with greater spontaneous cytokine release from PBMCs. These changes co-occurred with elevations in plasma testosterone levels. Similar effects were not observed for other sources of stress, nor were they found in women (with the exception of spontaneous cytokine release). These findings provide evidence that low childhood socioeconomic status has a lasting negative impact on multiple aspects of immune function, particularly in men.Early life stress increases one’s risk for health problems later in life, and many studies find that these effects are sex-differentiated. Here, we examined relationships between multiple sources of early life stress and adult immune function in humans across several functional assays. Adult participants provided retrospective information about their childhood (a) socioeconomic status, (b) household unpredictability, and (c) exposure to adverse experiences. Participants’ peripheral blood mononuclear cells (PBMCs) were then isolated for use in functional assays of immune performance: (a) tumor cell lysis by natural killer cells, (b) phagocytosis of  This propensity is found to operate independently of an individual’s adult economic circumstances7, suggesting that the impact of early life environments on health is long-lasting and not easily reversed. For example, one study found that exposure to childhood socioeconomic disadvantage predicted an increased risk of cardiovascular disease in a cohort of male physicians, despite their achieved adult socioeconomic status far exceeding that of the average American6.Over the last three decades, a considerable body of research has demonstrated that individuals exposed to stressors during childhood exhibit an increased risk for a number of chronic health problems later in life, including metabolic disorders7, the myriad environmental and biological pathways through which one’s early life experiences impact adult health outcomes are only beginning to be understood. Initial clues about contributors to this relationship have surfaced in research demonstrating a link between early life adversity and the tendency to exhibit an exaggerated inflammatory response to immunological stimulation8. Individuals exposed to abuse and parental absence during childhood also tend to have higher peripheral levels of inflammatory biomarkers in adulthood, such as C-reactive protein, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α)9. Moreover, many have proposed that chronic inflammation plays a mediating role in the relationship between early life trauma and adult disease states . Additional clues are found in research demonstrating that adults who report lower childhood socioeconomic status (SES) exhibit increased susceptibility to an experimentally-induced viral infection relative to those reporting higher childhood SES4. However, little is known about the broader immunological context in which such tendencies emerge  or the extent to which individual-level factors buffer or exacerbate the impact of adverse childhood environments.Despite the well-established link between childhood conditions and adult health7, many studies find these effects are sex-differentiated16. For example, early life exposure to infection and nutritional stress are often found to have an asymmetrically negative impact on men’s physical development and health compared to women’s18. Additionally, intervention programs aimed at improving health in the context of poverty are often disproportionately beneficial for boys compared to girls21. On the other hand, research also finds that certain early life stressors, such as maltreatment during childhood, more negatively impact the physical and mental health of women than men (e.g.16). Together, such results suggest that sex differences exist in health-related developmental plasticity, for better and for worse.One individual-level factor that is likely to moderate the relationship between early life environments and immunological function is biological sex. Indeed, although much research finds that developmental experiences can have a lasting impact on both men’s and women’s health12) and stronger canalization of bodily growth in women than men14. Others have proposed that sex differences in behavior play a role in this context, such as the greater tendency for men to engage in health-harming behaviors in stressful environments compared to women22. Further, research in the evolutionary sciences suggests that such differences reflect sex-differentiated energetic investments in mating effort vs. somatic development and repair34.Existing explanations for sex differences in susceptibility to the health consequences of early life stress include factors such as sex differences in hormone levels  What are the critical features of stressful early life environments that drive the association between childhood stress and adult immune function?, (b) How do the effects of these environmental stressors manifest themselves across qualitatively distinct aspects of immunological function?, and (c) Are these patterns sex-differentiated?a, the relative impact of three related, but distinct, sources of childhood stress on immune function were examined, including: (1) resource availability , (2) unpredictability36, and (3) adverse childhood experiences (ACEs37). Isolating the unique impact that each of these