Rousettus bat coronavirus GCCDC1 (RoBat-CoV GCCDC1) is a cross-family recombinant coronavirus that has previously only been reported in wild-caught bats in Yúnnan, China. We report the persistence of a related strain in a captive colony of lesser dawn bats captured in Singapore. Genomic evidence of the virus was detected using targeted enrichment sequencing, and further investigated using deeper, unbiased high throughput sequencing. RoBat-CoV GCCDC1 Singapore shared 96.52% similarity with RoBat-CoV GCCDC1 356 (NC_030886) at the nucleotide level, and had a high prevalence in the captive bat colony. It was detected at five out of six sampling time points across the course of 18 months. A partial segment 1 from an ancestral Pteropine orthoreovirus, p10, makes up the recombinant portion of the virus, which shares high similarity with previously reported RoBat-CoV GCCDC1 strains that were detected in Yúnnan, China. RoBat-CoV GCCDC1 is an intriguing, cross-family recombinant virus, with a geographical range that expands farther than was previously known. The discovery of RoBat-CoV GCCDC1 in Singapore indicates that this recombinant coronavirus exists in a broad geographical range, and can persist in bat colonies long-term. Human coronaviruses (HCoVs) have been studied for just over half a century, and are a priority for research as a consequence of recent high-profile disease outbreaks ,2. The fCoronaviridae is so named due to the morphology of the virion, which resembles a crown; thus, the name is derived from the Greek word for crown [The viral family or crown . Coronavor crown . Subgenoor crown . Coronavor crown . This poor crown . Cross-for crown . Viral ror crown .Rousettus leschenaultii) bats in 2016 in China [SARS-CoV, SARS-CoV-2, MERS-CoV and Rousettus bat coronavirus HKU9 (RoBat-CoV HKU9) are ancestrally related as bat-borne betacoronaviruses . RoBat-Cin China . This viEonycteris spelaea) population known to co-roost with Leschenault’s rousette [Following the discovery of RoBat-CoV GCCDC1 in Leschenault’s rousette in 2016, in Yúnnan, China, this virus was further detected in a lesser dawn bat  animal facilities. Trained laboratory personnel provided daily care for the animals according to the guidelines agreed upon by Duke-NUS Institutional Animal Care and Use Committee (2015/SHS/1088) and the Agri-Food and Veterinary Authority of Singapore. All sampling was noninvasive. Sampling was performed as previously described . In summExtractions and sequencing were performed as previously described . BrieflyRousettus bat coronavirus GCCDC1 was first detected in target-enrichment samples by read-mapping to Rousettus bat coronavirus isolate GCCDC1 356 (NC_030886) using CLC Genomics Workbench V11 . Further analysis for prevalence of the related strain was performed using shotgun data mapped using bbsplit with parameter adjustment of minid = 0.75 . ResultsMolecular phylogenetic analysis was performed with 100 bootstrap replicates, using the Maximum Likelihood method based on the General Time Reversible model in MEGA7 . The treLeschenault’s rousette and lesser dawn bat range data were obtained in shapefiles format from the International Union for Conservation of Nature and Natural Resources (ICUN) . Ranges Genomic evidence of RoBat-CoV GCCDC1 was detected in 72 of 206 samples that were collected from a captive colony of lesser dawn bats in 2016 and 2017, and characterized via shotgun sequencing. The virus was detected in five of six sampling dates in head, body, oral and rectal swabs , and thiThe genome of RoBat-CoV GCCDC1 includes a p10 gene from segment S1 of an ancestral orthoreovirus, inserted between the RoBat-CoV HKU9 nucleocapsid (N) and NS7a accessory genes A. The p1E. spelaea and R. leschenaultii species. A dinucleotide bias among strains of RoBat-CoV GCCDC1 has not been previously investigated, and we hypothesized that such a bias, if detected, may result from circulation in distinct bat host species. Upon investigation, we detected no significant difference in rho value or notable variation at the nucleotide level in the reovirus-derived segment p10 (E. spelaea (Singapore), E. spelaea (China) and R. leschenaultii RoBat-CoV GCCDC1  of this recombinant in the sampled cohort of captive lesser dawn bats. Moreover, the p10 segment of RoBat-CoV GCCDC1 Singapore is genetically distinct from known orthoreoviruses. Given that the necessary parental strains were not detected in the captive colony, we extrapolate from our observations that the recombinant is circulating in wild lesser dawn bats, and did not recently arise as a recombinant in the captive colony. Furthermore, we observe that the backbone of known RoBat-CoV GCCDC1 strains are closely related, and distinct at the nucleotide level from RoBat-CoV HKU9. The phylogenetic analysis of the full nucleotide sequence, as compared to previously published references for strains of GCCDC1 and RoBat-CoV HKU9 , illustrThe evolution of viruses of pandemic potential, such as bat-borne coronaviruses, is significant to public health due to the risk of spillover and subsequent sustained human-to-human transmission. Viral recombination in host reservoirs is a concern for public health, as these events can increase the potential for spillover. Here, we report genomic evidence of the recombinant RoBat-CoV GCCDC1 in a population of lesser dawn bats in Singapore. Interestingly, this recombinant virus strain exhibits genetic conservation, as compared to strains initially detected in Yúnnan, China. Therefore, we have demonstrated that a cross-family recombinant coronavirus persists in a captive colony of bats, and is similar at the nucleotide level to previously discovered strains, despite geographic and host differences. There is high similarity between RoBat-CoV GCCDC1 Singapore and previously reported strains by gene arrangement A, and hiThe virus was detected less frequently in the second year of the longitudinal study. It is unclear if prevalence of RoBat-CoV GCCDC1 was impacted during the first year of the study by unknown confounding factors. Development of a serological assay for RoBat-COV GCCDC1, paired with qPCR and/or high throughput sequencing (HTS), could expand our ability to understand the viral dynamics within bat colonies in the future. We hypothesize that RoBat-CoV GCCDC1 was able to persist in this lesser dawn bat colony, but, with no immigration or emigration, it was not continually shed at levels that were detectable by our methods. This work indicates the ability for the virus to persist long-term within a captive colony of lesser dawn bats. It is important to note that this study evaluated the population persistence of RoBat-CoV GCCDC1 in a captive colony, which is unique from previous longitudinal reports in wild-caught bats, which are exposed to variables such as weather changes or the ability to exchange genetic viral variants via dispersal and migration. The geographic distance and genetic similarity between strains provide insight to the possibility that this strain likely exists in the geographical region between Yúnnan province and Singapore. Additionally, unbiased biosurveillance assays could detect other, yet-to-be-discovered cross-family recombinants.Evidence of coronavirus infection has been detected in 198 bat species by a variety of methods, such as conventional polymerase chain reaction (PCR), quantitative PCR, serology, and HTS . CoronavE. spelaea extends across Southeast Asia, and it is unknown whether these populations are panmictic. As RoBat-CoV GCCDC1 continues to circulate in co-roosting populations of multiple bat species, the spike gene will be under pressure, as demonstrated in A continuation of sampling from Leschenault’s rousette and lesser dawn bat colonies in other geographic regions could reveal deeper insight into the circulation of RoBat-CoV GCCDC1 variants in wild bats. These bats inhabit regions between Singapore and Yúnnan province, China, and may also carry RoBat-CoV GCCDC1, based upon similarities in viruses detected among groups of the same species. For example, recent serological biosurveillance of lesser dawn bat populations in Singapore and Northeast India demonstrated that both populations had similar exposure to Asiatic filoviruses ,46. The Coronaviruses may emerge following random mutations permissive to infection of intermediate amplifying hosts, and/or recombination events that result in a large pool of variants that could infect humans, potentially without an intermediate host . Due to The datasets supporting the conclusions of this article are available in National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA), BioProject ID PRJNA561193 and under GenBank accession number MT350598.


