Bunyavirales, family Hantaviridae, have a world-wide distribution and are responsible for greater than 150,000 cases of disease per year. The spectrum of disease associated with hantavirus infection include hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) also known as hantavirus cardiopulmonary syndrome (HCPS). There are currently no FDA-approved vaccines or treatments for these hantavirus diseases. This review provides a summary of the status of vaccine and antiviral treatment efforts including those tested in animal models or human clinical trials.Hantaviruses, members of the order  Rattus norvegicus) has a widespread distribution coinciding with transportation of this rodent reservoir worldwide [Hantaviruses are the etiological agents responsible for two disease syndromes: Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) or hantavirus cardiopulmonary syndrome (HCPS) . Old Wororldwide . New Wororldwide . Human iorldwide .n and Gc), and the RNA-dependent RNA polymerase, respectively [Hantaviruses are enveloped, negative-sense, single-stranded RNA viruses with tripartite genome. The three segments are denoted as small (S), medium (M), and large (L) encoding for the nucleoprotein (N), glycoproteins  and SEOV with greater than 1.5 million cases between 1950 and 2007 [Apodemus flavicollis) is associated with severe HFRS disease in Europe while PUUV (carried by Myodes glareolus) is associated with a less severe disease in Europe, particularly Scandinavia and western Russia [Peromyscus maniculatus) infection. ANDV (carried by Oligoryzomys longicaudatus) is endemic in South America with 786 cases reported in Chile between 1995 and 2012 [Oligoryzomys nigripes). Sporadic cases of HPS have also been reported in Uruguay, Paraguay, Bolivia, Peru, Venezuela, and Panama [Reported cases of HFRS exceed 150,000 per year . The majand 2007 . Cases on Russia . In compn Russia  and 109 n Russia , the vasand 2012  and 1243and 2012 . In Brazand 2012 , with apand 2012 . Of thes® is a formalin-inactivated vaccine prepared by HTNV growth in suckling mouse brain. While eliciting strong responses immediately after the boost (97% seroconversion 30 days following the boost), only 50% of volunteers had neutralizing antibodies 1 year following the boost [® vaccination fails to demonstrate a statistically significant reduction in HFRS disease severity [HFRS cases in China have been reported in 29 of 31 provinces sparking the implementation of widespread preventative measures to include a vaccination strategy. Inactivated vaccines developed in cell culture have evolved to the bivalent vaccine for HTNV and SEOV made in Vero cells currently in use . Approxihe boost . Increashe boost ,22; howeseverity . Vaccinaseverity .Previous research has demonstrated that S and M or only M expressed gene products assemble into virus-like particles (VLP) that resemble hantavirus virions when examined by electron microscopy ,26. In CAnother VLP approach involves the insertion of foreign protein segments into the core protein of hepatitis B virus (HBcAg) to induce humoral and cellular responses . For hann, and Gc protein, recombinant proteins were generated and tested as potential vaccine candidates. Using a baculovirus expression system, recombinant N, Gn, and Gc proteins were used to immunize hamsters prior to HTNV infection [n, and Gc proteins independently and full protection was observed when hamsters were immunized with a combination Gn/Gc or N proteins. Also using a baculovirus expression system, truncated recombinant N proteins were developed indicating the amino terminal region is required for protection, harboring six of seven epitopes analyzed using bank vole monoclonal antibodies [Saccharomyces cervisiae that were protective in the PUUV/bank vole model [Escherichia coli cells. PUUV-infected bank voles were completely protected when immunized with PUUV N, partially protected when immunized with ANDV N, and not protected when immunized with DOBV N [Given the immunogenicity of the N, Gnfection . Partialtibodies . Similarle model . Cross-rh DOBV N . PUUV anSaccharomyces cervisiae. Immunogenicity, but not protective efficacy, was evaluated in two mouse models showing N-specific antibody titers 8 months following a 3-dose vaccination strategy, N-specific antibody induction across all IgG subclasses, and strong cross-reactivity against HTNV [Escherichia coli were coupled to the outer membrane protein A derived from Klebsiella pneumoniae (rP40) protected mice immunized 3 times from a DOBV challenge [German researchers used a similar approach to develop a His-tagged DOBV recombinant N proteins in nst HTNV . While imoniae rP protectehallenge .A double recombinant, chimeric molecular vaccine was developed by inserting HTNV S and M segment cDNA into vaccinia virus (VACV) . Protectn, Gc, or Gn + Gc were able to elicit robust CTL responses in mice and were protective when hamsters were challenged with a uniformly lethal dose of ANDV [Nonreplicating adenovirus vectors expressing ANDV N, G of ANDV . While pn and Gc was immunogenic and protective against a HTNV challenge in mice after 3 doses [Vesicular stomatitis virus (VSV) pseudotype virus containing hantavirus glycoproteins was examined as a potential vaccine strategy. A nonreplicating VSV pseudotype containing HTNV G 3 doses . Similar 3 doses . Interes 3 doses .n and lysosomal-associated membrane protein 1 (LAMP1) [c rather than Gn [n has the potential to achieve this goal.In an effort to prime a CD4+ T cell response, a Chinese group developed a DNA vaccine encoding the HTNV G (LAMP1) . LAMP1 a (LAMP1) . Similar than Gn . The oveDNA vaccines targeting the S and M segments of SEOV were generated by either cloning the gene segment into the DNA plasmid expression vector pWRG7077 or into a DNA-based Sindbis replicon or packaged Sindbis replicon vector ,53. IncrA Phase 1 clinical trial was conducted to evaluate the safety and immunogenicity of HTNV and PUUV DNA vaccine when administered alone or in combination to healthy volunteers using gene gun . There w® is also nearing completion, the final scientific report is in draft. Another Phase 1 clinical trial  of 48 healthy volunteers to evaluate the safety and immunogenicity of the ANDV DNA vaccine is ongoing with the first cohort starting the vaccination series. Finally, a Phase 1 clinical trial  of 72 healthy volunteers to evaluate the safety and immunogenicity of HTNV, PUUV, and HTNV/PUUV DNA vaccines delivered by intradermal and intramuscular delivery using the TriGrid Delivery System has been approved by an Institutional Review Board, but has not yet started recruiting. The evolution of hantavirus vaccine efforts is depicted in As of the writing of this manuscript, a Phase 2a clinical trial  of 120 healthy volunteers to optimize the dose of HTNV, PUUV, and HTNV/PUUV DNA vaccines delivered by intramuscular electroporation is nearing completion, the final scientific report is in draft. A Phase 1 clinical trial (S-15-40 2289) of 27 healthy volunteers to evaluate the safety and immunogenicity of HTNV, PUUV, and HTNV/PUUV DNA vaccines delivered by the PharmaJet StratisHerein, hantavirus disease treatments will be described based on the timing of drug administration in the hantavirus disease course. Drugs can be administered as either a postexposure prophylactic or a therapeutic. A postexposure prophylactic is administered after virus infection prior to the onset of viremia or clinical signs. A therapeutic is typically associated with a trigger-to-treat, either onset of clinical signs or detection of viremia. Candidate drugs evaluated in hantavirus animal models is described in Initially found to inhibit HTNV replication in vitro , ribavirA clinical trial for the treatment of HFRS using ribavirin has been conducted. In this trial, ribavirin treatment was assessed in 242 patients with confirmed HFRS in China . MorbidiA single study evaluated the efficacy of the iron-binding glycoprotein, lactoferrin, to prevent HFRS in suckling mice was done . While gETAR  is a nucleoside analog with antiviral activity against HTNV and ANDV in vitro . In vivoFavipiravir is a pyrazine derivative initially discovered for its anti-influenza properties. This compound was first evaluated in vitro against a panel of bunyaviruses and arenaviruses, having antiviral activity against both viral families . FavipirMechanisms of increased microvascular endothelial cell permeability due to hantavirus infection have been examined in vitro. ANDV enhances the permeability of endothelial cells in response to VEGF ,73,74 anHantaviruses preferentially infect endothelial cells and induce host responses to include a proinflammatory cytokine response. While not an antiviral, it was thought that limiting this aspect of the immune response to virus infection may confer clinical benefit. The first evaluation of this was done during the Korean War. Cortisone therapy was administered by either the intramuscular or oral route . There wIn Chile, a retrospective analysis of 22 HCPS patients treated with a high dose of methylprednisolone suggested that treatment reduced shock and mortality . This len and Gc [n or Gc alone is sufficient to prevent HTNV infection in hamsters. Protective efficacy of the monoclonal antibody HCO2 was confirmed in a subsequent HTNV/hamster experiment [In the 1980s, several monoclonal antibodies to HTNV were characterized describing neutralization sites on the glycoproteins, Gn and Gc ,83,84. In and Gc . This piperiment . The finperiment .Recently, researchers have shown that two recombinant monoclonal antibodies generated from an ANDV convalescent survivor with high neutralizing antibody titers protected ANDV-infected hamsters from lethal disease . In thisHuman convalescent plasma has been shown to protect animals in both the SNV/deer mouse infection model  and the To this end, the development of a standardized polyclonal antibody product using hantavirus DNA vaccines is currently in-progress. Hantavirus DNA vaccines have been shown to be immunogenic in rabbits, NHPs, ducks, and geese ,91,92,93The number of hantavirus disease cases each year warrants the development of medical countermeasures to combat infection from these viruses. However, the number of cases each year do not necessitate a widespread vaccination strategy outside of endemic areas except in the case of military conducting operations in these areas. Therefore, it is imperative to work towards the goal of a vaccine and an antiviral that could be used individually or in combination. An antiviral, such as a polyclonal antibody therapy, would provide instant immunity while vaccination could provide long-lasting immunity. Vaccination would also prevent the spread of ANDV, the only hantavirus with demonstrated person-to-person transmission. Antiviral treatment and vaccination is a strategy currently used for rabies virus infection that could be applied to hantavirus infections.The timing of treatment intervention presents a challenge for hantavirus disease, especially cases of HPS. Animal models have repeatedly shown that antivirals are not effective if administered after the onset of viremia. The prodrome phase can be difficult to differentiate from other febrile illnesses, hampering early intervention efforts. Continued work is required to determine if there are early host responses that can be used to support a hantavirus diagnosis.Additionally, the lack of an HFRS disease model that faithfully recapitulates the salient features of human HFRS disease is hampering medical countermeasure development efforts. Current infection models have utility for testing vaccines and antivirals in which sterile immunity can be used as an output. The unavailability of a model that can be used to evaluate antivirals targeting specific aspects of pathogenesis, postexposure prophylactics, or therapeutics is a major shortcoming of the field.The advancement of several vaccines into clinical trials is promising for the future use of these vaccines as licensed products or in emergency use. There are several path-to-licensure issues that must be overcome, reviewed in . These i


