Adolescents treated for self-poisoning with medication have a high prevalence of mental health problems and constitute a high-risk population for self-harm repetition. However, little is known about whether this population is also prone to injuries of other forms.Data were extracted from the Norwegian Patient Registry to include all incidents of treated injuries in adolescents aged 10–19 years who were treated for self-poisoning with medication during 2008–2011. This longitudinal approach allowed for the inclusion of injuries of various forms both before and after the index poisoning with medication. Gender differences and associations of injuries with recorded deliberate self-harm or psychiatric comorbidity at index poisoning were analysed. Forms of injury and psychiatric illnesses were coded according to the ICD-10 system.1497 adolescents treated for self-poisoning with medication were identified from the source database, including 1144 (76.4%) girls and 353 (23.6%) boys. For these 1497 adolescents a total of 2545 injury incidents were recorded in addition to the index poisoning incidents, consisting of 778 injury incidents taking place before the index poisoning and 1767 incidents taking place subsequently. Altogether 830 subjects (55.4%) had an injury treated either before or after the index poisoning. Injuries to the hand and wrist as well as injuries to the head, neck and throat were predominant in males. Females were more likely to repeat poisoning with medication, particularly those with psychiatric disorders.Adolescents treated for poisoning with medication represent a high-risk population prone to both prior and subsequent injuries of other forms, and should be assessed for suicidal intent and psychiatric illness. Non-fatal injuries are the leading cause of disability among children and adolescents worldwide . AlthougAccording to community studies –11, selfIn spite of interesting findings, previous studies on adolescent injury have often been based on data from local hospital records or cross-sectional surveys, lacked the details on types of injury, or focused on either accidental or intentional injuries alone . MoreoveThe study was based on all adolescents aged 10–19 years in Norway. Subjects of focus were adolescents who were identified from the Norwegian Patient Registry (NPR) as having received acute treatment for self-poisoning with medication from 2008 through 2011. The Norwegian version of the Tenth Revision of the International Statistical Classification of Diseases (ICD-10) has been used for medical diagnoses in the NPR. Identification of adolescents into study cohort was carried out in accordance with a previous study focusing on poisoning with medications [Variables of interest included all external injuries (S00-T65) that led to medical treatment in emergency and hospital services before and after the index medication poisoning. Injury diagnoses were further categorized by specific body regions according to the ICD-10 system as shown in the Tables Moreover, a supplemental diagnosis confirming intentionality in an injury, coded as X6n in the NPR, was regarded as a recorded deliberate self-harm. We should note, however, data describing the circumstances and the cause of injury were often insufficient in patient records, this diagnosis, i.e. deliberate self-harm (X6n), has been heavily underreported in the patient registry in Norway . Psychiax2 test was used to detect statistical differences between groups. Odds Ratios (OR) with 95% Confidence Intervals (CI) were estimated through general Logistic regression models. The analysis was conducted separately for each specific group of injuries and moreover by sex. The significance level was set to 0.05 and only tests being statistically significant were reported. All analyses were carried out on SPSS 22.0 or SAS 9.4 software for Windows.Number of injuries prior and subsequent to the index medication poisoning was counted and distribution by specific types of injury and by gender was explored through descriptive analysis. The associations of injuries with comorbid diagnoses of deliberate self-harm and psychiatric disorder at index poisoning were analyzed. A total of 1497 adolescents aged 10–19 years received medical treatment because of self-poisoning with medication from 2008 through 2011. These adolescents comprised 1144 (76.4%) girls and 353 (23.6%) boys with a female to male ratio of 3.24. Most of them (89.8%) were between 15 and 19 years old at the time of the index poisoning.Of the 1497 adolescents, 431 (28.8%) individuals were treated at least once for external injuries before the index medication poisoning, and 586 (39.1%) cases had at least one treatment because of injuries after the index poisoning during the observation period. In total, 830 (55.4%) adolescents  received hospital treatment at least on one occasion because of injuries or poisoning either before or after the index poisoning. Altogether 2545 injury incidents, in addition to the index poisoning with medication, were recorded, including 778 injury incidents of other types before the index poisoning and 1767 injuries of all types after the index poisoning.Figure The most frequently recorded injury form prior to the index poisoning was injury to the abdomen, lower back, lumbar spine and pelvis (S30-S39), present in 214 (14.3%) individuals and 12.8% of females and 19.3% of males in the cohort  and also a comorbid diagnosis of psychiatric disorder  in connection with their subsequent injuries within the observation period.At the index poisoning with medication, 668 (44.6%) adolescents received a supplementary diagnosis of deliberate self-harm with a significantly higher presentation in females than males . 508 (33.9%) individuals received a comorbid diagnosis of psychiatric disorder with no significant gender difference.Table Individuals diagnosed with a psychiatric comorbidity at index poisoning had significantly more injuries both prior and subsequent to index poisoning ; OR = 1.3, 95% CI: 1.0–1.6 (after)). These adolescents were also more likely to have injuries to wrist and hand before the index poisoning , poisoning with medication after the index poisoning , and to receive a diagnosis of deliberate self-harm both before  and after  index poisoning. Individuals receiving a comorbid psychiatric diagnosis at index poisoning were three times more likely to receive a psychiatric diagnosis at subsequent injuries .When males and females were analyzed separately Table , a supplFemale adolescents who received a comorbid diagnosis of psychiatric disorders at index poisoning were more likely to receive treatment for both prior  and subsequent  injuries of any form. Specifically, they more often had prior injuries to the wrist and hand (S60-S69)  and subsequent poisoning with medication and other substances (T40-T65) . More notably, these females were most likely to get a supplemental diagnosis of deliberate self-harm ; OR = 1.8, 95% CI: 1.3–2.5(after)) as well as a comorbid diagnosis of psychiatric disorder at injuries both before and after the index poisoning ; OR = 3.0, 95% CI: 2.1–4.2 (after)).To our knowledge, this is the first population study examining external injuries of various forms in a cohort of adolescents treated for self-poisoning with medication using data from a national patient registry. The results support our hypothesis indicating that: 1) external injury among the cohort adolescents was quite common and manifested itself in a variety of injuries, 2) the presence of both prior and subsequent injuries was highly associated with a comorbid diagnosis of deliberate self-harm or psychiatric illness at index poisoning, and that 3) there were significant gender differences in injury incidents and form of injuries.External injuries were prevalent in our cohort of adolescents, with over half of them having at least one injury before or after the index poisoning. About 30% of these adolescents had a record of treatment because of injuries in forms other than medication poisoning before the index poisoning – an insightful detail which has not been reported in related studies. In the meantime, nearly 40% of the cohort adolescents were treated for injuries of various forms after the index incident of medication poisoning.The commonness of injuries of other forms, seen in our cohort both before and after the index poisoning, supports the notion in previous studies  that metOur finding that less than half of the adolescents in the cohort received a comorbid diagnosis of deliberate self-harm at their index poisoning was clearly an underreporting of this diagnosis in the patient registry, as compared with the rates reported by studies –33 in whThe present study did not detect a highly distinct pattern of injuries between males and females in the cohort, but several findings separated the genders.Common forms of injury observed in males, both before and after the index poisoning, was injury to the abdomen, lower back, lumbar spine and pelvis (S30-S39) and injury to the head, neck and throat (S00-S29). Females on the other hand showed predominance of injury to the shoulder, arm and elbow (S40-S59). We cannot ascertain, however, whether this illustrates a difference in preferred method of self-injury or simply reflects gender differences in forms of non-intentional injuries likely happening in daily life.Interestingly, males in the cohort stood out as having significantly more injuries to the hand and wrist prior to the index poisoning. Community studies on self-cutting suggest that this behaviour was more frequent in females than males , 39, wheFemale adolescents diagnosed with a psychiatric disorder at index poisoning seemed to be particularly vulnerable to having sustained injuries both before and after the index poisoning and to receiving a supplemental diagnosis of deliberate self-harm (X6n) on these occasions. They were more often treated for injuries to the wrist and hand before the index poisoning and for repeat poisoning after the index poisoning. These findings support the argument of a possible switch of methods used for self-harm in this group of adolescents , 40, 42.Males in our cohort were significantly more likely than females to have a prior injury to the wrist and hand S60-S61) for which no comorbid diagnosis of deliberate self-harm (X6n) was given. This is an interesting finding given the high likelihood of such injury being a deliberate action. Females, on the other hand, were more likely to repeat poisoning with medication and also to receive comorbid diagnoses of deliberate self-harm and psychiatric disorder at their subsequent injuries. A possible reason for this could be that adolescents, especially boys, often attend sport activities, so their injuries on hand and wrist are commonly perceived as accidental instead of deliberate. Another possible explanation for the discrepancy might be that, from a clinical point of view, poisoning with medication occurs more often with intention as compared to injury to the hand or wrist . This is in line with previous research , 43 stat0-S61 forThe data obtained for the present study ensured the accuracy of episodic hospital contacts because of injury, but did not contain personal profiles such as socioeconomic status, detailed information on psychiatric diagnosis and other health related records that may have provided interesting insights. Also, for injuries treated in emergency clinics, the assessment of psychiatric illness and deliberate self-harm was not systematically carried out. This may impose an underestimation of the true prevalence of such comorbidities among injury incidents and limit the possibility to fully distinguish between self-inflicted injuries and injuries inflicted by others. Furthermore, since many mild injuries do not lead to medical care, this study could not generalize to those who do not present to emergency or hospital services after injury and does in no way reflect the incidence and frequency of injuries or deliberate self-harm among adolescents. In addition, data on type of medication used in self-poisoning was not available. It would have been of great value to identify whether the medications were prescribed or purchased over the count so that to inform strategies of control to prevent future self-poisoning.Despite of these limitations, the present study has several strengths. It is, to our knowledge, the first one that has examined a large cohort of adolescents with medication poisoning for their external injuries of various forms. Unlike studies relying on self-reports and clinical records, this study is based on precise data of patient records with a standardized diagnostic classification of injuries and comorbidities. The longitudinal nature of the data has enabled the possibility of looking into the details both retrospectively and prospectively.This study shows that adolescents with medication poisoning represent a high-risk group prone to both prior and subsequent injuries of various forms in need of hospital treatment. Possible switches in injury methods as well as gender differences in injury incidents and forms were observed. A male predominance of injuries to hand and wrist was found together with an increased likelihood of subsequent poisoning in females. Comorbid diagnoses of deliberate self-harm and psychiatric illness at index poisoning correlated strongly with prior as well as subsequent injuries in the cohort adolescents, especially among the females. With growing evidence for a positive effect of psychosocial assessment for patients treated for self-harm on repetition reduction , the pre


