It is well established that Indigenous populations are at a heightened risk of food insecurity. Yet, although populations (both Indigenous and non-Indigenous) are ageing, little is understood about the levels of food insecurity experienced by older Indigenous peoples. Using Australian data, this study examined the prevalence and correlates of food insecurity among older Aboriginal and Torres Strait Islanders. Using nationally representative data, we employed ordinal logistic regression models to investigate the association between socio-demographic characteristics and food insecurity. We found that 21% of the older Aboriginal and Torres Strait Islander population were food insecure, with 40% of this group exposed to food insecurity with food depletion and inadequate intake. This places this population at a 5 to 7-fold risk of experiencing food insecurity relative to their older non-Indigenous peers. Measures of geography, language and low socio-economic status were highly associated with exposure to food insecurity. Addressing food insecurity offers one pathway to reduce the disparity in health outcomes between Aboriginal and Torres Strait Islanders and non-Indigenous Australians. Policies that consider both remote and non-remote Australia, as well as those that involve Aboriginal people in their design and implementation are needed to reduce food insecurity. Like many Indigenous populations, the Aboriginal and Torres Strait Islander population in Australia has considerably poorer health outcomes when compared to their non-Indigenous peers, experiencing a range of health conditions at an earlier age of onset and considerably lower life expectancy ,2,3,4. TImportantly, many of the health conditions prevalent among older Aboriginal and Torres Strait Islanders are preventable, notwithstanding the levels of socio-economic disadvantage faced by this population. The Australian Institute of Health and Welfare 2016) estimates that the burden of disease in the Aboriginal population is 2.3 times of that experienced by non-Indigenous Australians [016 estimAn important proximate determinant and risk factor of poor nutrition outcomes is food insecurity. Food insecurity refers to the “limited or uncertain availability of nutritionally adequate and safe foods or limited or uncertain ability to acquire acceptable foods in socially acceptable ways” . A growiDespite evidence of heightened risk of food insecurity within Indigenous populations, little is known about the prevalence and correlates of food insecurity in the older Aboriginal and Torres Strait Islander population. However, studies have examined food insecurity among the older non-Indigenous population, concluding the strength of socio-economic factors in explaining food insecurity in later life ,15,16. TData for this study are from the 2012–2013 Australian Aboriginal and Torres Strait Islander Nutrition and Physical Activity Survey (NATSINPAS) conducted by the Australian Bureau of Statistics (ABS) from August 2012 to July 2013 . The surAn important advantage of the NATSINPAS is the geographic coverage of the survey, including remote and non-remote areas as well as discrete Aboriginal and Torres Strait Islander communities. Remote areas include very remote Australia and remote Australia under the Australian Statistical Geography Standard . Non-remNATSINPAS was part of the larger Australian Aboriginal and Torres Strait Islander Health Survey (AATSIHS). The sampling design of NATSINPAS sought to provide reliable estimates at the national level, as well as for remote and non-remote Australia. The in-scope population was divided into two broad populations. The first group included those living in discrete Aboriginal and Torres Strait Islander communities in remote Australia, defined as the ‘community frame’. The second group included the remainder of the in-scope population, referred to as the ‘non-community frame’. The Indigenous Community Frame (ICF) was used to target respondents for the community frame, and was built from a combination of Census data as well as data from the Discrete Indigenous Communities Database. The non-community frame was formed using Census data at a low geographic level (SA1). For some States and Territories, the non-community sample was disaggregated into non-remote and remote non-community areas. Survey weights at the person level were calculated by the ABS to calibrate the probability of selection into the survey relative to estimated resident population counts (less those in non-private dwellings).Data from NATSINPAS were collected under the Census and Statistics Act 1905. As NATSINPAS included a biomedical component, ethics approvals were sought at both the national level  and at the state and Territory level . Data for this study were made available by the ABS through the Universities of Australia agreement. NATSINPAS survey data are available to registered users of ABS microdata. n = 1062). Defining the sample aged 45 years and over as ‘older’ is common in studies of ageing among Aboriginal and Torres Strait Islander people for several reasons [In this study, we consider the prevalence and risk factors for food insecurity among older Aboriginal and Torres Strait Islanders aged 45 years and over  was asked, “In the last 12 months was there any time when you (or members of this household) ran out of food and couldn’t afford to buy more?” A follow up question was asked, “When this happened, did you (or members of this household) go without food?” Following other Australian studies, we use these two questions to create a variable which views food insecurity on a continuum . Those aTo model the association between socio-economic characteristics and food insecurity, we utilized Stata 14.0. As the dependent variable is ordinal, standard logistic regression is inappropriate. We utilized ordered logistic regression fitted by maximum likelihood . InitialAge: Measured in aggregate categories Marital Status: Married or not married. Unfortunately, more detailed items such as widowed, never married or separated were unavailable on the data set.Gender: Male or Female.Household Size: Categorized as 1, 2, 3 or 4, 5 or more.Household Composition: Whether the household includes Aboriginal and Torres Strait Islander members only, or with non-Indigenous members also present.Indigenous Language Speaking: Whether the persons speaks an Australian Indigenous language.Remoteness: Whether the household resides in remote or non-remote parts of Australia, as defined by the Australian Statistical Geography Standard (ASGS).Household Income: The measure of household income provided by the ABS was equivalized household income  and collapsed into deciles. This adjusted form of household income allows for welfare and financial wellbeing comparisons between households of different sizes and compositions.Self-Reported Health: A dichotomous variable indicating whether the person self-reported their health as being excellent or good versus fair or poor.Informed by the literature on food insecurity in Australia and using model selection techniques outlined above, variations in food insecurity were identified by a range of socio-economic characteristics. The final variables included in the model were:There is considerable socio-economic and demographic variation in exposure to food insecurity among older people . ImportaSimilarly, strong differences in food insecurity by Aboriginal household composition are observed. Those living in a household with both Aboriginal and non-Indigenous members have a food insecurity prevalence of about 7%, compared with 28% of those in Aboriginal only households. Overall, we identify several population sub-groups with a food insecurity prevalence rate of around 30%: Indigenous speakers in remote communities (37%), Aboriginal and Torres Strait Islander only households (28%), smokers (31%), persons in households with a large number of occupants (32%), non-married females (35%), and income earners in the lowest centile (31%).Although these descriptive results indicate significant differences in exposure to food insecurity by socio-economic characteristics, it is important to control for confounding effects to measure the association between each covariate and the probability of food insecurity. p < 0.10). In a similarly sized effect, households with both Aboriginal and non-Indigenous residents were about 60% less likely to experience food insecurity compared with Aboriginal only households, even with controls for geography .With a range of demographic and economic controls, English speaking and remoteness remain strong predictors of food insecurity . Those sNon-smokers remained 39% less likely to experience food insecurity  and persons in large households were at a high risk, particularly those in household with 5 or more occupants . Those in the top 20% of the income distribution were about 88% less likely  to suffer food insecurity and those in the 60–80 percentile were about 76% less likely  when compared to lowest income earners. Gender and social marital status also remains significant in the regression models. Compared to unmarried females, married females were about 48% less likely to experience food insecurity . Married males were 38% less likely to experience food insecurity relative to non-married females, but the estimate is only significant at the 90% level. Although several Australian studies have examined food insecurity in the older population, little is known about the prevalence and risk factors of food insecurity among older Aboriginal and Torres Strait Islanders specifically. In this paper, we have used nationally representative survey data to measure the prevalence and correlates of food insecurity among Aboriginal and Torres Strait Islanders aged 45 and over.Firstly, we find the prevalence of food insecurity among older Aboriginal and Torres Strait Islanders is high with about 21% reporting exposure, and 41% of this group reporting food depletion and inadequate intake. These estimates are in line with published results from the ABS showing that about 22% of Aboriginal and Torres Strait Islanders experience food insecurity compared to 4% of non-Indigenous Australians . The prevalence of food insecurity among older non-Indigenous Australians using a similar measure has previously been reported at 3% ,28. ThatThis finding is important for several reasons. There is now a significant body of evidence on the implications of food insecurity for individual health and wellbeing. International studies show exposure to food insecurity is associated with symptoms of depression and anxiety, multimorbidity, lower levels of self-reported health status, lower nutrient diets, a greater likelihood of reporting social isolation, long standing health