Primarily due to recent advances of detection techniques, microchimerism (the proportion of minor variant population is below 1%) has recently gained increasing attention in the field of transplantation. Availability of polymorphic markers, such as deletion insertion or single nucleotide polymorphisms along with a vast array of high sensitivity detection techniques, allow the accurate detection of small quantities of donor- or recipient-related materials. This diagnostic information can improve monitoring of allograft injuries in solid organ transplantations (SOT) as well as facilitate early detection of relapse in allogeneic hematopoietic stem cell transplantation . In the present review, genetic marker and detection platform options applicable for microchimerism detection are discussed. Furthermore, current results of relevant clinical studies in the context of microchimerism and SOT or allo-HSCT respectively are also summarized. Co-existence of genetically distinct/discordant tissues or cells is termed chimerism after the well-known Greek fusion creature. Based on the extent of the variant population, microchimerism is usually mentioned as a subgroup of chimerism in which the proportion of foreign cells or tissue does not exceed 1%. This proportion can readily be estimated in the circulation while quantitative comparison of major and minor populations is more challenging in cases of scattered cells or transplanted tissue.Based on origin, naturally occurring and therapy related microchimerism can be distinguished. Naturally occurring microchimerism refers to the bidirectional interaction between the genetically distinct mother and fetus(es) resulting in the long-lasting presence of small numbers (well below 1%) of fetal cells in the mother and vice versa. The interesting immunological consequences of these coexistences have recently been elegantly reviewed [The term therapy related microchimerism describes situations primarily related to transplantations and occasionally transfusions. Subsequent to solid organ transplantations (SOT) a well-defined organ coexists with the recipient which rarely involves mixture of cells. In contrast, variable recipient–donor cell ratios are characteristic for patients treated by allogeneic hematopoietic stem cell transplantation .The quantitative detection of chimerism in the circulation might serve as a biomarker with clinical relevance, which is based on polymorphic genetic structures (markers) similarly to those applied in forensic medicine for person identification. Large scale population studies such as the 1000 Genome Project characterized and classified millions of human variants in different populations allowing the identification of appropriate markers with high heterozygosity rate and discriminatory power. In the last decade, the development of highly sensitive, quantitative and accurate methods, e.g., digital PCR or next generation sequencing (NGS) enabled the precise quantitation of unprecedently low level molecular variants. As a result, the clinical application of these innovative technologies for microchimerism detection has started to enter the daily practice. In general, the paradigm of marker selection in transplantation dictates an allele choice as a marker that is not present in the dominant cell or molecule population: after SOT the donor should be marker positive and the recipient negative, while after allo-HSCT the opposite relationship is required.Chimerism could be investigated on cellular or on cfDNA level. The latter consists mainly of small sized (around 150 base-pairs) double-stranded DNA fragments resulting from apoptosis, necrosis, immune-mediated cell damage, or release of nuclear DNA into the circulation predominantly from hematopoietic cells . In the In the present paper, we provide an extensive review of the available information about microchimerism in the context of various forms of transplantation and transplantation-related advanced therapies with a special emphasis on the available markers and the multitude of detection platforms in this high sensitivity range.So far, more than 700,000 STRs (short tandem repeats) are characterized in human genome, for the most part they are considered as ‘junk DNA’, however some of them are located in protein coding region and associated with genetic disorders ,5,6. STRThese represent the most frequently observed variations in the human genome, around 1/1000 basepairs . In an aDIPs or INDELs are relatively short (up to about 50 bases in length), frequently biallelic variations located in non-coding regions of the genome displaying a lower mutation rate compared to STRs. As a consequence of the emergence of NGS technology and advanced software algorithms, the number of identified and characterized DIPs is rapidly increasing ,23,24. WEvidently, application of sex determining markers  are only useful and informative in sex-mismatched transplanted patients both in allo-HSCT and SOT. The amelogenin gene has an X and Y linked variant (AMELY and AMELX). The basis of variability in this case is a 6-basepair-long deletion segment found in X chromosome (AMELX). Utilization of Y specific markers, e.g., SRY, rely on the detection of a gene exclusively located on chromosome Y. Both sex-specific markers are informative in allo-HSCT when the recipient is a male and the donor is a female ,40,41, wAn ideal marker should be located in non-coding region ruling out the chance it is linked to genetic diseases, plays a role as genetic susceptibility factor or influences certain disease progression or response to therapy. It should be biallelic, differing by at least 2 basepairs, with a minimum allele frequency of at least 0.25 and a high level of heterozygosity (>0.4). Their chromosomal localization should be as distant as possible or on different chromosomes and they should also be located in sufficient distance from repetitive regions . When deAs allo-HSCT became widespread, an imperative need arose to repeatedly discriminate recipient and donor cells in the circulation after allo-HSCT for which various techniques have been developed. Initially, these approaches had limited sensitivity in the range of 10% recipient cells allowing only to characterize chimerism and not microchimerism. The most commonly used chimerism-detection methods in this time period were XY fluorescence in-situ hybridization and polymerase chain reaction (PCR) based amplification of variable number of tandem repeats (VNTR) or short tandem repeats (STR). The PCR-based technologies more recently predominantly use fragment analyses (FA) making it possible reach a sensitivity range of 1% still unsuitable for the reliable detection of microchimerism . SystemTo increase the sensitivity of chimerism monitoring in transplanted patients, several studies proposed real-time quantitative PCR (qPCR) method to detect single nucleotide polymorphisms (SNPs) or short deletions/insertions (DIP). QPCR allows 0.1% sensitivity quantification of the minor genotype ,37,38,44Digital PCR has initially been used to quantify low-copy number fetal DNA in maternal plasma . This inNext generation sequencing (NGS) is a massively parallel or deep sequencing, where millions of small fragments of DNA are sequenced in parallel affording an unprecedentedly high sensitivity. Sequenced fragments are pieced together by mapping individual reads to the human reference genome by bioinformatic tools. Several different NGS platforms exist with different chemistries and techniques. NGS can be used to sequence individual genes, targeting the whole exome or even the entire genome . This teg followed by a high speed centrifugation  [g, cfDNA concentrations were contaminated by genomic DNA [The volume of blood required for cfDNA analysis is usually between 6 and 10 mL. Concentrations in paired plasma and serum samples have revealed significantly higher cfDNA concentrations in serum than in plasma . This is,000× g) . In caseomic DNA .Two main types of cfDNA extraction systems have frequently been used: column-based systems and magnetic beads systems. Studies comparing cfDNA extraction methods and kits revealed substantial differences in total DNA yield . cfDNA sQuantitation of extracted cfDNA may be performed with several techniques such as fluoro-spectroscopy, Qubit  fluorometry and qPCR. In addition, quality control of isolated cfDNA can be performed by fragment analysis by capillary electrophoresis. Based on fragment size, the relative amount of nucleosome-protected cfDNA fragments with 140–160 base pairs can be compared to degradation-fragments with higher molecular weight DNA ,70. SingIn the context of solid organ transplantations (SOT), the meaningful use of the term microchimerism is only possible for cfDNA in the circulation since intact graft cells are unlikely to survive in blood. Appearance of low amounts  of donor hematopoietic (passenger) cells in the recipient’s circulation has been repeatedly observed after SOT and prompted speculations about a potential favorable role of these cells in graft acceptance. However, later clinical studies failed to find consistent associations and this cell based microchimerism also showed large inter-individual variations . This has outstanding significance as the overall frequency of graft related post-SOT complications—i.e., rejection—immunosuppressive treatment mediated toxicity or infection can reach 50% of all SOT cases . For theMonitoring of dd-cfDNA in plasma as a non-invasive indicator of graft damage after SOT was first suggested as early as in 1998 by the most outstanding expert of the field, Lo et al.  followedn = 50 for kidney or n = 20 for other solid organs respectively. As shown in Thus, we performed a focused literature overview of exclusively peer-reviewed papers addressing the potential role of plasma dd-cfDNA as a potential rejection marker in various SOT settings with an inclusion criteria of total cohort size equaling or exceeding As an overall conclusion, significantly positive role was exclusively found across all organs for plasma dd-cfDNA as allograft injury biomarker, no study was found concluding the lack of association. In kidney transplant, the paramount prospective trial of Bloom et al. called “Circulating Donor-Derived Cell-Free DNA in Blood for Diagnosing Active Rejection in Kidney Transplant Recipients” (DART) had the strongest impact with subsequent positive decision about health insurance reimbursement in the US. By using a cut-off value of 1.0% of dd-cfDNA these authors found that this biomarker had stronger discriminating power between ABMR and absence of ABMR compared to cell mediated rejection versus lack that of . The bion = 115) multicentric study demonstrated significantly elevated dd-cfDNA levels in acute rejection compared to transplanted patients in stable phase or those with hepatitis C infection [Heart transplant could be viewed as a pioneer of the field with an early study of a sizeable cohort from Stanford taking advantage of a large biorepository also containing plasma, allowing the selection more than a 100 sex-mismatched heart transplant cases  later confection . Similarnfection  with a mnfection  supplemenfection .n = 61) kidney transplanted cohort with limited potential as a general diagnostic biomarker [Besides plasma, alternative test materials have also been used to monitor cfDNA after SOT. Sigdel et al. observed rather large inter-patient variations of cfDNA in urine samples of a sizeable  from peripheral blood or bone marrow aspirates is a standard diagnostic procedure following allo-HSCT. The replacement of recipient cells by those of the donor called complete donor chimerism is an evidence for the successful engraftment and the reconstitution of hematopoiesis