environmental correlates of stress has on adult immune function will yield needed insights into the types of environmental interventions that are likely to have a lasting positive impact on adult health outcomes.To address question b, much of the previous work linking childhood environments to adult immune function has focused primarily on inflammatory processes10. In the current research, data were collected on a variety of immunological endpoints, many of which were measured at multiple time-points or at multiple effector-to-target (E:T) cell ratios. These measures included: (1) proliferation of peripheral blood mononuclear cells (PBMCs) in response to, and in the absence of, stimulation by lipopolysaccharide (LPS), phytohemagglutinin (PHA), and polyinosinic:polycytidylic acid (poly (I:C)) measured at 24, 48, and 72 h post-plating; (2) natural killer (NK) cell lysis of target tumor cells at 100:1, 50:1, 25:1, and 12.5:1 E:T ratios; (3) phagocytosis of opsonized Escherichia coli (E. coli) bioparticles; and (4) PBMC release of the pro-inflammatory cytokines interleukin-1β (IL-1β), IL-6, and TNF-α in response to LPS stimulation, as well as in the absence of stimulation , at 2, 24, 48, and 72 h post-plating. Additionally, plasma levels of total testosterone were also assayed to test whether diminished immune function co-occurs with increased investment in mating effort.Regarding question c, in line with the National Institute of Health’s recent policy on accounting for sex as a biological variable38, a number of steps were taken during study design, data collection, and data analysis to allow for targeted tests of sex differences in the impact of early life stress on immune function. These included recruiting equal numbers of men and women , testing interactions between each predictor and sex in all analyses , and clearly presenting sex-differentiated results , sex, and each dependent measure were tested in separate models for each environmental predictor. The results of these models are reported below as the primary analyses  were regressed on the childhood environmental factor, sex, and the interaction between sex and the childhood environment factor. Per convention, if no significant interaction was found, main effects were reported without inclusion of the interaction term. Where significant interactions were found, direct effects of the childhood factor(s) on the dependent immunological measures within each sex  were examined. Sex differences at high and low levels  of childhood factors involved in a significant interaction with sex in the initial models  were also probed. Given that the primary objective of the current study was to test whether the impact of childhood environmental factors on immune function differed by sex, only the simple slopes within each sex are reported in the main text. Regions of significance analysis can be found in the Supplementary Information and Figs. Given that the proliferation (time collected), NK cell cytotoxicity (E:T ratios), and cytokine release (time collected) data all contained nested structures, complex multilevel modeling (cytokine release data) and latent curve models  were used to analyze these data. First, unconditional models for each of the dependent measures  were tested to assess model fit to linear change across repeated measures . Note that while neither of these two childhood variables significantly predicted any immunological outcome in the primary models or models controlling for covariates, results of the partial effects model revealed significant interactions between each of these variables and sex in predicting PHA-stimulated proliferation. Further, there was a main effect of childhood unpredictability on spontaneous cytokine release by PBMCs. These results were not included in the main text as they only emerged in the partial effects model; full results can be found in the Supplementary Information. Additionally, no factors or interactions emerged as significant predictors of unstimulated PBMC proliferation or stimulated cytokine release in vitro.Results of all models revealed good model fit , phagocytosis of E. coli bioparticles, β =  − 0.64, SE = 0.27, t =  − 2.35, p = 0.02, and testosterone levels, β = 0.38, SE = 0.14, t = 2.73, p = 0.006.As predicted based on research indicating greater male susceptibility to adverse health effects of early life stress, results revealed significant interactions between childhood SES and sex in predicting NK cell cytotoxicity , and lower levels of testosterone, β =  − 0.24, SE = 0.06, t =  − 3.79, p < 0.001. For women, childhood SES did not significantly predict phagocytosis, β =  − 0.09, SE = 0.12, t =  − 0.22, p = 0.82, NK cell cytotoxicity , or testosterone levels, β =  − 0.11, SE = 0.13, t =  − 0.87, p = 0.38. Notably, although only reaching statistical significance at the highest E:T ratio , β = 0.21, SE = 0.09, t = 2.44, p = 0.02, but not the slopes of proliferation over time in these conditions (ps > 0.27). These effects were no longer significant after controlling for covariates  and should thus be interpreted with caution. Lastly, there was a main effect of childhood SES on the latent factor of spontaneous in vitro proinflammatory cytokine release by PBMCs , β =  − 0.22, SE = 0.07, t =  − 3.12, p = 0.002, but not stimulated cytokine release, β = 0.04, SE = 