problems and activity limitations, and a greater likelihood of reporting heart disease, diabetes, and high blood pressure ,35,36,37The high prevalence of food insecurity among older Aboriginal and Torres Strait Islanders is also important in the context of the gap in health outcomes that persists between Aboriginal and non-Indigenous Australians. ‘Closing the Gap’ is an Australian Government strategy whereby a key goal is to improve the health outcomes of Aboriginal and Torres Strait Islander populations. Specifically, the strategy seeks to reduce mortality by 2030 to levels experienced by the non-Indigenous population . AddressThe ‘Closing the Gap’ strategy recognizes that the likelihood of co-morbidities is higher in the Aboriginal population when compared to the non-Indigenous population . For exaThe prevalence of food insecurity is not evenly spread throughout the population and we find socio-economic factors are strongly associated with food insecurity and that specific demographic groups report higher rates of food insecurity. In the model of food insecurity formulated here, socio-economic factors are viewed as moderating food insecurity through the degree of resource-constrained access to the food supply.Specifically, we identify a number of population sub-groups with a food insecurity prevalence rate of around 1 in 3: Indigenous speakers in remote communities (37%), Aboriginal and Torres Strait Islander only households (28%), smokers (31%), persons in households with a large number of occupants (32%), non-married females (35%) and income earners in the lowest centile (31%). Differences in food insecurity status by socio-economic factors is consistent with studies in community dwelling non-Indigenous older Australians showing food insecurity differs by a range of socio-demographic risk factors, such as income, marital status, and smoker status ,15,16. TThe strength of socio-economic factors as risk factors in experiencing food insecurity is concerning given the continued economic deprivation experienced by many Aboriginal and Torres Strait Islanders. For example, considerable disparities in socio-economic outcomes persist between non-Indigenous and Aboriginal persons across all age groups through lower levels of education, employment and living in areas with greater socio-economic disadvantage, while also being at considerable risk of experiencing interpersonal racism ,42. Not Results presented here also underscore differences in food insecurity risks between remote and non-remote areas of Australia. Interestingly, we find that non-English speaking persons in remote areas (37.3%) are at double the risk of exposure to food insecurity than English speakers in either remote (19%) or non-remote (18.7%) Australia. Part of this disparity may reflect the lack of detailed geographical measures on NATSINPAS due to confidentialization. That is, non-English speakers are more likely to be resident in particularly isolated areas of remote Australia—with limited access to affordable food sources and health facilities. Notwithstanding, a considerable literature has emerged on the disparity in food prices between urban, regional, and remote regions in Australia. In one study, a 47% difference in prices was reported between remote and urban supermarkets for healthier foods . SolutioHowever, it was interesting to note that the likelihood of being food insecure did not differ between non- remote and remote English-speaking Aboriginal and Torres Strait Islanders with about 1 in 5 at risk of exposure to food insecurity. This is important as 75% of the Aboriginal and Torres Strait Islander population live in urban and regional areas . FurtherDespite the focus of recent programs, a greater awareness of the linguistic and cultural needs in remote areas is required for effective solutions to food insecurity. In a systematic review of Aboriginal food and nutrition programs, Browne and colleagues (2018) conclude that “the most important factor determining success of Aboriginal and Torres Strait Islander food and nutrition programs is community involvement in  program development and implementation” . As furtIn interpreting our results, it is important to consider the study’s limitations. Our findings potentially underestimate the degree of food insecurity in this population for several reasons. Firstly, the NATSINPAS population is limited to Aboriginal and Torres Strait Islanders residing in private dwellings and excludes a range of other vulnerable populations such as the homeless, those in poor health living in care facilities or hospitals. Indeed, there is a considerable need for further research on food insecurity risk in non-community based settings, among both Indigenous and non-Indigenous populations . SecondlNoting these limitations, this paper was the first to examine food insecurity among older Aboriginal and Torres Strait Islanders. We found that food insecurity is experienced by a sizeable minority of older persons (1 in 5), that 41% of this group go without food consequently and that specific demographic groups are at a considerably heightened risk. In total, older Aboriginal and Torres Strait Islanders are at about a 5–7 fold risk of experiencing food insecurity relative to their non-Indigenous peers. These findings offer information with which to identify food insecure persons and to inform nutrition programs in place to improve health and wellbeing. Existing studies note the importance of involving Aboriginal and Torres Strait Islander people in the management, design, and implementation of such programs and considerably more needs to be done in this area. However, nutrition programs alone are likely to be ineffective. The strength of socio-economic factors in explaining the prevalence of food insecurity, suggest that policies must improve economic and social wellbeing (through the ‘Closing the Gap’ strategy) in tandem with targeted nutrition programs and policies aimed at providing an affordable, healthy food supply. Recent evaluation studies, unfortunately, show slow progress with the ‘Closing the Gap’ strategy, with improvements in some but not all health and economic outcomes for Aboriginal and Torres Strait Islanders over the past decade ,59. Our findings further suggest solutions to addressing food insecurity in older Aboriginal and Torres Strait Islander populations need to focus on all geographical locations—not just on remote Australia, which has been the focus to date. In both remote and non-remote Australia, greater awareness of linguistic and cultural needs as well as the involvement and management of programs for and by Aboriginal and Torres Strait Islanders is required to improve levels of food security. Addressing food insecurity through these means offers one pathway for reducing nutrition related disease and co-morbidities, thereby assisting the ‘Closing the Gap’ strategy to achieve its aim of improving health outcomes for Aboriginal and Torres Strait Islanders.


Kidney development involves formation of nephrons intricately aligned with the vasculature and connected to a branched network of collecting ducts. Notch signaling plays multiple roles during kidney development involving the formation of nephrons composed of diverse epithelial cell types arranged into tubular segments, all the while maintaining a nephron progenitor niche. Here, we review the roles of Notch signaling identified from rodent kidney development and injury studies, while discussing human kidney diseases associated with aberrant Notch signaling. We also review Notch signaling requirement in maintenance of mature kidney epithelial cell states and speculate that Notch activity regulation mediates certain renal physiologic adaptations. The Notch signaling pathway is an evolutionarily conserved signaling pathway that has critical functions in embryonic development, tissue homeostasis, and disease. Unique features of the pathway include ligand and receptor expression in neighboring cells and a series of proteolytic cleavages of the receptor that lead to the expression of Notch target genes. After exit from the endoplasmic reticulum, the Notch receptors undergo processing in the Golgi where they are cleaved by furin-like convertases, resulting in a bipartite receptor that translocates to the plasma membrane . Notch rIn mammals, there are four Notch receptors (Notch1–Notch4) and five ligands [Delta-like  and Jagged (Jag1 and Jag2)]. These basic components, along with their downstream mediators orchestrate gene regulation in almost all tissues of multicellular organisms. Research conducted for over a century has contributed to our understanding of the mechanism of Notch regulated gene expression in a context dependent manner  signals from the metanephric mesenchyme to trigger ureteric budding from an epithelial tubular structure referred to as the Wolffian duct. The tip cells of the ureteric bud (UB) express high levels of Ret and GFRα1 receptors that are activated by GDNF , and facNephron formation occurs through a series of morphological changes that transform groups of cap mesenchymal cells that periodically exit the progenitor pool, into a tubular structure referred to as a nephron. Each nephron arises from an aggregate of cap mesenchymal cells, referred to as a pre-tubular aggregate (PTA) that forms below the UB tip. Mesenchymal aggregates epithelialize to form renal vesicles (RV) ,28, whicThe Notch pathway receptors and ligands have unique expression patterns within the developing kidney  35,38].,38.35,38RBPJ, or Notch1 and Notch2) sustain Six2 expression within the developing mouse kidney -S-phenylglycine t-butyl ester (DAPT) to inhibit Notch signaling  compartment of the RV closer to the UB occurs in Pax3-Cre;Notch2f/f kidneys just like in wild-type kidneys. However, the segment specific expression of Lhx1 is not maintained in the absence of Notch2 and the establishment of distinct nephron segments is defective from the RV stage onwards. Interestingly, Six2-Cre which drives Cre expression in the NPCs also reveals the requirement of Notch signaling in the establishment of the proximodistal axis within the RV and hence nephron segmentation, but only when both Notch1 and Notch2 are inactivated . Th. ThNotchelopment . A role d bodies  [42]. Th alleles .Notch1 and Notch2 along with a Rosa+/EYFP Cre reporter also results in defective nephrogenesis [Notch1 and Notch2 inactivation showed a deficiency in the expression of mature markers  of all nephron segments. The delayed inactivation of Notch1 and Notch2 using the Wnt4-Cre allowed for the initial segmentation of the RV along proximodistal axis but reduced the expression of these early stage nephron segment specific markers Lhx1 at later stages resulting in the failure to form normal