Transfusions of red blood cells (RBCs), platelets, and plasma are critical therapies for infants and neonates (particularly preterm neonates) in the neonatal intensive care unit, who are the most frequently transfused subpopulation across all ages. Although traditionally a significant gap has existed between the blood utilization and the evidence base essential to adequately guide transfusion practices in infants and neonates, pediatric transfusion medicine is evolving from infancy and gradually coming of age. It is entering an exciting era with recognition as an independent discipline, a new and evolving high-quality evidence base for transfusion practices, novel technologies and therapeutics, and national/international collaborative research, educational, and clinical efforts. Triggers and thresholds for red cell transfusion are accumulating evidence with current phase III clinical trials. Ongoing trials and studies of platelet and plasma transfusions in neonates are anticipated to provide high-quality evidence in years to come. This article aims to summarize the most current evidence-based practices regarding blood component therapy in neonates. Data on the use of specific components  are provided. We attempt to define thresholds for anemia, thrombocytopenia, and abnormal coagulation profile in neonates to highlight the difficulties in having a specific cutoff value in neonates and preterm infants. Indications for transfusion of specific products, transfusion thresholds, and current practices and guidelines are provided, and possible adverse outcomes and complications are discussed. Finally, the critical research knowledge gaps in these practices as well as ongoing and future research areas are discussed. In an era of personalized medicine, neonatal transfusion decisions guided by a strong evidence base must be the overarching goal, and this underlies all of the strategic initiatives in pediatric and neonatal transfusion research highlighted in this article. During their stay in the neonatal intensive care unit (NICU), the majority of extremely low-birth-weight (ELBW) infants receive at least one red blood cell (RBC) transfusion and many end up receiving multiple transfusions. These tiniest of recipients usually receive large volumes of blood products relative to their small size. High-quality research evidence guiding transfusion practice in this patient population has traditionally been lacking, but the pediatric and neonatal transfusion medicine research base has gradually been gaining momentum3. Triggers and thresholds for red cell transfusion are accumulating evidence with current phase III clinical trials. Ongoing trials and studies of platelet and plasma transfusions in infants and neonates are anticipated to provide high-quality evidence in years to come5.Blood transfusions can be critical supportive therapy for infants and neonates (particularly preterm neonates) who are frequently transfused7, but are also quite vulnerable to short- and longer-term issues resulting from transfusion-transmitted diseases, such as Babesia and Zika virus9. The true incidence of adverse outcomes from transfusions in neonates is not known. These complications tend to be underdiagnosed, especially in neonates with prior respiratory symptoms.Although transfusions overall are increasingly safe, neonates, especially extremely preterm neonates, are particularly predisposed to transfusion complications, including acute reactions such as transfusion-associated circulatory overload (TACO) and transfusion-associated acute lung injury (TRALI)This article aims to summarize the most current evidence-based practices regarding blood component therapy in neonates. Data on specific component use, thresholds, and guidelines are provided. Indications for the transfusion of specific products, including RBCs, platelets, and plasma, and possible adverse outcomes and complications are discussed. Finally, the critical research knowledge gaps in these practices as well as ongoing and future research areas are discussed.2. For such a critical therapy in neonates, one would expect the hemoglobin threshold for defining anemia as well as the threshold for transfusion to be well delineated in neonates. However, despite some studies attempting to study this, the lack of an optimal well-defined RBC transfusion threshold for neonates has been consistently recognized as a major gap in our understanding of neonatal transfusion5.RBCs are the most commonly transfused blood products in neonates. A majority of ELBW infants require at least one transfusion, and many receive multiple transfusions11. The definition can also be dependent on the outcomes being assessed. For example, preoperative hematocrit has been examined as a continuous variable and a level lower than 40% was found to be the ideal cutoff point (Youden J index) for predicting overall mortality12.One of the major problems with deciding when to transfuse is the lack of a clearly identified and agreed-upon definition of severe anemia in neonates. Owing to the absence of a universally accepted definition of anemia in neonates necessitating an RBC transfusion and to the wide age-specific reference range of acceptable hematocrit values, treatment thresholds are not standardized. In research settings, various strategies have been employed. Two randomized clinical trials comparing transfusion practices in preterm infants and other prospective studies have used the cutoff hemoglobin level of 8 g/dL or less for defining severe anemia in neonateset al., studying 100 hospitalized preterm infants with birth weights of 500 to 1,300 g stratified to two levels of hematocrit threshold for RBC transfusion, suggested that, although infants in the liberal transfusion group overall received more RBC transfusions (mean ± standard deviation 5.2 ± 4.5 versus 3.3 ± 2.9), in the restrictive transfusion group, the number of donors to whom the infants were exposed was not significantly different. They additionally reported that there was no major difference between restrictive versus liberal groups in the percentage of infants who “did not get transfusions at all” . Notably, infants in the restrictive group were more likely to have major adverse neurologic events, including (a) intraparenchymal brain hemorrhage, (b) periventricular leukomalacia, and (c) more frequent episodes of apnea (both mild and severe episodes), than the liberal transfusion group. This study raised the possibility that the practice of restrictive transfusions may indeed be harmful to preterm infants10.Although there is more convincing randomized controlled trial (RCT) data from adults supporting the use of restrictive transfusion strategies as compared with liberal transfusion strategies, the evidence in neonates is still highly conflicted. In 2005, an RCT by Bellet al. was followed by the Premature Infants in Need of Transfusion (PINT) study with contrasting results. This RCT included 451 infants with a primary goal to determine whether transfusion of ELBW infants at restrictive versus liberal thresholds had different rates of survival or morbidity at the time of hospital discharge. With a mean birth weight of 770 g and gestational age of 26 weeks, rates of the primary outcome (death before home discharge) were 74.0% in the low-threshold group and 69.7% in the high-threshold group . The study revealed that when a higher hemoglobin threshold in ELBW infants was maintained, a greater number of infants received transfusions, with little evidence of benefit11. It is important to note that the trial by Bellet al. and the PINT trial were both limited by not studying neurodevelopment beyond hospital discharge. Thus, the interpretation from these studies is restricted to the duration of hospitalization only and not the long-term effects. The shorter-term outcomes studied to date may be a poor proxy for the long-term neurological outcomes. There is limited knowledge on the effects of limiting oxygen delivery to the brain by withholding transfusions, and this remains an important gap in our understanding on the effects of RBC transfusions on neurodevelopment.The study by BellTwo ongoing and much-awaited definitive phase III transfusion studies in neonates are the Effects of Transfusion Thresholds on Neurocognitive Outcome of Extremely Low Birth Weight Infants (ETTNO) trial and the Transfusion of Prematures (TOP) trial to determine whether higher hemoglobin thresholds for transfusing ELBW infants resulting in higher hemoglobin levels lead to improvement in (1) survival (primary outcome) and (2) rates of neurodevelopmental impairment at 22–26 months of age.Age of transfused blood and outcomes in children. In children and neonates, as in adults, the age of stored RBCs and adverse outcomes has been a highly debated question. The Age of Red Blood Cells in Premature Infants (ARIPI) RCT showed no difference in outcomes in premature very-low-birth-weight (VLBW) infants requiring a transfusion when fresh RBCs (mean age of 5.1 days) versus standard blood bank practices (mean age of 14.6 days) were followed13. However, there were