after allo-HSCT. After myeloablative conditioning, residual recipient hematopoiesis may indicate graft failure and/or persisting disease. Remaining or re-emerging recipient cells in the circulation, called mixed chimerism, after transplantation is mostly associated with relapse in cases of hematopoietic malignancies, although long term mixed chimerism without relapse has been described . PersistThe dynamics of serial chimerism monitoring facilitates therapeutic interventions such as modulation of immunosuppression, donor lymphocyte infusions, chemotherapy, or second transplantation both in myeloablative and in non-myeloablative settings . Early, Analysis of STRs by fragment analysis (FA) is the most widely accepted choice of method for post-HSCT monitoring. Detailed recommendations are available for proper laboratory performance of STR chimerism monitoring . The majChimerism monitoring provides a surrogate biomarker for tumor-specific measurable residual disease (MRD). Chimerism testing is suggested to be applied in combination with MRD monitoring in malignant hematopoietic diseases ,100. In In recent years, the sensitive detection of MRD (reaching at least 0.1% or deeper limit of detection) gained increasing attention, as patients with MRD-positive morphologic remission showed similar outcome to patients with active disease ,101. IntSeveral papers indicated that the dynamics of microchimerism was more closely correlated with relapse in acute leukemia. The reappearance of recipient DNA after complete donor chimerism is a warning sign, and increasing recipient chimerism in two consecutive samples reliably forecast relapse ,103. Stap < 0.0002; microchimerism p = 0.0201). The introduction of sensitive chimerism monitoring allows early detection of recipient hematopoiesis, early intervention, and the decrease of patients proportions with mixed chimerism above 1% (23% before and 15% after the introduction of sensitive chimerism test) [Dynamics of chimerism also influence survival: complete donor and decreasing mixed chimerism present favorably, while increasing and stable mixed chimerism have adverse prognostic significance ,107. Recsm test) .Mixed chimerism, and also microchimerism, were found to strongly correlate with MRD-monitoring inasmuch, both biomarkers show similar time-course, when analyzed from the same sample type ,96,100. In hematology practice, special cellular therapies are becoming more and more frequent treatment options: examples are ‘microtransplantation’ in elderly AML patients, natural killer (NK) cell infusion or third-party donor antigen-specific T-cell infusions  . As thesIn a randomized, multicenter study of elderly AML patients, induction chemotherapy in combination with mobilized HLA-mismatched donor cells without GvHD-prophylaxis improved response rates with a low rate of mixed or complete donor chimerism (5/185) and severe acute GvHD (2/185). Microchimerism was detectable in 31% (8/26) sex-mismatched, informative transplants by SRY-qPCR (with up to 10 months follow-up) . MicrotrPlasma cfDNA markers as diagnostic applications have been intensively studied in the context of prenatal screening, solid tumors and SOT. The clinical usefulness of chimerism testing from plasma cfDNA in allo-HSCT has been sparsely reported. Initially, sex-mismatched allo-HSCT patients were screened to reveal the main cellular origin of plasma circulating cfDNA. Y-chromosome specific cfDNA was detected in plasma with a median of 6.9% in male patients with female donors, indicating the hematopoietic compartment as the main origin of plasma cfDNA .Plasma cfDNA was found to be more sensitive than cellular chimerism in detecting relapse in leukemia patients with complete donor chimerism in polymorphonuclear cells. All patients with clinical relapse  had more than 10% of recipient cfDNA in the plasma. Altogether 60% of patients displayed various levels of mixed chimerism in plasma, including an additional 16 patients with >10% mixed chimerism level and without clinical relapse .n = 3), when in peripheral blood cells complete donor chimerism was observed [A further study indicated that mixed chimerism was detectable in cfDNA in a higher percentage of samples than in peripheral blood cells after allo-HSCT. Interestingly, plasma cfDNA-based microchimerism was capable of detecting isolated extramedullar relapses (central nervous system, observed . Recipieobserved . Plasma observed .In the context of SOT, additional independent, sufficiently large cohort studies are needed to further confirm the diagnostic value of microchimerism characterized by dd-cfDNA proportion in plasma. A further goal of these studies can be the targeting patients with subclinical graft damage for which currently the only diagnostic tool is the invasive organ biopsy. Applicability of dd-cfDNA based microchimerism detection as valuable information in the clinical decision-making process regarding actual immunosuppression should also be tested in a prospective setting. Routine application and comparisons of alternative detection techniques may also be attractive for future research since a considerable number of transplantation diagnostic facilities may not have access to the most advanced NGS-based determinations.Although it is not widely accepted yet, monitoring of recipient cfDNA dynamics after allo-HSCT may be a valuable diagnostic tool to detect relapses at earlier time points, with special emphasis on extramedullary sites. Additionally, it can be utilized as a non-invasive biomarker for aGvHD activity. Further studies involving larger patient cohorts, as well as applying more sensitive methods than STR are necessary to address existing uncertainties regarding cfDNA chimerism testing in the allo-HSCT field.


Our recent Communications Biology research article revealed the genomic drivers and therapeutic vulnerabilities of sea turtle fibropapillomatosis tumors. Fibropapillomatosis is a debilitating tumorous disease afflicting populations of green sea turtles globally. While a virus is involved in the development of this disease, it is increasingly understood that the key trigger is linked to anthropogenic disturbances of the environment. The specific environmental co-trigger(s) has yet to be functionally confirmed. Here we outline the next steps required to advance our understanding of this enigmatic disease, to enable us to more effectively clinically combat it and to ultimately tackle its environmental co-trigger to halt and hopefully reverse the spread of fibropapillomatosis. This can create a snowball effect whereby emerging pathogens potentially accelerate the extinction of the species. Given the dire rate of habitat loss, biodiversity decline and unparalleled human-induced species loss (on a par with previous mass extinction events)7, it is imperative that we are increasingly vigilant in relation to endangered wildlife disease outbreaks. Indeed, human activities might be contributing to oncogenic processes in wild animal populations9. A host of tumorous diseases affecting wildlife populations are pathogen-induced11, as are at least 15% of human cancers13. One such pathogen-induced tumorous disease is sea turtle fibropapillomatosis, a virulent global epizootic  leading to tumors on turtles’ soft tissues and internal organs  is the most severely afflicted16. The number of stranded C. mydas exhibiting fibropapillomatosis has dramatically increased in recent years, within Florida and globally17. Fibropapillomatosis is ideally suited to in-depth molecular and clinical study given the large numbers of patients treated in rehabilitation facilities annually18.Fibropapillomatosis afflicts all seven sea turtle species, of which green sea turtles . Despite this low sample size we were able to gain important insight into the molecular mechanisms driving fibropapillomatosis tumors, identify the transcriptionally latent state of the fibropapilloma-associated pathogen, Chelonid herpesvirus 5 (ChHV5) within established tumors, and determine promising anti-fibropapillomatosis drug treatments. However, many mysteries still remain in relation to this disease. Therefore, we have greatly increased the number of tumors (and control samples) transcriptomically profiled in order to refine our drug target selection and answer a range of questions in relation to the environmental, host and viral drivers of this disease. We are particularly interested in improving our genomic understanding of early-stage tumors, post-surgical regrowth tumors and internal tumors.Highlighting the power of modern genomic and precision medicine techniques, the transcriptomic profiling that formed the core of our recent Communications Biology paper18.Understanding the genetic relationship between the numerous tumors occurring on individual turtles will have implications for the clinical treatment of the disease, as well as providing insight into the initiating mechanisms of the tumors. We are actively investigating whether these tumors are clonal  or if each tumor arises de novo (primary tumors) and has unique mutational profile and impact on distinct tissue types. We are also implementing detailed tumor (re)growth profiling to more robustly assess growth dynamics and the effect of therapeutic treatments22. Currently no treatment exists for turtles with visceral organ tumors, as the hard external shell (plastron and carapace) hampers surgical access to fibropapillomatosis-afflicted organs. Patients diagnosed with internal tumors are instead humanely euthanized, often after months of rehabilitation effort has already been expended.In terms of novel therapeutic approaches, because the vast majority of patients are simultaneously afflicted by tens or more tumors, we are prioritizing the identification of orally deliverable compounds to achieve systemic responses. We are also specifically seeking to identify therapeutic compounds for the treatment of internal tumors, which are currently inoperable. Internal tumors most commonly afflict the lungs, kidneys, heart, gastrointestinal tract, and liver25.In addition to determining host gene expression essential for the maintenance and growth of fibropapillomatosis tumors it will be crucial to further characterize the expression of ChHV5. We are currently determining the abundance and differential expression of ChHV5 viral transcripts across established tumors, early-stage tumors, post-surgical regrowth tumors, and internal tumors. Furthermore, we are employing unbiased deep-sequencing approaches to compare viral transcript expression levels with the overall level of ChHV5 DNA present within each tumor. Previous approaches to investigate ChHV5 load have primarily relied upon DNA-based PCR or qPCR of a small number of ChHV5 assays or targeted ChHV5 genome sequencing (e.g. enrichment of ChHV5 DNA by long-range PCR amplification)Together this research promises to shed further light on the viral and host molecular mechanisms responsible for the initiation, maintenance and regrowth of fibropapillomatosis tumors, enabling the design of rational treatment strategies.28.We are exploring the potential of prognostic and diagnostic biomarkers to improve the clinical care of fibropapillomatosis-afflicted sea turtles. Currently, x-ray, endoscopy, magnetic resonance imaging (MRI), or computed tomography (CAT) scans are required to diagnose internal tumors. To overcome their limitations in hard-shelled patients, including difficulty in detecting early-stage tumors, we are researching the diagnostic and prognostic value of qPCR-based blood-plasma biomarkers. We are investigating putative biomarkers for both the detection of internal tumors, and biomarkers capable of predicting ultimate rehabilitation outcome of individual turtles. Circulating tumor DNA in blood is a well-established source material for blood-based biomarkers for cancer detection, and is increasingly utilized in human oncology31 (e.g. deep sequencing or