In 2011, 982 million international touriststraveled around the world to visit friends and relatives, for business, leisure, orother purposes.2 Travelers however are now 10 times morelikely to die from injuries than from infectious diseases, which presents a relativelynew challenge for travel medicine.2Over the years, as travel patterns and destinations are changing, travel medicineis attempting to keep pace to reduce risk of diseases and adverse health events and tomake travel a healthy and enjoyable experience. With increasing availability ofimmunizations and prophylactic treatments, a change in morbidity and mortality patternshas been observed among global travelers. Infectious diseases now account for a verysmall proportion of reported deaths (<2%) among travelers.7 RTI was also the major reason to transfer US citizens out of acountry after non-fatal injuries.2Other causes of injury deaths among travelers include homicide, drowning, andsuicide.7Several studies have examined the causes of mortality among travelers and inthese studies injuries were found to be a leading cause of preventable deaths; andthe most common cause of injury deaths was road traffic injuries (RTIs).8 LMICs account for90% of the world’s fatal RTIs despite having only half the share (48%) of theworld’s vehicles.9 Thus,with increasing travel to LMICs, high-income travelers are exposed to a much higherrisk of RTI than in their home country . country . For ins3 while Tonellato and colleaguesreported a higher proportion of RTI deaths among travelers visiting LMICs of theAmericas.11 These findingsare reflective of the high burden of fatal RTIs in the LMICs of the world . The human tollof such events during travel is immeasurable—lives lost, families affected,and societies deprived of professionals. With more and more young individualsexploring the world through traveling, studying, volunteering, or researchingoutside their home countries, it is imperative that they are protected from alltravel-related harms including injuries.Of course, behind these numbers are real stories of aspiring youngindividuals like Aron Sobel, a US medical student who lost his life, along with 22other passengers while traveling on a bus in Turkey.2 Health educationis an essential but often neglected component of pre-travel consultation; providerstend to focus more on prevention of infectious diseases through vaccination andadministration of prophylactic medications.18 In addition,pre-travel consultations are brief and do not offer enough time to conduct educationand health promotion.17 With international travel soonreaching the 1 billion people traveling per year mark and growing, more effort isneeded to explore ways in which injury prevention can be adequately included inpre-travel consultation.One important strategy for protection is pre-travel consultation, which canplay an important role in injury prevention. A pre-travel consultation is expectedto include an assessment to identify potential risks at the travel site and fromtravel itself; risk communication aimed at discussing the risks identified duringassessment; and risk management through immunizations, prophylactic medications, andhealth education.Journal of Travel Medicine, Piotte and colleagues presentfindings from their study evaluating pre-travel consultation provided by primarycare physicians (PCPs) in France.18 They present the case of a 25-year-old man traveling alone fora 1-month trek in Peru for whom only 30% of PCPs recommended “repatriationinsurance.”18Higher risk of injuries is observed in young men and despite the travel itineraryand age-associated risk, fewer PCPs perceived injuries as a risk. In fact, PCPs weremore likely to recommend water, hand hygiene, and use of condoms than injuryprevention advice. Travelers themselves may also underestimate the risk of injuries,though this perception may change substantially post-travel.19An important prerequisite for communication is risk perception, and ifproviders and travelers do not perceive injuries as risks during travel they areless likely to discuss these or suggest preventive measures. In this issue of the13Some of these factors are preventable and pre-travel consultations can include afocused discussion on road safety measures and provision of resources to seek morespecific advice. Clear messages on the risks and how they can be reduced ought to bean important part of pre-travel consults (The higher risk of RTIs among travelers is caused by many reasons: variedmix of traffic, poor road conditions, unfamiliarity with traffic rules,unavailability of road safety measures—helmets, seatbelts, childrestraints—adventure-seeking attitude during travel, drinking and driving,speeding, lack of concentration because of exhaustion, jetlag, and cell phone usagewhen drivings, amongst others.consults .20 This may turn out to be the caseeven for injury prevention advice; therefore alternative approaches to communicationand development of factual materials will need to be explored. Further research canalso be conducted in the future to study if pre-travel injury prevention advice hasan effect on injury outcomes among travelers; this will provide a measure of realeffectiveness. In the meantime, injuries are a grave risk for travelers and wepropose that pre-travel consultations remain incomplete until they include injuryprevention.It has been observed that travelers do not adhere to all the pre-traveladvice that they receive for prevention of infectious diseases.


Plants are sessile organisms that have evolved exquisite and sophisticated mechanisms to adapt to their biotic and abiotic environment. Plants deploy receptors and vast signalling networks to detect, transmit and respond to a given biotic threat by inducing properly dosed defence responses. Genetic analyses and, more recently, next-generation -omics approaches have allowed unprecedented insights into the mechanisms that drive immunity. Similarly, functional genomics and the emergence of pathogen genomes have allowed reciprocal studies on the mechanisms governing pathogen virulence and host susceptibility, collectively allowing more comprehensive views on the processes that govern disease and resistance. Among others, the identification of secreted pathogen molecules (effectors) that modify immunity-associated processes has changed the plant–microbe interactions conceptual landscape. Effectors are now considered both important factors facilitating disease and novel probes, suited to study immunity in plants. In this review, we will describe the various mechanisms and processes that take place in the nucleus and help regulate immune responses in plants. Based on the premise that any process required for immunity could be targeted by pathogen effectors, we highlight and describe a number of functional assays that should help determine effector functions and their impact on immune-related processes. The identification of new effector functions that modify nuclear processes will help dissect nuclear signalling further and assist us in our bid to bolster immunity in crop plants. Plants are continuously challenged by biotic and abiotic stresses. As sessile organisms they have evolved refined mechanisms to deal with those threats that affect plant health or survival. Decades of intense genetic studies on the processes underpinning growth and reproduction, plant development, adaptation to stress as well as the mechanisms of immunity and susceptibility to pathogens has led to significant insights and critically unveiled common mechanistic themes in seemingly distinct processes . The emeMicrobe or Pathogen Associated Molecular Patterns (M/PAMPs)] or host-derived damage-associated molecules [Damage Associated Molecular Patterns (DAMPs)]. In this scenario, recognition of microbial patterns or the detection of host-derived damage signals leads to the rapid induction of signalling cascades that ultimately lead to the induction of defence gene expression and activation of plant defence responses. Collectively, these processes lead to enhanced immunity to most microbes designated as PAMP-triggered immunity (PTI)  TFs, thereby preventing NAC relocalization to the nucleus during infection and after PTI stimulation  , 28. Thiirulence . The molXanthomonas axonopodis was shown to target the host ubiquitin machinery. It interacts with Ubc13, a ubiquitin conjugating enzyme, avoiding K-63 linked ubiquitination required for DNA repair [Ubiquitination involves the reversible conjugation of ubiquitin to specific lysine residues in a target protein. Protein ubiquitination affects many processes and has been firmly connected with immunity-associated signalling events in plants , 31. In A repair . InteresA repair .A. thaliana E3 sumo ligase Siz1. siz1 mutants exhibit constitutive expression of pathogenesis-related (PR) and disease response genes leading to increased resistance to the bacterial pathogen P. syringae pv. tomato (Pst) DC3000 [Xanthomonas euvesicatoria was shown to target host nuclear sumoylation status. XopD was shown to catalyse the sumo hydrolysis of SIERF4, a tomato ethylene responsive transcription factor, causing its destabilization. The absence of SIERF4 avoids the transcription of ethylene (ET) defence genes required for immunity against Xanthomonas infection [Sumoylation is another PTM highly connected with nuclear plant defence mechanisms. This is evident based on the effects of the mutants of a nuclear ) DC3000 . XopD, anfection . It is wBesides inheritance of genes, the modification of histones, chromatin remodelling and DNA methylation provide another means by which plants can pass on beneficial (immunity related) traits to their progeny. N-terminal histone tails are post-translationally modified, e.g. by acetylation, methylation, phosphorylation, ubiquitination, by modifying enzymes recruited by transcription factors. These enzymes create a ‘histone code’, which causes specific changes in chromatin configuration and gene expression. Chromatin configuration is controlled by ATP-dependent chromatin remodelling complexes, which allow or prevent access to DNA by transcription factors to regulate essential cellular processes . These cP. syringae infection to help defence. This occurs by repressing the transcription factors WRKY38 and WRKY62, which normally negatively regulate the expression of PR genes [Arabidopsis methyltransferase SET DOMAIN GROUP8 is known to activate expression of nucleotide binding-leucine rich repeats (NB-LPR) genes, e.g. LAZARUS5 (LAZ5), which has an activating H3K36me3 mark enriched at the LAZ5 locus during infection [The occurrence of histone modifications at defence-related genes leads to transcriptional regulation of immunity during infection. Two of the best characterized histone modifications are acetylation and methylation. Histone H3/H4 acetylation, which is linked to gene activation, is controlled by histone acetyltransfereases and histone deacetylases (HDACs) . The AraPR genes . HistonePR genes . The Aranfection . Methylanfection .Cochliobolus carbonum HC toxin produced during maize infection inhibits HDAC activity, causing hyperacetylation in susceptible, but not resistant, maize plants [Arabidopsis tissue infected with P. syringae shows massive hypomethylation as a direct result of active demethylation [If plant defences are under some degree of epigenetic control, it is reasonable to assume that these processes would be targeted by pathogens. e plants . It is thylation . AlthougHyaloperonosporaarabidopsidis has been shown to interfere with plant immunity through the secretion of the nuclear localized effector HaRxL44. HaRxL44 decreases SA immune processes by interfering