concerns raised about the generalizability of the ARIPI study. The primary critique was that the mean duration of RBC storage in the standard-of-care group in the ARIPI study was not reflective of the mean storage duration in the US, in which the average storage age was reported to be higher (about 18 days). Furthermore, the hemoglobin thresholds for transfusion were not pre-specified or standardized, allowing for higher hemoglobin levels for each infant, and the storage solution and manufacturing of units in different anticoagulant-preservative solutions were not addressed in the study14. Currently, the Age of Blood in Children in Pediatric Intensive Care Units (ABC PICU) study, examining outcomes comparing transfused RBCs stored for not more than 7 days or standard-issue RBCs (expected mean RBC storage duration of 17–21 days), is awaiting completion. Evidence in older children from the Tissue Oxygenation by Transfusion in Severe Anemia With Lactic Acidosis (TOTAL) randomized clinical trial (290 children 6–60 months old) showed no difference in post-transfusion correction of mean lactate levels between young blood (median age of 8 days) versus older blood (median age of 32 days)15.Adverse outcomes from red blood cell transfusions18, retinopathy of prematurity (ROP)19, and chronic lung disease (CLD)21 as well as mortality. Interpretation of UK analysis of the Serious Hazards of Transfusion (SHOT) scheme data estimated the rates of an adverse outcome to be 18:100,000 red cells issued for children younger than 18 years and 37:100,000 for infants younger than 12 months compared with 13:100,000 for adults7. Thus, infants and neonates are disproportionately more predisposed to adverse effects from RBC transfusions than are other populations.RBC transfusions are based on the assumption that the transfusion leads to a direct and immediately effective increase in oxygen delivery to tissues. However, although the correlation with transfused red cell mass and improved oxygenation in neonates has not been proven, RBC transfusions do come with inherent risks for adverse complications in the recipient. Although many of these inherent risks are similar to ones in older children and adults, neonates have some specific adverse associations and outcomes potentially related to RBC transfusions, including the development of necrotizing enterocolitis (NEC), intraventricular hemorrhage (IVH)22. However, a more updated meta-analysis published recently which addresses the same question from 17 observational studies showed no temporal relationship between RBC transfusions and NEC23. This meta-analysis highlighted how the effect size for this relationship differed between matched case-control studies  and differed from those reported in cohort studies . This example and contrast in results based on the study type and methodology underscores the need for high-quality prospective evidence in the area.Meta-analyses/systematic reviews studying these relationships give contrasting results as more and more studies addressing the topics surface. As an example, in 2012, a meta-analysis of 11 retrospective case-control studies and one cohort study showed that recent exposure to transfusion was associated with NEC in neonates. Neonates who developed NEC were at overall higher risk of mortalityet al., in a prospective study assessing various predictors of adverse outcomes as time-varying co-variates, recently showed that among VLBW infants, severe anemia, instead of RBC transfusions, is associated with an increased risk of NEC, thus proposing the need for further studies to evaluate whether preventing severe anemia is more important than minimizing RBC transfusions24. Likewise, Goobieet al.12 recently suggested an association between preoperative anemia and postoperative mortality in neonates25.The important question, however, is whether the adverse effects are from anemia itself or from the transfusions, and methodologically sound prospective studies are required to answer this fundamental question. Patelet al. published the most comprehensive review and meta-analysis to date on the harmful effects and associations potentially attributable to RBC transfusions26. Together inclusive of 61 studies , this meta-analysis revealed no evidence of difference in rates of mortality between restrictive and liberal strategies for transfusion . Similarly, no differences were seen for other outcomes, including NEC, ROP, CLD, or IVH26.Within the transfused group as well, the outcome could vary depending on the hemoglobin transfusion threshold. Keir and StanworthThere are no consensus guidelines on how the transfusion practices require specific modification for neonatal use. The standardization of (1) definitions of adverse effects in neonates and (2) associations of RBC transfusion with many of these adverse effects in neonates, through an international consensus of experts, is essential.27.The majority of research studies thus far are limited by a small sample size; observational, retrospective, or a case-control design; or a lack of evaluation of time-varying exposures to study various outcomes. This underscores the need for prospective studies in which each documentation of anemia, subsequent RBC transfusion, and a specific outcome of interest can be prospectively and systematically evaluated28. The dire need of big data applications in transfusion medicine research29 is even more palpable in neonatal transfusion medicine28. This has been identified in scientific proceedings of the National Heart, Lung, and Blood Institute30. However, a few researchers have successfully employed some surgical databases for studying outcomes associated with transfusion and transfusion volumes31. In the absence of these large databases, methodologically sound meta-analyses and systematic reviews can provide key insights and ability to answer some questions26.One of the biggest impediments to advancement of neonatal transfusion research is that there are no vein-to-vein databases, registries, or networks that include neonatal RBC transfusion-relevant data with outcome or donor linkage to recipients in sufficient detail32.Estimates of the overall prevalence of neonatal thrombocytopenia vary from 1% to 5% of all neonates and is averaged at about 25% (range of 22–35%) among neonates admitted to NICUs. The most premature of infants have the highest risk of developing thrombocytopenia. Consequently, platelets are the second most commonly transfused blood component in neonates, next to RBCs9/L and is classified as mild (100–150 × 109/L), moderate (50–99 × 109/L), or severe (<50 × 109/L)33. However, the question is whether there is a standard definition of thrombocytopenia which can be applied to neonates of all gestational ages. The incidence of thrombocytopenia in neonates is extremely variable depending on the gestational age of the studied population. Whereas the overall incidence of thrombocytopenia in newborns at the time of birth is relatively low (less than 1%)34, among NICU patients, the incidence of thrombocytopenia has been reported to be inversely correlated to gestational age. Specifically, among neonates with a birth weight less than 1,000 g, it is estimated to be as high as more than 50%36. In 2009, Wiedmeieret al., in a large observational study on neonatal platelet counts, including data on about 47,000 infants , suggested a predictable increment in platelet counts since birth with an increase in gestational age, with a mean increase in platelet count of ~2 × 109/L per week increase in gestational age37. These findings suggest that there is not one single threshold to define thrombocytopenia in neonates and that different thresholds need to be used to define thrombocytopenia in preterm infants depending on gestational age37.Thrombocytopenia in neonates is traditionally defined as a platelet count of less than 150 × 1032. Several studies have found that a multitude of factors besides the severity of thrombocytopenia predict the bleeding risk in neonates, the degree of prematurity being a predominant factor. Thus, it will be important to develop improved tests to assess primary hemostasis and bleeding risk in neonates. At present, indications, thresholds, and rationale for prophylactic platelet transfusions to correct thrombocytopenia remain controversial and in need of a strong evidence base to guide safe platelet transfusion practices in neonates, especially the most premature infants.A clear relationship between low platelet counts and consequential clinical bleeding or major hemorrhage has not been established in neonates9/L) is not supposed to be detrimental. In an earlier study of prophylactic platelet transfusion thresholds, over 150 thrombocytopenic neonates less than 33 weeks’ gestation age  were randomly assigned to receive platelet transfusions to maintain the platelet count at either greater than 150 × 109/L or greater than 50 × 109/L in the first week of life. This study suggested that liberal platelet transfusion threshold (150 × 109/L) did not protect against IVH38. Another retrospective observational cohort study