Efflux was demonstrated to play an important role in MDR and many resistance phenotypes. Clinically relevant Burkholderia species are intrinsically resistant to ciprofloxacin but the soil Burkholderia species were not intrinsically resistant to ciprofloxacin. Using a phenotypic enzyme assay we identified the antibiotic specific inactivation of trimethoprim in 21 bacteria from different soils. The results of this study identified the importance of the efflux mechanism in the soil resistome and variations between the intrinsic resistance profiles of clinical and soil bacteria of the same family.Understanding the soil bacterial resistome is essential to understanding the evolution and development of antibiotic resistance, and its spread between species and biomes. We have identified and characterized multi-drug resistance (MDR) mechanisms in the culturable soil antibiotic resistome and linked the resistance profiles to bacterial species. We isolated 412 antibiotic resistant bacteria from agricultural, urban and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16–23 antibiotics, comprising almost all classes of antibiotic. The mobile resistance genes investigated, (ESBL,  Antibiotic resistance has developed over time from resistance to single classes of antibiotics to multi-drug resistance and extreme drug resistance. Until recently, antibiotics and antibiotic resistance were discussed in terms of treatments of infections and the prevention of successful treatment, respectively. The mechanisms of action of antibiotics and antibiotic resistance mechanisms have been studied almost exclusively in pathogenic bacteria. It is only in recent years that antibiotic resistance research has focused on the environment from which the antibiotics were initially extracted: soil microorganisms and the soil ecosystem. With an every decreasing supply of novel antibiotics and increasing resistance research has started to focus on investigating the natural antibiotic resistome and understanding the ecology and evolution of antibiotic resistance in the non-clinical environment in order to identify reservoirs of both known and novel antibiotic resistance mechanisms.Streptomyces, and an isolated cave microbiome Despite the belief that the soil antibiotic resistome bacteria play an increasingly important role in the evolution, development and spread of antibiotic resistance in humans and animals, there is little known about the natural bacterial resistome in soil. There have been many calls for more information about the natural resistome and these have also highlighted the importance of understanding the soil resistome in the preservation of antibiotics for the treatment of infections Culture based antibiotic susceptibility testing is the gold standard of antibiotic resistance testing in hospitals throughout the world. It is a relatively cheap and easy technique with little need for sophisticated or expensive equipment. Therefore, using susceptibility testing would enable the comparison of non-clinical data with clinical data. Antibiotic susceptibility testing also enables the phenotypic detection of as yet uncharacterized resistance mechanisms and complex resistance mechanisms such as efflux, which are frequently mediated by several genes. However, antibiotic resistance and antibiotic breakpoints have been defined within the context of their medical functions. Breakpoints define the thresholds of response of bacteria to an antibiotic in vitroThe bacteria that can be grown in the laboratory are only a small fraction of the total diversity that exists in nature. Approximately only 1% of bacteria on Earth can be readily cultivated Streptomyces and clinical pathogens blaCTX-M, qnrA and blaNDM as originating in the environmental bacteria Kluyvera sp., Shewanella algae and Erythrobacter litoralis, respectively Mechanistic commonalities between soil bacteria and clinical pathogens were first identified in the 1970s, including identical molecular aminoglycoside resistance mechanisms in The soil is a reservoir of antibiotic resistance genes, but not all resistance mechanisms are necessarily a threat to the continued use of antibiotics in all pathogens We aimed to identify the levels of culturable resistant bacteria, both antibiotic producers and non-antibiotic producers, and identify the roles of the different mechanisms of resistance such as efflux, novel enzymatic resistance mechanisms and selected plasmid mediated resistance genes in conferring multi-drug resistance within the soil bacterial community. We linked the bacterial phylogeny to the antibiotic resistance profiles in order to compare