The result demonstrated that TALENs potentially provide an effective strategy to remove latent MCMV in animals.Cytomegalovirus (CMV) infections are still a global health problem, because the latent viruses persist in humans and cause recurring diseases. Currently, there are no therapies for CMV latent infections and the therapies for active infections are limited by side effects and other problems. It is impossible to eradicate latent viruses in animals. HCMV (human CMV) is specific to human diseases; however, it is difficult to study HCMV due to its host specificity and long life cycle. Fortunately, MCMV (murine CMV) provides an excellent animal model. Here, three specific pairs of transcription activator-like effector nuclease (TALEN) plasmids  were constructed to target the MCMV M80/80.5 sequence in order to test their efficacy in blocking MCMV lytic replication in NIH3T3 cell culture. The preliminary data showed that TALEN plasmids demonstrate specific targeting and cleavage in the MCMV M80/80.5 sequence and effectively inhibit MCMV growth in cell culture when the plasmid transfection is prior to the viral infection. The most specific pairs of TALEN plasmids (MCMV3–4) were further used to confirm the negative regulation of latent MCMV replication and gene expression in Balb/c mice. The injection of specific TALEN plasmids caused significant inhibition in the copy number level of immediately early gene ( Studies on the functions of viral genes in human cytomegalovirus (HCMV) replication in vivo and understanding of viral pathogenesis are essential for developing novel drugs and strategies to treat the viral infections, but there are no suitable animal models for HCMV infection at present. Because HCMV proliferates in human cells specifically, grows slowly and has a very long lytic replication cycle in humans, it is quite difficult to study HCMV (genome size 235 kb) gene function and pathogenesis . HoweverAs a structural protein, the HCMV UL80 assembly protein is presumed to function in packaging DNA. A protease is encoded by the N-terminal region of UL80 that cleaves the assembly protein precursor at a site near the C terminus. The MCMV homolog (M80) of the assembly protein of HCMV (UL80) conserves the domain structure and cleavage sites present in HCMV UL80. MCMV is different in that the conserved CD1 and CD2 domains are separated by 100 amino acids, whereas in all other sequenced herpes viruses the two domains are separated by 80 to 84 residues . NI. NI23]. When the plasmid transfection was prior to the viral infection, the PCR products synthesized from the total DNAs targeted by TALEN plasmids were almost all the same A. HoweveHowever, if the viral infection was prior to the plasmid transfection, the PCR products synthesized from total DNAs targeted by TALEN plasmids were almost the same C. We couie-1DNA copy numbers were not detected. The results demonstrated that there were no viruses in the mice’s organs originally and no genome in Balb/c mice is homologous to the MCMV ie-1 gene.Three Balb/c mice were not infected with MCMV and were housed for latency establishment as negative controls. They were neither treated nor reactivated during the experimental process. We could not find any plaques from all five organs homogenates and For the plaque assay, it suggested no lytic viruses or only latent viruses were available in mice if the result was negative (no plaques detected). Otherwise, there were lytic viruses in mice if the result of assay was positive. For the following DNA copy number assay, if there were no DNA copies detectable this meant that there were no viruses; if the result was positive, this meant there were latent viruses in the organs.ie-1 DNA copy numbers were all about 102 , the DNA copies ranged from 10 to 102 in the livers, lungs, spleens, kidneys, and salivary glands. However, we found that the MCMV ie-1 DNA copies of the untreated group (Group 1) were more than those of the less specific treatment groups  and the nonspecific treatment group (Group 5) by about 10–100 folds in the livers, lungs, spleens, kidneys, and by about 3–10 folds in the salivary glands. Overall, we could conclude that the specific treatment group (Group 2) was the most efficient one to remove viral load in mouse organs.In The transfection efficiency of plasmids in NIH3T3 cells was about 20–25% in cell culture. We realized that an elevated level of transfection efficiency could increase the efficacy in inhibiting virus growth in cell culture. Despite this, we did not sort the transfected cells using flow cytometry for the studies in cell culture, because it was not feasible for us to do so in mice. To establish a more similar animal model, we used all the cells including untransfected and transfected cells for our ex vivo studies. To enhance the efficacy, multiple round injections for the formulation transfection during the treatment period are required in animal studies.5 pfu/mL within 1–3 days. However, if the plasmid transfection is prior to the viral infection, TALEN plasmid copy number might have increased and induced innate immune responses of host cells to secret cytokine or other factors to fight against invading viruses. Therefore, TALENs could inhibit virus growth by about 50–75% when the viral titer was 103–104 pfu/mL, and about 25% when the viral titer was about 105 pfu/mL.Cultured NIH3T3 cells are not specific for latency studies. If the viral infection is prior to the plasmid transfection, NIH3T3 cells are vulnerable to viruses because they do not have the same immune system as animals. In this case, TALENs can hardly protect the host cells from the viral infection, because the viral titer increases rapidly to about 105). The reasons might be that the amino acid-nucleotide recognition frequency is not absolute  and the transfection efficiency is about 20–25% in cell culture.In cell culture, the results for specific target and cleavage efficiency of TALEN plasmids reveal that some of the plasmids work well on MCMV M80/80.5 target sites. Specific TALEN plasmids also demonstrated their effects on the inhibition of virus growth, ranging from 25–75% depending on the viral titer. Although the decreasing level of viral titers was varied under different conditions, they had the same trend for the inhibition of virus growth. We also found that the higher the viral titer, the lower the effect of the TALEN plasmids on virus growth. Our findings indicated that the inhibition effect on MCMV is about 20–25% when the viral titer reaches the highest level  also worked well in reducing viral load, although they were less efficient than the specific treatment group (Group 2). This might be explained as follows. Firstly, during the multiple-round injection for TALEN plasmid treatment, recognition of foreign DNA in intracellular compartments or in the cytoplasm of host cells sends a signal of pathogenic invasion. In response, the innate immune DNA-sensing pathways start an antimicrobial type I interferon (IFN) (mainly IFNα and β) secretion to act against viruses. Acting in paracrine or autocrine models, IFNs stimulate intracellular and intercellular networks for regulating innate and acquired immunity in mice that are resistant to the viral infections ,33,34,35ie-1 DNA copies level. The viral latent infection in humans is a major barrier for effective treatment and also a long-term risk to the host. Although the mechanisms for inhibiting MCMV are still poorly understood, our studies demonstrate that the removal of latent MCMV in animals is possible using TALENs.Currently, it is impossible to eradicate latent CMV viruses in animals, although there are some effective drugs  for the treatment of active infections. Our data indicated that TALEN plasmids which could specifically target and cleave MCMV M80/80.5 ORF would effectively reduce the viral load in Balb/c mice, so that they resulted in the implicative decrease of


Intravenous immunoglobulin for the treatment of autoimmune encephalopathy in children with autism1 with great interest. It is undoubtedly important to communicate that children with autism may improve from IVIG treatment; this should be further investigated. In the study, the Cunningham Panel2 was used as a biological marker to predict improvement in the children. This immune panel comprises of five analytes: calcium calmodulin dependent kinase II activation (CamKII activation) and antibodies to dopamine receptors D1 and D2, β-tubulin, and lysoganglioside-GM1. CaMKII activation is regarded as positive if the activity exceeds 129, which was—in addition to one or more positive autoantibody titer—the prerequisite for IVIG treatment. The antidopamine D2L receptor antibody, the anti-tubulin antibody and the ratio of the antidopamine D2L to D1 receptor antibodies were chosen as predictors for treatment response. Improvement was based on the behavioral measures Aberrant Behavior Checklist (ABC) and/or Social Responsiveness Scale (SRS), in addition to parents’ reports.We read Connery et al. open-label case series entitled According to the outcome measures, 52–65% of the children were responders to IVIG. Unfortunately, the biomarkers and the two behavioral measures ABC and SRS were only acquired in 16 of the 31 children before and after IVIG treatment. However, based on this limited information, the authors, nevertheless, conclude that the Cunningham Panel predicts response to IVIG treatment with good accuracy. Yet, it is unknown if children with CaMKII activity below 130 would similarly improve from IVIG treatment, since the 17 patients reported to have such test results were not treated with IVIG. It could also well be that children with autism and normal levels of autoantibodies against dopamine receptors D1 and D2, β-tubulin, and lysoganglioside-GM1 may improve from IVIG treatment; this was not investigated in the study. Therefore, it remains unknown if any of the analytes in the Cunningham Panel can predict treatment response.4. In a Swedish study consisting of 53 patients (40 children and adolescents and 13 adults) with suspicion of having pediatric acute neuropsychiatric syndrome (PANS) and/or pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS), the property of the Cunningham Panel was investigated. To our knowledge, our study is the only independent study evaluating the Cunningham Panel hitherto published. Twenty-four of the patients met diagnostic criteria for PANS and/or PANDAS, whereas 29 did not. We also tested 27 healthy controls with the Cunningham Panel. Ten out of 21 (48%) healthy controls had elevated CaMKII activity as compared to 35 out of 53 (66%) of the patients. Moreover, at least one positive autoantibody titer was found in 17 of the healthy controls. In conclusion we found a so-called positive value on the Cunningham Panel in 86% of the healthy controls, as compared to 92% in the patients assessed for PANS and PANDAS.We have previously questioned the diagnostic value of the Cunningham PanelTo summarize, Connery et al. do not present any objective data in the causal pathway from symptoms to outcomes. Seveteen children with autism in the study were not treated with IVIG on the basis that their CaMKII was not elevated. Consequently, the effect of IVIG in children with autism and subthreshold levels of CaMKII activity is not tested. The reported improvements in the children with elevated CaMKII activity may stem from placebo response or the natural course, not necessarily from IVIG. Considering that the Cunningham Panel to a great extent is positive among healthy people, it seems premature to suggest that it can predict treatment response based on a small selection of children with autism in an open-label, uncontrolled study. At this point of time we cannot draw any quantitative conclusions about biomarkers from open label treatment studies of disorders like ASD.


