Chromogranin A (CgA (CHGA)) is the major soluble protein co-stored and co-releasedwith catecholamines and can function as a pro-hormone by giving rise to severalbioactive peptides. This review summarizes the physiological functions, thepathogenic implications, and the recent use of these molecules as biomarkers inseveral pathological conditions. A thorough literature review of the electronichealthcare databases MEDLINE, from January 1985 to September 2013, was conducted toidentify articles and studies concerned with CgA and its processing. The searchstrategies utilized keywords such as chromogranin A, vasostatins 1 and 2,chromofungin, chromacin, pancreastatin, catestatin, WE14, chromostatin, GE25,parastatin, and serpinin and was supplemented by the screening of references fromincluded papers and review articles. A total of 209 English-language, peer-reviewedoriginal articles or reviews were examined. The analysis of the retrospectiveliterature suggested that CgA and its several bioactive fragments exert a broadspectrum of regulatory activities by influencing the endocrine, the cardiovascular,and the immune systems and by affecting the glucose or calcium homeostasis. As somepeptides exert similar effects, but others elicit opposite responses, the regulationof the CgA processing is critical to maintain homeostasis, whereas an unbalancedproduction of peptides that exert opposing effects can have a pathogenic role inseveral diseases. These clinical implications entail that CgA and its derivedpeptides are now used as diagnostic and prognostic markers or to monitor the responseto pharmacological intervention not only in endocrine tumors, but also incardiovascular, inflammatory, and neuropsychiatric diseases. They are co-stored in secretory granulesand co-released with resident peptide hormones, neurotransmitters, or amines in responseto a variety of physiological and pharmacological stimuli. Nine members of the graninfamily have been presently described: chromogranins, namely chromogranins A (CgA) and B(CgB), and secretogranins, namely SgII, CgA (CHGA) is an acidic protein with a molecular weight of 48 kDa that iscomposed of 439 amino acids and expressed by several normal or neoplastic cells of thediffuse endocrine and neuroendocrine systems or by some cancer cells that can undergoneuroendocrine differentiation. Its name is derived from the original discovery inadrenal medulla CgA gene is located on chromosome 14q32.12, spans12 192 bp, and is organized in eight exons and seven introns. The derivedtranscript of 2 kb is translated into the 457 residues CgA protein of∼48–52 kDa molecular weight that undergoes post-translational processesand proteolytic cleavages by pro-hormone convertases. The CgA cleavages generate severalbiologically active peptides: vasostatins 1 and 2, chromofungin, chromacin,pancreastatin, catestatin, WE14, chromostatin, GE25, parastatin, and serpinin. Thescheme depicted in CgA DNA sequence and the corresponding derived peptides. The5′-UTR (259 bp) of the CgA mRNA and most of the signalpeptide of CgA correspond to the exon I. The β-granin represents the highlyconserved amino-terminal domain of CgA encoded by exons II–V and few amino acidsencoded by the exon VI. It encompasses several bioactive peptides: vasostatin 1 (VST1:hCgA1–76) and vasostatin 2 (VST2:hCgA1–113) have vasorelaxant andcardiosuppressive properties. Chromofungin corresponds to the sequence Arg 250–301), the catecholaminerelease-inhibitory and antihypertensive peptide catestatin, a 20 amino acid cleavageproduct that seems to be involved in pancreatic β-cell functions chromostatin, andthe 14 amino acid peptide WE14 (hCgA324–337). Thename of this molecule is derived from the presence of tryptophan (W) at the N-terminalposition and of glutamic acid (E) at C-terminal position; it modulates histamine releasefrom rat peritoneal mast cells and acts as auto-antigen in type 1 diabetes 403–428) regulates granule biogenesis inendocrine and neuronal cells by inhibiting granule protein degradation in the Golgicomplex and exerting a protective effect against oxidative stress. Serpinin's influenceon cardiac activity has recently been reported The human Both pattern and rate of CgA processing vary in a tissue-specific manner. In adrenalmedulla and anterior pituitary gland, rate and processing are low, while CgA isprocessed faster and more extensively in the endocrine pancreas and in gastrointestinaltissues. Proteolytic processing of CgA may also occur after its release fromneuroendocrine cells.In vitroin vivo studies2+. The abundance(∼2–4 mM) of CgA inside the granules contributes to makedense-core granules the major intracellular calcium reservoir 2+within secretory granules and the Ca2+ mobilizationinto the cytoplasm, through the activation ofIP3R/Ca2+ channels that are present onthe membranes of granules . S1–76) and 2(CgA1–113) represent the N-terminalfragments of CgA and exert a large spectrum of homeostatic actions, includingvasodilation, antifungal and antimicrobial effects, modulation of cell adhesion,and inhibition of parathyroid hormone secretion. The CgA processing intovasostatin peptides occurs both at the cell membrane level and in theextracellular matrix Vasostatins 1  and requiresC-terminal amidation to be active. Released with catecholamines from thesympathetic nervous system in stress situations, pancreastatin appears to beinvolved in the modulation of energy metabolism. Moreover, it influences multiplefacets of both carbohydrate and lipid metabolism decreasing glucose uptake (by∼50%) and increasing spillover of free fatty acids (by 4.5- to 6.4-fold) Pancreastatin was the first identified CgA-derived peptide The pancreastatin region of CgA gives rise to three genetic variants, one of which(Gly297Ser) substantially increases the peptide's potency to inhibit cellularglucose uptake. These observations suggest that hereditary alterations inpancreastatin's primary structure may give rise to interindividual differences inglucose and lipid metabolism.Pancreastatin also inhibits pancreatic and gastric exocrine secretion and also theparathormone release.Catestatin consists of a 21 amino acid peptide and acts at nicotinic cholinergicreceptors as a potent autocrine inhibitor of catecholamine secretion. Targetedablation of CgA locus in a mouse model results in severe hypertension that can berescued by administration of the catestatin fragment. Moreover, patients withhypertension display increased CgA in vitro intube formation assays, as well as in vivo in the mouse corneasystem Catestatin can induce cardiovascular responses at local as well as at systemiclevels The catestatin involvement in inflammation has recently been highlighted in termsof chemotaxis and induction of pro-inflammatory cytokines 347–419) consists of ahighly conserved CgA domain, described for the first time in the porcineparathyroid. Parastatin modulates parathormone release by porcine parathyroidcells at low plasma Ca2+ through an autocrinemechanism.Parastatin NETs represent a heterogeneous family of tumors with different morphological andclinical features originating from a variety of neuroendocrine cell types distributedubiquitously throughout the body. To date, CgA level, representing a constitutiveneuroendocrine secretory protein, is the most widely accepted biomarker, beingelevated in 60–80% of patients with NETs As CgA is much more stable than catecholamines in the circulatory system, itsplasmatic levels reflect the sympathetic tone and adrenomedullary system activity,that are altered in chronic heart failure, acute coronary syndrome, and hypertension.High CgA plasma levels are strictly associated with mortality risk after myocardialinfarction or acute coronary syndrome as well as heart failure while increasedcatestatin concentrations appear to improve post-ischemic recovery by reducing themyocardial infarct size and the increment of diastolic left ventricular pressureSerum CgA has been used as an early biomarker of disease severity in patientsadmitted with systemic inflammatory response syndrome The potential utility of CgA as a biomarker in neurological disorders has been onlyrecently established. In particular, decreased CgA levels have been detected in thecerebrospinal fluid of canonical, but not late-onset type II Alzheimer's disease,patients 194–213 fragment Recently, CgA has been supposed to be a potential biomarker of multiple sclerosis ascerebrospinal fluid from these patients evidenced a significant increase inCgAHelicobacter pyloriinfection may determine increased CgA levels, as a consequence of chronic elevationin serum gastrin levels Silent atrophic gastritis and gastritis due to In organ dysfunction such as renal and liver failures, the CgA levels in serum orplasma may also be markedly increased while slightly increased concentrations of CgAhave also been observed in ulcerative colitis and Crohn's disease,hyperparathyroidism, hyperthyroidism, and during menopause (probably due to theincreased sympathetic tone) and pregnancy It has recently been reported that CgA is released from human submandibular glandsand secreted into saliva Physical activity is associated with enhanced adrenergic tone. Recent studies haveshown that high-intensity exercise significantly increases plasma and salivary CgAlevels Plasma or serum sampling is broadly used for the laboratory determination of CgA in awide variety of endocrine and NETs. However, recent studies have analyzed the hypothesisthat detection of salivary CgA level may have a higher analytical and diagnosticperformance, as salivary sampling is non-invasive, rapid, and, different from thecirculating form, CgA in saliva is not bound to other proteins. Even though only fewpapers are available on this topic, data appear to suggest that, in physiologicalconditions, circulating and salivary CgA have different routes of secretion: indeed,salivary CgA peaks upon awakening and then quickly decreases to nadir after 1 h and ismaintained at a low level throughout the day, whereas plasma CgA did not show anycircadian rhythm in vivo administration have been registered to date. Experimentsperformed in genetically modified mice evidenced that catestatin inhibited thenicotine-induced catecholamine secretion, whereas its i.v. administration in ratsreduced pressure responses to the sympathetic activation and evoked a potentvasodilation The pleiotropic effects and the pathophysiological implications of CgA and its derivedpeptides seem to suggest that these molecules bear all the potentials to be therapeuticagents for several diseases. Nevertheless, no clinical trials on the effects of theirThis review summarizes the knowledge about CgA and functions of its cleavage productsemphasizing their importance in physiological and pathological conditions. It is worthnoting that some of the CgA-derived peptides can exert opposing effects, and therefore,the regulation of the CgA processing to generate diverse molecules under differentphysiological conditions is critical in order to counterbalance the effects and tomaintain homeostasis. The potential use of CgA as a pharmacological agent needs to beinvestigated to fill the current knowledge gap.Finally, the application of salivary samples could substitute CgA detection in plasma,for clinical purpose.The authors declare that there is no conflict of interest that could be perceived asprejudicing the impartiality of the research reported.This paper was supported by PRIN 2010–11 funds of the Italian Ministry ofEducation, University and Research.