with mediator function causing a shift towards JA/ET signalling, bringing about enhanced susceptibility to the pathogen [Arabidopsis transcription factor TCP14 , which has roles in plant development as well as regulation of defence genes [Xanthomonas species have been shown to use a novel class of effectors, known as transcription activator-like effectors, which function to bind specific host DNA sequences, resulting in the expression of host genes that aid pathogen colonization and infection [Transcriptional reprogramming forms a central component of the plant immune response on perception of a pathogen, whether it occurs during PTI or Effector Triggered Immunity (ETI). As part of a robust defence response against pathogen ingress, dramatic transcriptional changes occur, affecting up to 20% of the host gene complement . Transcrpathogen . The Arace genes , 50, hasce genes , 52. Thinfection , 54.et al. [Phytophthora sojae effectors (PsAvh18 and PsAvh146) inhibit biogenesis of small RNAs to suppress RNA silencing, while Navarro [P. syringae that can either suppress transcription of PAMP-responsive miRNAs or inhibit miRNA function.Similar to many other eukaryotes, plants use a sophisticated RNA interference (RNAi) machinery to control the expression of its gene repertoire and to combat viral infection by degrading viral RNA. Generally, plants use RNAi as a defence response in two ways: transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). During TGS, RNA is recognized by Dicer to produce small interfering RNAs, which are loaded into Argonaute to mediate defence through RNA silencing. PTGS on the other hand involves siRNAs being incorporated into the ARGONAUTE4 containing RNA-induced transcriptional silencing complex, which guides heterochromatin formation and methylation, acting as a positive regulator of plant defence , 55. Botet al.  showed t Navarro  identifiP. syringae infection in Arabidopsis [Alternative splicing (AS) is a nuclear process by which a single pre-mRNA is processed into multiple transcript isoforms, providing a source of diversity within eukaryotic transcriptomes and proteomes . AS has bidopsis , althougbidopsis . Althougbidopsis , which ibidopsis , 63.Arabidopsis, undergoes AS, and the truncated transcripts are required for its immune functions. Removal of RPS4 introns abolished RPS4 function, but transgenic Arabidopsis lines expressing intron-deficient and truncated transgenes of RPS4 showed partial resistance to P. syringae. This shows that the alternatively spliced variant of RPS4 has immune-related functions [S); and a truncated version lacking 13 of the 14 repeats of its LRR domain (NL). The NS transcript is more prevalent before and for 3 h after tobacco mosaic virus (TMV) infection, while the NL version is more prevalent 4–8 h post-infection. As in the case of RPS4, both transcripts of the tobacco N gene were shown to be required for full resistance against TMV infection [Arabidopsis RLKs: suppressor of npr1-1, constitutive 4 (SNC4) and chitin elicitor receptor kinase1 (CERK1). CERK1 is a chitin receptor, and in Arabidopsis mutants lacking SUA and RSN2, the chitin-mediated production of reactive oxygen species is reduced in contrast with the levels of growth of P. syringae in these mutants. This suggests that AS of CERK1 is required for its functions in immunity [RPS4, an R protein from unctions . AS of Rnfection . Anotherimmunity .Despite the importance of AS to plant immunity, no pathogen effectors are known to target this mechanism. Thus, a more in-depth understanding of AS events in plant defence responses and new ways of testing the action of effectors towards AS processes are yet required. Functional assays that report on AS induction or perturbation owing to effector activity could become crucial in understanding plant immunity and susceptibility.Alternative polyadenylation (APA) is another nuclear-based mechanism used to control gene expression at a post-transcriptional level. Immediately after transcription, pre-mRNAs are capped, spliced and cleaved, allowing the addition of a poly(A) tail to their 3′ ends. This tail is essential for mRNA stability, transport to the cytoplasm and recognition by the translational machinery. The occurrence of APA is dependent on the location at which the pre-mRNA is cleaved .Arabidopsis genes [APA appears to be highly present in plants. A deep-sequencing study showed that APA can affect up to 70% transcribed is genes . APA hasis genes .Arabidopsis RNA binding protein FPA, which regulates several APA processes, negatively regulates PTI processes through the immune-related protein ETHYLENE RESPONSE FACTOR 4 (ERF4). ERF4 undergoes APA on flg22 detection in Arabidopsis and FPA prevents it [Arabidopsis resistance protein recognition of peronospora parasitica 7 (RPP7) was shown to undergo APA to control its expression in a histone methylation-dependent manner [APA functions in plants are largely unknown with exception to its connections with flowering time control . Howevervents it . In anott manner . LargelyIt is without question that NGS sequencing and other -omics-driven studies have led to major leaps in our understanding of immunity-associated processes in plants. Given that access to -omics platforms is becoming cheaper, data analyses more straightforward and our understanding of general processes is becoming ever more detailed, we envision that the near future will see proliferation of targeted functional assays designed to identify specific effector functions and test their roles in immunity. We therefore will provide an overview of assays that have recently emerged, are derived from current assays or we deem to be achievable with existing technological platforms. These experimental strategies are summarized in One possible approach to identifying effector functions is the utilization of reporter assays for specific nuclear immunity processes A. A primThese examples highlight how specific reporter assays can be used to effectively identify effectors that target a given immune process. Importantly, these types of assays could be adapted to investigate other nuclear immunity processes highlighted in this article. In these scenarios, constructs that report on the occurrence or disruption of AS or APA could easily be implemented. Using this approach, it is possible that this will enhance our understanding of the nuclear immunity processes perturbed by pathogen effectors during the infection process.Arabidopsis, which have a specific phenotype associated with a determined cellular process. Plants over-expressing pathogen effectors could be phenotypically screened against known Arabidopsis mutants allowing the association of an effector with a cellular process , that indeed causes aberrant flowering development when over-expressed in Arabidopsis [Arabidopsis. SAP11 was then shown to target the host nucleus and to enhance colonization of the AY-WB insect vector Macrosteles quadrilineatus when over-expressed in Arabidopsis [Arabidopsis expressing SAP11 had similarities with plants over-expressing miR319, a microRNA induced by phosphate starvation responses. Identifying two over-expressing lines, which show altered phosphate starvation responses, suggests similar cellular processes are being targeted [Arabidopsis lines, over-expressing pathogen effectors, could yield important leads to infer effector function.A study in Phytoplasma successfully used such an approach. Phytoplasma are insect-transmitted bacterial pathogens that cause severe phenotypic changes in the host morphology to enhance the fitness of their insect vectors. These phenotypic changes include aberrant flower development and abnormal host tissue growth and were proposed to be caused by the action of effector proteins secreted by phytoplasma. Thus, 56 candidate effectors of Aster Yellows phytoplasma strain ‘Witches Broom’ (AY-WB) were expressed in bidopsis , 74. Anobidopsis . The phetargeted . Thus, wIn an approach that is akin to phenocopy assays, mutants with altered specific cellular process could be complemented with pathogen effectors and screened phenotypically. Complementation of mutant phenotypes would allow identification of direct links between effectors and the cellular mechanism they target.A. thaliana–H. arabidopsidis/P. syringae pathosystem, a large-scale Yeast-two Hybrid (Y2H) screen of 83 effectors from both pathogens suggested that effectors are likely to target a set of interconnected plant proteins, which are conserved between plant pathosystems [in vivo. We surmise that the deployment of -omics technologies to study effector activity in plants forms a complementary, but as of yet untested approach to study effector functions. Given the availability and decreasing cost of NGS technology, effectors and effector-triggered modifications can be dissected one by one to unveil the mechanisms of effector triggered susceptibility (ETS). Effectors can be expressed in planta, after which functional analysis could be pursued by means of proteomics  and NGS-based functional genomics  . A large number of studies have been focused on individual effectors. However, to fully understand plant–pathogen interactions, large sets of identified effectors need to be screened. In the osystems . The limhyl-seq) C. In Araesponses . Transcrl kinase . If implArabidopsis, several genes required for the progression of cell cycle were reported to affect the expression of plant R genes and contribute to ETI [We continuously acquire knowledge about the plant immune system and plant–pathogen interactions, but there is still a lot unknown. The interconnected nature of the plant immune system highlights the necessity to expand the toolset with which to interrogate plant immune signalling. A recent model suggests that activation of R genes results in the induction of reactive oxygen species (ROS) production and the modification of Red/Ox balance, causing growth inhibition . Moreovee to ETI , 80. Thee to ETI , PAMP ree to ETI . It was e to ETI . Finallye to ETI  and is te to ETI .Here, we have described a variety of well-known nuclear processes involved in plant immune responses. Because only few pathogen effectors that target these processes are known, we have suggested several approaches for the large-scale identification of effector function involved in nuclear immunity processes. It should be noted, however, that there are many other cellular processes/components that are thought to be involved in plant immunity. The increasing number of examples implies not only the complexity of plant immune system but also its pluri-sided and interconnected nature. For better understanding of plant–pathogen interactions, it is important to generate an authentic network view of plant immune signalling. This will allow us to dissect the critical processes, model them  and, subThe plant immune system is regulated by a vast signalling network, much of which is located in the host nucleus. The immune signalling network intersects with other key developmental and regulatory processes within the cell.Pathogens suppress host immunity by secreting arrays of proteins (effectors) that perturb host signalling and other cellular events.Next-generation sequencing and functional genomics approaches have helped (i) identify nuclear processes that underpin immunity and (ii) identify and group pathogen effector classes, some of which target processes that reside in the host nucleus.The availability of host and pathogen genome sequences, systems level information on immunity-associated processes and increasingly accessible next-generation -omics platforms, will inspire new functional assays to dissect the host immune system.