SSBs and nephrons [Lhx1 in the distal RV compartment and its requirement for Dll1 expression in distal compartment of the RV is suggestive that both Notch signaling and Lhx1 are critical for development of distinct cell types within the RV [Interestingly, delaying Cre expression to the pre-tubular aggregate stage just prior to RV formation, using the Wnt4-Cre to inactivate both ogenesis . Mutant nephrons . Downregnephrons . The expn the RV .Wnt4-Cre;Notch1f/f;Notch2f/f kidneys clearly implicates a critical requirement for Notch signaling subsequent to suppressing Six2 expression and allowing NPCs to exit the progenitor state. This later function involves the proper conversion of RVs to SSBs. At the cellular level this conversion step requires the sufficient proliferation of Jag1+ cells within the early stage nephrogenic bodies [Lhx1 and Hnf1b [The failure to form SSBs in the c bodies , and at nd Hnf1b .Notch1 alone or Notch2 alone within Six2 expressing cells allows for normal nephron formation but results in proximal tubular cysts  yields a variety of phenotypes, including hypoplastic kidneys, tubular cysts, and unilateral aplasia [In addition to patterning cell types within the collecting duct, altering Notch signaling affects ureteric bud branching. Overexpression of  aplasia . All the aplasia . HoweverWhile the role of Notch signaling in kidney development is well established (as reviewed above), the requirement for ongoing Notch signaling in the adult kidney is only beginning to be unraveled. Knowing the adult kidney functions of Notch signaling is important as therapies that target Notch signaling have been considered for treating various diseases including end stage kidney disease . Notch sNotch signaling is essential for podocyte development , but theDiseases such as focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy are caused by podocyte loss and dysfunction. In human biopsy samples and rodent models of type 1 and type 2 diabetes and of albuminuria and FSGS, Notch signaling is re-activated in the podocytes . This stAdditional studies support a role for injury induced increase in Notch signaling in adult kidney tubules promoting tubulointerstitial fibrosis (TIF). Injury induced by either Folic acid injection or unilateral ureteral obstruction (UUO) increased transcripts of some genes of the Notch signaling pathway. Pretreatment of mice with the gamma secretase inhibitor DBZ before initial insult with either folic acid or UUO reduced TIF progression. Further evidence of the involvement or requirement of Notch signaling came from reduction of TIF in tubular epithelial cell-specific knockout of RBPj, and from induction of ICN1 by a doxycycline inducible model that led to TIF. Four-week-old mice administered doxycycline in food for four weeks had an increase in Notch pathway genes and showed signs of fibrosis . Going fPpargc1a as a direct target of Hes1. A more recent study [Tfam) to be both differentially expressed in NICD1-overexpressing kidney tubules and a direct transcriptional target of RBPj. Similar to PGC-1α, Tfam was also down-regulated in NICD1-overexpressing tubules and in multiple models of kidney fibrosis. Tubule-specific knockout of Tfam led to altered mitochondrial structure and increased expression of profibrotic factors. In cultured renal epithelial cells, higher Notch signaling led to decrease in fatty acid oxidation, and this effect could be reversed by increasing Tfam levels. Taken together, the findings of these two studies indicate a role of Notch in perturbing metabolic pathways associated with cellular energy production to promote fibrosis.The downstream targets of Notch signaling in promoting kidney fibrosis are largely unknown. Recently, Han et al.  reportednt study  identifiAcute kidney injury (AKI) is triggered by a brief duration of injury that rapidly, within 1–2 days, results in loss of kidney function and decrease of urine output. AKI can be induced by a host of factors, including but not limited to surgery-related injuries, and other physiological events such trauma, stress, infection, and drug toxicity . One in de novo Sox9+ expression in response to injury. The Susztak group showed that these highly proliferative Sox9+ cells do not express any transcription factors commonly seen in the developing kidney, such as Lgr5, Pax2, and Six2, suggesting their progenitor nature. In vivo lineage tracing showed these Sox9-positive cells could give rise to all epithelia of the kidney except collecting duct and glomeruli. This cell population showed increased proliferation in the adult kidney after injury by either folic acid or IRI. Eventually, these cells led to tubule generation in different segments of the kidney [While many studies suggest a pathological role for increased Notch signaling following AKI as discussed above, recent studies reveal beneficial roles for Notch signaling activation following AKI. Following AKI-induced tubular epithelial damage, repair mechanisms are initiated to restore nephron structure and function. The repair is mediated by the neighboring less damaged tubular epithelial cells , but thee kidney . The expe kidney  and invoe kidney  make Note kidney . More ree kidney .The collecting duct segments of the kidney consist of two broad cell types, the principal cells (PCs) and the intercalated cells (ICs) intermingled with each other. These two cell types arise from a common progenitor, and proper functioning of the collecting duct system requires the relative ratio of these cell types be maintained throughout the tubule. These cell types are characterized by the presence of specific marker proteins. Differentiation of the ICs require the presence of the forkhead transcription factor Foxi1 , and sevOther studies point to a role for increased Notch signaling activity in the epithelial cells of the distal nephron segments to remodel the cell types in response to inactivation of the sodium chloride cotransporter (NCC). The distal nephron remodeling process is observed to involve induction of Jagged1/NOTCH signaling, and increased Hes1 levels. This then results in an increase in PCs within the cortical connecting tubule and replacement of acid-secreting alpha-ICs with pendrin expressing-ICs . Hence, Several developmentally important signaling pathways, such as Wnt signaling and hedgehog signaling are re-activated after injury (reviewed in ). Similade novo in genes coding for Notch signaling pathway components are associated with renal diseases  is an autosomal dominant disorder that can affect multiple organs of the body including the liver, heart, skeleton, eyes, and kidneys. ALGS is caused by heterozygous mutations in function ,101. Howfunction . Due to function . Accordifunction . To dateescribed . The majed [JAG1  and anotCH2 gene ,91. The JAG1. The ALGS associated renal anomalies include multicystic dysplastic kidneys, renal tubular acidosis, vesicoureteral reflux, obstruction, chronic renal failure, end-stage renal disease, acute kidney injury, renal lipidosis, renal artery stenosis, focal segmental glomerulosclerosis, duplex collecting system, and other renal diseases in a minor proportion [Renal anomalies are found in around 40% of the ALGS patients with mutations in oportion ,94. Curroportion , althougNOTCH2 gene, that results in the truncation of NOTCH2 protein and the absence of the c-terminal pest domain, which contains sequences necessary for the ubiquitinylation and degradation of NOTCH2 following association of the intracellular domain with RBPJ and Mastermind. The absence of the PEST domain is predicted to increase the half-life of NOTCH2 resulting in gain-of-function effects [Hajdu-Cheney syndrome  is a rare genetic disorder with prominent features including bone abnormalities and to a lesser extent renal and cardiac abnormalities among others . The dis effects . So far  effects ,107, and effects ,107. Sim effects . Brennan effects . Conside effects .JAG1 or NOTCH2 are associated with CAKUT. What is perhaps surprising is that mutations in other Notch pathways genes have so far not been associated with CAKUT. However, the genetic basis of CAKUT in most patients remains to be determined.Congenital anomalies of the kidney and urinary tract (CAKUT) includes a spectrum of anomalies affecting the kidney, the urinary tract, or both . CAKUT oDiabetic nephropathy is also called diabetic kidney disease (DKD), and is the leading cause of end-stage kidney disease and occurs in approximately 40% of patients who are diabetic. Diabetic nephropathy occurs due to hyperglycemia observed in diabetic patients, although the symptoms are quite variable between type I versus type II diabetes . Among tRenal carcinoma is one of the most commonly occurring cancers with nearly 270,000 cases diagnosed yearly and accounting for 116,000 deaths annually . Notch sRenal cell carcinoma can be further divided into at least three subtypes: Clear cell renal cell carcinoma, papillary renal cell carcinoma, and chromophobe renal cell carcinoma. Some of Notch signaling components are uniquely associated with each of these subtypes based on mRNASeq data of the patient samples . Low expNOTCH2 mutation may have no renal defects, or congenital cystic kidney disease or late adult onset kidney disease. This is similar to mouse lines with a sensitized level of Notch signaling during kidney development some of which develop small kidneys while others develop normal sized kidneys. The sensitized level of Notch signaling likely allows for normally insignificant genetic variations, or dietary changes, or environmental factors to affect the severity of the kidney disease. For example, normally a null mutation in one allele of Notch1 does not cause kidney disease in mice, however when we combine this sensitized level of Notch signaling with the conditional inactivation of Notch2 in the developing kidney nephron progenitors this results in development of multicystic hypoplastic kidneys. These mouse studies suggest that additional variations in the Notch signaling pathway genes could be present in some ALGS patients with severe kidney disease in addition to a mutation in JAG1 or NOTCH2. Additional genetic modifiers may also be present in genes involved in unique functions that Notch signaling mediates in the kidney of ALGS patients with severe kidney disease. The role of Notch signaling in the maintenance of adult kidney cell types, or in the distal tubule cell type remodeling to adapt to chronic dietary changes, or in renal epithelial repair following injury is suggestive that a secondary event in adults with reduced Notch signaling could trigger late onset of kidney disease in some ALGS patients.Mouse studies have revealed many functions for Notch signaling in kidney development, in the maintenance of adult kidney cell types and in the response to kidney injury. The myriad functions of Notch signaling may in part explain the high degree of variability in the onset and severity of kidney disease observed among individuals with the same genetic defect in the Notch signaling pathway. ALGS patients with the same