Bronchopulmonary dysplasia (BPD) is a major complication in prematurely born infants. Pulmonary hypertension (PH) associated with BPD (BPD-PH) is characterized by alveolar diffusion impairment, abnormal vascular remodeling, and rarefication of pulmonary vessels (vascular growth arrest), which lead to increased pulmonary vascular resistance and right heart failure. About 25% of infants with moderate to severe BPD develop BPD-PH that is associated with high morbidity and mortality. The recent evolution of broader PH-targeted pharmacotherapy in adults has opened up new treatment options for infants with BPD-PH. Sildenafil became the mainstay of contemporary BPD-PH therapy. Additional medications, such as endothelin receptor antagonists and prostacyclin analogs/mimetics, are increasingly being investigated in infants with PH. However, pediatric data from prospective or randomized controlled trials are still sparse. We discuss comprehensive diagnostic and therapeutic strategies for BPD-PH and briefly review the relevant differential diagnoses of parenchymal and interstitial developmental lung diseases. In addition, we provide a practical framework for the management of children with BPD-PH, incorporating the modified definition and classification of pediatric PH from the 2018 World Symposium on Pulmonary Hypertension, and the 2019 EPPVDN consensus recommendations on established and newly developed therapeutic strategies. Finally, current gaps of knowledge and future research directions are discussed.PH in BPD substantially increases mortality. Treatment of BPD-PH should be conducted by an interdisciplinary team and follow our new treatment algorithm while still kept tailored to the individual patient.We discuss recent developments in BPD-PH, make recommendations on diagnosis, monitoring and treatment of PH in BPD, and address current gaps of knowledge and potential research directions.We provide a practical framework, including a new treatment algorithm, for the management of children with BPD-PH, incorporating the modified definition and classification of pediatric PH (2018 WSPH) and the 2019 EPPVDN consensus recommendations on established and newly developed therapeutic strategies for BPD-PH. PH associated with BPD (BPD-PH) is characterized by abnormal vascular remodeling and rarefication of the pulmonary vasculature (vascular growth arrest),2 leading to increased pulmonary vascular resistance (PVR) and right heart failure. About 25% of infants with moderate to severe BPD develop PH4 that affects heart and lungs, greatly increasing mortality (47% of BPD infants die 2 years after diagnosis of PH).7Bronchopulmonary dysplasia (BPD) represents a major cardiopulmonary complication in survivors of premature birth. The incidence of BPD is on the rise despite/because of the advances in neonatal intensive care.9 and higher rates of tracheostomy, increased use of supplemental oxygen, feeding problems, and frequent hospital admissions.10 Young adults born preterm  are at increased risk for PVD, PH, and right ventricular (RV) dysfunction.11Compared to BPD without PH, BPD-PH is associated with suboptimal somatic growth and neurodevelopmental outcome,The increased survival of even the most immature infants makes infants with BPD-PH an ever-growing population among pediatric PH patients, requiring highly specialized care. Nevertheless, there is still a paucity of published data supporting the current management of children with BPD-PH.13 Finally, we discuss current gaps of knowledge and potential future research directions.In this article, we briefly review evolving concepts on the pathobiology of BPD-PH. We further discuss comprehensive diagnostic strategies, including the differential diagnosis of rather rare parenchymal and interstitial developmental lung diseases associated with PH. We provide a practical framework for the clinical management of children with BPD-PH, including established and newly developed therapeutic strategies, based on the 2018 World Symposium on Pulmonary Hypertension, and the 2019 updated consensus recommendations of the European Pediatric Pulmonary Vascular Disease Network (EPPVDN).14 Specifically, the lower limit of normal mean pulmonary arterial pressure (mPAP) was decreased from 24 to 20 mmHg.15 In adults, even mildly elevated mPAP values  were found to be independent predictors of poor survival.16 For consistency, this new definition of PH was also used in the pediatric WSPH 2018 document13 and the current 2019 EPPVDN guidelines,12 although the cut-off mPAP >20 mmHg remains arbitrary for children , postnatal hyperoxia-/hypoxia-triggered vascular remodeling, and the rarefication of pulmonary blood vessels (vascular growth arrest).22 However, this definition has recently been challenged and a new definition of BPD was proposed that includes infants on any form of respiratory support  at 36 corrected weeks (p.m.), even in the absence of supplemental oxygen use.23 The significance of this new, alternative BPD definition for the frequency and morbidity of BPD-PH still needs to be investigated, and has not been used yet for this review article. In any case, PH/PHVD might occur before the formal diagnosis of BPD can be made. Indeed, echocardiographic screening may help identify infants at risk for BPD-PH as early as 7 days of life.4Clinical suspicion of BPD-PH should be raised in every infant with a history of prematurity who requires supplemental oxygen and/or invasive or non-invasive respiratory support. Traditionally, BPD is defined clinically as supplemental oxygen use either at a corrected age of 36 gestational weeks (p.m.) or after 28 days of postnatal life in infants born <32 weeks of gestational age.12While BPD-specific changes are detected on conventional chest X-ray, the initial assessment of suspected PH is based on transthoracic echocardiography (TTE) Fig. . Here, o27 Screening for PH by TTE is indicated (1) if severe respiratory compromise is present in any preterm infant <28 weeks of gestation, (2) in any infant with established BPD at corrected age of 36 weeks gestation (p.m.) and before discharge, (3) in any infant with prolonged oxygen requirement, poor growth, unsatisfactory clinical improvement,12 and has also been suggested at 7 days of life to identify infants at risk for BPD-PH.4 Comprehensive TTE allows for complete initial assessment of cardiovascular anatomy and may also confirm RV pressure elevation through the measurement of the maximal velocity of the tricuspid regurgitation (TR) jet by continuous wave Doppler; TTE can detect impaired RV systolic function through qualitative assessment in several views, and quantitatively by means of a reduced age-related tricuspid annular plane systolic excursion as surrogate of longitudinal systolic RV function.12 Of note, TTE variables have different age-related reference ranges and variable impact on the accuracy of the diagnosis “PH” in children vs. adults, especially in young infants.24 The presence of a hemodynamically significant post-tricuspid shunt makes TTE estimation of PAP elevation less accurate.28TTE is an important diagnostic and monitoring modality.12 including those with BPD-PH, are listed in Table The relevant non-invasive imaging risk variables currently evaluated for infants and young children with PH,12 MRI offers the ability to assess pulmonary and systemic blood flow, pulmonary regional perfusion, PA and aortic diameters, (bi-) ventricular cardiac function, and myocardial tissue characteristics.29 More recently, lung MRI  was introduced to assess lung perfusion in PHVD.31 A recent study on MRI in BPD-PH revealed that the pulmonary artery to aorta ratio (PA/AO) increased with BPD severity. An increased PA/AO and MR-derived LV end-systolic eccentricity index (MR-LVEI) were associated with increased length of stay and duration of respiratory support in BPD infants.31 Both increased PA/AO ratio and MR-LVEI were associated with PH therapy during hospitalization and at discharge. The primary role of chest CT is to detect lung parenchymal disorders, thromboembolic disease, and vascular abnormalities, such as pulmonary vein stenosis, which may be causally linked to evident PH. The key role of chest imaging, including newer MRI techniques in differentiating interstitial and parenchymal developmental lung disease has been discussed elsewhere.32 The significant risk of sedation/general anesthesia in PH patients need to be balanced against the potential gain of information of MRI or CT studies, and their impact on the future therapy of the individual PH patient.12Magnetic resonance imaging (MRI) and computed tomography (CT) have become essential non-invasive imaging modalities in suspected or confirmed PH.13 exceptions may be made when the risk of the cardiac catheterization outweighs the potential benefits; for instance, severe hemodynamic instability (premature infants) or evidence of systemic vasculopathies. However, cardiac catheterization for the diagnosis and assessment of BPD-PH should be considered before introduction of a second PH-targeted medication; cardiac catheterization is advised if PH worsens under dual PH-targeted therapy or if an unsatisfactory response to PH-targeted pharmacotherapy is seen in any infant with PH at a corrected postnatal age of >3 months ,33 other features of an atypical response to therapy, and/or failure to thrive.34Cardiac catheterization is rarely necessary to make the diagnosis of BPD-PH early on. Although cardiac catheterization with acute pulmonary vasoreactivity testing (AVT) generally is recommended in pediatric PH prior to the initiation of pulmonary vasodilator therapy,12 However, the complexity of pediatric PH/PVD often requires individualized adaptations of these recommendations. A recent study found that AVT may aid in the assessment of disease severity and management of BPD-PH. In this cohort of 26 infants with BPD, 35% had positive AVT, which was associated with better long-term outcomes. AVT also distinguished higher from lower risk PH in infants with BPD better than baseline pulmonary hemodynamics.35 Others found that the severity of lung disease as assessed by impaired oxygenation at cardiac catheterization did not correlate with mortality while the presence of pulmonary vein stenosis was associated with death.33A systematic cardiac catheterization protocol for pediatric PH is available.12 Brain natriuretic peptide (BNP) and its N-terminal cleavage product (NT-proBNP) are secreted by cardiomyocytes in response to ventricular wall stress due to pressure overload and/or volume expansion. Both biomarkers exhibit similar relative stress-induced release and age dependency, but NT-proBNP has the longer half-life (118 vs. 18 min). In a meta-analysis of 25 small-scale pediatric studies, serum NT-proBNP was identified as significant prognostic factor in pediatric PH and suggested to aid in the longitudinal assessment of patients36 . Recent studies have also shown the applicability of serum NT-proBNP as a suitable biomarker in infants with BPD-PH.40 Based on our experience, especially in infants, absolute values of NT-proBNP have a high inter-individual variance, are strongly dependent on postnatal and gestational age, and thus have limited value in establishing the diagnosis or severity of PH, especially in young infants. However, longitudinal assessment of NT-proBNP in combination with echocardiography is very useful to assess disease progression and/or response to treatment.44Blood biomarker concentrations contribute to tailoring longitudinal medical care of PH patients.40Meanwhile, additional biomarkers are under consideration and may contribute to future PH management.12 Maximizing non-invasive respiratory support as much as possible in order to avoid mechanical ventilation and therefore aggravation of lung injury is advisable. However, atelectasis causes an increased ventilation/perfusion mismatch and local hypoxic vasoconstriction, and thus needs to be avoided. In addition, treatment with diuretics, that is, hydrochlorothiazide and spironolactone may be considered in infants with severe BPD.12 However, diuretic therapy in preterm infants remains controversial, given its negative impact on growth and the risk