Under the impression of the beauty of the frozen local rivers Saint-François and Magog, the researchers presented and discussed their recent data on the pivotal roles of mitochondria in various diseases, including neurodegeneration, cancer, schizophrenia, respiratory chain, and inflammatory disorders. In this way, the participants and attendees of the symposium got a broad overview of common and distinct mechanisms of mitochondrial (dys)functions underlying various diseases.On March 11Heidi McBride  was the first keynote speaker of the symposium. She presented mitochondria as very dynamic organelles, which tightly interact with an extended network of intracellular organelles, including the ER, lysosomes, early and late endosomes, and peroxisomes. Notably, mitochondria are able to bud vesicles. These mitochondrial-derived vesicles (MDVs) play important roles in maintaining mitochondrial quality control, and potentially contribute to peroxisomal function .One type of MDVs is targeted to the lysosomes. Here, MDVs are stimulated upon oxidative stress, and this machinery runs in parallel to mitophagy. MDVs targeted to lysosomes and mitophagy share some components, including the mitochondrial kinase Pink1 and the E3 ligase Parkin, which both are upon mutation causative for Parkinson’s disease de novo from the ER, as evidenced by the first appearance of the peroxisomal membrane protein Pex3 in the ER before reaching the peroxisomes. In contrast, in mammalian cells the default localization of Pex3 appears to be the mitochondrial outer membrane, suggesting that mitochondria contribute to the formation of peroxisomes. Previously, Heidi McBride resolved the cellular machinery of MDV formation towards the peroxisomes, which depends on the mitochondrial-anchored protein ligase MAPL In yeast cells peroxisomes are formed Denis Gris  analyzes the role of proteins involved in the innate immune response on the survival of neurons. He is working on members of the nucleotide-binding domain leucine-rich repeat-containing protein (NLR) family, which act as molecular switches that redirect multiple signaling pathways. He presented his work on NLRX1, the only member of the NLR family with mitochondrial localization sequence . Knock-down of NLRX1 in neuronal cells triggers necrotic cell death Ralf Braun . He demonstrated that accumulation of mutant ubiquitin impairs the ubiquitin-proteasome system (UPS), leads to pivotal mitochondrial impairment, culminating in apoptotic and necrotic cell death Data obtained from a yeast model expressing Alzheimer’s disease-associated mutant ubiquitin were presented from Julie St-Pierre demonstrated that mitochondrial metabolism is altered during the metabolicadaptations in cancer cells. She established unique methods enabling the metaboliteprofiling of cancer and control cells, including the targeted profile of allintermediates in the citric acid cycle Deregulation of cellular energetics is a hallmark of cancer. The second keynote speaker of thesymposium, PGC-1α regulates the metabolism of multiple tissues by promoting mitochondrial biogenesis, respiration, and reactive oxygen species detoxifying capacity. Several reports have shown that the expression of PGC-1α is decreased in breast cancer patients compared with normal tissues. However, PGC-1α expression is highest in HER2+ and triple negative breast cancers, subtypes that have the poorest prognosis Next, Julie St-Pierre investigated the metabolic target of metformin. Despite its common usage, the target of metformin has remained controversial. Using respirometry and metabolite profiling, she showed that cancer cells treated with metformin demonstrate increased glycolysis and impaired respiratory activities Verónica Dumit  presented her data using leaf extracts prepared from the aloe plant to trigger cell death in cancer cells but not in healthy control fibroblasts. She applied a quantitative proteomic analysis using stable isotope labeling in cell culture (SILAC) to determine protein alterations in cancer and healthy cells upon aloe extract treatment, and observed the downregulation of proteins involved in DNA replication and mitochondrial energy metabolism. Treatment with emodin, an anthraquinone component of aloe, resulted in similar results as compared to total aloe extracts. It leads to the downregulation of mitochondrial complex I subunits in cancer cells, and triggers mitochondrial fragmentation and ballooning . Emodin shifts isolated yeast mitochondria towards uncoupled respiration, affects the mitochondrial membrane potential, which then leads to impaired import of proteins into the mitochondrial matrix. Yeast cells adapted to fermentation are much more vulnerable to emodin treatment than yeast cells with high respiratory capacity. This effect might be comparable to cancer cells which prefer fermentation in contrast to control cells which demonstrate higher levels of respiratory activities. Both fermenting yeast and cancer cells can be protected from the detrimental effects of emodin by using the antioxidant N-acetyl-L-cysteine (NAC), demonstrating an important role of ROS in the execution of cell death. In summary, emodin triggers death of cells with an inefficient mitochondrial respiratory chain, such as cancer cells.Cécile Monpays  determined the mitochondrial activities in a mice juvenile two-hit model of schizophrenia. Schizophrenia is a chronic mental illness which affects 1% of general population and is characterized by different clinical symptoms with three core features, positive , negative (e.g. lack of motivation) and cognitive (e.g. attention deficit symptoms). These clinical symptoms are related to different neurochemical perturbations, among which are oxidative stress and energetic status disturbances. In schizophrenia, the imbalance between production of reactive oxygen species and activity of antioxidant enzymes points towards mitochondrial dysfunction. The colleagues of Cécile Monpays have developed a juvenile murine two-hit model of schizophrenia based on the combination of two environmental risk factors  to get a better understanding of the disease onset Paula Waters  gave an overview of clinical, biochemical and genetic aspects of mitochondrial RC disorders, dividing these into three broad classes  and presenting selected case histories illustrative of each.Defects in the structural and functional integrity of the mitochondrial respiratory chain (RC) can lead to significant mitochondrial disorders, with a wide variety of phenotypes. SURF1 gene which encodes the protein surfeit locus protein 1 (Surf1). This protein localizes to the mitochondrial inner membrane and is involved in the biogenesis of the cytochrome c oxidase complex (RC complex IV).The first class involves mutations in nuclear genes encoding protein subunits of the mitochondrial RC or encoding accessory proteins essential for assembly and stability of RC complexes. She presented the history of a family with children suffering from Leigh syndrome, caused by mutations in the POLG1. This gene encodes a subunit of the mitochondrial DNA polymerase γ, an enzyme with a pivotal role in mtDNA replication. The mutations affected mtDNA integrity, thereby impairing mitochondrial RC activities.The second class of mitochondrial RC disorders involves mutations in the nuclear genome which, in turn, affect the mitochondrial genome (mtDNA). Dr. Waters presented the history of an infant with Alpers syndrome, due to mutations in the nuclear gene The third class of mitochondrial RC disorders involves mutations in the mitochondrial genome. The example presented was the history of a child with NARP  caused by the m.8993T>C mutation, affecting a subunit of the mitochondrial ATPase complex (RC complex V). Complex issues facing families with pathogenic mtDNA mutations were highlighted. Mutations in the mtDNA demonstrate matrilineal transmission (mitochondria are only transmitted by the mother and not by the father) and therefore do not follow Mendelian inheritance. Heteroplasmy, i.e. a mixed population of mitochondria with wild-type and mutated mtDNA, can lead to incomplete penetrance, variable expressivity, and pleiotropy in this class of mtDNA disorders. Novel assisted reproductive technologies, designed to uncouple the inheritance of nuclear and mtDNA and thus to minimize the risk of transmission of mtDNA disorders, have recently been developed. These techniques, their potential clinical applications and wider implications, are currently the subject of active public discussion in many countries 2+ ions via plasma membrane channels. Daniel Serrano  presented data demonstrating that this is due to the inability of mitochondria in GIMAP5-deficient T-cells to sequester Ca2+2+ sequestration. GIMAP5 increases the capacity of mitochondria for Ca2+, highlighting the tight interconnection among the microtubule cytoskeleton and mitochondria in promoting survival of naïve T cells.The GTPase of the immune-associated nucleotide-binding protein 5 (GIMAP5) plays a pro-survival function in T-lymphocytes. Deletion of GIMAP5 results in spontaneous apoptosis in mature T-lymphocytes in rats, and impairs the entry of CaXavier Roucou  generated proteome databases including both the reference and the predicted alternative ORFs for different species, including humans and yeast. Based on these extended databases, he was able to identify numerous hitherto undetectable and unknown small proteins encoded by AltORFs Mature mRNA contains unconventional open reading frames (AltORFs) located in the untranslated regions or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames Ian Bates (Carl Zeiss Canada) briefly introduced super-resolution fluorescence microscopy. He reviewed existing super-resolution methods and discussed the advantages and disadvantages of existing techniques based on the particular scientific question. In particular live cell imaging is a challenge for super resolution imaging, since traditionally images either take a significant amount of time to acquire and/or require high laser powers, both conditions are not conducive to live cell imaging. Techniques which can both improve sensitivity and resolution are the ideal solution for maintaining cell viability yet at the same time reveal new biological information about dynamic processes seen in mitochondrial research in particular. This is the new frontier in super-resolution microscopy, and the power of these methods will bring new insights into the role mitochondria plays in various human disorders.2+ homeostasis. Therefore, it is hardly surprising that mitochondrial (dys)functions play important roles in various human disorders. In this symposium, the role of mitochondria was described in human patients as well as in mammalian cell culture, yeast, and transgenic mouse models for different diseases. It was fascinating to see how basic research resulted in the identification of mitochondrial-derived vesicular transport processes, which turned out to be relevant for neurodegeneration. Alterations in mitochondrion-localized metabolic processes and the activities of distinct complexes of the respiratory chain were discovered to be important in both cancer and neurodegeneration. Structural alteration of mitochondria and the switch of apoptosis to necrosis is relevant for inflammatory and neurodegenerative processes. In our next symposium, which will take place in Germany in 2016, we will continue our successful concept of bringing mitochondrial researchers together doing basic or clinical research, or working on diverse human disorders in different model systems. We trust that this was and will be a fruitful approach to think about common and distinct mechanisms of mitochondria in different pathophysiological conditions.Mitochondria are fascinating highly dynamic organelles which play important roles in many cellular processes, including ATP production via respiratory chain activities, amino acid metabolism, and Ca


However, the mechanisms that modulate hypoxia-induced RBC membrane fragility remain unclear. Here, we provide an overview of the role of RBC ATP release in the regulation of vascular tone and prevailing assumptions on the putative release mechanisms. We show importance of intravascular hemolysis as a source of ATP for local purinergic regulation of blood flow and discuss processes that regulate membrane propensity to rupture under stress and hypoxia.Intravascular hemolysis occurs in hereditary, acquired, and iatrogenic hemolytic conditions but it could be also a normal physiological process contributing to intercellular signaling. New evidence suggests that intravascular hemolysis and the associated release of adenosine triphosphate (ATP) may be an important mechanism for  For example, changes in partial oxygen tension  or large (20 μm) size they assessed effect of oxygenation on erythrocyte flow velocity and shear-induced deformability, respectively. These experiments revealed that O2 depletion increased the velocity of erythrocyte flowing through the microfluidic channel due to increased RBC membrane deformability. Viewed collectively, these data demonstrate that in addition to the increment of vessel diameter (see below), elevation of blood flow in microcirculatory beds under hypoxic conditions might be achieved via PO2-dependent regulation of erythrocyte deformability as a key determinant of blood viscosity , hypoxia affects vascular tone via release of ATP from RBCs that, in turn, leads to activation of P2Y receptors on endothelial cells, stimulation of NO production and NO-mediated vasodilation .Piezo 1 is a mechanosensitive non-selective cation channel expressed on RBC membrane. Gain-of-function mutations in Piezo 1 were linked to dehydrated hereditary stomatocytosis , and translocation of phosphatidylserine (PS) to the cell surface. The surface membrane content of sialoglycoproteins and sialic acids which accounts for majority of the negative surface charge of RBC membrane is also reduced in aged RBC contributing to altered RBC membrane mechanical and electrical properties, receptor-mediated cellular interactions, immune responses and survival  via activation of scramblase and inhibition of flipase. These [Ca2+]i-dependent events results in a collapse of membrane phospholipid asymmetry and cytoskeleton detachment  with downstream intermediates of intracellular signaling might be considered as a mechanism of triggering PO2-dependent erythrocyte responses. Indeed, in cell-free experiments it was shown that hemoglobin binds to the cytoplasmic domain of band 3 (cdb3)  with plenty of ATP for local purinergic signaling. If conductive release pathway would be involved what could be the mechanism allowing selective permeation of ATP but preventing massive influx of Ca2+ and Na+? Finally, much remains to be learned about the downstream intermediates involved in hypoxia-induced membrane fragility, membrane microdefects, lytic pore formation and associated ATP release.Several issues remain unsettled and require further investigations. Among them the question remains if besides hemolysis as the primary release mechanisms, are there conditions or stimuli that would induce regulated non-lytic ATP release from RBC and what pathway it could involve? What might be the contribution of young immature erythrocytes (reticulocytes) to such release? With their residual intracellular structures could they release ATP via exocytosis? While modest in relative terms (0.5–2.5%) these cells constitute a significant cellular pool (~4.5 × 10All authors listed have made a substantial, direct and intellectual contribution 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.