qPCR). By tracking the level of virus shed into the environment by each patient, we will better understand disease transmission dynamics. Furthermore, by correlating viral shedding to individuals throughout the various stages of the rehabilitation process, and individuals with varying tumor burdens and tumor aggressiveness18 we can begin to quantify whether there are certain times/states which promote enhanced viral shedding. Such profiling can also help inform clinical management , and potentially improve our understanding of how this disease spreads throughout the marine environment.We are also utilizing pioneering environmental DNA (eDNA) screening techniques to quantify the level of ChHV5 viral shedding in fibropapillomatosis-afflicted sea turtles. eDNA is an ultra-modern approach, whereby free DNA is isolated directly from an environmental sample  and subsequently detected by molecular approaches32 and in wild populations. A number of potential environmental co-triggers have been investigated, some of which correlate strongly with the disease incidence. However, no factor has yet been causatively proven to drive fibropapillomatosis tumor development16.While ChHV5 is likely a key driver of fibropapillomatosis, additional environmental co-triggers are required to drive fibropapillomatosis tumor formation in a laboratory setting14 suggest a possible role of UV radiation as an environmental co-trigger of fibropapillomatosis. This includes the differential regulation of Wnt and Metalloprotease signaling14 in fibropapillomatosis. These pathways are known to be regulated by UV exposure in human skin cells and canine cornea34. UV exposure has been linked to systemic immunosuppression36, which also occurs in turtles with fibropapillomatosis, although it is not yet determined whether this is a cause or a consequence of the disease16. Elevated UV exposure therefore holds the potential to induce immunosuppression in turtle skin, possibly enabling the rampant proliferation of ChHV5 and the crossing of an oncogenic threshold.While there are a number of putative environmental triggers that warrant further investigation, the correlation between elevated ultraviolet (UV) radiation levels and disease incidence, as well as the shared molecular drivers of fibropapillomatosis and human basal cell carcinoma37. Fibropapillomatosis tumors primarily occur on external regions, suggesting UV light may act as a direct mutagen contributing to oncogenesis, like with other human and wildlife skin cancers. UV exposure induces signature mutations in genes such as p53 (Tp53) and Patched (Ptch), two tumor suppressor genes responsible for human non-melanoma skin cancer37. Sunlight also acts as a tumor promoter by favoring clonal expansion of such mutated cells37. Expression of both Ptch genes are highly suppressed in fibropapillomatosis tumors14  to northern Florida, a distance of ~450 miles, despite neighboring juvenile green turtle populations running the length of the entire seaboard. Conversely, in a shorter time period the disease spread to distant C. mydas populations circum-globally, throughout equatorial and sub-equatorial zones15. UV levels reaching the Earth’s surface have increased in recent times both locally in Florida14 and globally43, potentially driving the spread of the disease away from the equator and increasing incidence in some longer-affected areas.Juvenile 45. While this process is globally ubiquitous, matching fibropapillomatosis incidence, it is most prominent in tropical and subtropical latitudes where sunlight is most intense and in areas where organic compound concentrations are highest (nearshore and estuarine waters). Therefore, this offers another potential mechanism through which UV could be indirectly contributing to fibropapillomatosis initiation.UV might also indirectly contribute to tumourigenesis by converting inshore chemicals into more potentent oncogenic forms. UV light is known to degrade organic compounds in natural waters (photolysis). Complex organic (often poly-unsaturated/aromatic) molecules can be broken down into smaller, more reactive compounds that can then react with each other forming different, potentially harmful compoundsWhether fibropapillomatosis tumourigenesis is triggered by UV directly (induction of genomic mutations) or indirectly , with the increases in UV exposure occurring globally it is likely that mechanistic findings will be highly relevant not only for sea turtle fibropapillomatosis but also other human and wildlife oncogenic diseases.13) or exacerbated by pathogens47. The key role of environmental co-triggers further complicates our understanding of pathogen-induced cancers. In humans, studying the initiating events that can go on to produce later cancers is difficult. Once assumed to be an issue for vulnerable cohorts (e.g. Kaposi sarcoma-associated herpesvirus drives tumor formation in immunocompromised patients)12, it is now understood that temporally distant viral events can sow the seeds of later cancers . HPV is also clinically associated with human non-melanoma skin cancer, including basal cell carcinoma50 which is the cancer type that transcriptomically most closely resembles chelonid fibropapillomatosis14. Furthermore, UV radiation and HPV infection are linked to human non-melanoma skin cancer, with UV-induced immunosuppression being a likely driving mechanism51. We urgently need tractable natural models of virally induced tumors to fully understand the role of pathogens in driving tumor formation and to determine environmental co-initiation mechanisms52. Sea turtles have a similar life-span to humans and have normally robust anti-cancer defenses, providing a more faithful comparative model of humans than common short-lived cancer models, such as rodents and zebrafish1. Fibropapillomatosis offers an intriguing new glimpse into a potentially devastating disease mechanism, revealing that an environmental co-factor is potent enough to enable a normally benign virus to cause disease of epidemic (epizootic) proportions in otherwise healthy juvenile populations. Intensive study of chelonid fibropapillomatosis will enable us to directly address the crucial question of how an environmental trigger is capable of conferring such potent oncogenicity on a clinically benign virus, an issue that potentially holds immense importance for our understanding of human and wildlife pathogen-induced tumors.Fibropapillomatosis provides an ideal opportunity to study pathogen and environmentally co-induced tumors. This will improve our ability to combat not only this sea turtle disease epizootic but also the ever-increasing number of wildlife and human cancers discovered to be pathogen-induced  can hopefully be fully assessed in the near future53. If Koch’s postulates are fulfilled, ChHV5 can finally be comprehensively claimed as the primary pathogenic agent driving tumor formation, albeit possibly still requiring environmental co-trigger(s) to unleash sufficient viral loads. Alternatively, if not fulfilled this will demonstrate that ChHV5 inoculation/exposure alone truly is not sufficient to drive this disease and that another trigger(s)/co-trigger(s) is essential to initiate fibropapillomatosis tumor growth. While it is possible that an alternative pathogen might be identified, the correlation of ChHV5 viral load to tumors and the lack of other pathogens in significant abundance within the tumors16 makes this less likely. The lack of a specific tumor associated virulent ChHV5 strain and the presence of ChHV5 in tumor-free populations16 demonstrate that the limiting factor in developing fibropapillomatosis is not just exposure to ChHV5 but also exposure to the elusive inshore environmental co-trigger(s).Interesting work is emerging regarding green turtle cell culture and the enigmatic ChHV5 virusAdvances in cell biology, environmental oncogenic trigger detection and assessment, and genomic medicine mean that now more than ever we are within sight of revealing the longstanding mysteries of the fibropapillomatosis disease epizootic. For our part, we will continue to pioneer advanced genomic and precision wildlife medicine approaches aiming to better understand, treat and ultimately prevent the burden of fibropapillomatosis on sea turtle populations.


Glossina palpalis gambiensis laboratory colonies was examined using 16S rRNA gene amplicon sequencing to evaluate the dynamics of the bacterial diversity within and between each G. p. gambiensis colony.Microbiota plays an important role in the biology, ecology and evolution of insects including tsetse flies. The bacterial profile of 3 G. p. gambiensis laboratory colonies displayed similar bacterial diversity indices and OTU distribution. Larval guts displayed a higher diversity when compared with the gastrointestinal tract of adults while no statistically significant differences were observed between testes and ovaries. Wigglesworthia and Sodalis were the most dominant taxa. In more detail, the gastrointestinal tract of adults was more enriched by Wigglesworthia while Sodalis were prominent in gonads. Interestingly, in larval guts a balanced co-existence between Wigglesworthia and Sodalis was observed. Sequences assigned to Wolbachia, Propionibacterium, and Providencia were also detected but to a much lesser degree. Clustering analysis indicated that the bacterial profile in G. p. gambiensis exhibits tissue tropism, hence distinguishing the gut bacterial profile from that present in reproductive organs.The three Our results indicated that age, gender and the origin of the laboratory colonies did not significantly influence the formation of the bacterial profile, once these populations were kept under the same rearing conditions. Within the laboratory populations a tissue tropism was observed between the gut and gonadal bacterial profile.The online version of this article (10.1186/s12866-018-1290-9) contains supplementary material, which is available to authorized users. Tsetse flies (Diptera: Glossinidae) are the sole vectors of the African trypanosomes that cause sleeping sickness in humans  and nagana in animals  , 2. ReceGlossina palpalis gambiensis (Gpg) from the Niayes area. The project was based on purchasing mass-reared sterile male tsetse pupae of a strain originating from Burkina Faso (BKF strain) from the rearing facilities at the Centre International de Recherche-Développement sur l’Élevage en zone Subhumide (CIRDES), Burkina Faso and the Slovak Academy of Sciences (SAS) in Slovakia. Both locations are rearing the same strain (BKF) that has been maintained for more than 35 years under artificial rearing conditions. Male pupae on the point of emergence were chilled to prevent emergence, irradiated and shipped twice weekly from CIRDES and SAS by air to Dakar, Senegal [In 2012, an SIT project was initiated in Senegal that aimed to eradicate a population of  Senegal , 8.Data from trial sterile male releases indicated high mortality and low recapture rate of the release sterile BKF males. In view of the higher mortality rates of the sterile males under field conditions in Senegal, an attempt was made at the Insect Pest Control Laboratory (IPCL), Seibersdorf, Austria, to establish a new colony from flies collected in the Niayes (SEN strain). Establishing and domesticating tsetse colonies is a slow and laborious process, so as soon as enough SEN material was available SEN males were outcrossed to BKF females to produce a line better adapted to rearing conditions. The introgressed flies were kept separate for four generations and then pooled to form the out-crossed colony (BKF-SEN strain). It was hoped that this BKF-SEN line would be better adapted to the harsh environmental conditions of Senegal. This colony was transferred to the SAS in Bratislava, Slovakia in mid-2012 for further colony build-up. In each generation, mating competitiveness tests were carried out in standard field cages where