for metabolic bone disease. Thus, indications and duration of diuretic treatment should be discussed in a multidisciplinary team, involving cardiologists, neonatologists, pulmonologists, and nutritionists. Infants with severe BPD have higher caloric requirements, which should be accounted for in their nutritional plans . Infants with BPD-PH are prone to a sudden elevation of PVR when undergoing stressful procedures (“PH crisis”) and often require sufficient sedation or even analgesia/relaxation before such procedures are performed in an intensive care setting. Administration of inhaled nitric oxide and phosphodiesterase-3 inhibitors, such as milrinone, which are frequently used for acute treatment of PH in infants , are beyond the scope of this review and discussed elsewhere.12Supplemental oxygen should be supplied when target oxygen saturations are >93% for infants with suspected and >95% for infants with proven PH Fig. .12 Maxim47 Of note, especially oral pulmonary vasodilator therapy may amplify ventilation and perfusion mismatch in any type of PH, especially in the setting of lung disease.The overall goal of therapy for PH patients, adult or pediatric, is (1) to induce pulmonary arterial vasodilation, (2) to pressure unload and support the RV, (3) to avoid coronary ischemia and heart failure, (4) improve clinical outcomes and quality of life, and (5) to improve signs and symptoms, and thus the aggravation of PH.15 PH-targeted therapy currently focuses on three main molecular pathways: the nitric oxide pathway, the endothelin pathway, and the prostacyclin pathway 15 is to treat the underlying lung disease.48 However, BPD-PH patients are likely to benefit from vasodilator treatment.49 A recent, retrospective analysis showed clinical and echocardiographic improvements with a survival rate of 95% during a median follow-up time of 2 years in infants treated with PH-targeted therapy for BPD complicated by PH.42Regardless of a patient’s age or type of PH group 1–5 PH),ay Table . While o–5 PH,ay 50 STARTS-1 and its extension phase, STARTS-2,51 were the first pediatric randomized, placebo-controlled clinical trials in PAH. The STARTS study demonstrated improvement in secondary outcomes among those PH patients on medium- or high-dose sildenafil, including functional class and PVR. Controversially, the STARTS trial detected an increase in mortality among those subjects receiving high-dose sildenafil vs. placebo group, which in 2013 led to a warning of the EMA not to use higher doses, and rejection of approval by the FDA. Nevertheless, sildenafil is—by most centers and experts—regarded as a safe and efficacious first-of-choice drug for PH/PVD in children, and its widespread use and recommendation in formal guidelines continues.42 Sildenafil at standard dose should be considered in children with BPD-PH as improvements of clinical status and PAP have been demonstrated for oral sildenafil therapy.41 Tadalafil is reported to be widely used in North America due to its reduced dosing frequency, compared to sildenafil. Common to all PDE5 inhibitors, gastroesophageal reflux is an often reported unwanted effect that is also highly relevant to neonates and infants.46 Riociguat is a novel oral agent with dual mode of action that acts in synergy with endogenous NO and directly stimulates soluble guanylyl cyclase, with only very limited pediatric experience.47Phosphodiesterase-5 (PDE5) inhibitors are the most commonly employed drugs acting on the nitric oxide (NO) pathway, namely sildenafil and tadalafil. PDE5 inhibitors inhibit primarily PDE5 (NO/cGMP pathway), thus inducing vasodilation through vascular smooth muscle cell relaxation, and also exhibit anti-proliferative effects.55 Nevertheless, monthly liver enzyme and function testing should be performed in children receiving bosentan.14 ERA treatment may also be considered in BPD infants.12 Macitentan provides an alternative to bosentan that shows a more favorable safety profile . Macitentan has been studied in a small prospective pediatric study and was shown to be safe and efficacious as add-on therapy.44 Off-label use of macitentan also has been reported in BPD-PH patients.56 Of note, bosentan lowers circulating sildenafil levels, while the other two ERA, ambrisentan and macitentan, do not.12Bosentan, an oral non-selective endothelin A- and B-receptor antagonist (ERA), counteracts the vasoconstrictive and mitogenic effects of endothelin-1 in PAH patients. Elevated liver aminotransferases may occur with bosentan use as a serious adverse event, but seem to be less frequent in children under 12 years than in adults and children ≥12 years of age (2.7% vs. 7.8%).2) and its analogs activate the PGI2receptor (IP receptor) and lead to vasodilation, inhibit vascular smooth muscle cell proliferation, and have anti-inflammatory effects. PCAs were developed which facilitate intravenous, but also subcutaneous, inhaled, and more recently oral delivery routes.12 As with other pulmonary vasodilators, PGI2 or PCAs may have off-target effects prompting side effects, including headache, jaw pain, nausea, vomiting, abdominal pain, diarrhea, and flushing. Inhaled PGI2 or PCAs may cause airway hyperreactivity,57 in addition to headaches and flushing. However, the use of especially inhaled PCAs has been reported in infants, for example, inhaled iloprost might be used as monotherapy or as adjuvant.58Selexipag is a new oral selective prostacyclin receptor (IP receptor) agonist, but not yet systematically studied in “BPD-PH”. The first prospective, pediatric multicenter study on PAH-combination therapy indicates that selexipag can be used safely even in small children (4–10 kg bodyweight), and that a positive drug response can be expected in half of the children treated with selexipag.43Prostacyclin  may facilitate decompressing a pressure-loaded RV in severe PH/PVD.12 Procedures for palliation of children with severe PH and RV failure include the creation of a right-to-left shunt with the aim to decompress the right heart and to increase cardiac output. These interventions decompress the right atrium only  or the RV (reverse Potts shunt). While the interatrial shunt resulting from BAS only secures some cardiac output in critical supra-systemic PAP elevations, the “endogenous” reverse Potts shunt  clearly decomprresses the RV and lowers RV pressure.In children with BPD, large left-to-right atrial or ventricular shunts are not well tolerated, and may be closed early if PVD is mild .60 Similarily, in acute deterioration, PGE1 may be used to keep the PDA open at the expense of hypoxemia in the lower half of the body.The resulting hemodynamic situation after both interventional procedures is similar to the physiology of Eisenmenger patients. In imminent RV failure, an existing PDA can be balloon-dilated and stented (endogenous Potts shunt), representing an elegant procedure to decompress a failing RV and may be considered in patients with supra-systemic PH refractory to any medical treatment, including combination PH-targeted therapy.62 Thus, preventive measures to decrease BPD frequency and severity are of pivotal importance in order to lower the BPD-PH burden among preterm infants. These preventive measures include, but are not limited to: less use of invasive mechanical ventilation and predominant use of non-invasive respiratory support strategies, minimally invasive surfactant application, avoidance of hyper- and hypoxia, caffeine, corticosteroids, and reduction of infections.66 Further research is required to determine the role of caffeine and corticosteroids on the developing pulmonary vasculature. The same is true for a possible contribution of a hemodynamically significant PDA (here: sole left-to-right shut) to PH and PVD in BPD infants. In addition, early biomarkers, detection, and treatment of PH + PVD might hold the potential to decrease BPD-PH disease severity  and systemic steroid use, hyperoxia, and inflammation/sepsis.ty Table .Table 4F70 A phase 1 study on the use of mesenchymal stem cell-derived extracellular vesicles in preterm neonates at high risk for BPD is underway (NCT03857841).BPD-PH represents a significant burden to pediatric PH programs, but has been relatively understudied to date. The current wide ambiguity regarding optimal diagnosis, treatment, and monitoring of disease progression creates several high yield opportunities for study and practice improvement Table . These i


Colorectal cancer (CRC) is one of the common malignant tumors, the incidence of which is on rise. LncHOTAIR, considered as an oncogene, contributed to the progression of a lot of cancers. However, the molecular mechanism and biological functions of the HOTAIR/miR-206/CCL2 axis have not been reported before. Here, our research aimed to explore HOTAIR/miR-206/CCL2 axis in CRC to demonstrate its role in predicting the poor prognosis of CRC. LncHOTAIR, miR-206 and CCL2 mRNA were detected in CRC tissues and cells by RT-PCR. The interactions among LncHOTAIR, miR-206 and CCL2 were explored by luciferase reporter assay, qRT-PCR, western blot and RNA interfere. Flow Cytometry Cell Analysis was performed to detect cell cycle and apoptosis as well as colony assay was prepared to test the cell proliferation. Immunohistochemical analysis was used to detect the CCL2 protein in CRC tissues. In our study, silence of LncHOTAIR by RNA interference could suppress the proliferation, migration and invasion of CRC cells. Mechanistically, LncHOTAIR downregulated miR-206 abundance which indicated that LncHOTAIR was considered as a competing endogenous RNA (ceRNA) by directly sponging miR-206 in CRC cells. In addition, further exploration suggested that miR-206 could inhibit the function of the downstream CCL2, the expression of which was repressed by LncHOTAIR/miR-206 signaling. Furthermore, we verified that the overexpression of CCL2 attenuated CRC cell proliferation, migration, invasion. Overall, this study firstly elucidated that LncHOTAIR played as oncogene in CRC via directly sponging miR-206 to activate the downstream CCL2, which would be considered as the novel therapeutic target in CRC. Colorectal cancer (CRC) is one of the common malignant tumors, the incidence of which is on rise Current studies have found that long chain non coding RNA (LncRNA) plays an important role in tumor progression. LncRNA transcribed by RNA polymerase II, which has obvious spatio-temporal expression specificity, lacks significant open reading frame, widely existing in nucleus and cytoplasm, so as not to participate in or rarely participate in protein coding Some researches focused on the HOTAIR/miR-206 axis in head and neck squamous cell carcinoma, ovarian cancer and breast cancer, however few researches concentrated on its function in CRC. miR-206 has been considered as the tumor suppressor in variety cancers, especially in CRC. In our study, we found that expression of HOTAIR was significantly increased in CRC tissues and cells. Loss function of HOTAIR repressed the proliferation and invasion and provoked the apoptosis of CRC cells, the deep mechanism of which may be it sponged miR-206, to activate the downstream CCL2, finally leading to the promotion of CRC progression. Thus, we intended to uncover the role of HOTAIR/miR-206/CCL2 axis in cell migration and invasion of CRC and the possible underlying mechanisms. This result explored the new mechanism of HOTAIR in CRC, which would indicate the new therapeutic target in clinical application.All the 32 CRC tissues and paired normal tissues, from the patients who has not gained therapy like preoperative chemotherapy or radiotherapy before, were obtained from specimen repository of Zhejiang Hospital from 2012-2017. Inclusion criteria included any case which were diagnosed and treated for stage II-IV colorectal cancer. Exclusion criteria included cases who received chemotherapy or radiotherapy as well as cases with missing data. Specimen has been frozen with liquid nitrogen after surgery or biopsy and kept at -80℃ until later use. This study achieved the permission from the ethics committee of Zhejiang Hospital. Prior to the usage of these clinical materials, every patient offered the informed consent. The different stage of