Immunosuppression for solid organ transplantation increases lymphoproliferative disease risk. While central nervous system (CNS) involvement is more rare, we noticed an increase in primary CNS (PCNS) disease. To investigate a potential association with the immunosuppressive regimen we identified all post-transplant lymphoproliferative disease (PTLD) cases diagnosed over a 28-year period at our institution  and all similar cases recorded in a United Network for Organ Sharing-Organ Procurement and Transplant Network (UNOS-OPTN) data file. While no PCNS cases were diagnosed at our institution between 1986 and 1997, they comprised 37% of PTLD cases diagnosed from 2011–2014. PCNS disease was more often associated with renal vs. other organ transplant, Epstein-Barr virus, large B-cell morphology and mycophenolate mofetil (MMF) as compared to PTLD that did not involve the CNS. Calcineurin inhibitors were protective against PCNS disease when given alone or in combination with MMF. A multivariate analysis of a larger UNOS-OPTN dataset confirmed these findings, where both MMF and lack of calcineurin inhibitor usage were independently associated with risk for development of PCNS PTLD. These findings have significant implications for the transplant community, particularly given the introduction of new regimens lacking calcineurin inhibitors. Further investigation into these associations is warranted. Post-transplant lymphoproliferative disease (PTLD) is a well-known complication of solid organ transplantation with both type and level of immunosuppression associated with risk for developing lymphoproliferative disease. Renal transplant recipients have been reported to have a relative risk of 12.6 of developing PTLD compared to the general population of developing lymphoma, while transplants requiring a higher level of immunosuppression, such as liver or combined heart/lung, have a relative risk from 30 to as high as 240  comparedPTLD may involve any organ with central nervous system (CNS) involvement in 10–15% of cases –8. For eIn our practice we noticed an apparent increase in the number of diagnoses of primary CNS (PCNS) PTLD. As immunosuppressive therapy has advanced, the association between the immunosuppressive drug regimen and the site at which lymphoproliferative disease develops has not been extensively investigated. We performed a retrospective review of all PTLDs diagnosed at our institution over the past 28 years and analyzed publicly available data collected by the United Network for Organ Sharing (UNOS) and Organ Procurement and Transplant Network (OPTN).We identified 177 cases of PTLD (Table The most common indications for kidney transplant among patients who developed PTLD were diabetes(9), hypertension(7), glomerulnephritis(6), obstructive uropathy(5) and systemic lupus erythematosus(5). Except for glomerulonephritis(6), IgA nephropathy(3) and cystinosis2), which were seen only in non-CNS PTLD patients, primary diseases requiring transplant were similar between PCNS and non-CNS PTLD patients . While many of these cases were seen in consultation, a similar trend was observed when only PTLD diagnoses made on in house surgical and autopsy specimens were considered . Given the increase in the percentage of PCNS PTLDs in relation to all PTLD diagnoses, which have remained relatively constant over the last 15 years, these findings are not merely a function of an increase in the number of transplants performed. Similarly, the number of total PTLD cases received in consultation has not increased over the past 15 years , and most of the classifiable lymphomas were large B-cell lymphomas Figure . Histopap < 0.005, Figure Irrespective of morphologic type, PCNS PTLD was associated with EBV (27/28) compared to non-CNS PTLD  in the year prior to and/or at the time of diagnosis . Patients who received MMF and CNIs together had an intermediate risk of developing PCNS disease, with an 18-fold increased odds compared to patients on CNIs alone , suggesting a partial protective effect of CNIs against the development of PCNS PTLD. There was no evidence to suggest a dose effect of MMF as a factor in these differences. Standard dosing regimens are recommended at our institution for adult patients, although they may be adjusted based on individual side effects or other factors. In adult patients where data were available, MMF dosing was similar between patients that developed PCNS and non-CNS PTLD and between patients who were taking MMF regimens with or without CNIs .While nearly all PCNS PTLD were associated with both MMF treatment and EBV, no direct relationship was seen between MMF, CNIs and EBV in non-CNS PTLD Figure . For exaA second analysis was performed using a larger, UNOS-OPTN STAR data file that included a total of 6966 PTLD cases, of which 4569 had associated immunosuppression data. While UNOS-OPTN data collection does not routinely include PTLD site, searching additional text fields identified 84 patients who were specifically recorded as having primary CNS involvement. Both PCNS and non-CNS cases were more common in men than women , which reflected the distribution seen in the overall transplant population (62%). The distribution of total solid organ transplant recipients according to ethnicity was 65% white, 18% black and 11% Hispanic with a similar distribution seen in both PCNS and non-CNS PTLD patients (Table p < 0.001). In addition, patients on MMF without CNIs were more likely to develop PCNS than non-CNS disease as compared to patients on MMF with a CNI , demonstrating a protective effect of CNIs against PCNS PTLD. Nonetheless, the majority of both PCNS and non-CNS PTLDs still arose in patients on regimens containing CNIs as these are the most common transplant regimens. Both CNIs (tacrolimus and cyclosporine) had a similar protective effect. In the absence of MMF, the odds of developing PCNS compared to non-CNS PTLD were unaffected by whether cyclosporine or tacrolimus was taken .As in the JHH dataset, patients in the UNOS-OPTN dataset who developed PCNS PTLD were more likely to have been taking MMF than patients who developed non-CNS PTLD Figure . PCNS PTKidney transplant recipients represented the highest fraction of PTLD patients within the UNOS-OPTN dataset (42%); however, similar to the JHH dataset, renal transplant recipients were relatively over-represented in PCNS PTLD . In addition, independent of the immunosuppressive regimen used, the odds of developing PCNS PTLD was significantly lower for both liver  and thoracic  transplants compared to kidney transplant. Gender  and ethnicity (black or other compared to white) did not significantly alter odds of developing PCNS PTLD in this model, although there was a borderline increase in odds of PCNS PTLD among patients aged 18–50 or > 50 compared to those < 18.Analysis of the UNOS-OPTN dataset using Firth's penalized regression method showed that the odds of PCNS PTLD were significantly increased when taking MMF as compared to not taking MMF . However, data thus far suggests that gene expression with EBV-associated lymphomas is not unique in elderly patients compared to patients younger than 50 years and common mechanisms may be involved .Regardless of potential mechanism, this is the first study to demonstrate a statistically significant association between the immunosuppressive drug regimen and PCNS PTLD. Our findings suggest that the decline in use of CNIs following the introduction of MMF in 1995 may have unmasked an inherent susceptibility of the CNS for immunosuppression-related lymphoproliferative disease.Identifying risk factors associated with PCNS lymphoproliferative disease is important given the challenges of CNS-directed therapy , 44 as wGiven the wide variability in immunosuppression regimens between institutions, these findings highlight the need to initiate multi-center trials to optimize graft function while minimizing toxicity and PTLD risk. Further investigation into potential drug interactions and strategies for the prevention of PCNS PTLD are warranted.Following institutional review board approval, the Johns Hopkins Hospital pathology database was searched from 10/1986 to 8/2014 to identify all in-house, autopsy and consultation cases for patients with a diagnosis of PTLD who had received a solid organ transplant. Bone marrow transplant patients were excluded as the majority were for a primary hematolymphoid disorder. Clinical, morphologic and phenotypic characteristics of these neoplasms were recorded where available, including patient age, sex, type and reason for transplant, date of transplant in relation to the timing of diagnosis, immunosuppressive regimen and survival. In all cases, the maintenance immunosuppressive regimen for one year prior to the onset of patient PTLD symptoms was verified and included in the analysis. A similar search was performed on a UNOS Standard Transplant Analysis and Research (STAR) file using OPTN data on all solid organ transplant recipients from 10/25/99 (when PTLD-specific data fields were first included) to 4/14/14.z-test. A logistic regression analysis on the UNOS-OPTN dataset was performed using Firth's penalized logistic regression. It was necessary to use a penalization due to the small number of identifiable cases of PCNS PTLD within the UNOS dataset. The regression included whether the drug regimen at the time of diagnosis or prior to diagnosis contained sirolimus, mycophenolate mofetil (MMF), or calcineurin inhibitors (CNIs) tacrolimus or cyclosporine. Age, gender, ethnicity and transplant type were included as potential confounders.Odds ratios with 95% confidence intervals (CI) were calculated by the Fisher's exact test to compare the association of PCNS and non-CNS disease with the drug regimen. Chi-squared tests were used to test associations with other parameters. Evaluation of PCNS compared to non-CNS PTLD incidence over time was done using a one-tailed, two-sample proportion


Despite adequate treatment, the abdominal pain and elevated creatinine continued to persist. Initial ascites fluid cultures returned back positive for growth of Enterococcus gallinarum. Empiric antibiotics were then substituted with ampicillin/sulbactam. Our case of Enterococcus gallinarum causing SBP is only the seventh case reported in the literature to date.We present the case of a 60-year-old Caucasian male with history of hepatitis C viral cirrhosis with portosystemic encephalopathy and ascites with evidence of spontaneous bacterial peritonitis (SBP) with absolute neutrophil count (ANC) of 944 cells/ Escherichia coli, Klebsiella pneumoniae, and Streptococcal pneumonia [The most common and life-threatening infection in decompensated cirrhosis is spontaneous bacterial peritonitis (SBP), with prevalence of 10%–35% and in hospital mortality rates ranging from 20 to 40% [µL blood. The patient complained of mild generalized abdominal pain without fever, chills, nausea, vomiting, chest pain, or dyspnea. His past medical history includes successful treatment of chronic hepatitis c virus with evidence of sustained virological response on admission given negative HCV PCR viral loads. The patient lives on a small farm and reveals frequent animal exposure to rabbits, squirrels, and dogs along with oral-to-oral contact through kissing all his pets. He denied any consumption of alcohol. The patient presented with recurrent ascites refractory to step one diuretics with furosemide 40 mg daily and spironolactone 100 mg daily. He was hospitalized three months prior for hepatic encephalopathy and ascites with no clear precipitant identified at that time. Laboratory results from three months ago were notable for creatinine of 1.05 and an overall MELD of 8. The patient struggled with issues of ascites  and portosystemic encephalopathy since that time. Computed tomography (CT) of the abdomen and pelvis with and without contrast revealed ascites, splenomegaly, and cirrhosis with portal venous hypertension and new-onset complete occlusion of the portal vein at the level of the confluence of the superior mesenteric vein  was initiated on day 3 of hospitalization including albumin human , midodrine, and octreotide. The patient's kidney function and urine output continued to decline despite HRS therapy and eventually required initiation of hemodialysis 1 day after the resolution of his SBP and then continued to require HD indefinitely.A 60-year-old Caucasian male with history of hepatitis C viral cirrhosis with portosystemic encephalopathy and ascites was admitted after routine therapeutic paracentesis (9.2 L removed) revealed evidence of spontaneous bacterial peritonitis (SBP) with absolute neutrophil count (ANC) of 944 cells/ Figures . Initial Enterococcus gallinarum causing SBP in a 60-year-old Caucasian man with hepatitis C viral cirrhosis is only the seventh case reported in literature to date [ Enterococci has been rising in the last decade resulting in treatment failures and poorer outcomes. Specifically, Enterococcus gallinarum is not known to cause human disease. It is suspected to have colonized the intestinal tract and have been acquired orally from prior exposure to animals [ E. gallinarum strains have a specific vanC gene that confers low-level vancomycin resistance [ Enterococcus gallinarum spontaneous bacterial peritonitis have been reported to date, with only one previously reported in a patient with direct animal exposure [Our case of to date –5. Despi animals . Infecti animals , 5. E. gsistance . Howeversistance , just prexposure –5. Enterococci, it is imperative to follow-up cultures and address these patient populations so appropriate antibiotics, such as ampicillin or gentamycin, can be swiftly initiated.Our case represents the importance of getting a thorough patient history, as it is necessary to highlight certain at-risk patient populations for enterococci infection, such as patients with significant animal exposure or those who are immunosuppressed. Since empirical third-generation cephalosporin therapy does not cover


Mediterranean pine forests display high resilience after extreme climatic events such as severe droughts. However, recent dry spells causing growth decline and triggering forest dieback challenge the capacity of some forests to recover following major disturbances. To describe how resilient the responses of forests to drought can be, we quantified growth dynamics in plantations of two pine species  located in south-eastern Spain and showing drought-triggered dieback. Radial growth was characterized at inter- (tree-ring width) and intra-annual (xylogenesis) scales in three defoliation levels. It was assumed that the higher defoliation the more negative the impact of drought on tree growth. Tree-ring width chronologies were built and xylogenesis was characterized 3 years after the last severe drought occurred. Annual growth data and the number of tracheids produced in different stages of xylem formation were related to climate data at several time scales. Drought negatively impacted growth of the most defoliated trees in both pine species. In Scots pine, xylem formation started earlier in the non-defoliated than in the most defoliated trees. Defoliated trees presented the shortest duration of the radial-enlargement phase in both species. On average the most defoliated trees formed 60% of the number of mature tracheids formed by the non-defoliated trees in both species. Since radial enlargement is the xylogenesis phase most tightly related to final growth, this explains why the most defoliated trees grew the least due to their altered xylogenesis phases. Our findings indicate a very limited resilience capacity of drought-defoliated Scots and black pines. Moreover, droughts produce legacy effects on xylogenesis of highly defoliated trees which could not recover previous growth rates and are thus more prone to die. Mediterranean forests are able to recover following major disturbances such as droughts by displaying high resilience  apparently well adapted to withstand drought stress  and Mediterranean (black pine) distributions to provide a measure of resilience in similar drought-prone forests. We capitalize on a drought-induced dieback caused by severe late-20th century droughts affecting Spain  and leading to growth decline and enhanced defoliation in pine plantations located in SE Spain  and black pine (Pinus nigra Arn.) in the 1970s, with most stands of each species located at approximate elevations of 1850–2000 m and 1700–1850 m a.s.l., respectively . Geological substrates are Paleozoic schist and quartzites leading to regosols soil types.The study area is located in the Sierra de Filabres, Andalusia, SE Spain  but presented contrasting defoliation degrees  is assumed to be a meaningful indicator of tree growth because it removes variation in growth attributable to increasing circumference and it is related to the transpiring crown surface  until becoming mature Mahmood, . This prMicro-cores were placed in Eppendorf tubes containing a mixed solution of formaldeid, acetic acid and ethanol (5:5:90) and stored as soon as possible at 5°C in order to avoid tissue deterioration. All samples were then processed within a maximum of 1–2 weeks after sampling. Micro-cores were sectioned using a sledge microtome  achieving samples 20-μm thick. Sections were mounted on glass slides, stained with 0.5% water solution of cresyl fast violet, fixed with Eukitt® and observed at 100–200x magnification under a light microscope (Olympus BH2).Four different xylogenesis phases were identified as follows  were recorded hourly and then converted to daily values  using a HOBO microclimate station  located in each pine stand  and ring-width indices. To detect time-dependent growth responses to climate, daily regional climate data were either averaged (temperature) or summed  at 10-day and 15-day long scales following Sánchez-Salguero et al. .x0.5-transformed) number of different types of tracheids along time, and checked the predicted values and residuals looking for signals of heteroscedasticity  were evaluated at 5-, 10-, and 15-day long time scales since daily dynamics of tracheid radial expansion have been described in Scots pine  in the case of D trees  as compared with trees presenting intermediate (I) or low (N) defoliation levels whose mean BAI values did not differ . These differences were not associated to tree size which did not differ between defoliation classes  dropped in all trees during the dry years 1994–1995, 1999, and 2005 Figure . These BDifferences in growth between defoliation classes could be traced back in time. Only the D trees showed a lower tree-ring width than the other defoliation classes considering the 1985–2008 period, and this difference was more evident in black pine  saplings under controlled conditions  trees showing dieback or in black spruce saplings experiencing imposed water deficit, respectively. However, the stem wood of defoliated silver fir trees showed a higher susceptibility to decay implying different lignifications processes , which particularly affects black pine, reduce radial growth but not the concentrations of non-structural carbohydrates  defoliated trees. Moreover, droughts produce legacy effects on xylogenesis of these severely defoliated trees which show irreversible growth decline and are prone to die.GG and RS performed the data collection in the field and contributed significantly to data analysis, discussing the results, and writing of the paper. JC conducted the statistical analysis and the manuscript redaction. RN has contributed to the study design and the manuscript discussion and approval.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.


One main interest in miRNA research is the investigation of their functional roles, which can typically be accomplished by identification of mi-/mRNA interactions and functional annotation of target gene sets. We here present a novel method “miRlastic”, which infers miRNA-target interactions using transcriptomic data as well as prior knowledge and performs functional annotation of target genes by exploiting the local structure of the inferred network. For the network inference, we applied linear regression modeling with elastic net regularization on matched microRNA and messenger RNA expression profiling data to perform feature selection on prior knowledge from sequence-based target prediction resources. The novelty of miRlastic inference originates in predicting data-driven intra-transcriptome regulatory relationships through feature selection. With synthetic data, we showed that miRlastic outperformed commonly used methods and was suitable even for low sample sizes. To gain insight into the functional role of miRNAs and to determine joint functional properties of miRNA clusters, we introduced a local enrichment analysis procedure. The principle of this procedure lies in identifying regions of high functional similarity by evaluating the shortest paths between genes in the network. We can finally assign functional roles to the miRNAs by taking their regulatory relationships into account. We thoroughly evaluated miRlastic on a cohort of head and neck cancer (HNSCC) patients provided by The Cancer Genome Atlas. We inferred an mi-/mRNA regulatory network for human papilloma virus (HPV)-associated miRNAs in HNSCC. The resulting network best enriched for experimentally validated miRNA-target interaction, when compared to common methods. Finally, the local enrichment step identified two functional clusters of miRNAs that were predicted to mediate HPV-associated dysregulation in HNSCC. Our novel approach was able to characterize distinct pathway regulations from matched miRNA and mRNA data. An R package of miRlastic was made available through:  MicroRNAs (miRNAs) represent small-single stranded RNA molecules that own the ability to bind to the complementary 3pression ,2. MiRNApression ,5. MiRNApression ,7. Dysrepression .et al. identified a set of core miRNAs implicated in HPV pathogenesis [miRNAs may act as oncogenes or tumor-suppressor genes, as demonstrated in various studies across many cancer types ,10,11, iogenesis ,26. Striin silico target prediction methods reside in the high false positive prediction rates and unspecificity for a given setup [Regulation of cellular processes by miRNAs was thought to be mediated through their target genes. It is thus worthwhile to infer target relationships of miRNAs in order to reveal their functional roles. The idea builds on two subsequent steps: (1) identification of mi-/mRNA gene interactions and (2) functional annotation of target genes. As for (1) obtaining mi-/mRNA gene interactions, both computational and experimental methods were used. Several bioinformatics algorithms have been proposed to predict miRNA targets: TargetScan uses a statistical model based on stringent seed pairing, site features and likelihood of preferential conservation , miRandaen setup . Other aen setup . Experimen setup  or High-en setup . The Staen setup . Other men setup , PAR-CLIen setup  or MIRZAen setup  used CLIWhile simultaneous measurements of entire transcriptomes including mRNAs and miRNAs have become relatively straightforward with high-throughput techniques, their integration is not trivial. Several methods have been proposed to integrate miRNA and mRNA data for the identification of mi-/mRNA networks. An intuitive approach for finding associations between miRNAs and mRNAs on expression level is correlation analysis ,38. Genep-values for targets of randomly selected miRNAs, revealing the intrinsic bias in the target prediction [To reveal functional properties of target gene sets, common