BKF-SEN males competed against BKF males for SEN females. The BKF-SEN strain progressively improved in mating participation with each generation. Once the BKF-SEN colony at the SAS reached a suitable size, shipments of sterile male pupae from BKF-SEN were sent to Senegal to assess its field performance. However, the performance of the BKF-SEN strain was worse than either the BKF or SEN strains in both the colony and under field conditions . It was Wigglesworthia, a mutualist providing the host with important nutrients and vitamins as well as contributing to immunity [Sodalis which is present in most tsetse populations. This symbiont has been mainly localized in the midgut and associated with trypanosome infection [Wolbachia, which has been found in natural populations of tsetse flies with wide infection rates ranging from 0 to 100% [Wolbachia strain present in Glossina morsitans morsitans (Gmm) can induce cytoplasmic incompatibility under laboratory conditions [Spiroplasma, representing a new class of tsetse symbiont found in G. fuscipes fuscipes (Gff), G. tachinoides, and G. palpalis palpalis (Gpp) [Spiroplasma displays a tissue tropism with Gff testes exhibiting higher density than in ovaries. Furthermore, in Gff a putative mutualistic role for Spiroplasma was acknowledged [The bacterial communities in tsetse flies vary among species and seem to play a vital role in biology, fitness, immunology, reproduction and vectorial capacity , 11. Tseimmunity –15. The nfection . The thi to 100% , 18. In Enterobacter, Enterococcus, and Acinetobacter species have been isolated from the guts of natural population of Glossina palpalis palpalis (Gpp) originating from Angola [Serratia glossinae, from the midgut of Glossina palpalis gambiensis (Gpg) insectary flies originating from individuals that had been field-collected in Burkina Faso [Gpp , Glossina pallicera  and Glossina nigrofusca flies (Enterobacter sp.) from Cameroon have been investigated using culture-dependent techniques [Glossina fuscipes fuscipes (Gff) harbour an unexpected high diversity of bacteria including Arthrobacter sp., Bacillus sp., Bacillaceae sp., Bacillales sp., Planococcaceae sp., Paenibacillus sp., Staphylococcus sp., Exiguobacterium sp., Burkholderia sp., Enterobacteriaceae, Pantoea sp., Morganella sp., Providencia sp., Pseudomonas sp. and Serratia marcescens [Gff, Gmm and G. pallidipes ) from Uganda was reported, based on multiple approaches, including deep sequencing of the V4 hypervariable region of the 16S rRNA gene, 16S rRNA gene clone libraries, and bacterium-specific quantitative PCR [Gff, Gpp and G. tachinoides (Gt), which belong to the palpalis subgroup of the Glossina genus, were infected with Spiroplasma [Enterobacteriaceae family, such as Klebsiella, Erwinia, Trabulsiella, Pantoea, and Serratia) of G. medicorum (Gmed), G. morsitans submorsitans (Gms), Gpg and Gt tsetse flies collected in Burkina Faso was identified by high throughput sequencing of the 16S rRNA gene [Gff, Gmm, and Gpal consists of additional bacterial species, such as members of Flavobacterium, Propiniobacterium, Brevundimonas, Aeromonas, and Rhodospirillales [Acinetobacter and Pantoea were detected in Gmm and Gpal, while sequences similar to Streptococcus were identified in Gmm, and Gff. Finally, sequences related to Shewanella and Pedobacter were found only in Gmm [Undoubtedly tsetse fly microbiota can be exploited since it is involved in several aspects of host biology and can be used for the development of innovative tools and strategies for vector and disease control , 19, 20.m Angola . Geiger ina Faso . Moreovechniques . Utilizircescens . On the tive PCR . Recentlroplasma . AdditioRNA gene . In pararillales . Also, sy in Gmm .rRNA gene to unravel the diversity of symbiotic bacteria in three laboratory populations of Gpg flies. Specifically, our research efforts focused on the investigation of microbiota in larval guts, and in guts and gonads of males and females collected at two age stages: (a) teneral and (b) 15-day-old insects from the three Gpg laboratory colonies. The aim was to assess the dynamics of bacterial diversity both within each Gpg laboratory colony and among them, in relation to the tissue distribution, insect age, gender and laboratory origin.In this study, we employed high throughput sequencing of the 16S Gpg laboratory colonies were investigated by 16S rRNA gene amplicon sequencing. In details, tissue samples (guts and gonads) from two developmental stages  originating from the three Gpg laboratory populations, as well as larval samples, were sequenced producing 3,469,061 reads after quality filtering. Overall, the three examined Gpg laboratory populations exhibited similar species richness and diversity indices based on number of operational taxonomic units (OTUs), Chao1 and Shannon indices  higher species-richness and diversity indexes when compared with adult guts . Similar statistically significant trend was observed among ovaries and adult guts, based on all indices   when compared with adult guts , based on the number of OTUs and the Chao1 indices  Fig. . Larval uts Fig. . Also, gGpg colony samples  higher diversity Simpson index when compared with older (15-day old age and 1-day old age) samples, as well as with female and male samples  , respectively  and larval tissue samples  and Sodalis , several other taxa  were detected. These include multiple members of the Gammaproteobacteria (such as Haemophilus and Providencia), the Bacilli  the Alphaproteobacteria , the Sphingobacteria , the Actinobacteria (such as Corynebacterium and Propionibacterium), the Deinococci (such as Thermus and Meiothermus), the Clostridia (such as Veillonella), the Armatimonadetes and the Bacteroidia (such as Prevotella) . This factor explained 86.7% of the total variance. The bacterial communities seem to be affected statistically by the developmental stage and age of the insects with the 1-day, 15-day, and the larval bacterial profile clustering separately . CAP ordinations were supported by significant traceQ_m’HQ_m statistics . Interestingly, the origin of the laboratory colonies does not seem to be a critical factor for the shaping of the bacterial profile in the gastrointestinal tract and the gonads of the insects  .Based on the β-diversity analysis, bacterial communities were strongly clustered according to the origin of the tissue distinguishing the gut bacterial profile with that from the reproductive organs Fig.   using a next-generation sequencing approach. Wigglesworthia and Sodalis were the most dominant taxa in all examined samples. Relative abundance was influenced by tissue type with Wigglesworthia being mainly dominant in adult gut tissues while gonads were characterized by the dominance of Sodalis. In larval gut samples, a balanced co-existence between Wigglesworthia and Sodalis was observed. Previously, Rio and colleagues showed that the two beneficial partners Wigglesworthia and Sodalis in a laboratory colony of Glossina morsitans morsitans exhibited well-regulated density profiles through host development and diverse disruptive events , while Wolbachia density displayed wide variability through the development, highlighting the importance of co-adaptive processes, the age of the association and the functional arsenal of symbionts [In the present work, we investigated the bacterial profile of three different ymbionts .Wigglesworthia and Sodalis), several other genera were detected including Acinetobacter, Bacillus, Brevundimonas, Enterococcus, Exiguobacterium, Flavobacterium, Klebsiella, Providencia, Pseudomonas and Staphylococcus, as reported in previous studies [Wolbachia was detected in the majority of the Gpg samples studied but in very low titers, while Spiroplasma was absent, in accordance with previous studies [Serratia was not detected in any examined Gpg samples, although this bacterium had been isolated from the midgut of insectary Glossina palpalis gambiensis flies originated from individuals that had been field-collected in Burkina Faso [In addition to the two dominant species , no statistically significant divergence in relative abundance was observed in relation to the origin of the three laboratory colonies. Both the maternally transmitted obligate and commensal dominant symbionts Wigglesworthia and Sodalis, as well as the environmentally acquired bacteria are not affected by the origin of the populations and present similar dynamics . This indicated a high degree of homogeneity for the three Gpg laboratory colonies in relation to their bacterial profile and the quantitative distribution of the bacteria that coexisted with them once they were established under mass-rearing conditions. In this respect, it is worth noting that the long domestication period for the BKF colony (> 35 years under laboratory conditions) did not have any impact on the bacterial profile as compared with the recently established SEN and BKF-SEN colonies. This might be due to the nature of the feeding type of these flies which were largely on sterile blood under laboratory conditions.Our data indicated that the origin of the three examined crobiota , althougDrosophila melanogaster different population backgrounds (genotype and origin) exhibited significant differences in the established host microbiome. This was regardless the long-term detrimental effect of diet [Bactrocera dorsalis displayed different bacterial diversity [Bactrocera dorsalis when field and laboratory populations were compared [Monochamus alternatus and Psacothea hilaris, [In  of diet . Differeiversity . This baiversity . Similarcompared . Additiohilaris, .Gpg laboratory colonies was based on the type of tissue examined. In particular, the gut samples displayed statistically significant lower species-richness than the gonads, which can be attributed to the sterile blood meal provided to the Gpg laboratory colonies which was considered as a limited bacterial source. A large-scale study showed that omnivorous insects harbor significantly higher level of bacterial diversity than stenophagous (carnivorous and herbivorous) insects [Our results indicated that the distribution of bacteria in  insects .Gpg laboratory colonies nor the relative abundance of the most dominant taxa in all analyzed samples. This is probably due to the standard rearing conditions applied to all laboratory colonies examined regardless of the developmental stage and it can be attributed to the unique lifestyle of tsetse fly [Gmm flies a significant increase of Wigglesworthia and Wolbachia was observed while the dynamics of the two symbionts remained stable through the other developmental stages [Gmm in teneral stage which allows bacterial proliferation [Spodoptera littoralis a similar significant reduction in bacterial diversity was observed during the development from egg to pupa, highlighting the effect the developmental stage has over the gut bacterial flora [Our study indicated that age and gender did not affect significantly neither the species richness/diversity of the etse fly , 32. Onll stages . This coferation . This adal flora .Anopheles gambiae, which is the main vector of human malaria (Plasmodium sp.) by the exploitation of isolations of the genera Enterobacter and Serratia with anti-Plasmodium activity [Although the bacterial diversity of natural populations and laboratory colonies of tsetse fly seem to be limited , 21, addactivity –37. HoweGpg laboratory colonies from IPCL were used  and positive controls were included. Amplicons were cleaned using Ampure XP beads , and re-suspended in 30 μl PCR-clean H2O. Products were quantified using the Qubit dsDNA High-Sensitivity assay , and an Agilent Bioanalyzer High-Sensitivity DNA chip . Samples were pooled at equimolar concentrations (300 ng per sample) for size-selection by Pippin-Prep , where a size range of 350–450 bp was extracted. Size-selected fragments underwent the same clean-up and quantification steps as above prior to sequencing. Sequencing was performed at IMGM Laboratories GmbH on an Illumina MiSeq platform using 300 bp paired-end read chemistry .Fusion primers U341F (5’-CCTACGGGRSGCAGCAG-3′), and 805R (5’-GACTACCAGGGTATCTAAT-3′) were used to amplify the V3-V4 of the 16S RNA gene . 