Pediatric patients with breast-related symptoms often initially present to the emergency department for evaluation. While pediatric radiologists are accustomed to evaluating acute infectious and traumatic etiologies, they may be less familiar with breast-specific findings. This study compares management recommendations of pediatric breast ultrasounds performed in the emergency setting between pediatric and breast imaging radiologists.This retrospective cohort study reviewed data from all pediatric breast ultrasounds performed in the emergency setting from a single academic institution from 1/1/14 to 12/31/19. During the study period, 12 pediatric radiologists with experience ranging from 1 to 33 years interpreted pediatric breast ultrasounds. Three breast imaging radiologists  retrospectively reviewed each case and recorded whether further management was recommended. Differences in recommendations were compared using Fisher’s exact test. Cohen’s kappa was used to assess agreement between subspecialty radiologists.k = 0.54). There was no significant difference in recommendations for further management between pediatric radiologists (22/75 [29.3%]) and the most experienced breast imaging radiologist (15/75 [20.0%]), p = 0.26.This study included 75 pediatric patients, with mean age 13 ± 5.6 years and malignancy rate of 1.3% (1/75). Pediatric radiologists and the most experienced breast imaging radiologist had moderate agreement in management recommendations (Recommendations for pediatric breast complaints in the emergency setting are comparable between subspecialties. While pediatric breast cancer is rare, malignancy remains in the differential diagnosis of breast-related symptoms in this population, especially those patients presenting with a palpable breast mass , 2. CurrPediatric patients with breast-related symptoms often initially present to the emergency department for evaluation. Unlike in the adult population, ultrasound is typically the initial imaging modality of choice for work-up instead of mammography due to the lack of ionizing radiation and higher sensitivity in dense breast tissue , 9. DepeTo optimize the breast care provided to pediatric patients in the emergency setting, adequate evaluation of breast complaints without missing suspicious lesions is vital for the initial work-up. Therefore, investigating the existence of discrepancies between management recommendations given by pediatric radiologists and breast imaging radiologists is relevant and can improve the quality of patient care. The purpose of this study is to compare management recommendations given by pediatric and breast imaging radiologists for pediatric breast ultrasounds performed in the emergency setting.This study has been reviewed and determined to be exempt by our institutional review board and Health Insurance Portability and Accountability Act (HIPAA) compliant.This retrospective cohort study reviewed data from all pediatric breast ultrasounds performed in the emergency setting for patients ages 0 to 18 years from a single, multisite academic institution from 1/1/14 to 12/31/19. Our institution’s Value Analytics team then extracted patient medical record numbers (MRNs) from Epic which met our inclusion criteria. At our institution, these studies are interpreted by pediatric radiologists. There were 12 pediatric radiologists who interpreted pediatric breast ultrasounds during the study period with experience ranging from 1 to 33 years. Potential covariates were extracted from Epic including patient age at the time of the exam, race, and specific pediatric radiologist interpreting the examination. Pediatric breast ultrasound report impression and management recommendations, follow-up instructions given by the emergency department at discharge, and patient follow-up outcomes were recorded by manual chart review for each patient by one of the authors.Three breast imaging radiologists  retrospectively reviewed each case independently and assigned a Breast Imaging Reporting and Data System (BI-RADS) category assessment. The breast imagers were only provided the patient’s age at the time of the exam and the indication for each ultrasound exam. They were not provided with the pediatric radiologist’s interpretation and recommendations and asked not to look at the report when reviewing each case.n = 17) or a BI-RADS 3, 4, or 5 assessments given by the breast imaging radiologists (n = 18). A BI-RADS 3 assessment is given when a finding is considered probably benign  and is closely monitored using a standard surveillance imaging protocol at 6, 12, and 24 months after initial BI-RADS 3 assessment examination ), no sociodemographic factor was significantly associated with non-adherence to follow-up recommendations on univariate analysis (Table /15), no Our study demonstrated that there is no clinically significant difference in management recommendations for pediatric breast ultrasound examinations in the emergency setting between pediatric and breast imaging radiologists. However, the majority of patients were non-adherent to follow-up recommendations by the pediatric radiologists after being discharged from the emergency department.Although the majority of pediatric breasts complaints are related to benign etiologies, the risk of malignancy is not negligible, especially when the presenting complaint is a palpable lump , 6, 7. TOur study had three masses which had discrepant management recommendations between the pediatric and breast radiologists. Per the ultrasound report, all three masses were described by the pediatric radiologists as being oval, circumscribed masses which likely represented fibroadenomas. The discrepancy in management recommendations may be related to the differences in how pediatric and breast imaging radiologists practice. Breast imaging radiologists’ practice patterns and management must follow the BI-RADS Atlas whereas pediatric radiologists who are interpreting breast examinations do not . Breast Prior literature has demonstrated that adherence to follow-up recommendations after discharge from the pediatric emergency department is poor , 18. OurOur study has a few limitations. This study was performed at a single, multisite academic institution and our small sample size could have affected the generalizability of our results. Larger sample size and multisite analysis may be warranted to confirm our findings and expose any disparities which may be associated with follow-up adherence rates. Additionally, we are unable to capture those patients who may have had follow-up performed at a different institution therefore patient non-adherence to follow-up recommendations is another limitation. Our malignancy rate may have been underestimated due to low adherence follow-up rates among patients with masses; however, given the very low incidence of breast cancer in the pediatric population, this likely would not have changed the overall conclusion of our study.In conclusion, management recommendations for pediatric breast complaints in the emergency setting are comparable between pediatric and breast radiologists. Initial evaluation of pediatric breast complaints can be adequately interpreted by pediatric radiologists in the emergency setting without missing suspicious lesions. However, there is a deficit in consistent follow-up of pediatric breast-specific findings initially evaluated in the emergency setting; therefore, more frequent and targeted interventions to promote follow-up adherence are required post-discharge from the emergency department to reduce the likelihood of a missed or delayed cancer diagnosis.


The low prevalence of Arterial Thoracic Outlet Syndrome (ATOS) and diffuse symptomatology have resulted in limited data on optimal treatment strategies and long-term outcome. The aim of this study was to report and evaluate a single center experience with the treatment of ATOS including midterm patient reported outcome.All patients treated for ATOS from 2004 to 2020 were retrospectively identified. Patients were divided into two groups based on presenting symptoms; ATOX group  and ATOS group (claudication symptoms). Baseline characteristics and treatment details were extracted from electronic patient files. A telephone survey was conducted to collect patients' follow-up data including a functional disability score using the Quick Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire. The primary endpoint of this study was symptom-free survival. Secondary endpoints were median QuickDASH scores during follow-up, postoperative complications and possible re-interventions.N = 9, ATOS N = 11). In the ATOX group, eight patients were primarily treated with catheter directed thrombolysis  or surgical thrombectomy (N = 3). All patients received staged thoracic outlet decompression surgery (TOD). In the ATOS group, 10 patients primarily received TOD and one patient was treated conservatively with physiotherapy. Seven ATOX patients and nine ATOS patients were symptom free at follow-up with a median QuickDASH score of 2.3 (IQR 12.5) and 2.3 (IQR 16.5) respectively. Ten complications occurred in the ATOX group; three bleeding complications, five re-occlusions, one arterial dissection and one occipital infarction. In the ATOS group five complications occurred; one perioperative bleeding complication, three re-occlusions and a stent fracture. Seven vs. five re-interventions were required in the ATOX and ATOS groups respectively.A total of 20 patients  were included and divided into two groups (ATOX The mid-term self-reported symptom free survival in both the ATOX as well as ATOS group seems acceptable while median QuickDASH scores in both groups indicate a very good functional outcome. This however comes at the cost of treatment related bleeding complications in especially the ATOX group presumably due to thrombolysis, and re-interventions required in almost one out of three patients. Arterial Thoracic Outlet Syndrome (ATOS) comprises approximately only 1%–2% of all thoracic outlet syndrome patients –8. Furthvia a transaxillary approach with a first and/or cervical rib resection combined with scalenectomy. When vascular reconstructive surgery of a subclavian aneurysm is required, a supraclavicular or paraclavicular approach is used. Subclavian aneurysms where defined as a >1.5 × increase in artery diameter. All symptomatic aneurysms where treated simultaneously with TOD, whereas asymptomatic aneurysms with <100% increase in diameter where left untreated.From 2004 to 2020, all patients referred to our tertiary vascular referral center in the University Medical Center Utrecht suspected of ATOS were retrospectively identified using diagnosis-treatment codes. All patients who met the ATOS diagnosis criteria established by the Society for Vascular Surgery, were included in this study  questionnaire was recorded in all patients , 10. TheThe primary endpoint of this study was symptom-free survival at last follow-up as reported by the patient either at their last hospital visit or in the telephone survey. Secondary endpoints were median QuickDASH scores at last follow-up, number of re-interventions, early (<30 days postintervention), and late complications (>30 days postintervention). Complications included arterial occlusions, bleeding complications according to the World Health Organization (WHO) bleeding score, and treatment related complications .Explorative and descriptive statistical analysis were performed using Statistical Package for the Social Sciences .N = 9) and patients with chronic arm claudication symptoms  , the majority was female (57%), and all patients were classified ASA 1 or 2. The median follow-up was 50.5 months (Range 7–198 months) and 17 out of 20 patients completed the survey including the QuickDASH questionnaire. In 10 patients subclavian artery compression was caused by a cervical rib, in four patients by a fusion of the first and second rib, and in the remaining six patients the compression occurred in an otherwise anatomically normal but narrow thoracic outlet. Initial symptoms at presentation were divided into two groups; patients with acute upper limb ischemia caused by an arterial occlusion  .N = 5) or surgical thrombectomy (N = 3) depending on the location and extend of the occlusion.  