To elucidate the role of syringing in assessing nasolacrimal duct (NLD) stenosis and non-anatomical functional NLD delay.Consecutive adult patients with epiphora attending a tertiary lacrimal clinic from June 2011 to March 2021 were reviewed. Cases with evidence of canalicular stenosis or other identifiable causes of epiphora were excluded. Following syringing, patients were investigated with dacryocystography (DCG) and dacryoscintigraphy (DSG). The sensitivity and specificity of syringing were evaluated using the combined findings on DCG and DSG.p = 0.17).A total of 289 symptomatic lacrimal systems  were included. More than one-third of cases with both normal DCG and DSG were noted to have some degree of reflux on syringing . The sensitivities were considerably low for NLD stenosis  and for functional NLD delay , of which 43.7% (95% CI 32.2–55.9%) and 54.3% (95% CI 45.7–62.7%) had full patency on syringing, respectively (Full patency on syringing was unreliable for ruling out NLD stenosis and functional delay. Furthermore, a positive syringing may be associated with functional NLD delay and cannot reliably differentiate it from stenosis. Lacrimal syringing is commonly used to investigate epiphora , 2, but Lacrimal investigations such as dacryocystography (DCG) and dacryoscintigraphy (DSG) can be used to evaluate the anatomy and function of the lacrimal drainage system, respectively , 9. HoweThe current study aimed to determine the sensitivity and specificity of syringing in NLD drainage impairment , as diagnosed by combined DCG and DSG findings.Data were collected retrospectively from consecutive adult patients with epiphora attending the Royal Adelaide Hospital lacrimal clinic from June 2011 to March 2021. The study received Institutional Review Board (IRB) approval and adhered to the tenets of the Declaration of Helsinki.Patients were excluded if they had puncto-canalicular obstruction/stenosis, eyelid malposition/paralysis, potential causes of reflex tearing, acute dacryocystitis, refluxable mucoceles, or previous lacrimal surgery. All patients underwent syringing as part of their clinical assessment. Trained, experienced oculoplastics surgeons carried out syringing. A lacrimal cannula on a 2-ml syringe was inserted 1–2 mm vertically through the punctum. The lid was distracted laterally by the operator’s finger and kept under tension. The lacrimal cannula was slowly advanced through the canaliculus until it reached a hard or soft stop. The cannula was held in place while syringing was performed under minimal pressure. The degree of reflux was recorded. As all suspected cases of canalicular or common canalicular obstruction (or stenosis) were excluded from this study (focusing on NLD impairment), the site of reflux was always the opposite punctum.Patients were subsequently investigated with DCG and DSG performed by trained radiologists. An experienced oculoplastic surgeon assessed the imaging studies.DCGs were performed with patients in the supine position. A drop of topical anesthetic (1% tetracaine hydrochloride) was instilled into the inferior conjunctival fornix of both eyes. The punctum was cannulated with a 27-gauge lacrimal cannula. Baseline X-ray images were taken, followed by real-time imaging during injection of contrast  through the cannula. This allowed digital subtraction of the pre-contrast image from post-contrast images. “NLD stenosis” was defined as having a duct diameter of less than that of the width of the lacrimal cannula tip on the X-ray image  but with patency . “NLD obA 10-ml drop of technetium-99 m pertechnetate was placed into both eyes with the patient sitting upright in front of a gamma camera. One-minute sequential images were taken over 45 min. At the end of the serial scanning, if the tracer has not sufficiently progressed to reach the nasal cavity in any eye, the participant was asked to clear their nasal passages, and a lacrimal massage was applied to both eyes. Another 45 min of a 1-min sequential scan was then subsequently acquired. The appearance of tracer at the lacrimal sac, lacrimal duct, or nasal cavity was recorded. Five minutes was used as the cut-off time-point to qualitatively determine normal versus post-sac (NLD) delay based on end-tracer location , 12.NLD drainage assessment was based on the combined DCG and DSG findings and categorized into four categories: Normal , NLD obstruction (NLDO) , NLD stenosis (NLDS) , or functional NLD delay (FNLDD) .The grading of the proportion reflux on syringing was utilized to define patency. In order to analyze the degree of reflux as a differentiating feature, the cut-off for defining a “positive”  syringing was set according to four different criteria and analyzed separately: “full patency” ; “80%  patency” (< 20% reflux versus ≥ 20% reflux); “50%  patency” (< 50% reflux versus ≥ 50% reflux); or “no patency” .Syringing specificity values correspond to the proportion of lacrimal systems with normal NLD drainage (DCG and DSG) and negative in the syringing test (true negatives).The syringing sensitivity values correspond to the proportion of lacrimal systems with NLD drainage impairment (DCG and/or DSG) and positive in the syringing (true positives).p-value of less than 0.05 was considered significant.Data were analyzed by the StatSoft Statistica software, version 10 . Proportions were compared by the chi-square test. A two-sided A total of 289 symptomatic lacrimal systems of 197 patients met the inclusion criteria: 85 patients had unilateral symptoms, 73 had bilateral and equal symptoms, and 29 patients had bilateral and asymmetrical symptoms (as per patient-reported symptoms). The mean age was 65.5 ± 14.9 years (range 18–94 years), and 130 (66%) were females.Overall, 43 (14.9%) lacrimal systems had normal drainage on DCG and DSG, 55 (19%) had NLDO, 64 (22.1%) had NLDS, and 127 (43.9%) had FNLDO.Table Of the 43 lacrimal systems demonstrating normal drainage on DCG and DSG, 65.1% (95% CI 50.2–77.6%) were fully patent (true negatives) on syringing. This denotes a moderate specificity when defining an abnormal syringing as any reflux, as more than a third of cases were falsely positive using this cut-off.The specificity improved when using syringing less stringent, “partial patency” (cut-off) criteria Table . Namely,NLDO manifested as (> 0%) reflux on syringing in 90.9% (95% CI 80.4–96.1%) of cases, representing a high sensitivity (true positives) at this syringing test cut-off. The sensitivity slightly declined in syringing’s partial patency (cut-off) criteria; however, it remained high .In cases of NLDS, the sensitivity of syringing was moderate  at > 0% reflux and was low across the less stringent cut-off definitions (Table p = 0.17 compared to the sensitivity in NLDS), 37.0% (95% CI 29.1–45.7%) at ≥ 20% reflux (p = 0.09 compared to NLDS), 17.3% (95% CI 11.7–24.8%) at ≥ 50% reflux (p = 0.004 compared to NLDS), and 7.1% (95% CI 3.8–12.9%) at 100% reflux (p = 0.06 compared to NLDS).The sensitivity of syringing in FNLDD was low across all cut-off definitions Table : 45.7%  failed to detect NLDS in 44% and FNLDD in 54% of cases. These results confirm that lacrimal syringing is unreliable for ruling out NLD stenosis or functional delay. Furthermore, full patency (or a negative syringing) is used by many to be a criterion for the diagnosis of non-anatomical FNLDD –16. Our The results also suggest that while any reflux may indicate pathology, it is also found in “normal” systems as defined by imaging. That is, the specificity of syringing was found to be limited, as approximately one-third of the cases that had normal NLD drainage on imaging were noted to have some degree of reflux on syringing. On the other hand, no patency on syringing was highly specific, with only one false-positive case. Taken together, no patency on syringing would suggest NLD impaired drainage with high confidence (98%), while partial (but not full) patency would suggest impaired drainage with lower confidence (65%).The detection of NLD stenosis and non-anatomical functional delay is particularly challenging in the clinical setting, and the role of syringing in these cases has not been clearly characterized to date . SyringiWhereas other studies have analyzed the relationship of syringing to DCG and DSG findings separately, to our knowledge, this study is the first to correlate lacrimal syringing to the combined (and complementary) findings on DCG and DSG , 12, 15.The correlation between syringing and lacrimal imaging  has been previously investigated; however, various definitions for full patency or a normal syringing were used. Some authors defined a fully patent syringing as less than 20% reflux , 12, othThese methodological discrepancies notwithstanding, our reported syringing general specificity 65%) and sensitivity for NLDO (91%) compare favorably with the figures reported by Nixon et al. [5% and seOne previous study compared syringing to DCG and DSG separately . The autThe strengths of this study include a large number of patients and the use of comprehensive imaging (DCG and DSG), allowing analysis of homogenous etiologies of drainage impairment . This study’s limitations firstly include its retrospective nature. Another limitation is using DCG and DSG as the diagnostic reference, as these modalities are themselves confined by investigative sensitivity and specificity constraints , 19, 20.Last, since syringing is a crude test, inter-tester variations in the exact pressure and subjective estimation of reflux (proportions) are possible. Nevertheless, in the current study, oculoplastic surgeons performed syringing, all trained under the last author (DS), who supervised and validated the technique. Thus, adherence to the same testing standards could be assumed for the entire study period. Furthermore, we categorized the estimated reflux to four cut-offs  which were scrutinized separately. Thus, grouping into categories (ranges) and not relying on “exact” proportions should minimize inter-tester bias.In conclusion, full patency on syringing was unreliable for ruling out NLD stenosis or functional delay. Hence, in patients with troublesome epiphora who are fully patent on syringing, imaging may determine the presence of NLD impairment and exclude those with normal systems which would be unlikely to benefit from intervention.We believe that based on the results, any reflux should be considered an abnormal test in the context of epiphora. Finally, a positive syringing may be associated with functional NLD delay and cannot reliably differentiate it from stenosis, and this may have implications for consenting patients regarding success rates of intervention.