Frontiers in Immunology Research Topic, leading international investigators in the field have contributed seven reviews, three minireviews, and four original research articles to this Research Topic to provide a comprehensive and timely examination of the role of chemoattractants in the tumor microenvironment (TME). The collection provides an updated overview of the most relevant issues related to the complicated interaction between chemoattractants and other mediators produced by host or tumor cells that contribute to tumor development, growth, metastasis, and immune escape.Chemokines and other chemoattractants induce directional migration and activation of leukocytes and of non-hematopoietic cells by stimulating specific G protein-coupled Receptors (GPCR). Since the term “chemokine” was officially accepted as the standard nomenclature for “chemotactic cytokines” in 1992, there have been over 100,000 manuscripts published and indexed in Pubmed, ~20% of which also include the search term cancer. In this Immune cells are fundamental in shaping the balance between a tumor-promoting or tumor-suppressive microenvironment. A fundamental role is played by macrophages, usually referred to as tumor-associated macrophages (TAMs). TAMs can stimulate proliferation of tumor cells, promote angiogenesis and fibrosis and suppress the anti-tumor immune response. In addition, the combined action of TAM at the primary tumors and the so-called metastasis-associated macrophages (MAM) in the metastatic sites promote the metastatic cascade.Argyle and Kitamura in this collection underscore the role of chemoattractants and of their receptors in TAM and MAM accumulation in primary and secondary tumor sites, highlighting the potential therapeutic role of targeting macrophage-recruiting chemokines to prevent malignant tumor development. One key determinant of monocyte recruitment and TAM accumulation provided by the CCL2/CCR2 axis. Indeed, different tumor types can produce CCL2, even though expression of this chemokine is regulated by different means. Consistent with these observations, loss of Ccr2 or CCL2 blockade inhibits TAM accumulation and is the most promising strategy for inhibition of immune suppression exerted by chemokines.The contribution by Ruytinix et al. discuss interesting observations indicating that macrophages recruited into tissues can be polarized toward cells able to produce pro-angiogenic and pro-fibrotic factors as well as to attract other immunosuppressive immune cells according to environmental factors, thus favoring tumor growth in primary site or seeding in distant organs. In addition to several growth factors, an important contribution on monocyte differentiation toward a pro-tumor phenotype is provided by chemokines, including CCL2 and CXCL12.Lepore et al. describes a pro-tumorigenic role for CXCL16/CXCR6 signaling in glioma progression pointing to a critical role in immune-suppression. In a GL261 syngeneic orthotopic implantation model, CXCR6-deficient mice survived significantly longer than WT counterparts, with significantly reduced tumor volumes. Using anti-CXCL16 neutralizing antibodies, the authors discovered that glioma-secreted-CXCL16 induced an immune-suppressive gene expression signature in primary microglial cells.An original article from Castriconi et al. Furthermore, by reporting evidence from the literature, Susek et al. revised the effect of CXCR1/2 and CXCR3, highlighting the importance of the formers in suppressive cell recruitment and of the latter in the generation of an effective T and natural killer cell anti-tumor response.Strategies able to inhibit macrophage recruiting or polarizing chemokines also create a permissive environment for immunotherapy, favoring activation of effector cells with anti-tumor activities. Nevertheless, activated CD8+ and NK cell populations rely on several receptors for their recruitment and infiltration and immunotherapeutic approaches are less effective in chemokine receptor deficient mice. Indeed, the distribution and phenotype of different NK cell subsets can be affected by specific types of tumor and its location and this often correlate to altered migration and homing. These and other aspects regulating trafficking and tissue localization of NK cells are discussed in this collection by Shin et al. provides a comprehensive examination of chemerin in cancer, with a focus on mechanistic preclinical studies and functional consequences of chemerin in tumors. An original research article by Pachynski et al. indicates that chemerin gene expression is significantly downregulated in human breast cancer, which the authors hypothesize to be part of an adaptive tumor evasion strategy. Chemerin overexpression by mouse EMT6 breast cancer cells suppressed tumor growth in vivo, which was associated with increased CD4+ and NK cell infiltration into the tumor and mechanistically dependent on NK cells.Since the first mechanistic study defining a protective role for leukocyte attractant chemerin in recruiting anti-tumor NK cells to melanoma lesions in 2012, there have been nearly 100 publications exploring the role of chemerin in cancer. The review by Many malignant tumors of non-hematopoietic origin express multiple chemoattractant GPCRs that increase the invasiveness and metastasis of tumor cells. In addition, chemoattractants also enable the interaction of tumor cells with host cells, thus promoting tumor growth and development of distant metastasis.Jacquelot et al. provides an in-depth look at the chemokines and chemokine receptors involved in melanoma progression. The expression of chemokine receptors by melanoma cells can be a determining factor in metastasis and survival outcomes, with CCR7, CCR10, and CXCR4 being particularly deleterious. The expression of certain chemokine receptors on blood or tumor infiltrating leukocyte subsets from melanoma patients or from preclinical studies can also be a determining factor in prognosis. The authors provide an up-to-date assessment of translational chemokine receptor targeting approaches in melanoma, noting the “double-edged sword” nature of this approach, in that targeting receptors expressed by melanoma may impair effective anti-tumor leukocyte functions.The review by Liubomirski et al.; Liubomirski et al., together point at the pro-inflammatory microenvironment, and at the Notch pathway, as targets for potential future treatments in TNBC. The authors found that TNBC from patient samples exhibited increased levels of Notch1 and Notch 3 and decreased Notch4 compared to luminal A breast cancers. Moreover, Notch1 expression correlated with TNF-alpha and CXCL8 expression. Notch 1 regulated the contact-dependent induction of CXCL8, and TNF-alpha stimulation led to activation of p65 and subsequently CXCL8 production. The authors conclude that the Notch pathway is a key mechanism for up-regulation of CXCL8 resulting in increased aggressiveness of TNBC.Triple-negative breast cancer (TNBC) is a subgroup of diagnosed breast cancer patients without targeted therapeutic options. Notch receptor expression and activation strongly correlate with the aggressive clinicopathological and biological phenotypes of breast cancer. Two articles by Salazar and Zabel reviews the ways in which chemokine receptor expression by tumor endothelial cells (TEC) can support cancer progression. TEC are highly heterogeneous and express a variety of chemokine receptors such as ACKR1, ACKR3, CXCR4, CCR2, CXCR2, and CXCR3. TME-derived chemokines contribute to the morphological and phenotypic dysregulation of the vascular endothelium, leading to pro-tumorigenic angiogenesis, vasculogenesis, intussusception, vessel co-option, and/or vascular mimicry. The authors speculate that chemokine receptors may be particularly promising targets for future vascular disruption therapies based on their restricted expression  and the potential for concomitant effects on leukocytes .In an intriguing change of pace from considering the role of chemoattractant receptors on tumor cells or leukocytes, Many chemokines are abundantly and concomitantly expressed in the TME and their function is regulated by complex mechanisms. In the latest years it has become clear that complexity is even higher because of the formation of heterocomplexes that exert antagonistic or synergistic effects on selected receptors.D'Agostino et al. have collected the available scientific literature and their own experience on the phenomenon of heterocomplex formation, concentrating their investigation in cancer. The possible outcomes of heterocomplexes between chemokines, as well as between chemokines and inflammatory molecules (such as HMGB1) on the shaping of the TME is discussed.Vilgelm and Richmond constructed a heat map showing prognostic associations between 25 individual chemokines and 12 different types of cancer. Certain chemokines contribute to establishing a “T cell-inflamed” TME that is associated with improved prognosis, particularly when checkpoint inhibitor treatments are administered. The authors also describe a variety of chemokine-based countermeasures that can be deployed to populate an “immunologically cold” tumor with anti-tumor leukocytes. Poeta et al. focus on the role of chemokines and chemokine receptors in cancer with considerations on the possibility to be targets for cancer immunotherapy with emphasis on the possibility to optimize the anti-tumoral potential of the immune system. They present an overview on the current use of antagonists or inhibitors of chemokine receptors to treat different type of tumors both in preclinical model and clinical trial.Given their role in the pathomechanisms of tumor progression, chemoattractant receptors and their ligands constitute targets for the development of novel anti-tumor therapeutics. Two reviews provide comprehensive insight into the role of chemokines and receptors in tumor pathobiology and targeted treatments. Using publicly available data from The Human Protein Atlas, It is our hope that this collection will serve to launch new studies that extend our understanding of chemoattractants in the pathomechanisms of tumor progression, and to inspire the discovery and development of new chemokine-focused treatments to make a real impact in the lives of cancer patients and their families.GB and BZ have both contributed substantially to the work and approved it for publication.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.