interposition bypass and the second by resection of the aneurysm with a direct end-to-end anastomosis. In the third patient the aneurysm was primarily left untreated, but required endovascular stenting after a dissection occurred  and one required additional postoperative stenting for a PTA resistant stenosis. One patient was treated conservatively with physiotherapy focused on posture improvement. Post stenotic aneurysms where found in four patients and all were treated conservatively without leading to TE complications during follow-up.In the ATOS group, 10 out of 11 patients were primarily treated with TOD; 10 N = 2), clopidogrel (N = 1), dual antiplatelet therapy (DAPT) (N = 1) and vitamin K antagonists (N = 5). (N = 2) to four months (N = 1) and lifelong antithrombotic therapy (N = 6).A total of 17 patients received postoperative antithrombotic therapy. In the ATOX group, all nine patients received postoperative antithrombotic therapy; acetylsalicylic acid (ASA) ((N = 5).  Therapy N = 3), clopidogrel (N = 3), DAPT (N = 1), and Direct Oral Anticoagulants (DOAC) (N = 1).In the ATOS group, eight out of 11 patients, including the stented patients, received lifelong antithrombotic therapy while 3 patients received no postoperative antithrombotics whatsoever. Prescribed were ASA (After a median follow-up of 50.5 months (range 7–198 months) a total of 16 patients (80%) reported to be symptom free at last follow-up. This was confirmed by the median QuickDASH scores of the whole cohort  and ATOX  and ATOS subgroups .  In the AIn the ATOS group one patient suffered from venous TOS on the contralateral side and underwent TOD. After 61 months of follow-up she remained symptomatic on both sides with a QuickDASH of 95.5. No clear substrate for her symptoms could be found despite extensive diagnostic analysis. The other symptomatic patient reported sporadic fatigue after intensive overhead labour after a follow-up of 24 months with a QuickDASH score of 20.5.A total of seven early complications occurred. In the ATOX group two puncture site bleedings after CDT (grade 2) occurred and one haematothorax (grade 3) that required coiling of the internal mammary artery. During this coiling procedure a dissection of an already known subclavian artery aneurysm was discovered and stented with self-expendable stents. This patient developed an ipsilateral occipital infarction with hemianopia three days later. On diagnostic imaging no clear embolus source was discovered. Finally, one patient suffered an early recurrent occlusion in-between surgical thrombectomy and TOD, which was successfully treated with CDT. In the ATOS group one patient developed a haematothorax after TOD that required thorax drainage.N = 4, ATOS group N = 3). All occlusions occurred while under antithrombotic therapy , and three out of seven were in-stent occlusions. In the ATOS group one stent fracture occurred over an insufficiently resected first rib  and/or were not placed in the thoracic outlet . Nonetheless, with the currently limited data available, we believe that stenting in the thoracic outlet should be applied cautiously and be considered a last resort option in patients with severe claudication symptoms  in this study . Based oThis study has several limitations. We only performed postoperative imaging on indication, including patients with conservatively treated post stenotic aneurysms, and were therefore unable to assess the postoperative arterial patency over time. However, we believe that symptom status and functional outcome measures are more insightful and relevant for the patient than standardized arterial patency assessment. Even for the patients with conservatively treated post dilatation aneurysms, standardized imaging might not be strictly necessary since none of these patients experienced TE complications in our series so far. Moreover, in a recent series that performed standardized follow-up imaging conservatively treated post stenotic aneurysms in ATOS patients, no further growth of the aneurysms or vascular complications were found . We therFurther limitations are the retrospective nature and small sample size. However, due to the low prevalence of ATOS, large prospective case series are hard to come by and can only be achieved through multicenter prospective registries. Despite these limitations, this study shows that the mid- to long term prognosis of ATOS seems favorable after adequate surgical and endovascular treatment. With the start of our multicenter TROTS registry on the treatment of thoracic outlet syndromes we will be able to expend and update this case series with prospectively collected data in the future.The mid-term self-reported symptom free survival in both the ATOX as well as the ATOS group seems acceptable and the median QuickDASH scores in both groups are very good. However, this comes at the cost of treatment related bleeding complications in especially the ATOX group, recurrent arterial occlusions and re-interventions were required in almost one of every three treated patients.


Children with chronic inflammation are often treated with glucocorticoids (GCs) and many of them experience growth retardation. It is poorly understood how GCs interact with inflammatory cytokines causing growth failure as earlier experimental studies have been performed in healthy animals. To address this gap of knowledge, we used a transgenic mouse model where human TNF is overexpressed (huTNFTg) leading to chronic polyarthritis starting from the first week of age. Our results showed that femur bone length and growth plate height were significantly decreased in huTNFTg mice compared to wild type animals. In the growth plates of huTNFTg mice, increased apoptosis, suppressed Indian hedgehog, decreased hypertrophy, and disorganized chondrocyte columns were observed. Interestingly, the GC dexamethasone further impaired bone growth, accelerated chondrocyte apoptosis and reduced the number of chondrocyte columns in huTNFTg mice. We conclude that TNF and dexamethasone separately suppress chondrogenesis and bone growth when studied in an animal model of chronic inflammation. Our data give a possible mechanistic explanation to the commonly observed growth retardation in children with chronic inflammatory diseases treated with GCs. JIA is characterized by increased systemic levels of pro-inflammatory cytokines, such as TNF and IL-6, not only affecting the joints but also the growth plate cartilage causing growth retardation in many children2. Although clinical studies of chronic inflammation in childhood show that long-term treatment with GCs may lead to irreversible growth impairment3, the underlying mechanisms are still unclear.Growth retardation affects many children with chronic inflammatory conditions, such as juvenile idiopathic arthritis (JIA), and many of these patients are treated with glucocorticoids (GCs)4. Locally, TNF and IL-1β synergistically cause growth retardation as confirmed in cultured fetal rat metatarsal bones by inhibiting chondrocyte proliferation and differentiation while inducing apoptosis of growth plate chondrocytes5. Apoptosis is linked to high expression of caspase-36, whereas PCNA7, and collagen X8 are decreased in conditions of growth retardation. In addition, Indian hedgehog also plays a crucial role in controlling proliferation and differentiation within the growth plate9. Systemically pro-inflammatory cytokines are known to suppress pituitary growth hormone (GH) release11. Nevertheless, it is unknown whether TNF overexpression can cause bone growth retardation, although it has been reported that anti-TNF treatment in JIA patients can rescue bone growth12. Similarly, studies in healthy mice have shown that GC treatment inhibits longitudinal bone growth by suppressing hedgehog-signaling, chondrocyte proliferation and differentiation while increasing cellular apoptosis14. However, it is still unknown how GCs affect bone growth in a disease model of chronic inflammation.Longitudinal bone growth occurs at the growth plate, a hyaline cartilage plate situated in the metaphysis at each end of long bones. Any disturbance within the growth plate cartilage may negatively affect longitudinal bone growth. TNF is a key player in chronic inflammation triggering other pro-inflammatory cytokines such as IL-1 and IL-617. Although TNF has been shown to suppress osteoblast activity and bone formation in young mice18, it is still unknown if TNF-overexpression may suppress growth plate chondrogenesis and longitudinal bone growth. To address this gap of knowledge, we characterized how growth plate chondrocytes and bone growth are affected in huTNFTg mice and how treatment with a GC, dexamethasone, modulates these effects.A limitation of previous experimental studies investigating GC effects on bone growth is that these have used healthy animal models and thereby provide no information on the effects in conditions of chronic inflammation. To address this, we used a model system of chronic inflammation induced by overexpression of human TNF in mice (huTNFTg). huTNFTg mice spontaneously develop arthritis and have previously been used to investigate the role of systemic TNF overexpression in different inflammation-driven pathologies15 was used in this study. Briefly, the huTNFTg mouse line expresses increased levels of human TNF resulting in the spontaneous development of fast progressing arthritis, a cachectic phenotype and shortened lifespan. We also used untreated age-matched wild type (WT) female C57BL/6 mice to measure femur bone length and growth plate height. The number of animals per group were decided based on power calculations.All animal experiments were