approaches were based on statistical enrichment testing to identify over-represented functional categories ,47,48. Fediction . A combiWe have set out to develop a method, called miRlastic, to infer miRNA functions in a data-driven manner using statistical inference of mi-/mRNA interactions, as well as functional characterization of the inferred network A.To obtain condition-specific mi-/mRNA interactions, we developed a statistical inference method taking into account matched miRNA and mRNA expression data of the underlying conditions together with prior knowledge of sequence-based predictions.We used a multiple linear regression model with elastic net penalty, which is a trade-off between lasso and ridge regression that accounts for both joint effects of several miRNAs on a common target B and co-On synthetic data, miRlastic outperformed commonly used methods and was especially suitable for low sample sizes. To functionally annotate miRNAs based on the inferred mi-/mRNA network, we introduced a local enrichment analysis that scores miRNAs given the underlying network structure with respect to functional annotations of target genes.We thoroughly evaluated miRlastic on a cohort of HNSCC patients provided by The Cancer Genome Atlas (TCGA). We inferred an mi-/mRNA regulatory network for human papilloma virus (HPV)-associated miRNAs in HNSCC. The resulting network best enriched for experimentally validated miRNA-target interactions, when compared to commonly used methods. Finally, the local enrichment analysis (LEA) procedure identified two functionally distinct clusters of miRNAs that were predicted to mediate HPV-associated dysregulation in HNSCC. An R package of miRlastic was made available through miRlastic .We downloaded Level 3 mRNA and miRNA RNA-seq data from the TCGA data portal. The mRNA Level 3 data consisted of gene expression measurements which were generated following the protocol previously described by the TCGA consortium . For anaradation . For eacradation  in orderradation , we alsoradation .X and Y as two matrices that contain miRNA s samples, with n and m represent the number of measured miRNAs and mRNAs, respectively. We denoted the regulatory interaction of miRNAs and their putative mRNA targets as a bipartite graph G. The bipartite graph G captures all putative mi-/mRNA interactions in G for the miRlastic inference method yielding an optimized graph, We defined ion 6.2)  and usedWe exemplary calculated pairwise Pearson correlation coefficients among all miRNA expression profiles from the HNSCC dataset, which were predicted by TargetScan with the common target C9orf85 A. Note tTo systematically analyze whether miRNA expression profiles were typically correlated when sharing a putative target, we next defined a measure of correlation strength Note that only the upper triangular matrix with Using the correlation strength measure G all connected j as To model the regulatory behavior of several miRNAs jointly modulating one common mRNA target The mi-/mRNA interaction can be modeled without feature selection by a linear regression model, separately for each mRNA as:As shown above, high co-expression among miRNAs is typical for miRNAs with functional similarity and thus, needs to be taken into account when performing feature selection. From correlation between miRNAs and their target gene, the effects of joint actions of miRNAs were detectable.For network inference, we next performed feature selection using penalized regression (elastic net ). We then levels . The βj λ was identified by 10-fold cross-validation using the glmnet package. For estimating the regression coefficients The parameter approach , which iα with respect to the potentially expected fraction of correlated predictor groups. The parameter To tune the elastic net penalty, we adjusted α allowed for an unbiased parameter tuning whereas lower values of The choice of the parameter z non-zero entries. In this case, edge weights were given by scaled negative regression coefficients. We transformed weights by To identify local, closely-connected functions within the inferred network, we designed a local enrichment analysis (LEA) procedure. We evaluated whether node arrangements were assigned to a certain term, describing e.g., a molecular function or biological process, occurred by chance or not. Proximity of two target gene nodes in the network were measured by their shortest path. The bipartite graph k and a and b is then called shortest path A path between two gene nodes was defined as m annotated genes for a specific term k with j in p-value indicating whether the values of p-values to assess a score j.Next, we scored local neighborhoods in the network. Let s (KEGG) , and letTo finally characterize the importance of miRNAs in p-value that is p-value for node B was not significant k, whereas node B is not. Given the p-values from the left-tailed Wilcoxon rank-sum test, we can calculate the scores of the two nodes M and B as We next compared the distributions of shortest distances to the background distribution that represents the shortest distances to genes, which did not overlap with the gene set assigned to the term C. We obsk and the genes in p-values (Bonferroni corrected) were below a significance threshold of Finally, we were interested in finding local enrichment of a set of terms given their associated gene sets. With glmnet package [The whole miRlastic pipeline was implemented within the R environment for statistical computing . For ela package . To calc package  in the i package  package.http://icb.helmholtz-muenchen.de/ mirlastic [MiRlastic can be downloaded as an R package from irlastic .σ. In addition to the miRlastic inference, we also applied correlation analysis and lasso on the generated profiles. For the correlation analysis, a synthetic miRNA was considered to be a true regulator of a mRNA if the adjusted p-value (Bonferroni corrected) of the negative Pearson correlation coefficient was below 0.05. For lasso, we used the miRlastic inference method with a fixed We set up a test environment: in each of the test runs, we generated a set of synthetic miRNA and mRNA expression values adapting to biological features. We modeled a set of miRNAs with expression values d miRNAs a. We genWe counted the number of falsely chosen miRNAs , since the measurements need to be performed twice. Here, we could show that the miRlastic inference was able to reliably identify true regulators with high specificity and sensitivity in a biologically reasonable synthetic test environment.et al. study [To understand HPV-associated miRNA-mediated gene regulation of HNSCC, we applied the miRlastic inference to the HNSCC TCGA data set. As HPV was shown to disrupt cellular differentiation in HNSCC ,63,64, wl. study , miR-363p-value of p-value of We next asked whether the network inferred by miRlastic was specifically enriched for experimentally validated mi-/mRNA interactions. Therefore, we collected experimentally validated interactions from StarBase and compared performance to three other existing methods, namely lasso and Pearson as well as Spearman correlation analysis. A Fisher’s exact test was conducted in order to determine whether the fraction of inferred and validated interactions was higher than expected from the prior target network (TargetScan) with respect to the number of inferred interactions C. For mily lower C. PearsoIn the previous sections, we described the network that resulted from the analysis of mi-/mRNA data from tumors of patients from the TCGA HNSCC cohort with known HPV status. To finally gain insights into the functional role of the identified mi-/mRNA pairs, we analyzed miRNA-target genes for local enrichment given the HNSCC network. For the application of LEA on the HNSCC mi-/mRNA interaction network, we downloaded 108 pathways from KEGG for gene annotations .In total, we obtained nine significantly locally enriched pathways . OverallWe next clustered miRNAs according to their functional miR score that reflected the strength of interactions of miRNAs with a particular pathway identified two clusters with similar pattern of miR scores across pathways .κB, which is involved in regulating chief processes governing tumorigenesis such as immune response, apoptosis, proliferation and angiogenesis [The two pathway MAPK- and neurotrophin-signaling pathway associated with both clusters suggesting that they were orchestrated by most miRNAs in HPV-associated dysregulation. Notably, MAPK-, RAS-, neurotrophin and toll-like receptor (TLR) signaling share common elements such as a series of MAPKs, protein kinase B (AKT) and most importantly the downstream located transcription factor NF-ogenesis ,73. Inteogenesis  and indeogenesis . This fiThe first miRNA cluster was characterized by associations of many miRNAs  mostly with the endocytosis pathway. Endocytosis was shown to be deregulated in cancer cells in such a way that cell surface molecules with growth advantage effects were recycled at higher rates while molecules with reduced growth effects or those making cells recognized by the immune system were removed from the cell surface .The second miRNA cluster was functionally broader and characterized by associations between hsa-miR-2355-5p/3p, -193b, -1910 and 3166 with cytokine-cytokine receptor (CCR) interaction, regulation of actin cytoskeleton, RAS signaling, apoptosis, toll-like receptor (TLR) signaling and signaling involved in regulating pluripotency of stem cells. Disruption of the apoptosis pathway is one of the hallmarks of cancer and is the result of bypassing cell cycle control in cancer cells, which, in turn, leads to uncontrolled proliferation of cells . DisturbThe oncoproteins E6 and E7 are known to bind several cellular targets, which have been described to regulate, among others, cytokine signaling, thereby providing a link between HPV-status and the “cytokine-cytokine receptor interaction” pathway . Cytokinversus HPV-HNSCC and, therefore, support plausibility of the results in the context of the biology of HPV+ HNSCC tumors.In all, the pathways identified applying miRlastic were in context with signaling disturbance known to be associated with HNSCC tumorigenesis. Moreover, the identified pathways were also known to be related to HPV-status of HNSCC, strengthening the validity of the generated mi-/mRNA regulatory network. Our finding suggested that the two clusters of miRNA-mediated target gene regulation were characterized by distinct signaling pathways deregulated in HPV+ We have demonstrated the power of coupling an miRNA-characteristic mi-/mRNA target inference method to a local enrichment analysis on the inferred network, in order to identify functional roles of miRNAs in a given experimental setting. The proposed target inference of miRlastic employed a linear regression model with elastic net penalization and negativity-constrained coefficient estimation. The choice of balancing between L1 and L2 was achieved by capturing the co-expression structure of miRNAs independently for each common target gene. The miRlastic inference was able to best enrich for experimentally validated interactions. The local enrichment analysis and subsequent clustering of functional miR scores across pathways, finally allowed us to assign functional roles to sets of miRNAs as given by short distances in the inferred networks between target genes of a selected pathway. The method was made publicly available as an R package. We applied mirRlastic to HPV-associated mi-/mRNA regulation in HNSCC data and were able to identify two miRNA clusters of each distinct functional roles. The HPV-associated analysis in HNSCC patients provided, via the identified mRNA targets, descriptive and mechanistic insights into the molecular phenotype of HPV-driven HNSCC and further validated the meaningfulness of the miRlastic method at a biological and clinical level. We used precursor miRNA expression instead of mature miRNA expression, which was used in previous applications of our approach ,45, and