Chronic wasting disease (CWD) is a contagious and fatal neurodegenerative disease and a serious animal health issue for deer and elk in North America. The identification of the first cases of CWD among free-ranging reindeer and moose in Europe brings back into focus the unresolved issue of whether CWD can be zoonotic like bovine spongiform encephalopathy. We used a cell-free seeded protein misfolding assay to determine whether CWD prions from elk, white-tailed deer, and reindeer in North America can convert the human prion protein to the disease-associated form. We found that prions can convert, but the efficiency of conversion is affected by polymorphic variation in the cervid and human prion protein genes. In view of the similarity of reindeer, elk, and white-tailed deer in North America to reindeer, red deer, and roe deer, respectively, in Europe, a more comprehensive and thorough assessment of the zoonotic potential of CWD might be warranted. Odocoileus hemionus), white-tailed deer (O. virginianus), North American elk (Cervus canadensis), and moose  are most commonly affected in the United States and Canada, and red deer (C. elaphus) and sika deer (C. nippon) in South Korea also have been affected  and in a free-ranging reindeer , a species not previously known to be affected by CWD in wild or farmed animals (Chronic wasting disease (CWD) is a fatal contagious prion disease of cervids that is found in the United States, Canada, South Korea, and most recently in Europe (C) into the abnormal and misfolded conformer (PrPSc) that is partially protease-resistant (PrPres). However, the molecular criteria for predicting zoonotic potential for prions are unclear. Consequently, approaches to understanding the zoonotic risk for prion transmission to humans have been empirical, involving in vivo and in vitro models  are well-characterized animal prion diseases affecting animals of consumption; scrapie affects sheep and BSE cattle. Human prion diseases occur as sporadic, genetic, and acquired forms; the variant form of Creutzfeldt-Jakob disease is zoonotic BSE, acquired through the oral route and having a long incubation period , analyzing white-tailed deer CWD specimens for the first time, and analyzing reindeer that have been experimentally infected with white-tailed deer CWD. The elk ML polymorphism at position 132 of Prnp is of particular interest because it corresponds to the methionine–valine (MV) PRNP codon 129 polymorphism in humans, which is itself a major genetic susceptibility factor associated with human prion disease (Capreolus capreolus), North American elk (a near relative of red deer), and, most important, reindeer might help in formulating risk assessments in Europe.In contrast to these in vivo approaches, we have used protein misfolding cyclic amplification (PMCA) to investigate the molecular compatibility of bovine, ovine, and cervid prions with full-length, glycosylated and glycosylphosphatidylinositol-anchored human prion protein (PrP)  elk, white-tailed deer, and reindeer frozen brain tissue from CWD-affected animals that had been confirmed positive by using a statutory diagnostic testing regime . Small pWe homogenized transgenic mouse brains in conversion buffer by using a glass on glass manual grinder and a conversion buffer made of 1× phosphate-buffered saline (PBS), 150 mmol/L of NaCl, 1% Triton X-100, and a complete protease inhibitor cocktail  to obtain a final 10% weight-to-volume solution. We cleared the homogenized tissue by using centrifugation as described previously  and boiled them at 100°C for 10 min. We loaded the samples on NuPAGE Novex  10% Bis-Tris gels  and subjected them to electrophoresis at 200 volts for 55 min. We transferred proteins to a polyvinylidene difluoride membrane by using 800 mA for 60 min  diluted 1/10,000 . We detected CWD PrP by stripping the Western blots  and reprobing them with a 6H4 antibody diluted 1/40,000 . We used ECL antimouse IgG, peroxide-linked species-specific F(ab′)2 fragment from sheep  as a secondary antibody diluted 1/25,000. We developed membranes by chemiluminescent detection using ECL Prime  and acquired digital images by using an XRS Bio-Rad system  with a CCD camera.We evaluated the presence of PrPres in the amplified sample after a defined period of Western blot image capture. The triplet pattern had to be absent from the unamplified sample tested under the same conditions analyzed in parallel.The criterion for conversion was 3F4 antibody detection of a pattern of 3 clear protease-resistant bands of the expected electrophoretic mobility and ratio for PrPg at 4°C as described previously (g for 1 h at 4°C). We resuspended the washed pellets directly in PMCA substrate before using them in a single round of amplification.We incubated brain homogenate from white-tailed deer and reindeer CWD specimens by using 20% sarkosyl (diluted in PBS) for 10 min at room temperature. We subjected samples to centrifugation for 1 h at 100,000 × res by using mAb 6H4. We detected similar levels of total PrP by using Western blotting among the elk specimens analyzed  but low levels in the other specimen (reindeer 2)  , 4.PRNP codon 132 MM elk CWD seeds in humanized transgenic 129 MM mouse brain substrate. Only those elk CWD samples that had readily detectable PrPres (as detected by 6H4 mAb) were able to produce human PrPres (detectable by the 3F4 mAb) after Western blot and proteinase K treatment, consistent with CWD PrPres playing a direct role in the misfolding process . Each of the CWD methionine homozygous (132 MM) samples resulted in human PrPres formation when used to seed the matched humanized substrate (129 MM). The heterozygous elk seeds did not result in any detectable conversion of the humanized 129 MM PrP substrate. When we incubated these same CWD brain homogenates with the heterozygous (129 MV) and homozygous (129VV) humanized substrates, we observed low levels of human PrPres formation but no obvious difference in efficiency between the CWD 132MM and CWD 132ML samples in the 129VV substrate  had roughly one tenth of the PrPres of the other sample, showing that the reaction with small amounts of reindeer PrPres are able to convert the humanized substrate. The PRNP codon 129 MV and VV genotype substrates also were readily susceptible to conversion by the reindeer seeds  to investigate the susceptibility of the human PrP to conversion to its disease-associated form by several animal prion diseases, including CWD  the presence of the 3F4 epitope on human but not cervid PrP. In effect, this degree of sensitivity means that any human PrPres formed during the PMCA reaction can be detected down to the limit of Western blot sensitivity. In contrast, if other antibodies that detect both cervid and human PrP are used, such as 6H4, then newly formed human PrPres must be detected as a measurable increase in PrPres over the amount remaining in the reaction product from the cervid seed. Although best known for the efficient amplification of prions in research and diagnostic contexts, the variation of the PMCA method employed in our study is optimized for the definitive detection of zoonotic reaction products of inherently inefficient conversion reactions conducted across species barriers. By using this system, we previously made and reported the novel observation that elk CWD prions could convert human PrPC from human brain and could also convert recombinant human PrPC expressed in transgenic mice and eukaryotic cell cultures  and the heterozygous (132 ML) seeds incubated with the other 2 human polymorphic substrates (129 MV and 129 VV). The presence of the amino acid leucine at position 132 of the elk Prnp gene has been attributed to a lower degree of prion conversion compared with methionine on the basis of experiments in mice made transgenic for these polymorphic variants (C by the full range of cervid polymorphic variants affected by CWD would be warranted.The contribution of the polymorphism at codon 129 of the human PrP gene has been extensively studied and is recognized as a risk factor for Creutzfeldt-Jakob disease (R. tarandus) in Norway . This finding suggests that CWD prions from reindeer could be more compatible with human PrPC generally and might therefore present a greater risk for zoonosis than, for example, CWD prions from white-tailed deer. A more comprehensive comparison of CWD in the affected species, coupled with the polymorphic variations in the human and deer PRNP–Prnp genes, in vivo and in vitro, will be required before firm conclusions can be drawn. Analysis of the Prnp sequence of the CWD reindeer in Norway was reported to be identical to the specimens used in our study (In light of the recent identification of the first cases of CWD in Europe in a free-ranging reindeer (ur study .Sc and PrPC is necessary for conversion and prion replication. Accordingly, polymorphic variants of the PrP of host and agent might play a role in determining compatibility and potential zoonotic risk. In this study, we have examined the capacity of the human PrPC to support in vitro conversion by elk, white-tailed deer, and reindeer CWD PrPSc. Our data confirm that elk CWD prions can convert the human PrPC, at least in vitro, and show that the homologous PRNP polymorphisms at codon 129 and 132 in humans and cervids affect conversion efficiency. Other species affected by CWD, particularly caribou or reindeer, also seem able to convert the human PrP. It will be important to determine whether other polymorphic variants found in other CWD-affected Cervidae or perhaps other factors (The prion hypothesis proposes that direct molecular interaction between PrP