performed at Biomedcode Hellas S.A.  and were approved by the BSRC Al. Fleming Institutional Committee of Protocol Evaluation in conjunction with the Veterinary Service Management of the Hellenic Republic Prefecture of Attica according to all current European and national legislation and were performed in accordance with relevant guidelines and regulations. The human TNF-over-expressing transgenic  mouse model  described previously15. Mouse health was monitored daily. Body weight and arthritis scores were recorded on a weekly basis as previously described19. Briefly, arthritis score was evaluated in the ankle joints by clinical assessment using a semi-quantitative score ranging from 0 to 3 based on the severity of joint swelling and grip strength; 0 = no arthritis, 1 = mild arthritis, 2 = moderate arthritis, and 3 = severe arthritis. We included 22 huTNFTg in this study and divided them into 4 groups: group 1 (n = 5) was males treated with saline; group 2 (n = 5) was females treated with saline; group 3 (n = 6) was males treated with dexamethasone (3 mg/kg); group 4 (n = 6) was females treated with dexamethasone (3 mg/kg). We also had 6 age-matched wild type (WT) females C57BL/6 mice treated with saline. After 4 weeks of treatment, all animals were sacrificed with CO2. Hind limbs from huTNFTg animals and WT animals were dissected for histopathological analysis. Femur lengths were measured by digital caliper and thereafter the bone was fixed in 4% formaldehyde for 24 h followed by decalcification in EDTA buffer for 3–4 weeks before dehydration and paraffin embedding. All bone sections were sliced with the same orientation, placing controls and treatment groups on the same slide.Starting from four weeks of age, the huTNFTg animals received daily subcutaneous injections with dexamethasone (3 mg/kg) or saline. The dose of dexamethasone was chosen based on a previous study from our group20. Briefly, after deparaffinization and rehydration, antigen retrieval was performed in sodium citrate buffer (10 mM pH 6.0) for 15 min at 75 °C. After retrieval, the slides were blocked with 2% serum (horse and goat) followed by incubation overnight at 4 °C with primary antibody (1:100). Sections were incubated with anti-caspase 3 antibody , anti-proliferating cell nuclear antigen (PCNA) antibody , anti-collagen X antibody  and anti-Ihh antibody . Sections were incubated for 1 h at room temperature with corresponding secondary antibody  followed by incubation with an avidin-peroxidase complex (Vectastain ABC-kit PK-6100) and visualized with 3,3' diaminobenzidine (DAB) (Dako K3468) development for 2–3 min. The slides were counterstained with Alcian Blue and dehydrated. Image J software (NIH) was used to quantify stained area and percentages of immunopositive cells in the growth plates.To analyze protein expression in the growth plate, immunohistochemistry was performed in serial sections of femur growth plates with minor modifications to what has been described before7. Images were photographed at 20 × magnification. Total growth plate height was measured as reported elsewhere21. Briefly, the measurement was performed from the proximal end of the resting zone to the chondro-osseous junction at the distal end of the growth plate. The height was measured at three different sites  of each growth plate and calculated as the average of three measurements. The height of the hypertrophic zone was measured in a similar way. The measurement was taken at the medial, central and lateral sites of the growth plate, from the first hypertrophic chondrocyte to the chondro-osseous junction, and the height was calculated as the average of three measurements. Hypertrophic cells were defined as cell more than 6 μm in height22. The number of hypertrophic cells were counted in the middle two-thirds of each growth plate and normalized by square millimeter. The size of hypertrophic cells was measured in 10 columns in each growth plate and calculated as the average of 10 measurements22. A chondrocyte column was defined as a column containing a minimum of 5 chondrocytes23. All measurements were done using the Image J Software by a person blinded to the experimental details.For histomorphometric analyses, serial sections (5 μm thick) of the femur growth plate were stained with hematoxylin–eosin which has been described beforeP values less than 0.05 were considered significant.Statistical significance of differences between groups was evaluated by 2-tailed Student’s t tests with 95% confidence intervals or Mann–Whitney Rank Sum Test. All data are expressed as mean ± SD or mean ± SEM. This study was performed in Biomedcode's animal facility, that is an independent wing of BSRC Al. Fleming animal facility, registered by the Official Veterinary Service of the Prefecture of Attica as Breeding, Experimental and Supplying Laboratory Animal Facility, under the Registration Codes EL 09 BIO 04, EL 09 BIO 05 and EL 09 BIO 06, respectively. All of the conditions of testing conformed to the Presidential Decree No 56/2013 Governmental Gazette No A’ 106 applicable in Greece, which is the implementation of the EEC Directive2010/63/EEC and were approved by the directorate of Agricultural and Veterinary Policy (DAVP) of the Attica Region . This in vivo study is in accordance with ARRIVE guidelines.p < 0.001)            or saline was administered for 4 weeks   Fig. A,B, whilol) Fig. C–E. Whenol) Fig. A, whereaol) Fig. B–D.FigurWe here present novel data clarifying the individual roles of GC treatment and/or inflammation on bone growth in a mouse model of chronic arthritis where human TNF is overexpressed (huTNFTg). In huTNFTg animals, bone growth and growth plate chondrogenesis were found to be suppressed compared to wild type controls. When treating huTNFTg mice with dexamethasone, although an anti-inflammatory effect was documented, bone growth was further impaired. Mechanistic studies showed that the dexamethasone treatment further increased chondrocyte apoptosis and decreased the number of chondrocyte columns when compared to untreated huTNFTg controls.25 and bone formation18, any effect on longitudinal bone growth and growth plate chondrogenesis has not yet been reported. Our primary finding was that bone growth was impaired in young mice exposed to increased TNF-levels throughout development. Although TNF has been earlier described to suppress the growth of ex vivo cultured fetal rat metatarsal bones, this effect was only achieved at a very high concentration11.To our knowledge, this is the first in vivo characterization of the effects of TNF-overexpression on bone growth and chondrogenesis. Although overexpression of TNF in mice has been associated with decreased body size26. Furthermore, disruption of Ihh signaling in Ellis-van-Creveld syndrome protein knockout mice has been shown to not only cause limb shortening but also reduced height of the proliferative and hypertrophic growth plate zones27. We report that chondrocyte columns were fewer and disorganized in the TNF-overexpressing mice were the expression of Ihh was also suppressed. A similar disorganized growth plate structure was earlier reported in mice with disrupted Ihh signaling which further supports a link between TNF and Ihh signaling in the growth plate cartilage27.We report that overexpression of TNF in mice not only suppressed bone growth, but detailed histomorphometry also showed reduced growth plate height where the hypertrophic zone was most affected. Besides reduced hypertrophic zone height, we also observed decreased size and number of hypertrophic chondrocytes suggesting that within the growth plate, these cells were most affected when overexpressing human TNF. We also report that chondrocyte proliferation was suppressed in huTNFTg mice. Since hedgehog-signaling is known to play a crucial role in controlling proliferation and differentiation within the growth plate cartilage, expression levels of Indian hedgehog (Ihh) were analyzed. We then found that the expression of Ihh was significantly decreased in the growth plate cartilage of the huTNFTg animals when compared to wild type controls. In a diabetic mouse model, elevated levels of TNF have previously been shown to directly suppress Ihh expression in skeletal stem cells suggesting a link between TNF and Ihh signaling which is in line of our present findings in huTNFTg mice28. The increased width of the growth plate has also been reported in conditions not associated with hypophosphatemia, such as abnormal IGF-1 metabolism29. Previously, it has been shown that glucocorticoids can not only impair GH-IGF-1 signaling30, but also cause hypophosphatemia in hospitalized patients31.To explore if an anti-inflammatory treatment with a glucocorticoid (GC) may rescue bone growth and chondrogenesis in an in vivo model of chronic arthritis, we also treated young huTNFTg mice with daily injections of dexamethasone or saline. We confirmed that dexamethasone not only suppressed the chronic arthritis but also restored the height of the total growth plate and the hypertrophic zone. However, despite these positive effects of the dexamethasone treatment, bone growth was further decreased compared to saline-treated huTNFTg controls. Interestingly, the observed increase in growth plate height in our study resembles the growth plate phenotype seen in hypophosphatemia27. Furthermore, dexamethasone treatment has been reported to suppress longitudinal bone growth by increasing chondrocyte apoptosis in the growth plates of treated mice13. These previous reports support our conclusion that increased chondrocyte apoptosis and disorganized chondrocyte columns may, at least partly, explain how dexamethasone further impaired bone growth despite suppressing the chronic arthritis in the huTNFTg mice. Importantly, body weight was not significantly affected in the young huTNFTg mice treated with dexamethasone suggesting that the observed growth suppressive effect of dexamethasone was not linked to poor general health.To clarify the underlying mechanisms for this unexpected finding, growth plate histomorphometry and immunohistochemistry was performed. We then found that despite successfully reducing inflammation, as verified by reduced arthritis score, the dexamethasone treatment was not able to restore chondrocyte proliferation in the growth plates of huTNFTg mice. Strikingly, the high apoptotic activity and disorganized chondrocyte columns observed in huTNFTg mice were further enhanced by the dexamethasone treatment. We therefore conclude that the anti-inflammatory effect of dexamethasone could not compensate for the negative effect of the GC treatment on growth plate. Instead the combination of TNF and dexamethasone further worsened longitudinal bone growth. A link between disorganized chondrocyte columns in the growth plate and impaired bone growth has earlier been reported in mice32, where growth plate height was increased despite bone length was decreased. Interestingly, it has also