examined infants with platelet counts of less than 50 × 109/L in the NICU, and preterm infants with platelet counts of less than 50 × 109/L in whom platelet transfusions were withheld did not suffer from serious hemorrhage, suggesting that lower platelet count thresholds may be safe for otherwise stable infants39. In a multicenter, retrospective cohort study of 972 VLBW infants from six US NICUs, the pretransfusion count was at least 50 × 109/L for 653 (65.4%) of 998 transfusions. While platelet transfusion decisions were taken with higher severity of illness, there was neither an association between the severity of thrombocytopenia and the subsequent risk for IVH nor a lower risk of IVH due to platelet transfusions 40. There is a need for RCT evidence to guide the optimal platelet transfusion decisions in premature neonates.There continues to be widespread heterogeneity in the pretransfusion thresholds for platelet count and overall platelet transfusion practices between various hospitals and countries. In general, moderate thrombocytopenia  instead of the platelet count as a transfusion trigger. The use of platelet mass-based NICU transfusion guidelines has been shown in few studies to be feasible as well as safe, as they were associated with fewer platelet transfusions overall and no recognized increase in hemorrhagic problems43. The NeoBAT score is being applied in prospective studies and trials of neonates which are mentioned subsequently. There are published algorithms which are helpful to direct the diagnosis and specific investigations of different etiologies of thrombocytopenia. Specific indications for platelet transfusions like neonatal alloimmune thrombocytopenia (NAIT) need a high index of suspicion for early diagnosis and prompt attention with specific requirements like antigen-negative platelets.The major clinical outcome of concern for neonates with severe thrombocytopenia is risk of major bleeding, including intracranial hemorrhage and specifically IVH and periventricular hemorrhage (PVH). Recently, a neonatal bleeding assessment tool (NeoBAT) was proposed aiming for standardized bleeding assessments in this high-risk population9/L and for stable children older than 4 months, prophylactic platelet transfusions should be given at a platelet count of less than 10 × 109/L. The guidelines admit that, given the lack of studies in pediatrics, the platelet transfusion indication for critically ill children or those with hematologic and oncologic malignancies who develop severe thrombocytopenia is based on adult study data44.The recent guidelines from the British Society of Hematology (BSH) recommend that for stable children younger than 4 months, prophylactic platelet transfusion should be given at a platelet count of less than 30 × 10Adverse outcomes from platelet transfusions. Platelet transfusions come with risks inherent to any blood component transfusion, including clinical errors in administration and incorrect blood component transfused. They also have significant risks of bacterial and septic infections due to storage at room temperature and risks of allergic reaction as well as TACO and TRALI due to the innate plasma component of platelet products.et al., in a study of 1,600 thrombocytopenic NICU patients, suggested that multiple platelet transfusions may be contributing directly to a small fraction of mortality in neonates45. These relationships remain associations at best and need to be validated in prospectively designed studies.Although the number of platelet transfusions might be a surrogate marker for level of illness and thus predictive of mortality, Baer7.Data from the UK’s SHOT national hemovigilance scheme against a population-based epidemiological study of transfused patients have indicated that transfusion-associated adverse events are disproportionate in the number of occurrences in the neonatal population as compared with older children and adults. Transfusion reactions are likely highly under-recognized and under-reported in neonates, especially the most premature infants46. Pathogen-reduced platelet products have been used in some European countries for over a decade. The published RCTs comparing the use of pathogen-inactivated platelets with conventional platelets did not specifically enroll neonates. There are few prospective observational studies which have found no evidence of major adverse events in infants (<1 year old). In neonatal patients treated with phototherapy devices with a peak energy wavelength of less than 425 nm, use of the INTERCEPT blood system for the preparation of platelet components is contraindicated owing to a potential for erythema from interaction between ultraviolet light and amotosalen (interceptbloodsystem.com)47. Other pathogen reduction products in the pipeline, such as Mirasol, a riboflavin (vitamin B2)-based system, are still in clinical trials. A recent Cochrane review of RCTs comparing the transfusion of pathogen-reduced platelets with standard platelets suggested no evidence of a difference in the incidence of clinically significant bleeding complication or all-cause mortality. The review also suggested that pathogen-reduced platelet transfusions increase the risk of platelet refractoriness and platelet transfusion requirements. Furthermore, evidence on subgroup differences in multiple transfusion trials between the two pathogen-reduced platelet technologies (Intercept and Mirasol) revealed that for all-cause mortality and the interval between platelet transfusions, Intercept was more favorable; however, these studies are not directly applicable to children/neonates48. Longitudinal studies are needed for both products and their use in neonates as well as older children.A pathogen-reduced platelet product (INTERCEPT) was approved for use in the US by the US Food and Drug Administration in 2014 and has been used in a small number of neonates worldwide49. Platelet trigger trials assessing clinically relevant outcomes are now under way in preterm neonates with thrombocytopenia. PlaNeT1 was an observational study of current practice and outcomes of platelet transfusions in neonates. PlaNeT2 (http://www.planet-2.com) is an ongoing randomized prophylactic threshold trial randomly assigning neonates to a 25 versus 50 × 109/L transfusion threshold. The NeoBAT bleeding assessment score is being used by the PlaNeT2 study. Thus far, treatments or research studies with thrombopoietic growth factors have not been undertaken in neonates. Besides studies of the platelet transfusion threshold, studies are needed to characterize the overall platelet function and the global hemostatic profile of neonates, especially preterm infants, as well as the use of thromboelastography (TEG) or thromboelastometry (ROTEM) to monitor hemostatic treatment to better predict their bleeding risk and thus direct prophylactic or therapeutic measures accordingly for IVH/PVH50.Study of platelet transfusion strategies to prevent or mitigate bleeding in neonatal and adult patients has been long outlined as clinical trial opportunities in transfusion medicine by the National Heart, Lung, and Blood Institute thinktank of experts51, we rely largely on expert opinion to make recommendations for plasma transfusions.Evidence guiding the transfusion of plasma, of all blood products, in neonates is the weakest. Although there are a few prospective multicenter studies exploring plasma use in neonatesThe neonatal coagulation system matures slowly after birth, and, depending on the levels of various factors at birth, the “normal” reference ranges in neonates and infants are quite different from those in adults. Even within the neonates, the reference ranges vary depending on the gestational age. However, owing to the lack of awareness of the different ranges, transfusion decisions in neonates are unfortunately made on the basis of the adult ranges or “cutoffs” used to define coagulopathy.52.A national audit of transfusion practice from the UK indicated that almost 50% of plasma transfusions are given prophylactically to neonates with “abnormal coagulation values” with no evidence of active bleeding, in order to prevent IVH, although there is very low-grade evidence to support the effectiveness of this practice. Likewise, there is widespread use of plasma reported in hospitalized children in the absence of demonstrated efficacy or effectivenesset al. and PlasmaTV investigators reported, in an international point prevalence study of plasma transfusions in critically ill children, that about one-third of the plasma transfusions given in a PICU setting are without any evidence of bleeding or an invasive procedure or surgery warranting a plasma transfusion and that only 22% were administered in case of critical bleeding53.Karama priori secondary analysis of a prospective observational study, investigators of the PLASMA TV study reported lower PICU mortality in the solvent detergent versus fresh frozen plasma (FFP)/frozen plasma . Based on these results, the authors suggested that solvent detergent plasma use in critically ill children may be associated with