The incorporation of DNA into plant genomes followed by regeneration of non-chimeric stable plants (transformation) remains a major challenge for most plant species. Forest trees are particularly difficult as a result of their biochemistry, aging, desire for clonal fidelity, delayed reproduction, and high diversity. We review two complementary approaches to transformation that appear to hold promise for forest trees. WUSCHEL and/or BABY BOOM have formed the basis of a high efficiency method for a variety of monocot genotypes and species. However, in dicots the utility of these genes as the basis of a robust transformation system has not been demonstrated. Many additional DGs appear capable of promoting regeneration that have not been systematically explored as transformation tools.Developmental genes (DG) may be useful tools for promoting transformation. DGs, which can act through a wide variety of developmental mechanisms to promote regeneration of transgenic cells, have been widely employed in model plants to promote embryogenesis and in some cases organogenesis. Following initial experimental demonstration in dicots, the DGs in vitro plant transformation systems are costly and must be customized for each new genotype and species, in vivo approaches to transformation hold much appeal. It is possible to produce stable transgenic plants by agro-inoculation of seeds or vegetative/floral buds, but as yet these approaches have not been used routinely in any plant species except for the Arabidopsis floral dip. We will discuss how the Arabidopsis system, and other in planta techniques, may be tailored for forest trees, taking into account variations in biology of different taxa.Because in vitro regenerability. Recent molecular evidence places these genes within a genetic regulatory network, connected by cascades and feedback loops of transactivation. Knowledge of these interactions, detailed in Supplementary Table 1 and Figure 1, can inform selection of individual genes and combinations of genes that may be most effective for improving regeneration. In this mini-review, however, we do not consider the many genes which may enhance regeneration via epigenetic mechanisms, or by affecting the rate of gene transfer or incorporation of DNA into the genome.When overexpressed, transcriptional or epigenetic regulators of embryo and meristem development  have been shown to confer improvements to Knotted-1, the first homeodomain transcription factor identified in plants , an Arabidopsis homolog of Knotted-1, was reported to lead to premature termination of shoot meristems and a phenotype of twig-like plants lacking lateral shoots (STM is regulated in part by a positive feedback loop between STM and the transactivator CUP-SHAPED COTYLEDON 1 (CUC1) , which was first identified as a regulator of embryogenesis when overexpression induced embryos to develop on leaves of Arabidopsis (A loss-of-function mutation of l shoots . Transcre effect . Directlbidopsis .WUSCHEL (WUS) loss-of-function mutants, which had similar phenotypes to stm mutants   . In Arabxpressed . WUS wasin vitro .WUS and STM are overlapping sets of diverse genes which balance differentiation and dedifferentiation to promote progressive development of meristems. These genes include members of the SE-associated receptor kinase (SERK) family, as well as enzymes for hormone biosynthesis, cell cycle regulators, and numerous transcriptional regulators, some of which function not only downstream, but upstream of WUS/STM (Downstream of  WUS/STM .WUS transcription is activated by a complex of factors including B-type ARABIDOPSIS RESPONSE REGULATOR (ARR) and HDIII-ZIP family proteins . In separate experiments, overexpression of ARR2 and ARR12 led to roughly twofold increases in number of shoots from callus in vitro  and numerous other poorly known genes  enhances formation of callus in addition to shoots (wind1 mutants to media without inducer (Supplementary Table 1).wn genes . ESR1 ovelopment . ESR2 is targets ; overexp targets . Unlike ts  3/5/7 are responsible for direct transactivation of WUS and indirect transactivation of both WUS and STM via CUP-SHAPED COTYLEDON (CUC) 1/2 . In Arabidopsis, overexpression of PLT5/7 enables cytokinin-independent shoot regeneration, although at a very low rate (low rate .BABY BOOM (BBM) overexpression confers the ability for cytokinin-independent in vitro somatic embryogenesis in Arabidopsis , enhances in vitro transformation and shoot regeneration in a variety of monocots, including rice, sorghum, and maize. Several completely recalcitrant maize inbred lines became responsive to transformation and regeneration when overexpressing either WUS or BBM homologs , overexpression of a KNOTTED-1 ortholog similarly promoted in vitro somatic embryogenesis  enabled in vitro somatic embryogenesis and efficient transformation of a genotype which was previously recalcitrant , and are thus in theory applicable to a large panel of genotypes and species. The target tissues are diverse, and can include secondary meristems. The induced somatic sector analysis (ISSA) approach is an example of what can be achieved in various tree species  . The train planta approaches to transformation hold promise to solve major problems in plant and tree transformation. DGs appear to hold most promise where a basic in vitro regeneration system is in place, thus might benefit from a large elevation of transformation efficiency using an established transformation pathway. It has to date been most effective in species with embryogenic rather than organogenic regeneration systems. In planta systems hold most promise where in vitro approaches are extremely difficult or impossible, thus alternate pathways are required. It will also be most easily pursued as part of a large-scale breeding program, enabling large numbers of floral buds to be treated and seedling populations screened for transformation and chimerism. In planta transformation of cambium and axillary/apical buds has been successful, but is prone to chimerism; research on germline transformation  may help to reduce this problem. In planta and DG overexpression approaches to efficient transformation might not be mutually exclusive; research is warranted to elucidate the potential for DGs to enhance in planta systems. Given the importance of regeneration as a bottleneck to transformation and gene editing of forest trees in research and application (In summary, both DG and lication , accelerin planta Transformation Methods in Forest Trees,” respectively, after extensive consultation with SS and GP. SS and GP revised the drafted text and prepared initial drafts of the sections “Introduction” and “Conclusion.”MN and AD drafted the sections “Developmental Genes as Tools for Transformation Improvement” and “Toward the Development of 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 purpose of this hermeneutic interpretive phenomenology study was to describe the shared meaning of compassion fatigue (CF) among registered nurses (RNs) who work in skilled nursing facilities (SNFs). The specific aims were to describe: 1) contributors  that cause symptoms of compassion fatigue, 2) associated physical and emotional symptoms, and 3) the short-term outcomes of unresolved compassion fatigue impacting nurses and patient care. CF can negatively impact patient outcomes, is associated with decreased quality of patient care, and can be a reason why nurses leave the profession. Eight participants were interviewed three times each, while concurrent data analysis helped to formulate mutual understanding of the phenomenon and informed subsequent interviews. Self-reflection, journaling, record keeping, and use of direct quotes enhanced trustworthiness. Four shared meanings were abstracted:1) I feel conflicted and that causes my CF; 2) physical and emotional manifestations of CF; 3) CF is infused in every aspect of my life; 4) we are trying to cope with CF. The participants shared their central desire to make a difference in the lives of their patients, which was of paramount importance. When participants felt they were unable to make the desired difference, they began to develop symptoms of CF. Symptoms were compounded when they experienced frequent patient deaths. A resiliency program specifically addressing the needs of SNF nurses, incorporating individuals and their organizations, could positively impact the nurses’ professional quality of life. Future research is needed to better understand CF and interventions specific to SNF nurses.


Leiomyomas of deep soft tissue are extremely rare and should only be diagnosed following adherence to stringent histological criteria, namely, the absence of nuclear atypia and of coagulative tumor necrosis. Whether extremely low counts of, or even any, mitotic activity are acceptable when making a diagnosis of leiomyoma in deep soft tissue sites is controversial. The morphology and immunophenotype of smooth muscle tumors in deep soft tissue are similar to their counterparts irrespective of topography. It is interesting to note that leiomyomas of deep soft tissue (extremity and retroperitoneum) are often hyalinized/sclerosed and calcified. However, the prediction of their behavior and correct codification is dependent on thorough, meticulous search for mitoses and necrosis. Leiomyomas of deep soft tissue in the extremity should be devoid of mitoses and “significant” cytological atypia. An occasional larger, slightly pleomorphic cell in the midst of bland spindle cells, can be regarded as insignificant atypia. If any mitotic activity and several atypical cells are encountered in smooth muscle tumors of deep soft tissue of the extremity, it would be prudent to invoke the appellation of smooth muscle tumor of uncertain malignant potential and advocate wide local excision and follow-up. In the past, the existence of leiomyomas of deep soft tissue was considered controversial. The prevailing view was that most, and perhaps all, smooth muscle tumors of deep soft tissue were malignant, although this was based on studies of smooth muscle tumors demonstrating both mitotic activity and cytological atypia. To date, there have been very few studies of well-differentiated smooth muscle tumors of deep soft tissue that display an absence of mitotic activity and cytological atypia. It is now thought that, although they are exceptionally rare, deep soft tissue leiomyomas do in fact exist –8 and caIn the literature, leiomyoma of deep soft tissue is scarcely mentioned, hence much of our knowledge regarding this entity is based on case reports and exiguous small case series , 10.The focus of this succinct review is on leiomyomas of deep somatic soft tissue.Leiomyomas of deep somatic soft tissue occur primarily in middle-aged adults, with a mean age of 37 years, and with equal distribution among the genders . Deep soGrossly, the majority of deep somatic soft tissue leiomyomas are circumscribed masses, with a tan-grey to white cut surface, and a mean size of 5.4 cm. Degenerative mucoid-cystic change is occasionally present .Microscopically, deep soft tissue benign smooth muscle tumors are often well-circumscribed, unencapsulated, occasionally multinodular, paucicellular spindle cell lesions . The lesWhile an important diagnostic tenet of leiomyomas of deep somatic soft tissue is that nuclear atypia is lacking in the majority, focal minimal atypia has been described in a few cases . MitosesIn 2001, Billings and colleagues analyzed the clinicopathological features of 13 highly differentiated smooth muscle tumors/leiomyomas of deep somatic soft tissue  to determine if it is indeed possible to define a biologically benign group of smooth muscle tumors at this site by using stringent criteria . Nuclearbona fide leiomyosarcomas of deep somatic soft tissue is usually higher than that in these 3 cases [Billings et al. cautioned that their study encountered three leiomyomas of somatic soft tissue with low levels of mitotic activity (1–4 mitoses per 50 HPF), all of which occurred in young individuals  . Follow- 3 cases , given t 3 cases . Thus, t 3 cases .Leiomyomas of deep somatic soft tissue are positive for smooth muscle actin (SMA), desmin, and caldesmon  and expeMED12 are indeed mutated in this particular group of benign smooth muscle tumors. Therefore, such diagnostic testing is not yet generally applicable in this histogenetic group.Genetics of deep soft tissue smooth muscle tumors are as yet poorly understood, and it remains to be seen if genes such as Differential diagnoses of benign smooth muscle tumors (leiomyomas) of deep somatic soft tissue include both benign and malignant entities.Angioleiomyoma is the most common peripheral soft tissue leiomyoma, typically occurring in the subcutis of extremities, particularly the lower leg, but also in the trunk wall and less commonly in the head and neck region. It usually forms a small 1-2 cm well-circumscribed, homogeneous rubbery nodule, and it is clinically notable for often being painful . HistoloEBV-associated smooth muscle tumor is a rare subset of smooth muscle tumor that occurs in immunosuppressed individuals. It is most common in patients with human immunodeficiency virus (HIV), many of whom are young, including children. EBV-associated smooth muscle tumors also occur in solid organ transplant recipients , who are usually middle-aged adults, and rarely in patients with a congenital immunodeficiency disorder . It can Smooth muscle tumors in soft tissue that contain both nuclear atypia and mitotic activity are generally designated leiomyosarcomas to denote their malignant (metastatic) potential . HistoloThese include nerve sheath tumors, fibrohistiocytic spindle cell lesions, and sundry spindle cell lesions with characteristic immunophenotypic profiles.The small number of cases with limited follow-up period reported in the literature precludes definitive conclusions regarding the significance of low mitotic rates in smooth muscle tumors of deep somatic soft tissue.In the study by Billings and colleagues, clinical follow-up information was available for 11 of 13 of their patients, with a mean follow-up period of 58.7 months . AlthougHowever, recurrence has been reported in a smooth muscle tumor of somatic deep soft tissue with a mitotic activity of 1 mitosis per 50 HPF . FletcheThese lesions are managed by wide local excision. Although 5 of 13 cases reported by Billings et al. had positive resection margins, tumor recurrences or metastases were not documented . The mosAs alluded to previously, leiomyomas of deep soft tissue segregate into two distinct clinicopathological groups, one group occurring in deep somatic soft tissue (the topic of this review) and the second occurring primarily in women in the pelvic retroperitoneum. For completeness, benign smooth muscle tumors (leiomyomas) of the pelvic retroperitoneum will be discussed briefly .Retroperitoneal leiomyomas occur preferentially in women during the perimenopausal period, usually in the pelvic retroperitoneum , and theMED12 gene have been reported in extrauterine leiomyomas, including at pelvic/retroperitoneal sites [HMGA2), three others had 8q rearrangements (the target gene of the 8q aberration was PLAG1), one tumor had deletion of the long arm of chromosome 7, del(7) (q22), and one had aberrations of chromosome bands 3q21∼23 and 11q21∼22 [MED12 but none of them had mutation in exon 2 of that gene [Similar to uterine leiomyomas , 24, mutal sites , 26. Recal sites . Three t11q21∼22 . All eighat gene . Thus, iThe striking female predilection of this type of leiomyoma suggests that they arise from hormonally sensitive smooth muscle and are functionally similar to uterine leiomyomas . The locThe diagnosis of leiomyoma in deep somatic soft tissue sites should be based on stringent histological criteria, specifically extremely low levels of, or even perhaps absence of, mitotic activity and the absence of nuclear atypia and coagulative tumor necrosis . Leiomyo


However, there have been no biosensors to monitor necroptosis. Development of a FRET biosensor that monitors necroptosis and generation of transgenic mice expressing such FRET biosensor might be useful to understand the mechanisms underlying the execution of necroptosis and also the consequences of necroptosis in vivo. In our recent study :4457), we developed a FRET biosensor for necroptosis, termed SMART (a sensor for MLKL activation by RIPK3 based on FRET). SMART is composed of a fragment of MLKL and monitors necroptosis, but not apoptosis or necrosis. Moreover, we recently developed a platform called Live-Cell Imaging for Secretion activity (LCI-S) to monitor protein secretion at the single cell level. This platform has enabled us to monitor the release of HMGB1 (High Mobility Group Box 1), one of the DAMPs, at the single cell level and reveals two different modes of the release of HMGB1 from necroptotic cells.Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL). While danger-associated molecular pattern (DAMP)s are released from dead cells and involved in various pathological conditions, the mechanisms underlying regulation of the release of DAMPs are not fully understood. Apoptosis and pyroptosis can be detected by several types of sensors such as Forster resonance energy transfer (FRET) biosensors, termed SCAT1 (a sensor for caspase 1 activation based on FRET) and SCAT3, respectively. These sensors have provided better understanding of pyroptosis and apoptosis  Various types of cells undergo necroptosis following stimulation with death ligands including tumor necrosis factor, Fas ligand, and TRAIL, or polyI:C, and viral infection. Among various molecules involved in regulation of necroptosis, RIPK3 and MLKL are essential key players  at the N-terminus that is a target of a necroptosis inhibitor, Necrosufonamide (NSA). NSA inhibits translocation of MLKL, but not oligomerization of MLKL. Taken that NSA also inhibited an increase in the FRET/CFP ratio of SMART, SMART might sense membrane translocation of oligomerized MLKL, but not the oligomerized MLKL itself. How do we integrate these findings and make a model for the mechanism underlying an increase in the FRET/CFP ratio of SMART? Notably, the interaction of SMART with RIPK3 was required for an increase in the FRET/CFP ratio of SMART and the majority of cytoplasmic MLKL translocated into the plasma membrane after necroptosis induction. Thus, once the oligomerized MLKL is released from activated RIPK3 and translocates to the plasma membrane, activated RIPK3 subsequently binds to induce conformational changes of SMART, increasing the FRET/CFP ratio  and glutamine at 343 (Q343), thereby preventing spontaneous oligomerization  that monitors cytokine secretion at the single-cell resolution. This system is composed of a cell culture chamber containing approximately 1,000 sub-nanolitresized microwells with 80 μm square in size -III complex is involved in membrane repair, virus budding, and multivesicular body formation. Recent studies have reported that MLKL-containing vesicles are released from dying cells. Taken that knockdown of components of the ESCRT-III complex such as


The growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis has a fundamental impact on glucose metabolism. Therefore, both untreated GH deficiency (GHD) and GH treatment (GHT) may be associated with some metabolic alterations, although the abnormalities of glucose metabolism have been investigated by relatively few studies as main outcomes.The present review summarizes the available data on glucose metabolism in children with GHD, providing an overview of the current state of the art in order to better clarify the real metabolic impact of GHD and GHT.Among all the existing studies, we evaluated all original studies that fulfilled our criteria for analysis reporting parameters of glucose metabolism as the primary or secondary objective.per se on glucose metabolism is quite homogeneous, with the majority of studies reporting no significant difference in metabolic parameters between GHD children and controls. Conversely, GHT proves to be more frequently associated with a subtle form of insulin resistance, while both fasting glucose and HbA1c levels remain almost always within the normal range.The reported impact of GHD The different methods to study glucose metabolism, the heterogeneity of the populations evaluated, the different doses of GH used together with the variable duration of follow-up may be responsible for discrepancy in the results. Long-term longitudinal studies having glucose homeostasis as their primary outcome are still needed in order better to clarify the real metabolic impact of GHD and GHT in children. Primarily, GH inhibits insulin-induced suppression of hepatic gluconeogenesis, thus increasing glucose production. In addition, it mainly act in stimulating lipolysis by providing free fatty acids (FFA) in order to switch metabolism from glucose and protein to lipid utilization (The growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis has a fundamental impact on metabolism  and GH treatment (GHT) may be associated with metabolic alterations and a common mechanism could be the increased flux of FFA caused by body composition alterations (in untreated GHD) or enhanced lipid oxidation secondary to the anti-insulin effect of GH (during GHT), respectively , 18.However, metabolic abnormalities associated with GHD have so far been evaluated only in a small number of children  and mostM-value (derived from euglycemic hyperinsulinemic clamp).PubMed was searched for studies evaluating the glucose metabolism in children with GHD and/or its changes during GHT. The search terms used were “growth hormone deficiency in children,” “growth hormone deficiency and glucose,” “growth hormone deficiency and diabetes,” and “growth hormone deficiency and metabolism.” We excluded from the analysis the review articles. We considered all original studies in English language published from 1970 to January 2018 which reported as the primary or secondary objective of the study at least one of the following metabolic parameters: fasting glucose, fasting insulin, glycosylated hemoglobin (HbA1c), homeostatic model assessment of insulin resistance (Homa-IR), the quantitative insulin sensitivity check index (QUICKI), the insulin sensitivity index (ISI), or the Specifically, we carefully evaluated more than 100 studies concerning children who have been treated with GH. We considered case–control prospective studies performed on consecutive patients (levels of evidence I/II). We excluded from the analysis all original studies which evaluated less than five patients, those with a follow-up of less than 1 month of treatment, studies on children treated with long-acting formulations of GH or treated with GH for indications other than GHD. The final analysis comprised 22 (for naïve GHD children compared to controls) and 24 studies (for children during GHT), that fulfilled our criteria for analysis.Low GH and IGF-1 levels are known to be predictors of unfavorable metabolic profile in healthy subjects –35 and iSubstantially, most of the studies did not show a significant impairment in glucose metabolism in naïve GHD children. The Table Fasting hypoglycemia and marked insulin sensitivity have sometimes been observed in GHD children due to diminished hepatic output through decreased gluconeogenesis or abnormal glucose mobilization . One of Partially in accordance with these data, more recently Stawerska et al. demonstrated comparable glucose and insulin levels between GHD children and controls but significantly lower insulin levels and higher insulin sensitivity in GHD children with lower IGF-1 bioavailability than children with higher IGF-1 levels .The insulin sensitivity related to GHD seems to decrease with age, probably due to the physiological effect of steroids production or the modifications of body composition during the time. Indeed, in normal children insulin action is physiologically reduced during puberty and insulin secretion is normally increased. The insulin resistance of puberty, which was found to be directly correlated with IGF-1 levels, leads to compensatory hyperinsulinemia which serves to amplify the anabolic effect of insulin during this period of rapid growth , 56. ForThese data contrast with those of