been shown that apoptosis is increased in the growth plates of mice with osteogenesis imperfecta, which potentially contributes to the reduction of the mineralized area in the hypertrophic zone33. These observations suggest similarities between dexamethasone induced bone growth suppression and growth impairment as seen in osteogenesis imperfecta, because in both conditions apoptosis is increased while growth plate height is also increased. Further, increased apoptosis in osteoblasts was observed in osteogenesis imperfecta where longitudinal bone growth is often decreased34. Similarly, GCs are known to increase apoptosis in osteoblasts35, which suggests that osteoblasts might also play a role in GCs induced growth retardation under chronic inflammation. However, more experimental evidence is required to address this complex question in the future. Another explanation might be that caspase-3 is not only related to cell death, but also to hypertrophy. It has been previously reported that activation of caspase-3 plays a key role in cardiac hypertrophy36. Similarly, another study showed a strong link between hypertrophy and caspase-3 expression in epiphyseal chondrocytes37. Altogether, the complex cross-talk between bone growth and caspase-3 expression and apoptosis needs to be further investigated.We observed that although the caspase-3 levels were increased after dexamethasone treatment, the growth plate height, including the hypertrophy zone, was increased, despite bone growth was impaired. A similar observation was earlier reported in a mouse model of osteogenesis imperfecta38. Also, the expression of IL-6 has been reported to be increased by TNF through NF-κB40. As both IL-6 and IL-1β have been described to be upregulated in TNF-overexpressing mice41, these pro-inflammatory cytokines may have contributed to the bone growth suppression and impaired chondrogenesis here reported in huTNFTg mice. A second limitation is the fact that bone length measurements and mechanistic studies were only performed at endpoint and therefore the timing of the different effects could not be further detailed. A third limitation is the relative low number of animals per group which makes sub-analyses of any sex differences difficult.There are several potential limitations in our study. First, despite TNF is one of the predominant cytokines in arthritis, there might be other pro-inflammatory cytokines upregulated as part of the cytokine response. We have previously shown that TNF and IL-1β act in synergy to cause growth retardation in cultured ex vivo fetal rat metatarsal bonesIn summary, this study for the first time shows how TNF and dexamethasone impair bone growth separately, and in an additive manner. TNF-driven chronic inflammation in mice is linked to suppressed chondrocyte proliferation and hypertrophy as well as increased apoptosis and disorganized chondrocyte columns in the growth plate cartilage. Dexamethasone treatment, despite efficiently suppressing the inflammatory activity, unexpectedly accelerated chondrocyte apoptosis and caused chondrocyte columns to become more disorganized in the growth plates and further suppressed bone growth. These results give a possible explanation to how GC treatment may further impair bone growth in children with chronic inflammatory conditions. When developing new effective treatments of chronic inflammatory conditions, our data highlights the importance of choosing therapeutic strategies with minimal effects on the growth plate cartilage thereby preventing bone growth impairment.Children with chronic inflammatory disorders are often treated with glucocorticoids and many experience growth retardation where the underlying mechanisms are not fully understood. In preclinical models, proinflammatory cytokines and glucocorticoids have individually been shown to impair bone growth, but how they interact is still unclear.This study supports a link between systemic inflammation and impaired bone growth in a mouse model of chronic arthritis where human TNF is overexpressed (huTNFTg). The data presented suggests that glucocorticoids, despite having an anti-inflammatory activity, further impair bone growth and chondrogenesis in huTNFTg mice. The study also provides mechanistic data showing that glucocorticoid treatment may further trigger chondrocyte apoptosis and loss of chondrocyte columns in the growth plates of huTNFTg mice.Our data provide a better understanding of possible mechanisms behind the growth retardation commonly observed in children with chronic inflammatory diseases. Our experimental data suggest that glucocorticoids may further impair bone growth and chondrogenesis when used to treat a chronic inflammatory condition. This study emphasizes the need of new anti-inflammatory treatment strategies that do not cause undesired apoptosis in the growth plate, which should reduce the risk of growth failure in children.


This cohort study evaluates the association between acoustic cry characteristics in preterm infants and developmental and behavioral outcomes at 2 years of age. Are acoustic cry characteristics in preterm neonates associated with developmental and behavioral outcomes at 2 years of age?In this cohort study of 363 preterm infants, acoustic cry characteristics were associated with clinically significant language and cognitive deficits, behavior problems, and a positive autism screen at age 2 years.These findings point to the potential use of acoustic cry characteristics in the early identification of preterm infants that are most at risk for longer term developmental and behavioral deficits. Acoustic cry characteristics have been associated with severe medical problems in newborns. However, little is known about the utility of neonatal acoustic cry characteristics in the prediction of long-term outcomes of very preterm infants.To evaluate whether acoustic characteristics of infant cry at neonatal intensive care unit (NICU) discharge are associated with behavioral and developmental outcomes at age 2 years in infants born very preterm.Infants born less than 30 weeks postmenstrual age (PMA) were enrolled from April 2014 through June 2016 as part of a multicenter (9 US university affiliated NICUs) cohort study and followed to adjusted age 2 years. Reported analyses began on September 2021. Data were analyzed from September 2021 to September 2022.The primary exposure was premature birth (<30 weeks PMA).Cries were recorded during a neurobehavioral examination administered during the week of NICU discharge. Cry episodes were analyzed using a previously published computerized system to characterize cry acoustics. Year-2 outcomes included the Bayley-III Composite scores, Child Behavior Checklist (CBCL) and the Modified Checklist for Autism in Toddlers (M-CHAT R/F), dichotomized using clinically significant cutoffs .Analyzed infants (363 participants) were primarily male (202 participants [55.65%]) and had a mean [SD] gestational age of 27.08 [1.95] weeks). Cross-validated random forest models revealed that cry acoustics were associated with 2-year outcomes. Tests of diagnostic odds ratios (DOR) revealed that infants who exhibited total problem behavior CBCL scores greater than 63 at age 2 years were 3.3 times more likely  to be identified as so by random forest model estimates relative to other infants (scores ≤63); this association was robust to adjustment for family-wise type-I error rates and covariate measures. Similar associations were observed for internalizing  and externalizing  scores on the CBCL, clinically significant language  and cognitive  scores on the Bayley-III, and a positive autism screen on the M-CHAT .In this cohort study of preterm infants, findings pointed to the potential use of acoustic cry characteristics in the early identification of risk for long-term developmental and behavioral deficits. The present study brings together a cry analysis system that leverages state-of-the-art signal processing with machine learning technology to take advantage of large and complex data sets to study associations with varied cry acoustic features including energy , pitch , formants , voicing , and fricatives . We studied preterm infant cries recorded before discharge from the neonatal intensive care unit (NICU) and developmental outcomes at age 2 years and hypothesized that acoustic cry characteristics were associated with neurobehavioral and behavioral deficits.The paradox in infant cry research is that early technologies to measure acoustic features of the infant’s cry used crude instrumentation; initially the sound spectrograph, which could only measure a few cry features, was used. Cry pitch became the harbinger of pathology because it is audible and high pitch is common in infants with medical problems.STROBE) reporting guideline.Infants were enrolled in the multicenter, observational Neurobehavior and Outcomes in Very preterm Infants (NOVI) study from April 2014 through June 2016 at 9 US university-affiliated NICUs. Parents of eligible infants were invited to participate at 31 to 32 weeks’ PMA, or when the attending neonatologist determined that survival to discharge was likely. Enrollment and consent procedures were approved by local institutional review boards. All mothers provided written informed consent. Sample size was determined by an a priori power analysis. This cohort study adhered to the Strengthening the Reporting of Observational Studies in Epidemiology (Inclusion criteria were: (1) birth at less than 30 weeks postmenstrual age, (2) parental ability to read and speak English or Spanish, and (3) residence within 3 hours of the NICU and Follow-Up clinic. Exclusion criteria included maternal cognitive impairment (inability to provide informed consent), maternal age of less than 18 years, maternal or infant death, and infants with major congenital anomalies.7 during the week of NICU discharge (± 3 days). The cries were elicited by handling the baby in a standardized format that is designed to increase the babies’ level of arousal. The stimuli are akin to cries that are elicited during routine caregiving such as a diaper change. Audio recordings were made using an Olympus direct to PCM digital voice recorder and saved in an uncompressed .wav audio format . Recorders were attached to the side of the infant’s crib at a standardized location and oriented toward the infant’s mouth. This allowed for a standard distance between the infant’s mouth and the recorder during the NNNS examination (approximately 8-9 inches from the infant’s mouth). Episodes of cry vocalizations were identified from these audio recordings. Cry episodes suitable for acoustic analysis were identified based on the absence of background noises that would interfere with the analysis . The identification of usable cries was based on reliability training from a previous study14 in which 89% agreement was established in identifying cries appropriate for acoustic analysis. The first suitable cry episode from