It is comprised of 23 peer-reviewed articles, including original research, reviews, opinion pieces, and case reports.The precise etiopathophysiology of most severe mental disorders, particularly schizophrenia spectrum and affective spectrum disorders, remains unclear but is thought to be a product of the intricate interplay among a number of risk factors. These include interactions between genetic, developmental and environmental factors, such as infections. Indeed, the evidence from many epidemiological studies carried out in different countries, especially those from Denmark, strongly associates both early and the frequency and severity of life-time infections with an increased risk of psychosis in later life, possibly through diverse immunological mechanisms. More recently, the continued discovery of antibodies against various neuronal cell surface proteins, such as anti-N-methyl-d-aspartate receptor (NMDAR) and gamma-aminobutyric acid beta receptor (GABAßR), directly links central nervous system (CNS) autoimmunity with dysregulation of glutamatergic and GABAergic neurotransmitter pathways to the neurobiology of acute psychosis in individuals with autoimmune encephalitis (AE). These findings, along with the rapidly emerging evidence of many other immunological abnormalities in most severe mental disorders, have made it clear that the nexus of psychiatry, neurology, and neuroimmunology represent a fruitful coalescence in understanding the pathogenesis of psychiatric disease. The interactive presentations on the link between immune dysregulation and various mental disorders during the 13 and 14th Psychoimmunology expert meetings in 2016 and 2018 at Ulm University in Germany  analysis in the standard diagnostic workup.Al-Diwani et al. Oxford UK, proposed using a syndrome-level taxonomy for isolated psychiatric syndromes associated with neuronal antibodies that can overlap with autoimmune encephalitis presenting with neuropsychiatric presentations; synaptic and neuronal autoantibody-associated psychiatric syndromes or “SNAps.” This pragmatic approach can serve as a reminder to consider early autoantibody screening for diagnosing SNAps, potentially permitting early diagnosis and management of these rare forms of autoimmune encephalitis that can mimic intractable severe mental disorders. Additional research is needed to investigate the therapeutic implications of SNAps and validate their presence as distinct clinical entities.Ellul et al. provided a thorough review of the clinical evidence supporting the existence of autoimmune psychosis as a distinct clinical entity among individuals with new-onset psychosis, highlighting the diagnostic challenges and therapeutic implications associated with this entity. The authors reviewed the clinical and biological features of autoimmune psychosis, including peripheral biomarkers of autoimmune dysfunction from dysbiosis to autoantibodies such as NMDAR antibodies, discussing the interplay between environmental and genetic factors.Najjar et al. summarized the clinical and experimental findings suggestive of the potential contribution of neurovascular unit dysfunction and blood brain barrier hyperpermeability to the neurobiology of schizophrenia. These include neuroinflammation- and oxidative stress-related neurovascular changes including endothelial dysfunction, leading to increased cross interactions between brain innate and peripheral adaptic immunities, thereby perpetuating harmful inflammatory and immune responses in the CNS. The authors concluded that these findings provide additional support for the mild encephalitis (ME) hypothesis of schizophrenia.Borroto-Escuela et al. reviewed the molecular data supporting the volume transmission hypothesis with specific relevance to NMDAR and its pathological allosteric receptor-receptor interactions that can lead to increased internalization and decreased NMDAR signaling. Combined with the triplet puzzle theory it is suggested that mild neuroinflammation is associated with formation of D2R heteromers, which in turn can enhance D2R promoter signaling, leading to schizophrenia-like symptoms.de Haan et al. reviewed the chronic self-sustaining immunological and inflammatory changes associated with neurodegenerative and psychiatric disorders. The findings link the cascading effects of neuroinflammation and autoimmunity to disturbances of cholinergic, dopaminergic, glutamatergic, histaminic, and serotonergic functions, relevant to the pathophysiology of neuropsychiatric disturbances associated with neurodegenerative and psychiatric disorders. Thus, these related neuropsychiatric disorders might benefit from novel immunotherapies.De Picker et al. presented a meta-review of recent quantitative systematic reviews and meta-analyses from 2010 to 2017 investigating the functional relevance of microglial activation-related immune signaling and brain plasticity to the pathophysiology of acute psychosis and schizophrenia. This review included data derived from translocator protein (TSPO) positron emission tomography, CSF analysis, and post-mortem studies, coupled with the results of clinical trials pertaining to the efficacy of various immuno-modulatory agents. They suggested that microglial activation and its downstream effects on the immune processes and neuroplasticity can influence the clinical presentation and the course of schizophrenia. However, the functional relevance of the cross talks between systemic and brain inflammation is less clear.Riedmüller and Müller and Müller and Riedmüller discussed the potential ethical implications of the ME hypothesis of schizophrenia that includes shifting our perspective on schizophrenia from being an incurable psychiatric syndrome to a chronic but a treatable neurological disease. This will lead to a newer theoretical conceptualization of schizophrenia that necessitates interdisciplinary care teams to diagnose and manage new-onset psychosis and schizophrenia. Moreover, this reform will have potential repercussions for the pharmaceutical industry and legal implications surrounding current compulsory treatment orders. It might also limit social isolation and decreases the burden and stigma associated with severe mental disorders.Kočovská et al. summarized the role of vitamin D deficiency as a potential environmental risk factor for three etiologically distinct disorders; multiple sclerosis, schizophrenia, and autism. The data suggest that vitamin D deficiency has a much more robust role in MS, compared with that in schizophrenia and autism. Further, Endres et al. investigated the prevalence of vitamin D deficiency in a 1-year cohort of adult inpatients with schizophreniform (n = 60) and autism spectrum syndromes (n = 23) at German tertiary care hospital, compared with that in control group . Severe deficiencies (< 10 ng/ml) were found more frequently in the schizophreniform (38.3%) and autism spectrum groups (52.2%), compared to that of control group (16.3%). These findings justify the need for a more frequent assessment of serum vitamin D levels in these disorders, and advocate for additional randomized clinical trials investigating the effectiveness of vitamin D supplementation in ameliorating psychiatric and behavioral symptoms.Endres et al. investigated the prevalence of CSF inflammatory abnormalities in a seven-year cohort study at the Freiburg University clinic. In 63 patients with bipolar disorder, CSF abnormalities, suggestive of mild inflammation, were found in 19% of patients. These include increased albumin quotients (12.9%), increased immunoglobulin (Ig) G indices (3.2%), oligoclonal bands (OCBs) in 1.6%, increased white blood cell count (1.6%). These findings further support the ME hypothesis of schizophrenia and autoimmune encephalopathy masquerading as severe mental disorder.Vogels et al. verified the previously reported findings of T-cell deficits and monocyte immune gene activation in a well-controlled cohort of 97 individuals with largely euthymic bipolar disorder. Notably, within the decreased T-cell populations, the counts of T-helper 17 and T-helper 2 were increased whereas T regulatory cell counts were decreased. Moreover, the circulating monocytes demonstrated an increased frequency of anti-inflammatory phenotypes. The authors conclude that T-cell deficits are likely a trait phenomenon while pro and anti-inflammatory factors are state dependent in individuals with bipolar disorder.Krause et al. showed significantly lower serum kynurenine level, and higher quinolinic acid/kynurenine (Qui/Kyn) ratio in a cohort of 32 individuals with major depression, compared to 20 healthy controls. Higher baseline kynurenine/tryptophan (KYN/TRP) ratio at baseline was predictive of remission and lower Qui/Kyn ratio following an add-on treatment with celecoxib correlated positively with remission. This study suggests that the measurements of above biomarkers might be useful in selecting individuals with major depression who are more likely to respond to anti-inflammatory agents.Ajdacic-Gross et al. analyzed epidemiological data from the PsyCoLaus, a large cohort study  in Switzerland, to explore the potential association between various early-onset anxiety disorders and the age at the onset of common viral childhood illnesses such as chickenpox, measles, and mumps. The authors found that only social phobia among early anxiety disorders was associated with delayed-onset viral infections, suggesting that common viral childhood infections of a delayed onset may increase the risk of social phobia, speculatively by infection-induced behavioral changes.Rahman et al. investigated the developmental effect of maternal immune activation (MIA), during early and late gestation, on glutamatergic signaling via NMDAR-related molecular changes in various brain regions  in adult rat offspring. The authors found that MIA can alter NMDAR indices, such as increased NR2A expression in cortical and hippocampal regions. This was more prominent in male compared with female offspring, irrespective of MIA gestational timing. They concluded that MIA-induced developmental molecular changes including increased NR2A expression in male offspring might trigger enduring vulnerability to impaired neuroplasticity and its consequent behavioral changes.Sommer et al. investigated the neural effects of the hydrogen sulfide , which is endogenously produced by enzymes utilizing cysteine in the peripheral and central nervous systems. The authors also conducted another experiment to analyze the effects of various antipsychotics on the expression of H2S forming enzymes in human cell lines. Local sodiumhydrogensulfide (NaHS) infusion alone into the hippocampus resulted in a significant increase in the hippocampal glucose and lactate levels, as well as glutamate release. Pretreatment with peripheral inflammatory lipopolysaccharide (LPS) was associated with a further increase in lactate, but without altering glutamate levels. While NaHS infusion was associated with a significant increase in hippocampal free radical formation, by contrast, LPS pretreatment was associated with reduced radical formation. Additionally, neuroleptics exhibited differential effects on the H2S forming enzymes, a finding that may be relevant to understanding the diverse functional effects of antipsychotic drugs.Mack et al. reported a case of a young woman presenting with a 25 years history of dominant psychiatric symptoms, ranging from depression to typical schizophrenia with fluctuating psychotic features. The psychiatric symptoms were poorly responsive to various traditional psychotropic interventions and required multiple prolonged psychiatric hospitalizations. Throughout the course of the illness, she had intermittently exhibited mild skin rashes thought to be related to possible underlying mixed connective tissue disorder. Indeed, one of her