In 2017, the WHO classification of tumours of the endocrine organs established the criteria for a NIFTP diagnosis. The present paper considers some aspects that are still debated or unresolved: the real incidence and clinical meaning of multifocal/multinodular lesions, the biological behaviour of micro-NIFTP, the sprinkling phenomenon and the corresponding modifications to the FNA reporting systems based on changes to the ROM. Moreover, the paper suggests possible scenarios for the clinical-pathological management of this entity. From the initial 1470 cases, a group of 68 NIFTPs was recruited in a 9 year-long period. The average age at diagnosis was 55 years. The average diameter of the lesion was 1.7 cm (0.1 cm–10 cm). In 41 cases (60.1%), the lesion was inserted in the context of a multinodular background. In 12 cases, the diagnosis was incidental and the pre- operative FNA was performed on a different target. In 10 out of 68 cases, there was a multifocal NIFTP; in 14.7% of patients, PTC-like nuclear features showed sprinkling phenomenon. The cytological revision allocated 21 cases (49%) to the SIAPEC TIR3 indeterminate class and a nuclear score 2 or 3 were identified in 25 smears. Multifocality is part of the spectrum of NIFTPs, that can arise in a multinodular background with variable sizes from microscopic lesions to very large ones. Cytopathological criteria such as an evaluation of the nuclear score may help the pathologists in promoting a NIFTP diagnosis in the preoperative setting. The diagnosis of NIFTP is performed only on surgical specimens and takes into account stringent histological criteria2. NIFTP should have a well-defined capsule, clearly separated from the surrounding parenchyma and without infiltration foci. The architectural pattern of the lesion is follicular, rendering it necessary to exclude the presence of true papillae. These kinds of tumours have nuclear characteristics that are similar to those of classical Papillary Thyroid Carcinoma (PTC). Despite the great utility related with the introduction of a more accurate terminology to define these indolent  lesions, different issues remain unresolved as of today. Firstly, a minimum (or maximum) size-limit of NIFTP is not defined, although the majority of the studies focused on lesions with dimensions of 1 cm or more5. Furthermore, no obvious data is available for bilateral  NIFTP lesions9. Another debated aspect regards the extent of the Papillary Thyroid Carcinoma-like (PTC-like) nuclear alterations necessary for the diagnosis. In particular, the so-called sprinkling phenomenon (heterogeneity of the PTC-like nuclear features within the same lesion) may be encountered in the routine practice with relevant diagnostic consequences10. Finally, fine needle aspiration (FNA) reporting systems are introducing some changes to the risk of malignancy (ROM) percentages of indeterminate classes, related with the NIFTP advent16. The present paper on a retrospective NIFTP series addresses the highlighted issues and suggests possible scenarios for the clinical-pathological management of this entity.The IV WHO classification of tumours of the endocrine organs in 2017 abolished the old diagnostic category of st 2010, 84 cases with diagnosis of EFVPTC (up to December 2016) and 22 NIFTP (since January 2017 to December 2018) were extracted. The original formalin-fixed paraffin-embedded (FFPE) and haematoxylin and eosin stained histological sections were re-evaluated according to the classification criteria proposed by Nikiforov et al. then introduced into the WHO classification of tumours of the endocrine organs of 2017, 4th edition, by a panel of expert thyroid pathologists 2. In particular, the following parameters were used as inclusion criteria.Presence of capsule, thin or thick, or clear demarcation of the lesion with respect to the surrounding parenchyma and absence of vascular or capsular invasion Fig. ;Figure 1Follicular growth pattern, with absence of papillae, absence of psammomatous bodies, solid/trabecular/insular growth pattern <30%, absence of necrosis and low mitotic index (less than 3 mitoses per 10 high power fields);2.PTC-like nuclear characteristics, with nuclear score (NS) of 2 or 3 Fig. . The nucNegative immunohistochemical BRAF ve staining  with confirmation of the wild type BRAF status by reverse hybrid allele specific oligonucleotide hybridization .From a retrospective series of thyroid surgical specimens (N = 1470), recruited at the Department of Pathology, San Gerardo Hospital, University of Milano-Bicocca, Monza, Italy since January 1incidental tumor; single/multifocal lesion; NIFTP showing “sprinkling” phenomenon. NIFTP was considered “multifocal” if two or more lesion foci were present in the same thyroid lobe or in both lobes. In the case of multifocal lesions, the maximum diameter was evaluated taking into account the lesion with the largest diameter. NIFTP was considered incidental if the preoperative fine needle aspiration (FNA) was performed on a different target.For the final clinical-pathological characterisation of the NIFTP series, the following parameters were recorded: sex of the patient; age at diagnosis; maximum tumor diameter; presence/absence of lymph node metastasis at presentation; mean, median and range of follow-up in years; uninodular/multinodular background ; 20. To confirm the representativity of the series, the incidence of the different diagnostic class was compared to a standard, expected, distribution taken from the study of Bizzarro et al.21 and to a target distribution of the international review by Bongiovanni et al.16. The cytological NIFTP NS was also calculated in blind from the histological results.The original cytological preparations that referred to the preoperative FNAs were retrieved. Traditional Pap-stained smears were diagnosed according to the 5-tiered 2014 Italian SIAPEC-IAP classification system, which is comparable to UK Royal College of Pathology system Fig. 17–20. ToStatistical analyses were performed using the open-source R software v. 3.5.0. A linear regression approach was used to evaluate the impact of diameters on an explanatory variable. To compare the evaluated NS on cytological and histological samples, and to compare the diagnostic classes and distribution of our data with an italian cohort population and a target distribution of an international review, the exact Fisher test was used. For each test, the level of significance was set equal to 0.05.All methods of the study were carried out in accordance with relevant guidelines and regulations; the protocol was approved by a ASST Monza Ethical Board . Appropriate informed consent was obtained from all subjects of the study.invasive EFVPTC due to capsular invasion being clearly present;17 cases were reclassified as 10 cases were reclassified as well-differentiated tumours of uncertain malignant potential  because capsular /vascular invasion was doubtful;22;4 cases showed focal papillaeFrom the EFVPTC group, 7 cases were excluded due to the unavailability of the BRAF status. Of the remaining 77 cases:24, in NIFTP Nikiforov’s study24 and in our series. The average age of the patients included in our series was approximately 10 years greater than that of the Nikiforov series, whilst it is slightly lower than the average age of the general population with a thyroid nodule. The average size of our NIFTPs does not differ from the general population, whilst it is lower than observed in Nikiforov’s series in which it can be noted that lesions smaller than 1 cm in diameter were not considered. Lymph node metastasis at presentation was absent in all cases 21  were reclassified as NIFTP due to satisfying all the required diagnostic criteria. Therefore, the study consisted of a group of 68 NIFTPs in a 9 year-long period . The average age at diagnosis was 55 years range 21–81 years). The average diameter of the NIFTP at diagnosis was 1.7 cm . In 28 out 68 cases (41%), the diameter was <1 cm; while in 4 patients >4 cm.Fig. . The FNA–81 yearscytological NS was calculated in 33 out of the 43 cases in which the FNA was performed appropriately on target because in 10 cases the morphological re-evaluation of the original slides was not available .non-invasive EFVPTC, the word “carcinoma” included in its name has nevertheless led clinicians to perform an aggressive treatment  for many years2. In 2016, an international group of pathologists proposed that the term non-invasive EFVPTC should be replaced with NIFTP, outlining the morphological inclusion criteria necessary to make this diagnosis2. The new terminology has been accepted and introduced in the 2017 WHO Classification of tumours of the endocrine organs1. In our study, we analysed a retrospective cohort of patients with a previously established diagnosis of EFVPTC and patients recruited after 2016 with a diagnosis of NIFTP. A team of thyroid-committed pathologists performed the morphological revision of the histological series to obtain a final homogeneous group of 68 cases. This was followed by the correlation with the preoperative cytological SIAPEC-IAP diagnostic class and the pathological re-evaluation of the original FNAs whenever available.Despite the indolent nature of the et al. that focused on a PTC population that contained 287 cased observed in an 8 year-long period, in the same routine practice, the follicular variant of PTC accounted for the 40% of the cases (115 out of 287)25. Due to the clearcut evidence of capsular invasion, the former carcinoma diagnosis was confirmed in a significant rate of patients (22%), while 10 out of 77 EFVPTCs were reclassified as WDT-UMP because capsular or vascular invasion was doubtful. In those situations, although the application of the morphological criteria was rigorous, doubts persisted especially with respect to the architectural criterion “complete capsule /distinct separation of the lesion from the surrounding parenchyma” because of initial/incomplete penetration phenomena (mushrooms). The board reconfirmed that a correct sampling of the lesion is essential for a complete morphological evaluation along with a scrupulous examination of the tumour-capsule interface/surrounding-tissue interface, such as the protocol proposed by Seethala et al.5. The diagnosis of NIFTP in our study occurred in a prevalent female population (74%) with an average age of 55 years, 10 years older than the multicentric series of Nikiforov’s that enrolled cases with diameter larger than 1 cm and were generally unifocal2. Effectively, these characteristics are related with an earlier age of diagnosis; moreover, the reference study on NIFTP started from second-level international institutions that selectively enroll neoplastic patients