the intrinsic resistance profiles of soil and clinical bacteria from the same bacterial order.Staphylococcus aureus (MRSA) and Acinetobacter, respectively. Multi-drug resistance is defined as resistance to three or more different classes of antibiotics.Four hundred and twelve antibiotic resistant bacterial isolates were cultured from ten soils from agricultural, urban and pristine environments . All isoBeta-lactam resistance ranged from 84% to 100% and was greater than 90% on average for the ten soils , indicatMulti-drug resistance conferred by efflux was identified in 83% of the soil isolates and 410 of 412 isolates were capable of conferring resistance to at least one class of antibiotics by efflux. Over 76% of the fluoroquinolone (levofloxacin and ciprofloxacin) resistance was mediated by efflux . In compThe phylogenetic profiling identified four phyla: Proteobacteria (81.7%), Firmicutes (6.3%), Actinobacteria (1.5%) and Bacteroidetes (1%) . The remPseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderia cepacia, Acinetobacter species, Achromobacter xylosoxidans, Serratia marcescens and Aeromonas species are intrinsically resistant to penicillin and the β-lactam antibiotics Stenotrophomonas species), Enterobacteriales (78% Serratia species) and Aeromonadales constituted 87% of the soil bacterial population within this study. The classes of antibiotics used to treat Pseudomonas species infections include the fluoroquinolones, amikacin, gentamicin and colistin as an antibiotic of last resort. Intrinsic resistance to these antibiotics has not yet been identified in clinical Pseudomonas species and levels of resistance remains relatively low It is important to factor in the role of intrinsic resistance in the levels of resistance, especially to the β-lactams. The pathogenic bacteria Burkholderia cepacia and Stenotrophomonas are intrinsically resistant to amikacin and gentamicin, and Serratia marcescens to amikacin. The soil bacteria belonging to the orders Burkholderiales, Xanthomonadales (Stenotrophomonas species) and Enterobacteriales  were also intrinsically resistant to the same aminoglycosides as their clinically relevant counterparts. In the soil bacterial populations the Bacillales, Pseudomonadales and the Aeromonadales were also intrinsically resistant to amikacin and gentamicin in contrast to clinical findings for the bacteria and the Enterobacteriales were additionally intrinsically resistant to gentamicin. Resistance to gentamicin was mainly mediated by efflux.In order to identify the impact of intrinsic resistance and efflux in the resistance profiles of the soil bacteria we separated the resistance levels according to most frequently identified bacterial orders . ResistaBurkholderia cepacia complex species of clinical bacteria Innate resistance to ciprofloxacin has been described only in the Pseudomonas aeruginosa are intrinsically resistant to the tetracycline class Burkholderia and Serratia marcescens species. Our results identified that in contrast to the clinical findings, these species in the soil populations were not intrinsically resistant to colistin and the Xanthomonadales and Aeromonadales bacteria were intrinsically resistant to colistin and was mainly mediated by efflux in all species, except the Xanthomonadales.Clinical in vitro by clavulanate. All β-lactam resistant isolates were tested for the presence of ESBL by comparison of growth on cefotaxime with growth on cefotaxime and clavulanate. Growth on cefotaxime but inhibition on cefotaxime and clavulanate suggested the presence of ESBL. Thirty isolates were ESBL positive, 50% of which were isolated from the urban soil S3. None of the plasmid mediated ESBL genes nor β-lactamase genes were detected in any of the isolates or the extracted soil DNA , Pseudomondales (6/221), Aeromonadales (5/31), Burkholderiales (5/34), unclassified (5/39), Actinomycetales (1/6), Bacilliales (1/26) and Xanthomonadales (1/28).The dissemination of extended spectrum β-lactamases (ESBL) and plasmid mediated quinolone resistance (PMQR) multi-drug resistant plasmids is causing increasing difficulty in the treatment and management of both community and hospital acquired infections. As these antibiotic resistance genes are thought to have their origins in environmental bacteria it was important to investigate the role of these genesin the soil resistome soil DNA . No inhiqnrA, qnrB, qnrS and qep genes by PCR. All isolates were negative by sequencing for the aac(6′)Ib-cr gene. The DNA extracted from five soils contained novel aac(6′)Ib genes, with between two and seven amino acid mutations in comparison to