0.05, t = 0.86, p = 0.39. Specifically, higher childhood SES predicted a diminished tendency to release proinflammatory cytokines in the absence of overt immunological stimulation. These results remained significant when controlling for both covariates and the effects of the other childhood environmental factors.In addition to these sex-differentiated results, significant main effects of childhood SES  were found predicting the intercept of PBMC proliferation  in response to stimulation by LPS, p-values in ascending order and comparing each p-value to the product of the alpha value  and the quotient of the rank divided by the total number of tests yielding a significant result . If the ranked p-value is less than the above product, the null hypothesis is rejected. This analysis revealed that all significant p-values passed this test  procedure. This method involves rank ordering the significant 10, particularly for men21. The current research sought to extend this work by addressing three key questions that have been heretofore unexplored in the context of a single investigation: (a) What are the critical features of early life stress that drive the association between childhood stress and adult health outcomes?, (b) How do these factors manifest themselves across qualitatively distinct aspects of immunological function?, and (c) Are these patterns sex-differentiated?See Supplementary Information for additional discussion. Considerable research indicates that early life experiences have a lasting impact on adult healthResults revealed that men exposed to lower childhood SES exhibited a diminished immune response across each measure of immunological function, as well as increased spontaneous pro-inflammatory cytokine release by PBMCs, compared to men with a higher childhood SES. Conversely, with the exception of the effects of childhood SES on stimulated PBMC proliferation and spontaneous cytokine release (both effects not sex-differentiated), the measured childhood environmental factors did not significantly predict immunological function in women. Further, with the exception of the relationship between childhood SES and stimulated PBMC proliferation, significant effects were not attenuated when controlling for adult SES , suggesting that these differences are primarily the result of developmental processes rather than current resource availability.10, as well as research demonstrating sex differences in key immunological outcomes28. Further, consistent with research and theory suggesting that androgens play an important in mediating sex-differentiated patterns of immunological investment44, results revealed that, in men, reduced immune performance co-occurs with higher testosterone levels. The results of the current research suggest that men who grow up in conditions of low SES may be more vulnerable to poor health outcomes later in life due to diminished investment in immune function. Moreover, the current results suggest that these differences might emerge in response to differential resource access, per se, as there was little evidence of a negative impact of childhood unpredictability or other adverse experiences  on immune function in either sex. It is important to note that others have found that the relationship between early life disadvantage and markers of poorer health  are driven by alternative features of such environments, such as abuse and parental neglect11, and that these factors may even more negatively impact the physical and mental health of girls relative to boys in certain contexts (see e.g.16). Accordingly, additional research is needed to understand the conditions in which each type of developmental stress is likely to negatively impact immunological development in each sex. Future studies may find, for example, that the timing of stress exposure influences both the magnitude of decrements in immune function, as well as differences in the extent to which men and women are affected. These questions could not be answered in the current study given its cross-sectional design and reliance on retrospective accounts of stress exposure across a wide period of early life.The present results are consistent with a growing body of research demonstrating a negative relationship between early life adversity on adult health45 and levels of these hormones are impacted by early life stress46.There are limitations of the current research that should be considered. First, although several components of immune function were measured in the current study, together they capture only a narrow range of the tremendous complexity that comprises immune function. Accordingly, future research might find that, in women, early life experiences play an important role in influencing other aspects of immune function, such as adaptive immunity. Further, the current research did not experimentally examine the proximate biological mechanisms driving the observed relationships between early life stress and adult immune function. One possibility is that glucocorticoids and other adrenal hormones ) play an important role in this context, as they have immunomodulatory propertiesIt is also important to consider that the majority of the participants in the current study were healthy college students. As a consequence, the low amount of variability in the adult SES variable may have