for the surgical treatment with higher aggressiveness or higher tumour-stage. On the other hand, our working group planned a monocentric study from a first-level general hospital that is primarily focused on benign thyroid pathology in patients with a more advanced age. In particular, a direct consequence was the unexpected high rate of not-solitary NIFTPs (60.1%). This percentage differs from other previously reported data that associated a NIFTP diagnosis to a “single nodule” clinical presentation; however more recent papers revealed more similar percentages with goiter or chronic pseudonodular lymphocitic thyroiditis as concurrent diseases8. We also recalled that a significant proportion of them were incidental (29% of multinodular cases) and a significant fraction subcentimetric (41%). The treatment of incidental thyroid tumours is still debated: some authors suggest a conservative surgical approach while Maturo et al. suggest total thyroidectomy because these tumours are commonly multifocal and/or bilateral and show occult lymph node metastasis27. In their study, Gartland and Lubitz reported that lobectomy and total thyroidectomy yield comparable oncologic outcomes for PTC measuring 1 to 4 cm, including cases of NIFTP30. Concerning the behaviour of the so-called micro-NIFTP (with a diameter of less than 1 cm), the literature showed a morphology and an indolent behavior very similar to that of the larger NIFTPs33. Micro-NIFTPs may occur as incidental findings in thyroidectomies performed for struma or other pathological conditions. On the contrary, for very-large NIFTPs (>4 cm) it was questioned whether the only surgical treatment without completion with radioiodine and TSH suppression therapy was a possible option, since the American Thyroid association guidelines suggests the combination of radioiodine therapy and surgical treatment34. Xu et al. reported the absence of disease recurrence in 37 patients with NIFTP over 4 cm in size that were not treated with radioiodine, and this result was confirmed by a study by Rosario et al.35. Therefore, NIFTPs of diameter greater than 4 cm are also characterised by having an indolent course after surgical treatment alone, which can therefore be the treatment of choice without the need to add a therapy with radioiodine and/or suppression of TSH. In our experience, we didn’t observe any recurrence or disease progression in both pre-2016 radioiodine-treated pT2/pT3 EFVPTCs and post-2016 radioiodine-free NIFTPs.Among the original EFVPTCs, NIFTPs accounted for 59.7% of cases. In a previous study by Pagni incidentalomas accounted for 45.2%25. Accidental diagnoses were more associated with size and a multinodular clinical presentation; both small (difficult to detect) and large  nodules were possible pitfalls25. In wide lesions (>4 cm) FNA- sampling only occurs in a limited area of the tumour, whilst the classical NIFTP diagnostic nuclear alterations may be present only in partial areas (sprinkling). In the pre-NIFTP era, Vanzati et al. have described cases of Follicular Variant of PTC (FVPTC) that, although at a lower magnification might appear as follicular adenomas (FA) or hyperplastic nodules, at high magnification presented microfollicles with PTC nuclear characteristics10. These modifications could be correlated with a genetic multifocality that is developing from a hyperplastic nodule or from a FA10. Compared to the multifocality parameter, 14.7% of our NIFTPs were plurifocal, as recently described8; 70% of these were bilateral. Multifocal NIFTPs are similar in appearance to multifocal classical PTC but, whilst some multifocal PTCs may derive from an intrathyroid lymphatic diffusion, this could not be true for NIFTP cases given that, by definition, they cannot exhibit lymphatic invasion  category (50%) and the second most frequent diagnostic class was suspicious . The remaining cases were distributed in the benign and malignant diagnostic categories, with a slight prevalence of the benign category compared to malignancy (14% Vs 5%).The more recent updating of the italian SIAPEC system for reporting thyroid pathology, encourage to use the sub-category Tir 3b with reference to NS2/3 when referring to the possibility of a NIFTP on cytological material 22. This would facilitate appropriate programming of surgical therapy. It should be noted that the percentages of NIFTP distribution within the indeterminate cytological diagnostic categories reported in the various literature studies are very variable: from 13% to 57% for the atypical cells of undetermined significance -AUS- /follicular lesions of undetermined significance -FLUS- category of the Bethesda System for Reporting Thyroid Cytopathology, from 13% to 82% for the follicular neoplasms (SFN/FN) category and from 13% to 35% for the suspicious of malignancy37. This variability is attributable to several factors, such as the lack of safe cytological criteria with reliability and high inter/intra-observer reproducibility, the lack of a defined diagnostic class in which to insert the NIFTP and the number of NIFTPs that are diagnosed at an institution. In fact, the comparison of our case study with that of another Italian centre that took into account a similar number of cases (43 versus 37), and with the review of Bongiovanni et al., did not show statistically significant differences 21. In this sense, there is a general agreement that it is not possible (nor it is desirable) to diagnose NIFTP on FNAs. The introduction of a semi-quantitative parameter, such as the NS may only allow to propose a suspicious of NIFTP on cytological preparations. In our series the number of cases with NS 2–3 were lower on cytological smears, since, in histology, a NS 1 is excluded by definition . Subsequently, NIFTP are frequently allocated to the indeterminate for malignancy cytological class (50%) with a surgical approach being selected, that seems to be justified by the NS trend. Brandler et al. compared the cytological characteristics of FAs, NIFTPs and PTCs on cytological smears, highlighting how, at this level, NIFTPs show characteristics in common with both the FA (microfollicular pattern) and the PTC , that are potentially indicative for this lesion if considered contemporaneously38. However, the diagnosis of NIFTP can currently only occur on histological sections because of the need for a scrupulous evaluation of the tumour capsule to rule out its invasion39.Comparing cases that had preoperative FNA performed on target lesions Vs incidental NIFTPs, diameters of these two subgroups showed a statistically significant difference . In a previous study of our group that investigated the problem of incidental diagnoses in a PTCs series, the frequency of sprinkling phenomenon should be considered carefully. Finally, although a NIFTP diagnosis may be incidental , cytopathological criteria such as the NS evaluation may help the pathologists in including NIFTP in the differential diagnosis of indeterminate cases in the preoperative setting and to more appropriately select the correct treatment.The NIFTP era is changing the approach to thyroid histopathology; physicians should envision a NIFTP diagnosis not only for solitary nodules, having important consequences both in the ecographic triage and in the pathological examination. Multifocality and bilaterality are part of the spectrum of NIFTPs, that can arise in a multinodular background with variable sizes from microscopic lesions to very large ones. In this sense, a close monitoring of the contralateral lobe is justified in patients that are not submitted to total thyroidectomy. Moreover, the heterogeneity of PTC-like nuclear features is a significant factor that may affect histopathology. The possible coexistence of two lesions and the so-called


Endometriosis is defined by the presence of endometrial-like tissue (lesions) outside the uterus, commonly on the pelvic peritoneum. It affects 6–10% of women and is associated with debilitating pelvic pain. Current management options are often unsatisfactory. Omega-3 polyunsaturated fatty acids (O-PUFA) have the potential to reduce the painful symptoms associated with endometriosis, reduce lesion size, preserve the patient’s ability to conceive, and have minimal side effects. We performed a two-arm, parallel double-blinded randomised controlled trial to inform the planning of a future multicentre randomised controlled trial to evaluate the efficacy of O-PUFA for endometriosis-associated pain.The primary objectives of the trial were to assess recruitment and retention rates. The secondary objectives were to determine the acceptability to women of the proposed methods of recruitment, randomisation, treatments and questionnaires, to estimate the variability in the proposed primary endpoints to inform the sample size calculation and to refine the research methodology for the future definitive trial.We recruited women with endometriosis from June 2016 to June 2017 and randomised them to eight weeks of treatment with O-PUFA or olive oil. Pain scores and quality of life questionnaires were collected at baseline and eight weeks. We calculated the proportion of eligible women randomised, and of randomised participants who were followed up to eight weeks. Acceptability questionnaires were used to evaluate women’s experiences of the trial.The proportion of eligible participants who were randomised was 45.2% (33/73) and 81.8% (27/33) completed the study. The majority of participants described their overall trial experience favourably and there were no adverse events in either group.Our pilot trial supports the feasibility of a future larger trial to definitively evaluate the efficacy of O-PUFA for endometriosis-associated pain.ISRCTN44202346).The trial was registered on the ISRCTN registry (registration number  Endometriosis is a chronic oestrogen-dependent inflammatory condition that affects around 176 million women worldwide . It is dOral Omega-3 polyunsaturated fatty acids   have potO-PUFAs play a role in the regulation of prostaglandins and cytokines, factors critical to endometriosis, by competing with Omega-6 PUFA to produce anti-inflammatory lipid mediators . ImportaIn a rat model of endometriosis, O-PUFA reduced lesion size and local prostaglandin and cytokine production . AdminisA large, prospective cohort study investigated dietary exposure and endometriosis risk in women, and concluded that increasing O-PUFA intake might reduce the risk of endometriosis . A dietaWe believe that it is important to determine the true value of O-PUFA for the control of, or potential elimination of, endometriosis-associated pain. A definitive evaluation of the efficacy of O-PUFA in the management of endometriosis-associated pain will