These cells have been demonstrated to be able to self-renew and undergo multipotent differentiation to give rise to all hormonal lineages of the anterior pituitary. Furthermore, evidence exists for their contribution to regeneration of the organ and plastic responses to changing physiological demand. Recently, stem-like cells have been isolated from pituitary neoplasms raising the possibility that a cytological hierarchy exists, in keeping with the cancer stem cell paradigm. In this manuscript, we review the evidence for the existence of pituitary stem cells, their role in maintaining organ homeostasis and the regulation of their differentiation. Furthermore, we explore the emerging concept of stem cells in pituitary tumours and their potential roles in these diseases.Evidence for the presence of pituitary gland stem cells has been provided over the last decade using a combination of approaches including  Pomc1) in corticotrophs , intermediate (IL) and posterior lobes (PL), with the former two being derived from Rathke’s pouch, an invagination of the oral ectoderm and the latter being a derivative of the overlying diencephalic neural ectoderm. The AL contains five distinct hormone-producing cell types: somatotrophs, thyrotrophs, lactotrophs, corticotrophs and gonadotrophs, which secrete growth hormone (GH), thyroid-stimulating hormone (TSH), prolactin (PRL), adrenocorticotropin (ACTH) and gonadotropins (FSH and LH), respectively , Tpit, wcotrophs . InteresThe pituitary possesses a remarkable capacity for plasticity in regulating the number and proportion of secretory cell types in response to physiological changes, such as puberty and pregnancy  and dispersed throughout the AL parenchyme. Work by Lepore in 2005 demonstrated that the culture of dissociated pituitary tissue in stem cell-promoting media resulted in the generation of adherent colonies, which express S100β and have the capacity to differentiate into hormone-producing cells  SOX2+ cells differentiated into all hormone-producing lineages. In addition, a proportion of traced cells express SOX2 and SOX9, but not any commitment or differentiation markers, suggesting self-renewal of the initially labelled SOX2+ cells  were rendered susceptible to diphtheria toxin-mediated ablation, revealed that the elimination of this lineage led to expansion of the SOX2+ stem/progenitor compartment, with increased numbers of double SOX2+/GH+ cells . Both stin vivo due to the effect of oestradiol and could differentiate to a corticotroph fate following acute adrenalectomy. Interestingly, the authors found that traced cells had a predisposition to becoming LH+ gonadotrophs and lactotrophs (PRL+), with a small number differentiating into somatotrophs (GH+) (As previously mentioned lineage tracing of putative PSCs has been performed by both our own (SOX2+ cell lineage tracing) and Lovell-Badge’s group (SOX2+ or SOX9+ cell lineage tracing)  has only recently emerged , these tPAs represent a varied group of tumours, can be familial or sporadic, and are classified according to their endocrinological activity , clinical manifestation, driver mutations and histological features  stem cell marker expression , (3) multipotency, (4) resistance to chemotherapeutic agents, (5) ability to initiate de novo tumours when transplanted into immunocompromised hosts  tumours  is another type of benign yet clinically aggressive pituitary tumour of Rathke’s pouch origin /+Hesx1 mice /+Sox2 mice   and by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children National Health Service Foundation Trust and University College London. S H holds a Wellcome Trust PhD Fellowship. J P M-B is a Great Ormond Street Children Hospital Charity Principal Investigator.All authors contributed equally to the writing of the manuscript. S H and S M generated the figures.


Alterations of mitochondrial DNA copy number (mtDNAcn) in the blood  appear associated with several systemic diseases, including primary mitochondrial disorders, carcinogenesis, and hematologic diseases. Measuring mtDNAcn in DNA extracted from whole blood (WB) instead of from peripheral blood mononuclear cells or buffy coat may yield different results due to mitochondrial DNA present in platelets. The aim of this work is to quantify the contribution of platelets to mtDNAcn in whole blood [mtDNAcn(WB)] and to propose a correction formula to estimate leukocytes' mtDNAcn [mtDNAcn(L)] from mtDNAcn(WB). Blood samples from 10 healthy adults were combined with platelet-enriched plasma and saline solution to produce artificial blood preparations. Aliquots of each sample were combined with five different platelet concentrations. In 46 of these blood preparations, mtDNAcn was measured by qPCR. MtDNAcn(WB) increased 1.07  per 1000 platelets present in the preparation. We proved that leukocyte count should also be taken into account as mtDNAcn(WB) was inversely associated with leukocyte count; it increased 1.10  per unit increase of the ratio between platelet and leukocyte counts. If hematological measurements are available, subtracting 1.10 the platelets/leukocyte ratio from mtDNAcn(WB) may serve as an estimation for mtDNAcn(L). Both platelet and leukocyte counts in the sample are important sources of variation if comparing mtDNAcn among groups of patients when mtDNAcn is measured in DNA extracted from whole blood. Not taking the platelet/leukocyte ratio into account in whole blood measurements, may lead to overestimation and misclassification if interpreted as leukocytes' mtDNAcn. Mitochondria are intracellular organelles involved in energy production through the process of oxidative phosphorylation (OXPHOS) that have their own genome (mtDNA), distinct from that in the cell nucleus (nDNA). Although the mitochondrial mass per cell varies with cell type and metabolic state, each cell type typically contains a fairly constant amount of mitochondria and accordingly, the number of copies of the mitochondrial genome is also constant, as it is linked to mitochondrial mass. Each miEither reduction or increase in the biogenesis or availability of mitochondria in the cells may be markers of primary mitochondrial pathology or of systemic pathology that affects mitochondrial biology. Although mtDNAcn can be studied in any tissue, blood is one of the most commonly used, as samples can be easily obtained. Alterations of mtDNAcn in the blood may be associated to primary mitochondrial disorders,11, whicFor the study of mtDNAcn, DNA is usually obtained from peripheral blood mononuclear cells (PBMC). In such samples, platelet contamination may lead to overestimation of mtDNAcn measurements since onThe aims of this work are to describe the effect of platelet count in mtDNAcn in whole blood, to evaluate the extent of mtDNAcn overestimation and misclassification on real epidemiological data, and to propose a correction formula to estimate leukocytes’ mtDNAcn [mtDNAcn(L)] from whole blood mtDNAcn [mtDNAcn(WB)], if hematological measurements of the original sample are available.MtDNAcn was measured in several artificial blood preparations that were experimental combinations of blood samples with platelet-enriched plasma and saline solution so that each sample was combined with five different platelet concentrations. The coefficients that relate hematological parameters with mtDNAcn, according to a theoretical formulation framework, were estimated statistically. Afterwards, mtDNAcn data from an observational study was used to describe the differences in value and subject classification between raw mtDNAcn measurements in whole blood and values corrected to approximate mtDNAcn(L) using the previously estimated coefficients.Fasting whole blood samples from 10 randomly selected (8 men and 2 women) participants of the Aragon Workers’ Health Study whose coAfter measuring blood cells count, each participant’s sample was processed to obtain 5 derived artificial blood preparations, so that each one contained a different proportion of leukocytes and platelets. DNA was subsequently extracted from the original samples and the derived preparations, and mtDNAcn was measured in the extracted DNA.For each participant, six 50 μl aliquots of whole blood were created and reserved. From the rest of the sampled blood, platelets were isolated to obtain platelet-enriched plasma. In brief, blood was centrifuged at 100 x g for 15 min at room