later relapses was associated with severe generalize exanthema that resolved rapidly with steroid and azathioprine. KB (the editor above) suspected that the psychosis is likely related to an underling systemic immune disorder for which treatment with azathioprine was maintained, resulting in a complete remission of the psychiatric syndrome for approximately 16 years. However, upon azathioprine discontinuation due to pregnancy, the patient developed a severe relapse of psychosis accompanied by severe diffuse skin rashes that required a 2 years inpatient psychiatric care. Treatment with various immunosuppressants, including belimumab, resulted in a full remission of both psychosis and skin rashes. The combination of systemic biomarkers for immune activation and mild CSF inflammatory abnormalities including the presence of OCBs led to the final diagnosis of atypical Lupus erythematosus with CNS involvement presenting with dominant psychiatric manifestations mimicking schizophrenia.Endres et al. report a case of steroid-responsive chronic psychosis associated with autoimmune thyroiditis mimicking antipsychotics-resistant schizophrenia, in the context of several clinical red flags that collectively pointed to underlying organic etiopathogenesis. These were intermittent electroencephalogram (EEG) slowing, mild temporal atrophy, and elevated thyroid antibodies. This case illustrates the importance of considering autoimmune psychosis in the differential diagnosis of secondary (organic) psychosis and to complete the relevant diagnostic workup to early identify this subgroup of immune-responsive psychosis. Endres et al. report another case with surprisingly rapid improvement of chronic schizophrenic symptoms under newly introduced antiepileptics after having identified respective suggestive EEG signs.Ong et al. presented a case of primary Sjögren‘s syndrome with dominant severe obsessive-compulsive together with depressive symptoms requiring psychiatric hospitalization. The diagnosis was eventually made based on the presence of serum biomarkers of Sjögren‘s syndrome coupled with CSF findings of mild inflammation. A few months of treatment with various immunosuppressants together with plasmapheresis resulted in a complete remission of all neuropsychiatric symptoms. The authors suggest that the clinical approach to the management of psychotropic–resistant obsessive-compulsive disorder should also include a careful search for biomarkers of inflammation and autoimmunity.Greenberg reports a case of severe psychotropic-refractory pediatric neuropsychiatric syndrome mimicking bipolar disorder thought to be linked to previously unrecognized infection, likely Bartonellosis. The presentation and the clinical course did not meet “pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections” (PANDAS) criteria. The patient exhibited a clinically meaningful response to antibiotics targeting Bartonella infection. This case illustrates that prior clinically unrecognized infections can serve as immunological triggers of secondary psychiatric illnesses.Klein provided a thorough review of the clinical and experimental evidence supporting the “viral hypothesis of schizophrenia.” The author suggests that HSV-1 infection of specific limbic brain regions such as hippocampal dentate gyrus among other factors is potentially mechanistically linked to psychosis in a subset of individuals with schizophrenia. The mechanisms potentially linking viral infections to the neurobiology of schizophrenia were highlighted. The author suggests that silencing the viral elements such as HSV-1, via either administering antiviral treatment or suppressing environmental factors that can influence viral expression such as stress can thereby potentially be curative in a subgroup of individuals with schizophrenia.In summary, the data from these reports strengthen the evidence linking immune dysfunction, autoimmunity, and neuroinflammation to various primary psychiatric illnesses including schizophrenia and affective spectrum disorders. Alternatively, they also show that brain autoimmune disorders such as autoimmune encephalitis and autoimmune encephalopathy can also present with diverse neuropsychiatric syndromes masquerading as severe mental disorders such as primary psychotic disorders unresponsive to traditional psychotropic and behavioral interventions. Thus, it is critical to obtain a detailed history, perform a thorough examination, recognize clinical features (red flags) suggestive of organic causes, and complete a relevant diagnostic workup to include screening for relevant neuronal antibodies in serum and CSF to exclude other organic causes, in order not to overlook immunological causes of new-onset secondary (organic) neuropsychiatric syndromes. Collectively, the above data also support the ME hypothesis of schizophrenia and severe mental illness , 2 and tKB prepared the intitial version of the Editorial, which was corrected and supervised and completed by SN and DB.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Postoperative remote lung injury is a complication following various surgeries and is associated with short and long-term mortality and morbidity. The release of proinflammatory cytokines, damage-associated molecular patterns such as high-mobility group box-1, nucleotide-biding oligomerization domain (NOD)-like receptor protein 3 and heat shock protein, and cell death signalling activation, trigger a systemic inflammatory response, which ultimately results in organ injury including lung injury. Except high financial burden, the outcome of patients developing postoperative remote lung injury is often not optimistic. Several risk factors had been classified to predict the occurrence of postoperative remote lung injury, while lung protective ventilation and other strategies may confer protective effect against it. Understanding the pathophysiology of this process will facilitate the design of novel therapeutic strategies and promote better outcomes of surgical patients. This review discusses the cause and pathology underlying postoperative remote lung injury. Risk factors, surgical outcomes and potential preventative/treatment strategies against postoperative remote lung injury are also addressed. There are more than 230 million surgical operations around the world each year . AlthougAcute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are two common forms of lung injury after surgery. Postoperative remote lung injury affects patient outcomes directly. In this review, the mechanisms of postoperative remote lung injury and its impact on surgical outcomes and prognosis will be discussed. Transfusion is often indispensable in surgery but can have detrimental effects after surgery, including infection and lung injury. According to the Food and Drug Administration, transfusion related acute lung injury (TRALI) is a severe event especially with platelet or plasma containing transfusions among the elderly  which isA study carried out by Blum et al. demonstrated that the incidence of ARDS in patients who have undergone non-cardiothoracic surgery is 0.2% . Kogan eRemote lung injury often occurs following major surgery due to traumatic injury to primary organs that triggers systemic inflammation  Fig. 1)1). Pro-iTraumatic tissue injury, for example during surgery, causes the release of inflammatory cytokines locally which then spread systemically and subsequently result in organ injury including lung injury. The release of cytokines can be detected in patients after cardiac surgery. Prondzinsky et al.  demonstrDAMPs are endogenous mediators that play critical roles in various diseases. It is thought that DAMPs are transported to the lungs via circulation from organs damaged perioperatively. Although the role of DAMPs in lung injury is not completely understood, several well-described DAMPs are involved in the pathogenesis of postoperative remote lung injury , 21.Injured cells and immune cells including macrophages release HMGB1 after surgery, which triggered the production of various pro-inflammatory cytokines. Various surgical procedures, including gastrointestinal surgery and CPB, may induce release of HMGB1 , 22. A sNLRP3 is a member of the NLR family that participates in inflammatory response under various cellular stresses. Upon activation, an inflammasome complex is formed which facilitates the maturation and secretion of IL-1β and IL-18, mediated by activated caspase-1 , 29. MorNLRP3 expression is increased in the lung following mechanical ventilation. Kuipers et al.  demonstrHaemorrhagic shock is capable of activating NLRP3 inflammasome. In lung endothelial cells, ROS derived from haemorrhagic shock-activated NAD(P) H oxidase induced inflammasome activation and IL-1β secretion. Inflammasome activation is amplified by ROS released by neutrophils which further enhance NAD(P) H oxidase activation . ImbalanHsp are another type of DAMPs released by cells under stress and noxious stimuli in a process known as stress protein response (SPR). SPR can be activated by hyperthermia and various environmental insults such as oxidative stress toxins . IncreasApoptosis in inflammatory cells and alveolar cells mediated ALI after haemorrhagic shock . In an iRecently, it was reported that regulated necrosis also participates in remote lung injury. Two different forms of regulated necrosis, necroptosis and parthanatos, were found to be present in lung injury after kidney transplantation in rats. TNF-α was responsible for activation of key elements of necroptosis and parthanatos in lung epithelial cells, whilst blocking their function with inhibitors alleviated remote lung injury .Postoperative remote lung injury shares the same pathological changes as ALI/ARDS. ARDS is characterized by increased permeability of the alveolar epithelium and capillary endothelium. The disruption of these barriers results in the accumulation of protein-rich fluid in the alveoli, causing pulmonary oedema.Taking remote lung injury following kidney surgery as an example, kidney injury after surgical operation causes the release of cytokines and chemokines as well as DAMPs which attract a large number of immune cells, such as polymorphonuclear leukocytes  and T cells into the alveolar space  Fig. 2)2). ActivOverall surgical outcome for patients who have developed lung injury after surgery is not optimistic. Kinugasa et al.  demonstrIn a retrospective single centre study conducted in Japan, 502 patients underwent oesophageal carcinoma resection were enrolled and their postoperative complications were analysed . The oveRecently, a meta-analysis discussed the relationship between postoperative lung injury and the incidence of mortality and mobility after abdominal or thoracic surgery . Among 3Postoperative remote lung injury and/or PPC are not uncommon following major surgical procedures , 59, 60.2 greater than 35% and SpO2 less than 95%. This study also indicated that the surgical lung injury prediction (SLIP) score is of inferior power in this high-risk population than SLIP-2, which is more accurate in diverse and acutely ill patients. However, due to variation in clinical settings in different centres, there is still no standardized prediction scale available worldwide. Furthermore, one recent study reported by Fernandez-Bustamante et al. showed that PPCs are common in patients in spite of protective ventilation settings [In 2010, a large prospective, multicentre study reported that 5% of patients developed PPC  re-evalusettings . They al2O is protective during one lung ventilation [Identification of risk factors allows clinicians to predict postoperative remote lung injury. However, efforts to improve ventilation strategies during surgery are also vital. Amongst patients without ARDS at the onset of ventilation, fewer patients develop lung injury under protective