require a large, multicentre randomised controlled trial (RCT). We therefore performed a two-arm, parallel double-blind randomised controlled pilot trial to inform the planning of a future multicentre RCT to evaluate the efficacy of O-PUFA for endometriosis-associated pain.th June 2016 to 16th March 2017. Data Collection was completed on 7th June 2017 Endometriosis diagnosed macroscopically by laparoscopy (performed at least two weeks prior to enrolment)A numerical rating average ‘worst’ pain score (NRS) of greater than or equal (≥) to four (see below for details).Women were excluded if they had one or more of the following:Unable to take/allergic to fish/O-PUFA/peanuts/soyabeanInsulin-dependent diabetesPregnancyTaking anticoagulantsBreast feedingUnable to give informed consentThe aim of this pilot was to establish feasibility of a future trial—it was therefore designed primarily to explore recruitment and retention rates. To achieve this, the aim was to recruit as many women as possible over a 12-month period. Based on estimates of the number of eligible patients, we postulated that we would recruit four to five patients per month (and recruit 40 patients in 12 months). Based on a recruitment rate of 50%, we estimated that 40 participants would give a 95% confidence interval (CI) of 39–61% .Eligibility for randomisation was based on the worst of four weekly numerical rating scale  pain scores collected by text messaging (‘run-in phase’). A score of four out of ten in two or more weeks was required. Texts were sent to the woman’s mobile phone, asking about average and worst pain, respectively, and the woman was asked to reply to the text message with her pain score, rating it from 0 for no pain at all, to 10 being worst pain imaginable. To capture known cyclicity in pain, the texts were sent once per week during the eligibility phase (weeks ‘minus four’ to ‘minus one’). If a woman had an NRS of greater than or equal to ≥ four in two or more of the pre-randomisation run-in weeks of the study, she was considered to be eligible for randomisation.The randomisation scheme was generated using tables, drawn up by an independent statistician, with 1:1 allocation ratio using blocks of four and six in a random pattern. The allocated treatment codes were sealed in consecutively numbered opaque envelopes. When a woman was randomised into the study, she received the treatment indicated in the next available envelope.Participants were randomly assigned to the intervention  twice a day) or a visually identical comparator  for eight weeks. The allocated treatments were taken for eight weeks. O-PUFA capsules have been used clinically in previous trials  at doses of 1000-4000mg daily (in divided doses of 1000mg or 2000mg) . HoweverRecruitment and retention rates.Estimation of the variability of responses to inform the primary outcome and sample size of the future RCT.Acceptability of proposed methods of recruitment, randomisation, treatments and questionnaires.Participants were given the questionnaires below (see published protocol for further details ) to compBrief Pain Inventory (BPI)12-Item Short-Form Health Survey (SF-12)Pain Catastrophizing Questionnaire (PCQ)TMPain DETECTSexual Activity Questionnaire (SAQ)Brief Fatigue Inventory (BFI)General Health Questionnaire-12 (GHQ-12)Work and Productivity ImpairmentQuestionnaire-specific Health Problem Version 2.0 (WPAI-SHP)An additional acceptability questionnaire was completed at the end of the study only. This questionnaire included questions about whether participants believed that they were receiving O-PUFA or olive oil, the acceptability of the allocated medication/ treatment regimens (and compliance) and on the acceptability of completing trial-specific procedures including the questionnaires. All questionnaires were anonymised and self-completed in private.Participants were provided with a treatment diary at the same time as their medication pack was dispensed. All medications other than the trial treatment taken during the treatment phase of the study were recorded in a treatment diary. This included prescription and non-prescription treatments, such as oral analgesics, contraceptives, vitamins, topical preparations and herbal preparations.Participants collected information about adverse events in their treatment diaries. They were also instructed to contact the clinical trial team at any time after consenting to join the trial if they had an event that requires hospitalisation or an event that resulted in persistent or significant disability or incapacity.We kept an anonymised electronic log of women who fulfilled the eligibility criteria, women who were invited to participate in the study, women who were recruited and women who left the trial early. Reasons for non-recruitment  were also recorded. We attempted to collect reasons for non-participation from women who declined to take part after previously providing contact details. During the course of the study, we attempted to document reasons for withdrawal from the study and loss to follow-up.A statistical analysis plan was agreed with the trial statistician  prior to a database lock. Estimates of recruitment and retention were to be calculated with their 95% Confidence Interval (CI). Where the denominator is small (<50), exact binomial confidence intervals will be calculated. Where the denominator is large the Normal approximation will be used. Change from baseline for each of the questionnaires and sub-questions were calculated for each patient, and these changes were compared between treatments using the t-test. Where the change from baseline was not normally distributed, the change was transformed on the log scale, and this was compared between treatments. All tests were by intention-to-treat and two-sided, with a 1% level of significance to make some allowance for multiple testing. Only patients with baseline and follow-up at eight weeks were included. Due to the small sample size, no attempts were made to impute missing data. Statistical analysis was performed using Microsoft Excel (ME) and SPSS software version 23. The number and nature of unanswered questions was recorded and explored.The trial was co-sponsored by the University of Edinburgh and NHS Lothian.The proportion of eligible participants who were randomised over 12 months was 45.2% ) and 81.8% ) completed the study. Seventeen participants were randomised to receive O-PUFA group and 16 participants were randomised to receive olive oil capsules. Examples of reasons for non-participation included ‘trying to conceive’, ‘strict vegetarian and will not accept fish oil, want only olive oil’, and ‘already taken the fish oil without much help’. Six participants were lost to follow-up, three from each treatment group.Supplementary tables  Tables sIn order to calculate the required sample size for a future RCT, we assumed that our primary outcome measure was the BPI severity score. From our results, we estimated a pooled standard deviation (SD) of 2.08. In order to detect a difference of 1 unit on the BPI scale, which is approximately a fall of 20% from the baseline mean, with a power of 80% and a significance level of 5%, we utilised the sample size equation for a difference in continuous data. This requires at least 69 analysable patients per group. Similarly, if we wished to use the interference score as the primary or co-primary outcome, the pooled SD is 2.18 and we would require 76 patients per group. However, we would also need to take loss to follow up into account (retention), and the recruitment rate in order to estimate the number of patients we would need to approach in order to achieve sufficient patients to achieve our primary outcome. A point estimate and 95% CI have been reported for recruitment and retention. As these figures (69 or 76) refer to the number of evaluable patients required, adjustments for retention will be made .The acceptability of the interventions was evaluated by calculating the proportion of participants who attended all visits and completed their treatments: 82.4%  of participants (14/17) who received O-PUFA and 81.3%  of participants (13/16) who received olive oil capsules attended all visits. The majority of participants described their overall trial experience favourably and rated it one or two on a five-point scale from positive to negative. Positive responses were recorded for the recruitment approach used, paperwork burden for participants and the experience of randomisation process of participants. Negative feedback was largely related to the size of the pills as some women experienced discomfort when swallowing the large capsules, time taken to complete the treatment diary and complexity of some of the questionnaires. The trial was not powered to detect difference in outcomes recorded by questionnaires.There was no report of any adverse event from any of the participant in either group.Our pilot trial demonstrates that a future definitive RCT to determine the efficacy of O-PUFA for the treatment of women with endometriosis-associated pain is feasible. Feasibility is supported by the fact that, over a 12 month recruitment period, we were able to randomise 33 women (45.2% of those eligible) to O-PUFA or comparator and obtain completed eight week follow up data in 27 women (81.8%).A recent comprehensive review of the recruitment and retention data of a cohort of 151 RCTs funded and published by the UK NIHR HTA Programme from 2004 to 2016 found that the average retention rate was 89% and the average proportion of eligible patients who are randomised was 70% . Whilst We were pleased to note that analysis of the data regarding the delivery of the trial was generally favourable. The limited negative feedback that we received was largely related to trial medication (large size of capsules) and treatment diary completion. Whilst the delivery of the medication cannot be altered, the other feedback has alerted us to the need to develop the option of a more accessible and user-friendly online treatment diary.The observed trend toward improvement in pelvic pain and quality of life scores in both treatment arms over the eight week intervention points to a strong placebo effect, which would require a much larger sample size, or the use of much smaller  effect sizes, to detect statistically significant differences between treatment groups. This suggests an effect from enhanced clinical interaction and trial participation on pain, psychological and physical well-being, and highlights a need for qualitative research to explore whether women experience a placebo effect which influences their experiences and reporting of pain and hence, potentially, trial outcomes. We are also aware that we used a potentially ‘active’ placebo. Olive oil contains oleocanthal which has been shown to act as a natural anti-inflammatory compound that has a potency and profile strikingly similar to that of ibuprofen . We planIn conclusion, we are now ready to capitalise on our pilot trial and secure funding to perform a definitive large multicentre double-blind randomised controlled trial to determine the efficacy of O-PUFA for the treatment of women with endometriosis-associated pain.S1 Consort checklist(DOC)Click here for additional data file.S1 TableSF-12 version 2 scores range from 0–100, where low scores are bad and high scores are good.