805R reRaw sequencing reads were de-multiplexed, converted to FASTQ, and the Illumina adapters were trimmed using Illumina standard algorithms. Paired-end reads were assembled, trimmed by length and further corrected for error and quality using the usearch -fastq_mergepairs option. All subsequent analyses were conducted in usearch version 10 . BrieflyQ_m’HQ_m statistics, and were performed with 9999 permutations within PRIMER6 + .Beta-diversity analysis was calculated using weighted Unifrac distances, and PCoA analyses were performed on the resulting distance matrix. These calculations and those for alpha diversity were performed in QIIME version 1.9.1 and PRIMER6+. PERMANOVA tests were employed to detect statistical differences. Overall similarities in bacterial community structures were shown using the unconstrained ordination technique of Principal Coordinate Analysis (PCA). Differences in community structure were viewed using the constrained ordination technique of Canonical Analysis of Principal coordinates (CAP), tested using the CAP classification success rate and CAP traceAdditional file 1:rRNA gene libraries from the amplicon sequence analysis of the three Gpg laboratory colonies. (DOCX 24 kb)Richness and diversity estimation of the 16S Additional file 2:Wigglesworthia, Sodalis and Streptococcus for the three laboratory colonies examined over time . (JPG 21 kb)Relative abundance of Additional file 3:Wigglesworthia, Sodalis and Streptococcus for the three laboratory colonies examined in relation to the gender and developmental stage . (JPG 15 kb)Relative abundance of Additional file 4:Gpg laboratory colonies of the three laboratory colonies examined. Variance explained by each PCoA axis is given in parentheses. (JPG 28 kb)Principal Coordinates Analysis (PCoA) of bacterial communities based on relative abundances of OTUs with ordinations from Additional file 5:Collection protocol and sample description. (DOCX 16 kb)


Despite modern therapeutic advances, the survival prospects of pancreatic cancer patients have remained poor. Besides being highly metastatic, pancreatic cancer is challenging to treat because it is caused by a heterogeneous array of somatic mutations that impact a variety of signaling pathways and cellular regulatory systems. Here we use publicly available transcriptomic, copy number alteration and mutation profiling datasets from pancreatic cancer patients together with data on disease outcomes to show that the three major pancreatic cancer subtypes each display distinctive aberrations in cell signaling and metabolic pathways. Importantly, patients afflicted with these different pancreatic cancer subtypes also exhibit distinctive survival profiles. Within these patients, we find that dysregulation of the phosphoinositide 3-kinase and mitogen-activated protein kinase pathways, and p53 mediated disruptions of cell cycle processes are apparently drivers of disease. Further, we identify for the first time the molecular perturbations of signalling networks that are likely the primary causes of oncogenesis in each of the three pancreatic cancer subtypes. Pancreatic cancer is the most lethal form of cancer. It has an extremely poor prognosis with less than 20% of patients surviving for more than one year following diagnosis , 2. FactA significant challenge to achieving better treatment outcomes has been the heterogeneity of pancreatic cancers. Underlying this heterogeneity is the vast array of somatic mutations that are acquired during oncogenesis, and the varied effects that these mutations have on cell signalling pathways , 7. ReceAiding in the discovery of novel drug targets has been the use of next-generation sequencing based analytical methods that simultaneously identify mutations in sequences and quantify the expression of all the cellular genes that might have an impact on cancer progression. In this regard, the Cancer Genome Atlas (TCGA) project, has performed a systematic genomic, transcriptomic and proteomic characterisation of matched healthy and cancerous tissue samples from thousands of individuals afflicted with a variety of cancers . This daKRAS as a hallmark of pancreatic cancers in that more than 90% of pancreatic cancer cases have mutations in this gene . We. Wede no members . Two of ut PHF24 .PI3KCA oncogene and its negative regulator, PTEN, occur in cancers of the colon, breast, and prostate: cancers where the co-occurrence of PI3KCA and PTEN mutations appears to both drive oncogenesis, and reduce anticancer drug sensitivity [To further investigate the degrees of alteration that are evident in the expanded CoMDP solution networks for the different PDAC subtypes, we mapped a combined dataset of mRNA transcript levels, protein expression levels, mutations and CNA onto these networks. We found that alterations in p53 and cell cycle checkpoint pathway genes were most apparent in C-PDAC tumours Figure  and 5C, sitivity , 42. Fursitivity , 44.Alterations in the p53 and cell cycle pathways are frequent in cancer, and here we found that such changes were apparent in 85% of the tumours examined . In addiABCC9; found altered in 21% of all in QM-PDAC tumours. Overall, we observed that 78% of all PDAC tumours harboured a genetic alteration in at least one ABC transporter gene. ABC transporter-mediated energy-dependent efflux of a multitude of unrelated classes of anticancer drugs across membranes is a major cause of multidrug resistance and chemotherapeutic failures during cancer therapy [Consistent with previous observations, we found mutations, CNAs and changes in mRNA transcription and protein expression levels for proteins that participate in various signalling pathways that have previously been associated with pancreatic cancer , 8, 10.  therapy , 48. TheTo link genomic changes to transcriptional events, we applied the Tied Diffusion Through Interacting Event (TieDIE) approach to reveal a protein interaction subnetwork that connects altered genes to transcription factors and their putative targets ; a netwoThe MAPK1 network was enriched with proteins whose associated mRNA transcription levels were significantly higher in QM-PDAC tumours compared to other PDAC subtypes. These proteins included EGFR (a receptor of the EGFR pathway that we found activated in QM-PDAC tumours), SOS1 and GRB2 , transcriptome data ; DNA copy number and mutation data (n = 179), and targeted proteome data . Not all types of data were available for all patients because of assay failures, incomplete specimen availability and quality of issues with certain samples. All data used in our analyses are available from the TCGA website; https://portal.gdc.cancer.gov/repository.We obtained data for 185 PDAC patients involved in the TCGA project. Besides treatment outcomes these data include: whole exome sequence  . X2K empWe evaluated the scope of genomic alterations in PDAC subtypes using significantly mutated genes and copy number alteration identifications obtained from MutSigCV and GISTIC2.0 outputs, respectively , 82. Datde novo driver pathways in cancer from mutation profiles [K = 5 to 11  to return mutated driver pathways for all K values (K = 10 were connected using experimentally verified protein-protein interactions to generate an intermediary network (To discover driver pathways based on the patterns of mutations associated with PDAC, we applied the CoMDP algorithm which employs a mathematical programming method to identify profiles . BrieflyK values . Genes i network . The solWe used PARADIGM-shift, a probabilistic graphical model approach that infers the activity of signalling pathway proteins by detecting differences in the expected activity of a protein on its downstream target relative to what is expected given it upstream modulator . We ran Genomic perturbations in PDAC subtypes were connected to associated transcriptional changes using TieDIE . This met-test and One-way ANOVA were used for continuous variables were appropriate. Statistical tests were considered significant at p < 0.05 for single comparisons, and Benjamini-Hochberg adjusted p-values for multiple comparisons.Except were stated otherwise all statistical analyses were performed in MATLAB 2017b. The Fisher’s exact test was used assess associations between categorical variables. The Wilcoxon rank sum test and Kruskal–Wallis test or independent sample Student


Ns) metric indicate that loess particles at saturation with respect to liquid water conditions are marginally more efficient than the mineral and soil dust values reported in the literature, but they have lower efficiencies than the predicted Ns efficiency of K-feldspar particles at supercooled temperatures greater than -38 °C.The heterogeneous nucleation of ice processes involving loess particles that influences the formation of mixed-phase clouds are poorly understood. Here, the ice nucleating ability of wind-blown dust or loess accumulated from the past glaciated area was investigated at three temperatures: -26, -30, and -34 °C and at below and above saturation with respect to liquid water conditions. Total six loess samples from different regions across Columbia Basin province, WA, USA were collected, dry dispersed, size-selected at mobility diameter 200 nm, and investigated for their ice nucleation efficiency. To understand the effect of atmospheric processing during long-range transport on their ice nucleating ability, similar experiments were also performed on acid-treated loess samples. Additionally, the ice nucleating properties of Arizona Test Dust (ATD) were investigated as a surrogate for natural mineral dust particles to test the experimental approach. Results show that treated particles have lower ice nucleation efficiency compared to untreated particles at all temperature and saturation with respect to liquid water conditions. Comparison based on ice-active site density ( Silt-sized terrestrial sediment or loess formed by the accumulation of wind-blown dust covers approximately 10% of the world’s land surface . Loess dPrevious studies concluded that individual, as well as a complex mixture of different, minerals are efficient ice nucleating particles (INPs) in different modes of ice nucleation , 11. HetIn this paper, we report the first results from an experiment designed to investigate the ice nucleation efficiency of bare and acid coated loess from Columbia Plateau province of the northwestern United States. To understand the implications of this loess variability towards ice nucleation, bulk surface samples were collected from six sites distributed across the plateau. These samples were size-selected based on their mobility diameter, and investigated for their ice nucleation efficiency at various temperatures and humidity conditions. To understand the effect of atmospheric aging, particularly chemical aging where condensation of acids may occur while particles are transported in the atmosphere, the samples were treated with acids, and the ice nucleation efficiency of these acid treated particles was investigated.Six undisturbed surface samples of loess from Columbia Plateau province were collected. No specific permissions were required for these locations because they are public. This field study also did not involve endangered or protected species. The location of the sampling site was based on the map that shows loess distribution across the province , 16. SeeLoess samples were sieved (Newark Superla) to eliminate any vegetation debris and particles larger than ~50 μm. ice) and relative humidity with respect to water (RHw) using Murphy and Koop (2005) . Retrieved July 3, 2019 from (TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.