limited our ability to detect effects of participants’ current socioeconomic circumstances on the key immunological outcomes. The present sample’s exposure to certain types of early life stress may also have been lower than that of the general population. Moreover, all of measures of early life stress used in this study were retrospective. Thus, a key direction for future research will be to examine, prospectively, the effects of childhood SES and multiple types of early life adversity on adult immune function in a more diverse sample using a longitudinal design. Such research would minimize the interpretability issues that arise from cross-sectional research using retrospective accounts of early life environments, which is a weakness of the current work. Future research may also benefit from utilizing longitudinal designs to track sex differences in the impact of early life conditions on immune function across multiple stages of development.The current research provides important evidence of sex differences in the impact of early life SES on multiple facets of immune function. Together, these findings suggest that, for men in particular, early life experiences become immunologically embedded in ways that may have a lasting impact on health. These results have important implications for interventions targeted at reducing the negative impact of early life disadvantage on health, and lay the groundwork for future research examining sex differences in relationships between childhood environments and immune function across the lifespan.Mage = 20.17 years, SD = 2.75). Restriction criteria were: (1) participants must have no history of chronic medical problems, including mental illness, (2) must be of healthy weight  < 30), (3) must be free from acute illness for at least two weeks prior to participation, (4) women must not be on hormonal contraceptives, (5) must be willing to abstain from steroidal and non-steroidal anti-inflammatory medications, exercise, and alcohol use for at least 2 days before participating, and (6) must fast the morning of the session. Additional characteristics of the sample are published elsewhere47. Women participated 4–7 days after the start of their most recent menstrual cycle . In exchange for participation, participants were given the choice of partial course credit or a $50 gift card.A total of 159 individuals from Texas Christian University or the surrounding community, participated in exchange for $25 or experimental research credit . The study was conducted in accordance with the protocol approved by the review board.On the day of their sessions, participants arrived at the laboratory at 7:30 AM, provided informed consent, and answered study compliance questions . Next, for the purpose of a larger study, participants completed a series of behavioral tasks and questionnaires. Finally, participants were led into an adjoining room, in which 85 mL of blood was collected by venipuncture into heparinized (or EDTA-containing) Vacutainer tubes .Escherichia coli (E. coli) bioparticles, (3) PBMC proliferation in response to, and in the absence of, mitogen/toll-like receptor stimulation, and (4) PBMC release of proinflammatory cytokines in response to, and in the absence of, mitogen stimulation.Peripheral blood mononuclear cells (PBMCs) were immediately isolated from whole blood using density gradient centrifugation in Ficoll Paque Plus ). PBMCs were used in four functional immunoassays: (1) natural killer (NK) cell lysis of target tumor cells, (2) PBMC phagocytosis of fluorescent dye-labelled l-glutamine, 1 mM sodium pyruvate, 100 U of penicillin/mL, 100 µg of streptomycin/mL, and 0.25 µg of amphotericin B/mL . In addition, plasma was collected at the time of blood processing and frozen at − 80 °C. Additional information about each assay follows, below.For each assay, PBMCs were brought to the plating density appropriate for the assay in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM 48. This measure was chosen to capture the extent to which participants felt they did or did not have access to resources during their childhood. Participants indicated their childhood SES by responding to three statements about their life before age 12  using a 7-point Likert scale . The items together yielded acceptable reliability (α = 0.81) and were formed into a mean composite, with a higher score indicating greater resource availability/a higher SES during childhood .First, participants’ childhood socioeconomic status (SES) was assessed using a previously validated scale36, consisting of three statements about chaos and uncertainty within the home . Participants reported agreement with how well each statement described their lives before age 12 using a 7-point scale . Together, the items yielded acceptable reliability (α = 0.74) and were formed into a mean composite with a higher score indicating greater experience with unpredictability during childhood .Next, childhood unpredictability was measured with a scale used in previous research37. This questionnaire assesses experience with adversity during childhood across five domains: abuse, family dysfunction, and peer, community, and collective violence/war. Given the nature of the present sample, which was primarily composed of college students, a truncated version of the survey was administered, removing