(DOCX)Click here for additional data file.S2 TableBPI scores range from 0–10, where low scores are good and high scores are bad.(DOCX)Click here for additional data file.S3 TableBFI scores range from 0–10, where low scores are good and high scores are bad.(DOCX)Click here for additional data file.S4 TableGHQ scores range from 0–12, where higher scores represent higher levels of mental distress.(DOCX)Click here for additional data file.S5 TableWPAIQ scores range from 0–100, where low scores are good and high scores are bad indicating greater impairment and less productivity.(DOCX)Click here for additional data file.S6 Table1 PCQ rumination scores range from 0–16, where low scores are good and high scores are bad. 2 PCQ magnification scores range from 0–12, where low scores are good and high scores are bad. 3 PCQ helplessness scores range from 0–24, where low scores are good and high scores are bad. 4 PCQ total scores range from 0–52, where low scores are good and high scores are bad.(DOCX)Click here for additional data file.S7 Table1 SAQ pleasure scores range from 0–18, where low scores are bad and high scores are good. 2 SAQ discomfort scores range from 0–6, where low scores are bad and high scores are good. 3 SAQ habit scores range from 0–3, where low scores are bad and high scores are good. 4 SAQ tired scores range from 0–3, where low scores are bad and high scores are good.(DOCX)Click here for additional data file.S8 TablePDQ scores range from 0–35, where low scores are good and high scores are bad.(DOCX)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.


Early warning systems (EWS) for river flooding are strategic tools for effective disaster risk management in many world regions. When driven by ensemble Numerical Weather Predictions (NWP), flood EWS can provide skillful streamflow forecasts beyond the monthly time scale in large river basins. Yet, effective flood detection is challenged by accurate estimation of warning thresholds that identify specific hazard levels along the entire river network and forecast horizon. This research describes a novel approach to estimate warning thresholds which retain statistical consistency with the operational forecasts at all lead times. The procedure is developed in the context of the Global Flood Awareness System (GloFAS). A 21-year forecast-consistent dataset is used to derive thresholds with global coverage and forecast range up to six weeks. These are compared with thresholds derived from ERA5, a state of the art atmospheric reanalysis used to run the baseline simulation for the years 1986–2017 and to give a best guess of the present hydrological states. Findings show that the use of constant thresholds for 30-day flood forecasting, as in the current operational GloFAS setup, is consistent throughout the entire forecast range in only 30% to 40% of the river network, depending on the flood return period. Findings show that range-dependent thresholds, of weekly duration, are a more suitable alternative to time-invariant thresholds, as they improve the model consistency as well as the skills in flood monitoring and early warning, particularly over longer forecasting range. GloFAS is designed to forecast large scale river flooding worldwide by detecting river reaches where predicted streamflow has significant probability to exceed model-consistent warning thresholds in a 30-day forecasting horizon . The est-ERA5, the latest generation global atmospheric reanalysis product by ECMWF, has become available in 2018 . Key fea-The approach currently used to produce a continuous meteorological climatology based on reforecasts generates streamflow statistics which are not fully consistent with those of the operational forecasts, hence possibly originating a bias in the resulting flood warning thresholds.-The extension in 2018 of GloFAS products to a 30-day forecast horizon urged the need to investigate whether thresholds derived from short-term forecasts  model version as the real-time ensemble forecasts (ECMWF-ENS), for the past 20 years. Similar to the operational forecasts, reforecasts have horizontal resolutions of 18 km for up to 15-day lead time and 36 km for longer forecast lead times up to 46-day. In this work we used the unperturbed ensemble member  of 6-week reforecasts with daily resolution, initialized once per week in the entire period of availability, coupled with the last year of operational forecasts. The collected dataset thus includes 1218 sets of reforecasts with global coverage, spanning 21 years between 21/11/1996 and 25/12/2017. More in details, each of the 21 years of data is composed by 58 weekly reforecasts starting in mid-November of the previous year, so that each day of each year is ultimately simulated in 6 different reforecasts with lead time between 1 and 6 weeks. As for ERA5, we extracted the same set of atmospheric variables and pre-processed them to obtain daily estimates of evapotranspiration, which were used as input of the hydrological model together with mean surface air temperature and precipitation.2.22 in area) across the globe using the WFDEI  and the mean annual precipitation (MAP), including spatial patterns of the differences between the two datasets. Due to their non-parametric nature, precipitation statistics are key diagnostic indicators that anticipate and explain possible differences between the corresponding hydrological model outputs. In this regard, Pmax is representative of extreme precipitation and is generally well correlated to floods e.g., , while M•While the ERA5 dataset is continuously extended in near-real time and therefore keeps growing in size, ECMWF reforecasts are regenerated every week using the latest IFS model version (which is updated more than once per year), yet only for the most recent 20 years. It follows that this 9-year gap will not be closed in the future unless a longer reference period is chosen at ECMWF for issuing reforecasts.•30 years are a commonly accepted duration for a reference climate, as defined by the World Meteorological Organization, as well as a recommended standard for extreme value analysis e.g., , hence iThe second part of the analysis is performed on flood thresholds based on ERA5 versus those based on reforecasts. To this end, we first ran a 32-year global hydrological simulation forced with ERA5 and extracted discharge time series at all points of the river network. Then, 1,218 sets of 6-week reforecasts, initialized once per week between 21/11/1996 and 25/12/2017, were used as input to run as many hydrological simulations, taking initial conditions from the corresponding date in the ERA5-based run. Such model runs forced by reforecasts include 58 different 6-week simulations per year, starting on the 21 November of the previous year, so that each day of each year is ultimately modeled in six different simulations, with forecast range spanning between 1 and 6 weeks. We applied Extreme Value Distribution (EVD) fitting to the annual maximum discharges extracted from the ERA5-based dataset as well as from the six datasets stemming from reforecasts, after reordering the data by weekly lead time. The first two years of the ERA5-based dataset were discarded to guarantee sufficient model warm-up, leaving 30 years of data for the EVD fitting, while in the case of reforecasts all 21 years of data were used, given that meaningful initial conditions were provided by the ERA5-based run. Such 9-year difference  in the length of the two datasets may produce discontinuities in the related flood thresholds, in case of substantial differences in the statistics of the extremes in those 9 years when reforecasts are not available. In view of the direct implications of this research with the operational runs of GloFAS we have opted to accept this potential discontinuities, for the following reasons:The chosen EVD fitting is based on the Gumbel distribution with the method of L-Moments , an effeIn the remainder, we refer to flood thresholds as all discharge values estimated from analytical extreme value distributions, corresponding to return periods equal or larger than 2 years. In reality, the occurrence of actual flooding depends on the flood protection standards along the river network, which are known to vary considerably from around 2 to several thousand years, depending on the exposure and on the country’s investment capacity . In the 3−1 at week 1, and 5 mm year−1 at week 4, 5, and 6.Statistics of precipitation were calculated over the world’s land area using ERA5  and refoThe map in Differences in the thresholds described above, based on different hydrological simulations, cause bias and hence inappropriate interpretation of ERA5 flood thresholds when they are used in the prediction of threshold exceedances along the forecast range. Such bias can be evaluated by inverting the EVD of reforecasts at each weekly lead-time, using discharge values corresponding to flood thresholds based on the ERA5 EVD fitting. An example for a return period of 20 years is shown in 4Results of our analyses show a complex spatial pattern of sign and magnitude of the differences between thresholds derived from a reanalysis dataset i.e., ERA5) and those derived from a forecast-consistent dataset . Differences do not appear to have clear linear dependence with a number of considered variables, such as return period of the flood thresholds, weekly forecast range, latitude, basin upstream area and mean discharge. Instead the pattern of such differences follows more that of the differences in precipitation statistics see  and 5, h and thosThe findings of this research indicate that range-dependent thresholds should be used and replace time-invariant thresholds to improve the consistency in flood monitoring and early warning, especially over longer forecast ranges. Unlike the constant-thresholds setup, the proposed approach would retain consistency along the entire forecast range with the operational streamflow simulations, which integrate the effect of three IFS model configurations with different spatial resolution. First, the ERA5 atmospheric reanalysis with grid