We sought to determine the association between mental stress-induced myocardial ischaemia (MSIMI) and quality of life (QoL) in patients with coronary artery disease (CAD) after coronary revascularization.This cohort study involved patients with high-risk MSIMI who received coronary revascularization between Dec 2018 and Dec 2019. Patients who screened positive for depression/anxiety were enrolled in this study. Mental stress was induced by the Stroop Colour and Word Test 1 month after coronary revascularization. All participants underwent single photon emission computed tomography (SPECT) scans at rest and under mental stress. MSIMI was defined as the presence of four abnormal SPECT phenomena. QoL was assessed using the Seattle Angina Questionnaire (SAQ) prior to treatment and 1 month after coronary revascularization.P = 0.005), and the proportion of deterioration in QoL was higher  in the MSIMI group than in the non-MSIMI group. Those with a deterioration in QoL had approximately twice the rate of MSIMI than those with an improvement in QoL .Of the 1845 consecutive patients who received coronary revascularization, 590 (31.9%) had depression/anxiety, and 205 agreed to accept the mental stress test. During the average observation period of 33 days, 105 (51.2%) patients exhibited MSIMI. All SAQ subscales showed significant improvement, except for QoL, in the MSIMI group. The QoL score was lower (− 0.2 ± 32.7 vs. 13.1 ± 29.9, Among patients with CAD who received coronary revascularization and had depression/anxiety, deterioration in QoL increased the likelihood of MSIMI. Hence, our results indicate that deterioration in QoL is a predictor of MSIMI.Trail Registration ChiCTR2200055792, retrospectively registered, 2022.1.20, www.medresman.org.cn Mental stress-induced myocardial ischaemia (MSIMI) is defined as an imbalance between myocardial oxygen supply and demand during mental or psychological stress. The data demonstrate that mental stress triggers transient myocardial ischaemia in 30–70% of patients with pre-existing coronary artery disease (CAD) –3. To daThe Seattle Angina Questionnaire (SAQ) is specific for CAD and has been widely accepted as a means of quantifying the outcome of CAD treatment on patients’ angina and QoL in trials . The SAQTherefore, this study was designed to examine the association between SAQ and MSIMI in high-risk patients with depression/anxiety prior to revascularization and whose CAD stabilized after at least 1 month of revascularization. In addition, the association between MSIMI and the benefit or drawback of CAD revascularization assessed by SAQ was evaluated.In the study of CAD with a high risk of MSIMI patients, 1845 patients with coronary angiography-confirmed CAD who received coronary revascularization treatment, including percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG), were screened between Dec 2018 and Dec 2019. The mental health status of all patients was assessed using the nine-item Patient Health Questionnaire-9 (PHQ-9) and the seven-item Generalized Anxiety Disorder-7 (GAD-7) severity measure. Those with screened depression/anxiety (PHQ-9 ≥ 5 and/or GAD-7 ≥ 5) were enrolled in this study. The study protocol was approved by the Medical Ethics Committee of Beijing Anzhen Hospital (NO. 2019001), and all participants provided informed consent prior to inclusion in this study.Mental stress was induced by the Stroop Colour and Word Test (SCWT), which was employed to assess the ability to inhibit cognitive interference. The SCWT has been well accepted for assessing patients with CAD and has been shown to exhibit good reproducibility and execution , 10. A s99mTc)-sestamibi SPECT imaging 1 month after PCI/CABG. All participants underwent two SPECT scans on two separate days: the first one at rest and the second one after mental stress. The patients received 99mTc-sestamibi at a dose of 20–25 mCi during the rest and stress phases. The SCWT mental stress test lasted approximately 5 min, and 99mTc-sestamibi was intravenously administered 1 min after the test started. Images were acquired using an e-Cam Duet gamma camera  60–90 min after the intravenous administration of 99mTc-sestamibi.Each participant underwent electrocardiographically gated technetium-99 m , end-diastolic volume (EDV), end-systolic volume (ESV), contractility, and myocardial thickness . Four abThe 19-item SAQ questionnaire, which measures five domains related to CAD, was used to assess angina frequency, physical limitations, angina stability, treatment satisfaction, and QoL. The scores ranged from 0 to 100, with higher scores indicating fewer symptoms and a better health status . The par2 test or Fisher’s exact test, whichever was appropriate. Each domain of the SAQ was evaluated using analysis of covariance before and after revascularization. Continuous variables were compared between groups using independent samples t test or Mann–Whitney-U Test. Within the same group, the differences in SAQ scores before and after revascularization were compared using a paired samples t test. For all other endpoints, a two-sided probability (p) value of 0.05 without correction for multiple testing was considered statistically significant. Logistic regression was used to identify the factors associated with MSIMI. The model was adjusted for coronary risk factors and imbalance factors before revascularization.Continuous variables were reported as the mean ± standard deviation or median (Q1/Q3) when appropriate, and categorical variables were reported as numbers and percentages. Concomitant medication was summarized and compared between the groups at discharge and after 1 month using the χOf the 1,845 consecutive patients with CAD who received PCI/CABG, 590 (31.9%) exhibited depression/anxiety, and 205 agreed to undergo the mental stress test. Overall, 105 (51.2%) patients exhibited MSIMI. The enrolment and evaluation process are shown in the flow chart , and 64 (31.2%) of the participants were women. The mental stress test was performed subsequent to an average observation period of 33 days (30/40) after coronary revascularization.Among 105 patients exhibiting MSIMI, 59 (56.2%) had reversible myocardial perfusion defects, 30 (28.6%) had an EF reduction of ≥ 5%, 27 (25.7%) had TIDs, and 13 (12.4%) had reverse redistribution. Furthermore, 82 patients had only one abnormal phenomenon, 22 patients displayed a combination of two abnormal phenomena, and 1 patient exhibited three abnormal phenomena. The characteristics of the 205 patients before revascularization in the MSIMI and non-MSIMI groups are shown in Table P < 0.05). Furthermore, EF was higher in the MSIMI group than in the non-MSIMI group . On the day of the mental stress screening, there were no significant differences in EDV, ESV, or EF between the two groups, except for the higher proportion of stress TPD in the MSIMI group. Moreover, non-invasively measured SBP, DBP, HR, and RPP were similar in the two groups both at rest and during the mental stress test , except for the QoL subscale in the MSIMI group. Although the QoL improved significantly in the non-MSIMI group after coronary revascularization, the score was numerically but not statistically lower in the MSIMI group .The five subscales measured using the SAQ questionnaire were similar between the non-MSIMI and MSIMI groups before revascularization. The only exception was physical limitations, which were significantly lower in the MSIMI group than in the non-MSIMI group , and fewer patients in the MSIMI group showed a significant improvement in the QoL subscale . Overall, QoL showed a consistently deteriorating trend in the MSIMI group but not in the non-MSIMI group.Table In the cohort that had CAD associated with depression/anxiety, the probability of MSIMI after 1 month of coronary revascularization was approximately two times higher in patients with a deterioration in QoL than in those with an improvement in QoL . Similarly, an improved QoL score of < 16 was associated with a twofold increase in the probability of MSIMI when compared with a score of ≥ 16   reported angina. It was found that only women who reported angina had MSIMI % reporte, 20, 21.The SAQ scores have been reported to be independently associated with 1-year cardiovascular events among patients with CAD and predict 1-year mortality and cardiac rehospitalizations , 24. TheThe main strength of our study is the selection of patients with a high risk for MSIMI. Previous studies have shown that depression/anxiety may be a risk factor for MSIMI , 20, 21.One of the key limitations of our study was the short follow-up period. The average follow-up time was only 33 days. In future research, we plan to follow up and obtain SAQ results at 3 months, 6 months, and 12 months to dynamically evaluate the association with MSIMI. In addition, we did not include the effects of MSIMI and major adverse cardiovascular events. In the future, we intend to follow-up on the events using repeat coronary angiography and psychological stress tests at 12 months.To the best of our knowledge, this is the first study demonstrating that deterioration in CAD-related QoL is associated with an increased likelihood of MSIMI among patients with CAD and depression/anxiety. Our results highlight the relationship between QoL and the likelihood of MSIMI.