Biopharmaceutics Classification System (BCS) Class II and IV drugs suffer from poor aqueous solubility and hence low bioavailability. Most of these drugs are hydrophobic and cannot be developed into a pharmaceutical formulation due to their poor aqueous solubility. One of the ways to enhance the aqueous solubility of poorlywater-soluble drugs is to use the principles of crystal engineering to formulate cocrystals of these molecules with water-soluble molecules . Many researchers have shown that the cocrystals significantly enhance the aqueous solubility of poorly water-soluble drugs. In this review, we present a consolidated account of reports available in the literature related to the cocrystallization of poorly water-soluble drugs. The current practice to formulate new drug cocrystals with enhanced solubility involves a lot of empiricism. Therefore, in this work, attempts have been made to understand a general framework involved in successful (and unsuccessful) cocrystallization events which can yield different solid forms such as cocrystals, cocrystal polymorphs, cocrystal hydrates/solvates, salts, coamorphous solids, eutectics and solid solutions. The rationale behind screening suitable coformers for cocrystallization has been explained based on the rules of five i.e., hydrogen bonding, halogen bonding , length of carbon chain, molecular recognition points and coformer aqueous solubility. Different techniques to screen coformers for effective cocrystallization and methods to synthesize cocrystals have been discussed. Recent advances in technologies for continuous and solvent-free production of cocrystals have also been discussed. Furthermore, mechanisms involved in solubilization of these solid forms and the parameters influencing dissolution and stability of specific solid forms have been discussed. Overall, this review provides a consolidated account of the rationale for design of cocrystals, past efforts, recent developments and future perspectives for cocrystallization research which will be extremely useful for researchers working in pharmaceutical formulation development. Biopharmaceutics Classification System (BCS) of drugs classifies drugs into four major categories  based onIn addition to BCS of drugs, Developability Classification System (DCS) of drugs also plays a significant role in determining the development of pharmaceutical formulations, especially the oral formulations based on its solubility in biorelevant media such as FaSSIF  and FeSSIF  rather than its solubility in buffers ,4,5. VerEnhancing aqueous solubility of poorly water-soluble drugs without compromising on stability is one of the major challenges faced by the pharmaceutical industries during drug discovery and development processes ,10,11,12Tremendous research is now being conducted in cocrystallization. Discovery of new cocrystals is now considered as one of the patentable invention . CocrystIn this review, we provide a consolidated account of information available in the literature on cocrystal engineering. We have discussed different aspects of cocrystallization technology such as the synthesis techniques, type of intermolecular interactions involved in cocrystallization, different types of solids obtained during cocrystallization, techniques to characterize cocrystals, different types of cocrystals and dissolution of cocrystals in aqueous medium.While cocrystal formation is the main desired outcome, an unsuccessful cocrystallization event might result in formation of different solid forms such as eutectics ,24,25,26As new forms of pharmaceutical solids are being produced with different type of guest molecules (solvents/water/solids), assigning an exact nomenclature for each of the solid was a hotly debated topic for many years ,45,46. Icrystalline materials composed of two or more molecules in the same crystal lattice’ . During grinding, the low molecular weight components (solid/liquid/gas phase) diffuse easily into the API crystal lattice  which caChadwick et al.  explaineNumerous reports are available in the literature where grinding (solid-state or liquid-assisted) methods were used extensively to produce cocrystals ,171,172.(b) Ultrasound-assisted CocrystallizationUltrasound has been widely used for inducing nucleation in solution and cocrystallizing small molecules. Ultrasound-assisted cocrystallization has several advantages over conventional solution crystallization. The mechanical energy released during passage of ultrasonic waves induces primary nucleation at lower supersaturation levels, thereby reducing the induction time and metastable zone width ,176. UltMorrison et al.  proposed(c) Cocrystal Generation Using MoistureRodriguez-Hornedo and coworkers  conducteFurthermore, Good et al.  attempteBatchwise variation in cocrystals quality can be rectified by means of continuous production by adopting a Quality-based Design (QbD) approachContinuous production of cocrystals is less labor intensive when compared to batch modeAs compared to batch process, the maintenance of uniform particle distribution throughout the entire production, monitoring of quality of cocrystals by Process Analytical Technology (PAT) and maintaining consistency in the quality of products obtained is easier for continuous productionContinuous production of cocrystals has gained much attention from pharmaceutical sectors ,181,182.Examples of different continuous cocrystal production methods are Twin Screw Extrusion , Hot-MelThough continuous cocrystallization production methods can be employed to give high yield of cocrystals, these methods have certain limitations in terms of reproducible cycling efficiency and accurate process control. Maintaining stability and quality of products when transferred from one unit operation to another  to make these products suitable for further use can be a challenge.(a) Solid-State Shear Milling (S3M) TechnologyRecently, Korde et al.  employed(b) Melt CrystallizationMelt crystallization has been adopted to produce cocrystals by several researchers ,191,192.Douroumis and coworkers  have con2-Based Methods(c) Supercritical CO2 as solvent or an anti-solvent instead of using liquid solvents serves as an excellent mean for large-scale production of cocrystals , drug (A) concentration can be expressed as a function of conformer (B) concentration using following equation,sp is the solubility product for dissolution of a cocrystal. PSDs are used to identify the regions for drug and conformer stability in terms of drug and conformer concentration . For a cDuring cocrystal dissolution in the aqueous medium, factors such as ionization of parent components , pH of t(a) Ionization of Parent Components and pH of Aqueous MediumCocrystal can be synthesized by cocrystallizing a neutral drug molecule with acidic or amphoteric coformer molecules or a zwitterionic drug with acidic coformer or a basic drug with acidic coformer molecule. As a result, the properties of a cocrystal designed for the same API but with different coformers also exhibit different solubility behavior mainly due to variation in ionization behavior of coformer molecules. Since ionization is highly dependent on pH, pH is another parameter which significantly affects the cocrystal solubility.2-FA (2:1), GBPL-4HBA (1:1), GBPL-GA (1:1), GBPL-4ABA (1:1) and GBPL-BA (1:1) cocrystals showed lower aqueous solubility than raw GBPL [max. max values exhibited by five different gabapentin-lactam cocrystals [max value, the five GBPL cocrystals showed lower solubilitythan the drug. Therefore, this point is called as ‘the Eutectic point’ or ‘transition point’ [max is also called as Gibb’s pH as it defines the thermodynamic stability of the cocrystal and above this pH the cocrystal is unstable [Maheshwari et al.  used Gabraw GBPL  and exhicrystals . Below pn point’ ,318. Theunstable .Thakuria et al.  has prov(b) Effect of Solubilizing AgentThough drug-solubilizing agents such as surfactants can increase the solubility of a cocrystal in an aqueous medium , at a ce(i)Dissociation of the cocrystal into amorphous or nanocrystalline drug clusters, which is represented as ‘spring’ effect (ii)Transformation of the amorphous or nanocrystalline drug clusters into a stable form through formation of a metastable phase by adopting Ostwald’s Law of Stages(iii)Attainment of higher apparent solubility and maintenance of optimal drug concentration in the aqueous medium, which is represented as ‘parachute’ effect .During dissolution, cocrystals exhibit maximum peak value in the drug concentration which is attained in a shorter time (such as less than 30 min) (spring effect). This high concentration is maintained for a longer period before ultimately decreasing to the equilibrium solubility level (parachute effect) . The sprDissolution of cocrystals can be influenced by several factors such as aqueous solubility of coformers (as discussed in (a) Crystal HabitCrystal habit of cocrystals can also influence its dissolution in dissolution medium. A drug can cocrystallize with a coformer molecule in various sizes and shapes depending on different crystallization conditions. A crystallization event can change crystal properties such as habit, polymorphism and size . The ter(b) pHCao et al.  showed tWhile cocrystallization can be used to enhance solubility of drugs, it can also be used to reduce the aqueous solubility of drugs . Though (a) Sulfacetamide CocrystalsSulfacetamide is a topical antibiotic used to treat conjunctivitis. Despite its therapeutic efficacy, its pharmaceutical use is limited because of physiological constraints such as tear flow, reflex blinking and drug loss. This drawback can possibly be eradicated by frequent dosing. However, frequent dosing in turn can lead to excess drug loading in patients. Therefore, reducing the aqueous solubility of sulfacetamide can help in getting rid of excess drug loading in patients without reducing the frequency of drug administration. Therefore, Nangia and coworkers  adopted (b) Fluoxetine HCl-Benzoic Acid (1:1) CocrystalFluoxetine HCl is an antidepressant drug available in the market with the trade name, Prozac. During a study carried out by Childs et al.  to prepa(c) Curcumin-phloroglucinol (1:1) CocrystalCurcumin is a natural phenolic ingredient with anticancer activity. Its therapeutic use is limited due to its poor aqueous solubility, and hence its poor bioavailability. Efforts have been made by several researchers to enhance its aqueous solubility by forming cocrystals ,72,328. (d) Lamotrigine-phenobarbital (1:1) CocrystalRecently, Kaur et al.  reportedManypharmaceutical cocrystals have been synthesized by several researchers to enhance the aqueous solubility of poorly water-soluble drugs . However, the number of cocrystals being used as a regular drug with the approval of FDA is very low. Also, the bioavailability studies for the synthesized cocrystals have been rare. At present, Entresto (used in the treatment of chronic heart failure), Lexapro (used as an antidepressant) and Depakote (used in the treatment of seizure disorders and manic depression) are the three pharmaceutical cocrystals being approved by FDA for clinical use. Cocrystallization of poorly water-soluble drugs is one of the novel ways to improve their aqueous solubility. A lot of research efforts are now focused on synthesizing cocrystals of poorly water-soluble drugs with appropriate coformers. The physicochemical properties of cocrystals such as melting point, aqueous solubility and hence their bioavailability depends upon the type of coformer used. Also, as mentioned earlier, the cocrystallization attempts do not always lead to a successful formation of cocrystals. At times, a eutectic or even a coamorphous solid is obtained. Therefore, choosing a correct coformer is of utmost importance. However, at present, conformers are either chosen based on empirical understanding or based on cumbersome methods requiring detailed analysis and calculations. Therefore, development of a new and fast coformer screening tool is necessary to screen coformers suitable for cocrystallization. Furthermore, efforts are also needed to develop a generalized understanding of intermolecular interactions that influence the cocrystallization outcome by employing supramolecular chemistry and crystal engineering principles. While a rationale design of a cocrystal can lead to a successful outcome at the end of cocrystallization, it is equally important to develop solvent-free cocrystal production methods such as melt crystallization. At present researchers are trying to develop continuous techniques for cocrystal synthesis. Further efforts in this direction will enable a large-scale production of cocrystals. Additionally, future research also needs to focus on stability of cocrystals. At present, very little information is available on the aspects related to the cocrystal stability such as conversion of cocrystals to drug polymorphs or degradation of cocrystals upon exposure to aqueous environment, pH or temperature, etc. It is also evident from the literature reports on cocrystal/eutectic/coamorphous/solid solution formulations reviewed so far that most of the solids prepared by cocrystallization have not been studied for their bioavailability, pharmacokinetics and pharmacodynamics. Mere production of cocrystals without investigating its efficacy, pharmacokinetic or pharmacodynamic behavior in biological system would end up in vain. Moreover, preclinical trials followed by clinical trials have needed to develop cocrystals into marketed product.