temperature. The upper layer was transferred into a new tube and was centrifuged once more at 200 x g for 10 min at room temperature. This supernatant, containing the platelets , was saved to be combined in different proportions with saline solution  and it wBlood samples and preparations were analyzed in a COULTER ACT 5diff analyzer (Beckman Coulter) to determine platelet and leukocyte counts. Internal quality controls of three levels were measured before the daily work. An external quality control from the Spanish Association of Pharmaceutical Analysts (AEFA) was processed every 4-months.For the experimental part, DNA was extracted manually from the blood samples and blood preparations with phenol-chloroform, according to the protocol described by Marcuello et al.. ConcentMT-CO2), and one for the nuclear gene for the A subunit of succinate dehydrogenase (SDHA), producing amplicons of 69 and 72 bp respectively. Each reaction contained 5 ng of DNA and 400 nM of each primer. Primer sequences and positions are indicated in Table A in Quantification of mtDNAcn relied on quantitative real-time polymerase chain reactions (qPCR) based on SYBR Green assays and performed in an ABI PRISM®7900 Real-Time PCR System (Applied BioSystems). In a single 384-well reaction plate, two sets of 10 μl reactions were performed in separate reaction wells, one for the mitochondrial cytochrome oxidase II gene (MT-CO2 to that of SDHA (mtDNA copy number—mtDNAcn) was used as a measurement of half of the number of copies of mtDNA in leukocytes. We use the term mtDNAcn(WB) to refer to the direct (uncorrected) measurements performed in whole blood samples or blood preparations in contrast to mtDNAcn(L) which would be obtained from measuring in leukocytes or derived with our correction formula (see below).The standard curve, which contained an equal number of copies for both amplicons (see below), was used as a reference for absolute quantification of each gene using the threshold cycle number (Cq) method. The thresholds were obtained by the default second derivate method on ABI PRISM . The ratio of the absolute quantity of MT-CO2 and one copy of the amplicon for SDHA[MT-CO2 gene (Table A in A PCR standard was created by ligating one copy of the amplicon for  for SDHA. A longenmtDNA) is the sum of the number of copies of mtDNA per leukocyte (mtDNAL) multiplied by the count of leukocytes (nL) plus the number of copies of mtDNA per platelet (mtDNAP) multiplied by the count of platelets (nP).In whole blood, the number of copies of mtDNA (nnDNA) is the number of copies of nDNA per leukocyte (nDNAL), assuming 2 in diploid cells (nDNAL = 2), multiplied by the count of leukocytes (nL).Similarly, the number of copies of nDNA (mtDNAcn) is calculated by dividing the number of copies of mtDNA (nmtDNA) by the number of copies of nDNA (nnDNA). Given that,The ratio of mtDNA  appropriately, we obtain:Replacing mtDNAcn(WB) with respect to mtDNAcn(L). We estimated the factor (K) by means of a statistical model:The term 0 will provide an estimation for mtDNAcn(L), assumed constant for our samples, and estimating β1 will provide an estimation for in which estimating βmtDNAcn(WB) is:Thus, the formula that we propose for correcting Data are presented as mean and standard deviation (SD). All statistical analyses were performed with the statistical package R (version 3.0.2). The coeWe calculated mtDNAcn(L) from mtDNAcn(WB) for the AWHS participants using our correction formula. We described the mean, standard deviation and percentiles, as well as cross-classification of participants in quintiles of both values. We report the percentage of participants that were classified in a different quintile by each value.The hematological values of the primary samples used for the experiment were within normal limits . In them3 platelets and it also showed an inverse association with leukocyte count (In the blood preparations supplemented with different amounts of platelet-enriched plasma (46 data points), mtDNAcn(WB) increased 1.01  per μl of platelet-enriched plasma added . The mtDte count .We built multivariate regression models to evaluate the influence of the platelets and leukocytes on mtDNAcn(WB), and we found a significant interaction between platelets and 1/leukocytes that was the only statistically significant term in that model for mtDNAcn(WB), consistIn the AWHS study (n = 3389) mtDNAcn(WB) was 68.25 (SD: 15.37) being higher in women than men and the ratio platelets/leukocytes, 33.08 (SD: 9.94) . After aIn our study, mtDNAcn was measured in whole blood as the ratio between mtDNA and nDNA using quantitative real-time polymerase chain reaction (qPCR) to study the influence of platelet count on mtDNAcn. We compared five sets of artificial blood preparations with different platelet concentrations and observed that the mtDNAcn did not only depend on platelet count but also on the platelets/leukocytes ratio.We found a significant influence of the number of platelets on mtDNAcn quantification. Similarly, Banas et al. and AndrThe main step forward in our study with respect to previous approaches,24,36,37Knez et al. measured mtDNAcn in buffy coat in a sample of Flemish population to describe correlations with clinical variables. ConsistMtDNAcn has been analyzed in whole blood in several clinical situations: chronic diseases such as breast cancer, diabetes mellitus or Huntington´s disease were associated with mtDNA depletion,19,30, wIn addition to this formula that allows correction for platelet presence in the original sample, there are several aspects that still need to be standardized in the technique of mtDNAcn measurement. Currently, methods for mtDNAcn quantification vary across laboratories and there are several pre-analytical and analytical factors that can affect the final measurement: sample conservation before DNA extraction, time between blood drawing and cell separation, anticoagulant used, DNA extraction method, PCR inhibitors, and DNA quantification method. Until sThe experimental design allowed comparing several platelet concentrations in preparations derived from blood from the same subject effectively factoring out biological variability to a great extent. Thus, most of the variation observed could be considered technical variation. Therefore, the effective sample size is 46, with data for 5 experimental conditions with 10 replicas (preparations) for almost each condition. These results are a starting point for further expanding this knowledge that will require measuring also natural variation in the platelets and leukocytes counts and mtDNAcn. Those measurements will need the evaluation of a larger sample, as well as double measurements of mtDNAcn in both leukocytes and/or PBMCs and whole blood. This work also has some limitations: Our formula estimates mtDNAcn in blood leukocytes as a whole, which include PBMCs and granulocytes and because of that the estimations cannot be interpreted as a direct replacement of PBMCs’ mtCNDcn. We did not do the parallel extraction of leukocytes and/or PBMCs, which would have provided more details in this regard. Also, DNA extraction was accomplished from the preparations, which were not regular blood, but 1/9 dilutions , but thiIn conclusion, both platelet and leukocyte counts from the sample are important for comparing the mtDNA content between different patients when mtDNA copy number is estimated from whole blood. Not taking the platelet/leukocyte ratio into account may lead to overestimation of leukocyte mtDNA content and misclassification, which may obscure the meaning of its variation as the non-corrected value depends on hematological differences to a non-ignorable extent. We propose a correction formula that may be used to mitigate this problem. This formula's coefficients are not final and future research will help in refining and validating them.S1 FileTable A, qPCR primers sequences. Table B, Multilevel mixed-effect models for mtDNAcn(WB) measured in blood preparations (n = 46). MODEL 1: Common slope and random intercept (per sample) MODEL 2: Correlated random intercept and slope (per sample) MODEL 3: Uncorrelated random intercept and slope (per sample) mtDNAcn: mitochondrial DNA copy number, WB: whole blood. Table C, Reclassification of AWHS cohort study subjects (n = 3389) between mtDNAcn(WB) quintiles and mtDNAcn(L) quintiles.(PDF)Click here for additional data file.