improved survival54. There are no dedicated studies about effectiveness/adverse effects of pathogen-reduced products in term and preterm neonates.In a follow-up of anCurrent expert opinion supports the “therapeutic” use of plasma either (1) with active bleeding or (2) concurrent to invasive procedures in patients at high risk of bleeding and those recognized to have an abnormal coagulation profile. An abnormal coagulation profile would be identified as the conventional coagulation parameters prothrombin time (PT) or activated partial thromboplastin time (aPTT) being significantly above the normal age and gestation adjusted reference ranges.44. BSH guidelines also advise that plasma not be used as a means for volume replacement or for prevention of bleeding  in neonates44. The BSH recommends that prophylactic FFP “not” be administered to “non-bleeding” children with minor prolongation of PT/aPTT, including prior to surgery, but it may be considered for surgery to critical sitesPlasma use in neonates comes with multiple risks: it could range from a simple allergic reaction secondary to plasma proteins or a febrile non-hemolytic reaction to severe life-threatening reactions, including TRALI, TACO, febrile reactions, and hemolysis, all likely under-recognized and under-reported in neonates.55. The neonatal study reported a dose-dependent effect with more than five times the odds of thrombosis in neonates who received greater than 50 mL/kg of plasma by day 5 of age. Raffiniet al., in a study of plasma transfusions in US hospitals, found that the rate of venous thrombosis in children (including neonates) receiving plasma was 10 times greater than the rate for hospital admissions without plasma transfusion52.Although plasma is usually transfused for its hemostatic properties, there have been reports of an association between plasma transfusion and venous thrombotic outcomes in neonatal as well as pediatric patients, with the coagulation factors and platelet-derived microparticles in plasma being considered responsible for the thrombotic effects56.The first necessary step in furthering research and evidence-based data to guide the indications for plasma use in neonates is the establishment of a well-validated and age-specific reference range for coagulation tests in preterm and term neonates and its variability with severity of illness. The ranges must be established for the classic tests, including PT/aPTT/international normalized ratio, as well as for the various global hemostatic assays, including TEG and ROTEM. The next step is having a validated bleeding prediction and assessment battery for neonates to standardize the actual bleeding. Correlation of the prolonged values with actual risk for clinically significant bleeding is another key step, and no correlation has been established so far. Finally, there is a need for RCTs to explore the prophylactic use of plasma in neonates deemed at high risk for bleeding or with abnormal coagulation tests. Hemostatic dysfunction is also prevalent among neonates with moderate-to-severe hypoxic-ischemic encephalopathy and is associated with an increased risk of bleeding and high transfusion burdenPediatric transfusion medicine is evolving from infancy and gradually coming of age. It is entering an exciting era with recognition as an independent discipline, a new and evolving high-quality evidence base for transfusion practices, novel technologies and therapeutics, national and international collaborative research, educational and clinical efforts, and enthusiastic and dedicated engagement from not just transfusion experts but also neonatologists, pediatric intensivists, pediatric surgeons, pediatric anesthesiologists, transfusion practitioners, and nursing personnel.Although harder to study as new transfusion technologies and therapies emerge, it is critically important to specifically study them in neonates, as they are the highest transfused subpopulation in pediatrics.We continue to rely on expert opinion to make many transfusion-related recommendations in infants and neonates, but with an increased focus on research in neonatal transfusion practice, this is anticipated to change and improve with time.


Enteric defensins likely play a key role in the management of the human microbiome throughout development. The functional and mechanistic diversity of defensins is much greater than was initially thought. Defensin expression and overall Paneth cell physiology likely plays a key role in the development of colitis and other inflammatory or dysbiotic diseases of the gut. As our understanding of enteric defensins grows, their potential as tools of clinical intervention becomes more apparent. In this review, we focus on the function and activity of Paneth Cell defensins and highlight their role in disease. In general, the term “antimicrobial peptide” is used to describe a polypeptide chain of 5–100 residues that has broad-spectrum antimicrobial activity at one or more sites throughout the body  of rhesus macaques but not yet observed in humans  and several types of epithelial cells. For our purposes, it is important to note all known enteric defensins in humans are alpha defensins, specifically Human Defensin 5 (HD5) and Human Defensin 6 (HD6).In humans, there are 17 described defensins found at various sites of the body  of the small intestine . CRS4C-1, a member of these mouse cryptdins, is stored in Paneth cells and secreted from them as non-microbicidal pro-CRS4C-1, which is cleaved into its active form in the intestinal lumen by matrix metalloproteinase-7 , suggesting Paneth cell defensins play a role in the suppression of pathogenic communities within the gut  is a condition following stem cell or bone marrow transplants in which the donor cells attack tissues of the recipient's body , is associated with abnormalities in gut microbiota; further, Paneth cell dysfunction is known to be associated with irritable bowel disease and other inflammatory conditions of the gut , or if Paneth cell abnormalities lead to changes in intestinal flora and associated intestinal inflammation. Evidence for microbe-driven ileitis comes from observations that transfer of dysbiotic microbial communities to germ-free mice induces symptoms similar to Crohn's disease, with loss of Paneth cell secretion of defensins following these inflammatory conditions , results in spontaneous ileitis  within the Paneth cells of individuals , MMP7 a defensing activator, as well as tight junction genes in the ileum. As previously shown this combination allows for increased gut permeability and microbial translocation, dysbiosis, endotoxemia, and systemic inflammation which underlie metabolic syndrome (Su et al., Due to their key role in the innate immune system, defensins show promise in many clinical applications.Tumor Necrosis Factor is a pro-inflammatory cytokine found in increased concentrations in the gut mucosa of patients with active colitis (Olsen et al., Defensins also show promise as sources of new antibiotics. HD5 has been artificially modified to enhance its antibacterial properties. It was found that substitution of Glu21 with Arg, and Thr7 with Arg greatly increased the antibacterial activity of HD5 despite hindering dimerization among individual peptides (Wang et al., HD5 may be implicated in the repair of epithelial tissues following damage. Treatment of murine wound beds with HD5 stimulates the migration of LGR stem cells into the wound bed, promoting epithelial repair and wound healing (Lough et al., Recently developed advances in the field of antimicrobial peptides have fueled new interest into properties of the human enteric defensins, HD5, and HD6 Stange, . Novel dRH and CD researched and reviewed in order to create the manuscript. SS oversaw and finalized the manuscript.The authors of this review were funded by the NIH: NHLBI on award 1R21HL126562-01A1.