SCGN), a regulator of synaptic transmission, as a new risk gene for ASD. Two heterozygous loss-of-function mutations in SCGN are presented in ASD probands. Deletion of Scgn in zebrafish or mice leads to autism-like behaviors and impairs brain development. Mechanistically, Scgn deficiency disrupts the oxytocin signaling and abnormally activates inflammation in both animal models. Both ASD probands carrying Scgn mutations also show reduced oxytocin levels. Importantly, we demonstrate that the administration of oxytocin and anti-inflammatory drugs can attenuate ASD-associated defects caused by SCGN deficiency. Altogether, we identify a convergence between a potential autism genetic risk factor SCGN, and the pathological deregulation in oxytocinergic signaling and immune responses, providing potential treatment for ASD patients suffering from SCGN deficiency. Our study also indicates that it is critical to identify and stratify ASD patient populations based on their disease mechanisms, which could greatly enhance therapeutic success.Autism spectrum disorder (ASD) affects 1–2% of all children and poses a great social and economic challenge for the globe. As a highly heterogeneous neurodevelopmental disorder, the development of its treatment is extremely challenging. Multiple pathways have been linked to the pathogenesis of ASD, including signaling involved in synaptic function, oxytocinergic activities, immune homeostasis, chromatin modifications, and mitochondrial functions. Here, we identify secretagogin ( Two antipsychotic drugs, risperidone and aripiprazole, are the only FDA-approved drugs for ASD patients;7 however, both have limited effects and the potential for significant side effects. Further understanding of the mechanisms of pathogenesis will be indispensable for the development of novel therapeutic strategies for ASD.Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental diseases characterized by deficits in communication and social interaction.10 So far, over 1000 ASD candidate genes have been included in the SFARI (Simons Foundation Autism Research Initiative) database (https://gene.sfari.org/autdb/GS_Home.do). However, the pathological mechanisms of these genes remain largely uncovered. In recent years, studies including genome-wide sequencing technologies have revealed a large set of ASD-risk genes,14 which are often associated with a number of biological functions/pathways, including synaptic structure and functions , chromatin modification , Wnt signaling , oxytocinergic signaling, immune function, and mitochondrial function, among other processes.17 One emerging concept in the field is that these pathways could converge on the regulation of a few key functional activities. For instance, the loss of synaptic adhesion molecule Nlgn3 impairs oxytocin signaling and disrupts normal translation. Conversely, pharmacologic treatment of Nlgn3-knockout mice resets the translation homeostasis and restores oxytocin responses.18 Therefore, dissecting the functions of ASD-risk genes may be essential to understand the heterogeneity of autism, and knowledge of their common and unique underlying mechanisms may help to develop therapeutic strategies with improved efficacy.The variable phenotypical presentations of ASD are mirrored by its complex causes. Genetic, prenatal, perinatal, and environmental factors may collectively contribute to ASD development. Nonetheless, 74~93% of ASD risk is estimated to be heritable.SCGN) is a gene conserved from fruit flies to humans and is expressed in multiple endocrine organs, including the pancreas, the gastrointestinal tract, the thyroid, the adrenal medulla, the adrenal gland, and the brain.20 It is highly enriched in pancreatic β-cells, where it controls insulin secretion21 and is similarly expressed by a subset of enteroendocrine cells in the gut,19 where it also facilitates hormone release. In the human brain, SCGN is highly expressed in the hippocampus and cerebellum, where it mediates the secretion of multiple proteins, including matrix metalloproteinase-2 (MMP2) and corticotropin-releasing hormone (CRH).23SCGN encodes a calcium-sensing protein that interacts with the SNARE core component SNAP-25 in a Ca2+-dependent manner to regulate membrane fusion events.24 In our recent study, we reported the crystal structures of SCGN in complex with SNAP-25, and showed that scgn was critical for neuronal development in zebrafish.25 SCGN deficiency has been linked to the pathogenesis of various disorders, including diabetes, schizophrenia and neurodegeneration, and more recently, autosomal recessive early-onset ulcerative colitis.30 However, the molecular functions of SCGN in the nervous system and its connection with neurological disorders are still unclear.Secretagogin  inherited from his mother, which resulted in a missense mutation in SCGN  inherited from his mother, as validated by Sanger sequencing  which retained the following intron and might subsequently lead to protein degradation or loss of function. Indeed, a minigene splicing assay demonstrated that the mutation identified from ASD0014 significantly altered the normal splicing of SCGN , the Autism Behavior Checklist (ABC), the Clancy Autism Behavior Scale (CABS), and the Developmental Quotient (DQ) tests. Among 168 cases that we have sequenced, two individuals were identified with mutations in CGN Fig. . The othing Fig. . BioinfoScgn-deletion using CRISPR/Cas9 gene editing technology. Remarkably, scgn knockout in both animal models exhibited shared phenotypes. We focus on our zebrafish data and discuss some common findings of mouse and zebrafish models here.To determine the role of SCGN in social behavior and neuronal development, we generated zebrafish and mouse models with 32 in which 4-month-old zebrafish  were placed in a tank with their swimming behaviors recorded and analyzed , followed by a second stranger fish group introduced to the upper right chamber for the next 5 min (novelty session). Behavior recording of the tested individual was conducted during the entire time  test to detect social preference and social novelty in two consecutive sessions within the same place.ime Fig. . Relativlty Fig. , as asseons Fig. . AltogetSCGN variants displayed delayed overall and neuronal development, we next investigated the roles of scgn in zebrafish brain development.32 Compared with scgn+/+ embryos, the size of the head was significantly reduced in scgn−/− embryos at 48 hpf  resulted in abnormally short and branched axons at 48 hpf, functionally similar to Scgn deletion. Importantly, co-injection of mRNA encoding human wild-type SCGN could effectively restore the axonal phenotypes, including the axonal lengths and branch numbers. Meanwhile, SCGN R79Q, capturing the zebrafish Scgn residue at this site, was also able to rescue the axonal morphologies, but the mutation R79W seen in our proband failed to rescue neuronal morphology  and deficient (scgn−/−) animals  of transcriptome analysis in mouse brain based on rlog values of all expressed genes showed that gene expression in Scgn-deficient animals was substantially different than in control mice  results indicated that oxytocin was expressed near the lateral edge of the neural tube in zebrafish embryos Fig. . Both ISoup Fig. . Oxytociyos Fig. . Zebrafiscgn deficiency, particularly the neuronal developmental impediment noted in zebrafish. We chose two anti-inflammatory drugs, dexamethasone (DXMS), a synthetic corticosteroid, and aspirin . We also tested carbetocin, a synthetic analog of oxytocin with an increased half-life. Zebrafish embryos were exposed to water with or without three different drugs from 1 dpf for 1 day  rescued the social interaction for 1/3 to 1/2 of scgn−/− zebrafish. Consistently, aspirin administration significantly reduced the expression of IL-6, IL-10, and IL-12 in scgn−/− zebrafish   in the hypothalamus was the focus of our analysis because of the high levels of SCGN expression and oxytocin production in this region.42 Social approach was analyzed first, using a three-chamber test  and sniffing times . No significant differences were detected between shScgn and shControl mice in terms of social preference of S1 or O, calculated by chamber times  and sniffing time  were both profoundly increased by oxytocin administration. Thus, treatment with oxytocin could improve the impaired social novelty behaviors of Scgn-deficient mice.Next, we performed a series of experiments to compare the behavior of shScgn and shControl mice.ors Fig. , c. Afteors Fig. . Similarors Fig. . As a coScgn knockout mice. Quantitatively, shControl and shScgn mice buried an average of 5 and 11 marbles, respectively . Also, shScgn mice spent more time self-grooming than the control mice, as shown by an average of 100 s for shScgn mice and 40 s for shControl mice  isoforms with pivotal roles in neuronal development.45 The network also illustrated 97 ASD-risk genes that were associated with the inflammatory/immune response pathway, including Tek, Fbn1, Dpp4, Igf1, Thbs1, Fgfr1, and Ptgs2. Specifically, Ptgs2 was found to be a common gene shared by the oxytocin pathway and the inflammatory/immune response pathway,47 which encodes cyclooxygenase-2 (COX-2), a key enzyme in prostaglandin biosynthesis normally induced during inflammation or by oxytocin. This analysis is consistent with our data that aspirin, an inhibitor of COX-2, attenuates social dysfunction in scgn−/− zebrafish larvae  in the 20 It has been reported that SCGN binds with SNAP-25 with high affinity and functions together to regulate hormone secretion.49 Remarkably, the deregulation of both genes has been linked with ASD.53 In this study, we further demonstrate that SCGN is critical for the secretion of oxytocin. Previous studies using single-cell sequencing demonstrate that SCGN + neurons express corticotropin-releasing hormone (CRH), but not oxytocin.23 Since CRH is a well-known regulator of oxytocin secretion,54 we suspect that SCGN controls the secretion of oxytocin, via CRH. Whether SCGN directly regulates the secretion of oxytocin or indirectly impacts its secretion via other hormones or neurotransmitters, still needs to be addressed. Notably, Nlgn3, another synaptic regulator linked to ASD, has been shown to regulate oxytocinergic signaling with the deletion of Nlgn3, resulting in reduced levels of oxytocin in mice.18SCGN is found primarily in pancreatic β-cells, the central nervous system, and enteroendocrine cells.57 Especially, an increase in the number of microglia in Scgn-deficient mice shows that neuroinflammation is linked to the oxytocin pathway in PVN. In response to SCGN deficiency, we identified the potential interconnectivity between oxytocin signaling and the inflammatory/immune response pathway. The fact that the administration of oxytocin analogs could attenuate pro-inflammatory gene expression suggests that disrupted oxytocin might function upstream of the impaired inflammatory pathway. This is supported by numerous studies showing that defects in the