ventilation compared to conventional ventilation . In a potilation .In recent years there has been an increasing understanding of the possible pathophysiological processes underlying the development of postoperative remote lung injury, with evidence to suggest that it may be possible to exploit this knowledge to reduce its incidence within the clinical environment.In vivo models of ARDS have demonstrated that various anaesthetic agents, including isoflurane, sevoflurane and desflurane, possess anti-inflammatory and cytoprotective effects –73. ThesIsoflurane is a commonly used volatile anaesthetic agent  and has Sevoflurane, another commonly used inhaled anaesthetic agent, has similarly been demonstrated the ability to ameliorate lung injury in vivo. In animal models of lung injury, sevoflurane has consistently demonstrated its protective properties by reducing deleterious histological changes, reducing wet to dry ratio and improving ventilation parameters –83. FurtPropofol has been shown to reduce the expression of a similar cytokine profile to sevoflurane, thus conferring a 2-fold increase in survival in an LPS model of lung injury by reducing pulmonary oedema and histological damage via its anti-inflammatory and anti-oxidative properties , 87. DexAnother therapeutic approach may be the use of xenon, a novel general anaesthetic agent, that has demonstrated anti-inflammatory and anti-apoptotic effects in models of acute lung injury following renal injury .Furthermore, numerous observational studies have attributed the judicious use of intravenous fluid in the perioperative period with a higher incidence of acute lung injury , 92. OneWhilst the scope for improvement in the incidence of postoperative remote lung injury is significant, there are a number of important unanswered questions. Currently, the vast majority of evidence underlying our current understanding of the pathogenesis of remote lung injury is through in vivo studies, with limited human studies.The off-license use of anaesthetic agents to ameliorate remote lung injury is inherently associated with risks in terms of safety and efficacy. Only a few human studies exist investigating the therapeutic effects of sevoflurane and propofol in ameliorating postoperative lung injury, with no overall significant identified differences in the incidence of ARDS, as well as conflicting biochemical changes reported between the studies –97. HoweThis highlights the importance of high-powered, controlled, clinical trials investigating previously purported in vivo markers of remote lung injury, such as IL-6, HMGB-1 and NF-κB, in human subjects. The aim of this should be to facilitate similarly high-powered, randomised controlled trials investigating the efficacy, safety and side-effect profiles of anaesthetic agents such as isoflurane, sevoflurane and dexmedetomidine within clinical practice, as well as conservative approaches such as non-judicious intravenous fluid administration. Furthermore, additional in vitro and in vivo research is required to investigate the development of novel agents targeting specific pro-inflammatory markers, such as HMGB-1, NLRP3 and Hsp, as these agents may confer additional therapeutic benefit in the future.Postoperative remote lung injury is not an uncommon condition clinically, and it is associated with adverse effects on overall patient survival. The pathological mechanisms are complex, involving cytokines and DAMPs released by injured organs or surgical sites. As a result, the normal function of the respiratory system is compromised, contributing to the development of lung injury. Due to the poor outcome associated with postoperative remote lung injury, predicting the population at risk is critical in order to facilitate lung injury prevention at the incipient stage. Approaches to ameliorate the pathological effects of postoperative remote lung injury are still required and necessitate further investigation, particularly utilizing well-powered, randomized controlled trials.


Radiation therapy (RT) is widely used in cancer care strategies. Its effectiveness relies mainly on its ability to cause lethal damage to the DNA of cancer cells. However, some cancers have shown to be particularly radioresistant partly because of efficient and redundant DNA repair capacities. Therefore, RT efficacy might be enhanced by using drugs that can disrupt cancer cells' DNA repair machinery. Here we review the recent advances in the development of novel inhibitors of DNA repair pathways in combination with RT. A large number of these compounds are the subject of preclinical/clinical studies and target key enzymes involved in one or more DNA repair pathways. A totally different strategy consists of mimicking DNA double-strand breaks via small interfering DNA (siDNA) to bait the whole DNA repair machinery, leading to its global inhibition. Radiation therapy (RT), in conjunction with surgery and systemic therapies , is a cornerstone of cancer care. About 50% of cancer patients receive RT . The priDNA lesions induced by RT activate the DNA damage response (DDR), which essentially involves post-translational modifications of proteins to activate downstream signaling pathways . DDR is RT induces a variety of DNA lesions. Approximately 10,000 damaged bases, 1,000 single strand breaks (SSBs) and 40 DSBs are produced per gray per cell , 22. SucFollowing RT, damaged bases induced by oxidative stress are repaired by the base excision repair pathway (BER) –31. In BTwo major pathways repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ) . HoweverIn mammalian cells, c-NHEJ . HR. HR40). The choice between the two major mechanisms for DSB repair (NHEJ and HR pathways) seems to be linked to several factors such as cell-cycle phase, chromatin context, or availability of key actors such as the Ku complex, 53BP1 or RAD51 , 65.−/− breast or ovarian cancer tumors, the inhibition of BER by PARP inhibitors leads to unrepaired DSBs and cell death  through association with DNA-PKcs , 123. A Checkpoint dysfunction represents a common molecular defect acquired during tumorigenesis , 127, unAnother target is the WEE1 kinase, which has been shown to be a major regulator of the G2-M checkpoint . This tyDNA repair proteins preserve the integrity of the genome; therefore, DNA repair targeting may enhance the tumor mutational burden, which may lead to the production of neoantigens and increased activity of anti-cancer T cells. Some clinical trials have been set to investigate the use of immune checkpoint inhibitors, notably by combining Dravulumab (anti-PD-L1) with PARP (NCT02484404), ATR (NCT02264678) or WEE1 inhibition (NCT02617277). To date, the interplay between radiation and the immune system is far from being completely deciphered, but several interesting facts have been reported. The cytotoxic action of radiotherapy on tumor cells provides T lymphocytes with tumor neoantigens and releases pro-inflammatory cytokines, thus promoting the immune response . The celTargeted therapies are beginning to demonstrate activity across a number of tumor types. The most promising approach toward improving the efficiency of a treatment and gaining a reliable response is to develop therapy combinations that decrease the chance of resistance and to treat resistance when it emerges. There has been a considerable renewed emphasis on new targeted treatments such as radiosensitizers that do not cause overlapping dose-limiting toxicities. Selection of appropriate targeted agents represents a challenge. As predicted, during preclinical and clinical trials, particular attention was paid to proteins involved in DNA repair pathways. Various strategies have been explored, ranging from specific protein targeting to global inhibition, and many DNA repair inhibitors have been developed. Up until now, only a few of them have reached the clinical stage, while even fewer have been tested in combination with RT. The several clinical trials currently underway will tell whether these new compounds can be tolerable and efficient.RT-induced lesions can be corrected by various DNA repair pathways. The intricacy of crosstalk between DNA repair pathways suggests that biomarker assays to determine the status of multiple DNA repair pathways could provide essential information on the sensitivity and resistance of cancer cells to repair inhibitors. Understanding these DNA repair pathways and identifying effective stratification biomarkers from the various DNA repair pathways that are specifically altered in some tumors would be required to characterize patients' responses to specific DNA repair inhibitors.JB and EC analyzed the literature and wrote the manuscript. PV and MD gave important intellectual input and carefully revised the manuscript. All authors approved the final manuscript for submission.MD is the cofounder of DNA Therapeutics/Onxeo, which is involved in DT01 development. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA-based immunization strategies have emerged as promising alternatives to conventional vaccine approaches. A substantial body of published work demonstrates that RNA vaccines can elicit potent, protective immune responses against various pathogens. Consonant with its huge impact on public health, influenza virus is one of the best studied targets of RNA vaccine research. Currently licensed influenza vaccines show variable levels of protection against seasonal influenza virus strains but are inadequate against drifted and pandemic viruses. In recent years, several types of RNA vaccines demonstrated efficacy against influenza virus infections in preclinical models. Additionally, comparative studies demonstrated the superiority of some RNA vaccines over the currently used inactivated influenza virus vaccines in animal models. Based on these promising preclinical results, clinical trials have been initiated and should provide valuable information about the translatability of the impressive preclinical data to humans. This review briefly describes RNA-based vaccination strategies, summarizes published preclinical and clinical data, highlights the roadblocks that need to be overcome for clinical applications, discusses the landscape of industrial development, and shares the authors’ personal perspectives about the future of RNA-based influenza virus vaccines. Influenza virus causes millions of illnesses and up to 650,000 deaths worldwide every year . ApproveVaccination with in vitro transcribed (IVT) messenger RNA (mRNA) was not widely considered as a viable approach until the early 2000s; however, several early studies using various types of mRNA vaccines demonstrated promising preclinical results ,8,9. SemEarly studies using naked (uncomplexed) unmodified RNA for immunization via the intramuscular route demonstrated some efficacy against various infectious pathogens  but subsVarious formats of RNA vaccines against influenza virus have demonstrated potent immunogenicity in preclinical models . MoreoveInfluenza virus infection is a major public health problem; thus, tremendous effort has been invested in improving current vaccines and creating new, more effective vaccines over the past decades. The need for a broadly protective/universal influenza virus vaccine, combined with the enormous infrastructure and research experience behind influenza virus research, and the relative ease of testing influenza virus vaccine efficacy in small animal models, have made it an optimal system for RNA vaccine development. Thus, influenza virus RNA vaccines (using IVT RNA) are among the most extensively studied infectious disease RNA vaccines, with studies dating back over 25 years . Two majThe majority of current SAM vaccines use a modified alphavirus genome that encodes the genes for the RNA replication machinery and the antigen of interest in the place of the viral structural protein-encoding