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Editorial on the Research TopicMetabolism As a Therapeutic TargetMetabolic reprogramming, also known as “Warburg effect,” is considered a distinct hallmark of cancer, although its relevance appears to be even more general, as all the identified hallmarks rely on metabolic changes. Indeed, decreased activity of Phase I enzymes, paralleled by enhancement of Phase II ones, is required for conferring resistance to ROS or chemically induced toxicity; metabolic enzymes are required to initiate vessel sprouting during angiogenesis; modifications of the autophagic apparatus eliminate defective organelles to reduce oxidative stress and enable tumor cells to cope with their high metabolic demand. Support for metabolism involvement in cancer also comes from the identification of germline mutations in metabolic genes of patients affected by hereditary cancers.Although the precise mechanisms responsible for the complex rewiring of metabolic circuitries remain poorly understood, it has become clear that they can be exploited as an Achilles’ heel for therapeutic targeting. Preclinical and clinical evidence, indeed, suggests that metabolism-targeting drugs can efficiently interfere with tumor progression. New strategies combining metabolic drugs with chemotherapy or targeted therapies are now under evaluation, with the hope that they can help overcome resistance onset, at the moment the main cause of therapeutic failure.As this is a recent and rapidly evolving field, with several aspects often controversial, the present research topic focuses on current achievements and future perspectives.Masgras et al. reviewed our present knowledge of the mitochondrial molecular chaperone TRAP1, a key regulator of mitochondrial bioenergetics in tumor cells that impacts neoplastic growth by downregulating both respiratory complex IV and succinate dehydrogenase (SDH). SDH inhibition causes accumulation of succinate and activation of the transcription factor HIF-1α, which promotes glutamine and glucose utilization. In their review, the authors describe the complexity of TRAP1 effects on tumor cells and the possibility of its interaction with other client proteins in cells. In this scenario, the possibility of connecting TRAP1 chaperone activity with oncogenic transduction pathways might elucidate the tumor type and the stage at which the metabolic rewiring prompted by TRAP1 is pivotal for neoplastic progression.Kowalik et al. illustrated the therapeutic potential hidden in the Pentose Phosphate Pathway and underlined that such potential is strongly limited by the lack of specific pharmacological inhibitors. Nevertheless, the evidence that increased hepatocyte glucose-6-phosphate dehydrogenase (G6PD) expression is an early event in animal models of hepatocarcinogenesis, is a feature unique to the tumorigenic process and discriminates human hepatocellular carcinoma (HCC) from peritumoral tissue, makes the search for reliable G6PD inhibitors very attractive in the HCC field. Indeed, the decreased NADPH generation upon inhibition of the oxidative PPP could (i) selectively eradicate cancer cells by decreasing their resistance to high intracellular ROS levels; (ii) increase, in conjunction with already-approved therapies , the susceptibility of cancer cells to anticancer drugs. The design of specific inhibitors targeting PPP might thus represent a useful therapeutic tool, in particular, for HCC.Taguchi and Yamamoto illustrates the Gianus behavior of NRF2. Both NRF2 inducers and NRF2 inhibitors are expected to function as anticancer drugs: while NRF2 inducers protect normal cells from carcinogens, NRF2 inhibitors suppress the proliferation of cancer cells displaying aberrant NRF2 activation. However, many questions related to NRF2 inducers and inhibitors must be answered before they can be applied in anticancer therapy. An emerging possibility is to use NRF2 inducers for cancer therapy in combination with conventional anticancer agents. In this regard, there is a concern whether long-term application of NRF2 inducers may eventually support the switch of cryptic cancer-initiating cells into real cancer cells.Activation of PPP has recently been suggested to be one of the mechanisms by which deregulated NRF2-KEAP1 signaling promotes cell proliferation and tumorigenesis. The work by Corazzari et al., although the UPR program is primarily a pro-survival process, sustained stress may results in cell death. In their review, Corazzari et al. discuss the role played by the UPR program in tumor initiation, progression, and resistance to therapy, highlighting the molecular mechanisms that regulate the survival/death switch.Perturbation of endoplasmic reticulum (ER) homeostasis results in “ER stress,” determining the activation of a program defined as unfolded protein response (UPR), whose primary aim is to restore the physiological activity of these organelles. As underlined by Morandi et al. investigates the metabolic rewiring that takes place in tumor cells undergoing epithelial-to-mesenchymal transition (EMT). EMT execution is sustained by the metabolic requirements of a fast-growing tumor, aiming at overcoming nutrients and oxygen supply limitation and colonizing secondary sites to secure the adequate support of energy and nutrients. Independently of the stimuli-inducing EMT, the key event is the loss of E-cadherin, whose expression is regulated by transcription factors such as Snail1, Slug, Twist, and ZEB1. However, increasing evidence links metabolic deregulation to the EMT program. Morandi et al. gathers the recent findings on EMT and metabolic reprogramming in cancer and discusses how targeting certain metabolic pathways/hubs may impact on EMT and thus on cancer progression.The review by Doerstling et al. summarizes the interplay between obesity and cancer metabolism. High serum levels of insulin and insulin-like growth factor 1, adipose tissue dysfunction, and nutrient-replete circulation are some of the mechanisms by which obesity supports malignant cell growth. The relationship between obesity and cancer is strengthened by the observation that by attenuating obesity-associated signaling pathways and activating nutrient stress responses, calorie restriction regimens reduce obesity-associated cancer risk. The adipose tissue is also characterized by high abundance of macrophages and other immune cells that increase expression of pro-inflammatory cytokines by adipocytes. In consequence, circulating levels of cytokines are quite high in overweight individuals. Notably, a highly significant epidemiologic association has been identified between high levels of circulating cytokines and cancer risk.Finally, the article by Overall, many aspects of cancer metabolism are still poorly known. The recently acquired knowledge that metabolic alterations sustain almost all the known hallmarks of cancer points at metabolism as a very interesting therapeutic target. Even though few specific drugs are currently available, it is expected that their number will rapidly increase, opening new therapeutic perspectives, likely in association with drugs targeting tumor drivers.The two authors are the editors of the research topic on “Metabolism as a therapeutic target.”The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Luminescence spectroscopy, microscopy and lifetime image mapping offers new insights into the bioreduction of Geobacter sulfurreducens with uranyl. VIO22+ by bacteria such as Geobacter sulfurreducens. The inherent luminescent properties of UVIO22+ have been combined with confocal fluorescence microscopy techniques and lifetime image mapping to report directly on uranium concentration, localisation and oxidation state in cellular systems during uranium bioreduction, suggesting that localisation of uranyl species on the cell membrane surface plays an important role and that extracellular biogenic features form alongside uranyl sorbed cellular species during early stages of the bioreduction. The use of confocal microscopy in tandem with lifetime image mapping offers both improved temporal and spatial resolution (nanoseconds to microseconds and sub-micron respectively) than more conventional X-ray based techniques and offers the potential to image redox reactions occurring in situ. Together, these techniques provide an excellent and sensitive probe to assess the coordination environment of uranium during bioreduction processes that are currently being considered for remediation strategies of redox active radionuclides present in contaminated land.We report a study of redox reactions of uranium in model conditions using luminescence spectroscopy, which with its ease and wide availability has the potential to offer new insights into a bioremediation strategy of particular interest – the enzymatic reduction of U Luminescence spectroscopy in particular combines high sensitivity, broad applicability, and low cost, making it an attractive option for studying metal oxidation states over more technologically demanding and/or restricted techniques such as X-ray diffraction, X-ray absorption and electron paramagnetic resonance techniques  which often require extensive sample preparation. It has also been combined with optical microscopy to form fluorescence microscopy, confocal microscopy, and, more recently, two-photon excitation microscopy2 and super resolution microscopy,3 which can provide both spatial and temporal data on a variety of chemical species in a biological setting.5The oxidation state of any metal is of vital importance when considering their impact on biological and environmental systems. Oxidation state determines the coordination geometry, bond strength, and Lewis acidity (and therefore the tendency to undergo oligomerisation) and underpins speciation of the metal ion. Additionally, many metal ions are involved in oxidative stress, which arises from the formation of reactive oxygen or nitrogen species and has been implicated in a wide range of diseases.6 Although there are concerns associated with its radioactivity and long half-life, the hazards of uranium are primarily due to its chemical toxicity.7 The dominant form of uranium under oxic, environmental conditions is UVIO22+, a potent nephrotoxin. The uranyl cation is also very soluble and hence mobile in groundwater