oxytocin signaling pathway can lead to pro-inflammatory responses.60 Children with ASD often have lower levels of oxytocin in their serum compared to their healthy peers.61 However, it is very controversial whether the administration of oxytocin could benefit autistic children. Several studies have shown that intranasal oxytocin can improve impaired social behavior in autistic children;63 a recent study, however, found that oxytocin did not confer any significant benefit.64 Possible reasons for these inconsistencies might include the lack of differentiation in the original serum oxytocin levels of participants and ineffective enrichment of the administrated oxytocin at the proper functional sites within the brain. In light of this, our study showed that oxytocin administration could improve social behavioral deficit behaviors in both SCGN mutant zebrafish and mice. Meanwhile, the importance of oxytocin was further addressed using the mouse model with a specific deficit in PVN, which primarily accounted for oxytocin production. It is possible that oxytocin administration may confer more benefits for autistic individuals presented with significantly reduced oxytocin levels, like the ASD probands carrying SCGN mutations that were studied here. Moreover, our findings suggest that a potential approach may also be the use of commonly prescribed anti-inflammatory medications, such as aspirin, for improving SCGN-deficiency-associated ASD symptoms. This may help to ease the financial burden and the adverse events associated with the current pharmacological use of dopamine-receptor and serotonin-receptor antagonists for ASD patients.It is noted that abnormal oxytocin signaling and inflammation regulation have been associated with many neurological disorders, in addition to ASD.Altogether, we have shown that functional disruption of SCGN results in autistic symptoms via abnormal pro-inflammatory responses and deregulated hormone secretion, suggesting that the administration of oxytocin and anti-inflammatory drugs as potential treatment for individuals with SCGN deficiency. In the era of precision medicine, these mechanistic studies will help to develop personalized treatments and to broaden the currently limited options for ASD therapeutic interventions.It was noted that the exact SCGN mutations were kept between the maternal parents as their sons, suggesting an incomplete penetrance probably resulted from the maternal-fetal effect rather than gender- or age-specific phenomenon. It seemed that the lack of maternal serum oxytocin during pregnancy served as the fundamental determinant for the neural development deficits of the fetuses, resulting in the collected ASD symptoms found in these probands. Retrospectively, the neural development of these SCGN mutation-carrying mothers were otherwise safe-guarded by the normal levels of oxytocin from the maternal grandmothers of these proband boys, who exhibited wild-type SCGN genotypes. This conjecture cannot be confirmed at present due to the limited pedigree information available and should be further addressed in future studies with larger cohorts.65 Impaired synaptic function and changes in axonal branching are among the important mechanisms of autism.66 We have not shown the data on axonal branching and dendritic spines and only showed the depletion of axonal length and branching in zebrafish.SCGN is a critical regulator of synaptic transmission.This work is approved by Medical Ethics Committee of Beijing Children’s Hospital, Capital Medical University (2018-k-62). Ethics approval was locally obtained for genetic analyses and/or data sharing for additional patients. Genetic analyses and patient inclusion were performed in accordance with the ethical standards of the GCP principles and relevant national laws and regulations. Informed consent for genetic analyses was obtained from all individual participants included in this study or their legal guardians.The included 168 patients were recruited from psychiatry outpatient clinics of the Beijing Children’s Hospital, Capital Medical University, and were diagnosed with autism by two independent psychiatrists. Enrollment occurred between July 2019 and January 2020. Written informed consent to participate was obtained from the parents or legal guardians of all of the participants (under the age of 10). Patients who had been diagnosed with ASD according to the criteria of DSM-IV. Patients with a definite history of craniocerebral trauma, neurological disease, and severe physical illness were excluded. The behavior and severity of patients were assessed with the Autism Behavior Checklist, Clancy Autism Behavior Scale, and Childhood Autism Rating Scale.scgn heterozygous (scgn−/−) and homozygous (scgn−/−) mutants; AB Wild-type (WT), Tg Hb9:GFP (WT) and Tg Huc:GFP (WT) stains.All experiments were approved by the Animal Welfare Center of West China Hospital of Sichuan University. Zebrafish (Danio rerio) were kept in accordance with the University of Sichuan institute animal welfare guidelines. The dark and light conditions were set for 10:14 h. Feedings were given twice per day. The following zebrafish strains were used: Scgn. Designed AAV delivers of shRNA targeting mouse Scgn  and shControl-gfp were obtained from Beijing Syngentech Co., LTD. Mice were separated into shControl and shScgn groups stochastically. Through a one-week adaptive phase, these mice were anesthetized with isoflurane (3.0% for induction and 1.5% for hold) and then fastened to the stereotaxic device (RWD instruments). shControl and shScgn AAVs were injected into the hypothalamus PVN nucleus ambilateral through an automatic microinjection instrument (World Precision Instruments). The coordinates of target area were as following: AP = − 0.94; ML = ± 0.25; DV = − 4.8. The injection was carried out with a speed of 0.1 ul/min, using a Hamilton needle . The needle was kept locally for 10 minutes after injection, then retreated slowly. Four weeks post-viral introduction, behavioral tests were performed. The staining of the marker for microglia cell was used Iba1 antibody (ab178846).All animal experiments followed the guidelines of the Institutional Animal Care and Use Committee at Chongqing Medical University. These studies were approved by the Institutional Review Board and Animal Ethics Committee of Chongqing Medical University. The shRNA was applied to knockdown 67 The biological materials were grounded, and the debris was removed using centrifugation. RNA was extracted with TRIzol reagent in combination with the RNeasy Mini Kit . Total RNA was separated by Plant Mini Kit (FOREGRNE), and then the Prime Script Reverse-transcription PCR kit (TaKaRa DRR014A) and 1 μg of total RNA were used to synthesize cDNA. Two hundred nanograms of resultant cDNA was used as a template for Real-Time PCR (Bio-Rad) with Real Master Mix Kit (Roche).The total RNA of zebrafish embryos at 48 hpf or brain tissues was collected for each sample.Zebrafish embryos (48 hpf) were collected, and the egg membranes were manually removed and then fixed with 4% PFA at −4 °C. Then, the heads of the embryos were cut off and soaked in the agar-sucrose mixed gel. Samples were fixed in the standard position. Agarose gel-containing samples were cut into cubes with a side length of 0.3 cm post-solidification. The gel cubes were dehydrated with 30% sucrose for 2 days. OCT embedded heads were frozen at −25 °C for 30 min prior to sectioning at 5 μm per slice and stored at −80 °C until use. Intracerebral apoptotic cells were examined by TUNEL assay. Sample slides were fixed with 4% PFA for 30 min, and permeablized by proteinase K and 0.1% Triton X-100. Following the protocols of TUNEL Apoptosis Assay Kit (Servicebio), samples were further balanced and stained with a reaction mixture and DAPI. Then they were rinsed with PBST, and sealed with quench-resistant sealing fluid. Lastly, Images were captured using a confocal microscope and analyzed using Image J software.Anti-pHH3 antibody  was used to identify proliferating cells. Similar to mentioned above, selected samples were permeabilized using 0.1% triton X-100-PBST, then circled with PAP PEN. After being terminated with buffer (1% goat serum and 3% BSA diluted by 0.2% triton-PBST), samples were incubated at 4 °C with pHH3 (diluted by 1:500). Second antibody and DAPI were incubated after 24 h according to the manufacturer’s protocol. After rinsing and sealing, images were captured and analyzed.68 Briefly, embryos at 48 hpf were fixed and permeabilized with proteinase K (10 mg/ml) for 20 min, and then fixed with 4% paraformaldehyde. Samples were then incubated with digoxigenin (DIG)-labeled antisense RNA probes (oxytocin) at 65 °C overnight. After the probes were washed out with formamide and SSCT concentration gradient, embryos were incubated with alkaline phosphatase (AP)-conjugated antidigoxigenin antibody (1:2000) at 4 °C overnight. NBT/ BCIP (Roche) staining was performed according to the manufacturer’s instructions.WISH analysis was carried out as previously described.Behavior was recorded using Ethovision XT13 software and a digital camera . All behavioral experiments were undertaken after 12:00 p.m. The experiments were carried out in a special room where the light and temperature were kept constant. There were no gender differences in behavior in our recording settings. Therefore, we used a mixed population of male and female adult zebrafish (around 4 months of age) in our experiments.Shoaling was recorded in 35 × 25 cm tanks filled with 8 cm depth of water. Adult fish were introduced into the box to acclimate briefly before being recorded for 10 min. We tracked the fish and measured the distances between every two-individual fish (inter-fish distance).33The test tank was subdivided into two average sections (left and right compartments). WT strangers were introduced into the left compartment, and the test fish were introduced into the right side. Then, the VMSP test  was carried out as described in ref. After anesthesia, zebrafish were dissected in PBS at 4 °C. Fish brain or blood was collected in EDTA tubes, centrifuged for 10 min at 4 °C, and then the plasma was immediately pipetted into 1 ml tube. Samples were immediately stored at −80 °C until tested for the quantification of the plasma oxytocin concentrations.The three-chamber sociability test was displayed as a classic model. Briefly, the social interaction test consisted of three 10 min stages. Mice were allowed to randomly accommodate the chamber for 10 min in the first stage. In the second stage, a novel object and an unfamiliar mouse (S1) were deposited in the right and left chambers, respectively. Test mice were set free to visit different chambers for 10 min. In the last stage, the novel object was substituted by another stranger mouse (S2), with the behavior of the experimental mice recorded for 10 min. Applied Noldus Observer software (Ethovision 11.5) was used to evaluate how much time it took in a single chamber of the three-chamber apparatus. Additionally, the social preference standard was determined by the digital divergence among the chambers with sniffing (S1 vs. object or S2 vs. S1) divided by total time.Test mice were put in a standard and transparent mouse cage independently to assess self-grooming. After a 10 min adaptive phase, the performance of mice was video recorded by a camera for 10 min. An experienced unbiased observer calculated the amount of time each mouse spent in self-grooming.Twenty black glass marbles were set at 4 × 5 on the surface of a 5 cm-deep packing inside a standard mouse cage (27 × 16.5 × 12.5 cm). Then the mice were individually placed into the cage, and the activity was observed for 30 min. Total marbles buried with > 75% in volume during the 30 min were calculated.2O) at 1:1 ratio, centrifuged at 4 °C, evaporated at 4 °C, and stored at 20 °C until recombination.69 The OXT levels in samples from different species were measured using corresponding ELISA Kits . All tests were repeated for three times. The IL-6 levels in plasma were measured using Elisa Kit (JL20268-96T), and IL-1β in plasma was measured using ELISA Kit (ab197742).Human or mouse serum, or zebrafish brain samples, were mixed with 0.1% trifluoroacetic acid water , which was diluted to a final concentration of 1 mg/kg in 0.1% DMSO 1 h prior to injection.70 The minigene vesctor (2 μg) was transfected into HEK293T cells using Hieff Trans Liposomal Transfection Reagent . Cells were incubated 48 h and then the RNA was purified with the Cell Total RNA Isolation Kit .The minigene RHCglo-SCGN was constructed as previously reported.http://hollywood.