genes ,36. The In summary, several proof-of-concept studies have been published about the efficacy of SAM influenza virus vaccines in preclinical models , but no A substantial amount of research has been invested into the development of directly injectable non-replicating mRNA vaccines. The first study that demonstrated efficacy after vaccination with mRNA was published in 1993. This seminal work showed that administration of influenza virus NP-encoding mRNA complexed in liposomes induced cytotoxic T cell responses in mice . InteresRecently published, highly efficacious influenza virus mRNA vaccines use unmodified or nucleoside-modified full-length HA-encoding mRNAs encapsulated in a LNP carrier. The LNP component of the vaccine has previously been shown to be efficacious for siRNA delivery . CureVacNucleoside-modified mRNA-LNP vaccines represent a new and highly efficacious category of RNA vaccines that can elicit protective immune responses against various pathogens ,46,47,48Coxiella burnetii [RNA vaccines have recently received significant attention and attracted massive academic and industrial investment. A limited number of medium-size biotechnology companies have successfully raised capital to develop innovative RNA vaccines . Both CureVac and Moderna have invested in internal production capabilities. Expected in 2018, CureVac’s “GMP III” facility will have the capacity to produce 10 million doses of GMP mRNA per year, while the commercial scale “GMP IV” facility is currently under construction and will be able to produce up to 30 million mRNA doses per year (www.curevac.com). The company envisions a “GMP V” facility that will be able to increase the capacity to 400 million doses per year . Other companies have also announced investments in developing RNA-based influenza virus vaccines; however, little is currently known about their research strategies  conducted by Moderna Therapeutics . Early results from this Phase I trial using nucleoside-modified mRNA-LNPs are discussed in rategies .Companies focused on developing RNA-based influenza vaccines aim to create a product that is superior to the currently available split (inactivated) or LAIV vaccines. Requirements for innovative, broadly protective influenza virus vaccines have been extensively discussed in the influenza vaccine scientific community  and the Currently used seasonal influenza vaccines show some level of protection against matched viruses but they offer little protection against drifted seasonal or pandemic viruses. A critical requirement for a new class of vaccine is to greatly improve efficacy and breadth of protection. Prophylactic RNA vaccines demonstrated heterologous protection in preclinical models , but tra5–7 PFU or TCID50) and it is in a liquid suspension or large droplet spray that induce only an upper respiratory tract infection after administration into the nares [Large-scale efficacy trials are expensive, and thus, an early indication of the level of protection and cross-protection against drifted strains could be provided by Controlled Human Influenza Virus Infection Models (CHIVIM). The utility of such a challenge system for predicting vaccine effectiveness depends on the nature of the challenge virus, its infectious dose, and route of administration. Currently available challenge viruses . Non-HA based vaccines or LAIV do not have a known correlate and need to rely on large-scale field efficacy trials. The most potent RNA vaccines induce very high HAI titers in small and large animals and HAI titers in the protective range in humans  has been shown to correlate well with protection in preclinical ,72 and cAs discussed in several recent publications ,57, an oCollectively, RNA-based vaccines represent a new vaccine class that can be used to effectively combat influenza virus. As noted, more data from clinical trials, including improved controlled human influenza virus infection models, and large-scale field efficacy trials will be critical to demonstrate the viability of this vaccine technology.


Pericytes, as a key cellular part of the blood-brain barrier, play an important role in the maintenance of brain neurovascular unit. These cells participate in brain homeostasis by regulating vascular development and integrity mainly through secreting various factors. Pericytes per se show different restorative properties after blood-brain barrier injury. Upon the occurrence of brain acute and chronic diseases, pericytes provoke immune cells to regulate neuro-inflammatory conditions. Loss of pericytes in distinct neurologic disorders intensifies blood-brain barrier permeability and leads to vascular dementia. The therapeutic potential of pericytes is originated from the unique morphological shape, location, and their ability in providing vast paracrine and juxtacrine interactions. A subset of pericytes possesses multipotentiality and exhibit trans-differentiation capacity in the context of damaged tissue. This review article aimed to highlight the critical role of pericytes in restoration of the blood-brain barrier after injury by focusing on the dynamics of pericytes and cross-talk with other cell types. A large number of people in developed societies are suffering from CNS problems burden them sensory, motor, and cognitive defects. BBB has the responsibility of maintaining homeostasis inside CNS by providing oxygen, nutrition, systemic regulatory factors, and removing metabolic wastes. Due to its complex and integrated structure, BBB delicately controls the crossing of chemicals on two sides of blood and brain. More importantly, BBB has a critical role in maintaining brain normal function in response to variations of multiple factors. Structurally, BBB is made up of different cell types, including ECs, pericytes, astrocytes, neurons, and microglia. The tight junctions between ECs strictly confine molecules transmission toward brain side Fig.  1–4].Fi–4.Fi1–4]On the blood side, BBB is paved by a united plane of ECs. The existence of tight junctions makes these cells to efficiently restrict molecules entrance toward the brain tissue. This characteristic in ECs is related to the exclusive expression of adhesion molecules such as claudins-3, − 5, and − 12. As a matter of fact, a main function of the BBB is to provide EC-EC integrity and various physical interactions between ECs with other resident cell types in neurovascular unit . In addiThe highest pericytes density exists in the CNS; thereby the brain has great potency for developing BBB integrity through the promotion of tight junction proteins between ECs –20. GeneA higher density of angiocrine factors and neurocine mediators are released via microvesicles in a paracrine manner such as brain-derived neurotrophic factor and β-nerve growth factor , 30. OnePericytes can regulate BBB resident cells function through secreting various proteins in BBB Table . The molThe primary response against oxygen and glucose deprivation is initiated via engaging STAT3, contributing to the control of metabolism and angiogenesis via bound regulated genes . In hypoFOXF2 expression which reduces pericytes differentiation . At. At51]. ectively .The pericyte-EC close physical contact is essential for controlling BBB integrity and function. Actually, it has been suggested juxtacrine contacts between both cell types are inevitable for normal development of the BBB . In addiIntegrity of the BBB could be reduced by pericytes upon the secretion of VEGF and MMPs , 48, 60.The migration of pericytes is deeply influenced by their interaction with ECs. Pericytes migration is preceded by the release of proteases and degradation of ECM . During Another factor termed TSP1 has paradoxical effects on ECs and pericytes. TSP1 prevents ECs migration while enhances pericytes mobilization. Actually, pericyte responsiveness to pro-migratory stimulation of PDGF is depended on the presence of TSP1. This effect was suppressed in TSP1 deficient pericytes (Fig. 2+ needed for pericytes motility and contraction [The balance of neural activity and cerebral capillaries blood flow guarantees proper function of the brain which termed neurovascular coupling. Pericytes motile identity introduces them as a prominent regulator of the BBB. During repeated seizures, neurovascular insufficiency occurs because of pericytes damage. Probably, mitochondrial injury underlies as a foremost cause of pericytes dysfunction following recurrent seizures which regulates intracellular Catraction . Here intraction , 82. Thetraction . Microgltraction  is a sugPDGFRβ/NG2 cells redistribution and higher immunoreactivity of PDGFRβ are observable in epileptic specimens. PDGFRβ signaling could be attributed as an enhancer of pericytes migratory activity , 85, 86.BBB resident pericytes show similarities with MSCs from the aspect of cell surface proteins expression and cellular behavior. Based on data from an experiment done by Tian et al., they showed treatment of MSCs, instead of pericytes in in vitro model of the BBB, with TGF-β or collagen type I elevated CD146 expression . The rol+ cells to neovascularization zones, so this mechanism can contribute angiogenesis after brain ischemia [Principally, two different aspects of pericyte-derived angiogenesis must be considered in the context of CNS. In brain tumors, the pro-angiogenic capacity of pericytes should be inhibited while during brain injury pro-angiogenic capacity of these cells is better to promote. The angiogenesis process is tightly controlled by both pro-angiogenic and anti-angiogenic factors . It has ischemia .The support of neurogenesis is another mechanism for pericytes toward regeneration. Pericytes might regulate neurogenesis indirectly through the synthesis of ECM proteins which take part in the presenting regulatory molecules to stem cell niche through tethering mechanism . In ischUpon the occurrence of brain pathologies, pericytes provoke immune cells to regulate neuro-inflammatory conditions. Even though, it was shown that pericytes are capable to represent microglial-like phenotype . NotablyAs pericytes have pluripotent individualities, so they have the capacity to trans-differentiate into other cell types such mesenchymal lineage , 22, neuBecause of particular characteristics, pericytes show a prominent role in protecting BBB during diseases which can be highly important for maintaining brain function . In addi