and biological systems, therefore remediation techniques focus largely on the reduction of UVIO22+ to the less soluble UIV cation.8 The inherent photophysical properties of the uranyl cation, arising from partially forbidden charge transfer transitions from oxo-based molecular orbitals to non-bonding, unoccupied f-orbitals,10 provide a convenient means of monitoring uranyl concentration, speciation, and movement without the need of additional imaging agents (such as dye probes). Despite recent time-resolved laser fluorescence spectroscopy (TRLFS) studies into the bioaccumulation of uranium,11 the use of optical techniques has been largely underutilised in favour of assay-based and scintillation techniques, to provide concentration data, and X-ray-based techniques such as EXAFS and EDX (coupled with electron microscopy), to provide structural data.Luminescence spectroscopy is an ideal technique for the study of uranium speciation. Since the development of nuclear power and weapons, containment breaches at all stages of the fuel cycle have led to elevated levels of uranium in the environment.VIO22+ to insoluble UIV-based mineral-type structures by endogenous bacterial populations.Here, we report the use of luminescence spectroscopy in combination with confocal fluorescence and phosphorescence microscopy and lifetime image mapping in the study of a process of great interest to the remediation of uranium in the environment – the bioreduction of UGeobacter sulfurreducens, is a Gram negative bacterium that is ubiquitous in subsurface soils. Geobacter sulfurreducens is well known to enzymatically reduce UVIO22+ under anaerobic conditions15 and was grown according to literature precedent.16 This reduction process has previously been studied by assay-based techniques to yield concentration data12 and EXAFS to give structural data,14 however many questions still remain on the exact enzymatic mechanisms responsible for the reduction process and despite its sensitivity and greater spatial resolution, luminescence spectroscopy remains underutilised in this field. Under the conditions required for bioreduction to occur 2, see ESI19 The luminescence lifetime could not be modelled adequately as a single exponential decay 34– (83%), UO2(CO3)22– (8%), and (UO2)3(CO3)66– (3%). As most uranyl carbonate complexes are known to be non-emissive at room temperature,21 it is likely that minor oligomeric and uranyl hydroxide species contribute disproportionately to the emissive properties of the system.The bacterium chosen for this study, al decay , and insGeobacter sulfurreducens a change in the emissive properties was observed , the primary product of microbial reduction of UVI with Geobacter sulfurreducens,26 confirmed by powder X-ray diffraction  and in more ‘geomimetic’ conditions in which nitrate, FeIII and MnIV may act as terminal electron acceptors.30 An alternative explanation may rest in changes in the uranyl speciation. If the most emissive UVIO22+ species was preferentially reduced by the bacteria32 a sharp decrease in emission intensity resulting from bioreduction may be followed by a gradual increase as the system re-equilibrates. This explanation, however, does not take into account the simplified uranium speciation profile at 77 K at which these results were obtained. A more likely explanation, it seems, is that the delayed rise in fluorescence intensity is due to the disproportionation of an unstable UVO2+ intermediate which is non fluorescent in the 450–600 nm spectral window employed. As enzymatic UVIO22+ reduction, a first order reaction with regards to [UVIO22+] occurs meaning the concentration of UVIO22+ and rate of reaction steadily decrease. At the same time, the concentration of UVO2+ increases and the rate of disproportionation, a second-order reaction, will increase quadratically. This may account for the fluctuations in the concentration, and hence emission intensity, of UVIO22+ as it is gradually reduced to UIVvia an unstable UVO2+ intermediate. Moreover, liquid scintillation counting experiments over the same time period show a linear decrease in total soluble uranium content, giving further weight to this argument  distribution of uranyl both within bacterial communities and with isolated single bacterium. The bioreduction reaction was carried out as described previously, with the only exception being that aliquots were placed directly onto a glass slide for observation by microscopy, which was carried out at room temperature. The microscope was equipped with a 405 nm diode laser; while this is not the peak excitation wavelength it does excite into the same uranyl absorption band as used in the spectroscopic experiments. Additionally a CCD-based spectrometer enabled the collection of steady-state spectra on objects observed under the microscope, full instrumental details can be found in the ESI.ca. 7–15 microns in size) that displayed intense fluorescence in the green-channel were clearly visible alongside the bacterial cells.Having shown that the inherent emission of the UVIO22+ species that have been sorbed to or complexed by biological material or precipitated out as amorphous or polycrystalline acetate, carbonate or hydroxide salts. This also supports the results of the spectroscopic experiments and suggests that the change in emission lifetime observed in the bulk solutions may be due to coordination of UVIO22+ to complexants released by the bacterial cultures. In samples prepared several hours after the introduction of uranium these features were no longer visible. Instead, the bright field images showed the Geobacter sulfurreducens cells surrounded by nanoparticulate matter that displayed no fluorescence or phosphorescence. It appears that as the bioreduction proceeds, and the solution-phase concentration of UVIO22+ decreases, any precipitated UVIO22+ was converted, possibly via re-dissolution, to the UIV product of the reaction, which formed the insoluble and non-emissive mineral uraninite (UO2).33Fluorescence lifetime image mapping (FLIM) and phosphorescence lifetime image mapping (PLIM) revealed that these features displayed long-lived emission, up to 130 μs, , left suGeobacter sulfurreducens, attention turned to the cells themselves. The rod-shaped, micron-sized cells were clearly visible in the bright-field image , resulted in a slight decrease in emission intensity upon passing through the Geobacter sulfurreducens cells. Although the spatial resolution of the FLIM and PLIM images do not enable the precise location of the uranium within the bacterial cells to be determined, these data may indicate that the uranium is principally associated with the cell walls and/or the outer cell membranes rather than incorporated into the cell. However, since the reduction in lifetime was not observed by spectroscopy in the bulk samples on short timescales (ns), it is likely that the surface bound species make up a very minor component of the total uranyl speciation. Further, gene deletion experiments alongside proteomic studies have suggested that both outer membrane and periplasmic c-type cytochromes play a central role in extracellular electron transport in the bioreduction of UVIO22+ with Geobacter sulfurreducens.38Furthermore, altering the excitation focus in the VIO22+ by Geobacter sulfurreducens over a 24 hour time period. The speed at which a spectrum can be obtained, and the minimal sample preparation involved has allowed the progress of the reaction to be studied in situ, importantly, at shorter time-increments than previously possible, providing supporting evidence towards the role of a UVO2+ intermediate. We have also demonstrated for the first time the viability of using the inherent fluorescent properties of the UVIO22+ to report directly on the localisation of uranium by fluorescence and phosphorescence lifetime image mapping without additional stains or imaging agents or the need for sample-altering preparation techniques such as fixation. Interestingly, long-lived phosphorescent extracellular inorganic-organic features are observed in the early stages (first hour) of the bioreduction process by both PLIM and FLIM techniques. Fluorescence microscopy of Geobacter sulfurreducens cells shows very little autofluorescence in the absence of uranium, however on the addition of uranyl acetate there is an immediate increase in emission associated with the surface of the cells, this suggests a previously unconsidered step in the enzymatic reduction process – the absorption of UVIO22+. The extremely short luminescent lifetime suggest that the uranyl is interacting with electron-rich species, potentially electron transfer proteins localised towards the periphery of the outer membrane of the Gram-negative bacterial cell, such as cytochromes (including c-type) which have been implicated in the reduction of uranium37 and those that are known to bind UVIO22+.40 The identification of extracellular material has suggested the applicability of this technique to the study of other bioremediation techniques such as biosorption, bioaccumulation and biomineralisation. This approach could provide new insights into the fate of uranium in more complex microbial–mineral–ground water systems, which are currently poorly understood. Further work will also aim at incorporating two-photon microscopy as a potential means of providing additional spectroscopy and spatial data.In summary, we have demonstrated the utility of readily available, facile luminescence spectroscopy in studying the bioreduction of U