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq_acc.html) and 5’ splice site (http://hollywood.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html). The sequence of the 3’ splice sites must be 23 bases long, including 20 bases in the intron and 3 bases in the exon. Similarly, the sequence of the 5’ splice sites must be 9 bases long, including the 3 bases in the exon and 6 bases in the intron.MaxEntScan method with the Maximum Entropy Model was used to assess the splice site strength for both 3’  following the manufacturer’s Recommendations. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform, and 150 bp paired-end reads were generated. Data analysis of transcriptome was performed as previously reported.71RNA-seq analysis was carried out in accordance with previous studies.https://sciex.com.cn/; MS, QTRAP System, https://sciex.com/) by Wuhan Metware Biotechnology Co., Ltd. PCA Unsupervised PCA  was performed by statistics function prcomp within R (www.r-project.org). The data was unit variance scaled before unsupervised PCA. Hierarchical Cluster Analysis and Pearson Correlation Coefficients The HCA  results of samples and metabolites were presented as heatmaps with dendrograms, while pearson correlation coefficients (PCC) between samples were calculated by the cor function in R and presented as only heatmaps. Both HCA and PCC were carried out by R package ComplexHeatmap. For HCA, normalized signal intensities of metabolites  are visualized as a color spectrum. Differential metabolites selected Significantly regulated metabolites between groups were determined by VIP ≥ 1 and absolute Log2FC (fold change) ≥ 1. VIP values were extracted from OPLS-DA result, which also contain score plots and permutation plots, was generated using R package MetaboAnalystR.The sample extracts were analyzed using an LC-ESI-MS/MS system  test and analyzed using Excel , ANOVA test using Graphpad Prism 8.0.2 for windows . Data are presented as the mean ± SEM or mean ± SD. P-value < 0.05 was considered statistically significant.Unless otherwise specified, statistical analysis was performed by using Student’s SUPPLEMENTAL Figure


Most justice-involved youth are supervised in community settings, where assessment and linkage to substance use (SU) treatment services are inconsistent and fragmented. Only 1/3 of youth with an identified SU need receive a treatment referral and even fewer initiate services. Thus, improving identification and linkage to treatment requires coordination across juvenile justice (JJ) and behavioral health (BH) agencies. The current study examines the comparative effectiveness of two bundled implementation intervention strategies for improving SU treatment initiation, engagement, and continuing care among justice-involved youth supervised in community settings. Exploration, Preparation, Implementation, Sustainment (EPIS) served as the conceptual framework for study design and selection/timing of implementation intervention components, and the BH Services Cascade served as the conceptual and measurement framework for identifying and addressing gaps in service receipt.Part of a larger Juvenile-Justice Translational Research on Interventions for Adolescents in the Legal System (JJ-TRIALS) Cooperative, this study involved a multisite, cluster-randomized control trial where sites were paired then randomly assigned to receive Core  or Core+Enhanced  intervention components. Youth service records were collected from 20 JJ community supervision agencies (in five states) across five study phases . Implementation teams comprised of JJ and BH staff collaboratively identified goals along the BH Cascade and used data-driven decision-making to implement change.Results suggest that Core intervention components were effective at increasing service receipt over time relative to baseline, but differences between Core and Core+Enhanced conditions were non-significant. Time to service initiation was shorter among Core+Enhanced sites, and deeper Cascade penetration occurred when external facilitation (of implementation teams) was provided. Wide variation existed in the degree and nature of change across service systems.Findings demonstrate the criticality of early EPIS phases, demonstrating that strategies provided during the formative exploration and preparation phases produced some improvement in service receipt, whereas implementation-focused activities produced incremental improvement in moving youth farther along the Cascade. Compares the effectiveness of two bundles of implementation strategies for improving substance use treatment initiation, engagement, and continuing care among justice-involved youth when cross-system collaboration is required;Combines two conceptual frameworks to guide study components and design: EPIS (selection and timing of intervention and data collection components) and Behavioral Health Services Cascade .Examines change across study phases, enabling comparisons with baseline service receipt rates and potential sustainment of gains;Includes data from 20 service systems in five US states, involving both juvenile justice and behavioral health agencies.A high proportion of youth involved in juvenile justice (JJ) exhibit problematic levels of alcohol and other substance use (SU). For this reason, and because SU services are linked to outcomes including reduced recidivism , 2, addrSubstance use is common among JJ-involved youth and is a primary factor associated with re-offending . ApproxiCascade frameworks depict a continuum where clients move through a series of service steps to maximize clinical gains –16. The A prominent model for promoting system change, Exploration, Preparation, Implementation, Sustainment (EPIS) , 18, serPrior research confirms the value and effectiveness of interorganizational relationships among agencies serving JJ-involved individuals , 22, 23.Data-driven decision making (DDDM) involves using data, collected within the specific context and in collaboration with stakeholders , to systematically evaluate the effectiveness of change efforts and to inform practice and policy within organizations and systems. DDDM has been adopted by different fields that include education, healthcare, and criminal justice –34 and cExternal facilitation of local implementation teams can be beneficial in supporting change, encouraging the adaptation of best practices in a manner that suits the particular qualities of the organization or system , 37. FacSystem change occurs within a broader context, yet information regarding inner and outer context factors that potentially predict treatment receipt among JJ youth is limited. An examination of Cascade outcomes during the baseline period of JJ-TRIALS (before the intervention began) found that elevated job stress, greater intra-agency communication, and more collaborative practices with community providers were associated with lower initiation rates, perhaps due to increased agency efforts to address problems . Such faN = 20). It was expected that participation in a Core set of strategies would result in improvement over time and that sites receiving Core+Enhanced strategies  would show greater improvement compared to sites in the Core-only condition. Change over time in service receipt was measured at the agency/community level through nested youth cohorts that correspond with five study periods and align with EPIS: Baseline, Pre-Randomization, Early Experiment, Late Experiment, Maintenance. The following Hypotheses were tested:H1: The percentage of youth receiving target services  will increase in both Core and Core+Enhanced conditions between Baseline and Late Experiment study phases.H2: Compared to Core sites, Core+Enhanced sites will have greater increases in the percentage of youth receiving target services between Baseline and Late Experiment study phases.H3: Among youth with a treatment referral, youth in Core+Enhanced sites will initiate services more quickly compared to youth in Core sites over time .H4: Youth in both Core and Core+Enhanced sites will progress further in the Behavioral Health Services Cascade over time .H5: Youth in Core+Enhanced sites will progress further in the Behavioral Health Services Cascade over time compared to youth in Core sites .The current study examined the comparative effectiveness of two bundled implementation intervention strategies for improving receipt of SU services along the BH Services Cascade. Prior reports using JJ-TRIALS data described Cascade outcomes covering the baseline period before intervention began , 13 and In addition to H1-H5, it was expected that means on Cascade outcomes would remain constant between Late Experiment and Maintenance study phases, after expert facilitation ceased (change driven by the research design).Data were from the JJ-TRIALS cooperative agreement, funded by the National Institute on Drug Abuse. JJ-TRIALS included six research centers, each working with six local justice agencies providing community justice supervision  in Florida, Georgia, Kentucky, Mississippi, New York, Pennsylvania, and Texas . ResearcN = 141 to 2017 per site; Core n = 56 to 1210; Core+Enhanced n = 59 to 970). Overall totals by intervention for these treatment sites randomly assigned in pairs were Core  and Core+Enhanced . Youth cohort assignment was based on date of entry into the JJ system. With regard to Study Phase, 3242 (36.1% of the sample) started in Baseline, 1894 (21.1%) started in Pre-randomization, 1399 (15.6%) started in Early Experiment, 1314 (14.6%) started in Late Experiment, and 1139 (12.7%) started in Maintenance .Youth records data were drawn from 20 JJ agencies (10 matched pairs) across five states . BecauseThe study protocol has been previously published . All sitThe Core+Enhanced arm included all components delivered as part of the Core intervention plus active facilitation of process improvement efforts . During Site Pairs referred to matched pairs of sites. Study Phase referred to one of five study periods corresponding to EPIS: 1 = Baseline (Exploration), 2 = Pre-randomization (Preparation), 3 = Early Experiment (Implementation), 4 = Late Experiment (Implementation), and 5 = Maintenance (Sustainment) . All phaYouth demographic and offense characteristics were extracted from justice case management records and included gender, race . Cascade Penetration represented the last Cascade stage that an individual achieved in their first referral to the JJ system: 0 = no Cascade stage, 1 = Screened/assessed, 2 = Identified Need, 3 = Referred, 4 = Initiated, 5 = Engaged, and 6 = Continued in Care.f2), where .02 is a small, .15 a medium, and .35 a large effect size, respectively.Because youth were nested within site and sites were randomly assigned to condition within each matched pair, a multilevel matched block analysis was used to test hypotheses using SAS PROC MIXED , with Si2; ω2) was used to evaluate significance, with the following designations: no effect (ω2 = 0), small effect (ω2 = 0.01), medium effect (ω2 = 0.06), and large effect (ω2 = 0.14). The partial ω2 showed that Race/ethnicity and Poverty had values greater than 0.03 . JJ Status and Maximum Charge Level showed a small effect size (at least 0.02). These covariates were used to control for case mix differences between the matched site pairs.Covariates . Cox proportional hazard model (SAS PROC PHREG) was chosen because it allows censored observations (treatment initiation status was unknown for some youth when the study ended), assumes a parametric form for the predictors, and allows for an unspecified form for the underlying survivor function. For interpretation of the estimated maximum likelihood (ML) weights, corresponding hazard ratios were computed by exponentiating the ML weights. While the hazard is defined as the slope of the survival curve , the hazard ratio compares the hazards for two categories (groups), that is, the relative survival experience of the two groups. For example, if the hazard ratio is 2.0, then the rate of achieving the event in one categorical group is twice the rate of achieving the event in the other group.N = 8988), 7817 (87%) received Screening, 2252 (25.1%) received a treatment Referral, 1763 (19.6%) Initiated, 681 (7.6%) Engaged, and 359 (4.0%) Continued in Care. With regard to average conditional probabilities, 78.3% of Referred youth Initiated, 38.6% of Initiated youth Engaged, and 52.7% of Engaged youth Continued in Care. On average, 16 days elapsed between JJ Intake and Screening , 130 days between Screening and Referral , and 156 days between Screening and Initiation . Percentages of youth receiving services were comparable to those reported in other studies of the full sample at baseline under the same Cooperative , but neither the main effect for Condition nor its interaction with Study Phase was significant. Therefore, there was no statistical support for Hypothesis 2; the increase between Early Experiment and Baseline did not differ significantly by condition.H2 examined differences between Conditions over time, with Site Pairs as the blocking factor. The estimates of the site variation for each treatment service variable (Initiated: 0.03 (0.02), Change across Study Phases relative to baseline is depicted in Fig. For H3, a Cox proportional hazard model was used to analyze time to treatment initiation on Site pair blocks, Condition, Study Phase, Condition x Study Phase, and covariates. Of the 2145 youth who initiated, 1773 had complete data on covariates; 1282 had an event (time to initiation); and 491 were right censored, with no initiation by the end of the data collection.χ2(9) = 538.59, p < 0.0001] and Condition  were significant, indicating that time to initiation  was significantly different for Core and Core+Enhanced  = 3.21, p = 0.52] nor Condition x Study Phase  were significant. Of the covariates, only positive urinalysis was significant .For this model, the Joint Tests showed Site Pairs , corresponded to a hazard ratio (HR) of 0.66. Therefore, there is a 33.9% reduction in achieving treatment initiation for Core compared to Core+Enhanced. The maximum likelihood estimated weight for Core+Enhanced was b = 0.41 (0.18), with a HR = 1.51. For Core+Enhanced the rate of achieving treatment initiation is approximately 1.51 times that for Core; that is, a 51.3% increase in achieving initiation. The HR for Core would suggest that the hazard  is two-thirds of that for Core+Enhanced. In summary, youth in Core+Enhanced initiated treatment more quickly than those in Core  = 1529.96, p < 0.0001, with site variance of 0.18(0.06), Z = 3.00, p < 0.0013) and residual variance of .9880 (0.01), Z = 66.96, p < 0.0001)]. The intraclass correlation suggested that 15% of Penetration variance was due to variation between treatment sites, indicating that multilevel analysis was appropriate.H4 and H5 assessed the maximum Penetration along the Cascade. Results of the base model found treatment site variance to be significant , probation , and positive urine  were significant; with each having a positive relationship to Penetration. The least square means  and Baseline and Late Experiment . The contrast between Baseline and Maintenance was not significant . Overall, results supported H4 that there would be a significant increase in penetration for Early and Late Experiment compared to Baseline.Findings for H4 (penetration) were similar to H1 (service receipt). The main effect for Study Phase was significant, documenting an increase in Cascade Penetration over time  and Study Phase x Condition were significant . In addressing simple effects for the significant interaction, post-hoc tests revealed one significant change from Baseline to Late Experiment  in the Core condition  and from the Baseline to Late Experiment . No significant change occurred between Baseline and Maintenance . Overall, there was statistical support for H5. While there was some increase in Penetration in Core, there was greater Penetration over time in Core+Enhanced.H5 extended the Penetration analyses to include Condition, Study Phase, and their interaction with Site pairs as the blocking factor, controlling for covariates. Study Phase [F4,799 = 11.40Despite considerable variability across justice agencies in Cascade-related practices, this investigation detected an impact of the JJ-TRIALS intervention on youth treatment service outcomes, adjusting for youth, justice, and county characteristics. Findings document that Core intervention strategies  were effective at increasing service receipt over time relative to baseline (H1). When comparing cohorts of youth who entered during different study periods, Initiation rates increased 5.7% from Baseline to Early/Late Experiment. Engagement and Continuing Care rates increased significantly between Baseline and Early Experiment periods and extended to later periods. There was no difference between Core and Core+Enhanced conditions on receipt of treatment services over time (H2), neither study phase nor its interaction with condition was significant. For H3, youth initiated treatment 51.3% faster in Core+Enhanced sites than in Core sites (H3).Findings also document that Cascade Penetration (last service stage achieved) increased from Baseline to Early and Late experiment in both conditions (H4), and the Core+Enhanced intervention  was more effective in Penetration across study periods compared to Core (H5). These data suggest that efforts to improve services along the Cascade are more effective when teams work iteratively and collaboratively with external BH providers and have the guidance, support, and accountability that external facilitation provides. This type of outer/inner context collaboration is one critical bridging factor that can lead to more effective system-level implementation efforts . It is wThis study extends prior research on SU Referral (under the purview of JJ) to services most often delivered by community partners. The pattern showing greater increases in referral rates over time in Core+Enhanced sites  was simiPerhaps the predominant finding was the ubiquity of site variability across jurisdictions, target services, and implementation over time. Observing between-site differences is more rule than exception for multisite behavioral intervention trials , at leasSite variability highlights the need to further understand change processes, including strategies used by implementation teams as they planned and orchestrated system change . For exaSustainment is a critical concern in implementation research, and beginning with sustainment in mind – as a critical goal – cannot be understated . While tIt is important to acknowledge potential limitations. First, it is not certain how much service receipt data were missing and for what reasons  , nor theSecond, the sample was not nationally representative, so findings may not generalize to other jurisdictions; however, youth and agency characteristics were sufficiently diverse and comparable to a nationally representative survey of JJ agencies . Third, Results from this study demonstrate that change is possible in complex service systems involving JJ and BH agencies. Improvement in SU treatment initiation occurred across Core and Core+Enhanced conditions, the former without external facilitation. Furthermore, Core implementation activities appear beneficial even when instruction is not specific to an evidence-based practice and target goals are allowed to vary along the Cascade. Better Cascade penetration occurs when external facilitation is provided, but wide variation exists in the degree and nature of change across service systems.Findings demonstrate the criticality of early EPIS phases from exploration through implementation, suggesting that Core strategies provided early on are effective at producing some improvement in treatment initiation rates. Implementation-focused activities in the Core+Enhanced condition (facilitation of implementation teams) are effective at incremental improvement in moving youth farther along the Cascade. Therefore, using a collaborative, multi-agency approach to system change, that utilizes a data-driven approach (with or without intensive facilitation), can be useful in improving SU treatment initiation rates, although substantial gaps remain in engaging youth in treatment after initiation. Focusing on improving SU identification and service receipt among justice-involved youth translates to benefits for public health and public safety.