A seven-year-old spayed female Labrador retriever presented for necropsy following an acute history of thrombocytopenia, anemia, leukocytosis and abdominal effusion. A 2 × 3 × 10 cm, cylindrical to tubular, mottled red-to-tan mass extended from the caudal pelvic cavity caudally and ventrally under the dermis along the caudal aspect of the left pelvic limb adjacent to the semimembranosus and semitendinosus musculature. Histologic examination of the mass revealed a singular central lumen lined by urothelium that multifocally transitioned into non-keratinizing, stratified squamous epithelium associated with few hair follicles and sweat glands. The lumen was surrounded by a dense collagenous stroma containing numerous, variably sized blood vessels. The combination of lesions was consistent with a diagnosis of incomplete urethral duplication associated with a dermoid cyst and vascular hamartoma. To the authors’ knowledge, this is the first report of an incomplete urethral duplication associated with a dermoid cyst within a vascular hamartoma. Urethral duplication is a rare congenital anomaly infrequently diagnosed in domestic animal species. A total of five cases of urethral duplication have been reported in veterinary medicine, with four cases reported in dogs ,2,3,4. UA seven-year-old spayed female Labrador retriever presented to the Utah State Veterinary Diagnostic Laboratory for necropsy following an acute onset of thrombocytopenia and anemia coupled with a peripheral neutrophilia, lymphocytosis and monocytosis. The history indicated a previous diagnosis of coccidioidomycoses that was being treated at the time of death. Abdominal ultrasound revealed an enlarged, granular liver with irregular liver margins. The urogenital tract showed no significant ultrasonographic abnormalities. In addition to fluconazole and amphotericin B treatment for coccidioidomycoses, the patient was receiving prednisone and myocophenolate for a presumptive immune mediated anemia and thrombocytopenia, and S-adenosyl-L-methionine (SAMe) for elevated hepatic enzymes prior to presentation for necropsy. The patient died naturally at home seven days after initial presentation.Gross necropsy revealed a hemoabdomen with approximately 1 L of serosanguineous fluid within the abdominal cavity, petechiation along the ventral abdomen and dark-red-to-purple viscous material within the entirety of the gastrointestinal tract. Originating within the pelvic fat lateral to the pelvic urethra and coursing caudally and dorsally within the subcutaneous fat overlying the left semimembranosus and semitendinosus muscles was a 2 × 3 × 10 cm, cylindrical to tubular, mottled tan-to-red mass that ended abruptly approximately one quarter to one third of the way down the pelvic limb. It was not determined at the time of necropsy if the mass directly communicated with the urinary bladder or urethra. The mass was grossly soft to spongy with a central slit-like lumen measuring up to 25 mm in diameter on cross section. The lumen continued down the entire length of the mass ending approximately 1 cm proximal to the blinded end within the pelvic limb.Microscopic evaluation of the mass revealed an unencapsulated, moderately cellular, well-demarcated expansile mass . The masAdditional gross and histologic diagnoses included cirrhosis, multiple adenohypophyseal cysts, meningioangiomatosis, splenic and bone marrow myeloid dysplasia, adrenal cortical hyperplasia and hypertrophy, lymphoplasmacytic gastritis and hemomelasma ilei. The cause of death in this dog was attributed to sequela associated with cirrhosis. Presumptive myeloid dysplasia was diagnosed on histopathologic examination of the bone marrow and splenic tissue. A complete blood count in the ante-mortem period was unavailable to confirm a concurrent diagnosis of myeloid leukemia.Urethral duplication is a rare lesion in veterinary medicine. This report outlines the first case of urethral duplication in a female dog. All cases of urethral duplication reported in veterinary medicine have been described in male dogs. In humans, urethral duplication is also rare, and is usually diagnosed at a young age, primarily in male patients. When diagnosed in female patients, urethral duplication is often coupled with additional urogenital abnormalities such as a prepubic sinus formation, abnormal urethral location within the vagina, duplication of the urinary bladder or renal abnormalities ,7. None Unlike previous reports of urethral duplication in dogs, the clinical history and medical record provided with this case did not indicate the presence of any previous urogenital abnormalities. Recurrent urinary tract infections have been previously reported with urethral duplication in a dog . There wAs with other cases of urethral duplication, multiple unrelated congenital anomalies were present in this dog. The mass ended blindly with a dermoid cyst. Dermoid cysts are rare lesions in domestic animals characterized by reduplication of haired skin including follicles and associated adnexa . DermoidThe association of a vascular hamartoma in conjunction with a duplicated urethra resembles the corpus cavernosum of a penis. The external genitalia of this female dog were grossly unremarkable. Specifically, the clitoris did not appear abnormally large or prominent. However, the gonads were unavailable for histopathologic evaluation due to a previous ovariohysterectomy. A singular case report in human literature of accessory female phallic urethral diverticulum formation described similar histopathologic findings that manifested clinically as an enlarged clitoris with urinary retention . The hisUrethral duplication remains a rarely diagnosed and poorly studied condition in veterinary medicine. While this condition is uncommon, urethral duplication should remain a differential diagnosis for cases of recurrent cystitis and should be evaluated in patients that present with disorders of sexual development.


Polyphenism describes the unique sub-type of phenotypic plasticity where the outputs are not continuous, but rather discrete and multi-stable, resulting in several distinct phenotypes on the same genetic background. Epigenetic regulation underpins the stable phenotypic divergences that exemplify polyphenism and their evolutionary origin. Here, we briefly summarize the apparent ubiquity and diversity of polyphenisms across the animal kingdom. We briefly review the best characterized models across taxa and highlight the consistent themes both in their epidemiology and what little we know about molecular mechanisms. Finally, we highlight work that supports the possibility that humans may have a subtle polyphenism at the level of metabolism.Phenotypic plasticity describes the capacity of a single genotype to exhibit a variety of phenotypes as well as the mechanisms that translate environmental variation into reproducible phenotypic modifications.  Phenotypic output is defined by DNA-sequence (genetics), chromatin regulation (epigenetics and cellular memory) and environmental variables , and their interactions . PhenotyDaphnia cucullata)  ; temperacullata)  and fishcullata) ; and envcullata) .Polyphenism is a special case of phenotypic plasticity where development exhibits reproducible bifurcations revealing multiple distinct outcomes . Environmental stimuli are the major external triggers of polyphenisms. Multiple physiological pathways, including epigenetic modifications during development, are believed to mediate and maintain phenotypic divergences that exemplify polyphenism. While polyphenisms are by definition discrete, their relative distributions within populations can be either highly stable  or highly variable to the extent that entire populations switch or oscillate from 100% of one morph to 100% of the other  . BecauseBelow, we highlight the key concepts of what we do understand of the epigenetic and genetic processes driving morph divergences, especially in animal kingdom.Camponotus floridanus), Indian jumping ant , and the honeybee (Apis mellifera).Eusocial insects are the epitome of polyphenism; the same genome gives rise to completely different morphologies and behaviors in a population. In this review, we will cover caste polyphenism in three insect species: the Florida carpenter ant  if the original queen dies . Data shDnmt3) in newly hatched larvae and in embryos strongly biases an individual’s outcome toward the queen fate . Such queens have fully developed ovaries compared to control-treated individuals (workers)  . These sDispersal polyphenisms are a sub-classification characterized by morph phenotypes that markedly influence the reproductive dispersal of an organism and thus the population (new cohort). A common example is winged versus wingless insect morphs that develop in heterogeneous environments . This alternative dispersal ability is likely advantageous for adapting new habitats and for maximizing focal populations while ensuring population spread and outbreeding under a wide range of environmental possibilities.Aphids are a diverse group of small insects with complex life cycles. Many of them undergo cyclic parthenogenesis and sexual reproduction. Sexual reproduction only occurs at the end of summer when temperatures fall or when daytime starts to shorten. Female aphids have an environmentally determined wing polyphenism (winged vs. wingless) during the parthenogenesis process . There aPristionchus pacificus (P. pacificus) is one of the most well-studied species of nematodes apart from Caenorhabditis elegans (C. elegans). They can be cultured on bacteria in the laboratory, but they live in a necromenic association with scarab beetles in the wild  stages. The life cycle of P. pacificus is approximately 4 days at 20°C, but under stressed environmental conditions, such as food shortage or high-population density, J2 larvae form dauers instead of developing into J3 larvae. In the necromenic association with scarab beetles, J2 larvae rest as dauer instead of entering J3 larvae on the insect and resume development into J3 larvae after the beetle’s death – in the carcass – to feed on the growing microbes.P. pacificus has versatile teeth-like denticles and mouth forms that can be either eurystomatous (Eu) or stenostomatous (St)  . The dorous (St) . The dorous (St) . Eu indious (St) ,D. The bous (St) . This beous (St) . These rStarvation and pheromones are two identified triggers that increase Eu individuals in a population . ConversThe nuclear hormone receptor, DAF-12, binds the steroid hormone Δ7-dafachronic acid (Δ7-DA). DAF-12 and Δ7-DA represent the first two molecules identified that specify mouth-form decision of individuals in response to starvation and pheromones. Loss-of-function of DAF-12 in dauer formation defective (daf-d) mutants, which are weakly responsive to starvation and unresponsive to pheromones, decreases the incidence of Eu formation. In addition, Δ7-DA treatment (DAF-12 ligand) decreases the incidence of Eu individuals as well .In a forward genetic approach, from Eu-form-defective mutant (eud-1), the authors identified Eud-1 as a developmental switch regulator. Eud-1 mutants do not produce Eu forms, while transgenic lines overexpressing eud-1 produce only Eu forms. One pheromone known to specifically regulate mouth dimorphism, diascaroside dasc#1, fails to turn eud-1 mutant into Eu forms when compared to the wild-type strain (98% Eu). This result indicates that the EUD-1 switch responds to external pheromones.In an epistasis study, Δ7-DA had no effect on the eud-1 transgenic line (100% Eu). This indicates that EUD-1 regulates mouth dimorphism downstream of Δ7-DA/DAF-12 . Still, P. pacificus as a model  and changes of external morphology and internal gonadal anatomy . Initialcyp19a1a in fish), an enzyme that converts androgens to estrogens . Naked mole rats exhibit two examples of discrete phenotypic variation. Like insects, strong phenotypic variation is associated with breeding social structure. Colonies have a single breeding queen that is larger, produces impressive numbers of offspring, and relies on servitude of colony workers for food and hygiene. If the queen of the colony dies, a few females may fight to become the new queen. When the new female begins her reign, she grows even though she is already an adult; a striking adult morphological plasticity that parallels ant gamergate plasticity and e genome . Interese genome . Thus fa+/D9Trim28), a non-sense mutation in the chromatin-interacting protein gene Trim28  stood out in which they exhibited exaggerated stochastic phenotypic variation specifically in body mass and adiposity  that appears causal in developmental bifurcation. Specifically, obese morphs exhibit reduced expression of neuronatin (Nnat), Peg3, Cdkn1c, and Plagl1. Deletion of either Nnat or Peg3 alone is sufficient to recapitulate the bi-modal adiposity phenotype, thus identifying a still small regulatory network of developmental switch genes in mammals and highlighting the stark potential for discrete epigenetic states in mammalian disease. To our knowledge, this work represents the first genetic proof of mammalian polyphenism, though we find evidence of obese morphs at frequencies of ~1–5% in most colonies examined.Our own group has demonstrated polyphenisms in the mouse. Building on ambitious experiments by the