In vitro studies primarily emphasize on simulating cellular uptake and toxicity of nanotubes on benign or malignant cell lines. On the other hand, in vivo studies focus on illustrating endpoints of serosal pathology in rodent animal models. From a molecular aspect, some nanoparticle categories are shown to be cytotoxic and genotoxic after acute treatment, whereas chronic incubation may lead to malignant-like transformation. At an organism level, a number of fibrous shaped nanotubes are related with features of chronic inflammation and MWCNT-7 is the only type to consistently inflict mesothelioma.Nanoparticles have great potential for numerous applications due to their unique physicochemical properties. However, concerns have been raised that they may induce deleterious effects on biological systems. There is accumulating evidence that, like asbestos, inhaled nanomaterials of >5 μm and high aspect ratio (3:1), particularly rod-like carbon nanotubes, may inflict pleural disease including mesothelioma. Additionally, a recent set of case reports suggests that inhalation of polyacrylate/nanosilica could in part be associated with inflammation and fibrosis of the pleura of factory workers. However, the adverse outcomes of nanoparticle exposure to mesothelial tissues are still largely unexplored. In that context, the present review aims to provide an overview of the relevant pathophysiological implications involving toxicological studies describing effects of engineered nanoparticles on mesothelial cells and membranes Nanoparticles are forms of matter with at least one dimension sized less than 100 nm. Compared to larger counterparts, they have advantageous physicochemical properties that render them suitable for a wide array of applications . For instance, Haniu et al. showed that three MWCNT types of distinct dimensions had differential effects on indices of oxidative stress, autophagy and plasma membrane permeability in MESO-1 cells   that occur in a fiber like shape resembling asbestos, have attracted remarkable attention. Depending on the amount of graphene sheets used to form a cylinder, they can be categorized in single and multi-walled (SWCNT and MWCNT respectively)  inflicts asbestos like pathogenicity characterized by granuloma, fibrous thickening and mesothelioma in the peritoneum of p53 heterozygous mice , that results in further recruitment of immune cells, mesothelial injury and chronic inflammation with foreign-body granuloma formation  and MWCNTs  for up to 4 months induced proliferation, migration and invasion similar to that of asbestos fibers. By the use of qtPCR, it was observed that MMP-2 mRNA expression increased 51-fold with SWCNTs and 23-fold with MWCNTs. In conformity with the qtPCR results, MMP-2 activity was also found to be upregulated at the protein level by three different methods, Western blotting, gelatin zymography and immunofluorescence. The key functional role of MMP-2 in CNT induced aggressive phenotypes was revealed with knockdown of MMP-2 by short-hairpin RNA plasmid, which reduced invasion (60–90%) and migration of MeT5A cells . It was then found that Ras is significantly upregulated in SWCNT exposed cells and that siRNA downregulation of Ras markedly mitigated their malignant like transformation. Amongst the H-Ras downstream targets, ERK1/2 was illustrated to be an important effector given that an ERK inhibitor suppressed invasion activity of treated cells in a dose dependent manner. Additional investigation revealed that cortactin and integrin alpha V were both overexpressed in cells incubated with CNTs and that cortactin was a downstream target of the H-Ras/ERK cascade  and asbestos for 4 months resulted in a change from a flattened to a spindled shape as well as a substantially augmented migratory activity compared to untreated controls , short  and tangled  MWCNT classes. Subsequently mice were euthanized at 1 and 6 weeks and their pleural cavity was lavaged to investigate the inflammatory response. At the 1 w timepoint only long CNT were associated with a small but significant increase in granulocyte content. By 6 w there was a marked elevation in total cell counts, LDH and protein levels owing to long CNT exposure. Pathological examination of the parietal pleura revealed lesions characterized by foreign body giant cells, granulocytes and collagen deposition. Moreover, CNTs were identified in sections of the diaphragm of long CNT treated mice, which was suggestive of the ability of long fiber-like nanotubes to translocate to the pleural cavity , MWCNT-7 (Mitsui-7) or crocidolite, five times over a 9-day period. At the end of the experimental period, rats were euthanized and pleural lavage and specimens of lung and chest tissue were collected. In line with Murphy et al., the cavity fluid contained increased amount of total cells, LDH and protein. Also, the authors reported evidence of pleural deposition as lavage cell pellets contained CNT and crocidolite control mostly located in macrophages. In further support of that notion, tissue samples had a small number of fibers that were found directly penetrating the visceral pleura into the pleural cavity. In addition, the visceral mesothelium of nanotube and crocidolite treated rats developed hyperplastic proliferative lesions [10 times higher proliferating nuclear antigen index (PCNA) compared to control] closely associated with inflammatory infiltrate and focal fibrotic response. On other hand, the parietal pleura showed no pathological alterations. To examine the role of inflammation in the induction of hyperplastic proliferative lesions, the authors exposed MESO-1 cells to conditioned media from CNT/crocidolite treated macrophages and to concentrated supernatants of pleural lavage. Incubation resulted in increased proliferation of mesothelial cells thereby suggesting that macrophage secreted mediators were important drivers of the response , followed by initiation of phagocytosis and upregulation of downstream cascades related to pro-inflammatory responses . At 2 mg/m3 the incidence of mesothelial hyperplasia and focal fibrosis of the parietal pleura was increased in male rats. Additionally, the incidence of focal fibrosis of the ventral pleura and the incidence of mediastinal inflammation were elevated in male/female and male animal groups respectively  short MWCNT were found penetrating the alveolar wall in tissue sections; and (ii) mediastinal lymph nodes were loaded with MWCNTs that accumulated time dependently. Hence, the authors postulated that short MWCNT either directly reached the visceral pleura or alternatively crossed through the stomatal pores and into the pleural cavity where they initiated inflammation before being cleared through the lymphatics. Intriguingly, short SWCNT were also found in mediastinal lymph nodes at 90 d, which suggests they may have followed a similar route. Discrepancies in the degree of pleural response can be explained based on the ability of short MWCNT to more efficiently migrate to the pleural cavity as compared to short SWCNT. In further support of this assertion, short SWCNTs were shown to substantially accumulate in the lungs instead, where they elicited a more pronounced effect that short MWCNT , which is indicative of their highly aggressive nature, and displayed homozygous CDKN2A/2B tumor suppressor gene deletions, a typical molecular abberration of asbestos induced mosothelioma. Based on these outcomes, it was suggested that reduced diameter and high rigidity are presumably attributes of fibrous nanotubes with increased carcinogenic potency , B , C  and D  of decreasing linearity. Rats that were administered MWCNT A, B, C, and D developed mesothelioma at a rate of 98% (high-dose group)/90% (low-dose group), 90/92%, 94/84% and 70/40% respectively. As for mean survival time, A, B, C, and D treated animals lived on average for 194 days (high dose group)/231 days (low dose group), 207/294, 265/415, and 585/666 respectively. For reference, the amosite asbestos group had a percentage of malignancy bearing animals of 66% with a mean survival time of 623 days. By ranking the CNTs based on the two indices of carcinogenic potential, the authors observed, in conformity with Nagai et al., that increasing linearity correlated with higher incidence and earlier progression of mesothelioma, which was predominantly of the sarcomatoid type. In addition, it should be noted that amosite and MWCNT induced mesotheliomas shared the same appearances and histological markers , which is closely associated with biopersistence, in the health hazard of nanotubes. Initially, the authors incumbated a panel of fibers for 10 weeks in Gambles solution, which