Resonance frequency signatures were processed using fast Fourier transform in an attempt to differentiate between RNA (control) and RNA (BC) via frequency response. RNA (BC) demonstrated a characteristic signal at 0.02 GHz, as well as a phase shift at 0.031, and 0.070 GHZ from RNA (control). These features could offer early BC diagnosis. This is the first time to describe an optical methodology based on nonlinear polarization as a facile principle to distinguish and identify RNA alterations in BC by their characteristic fingerprint spectral signature.Different cancers are caused by accumulation of numerous genetic and epigenetic changes. Recently, nonlinear polarization has been considered as a marvelous tool for several medical applications. The capability of nonlinear polarization, to monitor any changes in RNA's spectral signature due to breast cancer (BC) was evaluated. Blood samples, from healthy controls and BC patients, were collected for whole blood preparation for genomic total RNA purification. Total RNA samples were stimulated with a light-emitting diode (LED) source of 565 nm; the resonance frequency of investigated RNA samples was captured and processed  Nonlinear polarization has been considered as a marvelous tool for several medical applications, and the capability to monitor any changes in RNA's spectral signature due to breast cancer was evaluated by hyperspectral camera. Mutations in several other rare susceptible genes for instance TP53, STK11, PTEN, and CHEK2 were found to contribute to induction of breast cancer.2 The genetic information, crucial for the identity and function of each eukaryotic cell, is stored in the DNA.3 The process of transcription generates RNA by using the genome as a template, depending on certain functional genomic elements.4 Genetic information on DNA is encoded by the four bases adenine (A), guanine (G), cytosine (C), and thymine (T).5 Cancer genomes are highly enriched with mutational signatures, which may be regarded as a type of base substitution  or as the deletion/insertion of one or more bases.6 Exposure to UV light can cause adjacent pyrimidines to dimerize, while oxidative damage can cause several lesions that are mutagenic if not repaired.7 Moreover, some of the hydrogen atoms on each of the four bases can change their location to produce a tautomer.8 An amino group (–NH2) can tautomerize to an imino form (–NH). Likewise, a keto group can tautomerize to an enol form.5 These mutations in BC genomes cause corresponding RNA alterations and modifications in the order of the transcribed purine and pyrimidine bases.6,9Breast cancer (BC) is considered the leading cause of worldwide cancer deaths in females.10 Dielectric properties of macromolecules  can be determined from light absorption and emission.11 This means that a macromolecule exhibiting anisotropies in light absorption/emission, will exhibit characteristic polarization properties.12Macroscopic level investigation of polarized light effects on total RNA can reveal information about structural bonds, electronic states, electronic alignments, and dielectric properties.Macromolecular polarization properties include:13❖ Depolarization: the ability to depolarize incident polarized light.14❖ Di-attenuation: the dependence of light transmission on the incident polarization state.15❖ Polarizance: the ability to polarize un-polarized incident light.16❖ Retardance: the ability to generate a phase shift in the electric vector of incident polarized light.via partial separation of positive and negative charges.17,18 The macroscopic equivalence of dipole moment is the polarization P.19N, e, and d were number of dipoles per unit volume, electronic charge, and displacement, respectively.20,21When the oscillating electric field is applied on macromolecules; the electric field induces dipole moment 22 The induced dipole simply re-emits electric field at the same frequency known as Rayleigh scattering.23,24 Linear spectroscopy cannot secure consistent information about macromolecule chemical structure (i.e. RNA disorder). Nonlinear spectroscopy is related to the interaction of intense light with matter.25 Laser sources, can secure high light intensities to modify material optical properties.26 Light waves can then interact with each other, exchanging momentum and energy.27 This interaction of light waves can result in generation of new optical frequencies.28 Intense irradiance can excite many molecules to the high-energy state. This yields vibrations at all frequencies corresponding to all energy differences.29 Nonlinear relationship between the polarization and the electric field appears only with strong fields with generation of new electric field, with new frequencies 2ω1, 2ω2, |ω1 + ω2|, and |ω1 − ω2|.30 The phenomenon where 2ω1 and 2ω2 frequencies are produced is called second harmonic generation (SHG).31 Sum frequency generation (SFG) occurred at |ω1 + ω2|. Difference frequency generation (DFG) occur at |ω1 − ω2| as shown in 32The induced dipole moment can exchange energy, and can oscillate at resonant frequency with the incident electric field.33RNA as macromolecules with an alternating electron donating and accepting groups can offer characteristic signature when stimulated with intense laser beam.34 It could be considered as a way to study these molecules and answer how their structure controls the functional events inside the cell. These modalities may assist researchers in designing scenes and find out how numerous factors powerfully interact.35Hyperspectral imaging at RNA level may provide innovative nonlinear signatures to assess RNA functions; such signature can monitor any RNA mutation due to BC.Thus, the current study describes an optical methodology based on nonlinear polarization for spatial and spectroscopic characterization of RNA samples from BC patients and healthy volunteers to determine their spectral fingerprint signature. Latest advances in hyperspectral imaging offered unique capability for precise measurements of nonlinear polarization signature. Blood samples from five healthy controls and five BC patients were collected for whole blood preparation for total RNA purification. Total RNA samples were illuminated with LED source of 565 nm; characteristic resonance frequencies of investigated samples were captured and processed using hyperspectral camera.2.2.1Five newly diagnosed BC female patients, who haven't received any chemotherapy or radiotherapy, were recruited from the clinical oncology clinic, Ain Shams University Hospitals, Cairo, Egypt. Diagnosis was based on mammogram and cell biopsy. The study also included five healthy age volunteers to serve as healthy control. Informed consent was obtained from every patient and the study was approved by the Ethical Committee of Research, Ain Shams University Hospitals. Furthermore, the study was carried out in accordance with the regulations and recommendations of the Declaration of Helsinki.2.2For each participant, two blood samples were collected on EDTA vacutainers for whole blood preparation. Whole blood was used for complete blood count determination and total RNA purification.2.2.1Hemoglobin (Hgb), red blood cell (RBC) count, total leukocyte count (TLC) and platelet count determinations were performed using Z2TM Coulter Counter®, Analyzer, Coulter Electronics, USA.2.2.2Total RNA was extracted and purified using a QIAamp RNA blood mini kit  according to the manufacturer's protocol. Quantitation and purity assessment was performed using the NanoDrop® (ND)-1000 spectrophotometer .2.3Nonlinear polarization can offer wealth of information about chemical and physical properties of RNA macromolecule. This approach can find attractive biomedical applications. 10 mW laser source working at 656 nm was focused using microscopic lens to illuminate investigated total RNA samples. The scattered and the re-emitted radiations were collected using hyperspectral camera .Scattered and re-emitted resonance frequency signals were processed using custom digital processing algorithm for frequency and phase domain analysis. The frequency and phase responses were calculated to discriminate RNA (control), and RNA (BC). The difference in resonance frequency signature and phase shift could be ascribed to RNA gene mutations due to BC.2.4ap < 0.001, bp < 0.01, cp < 0.05 vs. RNA (BC) group. Statistical analyses graphs were drawn using Prism, version 8 .All statistical demographic analysis was performed using SPSS version 23 software package . Data are presented as mean ± standard deviation. Statistical significance between all groups was analyzed by using the 3.3.137The clinical and demographic data of the control and BC groups are summarized in 3.236 This signature could provide information about RNA mutation  related to BC. This approach could offer early diagnosis of BC as well as early treatment. Nonlinear resonance frequency response of investigated RNA samples, including RNA (control) and RNA (BC) are represented in ESI .The change in harmonic oscillation response could be correlated to chemical change and disorder of absorber size, location, and organization.It is obvious that each macromolecule demonstrated its characteristic nonpolarized resonance frequency signature. Resonance RNA (BC) frequency signature differ slightly from RNA (control). In an attempt to determine the spectral oscillation signature for investigated RNA samples; normalized resonance frequency signatures were calculated to eliminate the effect of polarized light intensity .Due to the changes in molecular electronic configuration, absorber size in the order (nm); the re-emitted signature will be in the range of visible and near IR.3.3RNA alterations are accompanied with changes in its optical and dielectric properties when stimulated with laser light. Resonance frequency radiation could offer a fingerprint signature of the macromolecule chemical structure and physical properties. Chemical structural arrangements that create differential absorption of different polarization states can exhibit light attenuation. The resonance frequency attenuation of investigated RNA samples is demonstrated in 3.4via frequency response change  and RNA (BC) e change .RNA (BC) demonstrated characteristic frequency signal from RNA (control). RNA (BC) demonstrated frequency band 0.004–0.01 GHz, and characteristic signal at 0.02 GHz. The phase change of RNA (BC) to RNA (control) was calculated from frequency signal as demonstrated in RNA (BC) demonstrated phase shift at 0.031, and 0.07 GHZ. These phase shift could be ascribed to change in chemical structure and dielectric properties due to BC.Statistical analysis of obtained results was performed; the histogram for the frequency distribution of 5 signal observations of RNA (control), and RNA (BC) response was calculated. The mean values for the RNA (control), and RNA (BC) are 0.0783 and 0.0852, respectively. The standard deviation for the RNA (control) and RNA (BC) are 0.0020 and 0.0027, respectively . It can 4.via their specific fingerprint nonlinear resonance frequency signature. RNA (BC) demonstrated characteristic frequency signal and phase shift from RNA (control). RNA (BC) demonstrated characteristic signal at 0.02 GHz; furthermore RNA (BC) demonstrated phase shift at 0.031, and 0.07 GHZ from RNA (control). We hope that our study will assist more researchers and pave the way for finding out how numerous factors powerfully interact in order to utilizing non-linear polarization in direct, fast, non-invasive diagnosis of BC.BC included RNA mutations with changes in RNA chemical structure, and physical properties . This is the first time to report an optical approach based on nonlinear polarization to identify RNA alterations in BC All experimental and investigation trials was approved and validated from “Ain Shams University” – Medical College – Ethics Committee.The authors stated and declare that there is no conflict or competing of interests.