Food products containing non-nutritive sweeteners (NNSs) instead of sugar have become increasingly popular in the last decades. Their appeal is obviously related to their calorie-free sweet taste. However, with the dramatic increase in their consumption, it is reasonable and timely to evaluate their potential health benefits and, more importantly, potential adverse effects. The main aim of this scoping review was to map the evidence about health outcomes possibly associated with regular NNS consumption by examining the extent, range, and nature of research activity in this area.We systematically searched Ovid MEDLINE, EMBASE and the Cochrane CENTRAL databases for studies on NNSs  using text terms with appropriate truncation and relevant indexing terms. All human studies investigating any health outcomes of a NNS intervention or exposure were eligible for inclusion. No studies were excluded based on language, study design or methodological quality. Data for each health outcome were summarized in tabular form and were discussed narratively.Finally, we included 372 studies in our scoping review, comprising 15 systematic reviews, 155 randomized controlled trials (RCTs), 23 non-randomized controlled trials, 57 cohort studies, 52 case-control studies, 28 cross sectional studies and 42 case series/case reports.In healthy subjects, appetite and short term food intake, risk of cancer, risk of diabetes, risk of dental caries, weight gain and risk of obesity are the most investigated health outcomes. Overall there is no conclusive evidence for beneficial and harmful effects on those outcomes. Numerous health outcomes including headaches, depression, behavioral and cognitive effects, neurological effects, risk of preterm delivery, cardiovascular effects or risk of chronic kidney disease were investigated in fewer studies and further research is needed. In subjects with diabetes and hypertension, the evidence regarding health outcomes of NNS use is also inconsistent.This scoping review identifies the needs for future research to address the numerous evidence gaps related to health effects of NNSs use.It also specifies the research questions and areas where a systematic review with meta-analyses is required for the proper evaluation of health outcomes associated to regular NNSs consumption. In the last decades, growing concerns about health and quality of life have encouraged people to avoid the consumption of food rich in sugar, salt or fat , 2. WithStevia rebaudiana-based products. Steviol glycosides, extracted from the plant Stevia include stevioside and rebaudioside A, but also other, less common glycosides  and [human studies]. Electronic searches were limited neither in time nor in language. Electronic searches were followed by hand searching of reference lists of relevant review articles and included primary studies. Electronic searches were updated in May Week 4 2017.Ovid MEDLINE (Titles and abstracts were screened for inclusion by a single reviewer (SL). Only clearly irrelevant records were excluded at this stage. All potentially relevant abstracts and full papers were screened for inclusion by two reviewers independently using an inclusion/exclusion form specifically developed for the purpose of this scoping review (SL and IT). In case of disagreement, the subject was discussed among the two reviewers until a mutual decision could be made. When this was not possible, a third reviewer (JM) was consulted. A data extraction sheet was designed and piloted. Then two reviewers (SL and IT) independently extracted the following data for each included primary study: 1) first author; 2) year of publication; 3) study location; 4) study design; 5) aim of the study; 6) main characteristics and size of the study sample; 7) main characteristics of intervention/exposure and control; 8) outcome measures with direction of effect.Intervention studies were classified as RCTs  or non-randomized controlled trials (non-RCTs), while observational studies were classified as prospective or retrospective cohort studies, cross-sectional studies, case-control studies, ecological studies or case reports/case series. Data sheets were compared and in case of differences in the extracted data, the relevant information was checked again in the study article and corrected.Data for each health outcome were summarized in tabular form and were discussed narratively. Bubble charts were used to highlight the main relationship among the types of NNS used in the studies as intervention/exposure, the health effects and the study types. Bubble charts are multi-variable graphs, whose plot points along a grid where the X and Y axis are separate variables .Additionally, the different colours of the plotted points represent a third variable (in our case they show the study type).For each included systematic review following data were extracted: 1) first author; 2) year of publication; 3) date of search; 4) databases searched 5) aim of the review; 6) study design of eligible studies; 7) main characteristics of eligible intervention/exposure; 8) outcome(s) eligible for inclusion.The flow diagram of the literature search (PRISMA Flow Diagram adapted for the scoping review process) is shown in Fig. In total, 24 systematic reviews  in 47 studies, while 4 studies investigated exposure to NNCSs. Cancer outcomes by type of exposure as investigated in primary studies are shown in Fig. Out of the identified 41 case-control studies reporting on the effect of NNSs on cancer, 32 assessed the relationship between NNS consumption and the risk of developing bladder cancer or urinary tract cancer. The results of these studies are controversial: 11 case-control studies describe a positive association between AS/NNS intake and bladder or urinary tract cancer risk –96, whilTwo case-control studies assessed the risk of brain cancer , 1Three prospective cohort studies investigated the risk of lymphomas or other hematological malignancies , 128, 1 The cross-sectional study described that breast cancer survivors compared to age-matched controls had significantly lower intakes of NNSs .In a systematic review by Cheungpasitporn et al. , the 4 iThe primary studies we found on the association of NNS consumption and the risk of developing chronic kidney disease were 3 prospective cohort studies (describing no association –138), 1 We found 16 intervention studies , 1 retrospective cohort study (with AS exposure) and 1 case-control study (with AS exposure) on the risk of developing diabetes. These studies are summarized in Table Among the studies investigating the exposure to AS, 2 prospective cohort studies , 163 andWe found 3 RCTs –169 withRCTs assessing the behavior and mood of essentially healthy children after they were given a preload of either an artificially sweetened or sugar-sweetened food or beverage found no consistent effect of ASs on behavior. Most of the interventional and observational studies investigating the effect of an AS preload on cognitive abilities in healthy children and adults demonstrated that there was no association between cognitive performance, measured by an array of tests, and the intake of ASs in different forms.Three studies (2 RCTs and 1 cohort study) investigated the effect of AS on depression and described an increased risk of developing depression symptoms or increased severity of symptoms in mood disorder patients –174. In Three cohort studies investigated the effect of AS consumption and preterm delivery –178, twoWe found 4 systematic reviews addressing the question whether NNS consumption has an unfavorable or favorable effect on body weight , 180–182A systematic review  focusingAnother systematic review  focusingWe found 31 interventional studies  control intervention, 1 describing an increase, while in 6 AS intervention studies no differences were observed between the two groups. There were 3 RCTs with a NNCS (stevia) intervention , 242. NoOf the 17 prospective cohort studies, 10 described a positive association  between NNS or diet beverage consumption and weight gain/increased BMI , 239, 3 Of the 17 cross-sectional studies, 12 described a positive –233, 241There are two main disease groups with a relatively wide literature of NNS intervention studies. In type-1 and type-2 diabetes patients, the effects of NNS use on diabetic control, including, but not limited to, blood glucose levels, postprandial blood glucose, and glycated hemoglobin (HbA1c), are widely investigated. We found 21 interventional studies , are required.Intervention studies on weight change focused mainly on the question whether NNS can be efficiently used in weight management. As part of weight loss intervention programs, more intervention studies would be required, to investigate the effects of NNS alone on body weight in both overweight, obese and normal-weight subjects. This would be especially important, since it is very difficult in observational studies to evaluate causality between NNS consumption and BMI/weight change and therefore results of these studies have to be interpreted with caution. A positive association between NNS consumption and weight gain in observational studies may be the consequence of and not the reason for overweight and obesity. Moreover, other factors, such as population characteristics, may influence the results of observational studies.In subjects with diabetes, the effects of NNS were investigated mainly on glycemic control. Because of the heterogeneous, if not contradictory results, a thorough analysis of these findings in a full-fledged systematic review including meta-analyses, subgroup and sensitivity analyses is needed and might help to resolve some of the ongoing uncertainties. Further studies on long-term patient-relevant outcomes in diabetes are required.The effect of NNS on lowering blood pressure in hypertensive patients should also be analyzed in a high quality systematic review and meta-analysis.Regarding NNCS, although Stevia is increasingly used as a sweetener, the number of studies on its health effects is limited as of now. Studies investigating the effects of NNCS on cancer or diabetes risk are completely lacking, while there are only few studies on weight gain and obesity risk. Clearly, there is a need for further research.Eligible NNS not addressed by any of the included primary studies were: neotame, alitame, neohesperidin DC, thaumatin and brazzein.The strength of our scoping review is its inclusion of all types of primary studies and systematic reviews which investigate any health effect of any NNS in any population. We are therefore able to present a comprehensive overview of the available scientific evidence on health effects of NNS.Our scoping review might be limited by the following factors. Firstly, the literature search was conducted in three major and comprehensive databases, but we might have inadvertently missed relevant studies listed in other databases. Secondly, the title abstract screening was conducted by one reviewer who might have inadvertently excluded relevant studies at the first stage of the screening. This limitation might be evened out by conducting the literature search in two steps. In the second step, relevant references for both topics were identified and the chances for including all relevant references in our review were increased.Detailed assessment of the study quality is not covered in a scoping review and was not conducted in the context of our scoping review. Therefore, information gathered on the health outcome includes only its direction of effect but no information on the internal or external validity of the study results.In our scoping review we found a large number of studies of different designs, investigating effects of different types of NNS in different populations on a variety of health outcomes.Systematic reviews to summarize the available evidence are already available Table . HoweverThere are systematic reviews, which also included key words for “diet soda” and “diet beverage” in their search strategy. There are several, primarily observational studies, where the exposure is defined as “diet”, which may indicate NNS-containing beverages, but further details are often not provided. Therefore, it is clearly a challenge when trying to synthesize the evidence to decide, how to deal with studies describing the intervention/exposure as “diet beverage”, “diet drink” or “diet soda” only. We also included such studies in this scoping review; however, it has to be mentioned that we did not include specific search terms for “diet” beverages/sodas in our search strategy, therefore the list of studies reporting on the effects of diet beverage etc. may be incomplete.Current evidence demonstrates that there is a need for both further primary research and high quality comprehensive systematic reviews including meta-analyses, to inform future recommendations about the health benefits and risks of NNS to advise and support health care practice and public health decision-making.This scoping review highlights the need for studies which investigate the long-term effects of individual sweeteners on some of the less well-researched health outcomes . Future studies need to be rigorous in design and conduct, with well-defined interventions (providing information on type and dosage of the non-nutritive sweetener) and controls. Study reports should include detailed descriptions of all methodological aspects to enable proper interpretation of the results.Systematic reviews are required for health outcomes with a large number of primary studies, but without conclusive evidence  to support the formulation of recommendations and to be able to decide whether further, well-designed primary studies are required.There are numerous gaps in evidence related to the health effects of NNS in both healthy and non-healthy populations. In healthy subjects appetite and short term food intake, risk of cancer, risk of diabetes, risk of dental caries are the most investigated health outcomes, all of them without any conclusive evidence. There is a need for well-conducted systematic reviews to quantitatively summarize results and assess their validity. Besides, there are numerous health outcomes, like incidence of headaches in association with NNS consumption, depression, Alzheimer’s disease, risk of preterm delivery, behavioural effects, cardiovascular effects or risk of chronic kidney disease, which were investigated in only few studies and further research activity is needed. A systematic review may also help to enable formulating recommendations for subjects with diabetes and hypertension on using NNS.