Snakebite envenoming is a major public health problem throughout the rural tropics. Antivenom is effective in reducing mortality and remains the mainstay of therapy. This study aimed to determine the cost-effectiveness of using effective antivenoms for Snakebite envenoming in Nigeria.Economic analysis was conducted from a public healthcare system perspective. Estimates of model inputs were obtained from the literature. Incremental Cost Effectiveness Ratios (ICERs) were quantified as deaths and Disability-Adjusted-Life-Years (DALY) averted from antivenom therapy. A decision analytic model was developed and analyzed with the following model base-case parameter estimates: type of snakes causing bites, antivenom effectiveness to prevent death, untreated mortality, risk of Early Adverse Reactions (EAR), mortality risk from EAR, mean age at bite and remaining life expectancy, and disability risk (amputation). End-user costs applied included: costs of diagnosing and monitoring envenoming, antivenom drug cost, supportive care, shipping/freezing antivenom, transportation to-and-from hospital and feeding costs while on admission, management of antivenom EAR and free alternative snakebite care for ineffective antivenom.We calculated a cost/death averted of ($2330.16) and cost/DALY averted of $99.61 discounted and $56.88 undiscounted. Varying antivenom effectiveness through the 95% confidence interval from 55% to 86% yield a cost/DALY averted of $137.02 to $86.61 respectively. Similarly, varying the prevalence of envenoming caused by carpet viper from 0% to 96% yield a cost/DALY averted of $254.18 to $78.25 respectively. More effective antivenoms and carpet viper envenoming rather than non-carpet viper envenoming were associated with lower cost/DALY averted.Treatment of snakebite envenoming in Nigeria is cost-effective with a cost/death averted of $2330.16 and cost/DALY averted of $99.61 discounted, lower than the country's gross domestic product per capita of $1555 (2013). Expanding access to effective antivenoms to larger segments of the Nigerian population should be a considered a priority. Snake bite is a major public health problem throughout rural communities in West Africa and leads to a significant number of deaths and disabilities per year. Even though effective antivenoms exist against the locally prevalent carpet viper and other poisonous snakes, they are generally not available in community settings, possibly because of their high acquisition cost. We evaluated the cost-effectiveness of making antivenom more broadly available in Nigeria by comparing the treatment costs associated with antivenom therapy against their medical benefit in reducing the risk of mortality. We find that the incremental cost effectiveness ratio (ICER) associated with making antivenom available in Nigeria was $2,330 per death averted and $100 per disability adjusted life year (DALY) averted. Both of these suggest that snakebite antivenom is highly cost-effective in Nigeria and they also compare very favorably against other commonly funded health interventions for which similar estimates exist. Since a substantial reduction in mortality and DALYs could be achieved at a relatively modest upfront cost, expanding access to antivenom to broader parts of the population should be a priority consideration for future investments in healthcare. Snakebite envenoming is a major public health problem among agricultural communities in the savanna region of West Africa Echis ocellatus) attacks, which accounts for at least 66% of all snakebites. However its range is primarily limited to the savannah regions of Nigeria Naja nigricollis), puff-adder (Bitis arietans), mamba (Dendroaspis polylepis), burrowing asp or stiletto snake (Atractaspis microlepidota), night adder (Causus maculatus) and very rarely boomslang (Dispholidus typus). With the exception of boomslang envenoming from them present with negative or normal clotting on 20WBCT, local swelling, necrosis and tissue reaction especially following puff adder, and paralysis particularly from Egyptian or forest cobra and mamba bites Most commonly, snakebite envenoming in Nigeria results from carpet viper  and polyspecific (EchiTab IgG Plus ICP) antivenoms The cost of the full antivenom treatment regimen was modeled as US$125 One-way sensitivity analysis and extreme value analyses were performed to test robustness and identify the most important variables influencing cost-effectiveness of an antivenom programme. Each base-case model input was varied independently according to the upper and lower limits from the relevant literature, and according to particular scenarios  mortality following envenoming by carpet viper and non-carpet viper snakes . ResultsUtilizing probability of blindness of about 0.01% with a disability weight of 0.552 yielded an ICER of $99.32/DALY averted while utilizing a probability of Post-traumatic Stress Disorder (20%) with a disability weight of 0.105 yielded an ICER of $101.44 Economic modeling has been helpful in determining the most cost-effective and optimum way of managing conditions prevalent in resource constrained settings such as poisoning With limited healthcare resources and competing priorities, it may be useful to compare our estimates to those reported in other therapeutic areas. The cost-effectiveness of first line antiretroviral therapy has been previously estimated at US$620 per year of life gained in Cote d′Ivoire In our sensitivity analysis, we estimate a cost/DALY averted of $76.08 if the model is restricted to carpet viper bites only. This is higher compared to an earlier study, which reported the cost effectiveness of antivenom following carpet viper envenoming at $10 per DALY averted The antivenom efficacy used in the decision tree was based on the systematic review and meta-analysis of studies conducted in Chad, Ghana and Nigeria (West Africa) The present study has several limitations. First and foremost, the effectiveness of carpet viper antivenom relative to no antivenom administration in our model was not based on evidence from randomized clinical trials, but rather, on evidence obtained in observational analyses. As mentioned previously, it would not be ethical to conduct such a trial with a non-treated control group. Furthermore, we apply the relative risk reduction estimated for antivenoms against carpet viper envenoming to the polyspecific antivenom for non-carpet viper envenoming in the base case, although we later relax this assumption in a scenario analysis. Also, we do not consider other potentially relevant disability outcomes that have been anecdotally reported among snakebite survivors such as malignant ulcers and pregnancy loss The results of our study suggest that making antivenoms available to treat snakebite is highly cost-effective in Nigeria. A substantial expected reduction in mortality and DALYs could be achieved at a relatively modest upfront cost, thus, expanding access to antivenom to broader parts of the Nigerian population should be a priority consideration for future investments in healthcare.S1 TextSupporting information on the estimation of specific cost inputs.(DOC)Click here for additional data file.


Grevillea, an Australian plant genus with 452 recognised species/subspecies and only 11 million years of evolutionary history. Here, we hypothesise that the apparent evolutionary success of Grevillea might have been triggered by the highly efficient use of key nutrients. The nutrient content in the seeds and nutrient-use efficiency during early seedling growth of 12 species of Grevillea were compared with those of 24 species of Hakea, a closely related genus. Compared with Hakea, the Grevillea species achieved similar growth rates (root and shoot length) during the early stages of seedling growth but contained only approximately half of the seed nutrient content. We conclude that the high nutrient-use efficiency observed in Grevillea might have provided a selective advantage in nutrient-poor ecosystems during evolution and that this property likely contributed to the evolutionary success in Grevillea.Several hypotheses have been proposed to explain the rich floristic diversity in regions characterised by nutrient-impoverished soils; however, none of these hypotheses have been able to explain the rapid diversification over a relatively short evolutionary time period of  For example, ant-dispersed Daviesia (Fabaceae) diversified into 127 species over c. 26 million years, whereas ant-dispersed Bossiaea (Fabaceae) diversified into 80 species within c. 10 million years of evolutionary historyGrevillea. With a relatively short evolutionary history of 11.2 million years and as the third largest plant genus in Australia after Acacia and Eucalyptus, Grevillea probably has one of the highest net speciation rates of all large Australian plant clades.Species in the family Proteaceae are highly adapted to and became diversified on the most nutrient-impoverished soils observed worldwide. Highly infertile soils have long been demonstrated to influence biological and physiological responses and speciation patterns4101112et al. suggested that high plant species diversity on infertile soils, particularly in species-rich biomes, is associated with functional diversity for nutritional strategies, including nutrient-use efficiencyGrevillea with those of the closely related genus Hakea, another Australian genus with 149 species that efficiently use seed nutrients during early seedling growthGrevillea.In southwestern Australia, seed germination and successful seedling establishment predominantly occurs following winter rains after summer fires. Seed-stored nutrients, particularly nitrogen (N) and phosphorus (P), are crucial for the emergence and early growth of seedlings, and the efficient use of these nutrients is critical for the successful establishment of seedlings in severely nutrient-poor and water-limited environments. Nutrient limitation has recently been proposed as a potential driver for variation in species diversityGrevillea  and Hakea , and Grevillea has a slightly higher median seed mass than Hakea (24.2 vs. 18.7 mg). The average seed masses of the 30 Grevillea species and 29 Hakea species used in the present study were statistically similar  pre-treated with smoke water exhibited a significantly lower germination rate of 32% Grevillea than in Hakea, the N:P ratios in both taxa groups were similar. Notably, the lower seed N and P concentrations in Grevillea relative to those in Hakea did not reflect dilution by seed mass because the tested species in the two groups had similar seed masses.The seed nutrient concentration in Hakea, the species in Grevillea achieved similar levels of seedling growth during the early stages. Indeed, both groups of plants expanded their first true leaves within a similar time, accumulated similar amounts of dry biomass, and diverted similar proportions of resources to the shoots and roots. Consequently, the results of this analysis suggest that compared with Hakea, Grevillea species use nutrients significantly more efficiently than do Hakea. This efficiency represents a clear ecological advantage over Hakea because Grevillea species can accumulate twice as much dry biomass and descend their roots three times deeper into the soil for every unit of nutrient in their seeds.With only half the seed nutrient content compared with species from the closely related genus Grevillea species can grow roots equal in length to those of Hakea and survive dry Mediterranean-type summers equally as well, and these characteristics provide a selective advantage to Grevillea compared