each examination was excerpted into an uncompressed .wav file for subsequent acoustic analysis. The extracted recordings only contained infant cries and were stripped of any identifiable information. Of the infants with 2-year outcome data, 428 had extractable cries .Cries of the preterm infants were recorded during administration of the NICU Network Neurobehavioral Scale (NNNS)Several acoustic features were used to characterize cry episodes, including energy, fundamental frequency, formants, utterances, voicing, fricatives, and signal quality. Briefly, energy measures sound pressure levels in decibels (which we hear as loudness) as air is expelled from the lungs. Related are utterances which can be long (>500 ms) or short (<500 ms). At the larynx, vibration of the vocal folds produces sound waves including the fundamental frequency (which we hear as pitch). This initial sound is then modified or filtered by the vocal track producing formants. Formants are characteristic features of the resonances of the space, . For example, a “C” note on a saxophone sounds different than a “C” note on a piano because the resonating chambers are different. Signal quality measures the reliability with which the cry analyzer can measure the acoustic characteristics. Voicing refers to sounds produced by vocal fold vibration. Sounds can also be unvoiced when the vocal folds are not involved but the sound is altered as, for example, with the tongue or lips. Fricatives characterizes how the sound is altered by friction due to constriction of the vocal tract.13 Infant medical risk was measured using an index,15 in which scores are computed as the sum of dummy variables indicating the presence of 4 morbidities ascertained prospectively16 from medical record reviews using Vermont-Oxford Network definitions17 of brain injury , bronchopulmonary dysplasia, severe retinopathy of prematurity, and necrotizing enterocolitis and/or culture positive sepsis.15 Race and ethnicity data  were obtained from maternal interview self-reports (preferred source), or medical record abstraction. We included race and ethnicity as part of standard, unbiased demographic information.Procedures and criteria for collecting maternal and infant variables have been previously described, including cranial ultrasonography readings, interpreted by centralized study neuroradiologists.18 Parents completed the Child Behavior Checklist (CBCL)19 problem scales and the Modified Checklist for Autism in Toddlers (M-CHAT R/F).20 These measures were dichotomized using clinically significant cutoffs .21Main outcome measures were completed at 2 years adjusted age (± 2 months). Assessments included the Bayley-III, administered by experienced examiners who were blinded to infant medical conditions in the NICU, and trained by certified Bayley-III trainers to reliability using standardized Bayley-III training protocols.18 Analysis proceeded in 2 phases. The first phase applied a cepstral-based acoustic analysis to extract acoustic parameters with a 12.5 ms frame advance. The second phase organized and summarized this information into cry utterances. The mean (SD) amount of crying analyzed per infant was 17.37 seconds (15.54). Long cry utterances were defined as a cry during the expiratory phase of respiration lasting at least 0.5 seconds. Short cry utterances were less than 0.5 seconds; short and long utterances show distinct acoustic characteristics and thus were analyzed separately. The automated cry analyzer produced 56 acoustic characteristics per cry utterance. Acoustic characteristics for a total of 4431 long (≥500 ms) and 10 270 short (<500 ms) utterances were processed, including a mean (SD) of 12.2 (12.2) and median (IQR) of 9 (6-14) long utterances and a mean (SD) of 28.3 (25.8) and median (IQR) of 20 (13-33) short utterances per child on average. For each acoustic characteristic, we computed infant-level means and infant-level signal quality . Infant-level counts of cry utterances were also computed (thereafter referenced as “utterances”). Next, the resulting set of candidate features was reduced, such that features with high rates (>40%) of missing data, near 0 variability, and high (r = 0.75) intercorrelation were removed, consistent with published guidelines for machine learning feature filtering.22As shown in the 23 package on R 4.2 24 using default parameters with exception of number of trees (10 000). Model estimates were evaluated using leave-1-out cross validation, such that the test-train split is repeated for every infant in the data set, random forest models are trained on data containing all except 1 infant, and the held-out infant is used to evaluate whether model estimates generalize to new data. Predicted probabilities generated using leave-1-out cross validation were used to create a receiver operating characteristic (ROC) curve, and pick an optimal probability cut point (based on the shortest distance to perfect prediction).25 The resulting binary estimates were used to compute diagnostic odds ratios (DOR),26 95% confidence intervals and P values . Area under the receiver operating curve estimates were also reported.26A cross-validated supervised machine learning model  was used to estimate developmental outcomes at age 2 using all available cry acoustics variables. Random forests were fitted using the ranger R27) to account for nesting of infants (level 1 unit) within study sites (level 2 unit). Consistent with prior work,29 the effect of confounding variables was adjusted using a stepwise method, where all covariate measures  were entered as level 1 fixed effects in the first step and the final estimate of our machine learning models was entered as a level 1 fixed effect in the second step. A (nonparametric) likelihood ratio permutation test was used to evaluate whether adding the final machine learning estimate to a model containing covariate measures led to a significant reduction in model deviance. The false discovery rate correction was used to maintain acceptable (α = .05) family-wise type I error rates. Data were analyzed from September 2021 to September 2022.A mixed effect binary logistic regression model was fitted (using glmer from the lme4 package21(541) = 6.25, P = .01, OR = 1.77; all other tests were nonsignificant.The study enrolled 704 infants, of which 556 completed the 2-year assessment . Of these, 363 completed the year 2 follow-up, had complete covariate data and valid cry data. Maternal and infant descriptive data of the final sample are shown in P = .04) that was robust to false discovery rate correction and adjustment for covariate measures. Similar, but nonsignificant tests of DOR were observed for other outcomes, including internalizing problem behavior , externalizing problem behavior , language deficit , cognitive deficit  and positive autism screen . As shown in P values. Finally, permuted likelihood ratio tests were significant only for model comparisons evaluating estimates of total problem behaviors.Model estimates of 2-year outcomes showed improvements over chance . Test of DOR  implied Examining the top 10 most important variables within each model (see eTable in Using a machine learning (random forest) approach along with contemporary signal processing methods, we have shown that acoustic cry characteristics are associated with some 2-year developmental and behavioral deficits in very preterm infants. Specifically, estimates of models trained using acoustic cry characteristics were associated with clinically significant total problem behavior scores, even after controlling for covariate measures or family-wise type-I error rates. Likelihood-ratio tests also indicated that adding estimates of random forest models trained on cry acoustics to a mixed model containing covariates significantly improved model fit, suggesting a unique association of acoustic cry characteristics with prediction of clinically significant total problem behavior scores at age 2. Similar (but nonsignificant) associations were observed for clinically significant language and motor composite scores on the Bayley-III, internalizing and externalizing scores on the CBCL, and a positive autism screen on the M-CHAT.32 and other at risk populations13 in the neonatal period but not to longer term developmental or behavioral deficits and typically researchers examined a limited subset of acoustic characteristics. Our findings point to the potential use of acoustic cry characteristics in the early (before NICU discharge) identification of which preterm infants are most at risk for longer term developmental and behavioral deficits and by perhaps to other at-risk and not at-risk populations.Previous work describing abnormal cry acoustics in infants with severe medical problems suggested that cry characteristics might be of diagnostic value in at-risk infant populations where developmental outcome is less clear. Cry acoustics have been associated with prematurityThe CBCL findings are noteworthy from a linguistic perspective. Human speech is divided into linguistic and paralinguistic components. The linguistic component refers to words and their syntax. The paralinguistic component is the prosodic features of pitch, loudness, melody, and intonation that convey the affective side of speech. Crying includes only prosodic features that are aligned with the behavioral and emotional problems measured by the CBCL. The effects that we found were on the internalizing problem score which includes anxious/depressed, withdrawn-depressed, and somatic complaint scales and the externalizing problem score that combines the rule-breaking and aggressive behavioral scales. This connection between cry as prosody and later behavioral and emotional problems could lead to the study of neural pathways related to the origins of mental health disorders.Our study had limitations. One limitation of this work is that we were only able to use cries from 363 of the 556 infants; however, there was only one significant difference between those included and those excluded. This suggests that we need to make improvements in cry collection and extraction. A second limitation is that this is a data driven approach and although the infants were drawn from 9 NICUs, this was still a single cohort study. The next step would be a hypothesis-testing study to determine if these acoustic cry models replicate in other cohorts. Additionally, although acoustic cry analysis may help identify which preterm infants are most at risk for adverse outcomes, these measures do not readily translate into specific intervention strategies.Despite its limitations, this work raises the possibility that cry analysis could be developed into a bedside, accessible, and non–labor-intensive machine-interpreted diagnostic similar to electrocardiographic readings. The ability to identify which infants are at highest risk for developmental and behavioral disorders could lead to the development of interventions to mitigate adverse outcomes.


Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes.n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis.Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate <0.05.Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.The online version contains peer-reviewed but unedited supplementary material available at 10.1007/s00125-022-05726-1. A recent report from the Diabetes Autoimmunity Study in the Young (DAISY) on prospective epigenomics of type 1 diabetes also included samples collected before seroconversion of the case participants to islet autoantibody positivity [DNA methylation at cytosine residues is one of the most important epigenetic mechanisms regulating gene expression. The modification converts cytosine to 5-methylcytosine, usually in the context of CpG dinucleotides. Differential methylation at the promoter or other regulatory elements affects gene expression in health and diseases . Most stsitivity .A peak in the appearance of islet autoimmunity occurs at an early age, between one and two years . We therIn this study we used the reduced representation bisulphite sequencing (RRBS) method to analyse umbilical cord blood DNA methylation associated with later progression to type 1 diabetes in a prospective cohort from the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. The aim was to detect DNA methylation patterns associated with later progression to type 1 diabetes. Such findings would be valuable for a better understanding of early mechanisms underlying the progression to type 1 diabetes related autoimmunity.The n = 43) who were diagnosed with type 1 diabetes during the DIPP Study follow-up or became persistently positive for at least two biochemical islet autoantibodies (in at least two consecutive serum samples) were compared with control participants (n = 79) who remained autoantibody-negative throughout the DIPP Study follow-up, i.e. up to 15 years of age or until their decision to discontinue participation in the study. Data until the end of year 2018 were included. Clinical data such as maternal insulin-treated diabetes, gestational weight gain and the child’s birthweight were used to adjust for potential confounding effects. The characteristics of the case and control participants are described in Table Case participants , IA-2A (insulinoma-associated protein 2 antibody), GADA (glutamic acid decarboxylase antibody) and ZnT8A (zinc transporter-8 antibody). Screening for classical islet cell antibodies was used as the only autoantibody screening method for children in the DIPP Study born before 2003, and, if positive, all other autoantibodies were measured from all previous and future samples from the child.The power analysis was performed on simulated bisulphite sequencing data using a tool developed by Lea et al   or persistent positivity for only one islet antibody (n = 4) were excluded from the study  bisulphite conversion efficiency, two were excluded due to missing clinical data, and five were rejected due to an inadequate amount or quality of DNA. Samples from individuals with transient islet antibodies  was estimated empirically through a permutation analysis [The library preparation steps were adapted from the RRBS protocol described by Boyle et al . An Illu package . As the analysis  , HLA risk class, sex, and PC1 and PC2 . After spatial adjustment, two adjacent CpG sites  on an intron of gene Plakophilin-3 (PKP3) showed weak evidence of hypomethylation in the case participants . We carried out pyrosequencing to validate five selected targets technically, but the results did not indicate differential methylation between the groups  and ages at diagnosis (range 1.6–18.8 years), who may represent different disease subtypes, the existence of which has been suggested by several studies during the past decade . For exaESM 1(PDF 1.01 mb)


Evidence on the predictors of common mental disorders using nationwide health registries are scarce in Norway. Identifying modifiable behaviours affecting mental health across the lifespan is paramount to develop tailored strategies to tackle mental illnesses. The aim of this study was to identify patterns of health-related behaviours in adolescence and their influence on anxiety and/or depression in adulthood.This was a prospective study based on data from the Trøndelag Health Study (HUNT) and health register data (N = 2061). Patterns of health-related behaviours were assessed according to physical activity, consumption of wholegrain bread, fish, fruit, vegetables, and sugar sweetened beverages and insomnia. Participant’s use of healthcare system for anxiety and/or depression was recorded at least once in the health registries. The patterns of health-related behaviors were identified through latent class analysis. Multivariable logistic regression was used to test the association between patterns of health-related behaviors and depression or/and anxiety.Four classes of health-related behaviors were identified: class 1 (15.2%), class 2 (36.0%), class 3 (24.2%), class 4 (24.6%). Adolescents with unhealthy behaviors  had 82%, 34% and 84% higher odds of depression and/or anxiety during adolescence and early adulthood than those from the healthy-related behaviors group (class 4).Our findings suggest that health-related behaviors are clustered among Norwegian adolescents. There was a meaningful association of the three patterns of unhealthy behaviors during adolescence with anxiety and depression in adulthood. Population strategies and policies aiming to tackle unhealthy behaviors among adolescents can positively impact on adult’s mental health.• Improving healthy behaviors during adolescence may reduce the burden of mental illness in adulthood.• Population strategies and policies aiming to tackle unhealthy behaviors among adolescents can positively impact on adult’s mental health.


De novo lipogenesis has been inversely associated with serum sex hormone-binding globulin (SHBG) levels. However, the directionality of this association has remained uncertain. We, therefore, studied individuals with glycogen storage disease type 1a (GSD1a), who are characterized by a genetic defect in glucose-6-phosphatase resulting in increased rates of de novo lipogenesis, to assess the downstream effect on serum SHBG levels.n = 10) and controls matched for age, sex, and BMI (n = 10). Intrahepatic lipid content and saturated fatty acid fraction were quantified by proton magnetic resonance spectroscopy.A case–control study comparing serum SHBG levels in patients with GSD1a (p = 0.041), while intrahepatic lipid content and intrahepatic saturated fatty acid fraction—a marker of de novo lipogenesis—were significantly higher in patients with GSD1a . In addition, there was a statistically significant, inverse association of intrahepatic lipid content and saturated fatty acid fraction with serum SHBG levels in patients and controls combined .Serum SHBG levels were statistically significantly lower in patients with GSD1a compared to the controls (Patients with GSD1a, who are characterized by genetically determined higher rates of de novo lipogenesis, have lower serum SHBG levels than controls. It has long been thought that sex hormone-binding globulin (SHBG) acts only as a simple carrier protein that regulates the bioavailable fraction of testosterone and other sex hormones . In the In vitro and animal studies have shown that carbohydrate-induced de novo lipogenesis is one of the mechanisms involved in the downregulation of SHBG levels . We haveG6PC gene encoding glucose-6-phosphatase  study  study , and (3)S] study . ParticiS] study . For theS] study . All stuS] study . All parAll participants had to be at least 18 years of age and were excluded from participation if they had any contraindications for magnetic resonance imaging (MRI) or were unable to give informed consent. Furthermore, as oestrogen-containing medication, which substantially affects serum SHBG levels, are relatively contra-indicated in GSD1a –21, cont1H-MRS) using a 3T clinical MR scanner , were performed as described previously [1H-MRS method. We previously showed that this fraction correlates well with de novo lipogenesis assessed by stable isotopes [For all participants, anthropometrics and quantification of IHL content by proton magnetic resonance spectroscopy . All results were considered statistically significant at p < 0.05. All statistical analyses were performed using IBM Statistical Package of Social Science (SPSS) version 25.0 for Windows .Continuous data are presented as median (interquartile range) and categorical data are presented as frequencies. Continuous variables were compared between GSD1a patients and controls by means of Mann–Whitney n = 10) and matched controls  are shown in Table 2). Age, sex distribution and BMI were, by design, comparable between GSD1a patients and controls   .Patients with GSD1a had statistically significantly lower levels of serum insulin compared to controls  GSD1a patients, and were notable outliers , or after stratification by condition in the GSD1a group , or in controls  . The SFA fraction was also statistically significantly inversely associated with lower serum SHBG levels in the overall population .In the overall population there was no statistically significant association between BMI or insulin with serum SHBG levels , which also results in higher intrahepatic glucose-6-phosphate levels and rates of de novo lipogenesis, albeit by a different mechanism. Similar to patients with GSD1a, individuals carrying the GCKR minor allele are characterized by lower fasting glucose and insulin levels, higher serum triglycerides, higher rates of de novo lipogenesis and a higher IHL content [GCKR minor allele is also associated with lower serum SHBG levels [The current findings are in line with large population studies that assessed the effect of a common variant in the glucokinase regulatory protein gene , the low serum SHBG levels may contribute to the higher prevalence of PCOS that has been observed in GSD1a , 32. SecThis study has several strengths and limitations. The use of GSD1a as a model for higher rates of de novo lipogenesis is a unique approach to study the direct effects of de novo lipogenesis on serum SHBG levels in an observational setting. However, the rare nature of this disease  resultsIn conclusion, in the present study we found that patients with GSD1a, who are characterized by genetically-determined higher rates of de novo lipogenesis, have statistically significantly lower levels of serum SHBG than controls.