CRC (Stage II-IV) was referred to previous article The human CRC cells lines including SW620, SW480, HCT116, HT29, LoVo, and the normal colon cell line, NCM460 were purchased from the Chinese Academy of Sciences (Shanghai Cell Bank). Cells were cultured in a complete RPMI1640 medium  containing 10% fetal bovine serum  and 1% double antibody , in a constant temperature incubator at 37℃ with humidified 5% CO2 atmosphere. When the cell density was more than 80%, trypsin was used for digestion and subculture and seed plate. Cell transfection referred to protocol of Lipofectamine 2000 . The small interference RNAs plasmid (siRNA) of HOTAIR and CCL2 according to the manufacturer's protocol as well as small interference RNA plasmid of CCL2 were purchased from Genechem . The miR-206 inhibitor, miR-206 mimics, mimic NC and negative control (NC) were synthesized by GenePharma . Transfection efficiency was determined by qRT-PCR.The detailed sequencings were listed in After 48 hours of transfection and culture, the total RNA was extracted by Trizol reagent  for qRT-PCR detection. The SYBR Green PCR Master One-Mix kit  and cDNA Reverse Transcriptase Kit  were purchased from ElifeBio . Real-time PCR 7300 System  was used for qRT-PCR detection. The reaction conditions were as follows: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 10 s, annealing at 60℃ for 30 s, and 35 cycles. Each group was repeated 3 times. Relative quantification of RNA expression was calculated using the 2-ΔΔCT method. All the primers of HOTAIR, miR-206, CCL2 and GAPDH were shown in Table S.4 / well, and the cells were cultured for 24 h, 48 h, 72 h and 96 h, as well as the cell viability was detected. After the cell culture medium was removed, the pre-configured cell counting kit-8  reagent was added, before the cell culture plate was placed in a constant temperature incubator for 30 min. The cell culture plate was placed in an enzyme labeling instrument to read the absorbance value at 490 nm measured using a microplate reader (Thermo Scientific). At last the proliferation ability of the cell was calculated.The transfected cells were inoculated on 96-well plate according to the density of 10Cell colony assay was prepared referring to a previous article 6 / well, and the apoptosis of the cells was detected after 24 hours of culture. The cells were collected by trypsin digestion and centrifugation, mixed with the binding solution, and then added with the Annexin V-FITC reaction reagent at the light avoidance reaction 10 min the propidium iodide (PI) staining solution again avoiding the light reaction 10 min, and finally adding the binding solution, the percentage of apoptosis was detected. The cell cycle distribution was analyzed by flowcytometry .The transfected cells were inoculated on the 6-well plate at the density of 10Cell invasion assays were measured by transwell chamber with Matrigel  according to the manufacturer's protocol. The detail of the assay referred to the previous study The expression of cell protein was detected after 24 hours of culture. RIPA lysate was used to extract the total cell protein and determine the total protein. Each group was treated with 100 μg protein for electrophoresis, as well as PVDF membrane was used for membrane transfer sealed at room temperature for 1 hour, then the membrane was incubated overnight with the first antibody at low temperature, and horseradish peroxidation was added the next day. The second antibody was incubated for 1 hour, and then the chromogenic solution was added for color development and photographed and analyzed. The antibody was listed in the After cut into 4-µm sections Tissues were deparaffinized and treated with EDTA (pH 9.0) to antigen retrieval in a microwave for 20 min. Autostainer Link 48 machine  was performed for staining. The detail of the assay referred to the previous study All experimental data from experiments were analyzed by GraphPad Prism 8.0 and results were shown as mean ± SD . Comparisons between two groups were made by Student's t-test. Multiple comparisons were made by One-way ANOVA. Survival analysis was analyzed via the Kaplan-Meier method. P < 0.05 was considered significant.1D, p < 0.01) and the paired normal colon tissue(n=41) by “UALCAN” database   as well as upregulated higher in stage IV and stage III than that in stage I and stage II by “Gepia” database (http://gepia.cancer-pku.cn/) . High expression of HOTAIR also showed the severe nodal stage  as well as the overall survival which meaned the poor prognosis . The cohort GSE 14333  and GSE 17537  from GEO database showed high expression of HOTAIR may suggest the poor disease free survival, which may be good proof of the validity of our result. In our research, from 32 CRC tissues and paired normal tissues, found HOTAIR also overexpressed in tumors in comparison with the normal . We also detected the HOTAIR expression in CRC cells to prove its high level of expression both in CRC tissues and cells . Moreover, we also found HOTAIR overexpressed in the CRC patients with stage III/IV compared with the stage I/II . Further exploration showed HOTAIR overexpression correlated with lymphnode metastasis  and liver metastasis . Furthermore, we aimed to explore the cut-off value of HOTAIR to establish its high or low expression in CRC. Our result found 1.967 was the threshold for HOTAIR (the relative mRNA levels of HOTAIR compared with GAPDH). Correspondingly mRNA levels of HOTAIR >1.967 was considered as “high” and those≤1.967 as “low” . We also found that the expression of HOTAIR mRNA significantly correlated with distant metastasis (P = 0.01), lymph node metastasis (P = 0.0001), MMR index (P = 0.034) and depth of invasion (P = 0.011) (Table 1).Previous studies have reported HOTAIR overexpressed in various cancer types 2G, mean survival: 32.0 months vs 38.0 months). High expression of HOTAIR also predicted the poor progressin-free survival . Furthermore, HOTAIR has been identified as the independent factor to impact the overall survival (Table 2).Patients with high HOTAIR expression revealed poor prognosis, especially the lower overall survival time compared with the patients with low expression . Moreover, CRC cells, silenced HOTAIR-1, were blocked at G0/G1 phase by flow cytometry cell cycle analyses . The CCK8 assay results uncovered that knockdown of HOTAIR could repress cell viability . Loss function of HOTAIR elevate the apoptotic rate, which was observed in si-HOTAIR-1-transfected CRC cells by flow cytometry apoptosis assay . Moreover, colony formation assay exhibited inhibition of LncHOTAIR suppressed SW620 cells proliferation . Afterwards, Transwell assay further indicated the silenced of HOTAIR decreased invasive cell numbers, which revealed HOTAIR would contribute to CRC metastasis .Due to the higher expression of HOTAIR in HCT116 and SW620 than other cell lines, we selected these two cell lines for the further research. The expression of HOTAIR was obviously decreased by qRT-PCR analysis after transfection with either Si-HOTAIR-1 or Si-HOTAIR-2, but the former showed much more power .Thus, we further aimed to detect their direct interaction . We constructed luciferase reporter plasmids of HOTAIR. Moreover, HOTAIR-silenced resulted in the increased expression of miR-206 by qRT-PCR . Meanwhile miR-206 may exert tumor suppressor in CRC due to its low expression in CRC tissues and cells . The expression of HOTAIR significantly decreased when SW620 transfected with the miR-206 mimic, whereas transfected with the miR-206 inhibitor repressed it significantly . Luciferase reporter, transfected into SW620 cell, containing exact or mutant miR-206 binding sites was prepared for further detection of the bonding effect between HOTAIR and miR-206, which showed miR-206 mimic significantly decreased the luciferase activity of the wild-type HOTAIR reporter plasmid, as well as the miR-206 inhibitor significantly increased it. However this effect was counteracted when mutant HOTAIR reporter plasmid was transfected . The CCK-8 assay showed cell proliferation in SW620 and HCT116 transfected with HOTAIR-silenced and cotransfected with miR-206 mimic was suppressed significantly compared with cotransfected with miR-206 inhibitor . Furthermore, the invasion in SW620 and HCT-116 transfected with HOTAIR-silenced and cotransfected with miR-206 mimic was significantly suppressed, but increased cotransfected with the miR-206 inhibitor . Taken together, these results conveyed that HOTAIR, functioned as the oncogene, could sponge miR-206 to provoke proliferation and invasion and suppress apoptosis in CRC cells.Emerging evidence demonstrates lncRNAs considered as ceRNA could regulate miRNAs in various cancer. Therefore, we aimed to verify whether HOTAIR could exhibit this function in CRC. We identified miR-206 as a most potential candidate via bioinformatics analysis and bioinformatics database . According to bioinformatics data, we screened out CCL2. Analysis of the 3'UTR sequence of the CCL2 gene by TargetScan revealed a putative miR-206 binding site . Then we explored the role of CCL2 in CRC, that it overexpressed in CRC tissues compared with normal ones by qRT-PCR . Moreover, the IHC staining in normal tissues, distinguished from the positive cells and the strong staining, was stronger in normal tissues compared with the almost no staining in CRC situ tissues especially in liver metastasis tissues . Due to the low expression of CCL2 in CRC tissues, we sought to explore the its role in regulation of phenotype of CRC. Knockdown of CCL2 mRNA could significantly accelerated the apoptosis of SW620 cells . miR-206 mimic could restrain the CCL2 mRNA and protein , in addition, knockdown of HOTAIR significantly augmented mRNA and protein level of CCL2 in colorectal cancer cells . Thus, we concluded that CCL2 was a direct target of miR-206 and positively modulated by HOTAIR in CRC cells.At first we selected the gene correlated with proliferation, invasion and apoptosis targeted by miR-206, which was predicted by  in HOTAIR-knockdown CRC cells. Moreover, miR-206 overexpression could effectively accelerate the inhibitory effects of HOTAIR knockdown on proliferation of CRC cells . Functionally, miR-206 attenuated the colony formation in HOTAIR-knockdown CRC cells . Furthermore, cell invasion of SW620 and HCT116 was significantly suppressed by HOTAIR-knockdown, additionally, miR-206 overexpression could accelerate this progression . Therefore, our results showed that HOTAIR could exert its oncogenesis effects on the growth and invasion of CRC via HOTAIR/miR-206/CCL2 axis.Although cell proliferation in SW620 transfected with HOTAIR-knockdown was significantly suppressed compared with control group. Subsequently, we validated whether miR-206 could exert its suppressive effects on CRC progression through HOTAIR/miR-206/CCL2 axis. Notably, miR-206 overexpression decreased the expression of CCL2 mRNA and protein  as well as referred to the high biological score, we speculated that miR-206 may be sponged by HOTAIR.miR-206, first found in skeletal muscle as well as considered to be related to the physiological and pathological process of skeletal muscle, has been identified as a tumor suppressor in many human malignancies, including breast cancer Based on the bioinformatics database (miRDB, Our study has confirmed the role of HOTAIR as oncogene in CRC tissues which associated with liver metastasis and advanced stage predicted the poor prognosis of CRC. Loss function of HOTAIR by RNA interference could suppress the proliferation, migration and invasion of CRC cells. Mechanistically, HOTAIR suppressed miR-206 abundance which indicated that HOTAIR directly sponged miR-206 in CRC cells. Further detection suggested that miR-206 could repress the function of the downstream CCL2, which also considered as the oncogene in CRC In conclusion, our research has illustrated that HOTAIR overexpressed in CRC which predicted poor outcomes of patients. HOTAIR provoked proliferation and invasion of CRC through sponging miR-206 and repression of CCL2. Hence, our study indicated that HOTAIR/miR-206/CCL2 would be the novel therapeutic target in CRC.Supplementary tables.Click here for additional data file.