with other organisms that might require additional nutrients in nutrient-impoverished soils.Seed-stored nutrients (N and P) are crucial for the successful establishment of seedlings in nutrient-impoverished landscapes. The rapid descent of the taproots to reach more reliable soil moisture reserves prior to the onset of summer droughts is critical for the survival of seedlings, particularly in Mediterranean climates with dry summer conditionsGrevillea relative to Hakea, which has much higher nutrient (N and P) concentrations, might allow Grevillea species to produce more seeds regardless of the post-fire regeneration mode. Because of challenges in gauging the size of the soil seedbank, systematic surveys of Grevillea seed production are rare, although Pickup et al. reported a large seedbank for Grevillea rivularis at 193 ± 73 seeds m–2Hakea speciesGrevillea. Interestingly, high propagule pressure has also been proposed as one of the mechanisms facilitating successful invasionSeed production is inversely proportional to the per-seed reserve of nutrients, particularly P, in nutrient-impoverished soilset al. reported that Proteaceae species such as Hakea species (Grevillea species were not included) from severely P-impoverished soils extensively replace phospholipids with galactolipids and sulpholipids during leaf development to achieve a high photosynthetic P-use efficiency, and proposed the “sulphur-for-phosphorus” hypothesiset al.Hakea species, although they rarely included Grevillea species; thus, additional research is required to test whether Grevillea have developed peak nutrient-use efficiency.Species in the Proteaceae family grow well in the world’s most nutrient-impoverished landscapes, particularly P-impoverished soils. Apart from diverse and efficient nutrient-acquisition strategiesGrevillea overcome the barriers imposed by the two most limiting factors to its development, low soil nutrient availability and periodic water availability, these plants can invade most habitats. Indeed, species of Grevillea have been observed throughout Australia. High nutrient-use efficiency has interacted with multiple factors, including myrmecochory, a predominantly sexual reproduction strategy, and a high seed output, to allow the Grevillea genus to diversify into a group with 452 species/subspecies within only 11 million years.It can be speculated that once Grevillea and Hakea used for the nutrient analyses and germination and seedling growth comparisons were acquired from Nindethana Seed Service . Genus-wide seed mass information was extracted from the Seed Information DatabaseHakea and 30 Grevillea, The seeds of Hakea and 27 Grevillea species, Hakea seeds were filled with 5 mL autoclaved deionised water, whereas those with Grevillea seeds (because of inherent dormancy) were filled with 5 mL 10% smoke water solution (Regen 2000 Smokemaster); previous studies have suggested that Grevillea seed germination is enhanced by smoke stimulationA total of 52 species , ten germinants (or fewer when available) were individually planted in PVC tubes (100 cm in length and 5 cm in diameter) with a substrate of washed white sand (with no nutrients) and placed in a hoop house. The seedlings were watered (20 mL) every 2 to 3 days and grown for 100 days (from late winter to spring 2014) prior to harvesting. The measurements taken at harvest were leaf number, shoot length, root length and fresh and dry shoot and root mass. The dry biomass of the shoot and root was recorded after the samples were oven dried at 70 °C for 48 hours.For each of the species germinated in the above-described germination phase  was defined as the amount of biomass produced or root length gained per unit of nutrient (mg) in the seeds. The nutrient-use efficiencies of How to cite this article: He, T. et al. High nutrient-use efficiency during early seedling growth in diverse Grevillea species (Proteaceae). Sci. Rep. 5, 17132; doi: 10.1038/srep17132 (2015).


Wilms’ tumor in the current era of multimodality treatment has promising outcome, but approximately 10-15% of the patients with favorable-histology, experience tumor progression or relapse. We hereby present an unusual case of repeated loco-regional recurrences in a patient with stage I intermediate-risk monophasic  Wilms’ tumor (WT) of pelvic kidney requiring aggressive therapy over a decade and lay emphasis on the importance of initial completion of therapy and need for long term follow-up. A one-year-old boy presented with lower abdominal lump, fever, and pain abdomen for one month. On abdominal examination a well-defined mass in the left lower quadrant was palpable. Radiological investigations including ultrasonography (USG) and contrast enhanced computed tomography (CECT) revealed mass in left ectopic kidney with no renal or inferior vena cava (IVC) thrombus . Left nephroureterectomy was done with para-aortic and para-caval lymph node sampling. IVC was palpated but no thrombus was found. Contralateral kidney was normal and there were no enlarged lymph nodes.Histopathologic examination (HPE) showed WT with predominant tubule formation, minimal atypia and mitosis (stage I). Sampled lymph nodes did not show any evidence of tumor. The patient was referred elsewhere for further adjunct therapy, where there was a considerable delay before any adjunct therapy was initiated. Repeat CECT done at this stage in radiotherapy department showed thrombus in IVC and right common iliac vein with para-aortic and iliac lymphadenopathy . After two cycles of VAC, family abandoned the therapy and was lost to follow-up. He returned with local relapse in retroperitoneal lymph nodes after 7 years. Partial calcified thrombus was present in sub-hepatic IVC on CECT. After 6 cycles of ifosfamide, cisplatin and etoposide (ICE Regime), retroperitoneal lymph node dissection (RPLND) was done; HPE showed no tumor.Fourteen months later, he again presented with mass in abdomen. Repeat CECT showed partial calcified thrombus in sub-hepatic IVC, and recurrence in mesocolic lymph nodes that was completely excised. It was followed by 6 cycles of IE chemotherapy and 20 Gy whole abdominal irradiation. HPE was again reported as epithelial variant of WT. Four months later, he presented with third recurrence -as mass abdomen (15×8×7cm) in left hemi pelvis posterior to the bladder. CECT showed partial calcified thrombus in sub-hepatic IVC . Tru-cut biopsy  again showed epithelial predominant WT. He received 6 cycles of paclitaxel based salvage chemotherapy followed by excision of the retro-vesical tumor and boost radiotherapy of 10.8 Gy to the pelvis. Six months later, he developed biopsy proven radiation nephritis and developed acute renal failure for which periodic hemodialysis was started. He succumbed to renal failure after few weeks at the age of 14 year.WT is the most common renal malignancy in children, and its treatment is one of the great success stories in pediatric oncology with cure rates approaching around 90%. This improved outcome has been the result of development of biological and clinical prognostic factors, which have guided for risk directed therapy. However, even with current multimodality treatment, approximately 10-15% of patients with favorable-histology (FH) disease and 50% of patients with anaplastic tumors experience tumor progression or relapse. These relapses are quite heterogeneous and though some can be effectively treated with the standard chemotherapy regimens, other with poor prognostic factors have dismal outcome.[1]Stage and histology are important prognostic factors and tumors with lower stage and favorable histology have better prognosis and lower relapse rate. Favorable prognostic factors after relapse include favorable histologic features, relapse occurring more than 12 months after the initial diagnosis, low stage (I or II) of primary disease, initial treatment with vincristine and dactinomycin only, few pulmonary nodules, and no previous irradiation of the tumor bed.[2] Index case complied with most of these favorable prognostic factors but it recurred each time. Though there was no palpable thrombus in IVC or in renal vein at the time of first surgery, but CECT done a month later revealed thrombus in IVC, which possibly could have been missed intra operatively. Surprisingly, even after early abandonment of adjunct therapy, he had event free survival for next seven years. This was followed by three loco-regional relapses in quick succession. Each time the tumor was excised completely and biopsy revealed favorable histology .As there is no fixed protocol developed by either group regarding the chemotherapy drugs, these recurrences were managed by giving ICE regime in the first recurrence and IE regime in second recurrence. Cisplatin was omitted in the second recurrence in view of preventing nephrotoxicity and ototoxicity. In the third recurrence taxane based salvage chemotherapy was given. Such treatment policy was adopted as there are no readymade protocols for management of multiple recurrences. In view of multiple recurrences, it was decided to give abdominal radiation to the tumor bed after discussing in tumor board meeting.This raises another important question as to what may be the cause of such behavior of this tumor. This emphasizes on the need to consider other biological markers, which could have role in such an adverse outcome. Tumor-specific LOH for both chromosomes 1p and 16q identifies a subset of FH Wilms’ tumor patients who have a significantly increased risk of relapse and death. LOH for these chromosomal regions can now be used as an independent prognostic factor together with disease stage to target intensity of treatment to risk of treatment failure.[3]A lot more has to be done regarding the standard protocol, dosing and regimens based on the site of recurrence and risk stratification.[4] Currently novel agents like topotecan have shown significant anti tumor activity in cases of relapse WT when given for 5 days daily over two consecutive weeks.[5] Bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody inhibits the growth of Wilms’ tumor, prevent metastatic spread, and even induce tumor regression has also shown promise. The index case repeated recurrence was either due to unfavorable biological markers or the calcified tumor thrombus in IVC acting as a source for each relapse. Inert tumor cells in the calcified IVC thrombus had possibly caused the repeated loco-regional recurrences of a low-risk localized monophasic WT.Source of Support: NilConflict of Interest: None declared


Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with target host defense proteins. Previous efforts to understand virulence largely relied on the prediction of secreted, small and cysteine-rich proteins as candidate effectors and thus delivered an overwhelming number of candidates. Here, we implement an alternative analysis strategy that uses the signal of adaptive evolution as a line of evidence for effector function, combined with comparative information and expression data. We demonstrate that in planta up-regulated genes that are rapidly evolving are found almost exclusively in pathogen-associated gene families, affirming the impact of host-pathogen co-evolution on genome structure and the adaptive diversification of specialized gene families. In particular, we predict 42 effector candidates that are conserved only across pathogens, induced during infection and rapidly evolving. One of our top candidates has recently been shown to induce genotype-specific hypersensitive cell death in wheat. This shows that comparative genomics incorporating the evolutionary signal of adaptation is powerful for predicting effector candidates for laboratory verification. Our system can be applied to a wide range of pathogens and will give insight into host-pathogen dynamics, ultimately leading to progress in strategies for disease control.Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus,  Puccinia graminis f. sp. tritici is the causal agent of wheat and barley stem rust. Wheat stem rust is a major threat to wheat production worldwide and hence to global food security, and has a long history of evolution to new virulent pathotypes in response to deployment of host genetic resistance  and non-synonymous substitutions (Nd) in genes is commonly used to assess selection. A Nd/Sd ratio <1 indicates purifying selection and functional conservation, Nd/Sd = 1 is consistent with neutral evolution, and Nd/Sd > 1 is indicative of diversifying selection or potential functional divergence.Advances in next-generation sequencing technologies are yielding a growing number of sequenced pathogen genomes, which allow a comprehensive and objective study of the evolutionary arms race between host and pathogen through the analysis of large-scale divergence genomic data. Genes undergoing purifying selection evolve slowly to maintain their conserved function. Weakened purifying selection occurs when a gene can mutate freely and randomly with little penalty because it has little or restricted functional significance. On the other hand, positive natural selection occurs when changes with functional consequences are favored as it produces high variability and adaptability. This is often observed in pathogenicity-related genes of microbes, which must adapt to the changing host environment and avoid immune recognition. The relative rates of synonymous  are available  analysis  genes control recognition of pathogens carrying specific avirulence (Avr) effectors in a gene-for-gene model. For several avirulence proteins, signatures of diversifying selection have been detected. For instance, the AvrL567, AvrP123, and AvrP4 genes are highly polymorphic in Melampsora lini and show evidence of diversifying selection consistent with an evolutionary arms race  to predict effector candidates in a pathogen genome that is highly capable of host adaptation. First, we use comparative information from publicly available fungal genomes to separate the predicted gene families into those that are specific to pathogenic fungi and into those that are found across pathogenic and non-pathogenic fungi. Both pathogen-associated and fungal gene families are then grouped using unsupervised clustering based on a broad set of 35 sequence-derived protein features to find putative effector protein clusters  is calculated by taking the average pathogen precision over its protein members. Gene families with P(i) > 90% were classified as pathogen-associated.For a gene family, k-means clustering method was applied to the pathogen-associated and fungal gene family representatives to find putative effector protein clusters across the gene families  were recorded. Putative effector clusters were chosen based on elevated levels of expression during infection using stem rust expression data. Stem rust expression data from Duplessis et al. (http://www.ncbi.nlm.nih.gov/geo/) as series GSE25020 and the tool GEO2R was used to identify differentially expressed stem rust genes by comparing in planta wheat infection to germinated urediniospores. Genes with fold change of at least two were reported as up-regulated during infection. P-values were adjusted using the Benjamini & Hochberg method, genes with p < 0.05 were reported as significant and genes with p < 0.00001 were labeled as highly significant. RNAseq expression data of isolated haustoria was compared to that of germinated urediniospores of wheat stem rust strain 21–0 and transcripts with normalized fold changes >2 were reported as haustorial up-regulated genes ].P. striiformis PST-78 .Melampsora lini  and per-domain hits per protein were recorded. Significant protein hits were kept if the combined domain hits for query and target cover more than 60% of the sequences, respectively. This ensured that for the subsequent diversifying selection analysis only reliable and well-conserved multiple alignments were used. Protein multiple sequence alignments were inferred using PRANK with the +F option  and 1141 fungal gene families not associated with pathogenicity.Lineage-specific expanded gene families in k-means clustering technique based on 35 sequence-derived protein features was applied  to all proteins in the secreted clusters. The pathogen-associated secreted cluster C6 has significant Pfam domain hits only for 53 of its 871 proteins (6.1%), which is expected as the vast majority of fungal effector proteins are known to lack functional annotation. On the other hand, the fungal secreted cluster C7 could be confidently annotated with Pfam domain hits for 327 of its 451 proteins (72.5%). Table We searched the Pfam database for significant hits  genes in the pathogen-associated gene family clusters, whereas up-regulation was detected for 1295 of 6536 (19.8%) genes in the fungal gene family clusters  can be observed across the clusters . In total, 10,213 stem rust genes with at least two orthologs across the Pucciniomycotina branch of the fungal kingdom were analyzed for signatures of site-specific diversifying selection. 4752 of these genes belong to the pathogen-associated gene families, 5177 belong to the fungal gene families and 284 genes are not part of gene families. In total, 387 of the 10,213 genes (3.8%) were found to show site-specific diversifying selection using two likelihood ratio tests of CODEML in the PAML software. Despite the higher number of genes in the fungal gene families, site-specific diversifying selection was predominantly detected across pathogen-associated gene families  from pathogen-associated gene families were predicted to undergo diversifying selection whereas only 11 up-regulated genes from fungal gene families are rapidly evolving. A more stringent significance threshold of p < 0.00001 returned 14 genes from pathogen-associated gene families and two genes from fungal gene families that are undergoing diversifying selection  in the fungal secreted cluster C7 is predicted to undergo site-specific diversifying selection, which indicates that these secreted proteins might have roles that do not require adaptation to the host or the environment. In contrast, the pathogen-associated secreted, cysteine-rich cluster C6 contains 15 up-regulated genes that are predicted to be undergoing diversifying selection. However, other pathogen-associated clusters such as C11 also contain a high number of rapidly evolving genes that are up-regulated during infection  can be a powerful and unbiased predictor for genes involved in host-pathogen interactions. Our diversifying selection pipeline can be applied to a wide range of pathogens for which divergence data is available and can give insight into the evolutionary processes between host and pathogen. In particular, our work indicates that elements of the genome linked to pathogenicity are evolutionarily dynamic, possibly in mechanisms relating to host adaptation or pathogen specialization. Therefore, effector candidates in plant pathogens that show signs of rapid evolution are promising targets for disease control in crop plants.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.


Subcutaneous emphysema (SCE) mimicking diaphragmatic hernia on X-ray is hitherto unreported and this case has been presented with the aim to alert the clinician about this unusual presentation of SCE to avert undue panic and initiate appropriate evaluation and management. This case also reemphasizes the importance of a thorough clinical examination, which probably could have revealed the subcutaneous emphysema earlier. A 1-month-old infant weighing 3500 grams was posted for Lumbosacral meningomyelocoele excision and repair under general anaesthesia. After completing the dural repair, the baby was given Valsalva maneuver to check for cerebrospinal fluid (CSF) leak. A sudden rise in peak airway pressure caused bilateral tension pneumothoraces that were recognized immediately and baby improved with thoracocenteses followed by bilateral intercostal drain insertion. Chest tube position was confirmed with C-arm.Baby was extubated on table and was shifted to the neonatal surgical intensive care unit. A repeat chest radiograph done next morning showed a well-defined longitudinal gas shadow extending from the left hypochondrium, across the diaphragm into the chest . The possibility of a diaphragmatic rupture on the left side  with herniation of bowel into the chest was considered. In lieu of barotrauma leading to bilateral tension pneumothorax, the possibility of traumatic diaphragmatic hernia was even more plausible. Clinically however, the baby was asymptomatic with no evidence of decreased air entry or bowel sounds on the left side of the chest on auscultation. Plain CT scan of the chest was done to confirm diagnosis.CT-chest showed an intact left hemidiaphragm with no diaphragmatic hernia. However, it showed subcutaneous emphysema extending from D4-D9 spine level on the left posterior thoracic wall  that was mimicking a bowel gas shadow on the chest radiograph. Retrospectively, a repeat clinical examination revealed a classical crepitus and subcutaneous emphysema over a 3 cm x 4 cm area on the left posterior lower thoracic wall (probably missed as the baby was nursed in the supine position and a bulky chest drain dressing). It resolved spontaneously in 24 hours, the chest drains were removed after 48 hours and baby discharged well.Subcutaneous emphysema (SCE), a fairly common clinical finding is categorized as a type of air leak syndrome, the others being pulmonary interstitial emphysema, pneumothorax, pneumomediastinum, pneumopericardium, pneumoperitoneum and systemic air embolism. Neonatal air leaks may present as a thoracic emergency requiring emergent intervention [1]. Emergent needle aspiration and/or tube drainage are necessary in managing tension pneumothorax or pneumopericardium with cardiac tamponade. When the air leak is asymptomatic and the infant is not mechanically ventilated, there is usually no specific treatment [1].Subcutaneous emphysema can occur as a result of trauma, iatrogenic, infectious causes or even spontaneously. The most common cause of air leak syndrome in neonates is inadequate mechanical ventilation of the fragile and immature lungs and its incidence is inversely related to the birth weight, especially in very-low-birth-weight and meconium-aspirated infants [1]. SCE is also a common occurrence after chest-tube insertion, thoracoscopic and laparoscopic procedures. Rare causes of SCE like gastric perforation [2], necrotizing enterocolitis [3], and fibre-optic endotracheal intubation [4] have also been reported in neonates. To prevent air leak syndrome, gentle ventilation with low pressure, low tidal volume, low inspiratory time, high rate, and judicious use of positive end expiratory pressure are the keys to caring for mechanically ventilated infants [1].Subcutaneous emphysema has a great importance due to its broad casualty, some causes being totally benign, but others potentially lethal. In addition, SCE can occasionally mimic other conditions like necrotizing fasciitis [5], meningitis [6], cellulitis [7] or diaphragmatic hernia clinically or radiologically leading to confusion, unnecessary interventions, panic or delays in management. SCE mimicking diaphragmatic hernia on X-ray is hitherto unreported and this case has been presented with the aim to alert the clinician about this unusual presentation of SCE to avert undue panic and initiate appropriate evaluation and management. This case also reemphasizes the importance of a thorough clinical examination, which probably could have revealed the subcutaneous emphysema earlier.Source of Support: NilConflict of Interest: None