Radetti et al., who through evaluation of QUICKI demonstrated a slight degree of insulin resistance in GHD children at baseline compared with controls , while oGrowth hormone treatment has been suggested to impair glucose metabolism because of the anti-insulin effect of GH and its direct effect on β-cell, although the real metabolic impact of GHT in children has not been established. The limitation of most studies is represented by evaluation of glucose metabolism through basal parameters and surrogate indices of insulin sensitivity, i.e., fasting glucose, fasting insulin, or Homa-IR, and not by the more reliable indexes derived from oral glucose tolerance test (OGTT), i.e., ISI Matsuda, or the gold standard technique to measure insulin sensitivity, the euglycemic hyperinsulinemic clamp , which iThe Table Fasting glucose is undoubtedly the parameter mostly used to evaluate glucose metabolism in almost all studies . In a recent study, Xue et al. reported a significant increase in fasting glucose in 60 children during 6 months of GHT, although in half of the patients a high GH dose (0.1 U/kg) was used . SimilarIn 2000, a large retrospective analysis of data more than 23,000 children reported that the incidence of type 1 diabetes did not differ from expected values, while the incidence of type 2 diabetes was higher  than reported in children not GH-treated, probably as a consequence of an acceleration of the disorder in predisposed individuals. However, it has to be highlighted that not only children with GHD were included in this analysis, but also children with other clinical conditions treated with supraphysiological doses of GH .In 2011 an analysis of data of 11,686 patients showed a slightly higher incidence of type 2 diabetes in GH-treated children than in the general population. Interestingly, most patients who developed diabetes had preexisting risk factors, like preexisting insulin resistance. Thus, monitoring of glucose before and periodically during GHT is recommended by the authors, especially for children with preexisting risk factors .Radetti et al. reported normalization of glucose tolerance in 5 out of 128 children who already presented impaired glucose tolerance before starting GHT , while uRecently, a large safety-monitoring study enrolling more than 54,000 children who were given GHT and were followed until GHT discontinuation showed no significant increase in incidence of diabetes compared to the general population , as wellHbA1c was only evaluated in 9 out of 24 studies and most of these (8 studies) showed no significant change in levels during GHT.Another metabolic parameter very frequently assessed during GHT is fasting insulin, evaluated in 18 out of 24 studies (75%). An increase in fasting insulin has been documented in 83.3% of studies and unchanged levels in 11.1% of studies. Lippe et al. showed comparable fasting insulin levels between GHD children and controls but with a concomitant increase in insulin secretion following OGTT in a group of 11 children after 6 months of GHT .Insulin sensitivity has been more frequently studied by surrogate indexes derived from fasting glucose and insulin levels, as Homa-IR or, less frequently, QUICKI.Homeostatic model assessment of insulin resistance was calculated in 15 out of 24 studies. Specifically, it proved to be unchanged in two studies (13%) , 66 and Quantitative insulin sensitivity check index was evaluated in very few studies  and it is proved to decrease during GHT in all of them , 63, 65.Fewer studies evaluated the degree of insulin sensitivity through evaluation of the more reliable ISI  and the majority of them  are concordant in detecting a reduction in ISI during GHT –65, 80.M-value, hence confirming a real deterioration in insulin sensitivity after GHT, though without evident changes in glucose tolerance  during 6 years of GHT. The results of this study suggested a positive influence of GHT on the β-cell secretory capacity, without a significant impact on insulin sensitivity . The DIoIt is important to highlight that, overall, the anti-insulin effect of GH seems to be prevalent during the first months of treatment and may be caused by a decline of peripheral glucose utilization and increase in insulin resistance  whereas,In synthesis, glycometabolic effects of GHT seem to be biphasic, leading initially to deterioration of glucose metabolism, but in the long-term an improvement of glucose handling is reported. These data can be explained by the long-term beneficial effects of GH on body composition, which probably may overcome the insulin-antagonist effect of GH in the long-term follow-up .Few studies have tried to identify any predictive factors of glucose alterations in children during GHT. Probably, the strongest predictors of the degree of insulin resistance and the development of diabetes during GHT are represented by the individual predisposition of patients or by the presence of preexisting diabetes risk factors , 74. IntM-value.Glucose metabolism in GHD children is not exhaustively and uniformly evaluated by the available studies. Fasting glucose and insulin are the parameters most frequently evaluated, followed by Homa-IR and HbA1c levels. Very few studies used other metabolic parameters, i.e., QUICKI, ISI, and per se on glucose metabolism is quite homogeneous, with the majority of studies reporting no significant difference in metabolic parameters between GHD children and controls. Conversely, GHT proves to be more frequently associated with a subtle form of insulin resistance that can be assumed to be the mechanism by which basal glucose does not significantly change after GHT despite the increase in insulin levels.In children, the reported impact of GHD M-value during clamp, a reduction in insulin sensitivity during GHT was almost always confirmed.The increase in insulin levels and, consequently, in Homa-IR during GHT probably does not indicate with any certainty a condition of insulin resistance, probably due to the inability of basal indexes to reliably assess insulin sensitivity. Indeed, the increase in Homa-IR, as well as the reduction in QUICKI, may just represent an expected consequence of GH-induced basal hyperinsulinemia and very few studies have compared data about the degree of insulin sensitivity in GHD children assessed by different indices . HoweverOverall, despite the decrease in insulin sensitivity, both fasting glucose and HbA1c levels remain almost always within the normal range. Therefore, the hyperinsulinemia documented during GHT can probably be compared to that observed during puberty , althougFor these reasons, to date there are no clear indications on routinely monitoring GHD patients receiving GHT with regards to the risk of diabetes. The only statements from the Growth Hormone Research Society are that “fasting insulin levels are not routinely measured” and “caution should be exercised when considering the decision of continuing GHT in conditions where there is a known risk of diabetes in the transition age” . Also a The different methods to study glucose metabolism, the heterogeneity of the populations evaluated, the different doses of GH used together with the variable duration of follow-up may be responsible for discrepancy in the results. Long-term longitudinal studies having glucose homeostasis as their primary outcome are still needed in order to better clarify the real metabolic impact of GHD and GHT in children.AC and CG contributed equally to this work.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the manuscript.


Saccharomyces cerevisiae. When DSB ends are occupied by KU, a central component of the NHEJ, the Mre11-Rad50-Xrs2 (MRX) nuclease complex (MRN in human), aided by the associated factors Sae2 (CTIP in human), initiates the resection process, inducing a nick close to the DSB ends. Then, starting from the nick, the nucleases Mre11, Exo1, Dna2, in cooperation with Sgs1 helicase (BLM in human), degrade DNA strand in both the directions, creating the 3′ ssDNA filament. Multiple levels of regulation of the break processing ensure faithful DSB repair, preventing chromosome rearrangements, and genome instability. Here we review the DSB resection process and its regulation in the context of chromatin. Particularly, we focus on proteins that limit DSB resection, acting as physical barriers toward nucleases and helicases. Moreover, we also take into consideration recent evidence regarding functional interplay between DSB repair and RNA molecules nearby the break site.In all the eukaryotic cells, nucleolytic processing (resection) of a double strand DNA break (DSB) is a key step to channel the repair of the lesion toward the homologous recombination, at the expenses of the non-homologous end joining (NHEJ). The coordinated action of several nucleases and helicases generates 3′ single strand (ss) DNA, which is covered by RPA and recombination factors. Molecular details of the process have been first dissected in the model organism  DSBs are classically defined as broken chromosomes, however uncapped telomere ends and reversed forks are bound and processed by the same factors. In this review we generally focus on broken chromosomes, although proteins, and mechanisms that we mention are active on whole types of DSBs.de novo telomere addition  or homology directed recombination (HDR). Both pathways are organized in distinct steps and sub-pathways, which involve the coordination of several factors and enzymes  of the DSB ends is a critical and finely regulated step to promote HDR over NHEJ. Indeed, the resection process generates an extended 3′-end ssDNA filament, which is then covered by RPA and the recombinase Rad51, depending on the sub-pathway , and Exo1 process a DSB whose 5′ ends are accessible. Alternatively, a DSB with blocked or chemically modified ends needs the activity of the MRX (MRN in human) complex to initiate the process . Indeed,The importance of regulating the DNA ends resection in DSB repair is underlined by the increasing list of factors participating in the reaction in human cells, including the oncosuppressor BRCA1  binds DNA ends in all the cell cycle phases, acting as a platform for the association of several factors involved in NHEJ  and its yeast ortholog Rad9 are important regulators of the DSB repair pathway choice  at the DSB is influenced by DNA:RNA hybrids?Other relevant questions regard the role and mechanisms of resection barriers at stall or collapsed replication forks. Indeed, transcription, and the DNA damage response are highly influenced by the chromatin architecture changes occurring during DNA replication.Remarkably, factors involved in DSB resection are deregulated in different cancers and genome instability syndromes, being also considered promising therapy targets. Indeed, the importance of all the factors involved in establishing and/or dampening resection barriers clearly emerged by treating tumor cells, which carry mutations in the BRCA1-axis, with the PARP1 inhibitor Olaparib. Notably, inactivation of the 53BP1-dependent resection barrier dramatically reduces the effectiveness of the treatment on BRCA1 defective cells, possibly leading to genome instability, poor prognosis, and cancer relapse (Lord and Ashworth, AP conceived the idea. All authors wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.