Malnutrition in liver cirrhosis is frequently underestimated. To determine if a patient is at risk of malnutrition, several screening tools have been established. However, most of them are not validated for patients with liver cirrhosis. Therefore, we compared the RFH-NPT  as the validated gold standard for malnutrition screening in cirrhosis patients with GMS , NRS-2002  and MNA-SF . Based on common validity criteria for screening tools, only the MNA-SF showed fair correlation (12/15 points) with the RFH-NPT, whereas NRS-2002 and GMS performed worse (6/15 points). Taken together, our results suggest that NRS-2002 and GMS are not suitable for screening of malnutrition in cirrhosis patients. A cirrhosis-specific screening tool like RFH-NPT should be used to assess malnutrition and to identify those at risk of malnutrition. In patients with liver cirrhosis, hepatic metabolic functions are impaired resulting in a variety of nutritional disorders including protein-energy-malnutrition (PEM) which occurs in 65–90% but is still frequently underestimated . HoweverIn clinical practice, malnutrition plays an important role for all hospitalized patients with different medical diagnoses, not only for patients with cirrhosis. In the current GLIM  criteria , the useIn the present study, we aimed to validate three general malnutrition screening tools  against RFH-NPT malnutrition in patients with liver cirrhosis.clinicaltrials.gov (NCT03080129) before the start of recruitment. The study was conducted after written informed consent according to the principles of the Declaration of Helsinki. Hospitalized men or women over the age of 18 years with clinical/ radiological/ histological diagnosis of cirrhosis were included in the study. Exclusion criteria were defined as hepatic encephalopathy > grade 2 and/or other cognitive disorder not allowing for informed consent or hepatocellular carcinoma stage BCLC C or D. After written informed consent, RFH-NPT [This analysis is a sub-analysis of a prospective cohort study on sarcopenia in patients with liver cirrhosis. The trial was approved by the research ethics committee of the Medical University of Graz (29-280 ex 16/17) and was registered at .gov NCT0080129 be RFH-NPT , NRS- 20p < 0.05 was considered statistically significant.Baseline characteristics were analyzed using descriptive statistics. Data are presented as frequencies, percentages and means with standard deviations (SD) for normally distributed variables. We performed statistical analysis by SPSS version 25 for Windows . Sample size was based on feasibility with an average yearly rate of 70 patients with cirrhosis who were screened for malnutrition by the dietary team and an 80% consent rate. All eligible patients with complete datasets for all four nutritional screening tools were included; The scores of each screening tool were normalized by scaling the rating values between 0 and 1. The normalized cut-off values for classifying malnutrition were calculated . Correlation between test results was assessed using Pearson’s correlation coefficient. Concurrent validity was determined by matching the RFH-NPT with the three other screening tools using the chi square test. Furthermore, sensitivity, specificity, positive and negative predictive values (PPV/NPV) and precision were calculated. For visualization of the differences between the various screening tools, Bland–Altman-Plot using differences versus averages were generated with a limit of agreement of 95%. Outliers, defined as values that fell outside the limits of agreement were defined. To evaluate the diagnostic accuracy area under the receiver operating curve (AUROC) was calculated. To determine predictive validity of the screening tools, the ratio between the odds ratios (ORs) and hazard ratios (HRs) were calculated. Kappa values were calculated for evaluating reliability. To rate validation results, published cut-offs were used. [209 patients were screened for participation between March 2017 and March 2019. 84/209 did not meet inclusion criteria or fulfilled an exclusion criterion. In total, 125 patients were screened for the risk of malnutrition by four different screening tools, 7/125 were excluded because of incomplete data. Data from 118 patients were included in the analysis. p = 0.163). Sensitivity was highest for MNA-SF (83%) compared to RFH-NPT, but precision was low (53%). GMS, NRS-2002 and MNA-SF had a high specificity  but a very low sensitivity  except for MNA- SF (83%). NPV were highest for MNA- SF (95%) and PPV for NRS-2002 (71%). Bland–Altman was calculated based on differences versus averages between RFH-NPT and MNA-SF, GMS as well as NRS-2002. Comparing bias, SD of bias and limits of agreement, MNA-SF showed the best agreement. The number of outliers was low for all comparisons. AUROC was calculated to evaluate the diagnostic accuracy and reliably between the different screening tools. The best AUROC was found between RFH-NPT and MNA-SF (0.834). To evaluate predictive validity of the screening tools, the ratio between ORs and HRs was calculated. Compared to RFH-NPT, MNA-SF and NRS-2002 were found to have the highest predictive value (2.159 and 2.932). MNA-SF had the highest Kappa coefficient (0.566) as measure for agreement in diagnosing malnutrition. An overview of the comparisons of the four screening tools is given in In our study population, we saw the highest correlation between the RFH-NPT and the MNA-SF based on Person´s correlation . Chi-square for comparison of concurrent validity of screening tools was applied in all patients enrolled. Only GMS did not differ significantly from RFH-NPT  and therefore has the highest accordance to RFH-NPT. An overview of the rating is illustrated in In our study, we validate three common general screening tools for malnutrition in liver cirrhosis by comparing them with the recommended liver disease specific screening tool (RFH-NPT). When using different statistical comparisons, MNA-SF has the highest rating score  and therefore the highest accordance to the RFH-NPT. MNA-SF gave false-negative results in comparison to RFH-NPT in only 17% whereas GMS and NRS-2002 have unacceptably high false-negative rates of 61% and 78%. False-positive results were obtained by MNA-SF in 16%, by GMS in 8% and by NRS-2002 in 2%. The requirements for a malnutrition screening tool are defined by the ESPEN guidelines for nutritional screening which should include the following four main principles: actual weight/BMI, recent weight loss, actual food intake and changes as well as disease process which accelerate nutritional deterioration . The assThe relevance of the diagnosis of malnutrition in patients with liver cirrhosis is highlighted in the new EASL Clinical Practical Guidelines for Nutrition in Chronical Liver Disease from 2019 .However, since malnutrition is common in all hospitalized patients , the useSince in reality, only one screening tool that fits all hospitalized patients with different clinical diagnoses will be used, choosing a disease specific screening tool for live cirrhosis is not a useful option. Since none of the tested screening tools performed sufficiently in cirrhosis, we suggest the following workflow to improve the screening accuracy when using the electronic patient record system: A ‘stopping rule’ should be implemented into the workflow when a patient is diagnosed with liver cirrhosis. A message to the person doing the screening should be generated to inform that this screening tool is not validated for cirrhosis and that a dietician should be consulted.


The mortality rate of all MS patients was 4% and a further 3% required invasive or non-invasive ventilation. When looking at the severe and fatal cases, it is particularly noticeable that patients without DMTs, with previous cardiovascular diseases, or with a severe degree of disability are at risk. Immunosuppressive therapy itself does not appear to be a substantial risk factor. Rather, it is reasonable to assume that the therapies could be protective, either directly, by mitigating the cytokine storm, or indirectly, by reducing the disease activity of MS.The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic is a challenge for all participants in the healthcare system. At the beginning of the pandemic, many physicians asked themselves what risk their patients, especially those with chronic diseases, were exposed to. We present an overview of all patients with multiple sclerosis (MS) and SARS-CoV-2 infection published in the literature so far. In total, there are publications on 873 SARS-CoV-2 positive MS patients and information on the outcome can be given for 700 patients. With regard to the different disease modifying therapies (DMTs), by far the most cases were described under anti-CD20 treatment ( The outbreak of the novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the resulting global pandemic has confronted the world and its health authorities with one of the biggest challenges in recent history. The rapid disease spread is especially impressive, as the virus has reached every country worldwide in less than six months . SARS-CoOne question is crucial for physicians of all specialties, namely which of their patients are particularly at risk during the pandemic? It has been shown that older age and obesity are the most important conditions that increase the mortality risk of SARS-CoV-2 patients . AccordiThe potential risk of patients with chronic autoimmune diseases, such as multiple sclerosis (MS), could not be fully clarified so far. MS is an immune-mediated central nervous system (CNS) disorder that requires immunosuppressive or immunomodulating disease-modifying therapies (DMTs). The inclusion of immunocompromised patients in the ‘high-risk population’ for COVID-19 is intuitive, as immunosuppression should make a person more likely to contract an infection and may complicate the disease course. However, analyses of large Chinese or Italian cohorts did not identify immunosuppression as a risk factor for disease severity in COVID-19 ,13. In an = 873) published in the literature so far. Besides detailed case reports, larger case series or studies were also considered. The outcome of patients is presented in relation to their respective DMTs.A large number of single case reports and case series have been published on patients with MS and SARS-CoV-2 infection, however, an overarching overview is lacking. Here we review the results of all MS patients with SARS-CoV-2 infection  was treated with anti-CD20 therapies, either ocrelizumab or rituximab. Another 100 patients (12%) received interferons or glatiramer acetate. They were summarized under the term “injectables”. Of the reported MS patients (n = 97), 11% were not treated with DMT. The remaining patients were distributed relatively evenly among the natalizumab, fingolimod, dimethyl-fumarate and teriflunomide groups (n = 29) or alemtuzumab therapy (n = 12) were reported.Of all reported patients the majority (e groups . Relativn = 28) of these patients died in total. The remaining 496 subjects were able to stay at home after SARS-CoV-2 infection had been diagnosed.Out of a total of 873 patients, published, detailed clinical outcome data were available for 700 patients . A totaln = 14), 50% occurred in untreated patients. We can conclude from the figure that individual patients treated with anti-CD20-therapies, natalizumab, injectables, DMF, fingolimod, or teriflunomide died of COVID-19. In each DMT group there were hospitalized patients, although they rarely required ventilation. By far the majority of the published patients were treated with anti-CD20 therapy. Of these, 147 patients (64%) did not require hospital treatment, 64 (28%) were hospitalized but did not require ventilation, 13 patients (6%) were ventilated and a total of 7 patients (3%) out of 231 died. In the literature 83 SARS-CoV-2 positive MS patients without DMTs are found. Of these patients 14 (17%) died and a total of 31 patients (37%) were treated in hospital. None of the subjects treated with alemtuzumab or cladribine had a severe course of the disease. The hospitalization rates were 24% (cladribine) and 14% , respectively. With regard to the other DMTs, a very similar number of severe and fatal courses could be observed. Only two patients  or one patient  of all published cases died. Thus, the death rates were 4% (teriflunomide), 3% , 2% (dimethyl-fumarate), and 1% (fingolimod and injectable). Non-fatal severe courses requiring ventilation were only described for fingolimod  and dimethyl-fumarate .n = 5), cladribine (n = 4), natalizumab (n = 4), rituximab (n = 4), and interferon beta 1-a (n = 1).A total of 51 SARS-CoV-2 positive MS patients had been published in detailed case reports or small case series . The repFor a total of nine patients, cardiovascular comorbidities such as arterial hypertension, dyslipidemia, obesity, or diabetes mellitus had been reported. Two patients either suffered from asthma or COPD and another 7 patients had other comorbidities, for example breast cancer, epilepsy, or chronic migraine. The totality of the published case reports showed a quite favorable outcome: twenty-two patients were able to stay at home, and 25 patients had to be hospitalized, but did not require non-invasive or invasive ventilation. Only three patients had to be treated in the intensive care unit including intubation or non-invasive ventilation and one patient died from COVID-19. Two of the four patients with a severe or fatal disease course suffered from cardiovascular comorbidities.The SARS-CoV-2 pandemic is a threat to many billions of people, many of whom are chronically ill. As neurologists, we are particularly interested in assessing the risk for those patients whose immune system is impaired by drug therapy. When we summarize the data of the literature regarding MS therapeutics and their potential risk in the context of the pandemic, it must be remembered that there is a certain publication bias. In general, more results are published for the highly effective and more innovative therapies, such as anti-CD20 treatment, than for the platform therapies. However, the numbers of serious or fatal cases remain surprisingly low overall. Considering all published cases of MS patients with COVID-19, the rate of fatal outcome is 4%. Even in the group of patients with anti-CD20 therapies, the rate of reported deaths is 3%. The proportion of patients undergoing intensive care is 6%. Similar or even better outcome results have been reported for patients treated with other MS therapeutics. It is noticeable that the group of untreated MS patients falls off quite markedly. Of the 83 people for whom the outcome was published, 17% died from COVID-19 and another 7% needed non-invasive or mechanical ventilation. These results might indicate that the MS therapy itself could even be protective or that the patients without immunomodulating therapy rather belong to the group of progressive MS patients and thus are more likely to show an advanced stage of the disease . This suIn addition to age, cardiovascular comorbidities have been identified as important risk factors for severe COVID-19 in recent studies ,80,81. IThis work has some limitations: First, this is a retrospective work that does not allow for causal conclusions. Secondly, we only describe the results published so far, but further analyses including multivariate logistic regression models have not been made and would exceed the scope of this paper. Finally, the already mentioned publication bias must be taken into account.In conclusion, it is important to address the risks of different patient groups in the context of the SARS-CoV-2 pandemic. It is reasonable to expect that patients with chronic autoimmune diseases such as MS, who often require immunosuppressive therapies, will be at a particularly high risk for severe COVID-19 course. However, the cases currently published in the literature show that even among these patients the death rate is comparatively low and that it is not the DMTs themselves that pose a risk; rather, age, co-morbidities and the severity of the underlying disease are much more important. The fact that patients without DMTs account for a significant proportion of all deaths suggests that DMTs may even be protective.