Whitelaw group intended to identify novel chromatin regulators in mice, we focused on chromatin regulatory mouse mutants with evidence of altered or enhanced phenotypic variation. One MommeD9  . BimodalTRIM28 is significantly reduced in adipose tissue of obese children. To mimic the analysis of +/D9Trim28 haploinsufficiency in mice, all individuals were grouped into high or low TRIM28 expressors based on adipose tissue mRNA levels and sub-grouped into obese or lean groups. Interestingly, in contrast to high TRIM28 expressors, obese children with low Trim28 also exhibited lower IGN1 gene expression when compared to equally TRIM28-low groups of lean subjects. These results recapitulated IGN1 downregulation in +/D9Trim28 haploinsufficient mice and are consistent with a conserved role for a TRIM28-IGN1 axis in human adiposity regulation. Perhaps most interesting, principal component analysis of transcriptomes from all the same individuals indicated that the components of the greatest variation across the cohort were not obese vs. lean, or male vs. female individuals, but rather TRIM28-low vs. TRIM28-high individuals indicating substantial differences exist between these two as of yet ill-defined categories of humans.Beyond epidemiology, our studies have also taken us to investigations of gene expression in a cohort of 22 lean and 18 obese children, aged ~2–10 years of age, a time of relatively tight environmental control in human lifespan . TRIM28 TRIM28 and IGN1 expressions are specifically decreased in the obese relative to lean isogenic co-twins. This indicates that TRIM28-IGN1 expression at the least correlates with epigenetic human obesity. If sufficiently powered they would be consistent with TRIM28-IGN1 insufficiency as a developmental gene switch.While these childhood cohort studies intrigue, they remain poorly genetically controlled. Adipose tissue microarray data from 13 discordant monozygotic (MZ) twin pairs, each comprising one obese and one normal co-twin , indicatPolyphenism is conserved in species from insects to nematodes, and evidence is mounting that it extends into many mammalian species.The naked mole rat eusocial phenotypic plasticity, whose phenotypes are still malleable after adulthood, is reminiscent of seasonal phenotypic plasticities in coat colors of arctic animals and in antler growth. It is similarly reminiscent of physical attributes associated with alphas in pack animals, a social and physiological divergence that is widespread across the mammalian kingdom. The blurriness between the concepts of polyphenism and hierarchy-associated phenotype reflects our limited understanding of the interplay between environment  and the molecular regulation of developmental switches. It will be intriguing to see the overlap and contrasts in molecular mechanisms that beget adult seasonal/hierarchical phenotypic plasticities. The seemingly more stable developmental switches likely reflect analogous molecular principles at earlier developmental time points and therefore with much farther reaching and stable developmental consequences. Will we find strong concordance in molecular principles between socially reinforced phenotypic differences , robust nutritionally conditional systems , and classical developmental switch polyphenisms? Considering what we know of the enormous diversity that nature presents, there is no reason that these concepts should be mutually exclusive.+/D9Trim28 mice show a reduction in the frequency of obese morphs with increased housing density and reduced environmental temperatures, these have yet to be formally proven and the mechanistic nature of the triggers needs to be investigated. When and how are the developmental switches regulated? Trim28 loss has been linked to the dysregulation of imprinted genes during development (NNAT, as one gene of the IGN1 network, has been correlated with human obesity (NNAT locus are associated with susceptibility to severe forms of obesity in human children and adults (NNAT gene expression is reduced in subcutaneous adipose tissue from obese humans (TRIM28/IGN1 axis has been defined as the core developmental switch, the upstream signaling linking this axis to the external environment, and the epigenetic regulation that builds bi-stability into the system are the key future questions. Trim28 links DNA methylation, repressive histone modifications, and transcriptional repression. Our own observations indicate that −/+Dnmt3a mice show not only excessive phenotypic variation (The environmental and mechanistic underpinnings that contribute toward polyphenism in mammals are only beginning to be elucidated. Although we have anecdotal evidence that elopment . IGN1 geelopment , developelopment , suggest obesity  and very obesity . Two sind adults . Furthere humans . Furtherariation  but alsoA further understanding of the mechanistic principles across species will in the long run solidify our understanding of phenotypic diversity across species, kingdoms, and even across evolution. Hopefully deeper understanding of these magnificent processes can enlighten us equally regarding the non-genetic drivers of human disease.C-HY and JP contributed to the conception and design of the review. C-HY wrote the first draft of the manuscript. All authors revised, read, and approved the submitted version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Biliary cysts are rare cystic dilatations of the biliary tree. Biliary cysts are positively associated with several significant complications, amongst them, cholangiocarcinoma befalls the most dreadful one. The elevated incidence is 20-30% in the unresected cyst and 0.7% in resected cysts. Magnetic resonance imaging (MRI) scan, magnetic resonance cholangiopancreatography (MRCP) or a contrast-enhanced computed tomography (CECT) is applied for the initial diagnostic study but the ultimate diagnosis ordinarily requires the tissue biopsy. Currently, the sole curative option involves the complete surgical resection of the lesion, with standard chemotherapy and active radiation applied as an alternative for the unresectable tumors. Despite the curative surgery the percentage of eternal recurrence of the tumor indefinitely persists, and effective post-surgical surveillance is reasonably demanded. We report a case of 29-year-old female with local recurrence of cholangiocarcinoma in a previously resected biliary cyst type I. The curative resection of the choledochal cyst only minimizes the considerable risk of the possible development of future cholangiocarcinoma but it does not completely prevent it. The appropriate follow-up for potential patients who have been typically treated for a biliary cyst is unclear. The lethal course of cholangiocarcinoma is believed due to its slow asymptomatic growing phase. Therefore, to adequately screen for malignancy, periodic imaging along with annual liver tests represents a reasonable approach to prevent the possible development of this appalling complication. Biliary cysts, formerly known as the choledochal cysts, are rare cystic dilatations of the biliary tree with an elevated incidence of 1:100,000. The likely female to male ratio is invariably 4:1 with a marked predilection for Asian ethnicity . LatterlA 29-year-old female of Asian ethnicity presented to the medical outpatient department with chief complaints of progressive jaundice and continuous dull pain in the right hypochondrium from the past seven months. She also reported an unintentional weight loss of 22 lbs during the corresponding period. On physical examination, she was severely icteric and cachectic with an abdominal examination revealing tender hepatomegaly and splenomegaly. On chest auscultation, she invariably had bilateral coarse basal crepts. Blood investigations precisely revealed an obstructive pattern of liver enzymes with raised serum bilirubin of 21.9 mg/dl with direct 20.0 mg/dl, alkaline phosphatase (ALP) 1854 U/L and gamma-glutamyl transferase (GGT) of 2022 U/L with markedly deranged prothrombin time (PT) and activated partial thromboplastin time (APTT). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and all other routine investigations were normal. Ultrasonography (USG) of the abdomen showed an enlarged liver with focal lesions with no hepatic biliary dilatation.Previous surgical history was significant for biliary cyst diagnosed four years back. Magnetic resonance cholangiopancreatography (MRCP) report at that specific time showed a 2.3 cm fusiform dilatation of common bile duct, positively confirming the potential diagnosis of biliary cyst type I Figures , 2.She underwent complete cyst resection and cholecystectomy with Roux-en-Y hepaticojejunostomy. A biopsy at that particular point showed no signs of malignancy. Her immediate postoperative course was unremarkable and no post-operative surveillance was routinely done.A contrast-enhanced computed tomography scan (CECT) and magnetic resonance cholangiopancreatography were planned. They both suggested multiple masses in the liver that were obstructing the common hepatic duct resulting in jaundice with metastatic spread to regional lymph nodes and lungs Figures -5.For the histopathological confirmation of the cancer type, image-guided fine needle aspiration cytology (FNAC) was performed adequately. The biopsy sample sufficiently revealed malignant neoplastic lesions with prominent glandular differentiation positive for cytokeratin 7, 19 and CDX-2 suggesting metastatic adenocarcinoma most likely cholangiocarcinoma.Liver resection was not possible due to the widespread metastatic disease at official presentation, and she was cautiously started on intensive chemotherapy. Her condition deteriorated after her fourth chemotherapy session, resulting in multi-organ failure. Palliative care was adequately provided but she expired within a few abysmal weeks due to the potential complications of organ failure.Biliary cysts are rare cystic dilatations of the biliary tree that may occur singly or in multiples throughout the intra-hepatic or extra-hepatic biliary channels. Formerly the biliary cysts were popularly known as the choledochal cyst due to their possible involvement of the extra-hepatic bile ducts. However, the standard classification was carefully revised in 1977 by Todani et al. to appropriately include the intrahepatic cysts . This clThe characteristic presentation of a biliary cyst traditionally includes an abdominal mass that presents with abdominal pain and jaundice, but only a few patients present with the classic symptoms . Most paPossible complications of biliary cysts principally result from stasis and typically include cholangitis, choledocholithiasis, recurrent pancreatitis, cirrhosis, and portal hypertension . HoweverWhile the carcinoma risk decreases with prior resection of the cyst, these selected patients continue to be at considerable risk. The elevated incidence of progressively developing cholangiocarcinoma in the remaining biliary tree compared to the general population remains high in these selected patients. Post-excisional malignant disease is typically seen in 0.7 to six percent of compelling cases . This caWe reasonably conclude that along with prompt diagnosis and complete resection of a biliary cyst, regular follow-up is necessary to prevent the gradual development of cholangiocarcinoma. Successful surgery adequately provides a unique possibility for the cure. However, only a minority of patients presents with an early stage disease and are independent candidates for curative resection. Furthermore, the likely outcomes after curative resection are unsatisfactory, particularly with node-positive disease. Annual imaging with serial MRI, MRCP, CECT or intraductal ultrasound along with successive liver function tests, can be helpful to identify malignant changes at a primitive stage in these susceptible patients.