resolution of 31 km, used in the baseline run from which hydrological initial conditions are taken. Second, medium-range forecasts up to 15 days with grid resolution of 18 km, which are primarily governed by atmospheric initial values. Third, extended-range forecasts with ocean–atmosphere coupling from 16 to 46 days with grid resolution of 36 km, where the sea-surface temperature and associated heat and moisture start having non-negligible influence on the atmospheric evolution .Traditionally, flood thresholds used in short-term forecasting and early warning are based on observations or reconstructed extreme value distributions based on long term simulations . On the To the authors’ knowledge, no operational system for medium-range flood early warning uses range-dependent thresholds to detect upcoming severe events. The concept of range-dependent thresholds may initially be difficult to accept for users, as it eliminates the univocal link between discharge thresholds and their probability of occurrences. In other words, any given discharge peak would correspond to a different probability of occurrence depending on when it is forecast in the future. Yet, the proposed approach is more correct and consistent with the data, hence it is likely to improve the estimation of the magnitude of upcoming extreme events over longer forecast ranges.5In this research we have tested a new method to estimate discharge thresholds for medium-range flood forecasting, for potential use in the Global Flood Awareness System. We have provided evidence that flood thresholds for medium-range forecasting can take on significantly different values from thresholds derived from the reference baseline run forced by ERA5 data, used to initialize the forecasts. In addition, thresholds often vary throughout consecutive weekly forecast ranges with a complex pattern of change, mostly driven by differences in the atmospheric datasets used as forcing to the hydrological model. Therefore, we propose a modified framework for the early detection of floods, based on range-dependent flood thresholds, which are able to compensate for forecast drifts in the extreme values, particularly useful over long ranges. Upcoming work, ahead of the operational implementation, will involve extensive testing and skill evaluation of the operational ensemble forecasts, to assess the limits of predictability of the system and compare the system skills to the current operational version. This represents a key step in the evolution of GloFAS which may ultimately lead to the extension from the current 30-day to a 6-week forecasting range. It is worth noting that flood thresholds based on reforecasts must be updated regularly as new IFS cycles are released, to retain consistency between streamflow predictions and warning thresholds and improve the detection of severe floods along the entire forecast horizon.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Since the dissemination of individual-level genomic information is strictly controlled, it will be useful to construct population-level allele frequency panels with easy-to-use interfaces. In the Tohoku Medical Megabank Project, we sequenced nearly 4000 individuals from a Japanese population and constructed an allele frequency panel of 3552 individuals after removing related samples. The panel is called the 3.5KJPNv2. It was constructed by using a standard pipeline including the 1KGP and gnomAD algorithms to reduce technical biases and to allow comparisons to other populations. Our database is the first large-scale panel providing the frequencies of variants present on the X chromosome and on the mitochondria in the Japanese population. All the data are available on our original database at  A new database provides information on the frequency of genetic variations within 3552 Japanese individuals, and facilitates comparisons with other populations. The reference panel, constructed by Kengo Kinoshita of Tohoku University, Sendai, and colleagues in Japan is also the first large-scale database to provide genetic variation frequency information on the X chromosome and mitochondrial DNA in the Japanese population. The methods used to sequence the genetic data are similar to those used in other large databases, allowing comparisons with other populations. The population size and methods used to compile the database overcome limitations in previous Japanese reference panels. This and similar databases that catalog genetic variations within populations can improve efforts towards personalizing healthcare and contribute to the study of human population genetics. The database is publicly available online. Since individual-level genomic information is classified as personal data, access to it is strictly controlled. Therefore, allele frequencies have been published in the form of reference panels4 to clarify population-level differences.It is of fundamental importance to catalog the genetic variation in a general population to realize personalized healthcare and personalized medicine. Since different populations show divergent genetic variations, population-specific analyses based on large cohorts are required6 by the Tohoku Medical Megabank (TMM) Project7. A subsequent version was published in 2016 that included 2049 individuals8, and one distributed in 2017 included 3554 individuals. These reference panels were used for various other projects. For example, the IRUD project (Japan’s Initiative on Rare and Undiagnosed Diseases9) used the reference panel to reduce the discovery of false positive single nucleotide variants (SNVs) during the exome analyses of undiagnosed patients. In another project, CYP SNVs included in the reference panels were selected and analyzed systematically for their effect on drug metabolism12. As seen from these examples, the previous versions of the reference panels worked well; however, there are some limitations. One of the limitations was the lack of multiallelic sites as predicted by the infinite site model13. Following standard practice, multiallelic sites were removed from the previous frequency panels, which resulted in a lack of high frequency alleles in the reference panel. Since human genomes are now considered to have accrued a large number of mutations due to a rapid expansion of the population size, the analysis of multiallelic sites should prove interesting from the perspective of human population genetics. However, we hope that this will be described elsewhere. Another limitation of our analysis was the gradual obsolescence of the 1KJPN pipeline. When we constructed 1KJPN, several analysis pipelines were used, but now large-scale analyses, such as the 1000 Genomes Project (1KGP)14 and the genome Aggregation Database (gnomAD)15, use virtually equivalent protocols for variant calling. The difference in pipelines can make it difficult to compare the allele frequencies of different populations. Thus, we decided to perform a reanalysis of variant calls and construct a new reference panel for the Japanese population. In this paper, we will describe some details of our new panel construction by using a pipeline similar to the 1KGP and gnomAD pipelines. We also report the variant frequencies of the X chromosome and those of mitochondria, which makes this the first such report to do so on a large scale for the Japanese population.Accordingly, a large allele frequency reference panel based on the genomes of 1070 Japanese individuals was first published in 201416. Finally, 181 samples came from individuals recruited by the National Hospital Organization Nagasaki Medical Center. Written informed consent was obtained from all the participants.Data were obtained from 3552 individuals in Japan [Supplementary Table Participants’ declared age and their sex as determined from X and Y chromosome sequencing are presented in Supplementary Table 5. Briefly, genomic DNA extracted from the buffy coat was fragmented by sonication to an average target size of 550 bp. After library quantification using the quantitative MiSeq method17, sequencing was performed on the HiSeq 2500 system (Illumina). The TruSeq Rapid PE Cluster V1 and SBS Kits (1 sample per flowcell) and the TruSeq Rapid PE Cluster Kit V2 and SBS Kit (2 samples per flowcell) were used for 162-bp paired-end (162PE) and 259-bp paired-end (259PE) protocols, respectively.Library preparation and sequencing were performed as described earlier with minor modifications14 and gnomAD15, adopted the same workflow. Although we used an original re-sequencing workflow for 1KJPN5, 2KJPN, and 3.5KJPNv1, we decided to use a more common pipeline to build 3.5KJPNv2 to allow for comparisons of allele frequencies between different populations. We customized three steps in the GATK Best Practices workflow: (1) the choice of the reference genome, (2) the use of base quality score recalibration (BQSR), and (3) the joint genotyping step.We employed a workflow known as the GATK Best Practices workflow, which is becoming the standard procedure globally for whole-genome re-sequencing analysis. Several recent large-scale genome analyses, such as the 1000 Genomes project14.The FASTQ files of each sample were aligned to a set derived from the human reference genome (GRCh37) that contains the revised Cambridge Reference Sequence (rCRS), unlocalized/unplaced contigs, human gammaherpesvirus 4 sequence (NC_007605), and a decoy sequence (hs37d5). Two pseudoautosomal regions (PAR1 and PAR2) on the Y chromosome are masked as N. This reference genome sequence is referred to as hs37d5.fa and is the same reference sequence as that used in the 1000 Genomes project Phase 218 version 0.7.12 and sorted by their coordinates using the SortSam program included in Picard19 version 2.10.6. BWA-MEM was run at “-K 10000000”, in addition to the default options to reduce any differences when we performed calculations with multiple threads. Thereafter, duplicate PCR reads were removed by using the MarkDuplicates command in Picard. The output was written into a BAM (Binary Alignment/Map) format file. Such files will be referred to as the baseline BAM files in this study.FASTQ files were aligned with hs37d5.fa using BWA-MEMAlthough the GATK Best Practices workflow recommends that the BQSR step be carried out after the mapping, we did not do so for the following reasons. Before analyzing our full dataset of 3,552 samples, we evaluated the effect of BQSR on our dataset. For this purpose, we randomly selected 100 samples from our dataset and re-sequenced them using BQSR as described in GATK Best Practices. We also performed re-sequencing without the BQSR step. Finally, we performed SNP array analyses on both sets of 100 samples. In other words, we checked concordance among two kinds of