Data on the comparative effectiveness of Diabetes Prevention Programs (DPPs) in the workplace are limited.Between September 2015 and July 2016, employees of the City and County of San Francisco who were at risk for type 2 diabetes (N = 158) were randomly assigned to one of 2 DPP-derived programs recognized by the Centers for Disease Control and Prevention: an in-person YMCA-DPP (n = 78) or an online virtual lifestyle management DPP (VLM-DPP) offered through Canary Health (n = 80). The primary outcome was change in body weight assessed at 6 and 12 months. Follow-up ended in August 2017.Both the YMCA-DPP and VLM-DPP yielded a significant reduction in percentage body weight at 6 months. For the YMCA-DPP, mean percentage change at 6 months was −2.70%  and at 12 months was −2.46% . For the VLM-DPP, mean percentage change at 6 months was −2.41%  and at 12 months was −1.59% . The mean between-condition difference at 6 months was −0.25%  and at 12 months was −0.84% . No significant differences were observed between conditions. The YMCA-DPP had a slightly higher reduction in waist circumference than VLM-DDP at 6 months . Participant engagement, expressed as mean number of completed core program sessions, was significantly higher for the YMCA-DPP than the VLM-DPP. Participants of the YMCA-DPP completed an average of 10.2 sessions , and participants of the VLM-DPP completed an average of 5.9 sessions . The adjusted mean between-condition difference was 4.2 sessions .Both the YMCA-DPP and VLM-DPP yielded weight loss at 6 months, which was maintained at 12 months in the YMCA-DPP. The workplace may be an effective setting to offer DPPs. To our knowledge, this is among the first randomized controlled trials to evaluate lifestyle programs recognized by the Centers for Disease Control and Prevention’s Diabetes Prevention Recognition Program, implemented in the workplace, and delivered by trained staff from the recognized organizations.In this trial, 158 City and County of San Francisco employees at high risk for type 2 diabetes were randomly assigned to an in-person YMCA program or an online virtual lifestyle management program to assess the effectiveness of both programs on weight loss among its participants.Weight loss of participants of both programs was significant at 6-month follow-up, but the amount lost did not differ significantly between the 2 programs. We found that the workplace may be an effective venue in which to offer Diabetes Prevention Programs.An estimated 84 million people in the United States have prediabetes ,2. The DThe Centers for Diseases Control and Prevention (CDC) developed the National DPP  with theGiven the availability of DPP-derived lifestyle interventions and the high costs of diabetes, employers are increasingly considering offering type 2 diabetes prevention programs to their at-risk employees. Because of its convenience, the workplace holds promise for reaching adults who have barriers , including individuals with fewer socioeconomic resources, that may prevent them from engaging in DPPs –10. HoweResearchers at the Kaiser Permanente Northern California Division of Research collaborated with the City and County of San Francisco (CCSF) Health Service System to conduct a randomized controlled trial comparing the effectiveness on weight loss of 2 DPRP-recognized lifestyle programs implemented in the workplace setting: 1) YMCA-DPP , an in-pThis study was approved by the Kaiser Permanente Northern California institutional review board. All participants provided written informed consent.The CCSF Diabetes Prevention Trial was a 2-arm, parallel, randomized controlled trial to compare the effectiveness of the YMCA-DPP and VLM-DPP in 7 worksites of the CCSF. Study staff enrolled CCSF employees from each worksite location in waves. At each worksite, enrollment lasted approximately 1 month. Participants were enrolled in September 2015 at the first worksite and in July 2016 at the seventh worksite. Follow-up ended in August 2017.2) of ≥25.0 kg/m2 or ≥23 kg/m2 if Asian. Criteria for exclusion were having a major health condition , not having regular access to a computer or the internet, current enrollment in another weight loss program, or plans to move or change departments in the next 12 months.Participants were CCSF employees. Department contacts from the 7 participating locations promoted the study using flyers, department-wide emails, announcements at meetings, and information on the CCSF website. Interested CCSF employees attended an onsite open information session where research staff explained the 2 DPP interventions; described what participation in the research study would entail; conducted eligibility screening; and assessed height, weight, and waist circumference. Employees were considered eligible based on results from a CDC prediabetes screener , which dEligible employees were asked to attend an orientation session 1 week later, during which research staff further explained the study. Employees who were interested in participating provided written informed consent and completed surveys.2 or ≥30 kg/m2), sex , race/ethnicity , and job type .The study project manager randomly assigned employees to either the in-person YMCA-DPP delivered at the worksite or the online VLM-DPP. An adaptive randomization procedure  was usedAll participants were given the goal of losing 5%–7% of their body weight through dietary change and participation in 150 minutes of moderate-intensity physical activity per week.The YMCA-DPP, an in-person intervention of 16 weekly, 1-hour group sessions (core curriculum) and 3 biweekly and 5 monthly group sessions (maintenance curriculum) , was helParticipants assigned to the VLM-DPP, an online program offered through Canary Health , first rAssessments were conducted by trained research staff blinded to intervention assignment at baseline, 6 months, and 12 months. Data on demographic characteristics and information on whether a participant’s job was office-based or not office-based  were collected at baseline. Height was measured at baseline using a Seca stadiometer (Seca GmbH). Weight and waist circumference were measured at baseline and at 6 months and 12 months. Weight was measured using a Tanita scale (Tanita Corporation). All body measurements were taken according to standard protocols  with parDiet and physical activity were assessed at baseline and the 6-month and 12-month follow up. The Block Fat Screener was used to assess diet , and theThe primary outcome was body weight expressed as change in the amount of body weight in kilograms and percentage change in body weight. Secondary outcomes included changes in waist circumference; physical activity, expressed as duration and intensity per week; dietary fat intake; and participant engagement, expressed as mean number of intervention sessions completed and the proportion of participants who completed 4 or more sessions.The study sample of 80 individuals per intervention condition provided 80% power to detect as small as a 1.8% difference in mean percentage weight loss between intervention conditions, assuming a standard deviation for percentage weight loss of 4% ,10. In aLinear mixed effects regression techniques were used to model change in weight, percentage change in body weight, waist circumference, physical activity, and dietary fat intake, as a function of intervention condition and time, controlling for baseline variables used in the adaptive randomization procedure  . AdditioP < .05). Baseline characteristics of randomly assigned participants did not differ significantly by intervention condition (P < .05). Intervention engagement differed by condition; 71 (91%) participants in the YMCA-DPP and 59 (74%) participants in the VLM-DPP completed 1 or more sessions (P = .005).A total of 351 CCSF employees attended the information session at their worksite, and 158 (45%) were randomly assigned to either the YMCA-DPP (n = 78) or the VLM-DPP (n = 80) . Employeondition . For folondition . ParticiIn the analysis using multiple imputed measurements, during the follow-up period, participants in the YMCA-DPP lost more weight on average than those in the VLM-DPP; however, this difference was not significant, with an average adjusted mean between-condition difference of −0.21 kg  . Both thMean total calories, dietary fat intakes, and saturated fat intakes decreased significantly in both intervention conditions at 6 and 12 months; however, no significant condition differences were observed . NeitherThe mean number of completed core sessions was significantly higher for the YMCA-DPP  than for the VLM-DPP ; the adjusted mean between-condition difference was 4.2 sessions . Participants in the YMCA-DPP were significantly more likely than those in the VLM-DPP to complete 4 or more of 16 core sessions .To our knowledge, this is among the first studies to evaluate the comparative effectiveness of 2 DPP-derived lifestyle programs recognized by the CDC DPRP and delivered by trained staff from the approved organizations. In this 2-arm, parallel, randomized controlled trial of 158 employees who were at high risk for type 2 diabetes, we found no significant differences in weight loss across 12 months of follow-up between those randomly assigned to receive an in-person lifestyle program delivered at the workplace or an online lifestyle program delivered at participants’ convenience. Although we found no significant between-condition differences, both the YMCA-DPP and the VLM-DPP yielded significant reductions in body weight and percentage change in body weight at 6 months. Body weight reductions remained significant at 12 months among participants in the YMCA-DPP but not the VLM-DPP. The YMCA-DPP also resulted in reduced waist circumference at 6 months, although the finding was not significant.Several DPP-derived lifestyle programs implemented at the workplace have been evaluated using a randomized controlled design; however, most of these interventions were delivered by research staff. In the CCSF trial, we observed a lower percentage of weight loss (from 2.4% to 2.7% at 6 months and 1.6% to 2.5% at 12 months) than that observed in previous randomized controlled trials of lifestyle interventions in workplaces. In a trial conducted at a university worksite in Ohio, participants assigned to a DPP-derived lifestyle intervention had a mean percentage weight change from baseline to 7 months of 5.5% . In a trA strength of this study was our ability to randomly assign employees and thereby reduce selection bias. Using the CDC prediabetes screener was also a strength because it allowed nonclinical staff to easily identify employees at risk for type 2 diabetes and invite them to participate. An additional strength was the inclusion of a racially and ethnically diverse population of men and women, particularly given that most trials of technology-based DPP interventions conducted to date have been among primarily white populations . Study lIn this real-world setting, it was challenging to ensure participant engagement in each intervention program, especially for the online program. Engagement and retention in the program were greater in the YMCA-DPP, where participants were more likely to start the program and completed more sessions than participants in the VLM-DPP. Given the importance of strong engagement to successful program outcomes , these rSince the conclusion of this trial, the CCSF has begun offering an in-person DPP-derived lifestyle program to all employees at risk for type 2 diabetes. Given the lessons learned from this trial, city officials are prioritizing recruitment and engagement activities as well as the inclusion of settings that employ workers with nonoffice jobs. As evidenced by this study and previous studies ,10, workIn conclusion, both CDC-recognized, worksite-setting lifestyle programs  assessed