the closest previously described protein or amino acid sequence in GenBank. These novel gene sequences have been deposited in GenBank . The DNA extracted from soils S1 (agriculture), S7 (urban) and S10 (urban) contained the same novel aac(6′)Ib gene (KC916938). Soils S4  and S9  each contained another novel aac(6′)Ib resistance gene containing two and three amino acid mutations, respectively, in comparison to their closest described amino acid sequence in GenBank.One hundred and four isolates resistant to the fluoroquinolones, levofloxacin and ciprofloxacin, and the total DNA extracted from the soils were screened for the presence of PMQR genes . All fluAntibiotic inactivation assays were performed in order to identify novel mechanisms of antibiotic resistance in the soil resistomes . InactivIn order to analyze the soil antibiotic resistome, we investigated soils closest to human activities i.e. farming and urban, and compared these to a pristine soil population. While culture techniques will always bias the population of bacteria under study they have the advantage of being able to detect phenotypic resistances, including efflux mediated resistance and novel resistance mechanisms, and the resistance mechanism can be investigated in further detail. Culture techniques are also the gold standard for clinical testing of antibiotic resistance levels and certain resistance mechanism such as efflux or ESBL.The resistance levels to almost all classes of antibiotics were extremely high. We identified the multi-drug resistant nature of soil bacteria, with greater than 80% of all isolates resistant to 16–23 antibiotics. Antibiotic resistance of bacterial pathogens consists of acquired resistance mechanisms and intrinsic resistance mechanisms. In order to elucidate the resistance mechanisms responsible for these high levels of resistance we investigated the bacteria and soil DNA for different resistance mechanisms, including efflux, production of ESBL and the presence of PMQR genes and antibiotic inactivation.Pseudomonas aeruginosa). We expected that the intrinsic resistance patterns of the clinically relevant species would correspond to the soil bacteria, in particular for bacterial pathogens that originated from soil, such as Stenotrophomonas maltophilia, Burkholderia species and Pseudomonas species. Resistance to the β-lactam and macrolide classes of antibiotics is mediated by intrinsic resistance in these clinically relevant species and soil bacteria. However, the pattern of intrinsic resistance of the soil bacteria of this study to the aminoglycosides, ciprofloxacin , tetracyclines, and colistin were not those associated with the corresponding pathogens. In some cases the soil bacteria were not intrinsically resistant , and in others they were intrinsically resistant to antibiotics used to treat pathogenic bacteria . Burkholderia species and Stenotrophomonas maltophilia are intrinsically resistant to all aminoglycosides, attributed to poor permeability and putative efflux Pseudomonas and Stenotrophomonas have their origins in soil, their closely related soil species do not necessarily contain the same intrinsic resistance mechanisms.Intrinsic resistance in pathogenic bacteria has been well characterized in order to ensure that the most appropriate antibiotic therapy is administered Pseudomonas species is most frequently associated with alterations in the outer membrane lipopolysaccharide (LPS) Yersinia species but has not been well characterized Multi-drug resistance was most frequently mediated by the efflux resistance mechanism and was identified in non-antibiotic producing bacteria. Resistance to colistin and tetracycline in particular was frequently mediated by efflux. Resistance to colistin in clinically relevant qep or recently a resistance-nodulation-cell division/multidrug resistance efflux mechanism Bacteria have the ability to survive in antibiotics due to their evolution in the presence of natural antibiotics over millennia but in our study this also holds true for the synthetic antibiotics, which have not been present in soils. These soil bacteria have developed or utilize universal mechanisms of resistance to overcome the inihibtion by natural antibiotics or toxins, which also work efficiently against many classes of antibiotics blaNDM β-lactamases in the soil DNA is similar to findings from a study conducted in another European site (Cardiff wastewater treatment plant), which also contained no blaNDM genes but are in contrast to environmental sites in New Delhi, India, where blaNDM was detected in two of 50 drinking-water samples and 51 of 171 seepage