Patent ductus arteriosus (PDA), the most commonly diagnosed cardiovascular condition in preterm infants, is associated with increased mortality and harmful long-term outcomes . Although pharmacologic and/or interventional treatments to close PDA likely benefit some infants, widespread routine treatment of all preterm infants with PDA may not improve outcomes. Most PDAs close spontaneously by 44-weeks postmenstrual age; treatment is increasingly controversial, varying markedly between institutions and providers. Because treatment detriments may outweigh benefits, especially in infants destined for early, spontaneous PDA closure, the relevant unanswered clinical question is not whether to treat all preterm infants with PDA, but whom to treat (and when). Clinicians cannot currently predict in the first month which infants are at highest risk for persistent PDA, nor which combination of clinical risk factors, echocardiographic measurements, and biomarkers best predict PDA-associated harm.Prospective cohort of untreated infants with PDA (n=450) will be used to predict spontaneous ductal closure timing. Clinical measures, serum  and urine  biomarkers, and echocardiographic variables collected during each of first 4 postnatal weeks will be analyzed to identify those associated with long-term impairment. Myocardial deformation imaging and tissue Doppler imaging, innovative echocardiographic techniques, will facilitate quantitative evaluation of myocardial performance. Aim1 will estimate probability of spontaneous PDA closure and predict timing of ductal closure using echocardiographic, biomarker, and clinical predictors. Aim2 will specify which echocardiographic predictors and biomarkers are associated with mortality and respiratory illness severity at 36-weeks postmenstrual age. Aim3 will identify which echocardiographic predictors and biomarkers are associated with 22 to 26-month neurodevelopmental delay. Models will be validated in a separate cohort of infants (n=225) enrolled subsequent to primary study cohort.The current study will make significant contributions to scientific knowledge and effective PDA management. Study results will reduce unnecessary and harmful overtreatment of infants with a high probability of early spontaneous PDA closure and facilitate development of outcomes-focused trials to examine effectiveness of PDA closure in “high-risk” infants most likely to receive benefit.ClinicalTrials.gov NCT03782610. Registered 20 December 2018.Supplementary Information accompanies this paper at 10.1186/s12887-019-1708-z. Patent ductus arteriosus (PDA), the most commonly diagnosed cardiovascular condition in preterm infants, is associated with an eight-fold increase in mortality  and multDespite association with worsened outcomes in preterm infants, PDA closure has become increasingly controversial among cardiologists and neonatologists , with trNonsteroidal anti-inflammatory drug treatment is linked to nephrotoxicity , acute rStill, treatment of some infants with symptomatic PDA is necessary . A subseThe American Academy of Pediatrics and experts on PDA in preterm infants agree that future outcomes-based randomized clinical trials are needed to determine the effectiveness of selective pharmacological and/or interventional treatments for “high-risk” infants with PDA , 13, 51.The objectives of this study are to use a prospective cohort of untreated preterm infants with PDA to accurately predict the timing of spontaneous ductal closure  in preterm infants with PDA, and the identification of measurable echocardiographic predictors and biomarkers that are present in the first postnatal month that are associated with mortality, chronic lung disease (CLD), and long-term neurological impairment. These results will ultimately improve neonatal patient care by informing the design of outcomes-focused randomized clinical trials that will examine the effectiveness and timing of PDA closure in those “high-risk” infants most likely to receive benefit .A number of clinical , echocarWe will recruit a cohort of <30-weeks gestation preterm infants consecutively enrolled using the following inclusion and exclusion criteria:0/7 weeks) and 296/7 weeks of gestation, inclusiveInfants born between 23-weeks + 0 days  within 72-hours of birthPDA noted on initial screening echocardiogram at <72 postnatal hoursLife-threatening congenital abnormalities including congenital heart disease Infants whose parents have chosen to allow natural death (do not resuscitate order)primary study cohort (n=450) is ongoing  with a goal of 2 years. Because prediction models should be validated in a population separate from that in which they were created, inpatient recruitment of a validation cohort (n=225) will begin immediately following primary study cohort enrollment completion. Infants will remain in their respective cohort until follow-up at 22 to 26-months corrected age.Cohort entry will occur within 72 hours after birth, a time of PDA patency in >95% of infants born at <30-weeks gestational age . The resRecruitment will take place within the Nationwide Children’s Hospital (NCH) Neonatal Network, one of the largest neonatal intensive care networks in the United States. The NCH Neonatal Network is comprised of 9 hospitals in central Ohio within a 20-minute drive from the NCH research campus, has 268 neonatal beds, and had >420 admissions of infants born at <30-weeks gestation in 2016. Patient demographics, including sex and race/ethnicity, are largely representative of the US population. All sites share clinical-practice guidelines, are staffed by NCH neonatologists, and electronically transmit echocardiographic images to NCH for cardiologist interpretation.th-75th percentiles: 35.4 - 41.4 weeks].The NCH Neonatal Network is committed to a guideline-driven approach for the care of infants born at <30-weeks gestation . The NCHPatient demographic, clinical, and sample data will be collected in REDCap (Research Electronic Database Capture), a robust, secure web-based electronic capture tool that allows for real-time data entry with embedded logic and range checks, skip patterns, and missing data alerts to ensure quality control, while minimizing the amount of missing data, and assigned a study ID number . Study dThe clinical cardiac examination will be performed consistent with standards outlined by the American Heart Association and American College of Cardiology . To ensuper NCH Neonatal Network standard of care) in the outpatient neonatal-cardiology clinic. All echocardiograms will be performed with a dedicated research ultrasound . Images will be obtained with the narrowest sector angle to maximize frame rate  for optimal image quality  for gestational age, lowest mean blood pressure, lowest temperature, pO2/FIO2, lowest serum pH, presence of multiple seizures, and low urine output) [Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Neonatal Research Network (NRN) Extremely Preterm Outcomes Prediction Tool variables [We will include variables routinely collected in preterm infants within the first 12-hours postnatal that comprise the Score for Neonatal Acute Physiology with Perinatal Extension-II  . This sc output)  of morta output) , 76, CLD output) , and lon output) , 79. We ariables . These variables .Aim 2), early risk variables from the best clinical prediction model for bronchopulmonary dysplasia (BPD) severity  will be included [2). Pulse oximetry continually measures peripheral capillary oxygen saturation throughout NICU hospitalization as standard of care. We will incorporate the daily oxygen saturation index , which has been validated as a non-invasive alternative to oxygenation index for assessing neonatal hypoxic respiratory failure severity [To determine which early echocardiographic parameters and biomarkers in preterm infants with PDA are independently predictive of respiratory illness severity at 36-weeks PMA  local infants with echocardiography-diagnosed PDA who were born 23 to 30-weeks gestation and who had at least 2 echocardiograms to allow PDA status verification (open/closed) over time (n=244)  to reduce the frequency of intraventricular hemorrhage. Since indomethacin prophylaxis may contribute to PDA closure in some infants by modifying the effect of determined predictors on PDA-associated outcomes , we willThe clinical signs associated with PDA shunting depend on shunt magnitude and the compensatory ability of the immature myocardium to handle the additional volume load . HistoriTraditional echocardiography is the gold-standard for PDA diagnosis and provides assessment of ductal diameter, shunting pattern, and volume . EchocarTissue Doppler imaging can provide quantitative measures of the velocity of myocardial contraction and relaxation throughout the cardiac cycle as a reflection of cardiac performance. Because both systolic and diastolic information are contained in a single tracing, simultaneous isovolemic contraction and relaxation time intervals can be obtained. Tissue Doppler imaging can also be used to calculate a myocardial performance index that reflects both systolic and diastolic global function . In oldeTissue Doppler images will be obtained from the apical 4-chamber view with the point of interest along the annulus at the following 3 segments: right ventricle free wall; interventricular septum; and left ventricle free wall. Tissue Doppler imaging tracings will demonstrate a systolic wave, early and late diastolic waves for each segment, and simultaneous isovolemic contraction and relaxation time intervals.Myocardial deformation imaging assesses strain  and strain rate (deformation over time) –112. In Right and left ventricle myocardial deformation imaging strain will be assessed using two-dimensional, apical 4-chamber images , 113. Th. The selected biomarkers appear uninfluenced by gestational age, birth weight, and antenatal and postnatal events, making them ideal for evaluating PDA-related myocardial damage and hemodynamic status in preterm infants [If either the clinical exam or biomarkers can replace the more costly and labor-intensive echocardiographic techniques at specific postnatal time-points in some predictive models (each echocardiogram is >10-times the cost of the proposed biomarker tests), health care costs would be greatly reduced . Interes infants , 124–136Weekly blood and urine samples for biomarker analysis will be collected within one calendar day of the echocardiogram for the first 4 weeks postnatal. Blood draws (0.5 mL) will be collected concurrently with weekly nutrition labs that are obtained on all premature infants per local standard of care. Blood samples will be capillary  or arterial if a pre-existing indwelling line is present. Cotton balls will be put in the diapers of enrolled infants to collect urine. De-identified and labeled blood and urine samples will be transported on ice to the Center for Perinatal Research at NCH where samples will be centrifuged, and frozen in a dedicated freezer at −70°C for batch analysis. We have locally validated the serum and urine biomarkers for our specific