The literature on non-genetic peripheral biomarkers for major mental disorders is broad, with conflicting results. An umbrella review of meta-analyses of non-genetic peripheral biomarkers for Alzheimer’s disease, autism spectrum disorder, bipolar disorder (BD), major depressive disorder, and schizophrenia, including first-episode psychosis. We included meta-analyses that compared alterations in peripheral biomarkers between participants with mental disorders to controls  and that assessed biomarkers after treatment . Evidence for association was hierarchically graded using a priori defined criteria against several biases. The Assessment of Multiple Systematic Reviews (AMSTAR) instrument was used to investigate study quality. 1161 references were screened. 110 met inclusion criteria, relating to 359 meta-analytic estimates and 733,316 measurements, on 162 different biomarkers. Only two estimates met a priori defined criteria for convincing evidence . Of 42 estimates which met criteria for highly suggestive evidence only five biomarker aberrations occurred in more than one disorder. Only 15 meta-analyses had a power >0.8 to detect a small effect size, and most (81.9%) meta-analyses had high heterogeneity. Although some associations met criteria for either convincing or highly suggestive evidence, overall the vast literature of peripheral biomarkers for major mental disorders is affected by bias and is underpowered. No convincing evidence supported the existence of a trans-diagnostic biomarker. Adequately powered and methodologically sound future large collaborative studies are warranted. However, unlike other medical fields, there is an acknowledged ‘translational gap’ in psychiatry3. In parallel, the field of biological psychiatry aiming to provide a neurobiological basis for current mental disorders, has provided contrasting results, even in pivotal biomarkers4. Hence, the diagnosis and clinical management of major mental disorders is still entirely based on psychopathological knowledge, while the treatment of mental disorders remains predominantly based on ‘trial and error’, albeit within the confines of fitting evidence-based prescription to a clinical profile5.One of the overarching goals of the emerging field of precision psychiatry is to incorporate advanced technologies to provide an objective data-driven personalized approach to the diagnosis and treatment of mental disorders6. In particular, the literature on non-genetic peripheral biomarkers has grown exponentially, with the publication of several systematic reviews and meta-analyses12. The identification and validation of biomarkers for mental disorders are thought to be crucial steps in the development of precision and biological psychiatry, and its ultimate incorporation in the current landscape of psychiatric care is expected to follow1. However, this change is not translating into meaningful modifications in clinical practice.Over the past two decades the field has witnessed a remarkable increase in interest on biomarkers for mental disorders13. In this respect, it has been proposed that similarly to genetic14 and neuroimaging16 biomarkers, alterations in peripheral biomarkers for major mental disorders may be shared across distinct diagnostic categories, and thus may have a transdiagnostic nature6. However, what is a trans-diagnostic construct in psychiatry remains debated, and no study has properly assessed the trans-diagnostic nature of any biomarker with a methodologically sound approach17.Several reasons may contribute to the contrast between the overall volume of this literature and the limited applicability of peripheral biomarkers in current psychiatric practice. For instance, it has been proposed that conventional psychiatric diagnoses based, for example, on the Diagnostic and Statistical Manual for Mental Disorders (DSM) may lack biological validity18. Umbrella reviews are particularly suited to uncover these biases19, as previously demonstrated with respect to peripheral biomarkers for depression20, bipolar disorder (BD)20, and schizophrenia21. However, those previous umbrella reviews have only addressed studies that have differentiated participants with a specific mental disorder and healthy controls, and not changes in peripheral biomarkers following treatment for these disorders. Moreover, those umbrella reviews focused on only one mental disorder each.In addition to the lack of consensus on how to define a trans-diagnostic construct, a core reason for this translational gap even in a single disorder may be due to the presence of several biases including large heterogeneity, an excess significance bias, as well as a selective reporting of statistically significant  findings without proper adjustment to multiple confounders. An Umbrella review systematically evaluates and collects information from multiple systematic reviews and meta-analyses on all outcomes of a given topic for which these have been performedThus, the current work provides a comprehensive umbrella review of meta-analyses of peripheral biomarkers for major mental disorders related to high prevalence and burden, namely Alzheimer’s disease (AD), autism spectrum disorder (ASD), BD, major depressive disorder (MDD), and schizophrenia, including also first-episode psychosis (FEP) stage. We aimed to re-assess the presence of bias in this literature and identify biomarkers that would be supported by most convincing evidence. In addition, we aimed to identify shared and unique alterations in biomarkers for those major mental disorders among those supported by either convincing or highly suggestive evidence. In the current analysis, we considered both studies that investigated abnormalities in peripheral biomarkers of mental disorders compared to controls  and ones that assessed alterations in the levels of peripheral biomarkers after treatment .22. The PubMed/MEDLINE database was searched from inception to February 17, 2019 for all available meta-analyses non-genetic peripheral biomarkers for major mental disorders. This search strategy was augmented through (1) handsearching the reference lists of included articles and (2) tracking citations of included articles through the Google Scholar database. The search string used in the current umbrella review was developed by a professional librarian and is available in the We conducted an umbrella review, which is a systematic collection of multiple systematic reviews and meta-analyses done in a specific research topicInternational Classification of Disease (ICD) or the Diagnostic and Statistical Manual of Mental Disorders (DSM). We also considered studies in which a probable diagnosis of a major depressive episode was established through a validated screening questionnaire as well as studies in which a diagnosis of FEP was based on clinical assessment by a mental health care provider. We excluded the following types of studies: (1) systematic reviews without a meta-analytic synthesis of the evidence; (2) animal studies; (3) studies of other types of biomarkers ; (4) studies that included participants with two or more diagnoses; (5) studies that included participants with other primary psychiatric diagnoses (e.g. anxiety disorders); (6) studies that investigated biomarkers for other purposes 23; (7) studies conducted in pediatric samples (except from ASD and FEP); and (8) if there was more than one meta-analysis for the same biomarker in the same population, we considered only the largest MA .We included meta-analyses published in peer-reviewed journals that assessed and synthesized studies on peripheral biomarkers for adults with AD, ASD, BD, MDD, Schizophrenia, including FEP. We included studies in which biomarkers were assayed in participants with a specific mental disorder compared to controls , as well as ones which assessed changes in peripheral biomarkers in any of those disorders after treatment . Studies published in English were considered for inclusion. This decision was made because most well-designed systematic reviews and meta-analyses are published in English. We included studies in which diagnoses of mental disorders were conducted by means of a validated structured interview based on standard diagnostic criteria such as the For each eligible reference, we extracted the first author, year of publication, specific diagnoses assessed, as well as the number of included studies. We also extracted the summary effect size (ES) measure of each meta-analysis considering the ES used in each study. When available, the following variables were extracted at a study-level: number of cases, number of controls, sample size, ES, and study design. In each eligible reference, we only included the primary analyses due to the expected large amount of evidence. However, when included references provided details on the mood state of participants (e.g. mania or bipolar depression), we also extracted this information at an individual-study level.24. Due to the anticipated high heterogeneity observed in meta-analyses of peripheral biomarkers for major mental disorders, random-effects calculations were considered in this review. When ESs were not provided as standardized mean difference (SMD) metrics , we converted the primary ESs to SMD25. We also estimated the 95% prediction interval, which accounts for between-study heterogeneity and assesses the uncertainty of the effect that would be expected in a new study addressing the same association26. For the largest study included in each meta-analytic estimate, we calculated the standard error (SE) of the ES. If the SE of the ES is <0.1, then the 95% CI will be <0.20 . We calculated the I2 metric to quantify between-study heterogeneity. Values ≥50% and ≥75% are indicative of large and very large heterogeneity, respectively27. To assess evidence of small-study effects, we used the asymmetry test developed by Egger et al. 28. A P-value <0.10 in the Egger’s test and the ES of the largest study being more conservative than the summary random-effects ES of the meta-analysis were considered indicative of small-study effects20. We also annotated whether the association reported in each meta-analytic estimate was nominally significant at a P < 0.05 level as well as at a P < 0.005 level. The level of P < 0.005 has been proposed as a more stringent level of significance that could increase the reproducibility of many fields29.Data were analyzed from March 1, 2019 to October 10, 2019. We estimated ESs and 95% confidence intervals (CIs) using both fixed and random-effects modeling30. Finally, we also assessed evidence of excess of significance bias with the Ioannidis test31. Briefly, this test estimates whether the number of studies with nominally significant results  among those included in a meta-analysis is too large considering their power to detect significant effects at an alpha level of 0.05. First, the power of each study is estimated with a non-central t distribution. The sum of all power estimates provides the expected (E) number of datasets with nominal statistical significance. The actual observed (O) number of statistically significant datasets is then compared to the E number using a χ2-based test31. Since the true