Inactivated polio vaccine (IPV) is now the only source of routine type 2 protection. The relationship, if any, between vaccine-induced type 2 humoral and intestinal immunity is poorly understood.Two clinical trials in five Latin American countries of mixed or sequential bOPV-IPV schedules in 1640 infants provided data on serum neutralizing antibodies (NAb) and intestinal immunity, assessed as viral shedding following oral mOPV2 challenge. Analyses with generalized additive and quantile regression models examined the relationships between prechallenge NAb titers and proportion, duration and titers (magnitude) of viral shedding.10 stool viral titer over 4 post-challenge assessments. Day 28 post-challenge shedding was 13.4%  lower and the Day 21 post-challenge median titer of shed virus was 3.10 log10  lower for subjects with NAb titers at the ULOQ as compared with LLOQ on day of challenge. Overall, there was a weak but significant negative relationship, with high NAb titers associated with lower rates of viral shedding, an effect supported by subset analysis to elucidate between-country differences.We found a statistically significant (p < .0001) but weak relationship between NAb titer at the time of mOPV2 challenge and the Shedding Index Endpoint, the mean logTaken alone, the weak association between pre-challenge NAb titers following IPV or mixed/sequential bOPV/IPV immunization and differences in intestinal immunity is insufficient to predict polio type 2 intestinal immunity; even very high titers may not preclude viral shedding. Further research is needed to identify predictive markers of intestinal immunity in the context of global OPV cessation and IPV-only immunization. Although the per-dose effectiveness of IPV in producing humoral immunity as measured by seroconversion and neutralizing antibody (NAb) titers has been well established, its relationship to primary intestinal mucosal immunogenicity is limited and less clearly understood. Of interest, in relation to the global switch from tOPV to bOPV is the impact on type 2 intestinal immunogenicity from one or more dose(s) of IPV. Recent randomized controlled trials exploring bOPV–IPV schedules followed by mOPV2 challenge have concluded that although regimens including IPV reduce the duration and titer of viral shedding, they tend to be associated with limited overall impact on virus shedding, particularly during the time that virus excretion peaks, at around 7 days following oral challenge As there are often significant variations in levels of serum NAbs within vaccination regimens, we used data on polio type 2 circulating antibodies and virus excretion dynamics obtained from recent randomized controlled trials conducted in Latin America to directly explore a potential relationship between individual pre-challenge serum NAb levels and intestinal immunity that should add value to the evidence base on the new schedules of polio vaccination.2Data were derived from two recently published randomized controlled trials performed in 2013–2014: study IPV001, performed in Colombia, the Dominican Republic, Guatemala and Panama, and registered on clinicatrials.gov as NCT01831050 10 CCID50/g) collected on days 7, 14, 21, and 28 post-mOPV2 challenge. Viral titers were assessed in stool samples that had detectable OPV using RT-PCR, with a viral titer of 0 recorded for samples negative for OPV. Defined in this way, the SIE captures both the magnitude and duration of viral shedding. Also measured were the titers of serum neutralizing antibody (NAbs) immediately prior to mOPV2 challenge. All subjects from both studies with sufficient data available to compute the SIE and pre-challenge NAbs were used for this analysis. Both the SIE and NAb titer values are subject to censoring at their respective lower and upper limits of quantitation  as the assay workflows were set up for specific ranges of dilutions and values outside of these ranges were not captured, resulting in the described LLOQ and ULOQ. If a positive sample had a titer below the lower limit of quantitation (LLOQ), this limit was used for the data point. All other limits of quantitation were used as data points for values exceeding them.The primary endpoint used for this analysis was a shedding index endpoint (SIE) 2.1Regression models were fitted to endpoints using NAb titer and/or nominal stool collection day as predictors to describe the relationship between the likelihood and extent of viral shedding as a function of NAbs on day of challenge. Shedding positivity at each day post-challenge was modelled using an additive generalized linear model (GAM), implemented with the mgcv package The titer of shed virus at each post-challenge day was modelled using median regression, implemented in the quantreg package for R 2). For the upper limit of quantitation (ULOQ), a shifted exponential model was assumed, with shift equal to the upper limit (10.5 log2) and a rate of 0.7. The rate was selected by manually tuning the resulting distribution to align with Sabin-2 NAb data from IPV vaccinees obtained from an assay with a higher ULOQ used in another study To evaluate inter-country differences in the relationship between pre-challenge type 2 serum NAbs and viral shedding, due, perhaps, to different levels of passive type 2 exposure in the different study locations, a subset analysis of IPV001 data only was performed. IPV002 data were excluded to avoid confounding of country with regimen and timing; as randomization in IPV001 was balanced across country, the influence of regimen is not confounded with country in this subset. For all models, the NAb predictor was used in two different methods. The first considered the observed NAbs, using the limits of quantitation as observed data. The second method involved posing a pair of models for values above and below the limit of quantitation, respectively, as a sensitivity measure for the influence of this censoring. This second method involved repeatedly simulating the censored values from the models, re-fitting the regression model while reusing the observed (uncensored) data, and aggregating the results. For the lower limit of quantitation, it was assumed that NAb titers were uniformly distributed between 0 and the lower limit , which is itself significantly better than the null model (p < .0001). The interaction between NAb titers and study day, however, was not supported by the data (AIC difference of 54 units), indicating similarity in the shape of the relationship between the two variables across post-challenge time point. Comparison with a model which omits the smoothing term in favor of a linear relationship indicates the nonlinear smoothing term is a significant component of the model (p < .0001). The results are supported by repeat testing during the censored-data simulation procedure.2 and 10.5 log2. This model produces estimated differences in the probability of shedding positivity between NAb LLOQ and ULOQ of 2.7%  for Day 7, 6.7%  for Day 14, 10.8%  for Day 21, 13.4%  for Day 28, with a median AUC of the ROC of 0.69 across simulations, compared with 0.67 for the model which includes a factor for study day, but excludes pre-challenge NAbs.Predictions from the model fit are described in 3.2Modelling titer of shed virus as a function of NAb titers is depicted in Not surprisingly, the model predicts monotonically decreasing viral shedding with advancing study day for each fixed value of NAb titer, which is again consistent with univariate descriptive results , with higher median viral titer at the NAb LLOQ compared with the ULOQ for Days 14  and 21 .3.3Neutralizing antibody titer is a statistically significant predictor of the SIE (p < .0001 vs null model). The estimated relationship is depicted in 10 CCID50 per gram of stool, and explains a modest 3.0% of total variation in median SIE.The model suggests a detectable, but weak negative relationship exists between extent of viral shedding over 28 days and NAb titers on the day of mOPV2 challenge. The model fit obtained by considering censored values as observed A more clThe subset analysis of SIE among only IPV001 participants yielded negative estimated relationships of SIE with pre-challenge type 2 serum NAbs which were generally stronger than in the combined-data analysis, evident by larger differences in median SIE across the range of NAb LLOQ and ULOQ . The int4Withdrawal of type 2 OPV and introduction of IPV into routine immunization programs are the first steps in the subsequent removal of all OPV that should eliminate the risks of vaccine-related poliovirus disease and circulation. Improving our understanding of the relationship between development of serum antibodies and mucosal immunity is important to characterize the risk of virus spread in populations immunized only with IPV and plan appropriate response strategies.Post-vaccination serum neutralizing antibody titers of 4–8 are widely accepted as correlates of protection against paralytic poliomyelitis We found a weak negative relationship between humoral antibodies and excretion of shed virus. The estimated probability of shedding for each of 4 weeks post-mOPV2 challenge declines gradually with increasing NAb titers, but the observed difference in shedding probability across the entire measured range of NAb titers at Day 28 peaks at approximately 13%, a relative decline of approximately 30% from the estimated probability at NAb LLOQ. In contrast, the estimated median titer of shed virus at 3-week post-challenge exhibits substantial variation across NAb titers – a decline of approximately 93% from NAb LLOQ to ULOQ. This difference in median viral shedding across the extremes of pre-challenge titer is significantly lower on Day 14, and disappears by Day 28, where median shedding has dropped to 0 (more non-shedders than shedders) for all levels of pre-challenge NAb.The SIE, the combination measure of duration and quantity of shed virus, is estimated to decline gradually with increasing pre-challenge NAb titer. Across the range of pre-challenge NAb titers, the predicted relative decline in SIE is estimated to be approximately 22%. Results from IPV001 Group 2 (bOPV only) vs Group 5 (bOPV plus two IPV) While the relationships estimated here are detectable, consistent, and statistically significant, they do not account for a substantial amount of variability in shedding characteristics. Measures of model fit, including the ROC AUC for shedding positivity and in particular the proportion of variation explained by median regression for weekly viral titers and SIE, indicate some significant unexplained variation in viral shedding remains beyond that explained by pre-challenge NAb titers. Heterotypic protection from bOPV could not be rigorously assessed from these combined data. However, previously published results from IPV002 showed subjects given three doses of IPV but no bOPV shed virus in greater quantities than those given fewer IPV doses along with bOPV In comparison with viral shedding results from the bOPV–IPV regimens considered here, subjects from IPV001 who received three doses of tOPV (without IPV) shed very low quantities of type 2 virus . These sOverall, the weak relationship we found in this exploratory analysis serves to support the potential distinctiveness of humoral and mucosal immune responses in the new endgame mixed or sequential bOPV-IPV schedules the and underscores the importance for further exploration of the dynamics of intestinal mucosal immunity in such schedules and settings.One limitation of our analysis is that the SIE is an imperfect endpoint because it is a mean of potentially censored values. Regression for the median SIE was employed to mitigate this weakness. The SIE, however, remains a meaningful endpoint that captures both the extent and duration of viral shedding in a single index and is thus important from an epidemiological standpoint to assess risk of viral shedding and transmission. The heterogeneity of different schedules, populations and social and epidemiological factors in the countries where the trials were conducted also introduced uncontrolled variation in the overall assessment. The use of censored NAb (predictor) values is a weakness of the regression modeling. The use of a simulation method to assess the sensitivity of results to this censoring serves to mitigate this weakness, although the model for censored values chosen here is a simple one, and other choices may also be reasonable. Other unmeasured and unknown confounding variables may exist, but the most important known potential confounders, heterotypic intestinal immunity conferred by bOPV administration and potential differences in exposure to circulating type 2 virus across the region, have been investigated, and it appears that neither is associated with artificially strengthening the observed relationship between pre-challenge type 2 serum NAbs and measures of intestinal immunity based on viral shedding.5High serum NAb titers against poliovirus type 2 at the time of mOPV2 challenge among IPV vaccinated infants are only weakly associated with lower viral shedding. Thus, NAb titers at 4 weeks post-final vaccination are an insufficient correlate of intestinal immunity. The impact of the magnitude of reduction in shedding associated with high NAb titers remains unclear. Research directed toward defining stronger correlates of polio intestinal immunity should facilitate better understanding of population vulnerability to poliovirus transmission in the final phases of polio eradication.6Bill & Melinda Gates Foundation (BMGF). ASB and JFM are full-time employees of the BMGF and contributed to the study design, data interpretation and writing of the manuscript. Other authors confirm they have no conflicts of interest to declare.This study was funded by the All authors contributed to the design and/or conduct of the studies, and the elaboration of this manuscript, and agree to the submission to the journal.