samples blaNDM-1 infections in Switzerland were isolated from three patients in Geneva university hospitals between 2009 and 2010 blaNDM positive bacteria.None of these ESBL or quinolone mediated plasmid mediated resistance genes were present in either the bacteria or the DNA. These results suggest that these resistance genes are either location or sample type specific or in extremely low numbers in the environment. The lack of This study identified the first inactivating resistance mechanism against trimethoprim in 21 different bacterial isolates from five different soils, comprising farmland and urban environments. Although previous studies, restricted to cave bacteria and Actinomycetes, have investigated soil bacteria for trimethoprim inactivating enzymes they did not result in their identification There have been many calls for more information about the natural resistome and these have also highlighted the importance of understanding the soil resistome in the preservation of antibiotics for the treatment of infections Serratia marcescens and Bacillus species are intrinscially resistant to colistin but were not intrinscially resistant to colistin in the soil population. The results of this study identified that the intrinsic resisome of soil bacteria is in contrast to that of their closely related clinically relevant species. These results suggest that the evolution of intrinsic resistance in soil and clinical bacteria has not developed along the same lines, although there may be similarities in certain resistance genes detected in both environments.While soil bacteria are naturally resistant to many different classes of antibiotics, natural, snythetic and semi-synthetic, the resistance mechanisms are most frequently the non-specific efflux or intrinsic resistance mechanisms. The efflux mechanisms could be present on mobile elements or may be transferred to mobile DNA and cause increased difficulties in the treatment of bacterial pathogens. We have identified that the patterns of intrinsic resistance of the soil bacteria differ from the clinical isolates of the same phyla. Clinical isolates of Burkholderiales are intrinsically resistant to ciprofloxacin and colistin but soil isolates of the order Burkholderiales were not intrinsically resistant to ciprofloxacin or colistin. Similarly, clinical isolates of The conclusions of this study are that there was a high level of multi-drug resistance in soil bacteria to a wide variety of antibiotics and was not dependent on the soil use. This MDR was most frequently conferred by efflux, which if present on or transferred to mobile elements could cause increased difficulties in the treatment of human bacterial pathogens. These results can be used to enhance the understanding of the emergence and dissemination of novel antibiotic resistance from the natural reservoir to the clinical setting, which may aid the development of inhibitors of resistance mechanisms and resistant bacteria.The following antibiotics and antibiotic resistance mechanism inhibitors were used in this study: Penicillin, dicloxacillin, amikacin, cephalexin, kanamycin (Carl Roth GMBH and CO. KG), gentamicin, sisomicin, streptomycin, vancomycin (Carl Roth GMBH and CO. KG), levofloxacin, ciprofloxacin, sulfamethizole, nalidixic acid, chloramphenicol, d-cycloserine, cefotaxime, novobiocin, trimethoprim, sulfisoxazole, tetracycline, erythromycin, colistin, rifampicin, cefotaxime, clavulanic acid, phenylboronic acid and carbonyl cyanide 3-chlorophenylhydrazone (CCCP). All antibiotics and chemicals, except kanamycin and vancomycin, were obtained from Sigma-Aldrich Chemie Gmbh, Buchs SG, Switzerland. All antibiotic solutions were prepared according to manufacturer’s instructions.The soils were collected from four agricultural sites, four urban sites and two pristine environments as described in The antibiotic resistant bacteria were isolated as previously described The antibiotic susceptibilities were investigated as previously described Twenty three different antibiotics were tested. The ESBL phenotype was determined by resistance to cefotaxime and susceptibility to cefotaxime (20 µg/ml) with clavulanic acid (4 µg/ml). Phenotypic efflux resistance was defined as resistance to the antibiotic and susceptibility to the antibiotic when carbonyl cyanide 3-chlorophenylhydrazone (CCCP) at 20 µg/ml was added to the agar. Growth of 152 isolates were inhibited by CCCP at 20 µg/ml. Inhibition of these isolates by CCCP were tested at doubling dilution concentrations of 0.5 µg/ml to 10 µg/ml. These isolates were not inhibited by CCCP alone at a final concentration of 1 