Although the fascia iliaca compartment block (FICB) seems to be an ideal technique for femoral neck and shaft fractures occurring in resource-poor settings, it has been unclear how effective it is when used by emergency physicians (EP) with little training in the technique, using equipment, medications and methods that differ from those commonly available in developed countries. This series was designed to demonstrate that EPs in a resource-poor setting can provide effective analgesia for femur fractures with anatomic landmark-guided FICBs, clinician-compounded lidocaine-epinephrine , and a standard injection needle.Over a three-month period, patients ≥12 years old presenting to the emergency department with hip or femur fractures and a Likert visual analogue scale >4 had an EP-administered FICB. EPs used a standard intramuscular needle and a lidocaine-epinephrine solution they compounded at the bedside and located the injection site using only anatomic landmarks. EPs evaluated the patient’s pain level at 30 minutes and at two hours post-FICB. We also reviewed articles since 2016 that describe the FICB.We enrolled a non-consecutive sample of 10 patients in the case series. Five had femoral neck (hip) fractures and five had femoral shaft fractures. All patients had a reduction in their pain levels after the FICB. On average, the block took effect about three minutes after injection. At 30 minutes all patients reported clinically meaningful pain reduction. The analgesic effect of the compounded agent lasted approximately 200 minutes. No adverse effects were reported. No published journal articles about FICB since 2016 were from resource-poor settings, and only one was from a developing country.This series suggests that the FICB is effective even when performed with the minimal materials that are usually available in resource-poor settings. Methods such as this, which use simplified clinical tests and techniques applicable in resource-poor settings, can assist global emergency medicine (EM). We can assist global EM by similarly finding methods to simplify useful clinical tests and techniques that can be used in resource-poor settings. Patients with femur and hip fractures commonly present to emergency departments (ED). Promptly providing analgesia reduces the significant physical and psychological effects on patients. It speeds their operative management, lessens the length of hospitalization,1Building on Sharrock’s accidental discovery of the fascia-iliaca compartment block (FICB) in 1980,6While numerous publications have demonstrated the block’s efficacy in relieving pain for patients with acute femur fractures, the methods relied on equipment and medications not available in most facilities outside the developed world and few have originated from resource-poor facilities in developing countries. Resource-poor settings, which often have only parenteral opiates to provide analgesia after acute fractures, are where FICBs may prove most useful. This paper reviews recent publications that describe FICB settings and techniques and uses a case series from a resource-poor hospital ED in a developing country to demonstrate the block’s efficacy using basic equipment, inexpensive and readily available medications, and novice practitioners.The convenience study sample was patients ≥12 years old with moderately and severely painful  injuries to the hip and femur. Patients were excluded if they had a history of allergies to local anesthetics; a history of femoral bypass surgery; signs of an infection at the FICB injection site; a bleeding diathesis (known history or currently evident); or a history of second- or third-degree heart block. EM faculty and resident physicians performed all the blocks. The Medical Advisory Council  approved this study. All participants or their surrogate decision-makers provided informed consent.Prior to the study, participating emergency physicians (EP) attended a short teaching session, received a printed protocol with anatomical illustrations, and were shown the procedure for compounding the lidocaine-epinephrine mixture. After identifying appropriate patients with a VAS pain scale ≥4, the EPs obtained informed consent. They then completed a neurovascular examination of the affected limb and prepared the lidocaine-epinephrine 1:100,000 solution (1 milligram [mg] =1 milliliter [mL] 1:1000 epinephrine/100 mL lidocaine). Compounding the solution at the bedside, they added 0.2 mL of epinephrine (1:1000) to 20 mL of lidocaine 1% (10 mg/mL). They aspirated the mixture into a sterile syringe attached to a 21-gauge, 2-inch intramuscular injection needle.8What do we already know about this procedure?Fascia Iliaca Compartment Blocks (FICB) are ideal for providing analgesia to emergency department (ED) patients with femoral fractures but are rarely used in resource-poor settings.What makes this case series reportable?Emergency physicians in a resource-poor setting successfully did FICBs using only anatomical markers, clinician-compounded analgesic and standard injection needles.What is the major learning point?FICB blocks can be successfully performed  with the minimal equipment and materials present in any ED.How might this improve emergency medicine practice?This study (the first from a developing country since at least 2016) should encourage clinicians in similar settings to use the FICB despite lacking high-level resources.With the patient supine, the inguinal ligament was identified, the femoral artery palpated, and the skin cleaned with iodopovidone. The needle was inserted perpendicular to the skin at a point one centimeter (cm) below the juncture of the lateral and medial 2/3 of a line from the pubic tubercle to the anterior superior iliac spine . Using tWe searched PubMed for publication dates between 2016 and mid-May 2018 using the following search strategy: “Best Matches” for “fascia iliaca block” OR “iliofascial block”: [“fascia iliaca block”[All Fields] OR ]. The papers were then culled for non-review papers in English that had information describing their setting and preoperative-analgesia FICB protocol using a single (non-continuous catheter) injection for acute femur fractures.Ten patients aged 12 to 93 years (median 59 years) had FICBs using bedside-compounded lidocaine-epinephrine. Five patients were males; five had femoral neck and five had femoral shaft fractures. Seven EPs performed the blocks: five residents and two faculty. All seven initially performed the block for the first time. Medication preparation and block performance generally took less than five minutes. Repeat pain levels were measured at 30 minutes and 120 minutes post-block. All patients experienced clinically relevant pain reduction post-block at both time points. VAS pain scores averaged 7.1 (range 5–9) on presentation, 2.7 (range 0–4) at 30 minutes post-block, and 0.6 (range 0–2) at 120 minutes. The average duration of analgesia was 200 minutes. No adverse events were reported from the blocks. Most study patients were admitted to the operating room (OR) or ward before the block lost effect. For the few who remained in the ED, an additional FICB was administered. One patient refused to be transferred to the ward before he received a second FICB.We identified 10 articles using the PubMed search. One was in Chinese, one described a femoral nerve block, and one had insufficient information. The Since 2016, all journal articles describing the use the FICB for analgesia after acute fractures have originated from resource-adequate facilities; all but one are from developed countries. In contrast, the current case series is from a resource-poor teaching hospital in a developing country, a situation in which the FICB is particularly useful. Resource-poor settings have less monitoring available for patients who have been given mind-altering analgesics, surgery may be significantly delayed due to a lack of staff or equipment, and inpatient admission delays may require EPs to repeatedly administer analgesia over several days. Yet clinicians in these facilities may hesitate to do FICBs without the equipment and medications most commonly described for the procedure. We conducted this case series to demonstrate that the FICB can be performed successfully with limited equipment, medication, and training.Of the seven PubMed articles that met the criteria, only one was from a developing country.The loss-of-resistance FICB using anatomic landmarks does not require ultrasound guidance, since its safety relies on the distance of the thigh’s major neurovascular structures from the injection site. Neither the study that first described the FICB nor the initial studies demonstrating its utility for acute fractures in EDs used ultrasound guidance, although they had high success rates.A common recommendation for performing the FICB is to use a blunt 21G or a Tuohy  needle to more easily recognize passage through the tissue planes. Three of five articles described using a Tuohy needle, while another used an echogenic needle with ultrasound guidance. In our setting, neither recommended needle was available. We successfully used an intramuscular injection needle.As the reviewed articles demonstrated, several long-acting local anesthetics work well for single-injection (as opposed to catheter-infusion) FICBs. Cost and logistics prevent our ED from stocking anything but plain lidocaine. To produce long-acting anesthesia, we added epinephrine to lidocaine, often doubling its anesthetic duration.No inexperienced clinicians in the reviewed publications performed FICBs, and only two had EPs doing the block. As in our setting, clinicians in resource-poor EDs often must perform novel techniques based on written, oral, or video descriptions. The blocks performed by our ED staff and resident physicians were successful, although they had learned the procedure using written and oral descriptions.This study had two major limitations: only 10 patients met the study criteria; and the IRB did not permit sham injections for a control group. Nevertheless, the high success rate our EPs had with the FICB using inexpensive and readily available materials is consistent with the results in studies with the normally recommended equipment and medications.EM faculty and residents in a resource-poor ED successfully reduced the pain from femoral fractures by performing FICBs using only anatomic landmarks, physician-compounded lidocaine-epinephrine, and a standard injection needle. This demonstrates that physicians in resource-poor settings can safely and quickly perform the FICB with little training, using the equipment and medications they typically have available. Using the FICB in resource-poor, often rural settings also reduces risks related to opioid use and provides an excellent method to ease the pain of patients transferring to higher levels of care.Documented patient informed consent and/or Institutional Review Board approval has been obtained and filed for publication of this case report.