the collective violence, sexual abuse, and secondary household abuse questions, and leaving a total of 20 items. Continuous items were recoded as binary, per convention, and all items were then summed into a composite measure of cumulative exposure to adverse experiences during childhood .Finally, participants’ experience with other types of adversity during childhood were also measured using the well-validated Adverse Childhood Experience International Questionnaire (ACE-IQ), developed by research teams from the World Health Organization51Cr-release assay. K-562 target tumor cells  were grown inside T-25 flasks  in RPMI-1640 growth medium supplemented with 10% FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 U of penicillin/mL, 100 µg of streptomycin/mL, and 0.25 µg of amphotericin B/mL  and incubated at 37 °C, 5% CO2, and 100% humidity.NK cell cytotoxicity was measured using the classic 51Cr  for one hour before plating them at a density of 1 × 104 cells/well in a 200 µL final volume into Corning V-bottom plates . PBMCs were plated with target tumor cells, in triplicate, at the following effector:target (E:T) cell ratios: 100:1 (1 × 106 PBMCs/well), 50:1 (5 × 105 PBMCs/well), 25:1 (2.5 × 105 PBMCs/well), and 12.5:1 (1.25 × 105 PBMCs/well). Spontaneous/background lysis controls  and maximal lysis controls ) were plated in sextuplicate. Plates were incubated for four hours at 37 °C, 5% CO2, and 100% humidity.Target tumor cells were labeled with 1 µCi 51Cr release on a CAPRAC-t gamma counter . Percent maximal lysis of target tumor cells by participant NK cells was calculated at each E:T ratio by dividing 51Cr release by maximal release after subtracting spontaneous release from both values and multiplying by 100.After brief centrifugation of the V-bottom plate, supernatants were collected into glass scintillation vials and quantified E. coli BioParticles  that were opsonized using 1 mg/mL of manufacturer-provided opsonization buffer. PBMCs were plated in triplicate with E. coli bioparticles into BrandTech black, flat-bottom microplates  at a density of 5 × 105 cells/well in a 200 µL final volume. Negative controls (bioparticles plated with media only in 200 µL volume) and positive controls ) were plated in triplicate. Plates were incubated for two hours at 37 °C, 5% CO2, and 100% humidity before being read on a fluorescence plate reader  at FITC dye settings of 490 nm excitation/520 nm emission. Percent maximal fluorescence was computed by dividing experimental fluorescence by maximal fluorescence , after subtracting fluorescence in the negative controls from both, and multiplying by 100.The phagocytic capability of participants’ PBMCs was assessed using fluorescent pHrodo Green 5 cells/well in a 200 µL final volume for four conditions: (1) with media only , (2) with 1 µg/mL of lipopolysaccharide (LPS) obtained from E. coli , (3) with 5 µg/mL of phytohemagglutinin , and (4) with 50 µg/mL of polyinosinic:polycytidylic acid (poly (I:C); high molecular weight; InvivoGen, San Diego, CA). Plates were incubated at 37 °C, 5% CO2, and 100% humidity.PBMCs were isolated as described above, and plated into Falcon 96-well tissue culture plates . PBMCs were plated in triplicate at a final density of 2.5 × 10Approximate cell density was measured at three time-points: 24, 48, and 72 h post-plating using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay . Each plate was read on a plate reader  at 490 nm.5 cells/well, in a 200 µL final volume. PBMCs were plated both in media only (unstimulated condition), as well as with 1 µg/mL of LPS, obtained from E. coli , and were incubated for up to three days at 37 °C, 5% CO2, and 100% humidity. Cell culture supernatants were collected at 2, 24, 48, and 72 h post-plating, and then stored them at − 80 °C until assays were conducted.PBMC release of proinflammatory cytokines was measured in vitro both in response to LPS stimulation, as well as in the absence of stimulation. After isolation, PBMCs were plated in triplicate at a density of 2.5 × 10Cell culture supernatants were later thawed and assayed in duplicate for levels of a trio of proinflammatory cytokines: interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) using a MILLIPLEX MAP Human Cytokine Panel magnetic bead kit , and read using a Luminex MAGPIX fluorescent detection system  and xPONENT software . Intra-assay coefficients of variation (CVs) were 8.20% (IL-6), 6.97% (IL-1β), and 5.98% (TNF-α). Inter-assay coefficients of variation (CVs) were 17.27% (IL-6), 10.53% (IL-1β), and 11.62% (TNF-α).47). No compromised samples were assayed, and thus data from these samples were not included in any analysis. All other biological samples from these participants  were stored elsewhere and were thus unaffected by the freezer failure.Due to a freezer failure on November 5, 2016, cell culture supernatant samples were compromised for 32 participants . Plates were read on a plate reader  at a wavelength of 450 nm. The intra-assay CV was 2.48% and the inter-assay CV was 13.45%.Several variables that may influence relationships between early life conditions, sex, and immune function were measured. These included race, age, exercise, sleep, BMI, stress, loneliness, recent illness, season, and adult SES. See Supplementary Information for more details.Supplementary Information.