patient population.To avoid excessive blood collection in preterm infants two serum natriuretic peptide biomarkers were selected a priori: beta-natriuretic peptide and N-terminal pro-B-type natriuretic peptide, which have been shown to predict likelihood of spontaneous PDA closure, respiratory outcomes, mortality, and neurodevelopment , 137–140Ductal shunting may decrease renal and mesenteric bed perfusion , 143. WeTo estimate the probability of spontaneous PDA closure and predict the timing of ductal closure using echocardiographic, biomarker, and clinical predictors obtained within the first postnatal month.primary outcome for Aim 1 is documented PDA closure by 36-weeks PMA.Until documented PDA closure, echodardiograms will be conducted weekly for the first 4 weeks postnatal and biweekly thereafter, between study entry and 36-weeks PMA on all inpatient study participants. Based on local pilot data, we expect 15% of PDAs  to remain patent at NICU discharge. All preterm infants with PDA at discharge will follow-up with the pediatric cartiologist at the dedicated neonatal-cardiology clinic within The Heart Center at NCH . Per local standard of care, these infants will receive echocardiograms at 2-3 month intervals until documented closure; any infants with a persistent PDA at 22 to 26-months postnatal age (~5% of those discharged with a persistent ductus) will undergo catheter-based closure to prevent long-term complications of PDA   Table . Such coAn infant’s daily respiratory support modality record at 36-weeks PMA closely correlates with important long-term respiratory and neurodevelopmental outcomes , 157. CLWe expect to accurately predict the probability of mortality or requirement for supplemental oxygen or positive-pressure support at 36-weeks PMA using a combination of echocardiographic measures, biomarkers, and clinical measures from the first 4-weeks postnatal. We will develop a clinically useful prediction model that will allow clinicians and those conducting clinical trials to estimate by week in the first 4 postnatal weeks, the probability of mortality or moderate/severe CLD (BPD) development. We will also evaluate the impact of spontaneous PDA closure and duration of PDA exposure on the 36-weeks PMA outcomes. Early prediction of PDA-associated complications will be crucial to future trial development to determine the population of infants who may require interventional PDA treatment and the ideal timing for such an intervention.To determine which echocardiographic predictors and biomarkers in preterm infants with PDA within the first postnatal month are associated with 22 to 26-month neurodevelopmental outcomes.The extent to which PDA persistence and/or severity contribute to childhood neurodevelopmental impairment is not fully known. The high-risk subset of children who undergo surgical PDA ligation also have the highest probability of neurodevelopmental impairment. Prior to ligation, they have altered cerebral perfusion , 160 andprimary outcome) and gross motor, fine motor, cognitive composite, and language composite scores (secondary outcomes) at 22 to 26-months corrected age (=age since birth–number of weeks born before 40-weeks gestation) . In addition, we will examine the effect of PDA duration as a specific feature of 22 to 26-month neurodevelopmental impairment to more thoroughly evaluate the contribution of extended PDA exposure to these outcomes  and PDA, in addition to individual features.A multivariable prediction model for PDA closure risk will be developed in the training cohort (n=450) based on verified features identified by previous work and our hypothesized new covariates of interest . The test/validation set that will be recruited in the year following the initial cohort is expected to consist of 225 neonates. Hierarchical clustering will be used to display the unique patterns of PDA closure based on the top echocardiographic, biomarker, demographic, and clinical features. We hypothesize that in addition to echocardiographic and serum biomarker measures, the magnitude of respiratory support in the first 4-weeks postnatal is an important predictor of PDA closure. Higher respiratory support may predict delayed closure and modify the effect of echocardiographic and serum biomarker measures of PDA intensity on closure probability. Therefore, we will examine interactions between respiratory measures  will be used to select the top predictors of PDA closure risk. The “inner-run” cross-validation and the area under the receiver operating curve (AUC) will be used to choose the optimal tuning parameter. Based on the optimal tuning parameter, the selected model prediction accuracy and sensitivity/specificity will be evaluated based on the test set. An overall receiver operating curve and AUC based on all test set samples will be plotted and estimated after running all 10 folds. The aforementioned cross validation procedure will be replicated 500 times and the distribution of the overall model performance  will be obtained. If the average AUC is acceptable (≥0.70) and the variance is limited (0.05), the final model will be estimated with the entire test set.training stage will be validated in an independent validation, derived from approximately 225 participants in the cohort recruited the year following enrollment of the initial training cohort. Model prediction accuracy will be evaluated by AUC, with an AUC performance ≥0.70 of interest for further exploration in larger cohorts.The final predictive model formulated at the As in Aim 1, we will focus on building predictive models. For the composite outcome of mortality or supplemental oxygen or positive-pressure respiratory support at 36-weeks PMA, we will adopt the same structure as in Aim1, by utilizing the training (450 patients) and validation cohort, recruited thereafter.primary outcome) or mortality (secondary outcome). A ten-fold cross validation procedure will be iterated 500 times to develop the optimal diagnostic model. Penalized logistic regression  will be utilized to predict our composite outcome and select the top predictors of mortality and CLD risk. We will choose the optimal tuning parameter, determine prediction accuracy and sensitivity/specificity, and estimate the final model with the entire test set as described above.Together with clinical features that include respiratory severity and other markers of clinical severity of illness Table , longituprimary and secondary outcomes) will be fit to the validation cohort, and the model fit summarized as previously detailed.Given our interest in both the impact of PDA duration and the additional benefit of PDA closure on outcomes , we will force these two measures (PDA duration and closure) into the model and subsequently compare model fit and accuracy to the models of best predictors that exclude them. Furthermore, we will explore interactions/effect modification between respiratory and other risk factors and PDA measurements  to predict outcomes. Final models , we will also explore models that will jointly model neurodevelopment outcomes and time to loss or death with a Cox survival model. We will follow the same general framework for model development as previously described, using a joint random-intercept Cox model .Aim 1) following enrollment (upwards of 80-85% of the 450 in the training stage), and the relatively limited set of potential covariates, we expect our resulting classification from the training set to have a tolerance/accuracy of at most 0.05 of the optimal accuracy. An accuracy of 0.05 can be interpreted as the expected accuracy of the classifier to be within 5% of the "best" possible accuracy achieved with a binary classifier. Moreover, with 10-fold cross validation, we expect that power will be high (over 80%) with two-sided type I error of 5% with the expected 450 patient target accrual and modest hazard ratios between 1.5-2.0.Sample size in the training/model building stage is based on the justification according to Dobbin et al.  and PangAim 2 primary outcome event, we expect our resulting classification from the training set to have a tolerance/accuracy of at most 0.05 of the optimal accuracy using the justification according to Dobbin et al. [Assuming approximately ¼ of patients (n=112) will experience the n et al. , 179We do not anticipate missing covariate data since we will prospectively record data. However, should substantial covariate data be missing, we will employ multiple imputation techniques.The strengths of this investigation include a prospective cohort design within a unique population of untreated infants with PDA, and innovative modeling using advanced echocardiography, biomarkers, and clinical measures to accurately predict not only spontaneous PDA closure timing, but which infants with persistent PDA are at the highest risk for worsened PDA-associated outcomes . This novel approach will fulfill a critical American Academy of Pediatrics clinical research objective  by deterThe results from this study will immediately inform clinical decision-making and, as a critical step to designing trials that will enroll appropriate participants in randomized trials, will ultimately lead to improved outcomes for preterm infants with PDA. Our weekly prediction models for the first postnatal month, the period when pharmacologic and early interventional closure decisions must be made, will aid practicing clinicians to avoid pharmacologically treating infants unlikely to benefit, and randomized-trial planners to develop evidence-based trial designs to determine whether pharmacologic and interventional PDA closure is beneficial in specific prenatal infant populations, and when PDA treatment is most effective. Because of our diverse population of <30-weeks gestation preterm infants within both delivery and referral hospitals’ NICUs in one of the largest allied NICU networks in the United States, we anticipate findings will be applicable to similar preterm infants with PDA throughout the U.S. and developed world.Although this study uses a prospective cohort and rigorously designed observational study design to answer proposed questions that cannot be answered with a randomized trial, but that are crucial to future PDA trial development, confounding is a potential problem . We willAim 2 we will employ a primary mortality or CLD composite outcome at 36-weeks PMA, the accepted practice among neonatal randomized trialists. For Aim 1 (spontaneous closure) outcomes we will employ Fine and Gray subdistribution hazards models, which will allow us to account for competing mortality risks and limit the analysis to observed follow-up time. In Aim 3  we will explore using joint models to account for drop out and potential death.Early mortality precludes PDA-closure and development of PDA-associated outcomes. Our team has experience in applying composite outcomes and guarding against mortality-associated biases, such as for immortal-time bias, in the preterm infant population , 180. FoAdditional file 1: Table S1. World Health Organization Trial Registration Data Set. (PDF 94 kb)