Higher levels of moderate-to-vigorous physical activity have been associated with a lower risk of diabetes, but less is known about how daily step counts (steps/day) are associated with diabetes risk. Therefore, we examined the association of steps/day and step intensity with incident diabetes.We included 6634 adults from the population-based prospective cohort Hispanic Community Health Study/Study of Latinos (HCHS/SOL) (2008–2017). Cox proportional hazard models that accounted for complex survey design and sampling weights were used to estimate the association of baseline accelerometer-assessed steps/day and step intensity with 6-year risk of incident diabetes as hazard ratios (HR) and 95% confidence intervals (CI). We further examined whether the percent of intense steps at a given accumulation of steps/day was associated with diabetes risk, and if associations were modified by specific cohort characteristics.> 100 steps/min). Over 6 years of follow-up, there were 1115 cases of diabetes. There was a suggestive lower risk of diabetes with more steps/day– adults had a 2% lower risk per 1000 steps/day ). Inverse associations between average steps/day and diabetes incidence were observed across many cohort characteristics, but most importantly among adults at high risk for diabetes – those who were older, or had obesity or prediabetes. Adults who accumulated 17 min/day in brisk ambulation compared to < 2 min/day had a 31% lower risk of diabetes ). A greater percent of intense steps for a given accumulation of steps/day was associated with further risk reduction.The average age of cohort members was 39 years and 52% were female. Adults had an average of 8164 steps/day and spent 12 min/day in brisk ambulation (Adults who accumulate more daily steps may have a lower risk of diabetes. Accumulating more steps/day and greater step intensity appear to be important targets for preventing diabetes.The online version contains supplementary material available at 10.1186/s12966-022-01284-2. In the United States (US), over 26 million adults 9.8%) have a diagnosis of type 2 diabetes and 91.8 million 37.6%) have prediabetes 7.6% have. Higher % have a > 100 steps/min is suggested to be a moderate intensity activity or greater  [Total step volume (steps/day) includes steps accumulated at a light, moderate, and vigorous intensity . The ratvalents) . Four lovalents) –19. In tvalents) . In threvalents) , 18. Thevalents) , 20, 21.valents) , 23 and valents) , 24–26.Given the limited information on how stepping is associated with diabetes, we examined how steps/day were associated with the risk of diabetes in the Hispanic Community Health Study/Study of Latinos (HCHS/SOL). Furthermore, we examined if specific cohort characteristics  modified the association between steps/day and diabetes. Because steps/day is a combination of light, moderate, and vigorous intensity steps, we further examined if step cadence and stepping pattern (bouted stepping) were associated with the risk of diabetes. We further explored the impact of step intensity by examining if achieving the same steps/day at a greater percent of intense steps compared to a lower percent of intense steps was associated with a lower risk of diabetes. Chen et al.  observedThe HCHS/SOL is the largest population-based cohort of Hispanic/Latino adults from four US metropolitan areas  , 28. A tAt baseline, participants were asked to wear an Actical accelerometer  during waking hours for 1 week. The Actical was attached to a belt on the right hip and captured accelerations in 1-min epochs. Non-wear was defined using the Choi algorithm . Partici> 40 steps/min (purposeful steps and faster movement), > 70 steps/min , and > 100 steps/min  [> 40 steps/min, > 70 steps/min, and > 100 steps/min). A bout was defined as > 10 consecutive minutes above the specified cadence while allowing for interruptions of up to 20% of time below the cadence threshold and less than 5 consecutive minutes below the threshold [> 100 steps/min, termed ‘intense steps’, to examine the contribution of intensity when holding steps/day constant.Steps were determined by the manufacturer’s step algorithm. The Actical step function has performed well in validation studies, although it may undercount steps at slow walking speeds –34. We aovement) . We derihreshold . The bouhreshold . Interruhreshold . We calc> 126 mg/dl, non-fasting plasma glucose of > 200 mg/dl, 2-h postload oral glucose tolerance test (OGTT) > 200 mg/dl, or glycosylated hemoglobin (HbA1c) > 6.5% [We examined two definitions of incident diabetes in order to enhance comparisons of our results with other studies that have used various diabetes definitions. The first definition was based on three criteria: 1) self-reported diagnosis of diabetes, 2) self-reported use of diabetic medication, or 3) laboratory-tested fasting plasma glucose ) > 6.5% . The secWe included the following covariates assessed at baseline in our analysis: age, sex, Hispanic/Latino heritage, HCHS/SOL field center, education, marital status, employment, years lived in the US, self-rated general health, mobility limitations, cigarette packyears, alcoholic drinks per week, energy intake, the 2010 Alternative Healthy Eating Index (AHEI-2010), body mass index (BMI), insulin resistance as measured by homeostasis model assessment of insulin resistance (HOMA-IR), prediabetes, and report of occupational physical activity, with description of the measures in the n = 2541), did not wear the accelerometer at Visit 1 (n = 732), were not adherent to accelerometry , experienced an accelerometer malfunction (n = 62), and were missing any covariates used in analysis (n = 515). We trimmed the top and bottom 1% of the steps/day distribution in order to remove extreme outliers (n = 54). After exclusions, the analytic sample consisted of 6634 adults.Of the 16,415 enrolled participants, 11,623 attended Visit 2. Of the Visit 2 participants, we excluded those who had diabetes at baseline  and 95% confidence intervals (CI). Follow-up time was calculated as time from baseline to occurrence of incident diabetes, end of follow-up, or death, whichever occurred first. The time scale for all models was time since baseline. We tested the proportional hazards assumption by examining interactions between each step metric and follow-up time and the assumption was met for all models. We further estimated incidence rates (IR) per 10,000 person-years with Poisson models that accounted for the complex survey design and survey weights. All analyses were conducted with SAS version 9.4 and Stata version 15.0.> 12,500 steps/day (highly active)). We classified peak 30-min cadence into four categories [> 100 steps/min ). We separately examined minutes spent above > 40, > 70, and > 100 steps/min and specified categories of minutes spent at different cadence as quartiles. For minutes spent in bouts, we separately examined bouted minutes above thresholds of > 40, > 70, and > 100 steps/min. We included a category of no bouts and specified remaining categories based on tertiles. For all step metrics we estimated a p-value for trend by specifying each step metric as an ordinal variable in models. All models were adjusted for age, a quadratic term for age, sex, Hispanic/Latino background by HCHS/SOL field center specified as a 17-level categorical variable, education, married/partner status, employment status, years in the US, self-rated general health, mobility limitations, cigarette pack years, alcoholic drinks per week, energy intake, AHEI-2010, and accelerometer wear time. We conducted sensitivity analyses by further adjusting for BMI, which we expected to attenuate the association as a mediator.We examined continuous and categorical measures of all step metrics. First, we examined the dose-response association of steps/day, peak 30-min cadence, and minutes spent at different cadence with incident diabetes. For each step metric, we tested for non-linearity by specifying models with restricted cubic splines with knots at the 25th, 50th, and 75th percentile of the step metric distribution. For all step metrics, the spline terms were not significant suggesting the relationship between each step metric and diabetes was not curvilinear . In modetegories  (< 60 st> 50 years), sex , occupational physical activity , obesity , insulin resistance as measured by HOMA IR , prediabetes status , and Hispanic/Latino background . A likelihood ratio test compared a model with an interaction term between steps/day and the modifier to one without the interaction term and a p-value < 0.10 suggested the model with the interaction term was a better fit [n = 3799).We further explored the role of intensity by examining if having a greater percent of intense steps at a given steps/day was associated with a lower risk of diabetes than having a lower percent of intense steps at the same steps/day . All comparisons were made to the referent of the 10th percentile of steps/day and percent of intense steps (3400 steps/day and 1%). Last, we examined if the relationship between continuous steps/day and diabetes varied by age  of follow-up, there were 1115 12.5%) and 740 (8.2%) incident events of diabetes based on the two diabetes definitions. Generally, incidence rates and HRs were lower with greater steps/day; however, in the majority of analyses, the confidence intervals included the null value ) lower risk of diabetes (three criteria definition) compared to those who accumulated less than 2 min/day. For both diabetes definitions, the HR estimates were generally lower with more time spent in bouts at each cadence but the confidence intervals included the null value for all analyses ) lower risk of diabetes per 2000 steps/day (2% per 1000 steps/day) . With diteps/day . AlthougIn addition to these studies on steps/day, there has been consistent evidence on the inverse association of self-reported physical activity with diabetes risk, but more evidence is needed on the dose response relationship , 6. A reIt is hypothesized that physical activity may lower the risk of diabetes through several biological mechanisms. Greater amounts of physical activity may reduce adiposity, a risk factor for diabetes . AdditioDiabetes risk is higher among older age groups and those with prediabetes or obesity , 20. We > 39 min and > 17 min at > 70 and > 100 steps/min respectively, cadences described as slow and brisk walking [> 40 steps/min and did not find strong associations between these cadence measures and the risk of diabetes [Our results regarding cadence, a proxy for step intensity, suggest that a higher peak 30-min cadence and more time at a faster cadence were associated with a lower risk of diabetes. Peak 30-min cadence has been described as a summary of an individual’s best natural effort . Adults  walking , were asdiabetes . The OPAdiabetes . Two crodiabetes , 52 withdiabetes . Few studiabetes , 54.In addition to observing a potential lower risk of diabetes with more steps/day, our results suggest that the risk of diabetes was even lower when these steps were accumulated at a higher intensity. Adults who accumulated 7000 steps/day with 10% versus 30% being intense steps (≥100 steps/min) had a 10% versus 18% lower risk of diabetes, respectively, compared to those accumulating 3400 steps/day with 1% being intense steps. At 7000 steps/day, 10 and 30% of intense steps are equivalent to accumulating 700 and 2100 steps at a brisk walk or faster pace which can be reached by taking a 7 min and 21 min brisk walk. In support of our finding, Strain et al. , in the A strength of our study is that we focused on steps/day, an easy-to-understand metric  that hasOur analysis is unique, as it was conducted with the largest longitudinal Hispanic/Latino cohort. The Hispanic/Latino population accounts for 18% (60 million) of the US population  and is pThere are limitations to our analysis to be considered. Reverse causality cannot be ruled out as we are unable to determine exactly when diabetes developed and adults who developed diabetes close to baseline may have lower steps/day. We used one measure of steps/day and cadence and acknowledge that activity patterns may change over time. However, data from the Women’s Health Study examined steps/day over 3 years and found that a 7-day accelerometer collection of steps/day was a reasonable estimate of longer-term physical activity . Steps/dIn conclusion, engaging in more steps/day and taking more steps at a brisk walk or faster pace is potentially associated with a lower risk of diabetes among US Hispanic/Latino adults. Our results suggest that adults had a 2 to 3% lower 6-year risk of diabetes per 1000 steps/day -- any amount of stepping was associated with a lower risk of diabetes but greater risk reduction was achieved by taking more steps/day. The inverse association between steps/day and diabetes risk was observed across many cohort characteristics, most prominently among adults at high risk for diabetes – those who were older, or had obesity or prediabetes. Accumulating the same steps/day at a greater percent of intense steps was associated with further risk reduction. Our results suggest adults can lower their risk of diabetes by taking more steps/day at any pace, but should engage in brisk walking for part of their day to gain the greatest benefit.Additional file 1.