ES of a meta-analysis cannot be precisely determined, we considered the ES of the largest dataset as the plausible true ES. This decision was based on the fact that simulations indicate that the most appropriate assumption is the ES of the largest dataset included in the meta-analysis32. Excess significance for a single meta-analysis was considered if P < 0.10 in Ioannidis’s test and O > E20. We graded the credibility of each association according to the following categories: convincing (class I), highly suggestive (class II), suggestive (class III), weak evidence (class IV), and non-significant associations  or a medium . We used the method described in detail elsewherens Table .c (credibility ceiling) that the true ES is in a different direction from the one suggested by the point estimate33. The pooled ESs were estimated considering a wide range of credibility ceilings. All analyses were conducted in STATA/MP 14.0  with the metan package.For evidence supported by either class I or class II evidence, we used credibility ceilings, which is which is a method of sensitivity analyses to account for potential methodological limitations of observational studies that might lead to spurious precision of combined effect estimates. In brief, this method assumes that every observational study has a probability Assessment of Multiple Systematic Reviews (AMSTAR) instrument, which has been validated for this purpose35. Scores range from 0 to 11 with higher scores indicating greater quality. The AMSTAR tool involves dichotomous scoring (i.e. 0 or 1) of 11 items related to assess methodological rigor of systematic reviews and meta-analyses . AMSTAR scores are graded as high (8–11), medium (4–7) and low quality (0–3)34.The methodological quality of included systematic reviews and meta-analyses was also appraised using the 139, and 60 references were excluded with reasons  had an estimated power >0.8 to detect a medium ES Table .I2 < 50%) was found in 65 meta-analyses (18.1%), whilst 294 (81.9%) meta-analytic estimates had evidence of large heterogeneity . The prediction interval crossed the null value in 341 (94.9%) meta-analytic associations, while prediction intervals of 20 (5.0%) meta-analyses did not cross the null value  meta-analyses, whilst evidence of excess of significance bias was verified in 74 (20.6%) meta-analytic estimates Tables .P < 0.005) compared to controls87. Participants with schizophrenia presented decreased Vitamin B6  levels relative to controls123. In addition, 42 (11.7%) meta-analytic estimates were supported by class II evidence, of which 3 were derived from within-group meta-analyses -2 receptor (sIL-2R) levels were increased in MDD and in schizophrenia relative to controls8. Moreover, levels of antibodies against the N-methyl-d-aspartate receptor (NMDA-R) were elevated in BD and in schizophrenia relative to controls85. Brain-derived neurotrophic factor (BDNF) levels were decreased in AD and in MDD110. Furthermore, levels of insulin-like growth factor-1 (IGF-1) were elevated in bipolar mania and in MDD relative to controls84. The remaining findings supported by type II evidence were unique to a single disorder (Table Only 2 (0.5%) meta-analytic estimates exhibited class I evidence 83, 119). In euthymic BD participants there was an increase in basal cortisol awakening levels  survived 10% credibility ceilings Table .Table 2SQualitative methodological appraisal of eligible meta-analyses through the AMSTAR tool revealed that 49 references were classified as high, 58 as medium, and 3 as low methodological quality, respectively Table . The oveOur umbrella review provided an up-dated synthesis of the literature of non-genetic peripheral biomarkers for major mental disorders. We included data from 733,316 biomarker measurements. However, in this vast literature only two associations met a priori defined criteria for convincing evidence, whilst 42 meta-analytic estimates met criteria for highly suggestive evidence. This collaborative effort found compelling evidence that overall the literature on non-genetic peripheral biomarkers has a high prevalence of different types of bias. In addition, this umbrella review provides relevant insights for the conduct of further studies to investigate the associations supported by most convincing evidence. It should also be noted that overall the methodological quality of eligible meta-analyses as assessed with the AMSTAR tool was high, which provides further credibility to our quantitative grading of findings.87. This finding indicates that the hypothalamic–pituitary–adrenal (HPA) axis is disrupted in BD on a trait-like basis. This suggests that the HPA axis could be targeted in BD140 to improve cognitive function, which may be compromised even during euthymic states142. In addition, participants with schizophrenia exhibited decreased vitamin B6  levels compared to controls123. This suggests that individuals with schizophrenia may present aberrations in the one-carbon cycle where pyridoxal is a main metabolic component. An alternative explanation might be the poor nutrition which frequently affects people with schizophrenia98. This finding is consistent with a recent systematic review and meta-analysis which provided preliminary evidence that adjunctive pharmacological interventions targeting the one-carbon cycle may improve negative symptoms in schizophrenia .Associations supported by convincing evidence merit discussion. First, euthymic participants with BD exhibited a high cortisol awakening response relative to controls17. We found peripheral elevation on the acute phase reactant, CRP, in BD (both during euthymia and mania) as well as in MDD providing evidence that these disorders are at least partly associated with peripheral inflammation. In addition, the s-IL-2R was increased in both MDD and schizophrenia relative to controls. It is noteworthy that IL-2 is a key cytokine involved in the development, survival and function of regulatory T cells (TRegs)147, and it has been recently proposed that aberrations in “fine tuning” immune-regulatory mechanisms may contribute to the pathophysiology of both MDD and schizophrenia149. Antibodies against the NMDA-R were increased in BD and schizophrenia. This finding is consistent with the existence of autoantibodies against the GluN1 subunit of this receptor in patients with psychotic manifestations151. Furthermore, lower serum BDNF levels were observed in participants with MDD and AD relative to controls. This finding is consistent with the “neurotrophic hypothesis” of depression152, while parallel lines of evidence suggest that aberrations in BDNF signaling may contribute to neurodegeneration in AD153. Finally, lower levels of IGF-1 were observed in bipolar mania and MDD compared to controls. This finding is consistent with the modulatory role of glucose-related signaling including the trophic molecule IGF-1 in hippocampal plasticity154. In addition, preclinical evidence suggests that IGF-1 may be involved in the pathophysiology of affective disorders156.Importantly, only five biomarkers were found to be significantly associated with more than one mental disorder. Also, the highest class of evidence for these biomarkers was II. Moreover, no study applied a methodologically solid approach to assess the trans-diagnostic nature of any biomarker158. In addition, IL-6 levels decreased after treatment with antidepressants. These data are consistent with preclinical findings which show that antidepressants have anti-inflammatory properties and may also inhibit M1 microglia polarization159. Finally, lipid peroxidation markers increased after antidepressant drug treatment for MDD.There is an emerging body of literature investigating the putative role of non-genetic peripheral biomarkers for the prediction of treatment response in major mental disorders. Surprisingly, no such biomarkers met criteria for convincing evidence, while only three biomarkers met criteria for type II evidence. Adiponectin levels in schizophrenia decreased after treatment with second-generation antipsychotics. This is an interesting finding since hypoadiponectinemia has been associated with a wide range metabolic diseases which are common untoward effects of these drugs20. This may undermine the progress and reliability of this particular field and of neuroscience in general through the generation of spurious findings160. The “true” ESs of most non-genetic peripheral biomarkers may be expected to be small, similarly to those reported in the genetic literature. Therefore, the design of large, multicenter studies with an open pre-registered protocol, or the creation of Consortia, may be a crucial step to assess the role of peripheral biomarkers in the diagnosis and treatment of major mental disorders within the framework of precision psychiatry1, as the model adopted by the Enigma neuroimaging group161, or similarly to other large collaborative initiatives162. Likewise the creation of biomarker scores using a similar rationale as for the generation of polygenic risk scores may ultimately be a next step in this field.It is worth noting that only 15 meta-analytic estimates had a power >0.80 to detect a small ES. In addition, previous umbrella reviews indicate that the vast majority of peripheral biomarker studies are substantially underpowered163. In addition, methodological differences of individual studies included in the assessed meta-analyses may also contribute to heterogeneity. Those include, for example, the time of sample selection as well as measurement properties of the assays (e.g. intra-assay and inter-assay coefficients of variation). Guidelines to standardize the collection and measurement of peripheral biomarkers in psychiatry have been recently proposed164. Furthermore, differences in sample selection across individual studies might have contributed to the observed heterogeneity in some meta-analytic estimates. For example, illness stage and disorders in which mixed presentations are common  might have contributed to heterogeneity across some included meta-analyses. In addition, approaches to subtype major mental disorders according to frameworks such as the NIMH Research Domain Criteria may help to decrease the heterogeneity of this literature in the future through the study of biologically valid and more homogenous phenotypes165.It should also be noted that large statistical heterogeneity was verified in most included meta-analytic estimates (81.9%). Although this is considered a relevant indicator of bias in this literature, it may also reflect genuine heterogeneity, which may occur both within and between major diagnostic categories17, as well as the subtyping of these disorders into more biologically valid phenotypes, and enough statistical power may improve the reliability and reproducibility of this field, which is of relevance for the translation of biological and precision psychiatry into practice.This umbrella review of non-genetic peripheral biomarkers for major mental disorders revealed that this literature is fraught with several biases and is underpowered. Nevertheless, two associations supported by convincing evidence and 42 associations supported by highly suggestive evidence were verified. Most associations supported by either convincing or highly suggestive evidence pertained to a single disorder. Future multi-centric studies with a priori publicly available protocols, with an ad-hoc methodology to assess the trans-diagnostic nature of biomarkersSupplementary Online material