The relaxed phase-matchingconditions in a WS2 monolayer enable the simultaneous acquisitionof the spectra resulting from both four-wave mixing (FWM) and sum-frequencygeneration (SFG) nonlinear processes while varying the time delaybetween the two ultrashort pulses. Next, we introduce an adjusteddouble-blind FROG algorithm, based on iterative fast Fourier transformsbetween two FROG traces, to extract the intensity distribution andphase of two ultrashort pulses from the combination of their FWM andSFG FROG traces. Using this algorithm, we find an agreement betweenthe computed and observed FROG traces for both the FWM and SFG processes.Exploiting the broad-band nonlinear response of a WS2 monolayer,we additionally characterize one of the pulses using a second-harmonicgeneration (SHG) FROG trace to validate the pulse shapes extractedfrom the combination of the FWM and SFG FROG traces. The retrievedpulse shape from the SHG FROG agrees well with the pulse shape retrievedfrom our nondegenerate cross-correlation FROG measurement. In additionto the nonlinear parametric processes, we also observe a nonlinearlygenerated photoluminescence (PL) signal emitted from the WS2 monolayer. Because of its nonlinear origin, the PL signal can alsobe used to obtain complementary autocorrelation and cross-correlationtraces.The precise characterizationof ultrashort laser pulses has beenof interest to the scientific community for many years. Frequency-resolvedoptical gating (FROG) has been extensively used to retrieve the temporaland spectral field distributions of ultrashort laser pulses. In thiswork, we exploit the high, broad-band nonlinear optical response ofa WS Since the realizationof ultrashort laser pulses, there has beenmuch interest in accurately retrieving their temporal intensity distribution.Ultrashort laser systems readily produce pulses with a pulse durationthat is too short to be directly measured with even the fastest photodiodes.Therefore, indirect autocorrelation methods are used to estimate thelaser pulse duration, where the pulse interacts with itself and thetime delay between two copies of the pulse is varied. However, autocorrelationmethods intrinsically cannot provide the full pulse information, thatis, spectral resolution is needed to retrieve the spectrum and time-dependentphase of the pulse.2 In a FROG measurement, two ultrashort laser pulses are combinedin a nonlinear medium. The spectrum of the nonlinear signal, generatedvia parametric optical processes, is recorded as a function of thetime delay between the pulses, resulting in a FROG trace that containsboth spectral and temporal information. FROG has been implementedusing different optical setups and nonlinear processes including collinearsetups, where the laser beam paths overlap, for second-harmonic generation(SHG),3 third-harmonic generation (THG),4 and noncollinear setups.5 In an autocorrelation FROG measurement, two copies of the same pulseare combined inside a nonlinear medium. Alternatively, in a cross-correlationFROG measurement, a known reference pulse is combined with an unknownpulse to generate nondegenerate signals such as sum-frequency generation(SFG) or four-wave mixing (FWM).9One of the most investigated and commonlyused techniques to characterizeultrashort laser pulses is frequency-resolved optical gating (FROG).12To retrieve the pulse information of two unknownultrashort pulsesat different frequencies, the measurement of a FROG trace based ona single nondegenerate nonlinear process will not contain enough information.Therefore, cross-correlation measurements using nondegenerate nonlinearprocesses depend on a known reference pulse, a priori. With this method,a full characterization of two individual pulses still involves twomeasurements: an autocorrelation FROG measurement to retrieve theintensity distribution and phase of the reference pulse and the actualcross-correlation FROG measurement. The pulse shapes of two independentlaser pulses can also be retrieved using multiple FROG traces basedon different nondegenerate processes that are simultaneously measured.12 Atomically thin transition-metal dichalcogenide (TMDC) materialsalso show a remarkable high nonlinear surface response,15 along with an increase in absorption for photon energies above thesemiconductor band gap.16 In addition tothe background-free signals and the broad-band nonlinear response,TMDCs are also very promising materials for ultrashort pulse characterizationdue to their large atomically flat surface, which allows for easybeam alignment and transmission measurements. Since WS2 and related TMDC materials have large nonlinear susceptibilitiesover the whole visible wavelength range, they can be used to characterizelaser pulses with a wide variety of wavelengths. Different types ofTMDC monolayers or multilayer materials with different band-gap energiessuch as WSe2, MoS2, and MoSe2 couldbe selected depending on the wavelength of the ultrashort laser pulsesand nonlinear processes employed to generate the FROG trace. Furthermore,different combinations of nonlinear FROG signals can be selected dependingon the characterization demands at hand, specifically those relatedto the laser wavelengths.The most commonly used nonlinear media in FROG systems arenonlinearcrystals due to their high nonlinear coefficients and broad frequencyrange of transparency. However, phase-matching requirements have tobe satisfied in these nonlinear crystals, which can make experimentalimplementation difficult and restrict the frequency range of applicability,making simultaneous measurements of different nonlinear processesimpractical. Nonlinear surface processes have a shorter interactionlength and can overcome these phase-matching requirements. Therefore,nonlinear media with a large nonlinear surface response provide aroute to generate and exploit multiple nonlinear processes simultaneously.The nonlinear surface response of plasmonic nanoantennas has beenused for ultrashort pulse characterization.2 could be further and selectively enhanced usingmore complex layered WS2 structures18 or combining WS2 with plasmonic nanostructures.19 Recently, FROG characterization of a singlepulse using the SHG process has been performed on a WS2 monolayer.20Their fabrication process is relativelyinexpensive and robustcompared to plasmonic nanostructures. Nonlinear signals generatedin WS2 monolayershave another interesting property as nonlinear media that can be exploitedfor ultrashort pulse retrieval. The nonlinear generation of light-inducedelectron–hole pairs, the so-called excitons, can be potentiallyutilized in an autocorrelation measurement as its spectral width remainsconstant. The band structure of WS2 monolayers have a directoptical band gap at the K and K– points allowingfor the (nonlinear) generation of excitons.21 The radiative recombination of these excitons to the ground statecan be observed as a photoluminescence (PL) signal.15In addition to theiratomic thickness, WS2 monolayer over a broad spectral range to characterizetwoultrashort laser pulses by measuring FROG traces based on the SHG,SFG, and FWM nonlinear processes. The spectra of the SFG and FWM processesare simultaneously recorded as a function of the time delay betweentwo pulses with different wavelengths using a collinear optical setup.With a newly developed adaptation of a double-blind FROG algorithm,we precisely retrieve the pulse shape of two laser pulses at two differentwavelengths, E1 and E2, using experimental FROG traces based on FWM and SFGthat were simultaneously measured. We validate the pulse shape ofone of the fundamental pulses using a separate SHG FROG measurementand by utilizing autocorrelation traces generated via nonlinear photoluminescence.In this paper, we exploit the high nonlinear responseof a WSE1 and E2 from nondegenerate FROG traces,we use an iterative retrieval algorithm based on the common pulseretrieval algorithm (COPRA), recently developed by Geib et al.22 The nonlinear process spectra of a measurednoncollinear FROG trace can be defined as23E is theelectric field of the laser pulse, SFG = ω1 + ω2) and FWM (ωFWM = 2ω1 – ω2) processesas observed in our experiment, where the delay time of E2 is varied with respect to E1, can be derived asE1 and E2 so thatthe calculated FROG trace I matches the measuredtrace, Imeas, as a nonlinear least-squaresproblem, further described in Geib et al.22 For a nondegenerate cross-correlation retrieval algorithm, one ofthe pulses is optimized, while the second pulse acts as a referencepulse. In this work, we modified the COPRA retrieval algorithm tooptimize the pulse E1 or E2 for both experimental traces, Imeas,SFG and Imeas,FWM, while thesecond pulse acts as the reference pulse, further described in the Supporting Information.To retrieve the complex pulse shapes E1 and E2, we utilize an iterativeoptimization scheme. Here, we start with two Gaussian pulses withcentral wavelengths λ1 and λ2 asour initial guess. Next, we start optimizing E1 for Imeas,SFG and Imeas,FWM while keeping E2 asa fixed reference pulse. Since an initial guess of E2 is used as a reference pulse, the pulse retrieval of E1 is not completed but the retrieved pulse shapeof E1 is still a better approximationthan the initial guess. In the next step, the newly retrieved approximationof E1 is used as a reference pulse inthe retrieval of E2 resulting in a betterapproximation of E2. This process is iterateduntil the improvement of the normalized trace error between the measuredand the retrieved FROG traces between iterations falls below a predefinedvalue.To simultaneouslyretrieve two unknown pulses In this work, we retrieve thepulse intensity distribution andphase of two femtosecond laser pulses with different central wavelengths.A femtosecond laser oscillator (Spectra-Physics Tsunami) generatespulses with a central wavelength of 775 nm. Part of this laser beamis used as our first laser pulse, while a fraction of it is used topump an optical parametric oscillator  thatdelivers the second laser pulse at 1200 nm. The pulse distributionsof the 775 and 1200 nm laser pulses are retrieved by simultaneouslymeasuring two cross-correlation FROG traces based on SFG and FWM signalsusing the optical setup sketched in 2 monolayer flake using a microscopeobjective (Olympus UPlanSapo) with a numerical aperture of 0.95. TheWS2 monolayer is mechanically exfoliated from commerciallyavailable bulk WS2 onto a glass substrate . SFG = ω775 + ω1200, and FWM process around 572 nm, where ωFWM = 2ω775 – ω1200. In addition,a peak can be seen around 600 nm originating from the second harmonicof the 1200 nm pulse, where ωSHG = 2ω1200. These nonlinear signals have been well studied in WS2 and other 2D-layered materials.15 Finally, abroader peak at 625 nm is observed, which can be attributed to thePL signal of the WS2 monolayer. Note that the fundamentallaser pulses with wavelengths of 775 nm and 1200 nm have a photonenergy that is below the band gap of a WS2 monolayer. Withthese wavelengths, the band gap can only be excited via