Flowering is the result of the coordination between genetic information and environmental cues. Gene regulatory networks have evolved in plants in order to measure diurnal and seasonal variation of day length (or photoperiod), thus aligning the reproductive phase with the most favorable season of the year. The capacity of plants to discriminate distinct photoperiods classifies them into long and short day species, depending on the conditions that induce flowering. Plants of tropical origin and adapted to short day lengths include rice, maize, and sorghum, whereas wheat and barley were originally domesticated in the Fertile Crescent and are considered long day species. In these and other crops, day length measurement mechanisms have been artificially modified during domestication and breeding to adapt plants to novel areas, to the extent that a wide diversity of responses exists within any given species. Notwithstanding the ample natural and artificial variation of day length responses, some of the basic molecular elements governing photoperiodic flowering are widely conserved. However, as our understanding of the underlying mechanisms improves, it becomes evident that specific regulators exist in many lineages that are not shared by others, while apparently conserved components can be recruited to novel functions during evolution. Several plant species measure day length to start specific developmental switches, e.g., the transition to reproductive growth, during the most appropriate times of the year. Seasonal variation of day length provides a fundamental parameter to synchronize developmental changes, because it is not subject to fluctuations like other environmental cues such as temperature.Plants can be categorized as long day (LD) or short day (SD) species, depending on the photoperiod most effective at triggering reproductive growth. When day length exceeds a specific critical threshold, flowering is promoted in LD plants, whereas SD plants flower in response to reduction of day length below a critical threshold. Such thresholds are characteristic of each species and largely determined by the region where the species originated and first adapted. Plants growing at low tropical latitudes tend to flower in response to exposure to long nights, whereas species adapted to higher latitudes promote flowering during seasons characterized by LD, indicative of the warm days of spring and summer. Plants adapted to temperate regions that germinate before winter, often also need to satisfy a vernalization requirement  to become competent to respond to photoperiodic induction. Additionally, many plants can promote flowering even after long exposures to non-inductive photoperiodic conditions, indicating a facultative response to day length and the existence of floral promoting stimuli that can bypass the requirement for specific conditions. Therefore, plant interactions with its growth environment can be complex, and gene networks have evolved that respond to changing seasonal parameters.In crop species, responses to day length have been extensively manipulated, creating varieties that can grow, flower and set seeds at latitudes outside of the range occupied by the wild progenitor. Artificial adaptation to broad latitudinal ranges has been a key step during domestication of several species, allowing cultivation and diversification in many regions of the globe. Natural genetic variation has offered the substrate for human selection and remarkably, many domestication loci encode orthologous genes in distantly related species providing a molecular perspective to look at conservation and evolution of pathways regulating flowering.Here, we will summarize recent advances in understanding photoperiodic flowering regulation in crop species, focusing on cereals. Starting with the tenets established using Arabidopsis as model system, we will discuss how conserved and unique elements have been deployed to evolve flowering networks of LD and SD plants and how they control production of a florigenic systemic signal in leaves.Table 1).Photoperiodic flowering has been mostly studied using the dicot Arabidopsis, through which core genetic and molecular mechanisms at the base of the process have been characterized . ArabidoCDFs encode transcriptional repressors that limit expression of the CONSTANS (CO) zinc finger transcription factor, a central regulator within the photoperiodic flowering pathway (FLOWERING LOCUS T (FT), component of the systemic florigenic signal  and FLAVIN BINDING KELCH REPEAT F-BOX PROTEIN 1 (FKF1) proteins are expressed at the end of the light phase and interact in a light-dependent fashion . The res pathway . Besides pathway , alterna pathway , photore pathway , as well pathway , hormona pathway  and post pathway . However pathway . During  pathway . Consequc signal .FT mRNA abundance are mediated by several classes of protein interactors that include transcription factors and transcriptional co-regulators, photoreceptors, histone-like proteins, and ubiquitin ligases .The effects of CO protein on the levels and rhythmicity of GI, FKF1, the CDFs, CO, and FT exist in rice and have been partly linked in a cascade that resembles the photoperiodic pathway of Arabidopsis (Table 1). The OsGI and OsFKF1 proteins can interact with each other and with a CDF protein, OsDOF12, similarly to their Arabidopsis homologs , a homolog of FT, under LD while having no impact on transcription of Heading date 1 (Hd1), a homolog of CO. Thus, the function of OsDOF12 is opposite to that of Arabidopsis CDFs, effectively promoting flowering  was later cloned and shown to encode a B-type response regulator , Hd1, and Pseudo Response Regulator 37 (PRR37) .Cloning of og of CO . Howeveratitudes . A seconegulator . Ehd1 inegulator , and to egulator . Under S species . Under L (PRR37) . The Hd17) . Both proteins encode mobile leaf-borne systemic signals, but whereas Hd3a is required only under SD to induce flowering, the RFT1 protein is expressed and can promote flowering under both SD and LD  is a SD plant evolved in Africa, in the Sudan region. Six major QTLs controlling flowering time and termed Maturity loci (Ma1–Ma6) have been detected in sorghum. Almost all QTLs have been identified as photoperiodic flowering regulators and their study is demonstrating the strong homology occurring between the sorghum and rice pathways (Table 1).Sorghum  and SbGhd7 (Ma6). These genes encode flowering repressors that limit mRNA expression of downstream targets under LD, including Ehd1, SbFT, and SbZCN8   . The floppressor . AdditioSbCO transcription mediated by SbPRR37 has also been investigated. The data suggest that SbPRR37 modulates SbCO expression at dawn, promoting its transcription under LD, whereas under SD SbCO expression seems not to depend upon SbPRR37  could promote flowering when constitutively expressed in Arabidopsis  was domesticated in central Mexico from Teosinte, which is a SD plant. The first flowering gene cloned in maize was INDETERMINATE 1 (ID1): plants with mutations in this gene delay the floral transition and produce aberrant inflorescences . Although a maize Ehd1 homolog has not yet been found, the high homology between ID1 and OsEhd2 could suggest a similar regulatory mechanism, possibly indicating the existence of a ZmEhd1-like protein subject to similar regulation. Indirect evidence supporting this view is that the CCT-domain transcription factor ZmCCT shows sequence homology with OsGhd7, and encodes a strong LD flowering repressor . Mutations in ZmCCT cause early flowering and have been artificially selected to expand maize cultivation to higher latitudes  and GIGANTEA2 (GI2)  and ZCN8 (homolog of Hd3a), both of which displayed increased expression in the gi1 background  . In Arabckground . These d of AtGI . Whetherlowering .FT-like genes, ZCN8 was identified as the strongest candidate for the maize florigen  and barley (Hordeum vulgare) were domesticated in the Eastern Mediterranean region, in areas characterized by the alternation of cold and warm seasons. These cereals have evolved mechanisms to prevent flowering when temperatures are low, to protect the meristem from cold damage. Flowering is promoted after exposure to vernalizing conditions, when plants resume growth in the spring. During domestication, some cultivars of these species have lost sensitivity to vernalization and, depending on the response to cold, they could be classified as winter or spring types. Winter-types have an obligate vernalization requirement. Such response is controlled by the VERNALIZATION (VRN) loci  and APETALA1 (AP1) of Arabidopsis, whereas VRN2 is a floral repressor sharing sequence similarity to Ghd7 of rice (Table 1). Under low temperatures, the expression of VRN1 is induced and the protein directly binds to the promoter of VRN2 to reduce its expression during vernalization  loci . VRN1 islization . Dominanon areas .VRN2 is required to induce VRN3 expression during the floral transition. VRN3 proteins  are homologs of the Arabidopsis and rice florigens, and move to the apical meristem to promote flowering upon exposure to warm temperatures and LD  gene has been described as the major factor controlling sensitivity to day length in wheat and barley . Interestingly, overexpression of HvCO2 was recently shown to increase expression of VRN2 under LD and SD in a winter variety (VRN2 repressor, overexpression of HvCO2 could still promote flowering, likely through a VRN2-independent pathway. The data might suggest that HvCO2 mediates a floral repressive function through VRN2, to limit FT expression. Whether barley orthologs of Hd1 display dual functions similarly to rice Hd1 awaits further testing. The use of mutant resources and possibly of edited alleles might help to address this issue.Homologs of d barley . The TaHs system . In barl by PPD1 . Thus, b variety . DespiteEhd1 and Ghd7, in others. A common theme appears to be the requirement for upstream master regulators to control expression of FT-like genes, but their number and relative contributions to heading time broadly varies between species. While in Arabidopsis, CO acts as central and primary regulator of FT, CO homologs in crops are coupled to parallel pathways largely sharing the workload, and FT expression often strongly depends on additional regulators.The examples discussed above illustrate the flexibility of photoperiodic flowering networks and how adaptation to distinct environments modifies their topology. Major changes include the integration of vernalization modules in some networks and the recruitment of non-shared regulators, such as Efforts will be needed in the future to isolate all components of the networks in crop species, many of which are still to be cloned. Quantification of transcripts offers a rapid way of determining relationships between genes, but provides only limited information on protein expression or biochemical function. Finally, molecular networks are starting to be built, based on protein–protein or protein–DNA interactions especially in rice. Expanding these efforts toward other crops will prove necessary.Figure 1. JG-A wrote the temperate cereals section. FF revised the manuscript.FF and VB organized the manuscript and wrote the Arabidopsis and rice sections. MC wrote the maize and sorghum section and prepared 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.