contains electrolytes present in biological systems and has a pH close to that inside macrophages. Most tested CNT and asbestos were durable, however, a 12.4 μm class showed a gradual loss of mass (30%) that ceased after 3 weeks of incubation. Electron microscopy subsequently confirmed that the proportion of long fibers was significantly reduced compared to that of the briefly exposed CNTs. Intraperitoneal injection of the intact fraction at 50 mg induced an inflammatory response in 24 h that did not subside by 7 days and also a fibrotic response at that timepoint. Degraded nanotubes (avg. length 11.1 μm) on the other hand were associated with lesser responses. In light of the above, the authors maintained that fibrous CNTs may inflict asbestos like pathology in mice, that can be however be avoided under the condition that the material has insufficient durability  that can alter their toxicity  Exposure is performed via bolus deliveries of test nanomaterials whereas actual exposure occurs gradually over a long period of time and probably is smaller doses; (ii) deposition to the lung parenchyma via these methods is probably heterogeneous as compared to nose-only inhalation administration which could better mimic the physiological route of breathing in rodents; and iii) the dispersion of liquid-suspended nanoparticles is often inferior to that of particles in the form of dust. Taken together, instilled CNTs, primarily at higher doses, are likely to overwhelm lung defenses and clearance mechanisms thereby producing aggravated responses that would not be seen with inhalation studies  and biopersistence. However, it does not include a complete mechanistic analysis of the complex pathway of nanotube induced adverse effects. Special emphasis should now be placed on elucidating processes such as the route and mechanism of translocation at the pleura as well as the molecular background of CNT toxicity at this site. The dependence of pleural translocation on time, dose and physicochemical nanoparticle properties requires further elucidation. One of the most challenging issues in this effort is to assess the toxicologically active fraction in the pleura, which is ideally defined by the fibers that remain in the pleural cavity evading clearance mechanisms  in order to address the questions that still need to be answered, ethical concerns could arise combined with cost and time burden. Rodents do not accurately mimic human physiology and pathophysiology in terms of exposure, injury and clearance of nanoparticles. In that aspect a gradual swift to well characterized relevant human It is currently urgent that further studies are performed to elucidate the risk of nanoparticles on mesothelial cells and membranes and specifically the pleura. At the moment, only MWCNT-7 is consistently shown to induce mesothelioma whereas other classes of fibrous shaped CNT as well as silver and nickel nanowires are associated with pathological features of inflammation in rodent animal studies. In contrast, tangled/aggregated CNTs are more likely to behave like granular particles with negligible carcinogenicity.Toxicological studies reporting:Adequate characterization  of engineered nanoparticles.In vitro endpoints using mesothelial cell lines .In vivo endpoints of rodent mesothelial membranes .“nanoparticles AND (pleura OR peritoneum OR pericardium)”“nanoparticles AND ”“nanotubes AND (pleura OR peritoneum OR pericardium)”“nanotubes AND ”“nanotubes AND mesothelial”Two authors (SS and SZ) performed the literature search in PubMed between August 5st and August 10th 2017. Papers not in English were excluded via the use of the corresponding filter. Eventually, the final yield was 508 unique papers amongst which 55 were eligible. Author names, publication year, nanoparticle class and dimensions, experimental model, exposure conditions, assays and results were recorded. With regards to software, Mendeley and Microsoft Word were utilized for literature management and manuscript preparation respectively.SS: Performed the literature research and wrote the manuscript; CH: Carefully read the manuscript for important intellectual content; KG: Carefully read the manuscript for important intellectual content; SZ: Conceived the review, performed the literature research and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Apoptosis, a form of programmed cell death, is a highly regulated process, the deregulation of which has been associated with the tumor initiation, progression, and metastasis in various cancers including breast cancer. Induction of apoptosis is a popular target of various therapies currently being tested or used for breast cancer treatment. Thus, identifying apoptotic mediators and regulators is imperative for molecular biologists and clinicians for benefit of patients. The regulation of apoptosis is complex and involves a tight equilibrium between the pro- and anti-apoptotic factors. Recent studies have highlighted the role of miRNAs in the control of apoptosis and their interplay with p53, the master guardian of apoptosis. Here, we summarize and integrate the data on the role of miRNAs in apoptosis in breast cancer and the clinical advantage it may offer for the prognosis or treatment of breast cancer patients. BRCA1, BRCA2, and other breast cancer susceptibility genes, alcohol intake, endogenous estrogen levels, use of hormone replacement therapy, radiation exposure to the breast, obesity, and race/ethnicity  have emerged as critical regulators of apoptosis. miRNAs are small non-coding RNAs (~18–25 bases) regulating gene expression at the post-transcriptional and/or translational levels. Traditionally, they are known to bind to the 3′ untranslated region of the target transcript to bring about transcript degradation or inhibition of translation depending on the extent of complementarity Bartel, . HoweverThe generation of miRNA profiles of normal or patient samples using microarray, qPCR, or deep sequencing technology and its analyses has helped in identifying useful clinical correlations of miRNAs with potential applications in cancer diagnosis, prognosis or therapy Hammond, . Severalin vitro experiments with BCL2 overexpression or silencing in breast cancer cells confirm its pro-survival effects accounting for current pre-clinical and clinical trials involving BCL2 silencing for breast cancer treatment , were identified as direct targets of miR-26a. MCF7 xenografts with exogenous miR-26a show that a decrease in expression of both MTDH and EZH2 is accompanied by an increase in apoptosis  or the protein  levels in breast cancer based on analyses using genes to system breast cancer (G2SBC) database  were found to be associated with regulation of apoptosis based on functional annotation clustering using DAVID microarray software  to influence apoptosis in breast cancer. miR-504 was shown to directly target p53 and thus, inhibit p53 dependent apoptosis in breast cancer  indicating that its levels are kept under tight control in breast tissue. Overexpression of miR-24, 34a, and 16 reduces BCL2 expression that leads to apoptosis through disruption of the mitochondrial membrane potential and the release of cytochrome C  and mitochondrial (intrinsic) pathways. It was found to be a direct target of miR-221/222 in breast cancer  and Her2+ve patients receive trastuzumab (herceptin), a monoclonal antibody against Her2, included in the chemotherapy regimen Osborne, . While tSeveral mechanisms of chemoresistance in breast cancer have been reported in breast cancer such as upregulation of the ATP-binding cassette (ABC) transporters, drug inactivation or detoxification, alterations in genes related to cell cycle, apoptosis or DNA repair enzymes, epigenetic modifications, and activation of signaling pathways related to the progression of cancer , predictive [which are involved in response (sensitivity/resistance) to conventional breast cancer therapeutic strategies] or prognostic  potential , successfully completed phase I clinical study and is undergoing phase II clinical trials for treatment of chronic hepatitis C virus infection  and undesired (autophagy and necrosis) forms of cell death for breast cancer treatment. Nevertheless, miRNA delivery issues are slowly getting resolved, miRNAs are also being considered as means to target multiple aberrant pathways and certainly preclinical and clinical trials of some of these miRNAs look promising. The pool of apoptomiRs in breast cancer is likely to get bigger with advanced technologies such as large scale miRNA functional screens using 3D tumor models, thus giving us more probable candidates as novel targets for breast cancer therapy.RK conceptualized the theme of the review and has written the review. SS compiled all the data on pro- and anti-apoptotic miRNAs and their correlation to drug resistance and has contributed in writing the review. PP and SA compiled the data of miRNA and p53 regulatory loop and contributed toward writing this section.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer KT and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.