genotyping results: (i) genotyping results obtained after the incorporation of BQSR and (ii) results obtained without BQSR.Variant calls for each baseline BAM file were made by using the HaplotypeCaller program included in GATK version 3.7, resulting in the generation of genomic VCF (GVCF) files. These were used to perform multisample joint genotyping in the following steps. After generation of GVCFs for all samples, joint genotyping was performed using the GenotypeGVCFs program included GATK version 3.7. Joint genotyping of large samples usually takes a large amount of computational time, but it was not feasible for us to perform joint genotyping of 3500 samples at the same time. To overcome this difficulty, we divided the autosomes into 3 Mb chunks and performed joint genotyping of each chunk across all the samples. After all the chunks were processed, they were concatenated to produce the whole autosome. To avoid edge effects that may be introduced by chunk splitting, we made overlaps of 1 kb between adjacent chunks and checked the concordance of variant calls in the overlapping regions. If discordant variant calls were found in the overlapping regions, we removed them. In total, we found 470 discordant variants on the autosomes, and they were not included in the results.After merging the chunks and checking their concordance, we applied the Variant Quality Score Recalibration (VQSR) filter. The GATK resource bundle was used as known site information for the VQSR step. We opted for SNV filtration based on the following VQSR scores: QD , MQ , MQRankSum , ReadPosRankSum , FS (strand bias estimated using Fisher’s exact test), SOR (strand bias estimated using the symmetric odds ratio test), DP , and InbreedingCoeff (likelihood-based test for the inbreeding among samples). For INDEL filtration, we excluded the MQ and MQRankSum scores from the preceding list. Finally, we collected the SNVs and INDELs that passed the VQSR step. The numbers of SNVs and INDELs found on the autosomes and the X chromosome are shown in Table 22. To check the pseudoautosomal nature of XTR, we observed heterozygosity of the X chromosome for male samples by SNP array analyses. We found significantly higher heterozygosity in three regions, including the XTR, in the Japanese population  on the X and Y chromosomes that have similar sequences. Thus, variant calling for these regions should be performed with different ploidy settings according to the sex of each sample. The sex of each sample was determined using PLINK 1.9b05). Since the female samples have two X chromosomes, we treated their reads as having originated from the diploid genome and performed variant calling for them just as we did for the autosomes. For the male samples that have one X chromosome and one Y chromosome, we performed variant calling for PAR and non-PAR reads using different ploidy settings. We treated the PAR reads as having originated from the diploid genome, and non-PAR reads as having originated from the haploid genome. The existence of at least two PARs, called PAR1 and PAR2, has been accepted by most international genome projects. However, there is some discussion about the existence of another similar pseudoautosomal region called the X-transposed region (XTR).9b05. SiWe constructed X chromosome allele frequency panels for the two ploidy settings by generating the GVCF files and by joint genotyping as described before. We also used the GATK resource bundle as known site information for the VQSR step.Since the mitochondrial DNA is circular, we analyzed data originating from the mitochondrial genomes by converting the circular DNA sequence into two linear DNA sequences by inserting a breakpoint within it. One of the linear DNA sequences was the same as that used by rCRS, while the other was generated by shifting the breakpoint by 10,000 bases. The shift was introduced to avoid any edge effects at the breakpoint on the variant calls. First, we extracted unmapped reads and reads aligned onto the mitochondrial genome from the baseline BAM files. Thereafter, we realigned them onto the two linear mitochondrial genomes using BWA-MEM version 0.7.12. Afterwards, we used GATK HaplotypeCaller version 3.7 to detect the variants. Mitochondrial genomes are known to be heteroplasmic. However, we ignored this consideration while building the current version of the variant panel because our focus was on determining the major variants in the first step of analyses for the Japanese population. Therefore, we treated the mitochondrial DNA as haploid when we performed the variant calls.20 version 4.3t while using GENCODE21 release 28. GENCODE release 28 is not provided as a prebuilt database for snpEff. Therefore, we manually converted the GTF file downloaded from the GENCODE website to a snpEff database according to the instructions given in the snpEff online manual. In addition to GENCODE gene annotations, rs numbers of each variant were resolved by SnpSift22 version 4.3t while using dbSNP23 release 150.Variants found from 3,552 individuals were annotated by snpEffFrom the baseline BAM files of each sample, we collected mean depth metrics for each base. We refer to the dataset as the “Genome Accessibility” dataset. Mean depth metrics helps us to identify genome regions where alignment of short reads could not be performed well, mainly due to complexities in the genome sequences. The genome accessibility dataset could be used for two main cases: (i) filtering of variants by depth information and (ii) supporting evidence of the absence of variants in regions when no variants are discovered in the region.24 1.6 was used to retrieve depth information from each BAM file, and an in-house Python script was used to obtain final mean metrics.For this purpose, we calculated the mean depth from 500 samples (162PE protocol) and 445 samples (259PE protocol) for each base. To clarify regions where alignment of short reads could not be performed, we calculated mean depth by using all reads aligned to reference genome regardless of mapping quality (MAPQ) of reads and by using the reads with MAPQ >= 20. During the alignment step, sequence reads mapped onto multiple regions on the reference genome will be assigned a low MAPQ value , and the difference in mean depth values obtained by the two calculations will reflect whether multimaps of reads are likely to occur. SAMtoolsWe performed whole-genome re-sequencing analyses as described in the Methods section and collected the SNVs and INDELs that passed the VQSR step. The numbers of SNVs and INDELs found on the autosomes and the X chromosome are summarized in Table 25 for 1036 samples. As a result, we did not observe significant differences in concordance between genotyping results obtained after the incorporation of BQSR and results obtained without BQSR, according to the markers present in the SNP array , by Japonica Array version 2 (420 samples), by Illumina Omini2.5 (3399 samples) or by Illumina Omni Express Exome (408 samples). In short, all of the samples included in 3.5KJPNv2 had both whole-genome sequence data and genotyping results by some SNP arrays. We observed that most samples have a high concordance (>=99.0%). Supplementary Fig. http://www.repeatmasker.org/). A previous study26 suggested that the complexity of the genome sequence can affect the accuracy of short-read aligners, and thus it would be difficult to perform short-read sequencing analyses. Regions (ii) and (iii) contained approximately 20 SNVs in total, all of which were located around the HLA region (HLA-DQA1 gene and HLA-DQB1 gene). Again, these are known to be difficult regions for short-read analyses due to their high diversity28. In both cases, we think that most of the outliers resulted because of poor alignment of the short reads. Some of these outliers may be resolved upon reanalysis with next-generation long-read sequencers.We compared the allele frequencies obtained by us as part of 3.5KJPNv2 to those of the whole East Asian population (EAS) obtained by the genome aggregation database gnomAD project. Here, we have shown the results for chromosome 6. As shown in Fig. 29 consisting of 2000 Japanese individuals, independent of our samples Fig. We also compared our reference panel with the RIKEN reference panelWe also checked the distribution of Ts/Tv metrics for 3.5KJPNv2 and for the 1000 Genomes for each chromosome. In Fig. 5.To observe the population structure in 3.5KJPNv2, we created a PCA plot for individuals included in 3.5KJPNv2 and the East Asian populations included in the 1000 Genomes Project. For the East Asian populations, we used the 1000 Genomes Project Phase 3 genotype data, available in the VCF format, for the following populations: CHB , JPT , CHS (Southern Han Chinese), CDX , and KHV . We obtained a combined genotype dataset by converting the genotype dataset of 3.5KJPNv2 and the dataset of East Asian populations by PLINK. During the conversion, variants with MAF < 0.01 or HWE < 1.0e-5 were removed. The two resultant BED files were combined on commonly existing variants. For the combined dataset, we removed variants with MAF < 0.05, HWE < 0.05, or those with a missing rate > 0.01. PCA was applied after LD pruning for the remaining variants with PLINK by selecting the “--indep-pairwise 200 4 0.1” option. We used the same PLINK parameters as Nagasaki et alIn the PCA plot Fig. 30. From jMorp release 201806 , genomic variant data have been added, and the latest version 201902  is where allele frequencies of all the genomic variants can be examined through the web interface. Adding genomic variant information further enhances multilayer omics analysis. Details of the web usage are described in the tutorial section of the web page at https://jmorp.megabank.tohoku.ac.jp/201902/help/tutorial.3.5KJPNv2 is distributed from jMorp (Japanese Multi Omics Reference Panel) with a web interface. jMorp was originally published as a database of metabolites and proteins in plasma obtained from volunteers in ToMMo, which was already described by Tadaka et alhttps://humandbs.biosciencedbc.jp/en/) with accession code hum0015.v3 by the National Bioscience Database Center (NBDC) of the Japan Science and Technology Agency (JST) to ensure accessibility, preservation and stability of the 3.5KJPNv2 datasets.3.5KJPNv2 is available at the jMorp website with web interface, and the raw data in VCF  format was also registered at the NBDC Human Database (Individual’s sequence data and genotyping results from which allele frequency dataset is constructed and validated are available upon request after approval of the Ethical Committee and the Materials and Information Distribution Review Committee of Tohoku Medical Megabank Organization.Supplemental Material