Long bone defects represent a clinical challenge. Bone tissue engineering (BTE) has been developed to overcome problems associated with conventional methods. The aim of this study was to assess the BTE strategies available in preclinical and clinical settings and the current evidence supporting this approach. A systematic literature screening was performed on PubMed database, searching for both preclinical  and clinical studies. The following string was used: “(Scaffold OR Implant) AND .” The search retrieved a total of 1573 articles: 51 preclinical and 4 clinical studies were included. The great amount of preclinical papers published over the past few years showed promising findings in terms of radiological and histological evidence. Unfortunately, this in vivo situation is not reflected by a corresponding clinical impact, with few published papers, highly heterogeneous and with small patient populations. Several aspects should be further investigated to translate positive preclinical findings into clinical protocols: the identification of the best biomaterial, with both biological and biomechanical suitable properties, and the selection of the best choice between cells, GFs, or their combination through standardized models to be validated by randomized trials. Traumatic long bone defects still represent a clinical challenge for orthopaedic surgeons. In fact, a critical size defect requires invasive surgical procedures to reconstitute the structural integrity of the collapsed bone. This does not provide fully satisfactory results entailing a significant socioeconomic burden , 2. DespThe concept of bone tissue engineering (BTE) has been developed to overcome problems associated with conventional methods. The typical paradigm of BTE is constituted by the four biological prerequisites which include osteogenic cells, osteoinductive stimulus, osteoconductive matrix scaffolds, and mechanical environment (the diamond concept) . These pThe main objective of this systematic review was to assess the BTE strategies available in preclinical and clinical settings, in order to analyse the current evidence supporting the use of this approach for the treatment of long bone defects.A systematic literature screening was performed by two independent reviewers (GS and NG) on the PubMed database, searching for both preclinical and clinical studies on 3D synthetic scaffolds with organized structures for long bone defects developed to treat defects of the upper/lower extremities. In particular, the research criteria included studies published in English language until February 2017. The following string was used: “(Scaffold OR Implant) AND ” . Among pThe PubMed search analysis retrieved a total of 1573 articles and, following the inclusion criteria, 51 preclinical studies –69 and 4In the past, few years there has been a progressive increase in the number of publications for scaffold treatments in the preclinical field, as shown in Concerning the scaffolds alone application , only 1/The use of cells was described in 22/39 papers; 2/22 of which used bone marrow concentrates (BMC), 19/22 expanded bone marrow MSCs (BMSCs), and 1/22 expanded adipose tissue MSCs (ADMSCs). Overall results showed that the use of cells  in combination with scaffolds had an additional positive effect compared to the scaffold alone, with the ability to heal segmental defects with improved bone regeneration, in terms of radiological and histological results.β-3. Results of 5/8 articles reported positive effects, mainly emphasizing that GFs largely assisted the healing process of critical sized defects due to their osteoinductive properties. On the other hand, 2/8 papers, 1 on freshly extracted BMPs from bone and 1 on rhTGFβ-3, reported inferior and similar results, respectively, when compared to ABG treatment.The use of scaffolds with GFs augmentation was reported in 8/39 papers. Out of these, 7/8 papers reported the use of BMPs  and 1/8 paper reported the use of rhTGFFour papers out of 39 compared the use of different cell sources, or cells versus GFs. Among these, 2 papers reported superior results for BMSCs when compared to PRP or BMC augmentation. One paper showed no significant differences between BMC and BMP-7 added to a HA scaffold, with better results compared to the scaffold alone and similar results compared to ABG. Interestingly, one paper comparing the effects of PRP, mesenchymal progenitor cells (MPCs), orofacial skeleton osteoblasts (mOBs), and axial skeleton osteoblasts (tOBs) found that the MPCs group produced better results when compared to other biological enhancers.The use of cells and GFs in combinations was reported in 5/39 papers with different study designs, which prevents us drawing an overall conclusion. The combination of scaffolds, cells, and GFs (either PRP or AdBMP-7) provided superior results compared to the scaffold/cell construct in 2/5 papers. On the contrary, 2/5 studies showed worse results for scaffold/BMSCs/PRP compared to a scaffold/BMP-7 construct. Finally, one study showed superior results combining 2 cell sources, such as OBs (osteoblasts cells) and ECs  compared to the use of a single cell type (ECs).The literature search identified 4 clinical papers that met the inclusion criteria. Two of the 4 clinical trials used scaffolds without any augmentation and the other 2 reported a cell augmentation approach. An HA scaffold was used in 3/4 papers followed by beta-TCP in 1 study.In 2000 Werber et al.  presenteIn this systematic review, the interest on the scaffold based strategy to treat long bone defects was documented by the great amount of preclinical papers (even though highly heterogenous) published over the past few years, showing overall promising findings in terms of radiological and histological evidence, with the ability to treat segmental defects in large animal models. Unfortunately, this in vivo situation is not reflected by a corresponding clinical impact of this treatment approach, with few published clinical papers, highly heterogeneous and presenting small patient populations.In orthopaedic surgery, critical size bone defects derived from nonunion, trauma, or tumours are a challenging problem, from both a social and economic perspective. In fact, in Europe, the total cost of treatment of nonunion defects is between 10000 and 100000 € per patient, with an amount of around 1 million bone operations every year . Bone grIn the last 20 years, the number and variety of biomaterials developed for the treatment of segmental bone defects have been increasing, especially in preclinical setting. This review was focused on solid biomaterials, with 3D scaffolds that can mimic bone structure and composition when implanted in vivo into the defects, with results documented in studies on large animals (to better reproduce human conditions) and in clinical settings.HA and ceramic calcium phosphates, such as TCP that resemble mineral components of bone, are the most used materials in both preclinical and clinical settings, offering a biological response similar to that of natural bone . This abAnother interesting aspect in the field of biomaterials is related to the diamond concept, which involves a combined approach by combining osteoinductive factors (cells or GFs) with 3D scaffolds and was the most investigated option in preclinical settings. Due to the heterogeneity of these studies , it is difficult to draw a conclusion about the best augmentation procedure able to enhance bone healing. In fact, overall positive results have been reported, but only few papers compared the different augmentation strategies in the preclinical model, thus leaving literature findings inconclusive. Among GFs, the most exploited strategy involves the BMPs family, whose discovery dates back to the end of the nineteenth century and drew an increasing attention in the scientific community, with a large literature including also the overall good results of the clinical application, although documented adverse events and some controversial reported outcomes limited their impact in the clinical practice . While oThe clinical scenario does not reflect the high research activity documented by the preclinical literature: only 4 papers were found, all with a small patient number, different study design, and heterogeneous pathologies treated. The search identified 2 clinical trials using scaffolds without any augmentation, while another 2 reported a cell augmented scaffold approach. Among different sources of MSCs, bone marrow was the most commonly used for BTE in orthopaedic surgery , 76. BMSThis systematic research of the literature documented a growing interest in scaffold based approaches applied in preclinical settings to promote tissue regeneration in long bone defects of critical size. However, this evidence did not translate into a similar interest in the clinical scenario, characterized by only 4 papers published, with low quality and heterogeneous study designs. Several interesting aspects have been underlined by preclinical literature, in particular with regard to the benefit of an augmentation strategy to enhance the regenerative potential of the biomaterial. These should be further investigated in order to translate positive preclinical findings into clinical protocols: first of all, to identify the best biomaterial for long bone defects, with both biological and biomechanical suitable properties, and then to select the best choice between cells, GFs, or their combination, in order to provide the best treatment option for patients affected by long bone defects.


Cancer is a genetic disease, but extreme degrees of heterogeneity in the mutations are found in tumors of different organs or tumors from a single organ. Only a small proportion of these mutations are drivers of neoplastic behavior, which are the logical targets for precision therapy. However, because of the genetic heterogeneity, probably no two human tumors have ever been alike. This implies that one must obtain specific mutational data from each tumor to plan rational therapy, rather than use the same treatment for all tumors arising in a specific organ.KRAS in the case of most pancreatic cancers) or the inactivation of a tumor suppressor gene . However, a considerable number of tumors are initiated by the loss of a DNA repair mechanism, including base excision repair, nucleotide excision repair, homologous recombination repair, DNA mismatch repair (MMR), and others. Inactivation of DNA repair systems permits the accelerated accumulation of activated oncogenes or inactivated tumor suppressors. Moreover, inactivation of each of these systems produces a recognizable “signature” mutational pattern in its wake.Some tumors are initiated by the activation of proto-oncogenes , affected patients are born with no MMR activity, a normal phenotype, but have an extremely high risk for hypermutated childhood cancers [About 15% of colorectal cancers (CRCs) begin with the inactivation of the DNA MMR system, followed by a cascade of mutations in genes that are the actual drivers of neoplastic behavior. Loss of the DNA MMR system is not, BRCA2, EGFR, and NTRK. For each gene, they found that such mutations were significantly over-represented in dMMR CRCs, compared to CRCs with proficient MMR activity, and then confirmed their findings using a publically available database (COSMIC). Intriguingly, they found inactivating BRCA2 mutations in 50% and 38% of dMMR CRCs compared to 14% and 6% in non-dMMR tumors respectively in these databases. Moreover, these mutations were preferentially found in simple repetitive sequences  which are surprisingly abundant in the exons of BRCA2 [In this issue of Oncotarget, Deihimi and colleagues have used two databases to determine the mutational spectra in CRCs associated with DNA MMR deficiency (dMMR) . They beBRCA2 mutations in dMMR CRCs can further add to the therapeutic armamentarium remains to be seen, but is implied by the data in this paper. Poly(ADP-ribose) polymerase (PARP) inhibitors are particularly efficacious against BRCA1/2 mutated breast and ovarian cancers, and it is tempting to speculate that these drugs could be used to treat dMMR CRCs with somatic BRCA2 mutations. In fact, recent data in ovarian cancer suggest that BRCA1/2 mutations by themselves are associated with high neoantigen loads and increased PD-1 and PD-L1 expression in ovarian cancer [These findings raise intriguing questions about the development of appropriate therapy for dMMR CRCs. Recent data have shown that immune checkpoint blockade (ICB) with anti-PD-1 antibodies has particular efficacy against hypermutated tumors due to the fact that the underlying dMMR problem inherently generates a diverse array of immunogenic frameshift peptides . Whethern cancer . Such fin cancer  has genen cancer  with ICBEGFR-mutated lung adenocarcinomas. Likewise, as discussed by the authors, small molecule inhibitors targeting oncogenic somatic NTRK rearrangements are under development, though it is unknown whether they would be similarly effective against missense NTRK mutations.Furthermore, many of the same somatic EGFR mutations observed in the dMMR CRCs in this study  are knowBRCA2, EGFR, or NTRK mutations, it is important to remember the humbling lessons from targeted therapies against BRAF V600E mutations, where profound successes (with RAF and MEK inhibition) in BRAF V600E-mutated melanoma were followed by comparably meager activity in BRAF V600E-mutated CRC due to underlying biologic differences between cancer types [While the findings from Deihimi and colleagues  lead nater types . What isNonetheless, this work represents a substantial step forward in our rational understanding of how tumors develop and raises exciting questions about extending such insight to guide precision therapy. Deep sequencing of the DNA of all advanced cancers may soon become standard of care, and understanding the complex interactions between defective DNA repair mechanisms, acquired driver mutations, and mechanisms of primary and acquired therapeutic resistance will be the key to truly recognizing the promise of personalized, targeted therapy.