multiphotonexcitation. Therefore, the PL signal observed here is emitted by nonlinearlygenerated excitons.In both optical measurementschemes, motorized delay stages areused to control the time delay between the two laser pulses beforefocusing the beams on a WSalso ref ). The em2 monolayer.The power dependency on the nonlinear response is more extensivelydescribed in Hernandez-Rueda et al.15 Forboth setups, the time delay is created by a motorized delay line inthe 1200 nm beam path with spatial delay steps of 400 nm, equivalentto ∼1.3 fs. The spectrometer is calibrated by a rigid shiftof the recorded spectrum. To reduce experimental noise, we fit a Gaussianshape over the nonlinear signals, and afterward, we interpolated thedata onto a 1024 by 1024 matrix for the pulse retrieval algorithm.The nonlinear spectra are recorded for severaltime delays betweenthe pulses to obtain the experimental FROG traces with an exposuretime of 1 s. Here, we use an optical power of 2.4 mW for the 1200nm laser beam and an optical power of 22.6 mW for the 775 nm beamthat are below the damage threshold of the WSx-axis, the wavelength of the nonlinear spectrum is plotted,and on the y-axis, the delay time between the twolaser pulses is plotted. The top-left and middle panels depict theFWM and SFG traces, respectively, that are simultaneously collectedfrom the same spectrum for each time delay using the optical setupsketched in The top row of E1 and E2, usingthe algorithm described in the Theory section. We initialize the pulseretrieval routine using two guess pulses with a Gaussian intensitydistribution in the frequency domain, with a preset bandwidth of ∼16.7 nm full width at half-maximum (FWHM) and central wavelengthsat 775 and 1200 nm. We first retrieve E1 with E2 as a reference pulse and thenretrieve E2 using E1 as a reference pulse and iterate these two retrieval algorithms15 times.The collected FROG tracesof the SFG and FWM processes can nowbe used to extract the pulse intensity distribution and phase, R, that can be calculated from the measurement and retrieved traces.This normalized trace error is equivalent to the commonly used FROGerror  because the photon energies at the fundamental wavelengthsare smaller than the exciton band gap.25 The intensity of the photoluminescence thereforealso changes with the time delay between the laser pulses due to thecontribution of nondegenerate multiphoton excitation pathways, allowingfor direct (cross-)correlation measurements.Nonlinear excitation pathways similar to those in SFG, SHG, andFWM mechanisms can mediate the generation of excitons through nonparametricprocesses. For frequencies above the band gap, light is absorbed;subsequently, the radiative recombination of the A-exciton also causesthe characteristic photoluminescence signal at 625 nm, as observedin 2 monolayer sample, as illustratedin 1200, 2ω775) and nondegenerate excitationpathways . For long time delays,for which the individual pulses do not overlap in time, the PL signalis solely generated by degenerate multiphoton absorption mechanisms.In AFWHM, of 227 fs. This corresponds to a pulseduration, ΔτpFWHM, of 161 fs assuming a Gaussian pulsedistribution of the 1200 nm pulse. This pulse duration is in closeagreement with that extracted from the nondegenerate FROG method andthe SHG FROG method.2 monolayer due to its relaxed phase-matching conditionsfor ultrashort pulse characterization. Using a collinear optical setup,we measure both degenerate (SHG) and nondegenerate  nonlinearsignals simultaneously by illuminating a WS2 monolayerwith ultrashort laser pulses at two different wavelengths. We measurethe nondegenerate nonlinear signals as a function of the time delaybetween the laser pulses to perform collinear (cross-) correlationFROG measurements. Using a novel adaptation of the COPRA FROG algorithm,we retrieve the complex pulse distribution of both laser pulses. Anagreement is found between the retrieved pulses from the degenerateand nondegenerate FROG retrieval methods. We demonstrate the advantagesof the simultaneous measurement of multiple nonlinear processes forultrashort pulse characterization facilitated in 2D materials. Inaddition to the nonlinear parametric signals, we also measure thenonlinearly generated PL signal from the WS2 monolayer.The high nonlinear surface response over a broad range of wavelengthson an atomically thin TMDC allows for the implementation of novelretrieval methods simultaneously using multiple (non)degenerate nonlinearsignals and PL emission, i.e., by also recording the spatial chirpof the nonlinear spectrum.26We have exploited thebroad-band nonlinear response of an atomicallythin WS


Myelofibrosis (MF) is an acquired clonal hematopoietic stem cell disorder associated with debilitating constitutional symptoms, extramedullary hematopoiesis resulting in splenomegaly, and a propensity to transform to a blast phase/acute myeloid leukemia (AML). The discovery of JAK inhibitors (JAKi) has been pivotal in the treatment of symptomatic MF by reducing spleen size, and alleviating cytokine-related symptom burden . DespiteProspective trials of JAKi therapy provide little information after patients discontinue therapy and safety specific follow up is completed. Although survival following ruxolitinib cessation is poor, in the range 13–16 months, the clinical course and reasons for JAKi failure in MF patients are not well characterized –7. CriteWe conducted a retrospective study analyzing a molecularly annotated, mature dataset of MF patients treated with JAKi followed in a prospective MPN registry (NCT02760238) at Princess Margaret Cancer Centre. We evaluated the impact of baseline clinical and molecular factors on clinical outcomes and therapy failure. We characterized different patterns of JAKi failure according to consensus criteria of the Canadian MPN Group  was predictive for OS in model 1, and JAKi failure in both models.During the course of the study period 85 (75%) patients died with median follow-up in survivors of 74 (range: 21–120) months. A total of 107 (95%) patients experienced JAKi failure; and cumulative incidence of JAKi failure at 1, 3, and 5 years was 34%, 71%, 87%, respectively  or loss  of spleen response ; cytopenias ; AP/BP transformation ; non-hematological toxicity , and second malignancy . For all patients (n = 107), median [95%CI] OS following JAKi failure was 13.6 [9.0–19.6] months. Median survival by pattern of failure was: 4.1 [1.0–5.3] months for AP/BP; 17.5 [6.5–27.0] months for cytopenias; 21.8 [11.7–44.5] months for loss of/suboptimal spleen response; 0.3 [0.3- not reached] months for second malignancy and 13.6 [0.2–24.6] months for non-hematological toxicity (p < 0.001). There was no association between baseline mutations, number of mutations, or number of HMR mutations and pattern of JAKi failure  or after at least 3-years sustained clinical response to JAKi therapy (n = 6). Of the six patients with ongoing benefit from therapy: three patients had the same variants detected, two patients had dropout of mutations, and one patient had two emergent mutations  and dropout of JAK2.Analyis of paired sequencing was performed on 55 patients Fig.  who had JAK2 (n = 4), and TET2 (n = 4) the most commonly observed. A total of 29 emergent mutations were observed in 19 (39%) patients. The most common emergent mutations were in KRAS (n = 4) and ASXL1 (n = 4); with RAS pathway genes  and HMR the most common class of emergent mutations occurring in 9 (47%) patients each. Emergent mutations were more frequently observed in patients with JAKi failure due to AP/BP  than failure due to cytopenia ; while there was no significant difference when compared to patients with failure due to loss or lack of spleen response , non-hematological toxicity , or secondary malignancy . The median overall survival following JAKi failure was significantly shorter in those with emergent mutations compared to those without  patients had no change in observed variants. Dropout of 18 previously identified variants in 12 (24%) patients was observed; with dropout of CBL and total number of mutations predict OS independent of MIPSS risk category. A shorter time to JAKi failure was predicted by ECOG, RBX Tx, and CBL mutation; though not by MIPSS or total number of mutations. These results add further evidence and validation for the consideration of CBL mutations in future revision of the definition of high-risk MF [This study provides further understanding of the clinical and molecular outcomes following JAKi failure. Our analysis differs from previous studies looking at outcomes after JAKi discontinuation, as we used standardized JAKi failure definitions as opposed to relying on drug discontinuation as the sole indicator of failure. Despite this key difference, the overall survival following JAKi failure is poor and similar to previous reports –7. In MV-risk MF .Our study has expanded on previous research by describing and analyzing clinical features, correlates, and outcomes according to the pattern of JAKi failure. Patients who develop AP/BP or non-hematological malignancy on JAKi have a dismal prognosis; while outcomes following spleen progression or cytopenias have comparable outcomes. It is also important to note that in clinical practice, these failure reasons do not occur in isolation. For example, a patient may develop cytopenias requiring a dose reduction in JAKi and as a result the patient then loses their spleen and/or symptom response. Adherence to standardized criteria of JAKi failure will help in early recognition of the pattern of failure, facilitate clinical trial enrollment, and understanding of comparative effectiveness of novel agents.The emergence of mutations in our cohort was common, occurring in 37% of patients. This contrasts with previous reports from Lundberg et al., which detected only two new mutations in chronic MPN patients during 133 patient-years follow-up [ASXL1 mutations (21% of patients with emergent mutations) similar to a study from MD Anderson [KRAS). Differences between these cohort studies may be due to use of JAKi failure rather than drug discontinuation as our endpoint; or the larger panel of genes evaluated by NGS used in our study in particular with the inclusion of CBL [The clinical significance of clonal evolution as evidenced by the emergence of new mutations on paired analysis is an area of ongoing research. Consistent with previous reports, our data demonstrate that variant emergence is associated with inferior survival following JAKi failure , 14. BasAnderson , but then of CBL , 15. OurIn conclusion, we demonstrate that outcomes following JAKi failure are significantly correlated with the pattern of failure. Patients who transform to AP/BP have dismal outcomes, where as those with sub-optimal or loss of response or significant cytopenias have similar outcomes. Baseline molecular signatures did not predict the pattern of JAKi failure; however, development of emergent mutation at time of JAKi failure is observed more frequently with AP/BP disease.Supplementary material