µg/ml. Therefore, efflux in these isolates was defined as resistance to an antibiotic and susceptibility to the antibiotic when 1 µg/ml CCCP was added.The total soil DNA from each of the ten soils was extracted using the Mo Bio PowerSoil® DNA Isolation Kit, Mo Bio Laboratories Inc. .TEMbla, SHVbla, OXA, blablaCTX-M Groups 1, 2, 8, 9 and 25, VEBbla, PERbla, GESbla ESBL resistance genes aac(6′)Ib-cr, qnrA, qnrB, qnrS and qep resistance genes using previously described primers and parameters aac(6′)Ib-cr PCR products were sequenced using the PCR primers. The DNA samples extracted directly from the soils were additionally screened for the presence of NDM-1bla. The NDM-1bla PCR primers were NDMF 5′ GAAGCTGAGCACCGCATTAG 3′ and NDMR 5′ TGCGGGCCGTATGAGTGATT 3′. The annealing temperature was 55°C and the PCR product of the positive control was approximately 800 bp. All resulting PCR products were sequenced. Positive controls were included in each PCR run except for VEBbla, PERbla, GESbla.Phenotypically ESBL positive isolates and the extracted total DNA from the ten soils were screened by PCR for the All new data has been deposited in GenBank with the accession numbers KC916938, KC916939, KC916940.CAGGCCTAACACATGCAAGTC 3′ and Bact_1389r 5′ ACGGGCGGTGTGTACAAG 3′ TCCTACGGGAGGCAGCAGT 3′ and 16SR 5′ GGACTACCAGGGTATCTAATCCTGTT 3′. 16S rRNA gene amplicons were sequenced and classified using Greengenes The 16S ribosomal RNA (rRNA) genes of all isolates were amplified using bacterial 16S primers: Bact_63f 5′ Escherichia coli ATCC 25922 or Staphylococcus aureus ATCC 25923 inocula were prepared to a 0.5 McFarland standard and 80 µl was spread on LB agar plates containing 20 µg/ml of penicillin, cefotaxime, kanamycin, tetracycline, gentamicin, amikacin, colistin, ciprofloxacin, levofloxacin, nalidixic acid, trimethoprim, rifampicin, erythromycin, vancomycin, streptomycin or chloramphenicol. Wells were cut aseptically in the antibiotic agar. Plugs corresponding to the same diameter as the wells were cut aseptically from the resistant bacteria plates and inserted into the wells of antibiotic containing plates. The plates were incubated at 37°C for 1–2 days. A circle of growth around the plugs, which reduced in cell density in relation to its distance from the plug, indicated antibiotic inactivation.The enzyme inhibition assays were performed using a plug assay. Figure S1The antibiotic resistance profiles segregated according to soil for all antibiotics. The antibiotics are color coded according to class.(TIF)Click here for additional data file.Figure S2The numbers of antibiotics each soil bacterial community is resistant to as a percentage of the total bacterial popoulation within the given soil.(TIF)Click here for additional data file.Figure S3The quinolone antibiotic resistance profiles of the soil bacteria separated according to soil sample.(TIF)Click here for additional data file.Figure S4The aminoglycosides antibiotic resistance profiles of the soil bacteria separated according to soil sample.(TIF)Click here for additional data file.Figure S5The tetracycline antibiotic resistance profiles of the soil bacteria separated according to soil sample.(TIF)Click here for additional data file.Figure S6The colistin antibiotic resistance profiles of the soil bacteria separated according to soil sample.(TIF)Click here for additional data file.


Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the selective and progressive loss of upper and lower motor neurons (MNs) of the brainstem, spinal cord and cerebral cortex. Key discoveries in the field of ALS have exponentially increased since it was first described in 1869 by Jean Martin Charcot, and the identification in 1993 of the first ALS-causing mutations in the gene encoding the well-studied antioxidant enzyme Cu, Zn superoxide dismutase (SOD1). The etiology of sporadic ALS largely remains unknown and the mechanisms of MN degeneration are still being investigated. The only FDA drug approved for the treatment of ALS, riluzole only modestly benefits patients, but multiple drugs are currently in the development pipeline and in human ALS clinical trials. It has become obvious that the lack of understanding of the precise mechanisms of motor neuron degeneration presents a major obstacle in the development of effective therapies for ALS. Altered calcium homeostasis and calcium signaling pathway activation is one potential mechanism that accounts for at least three major and interrelated toxic pathways: oxidative stress, mitochondrial dysfunction and neuroinflammation in neurodegenerative diseases such as ALS.2+ homeostasis and mitochondrial signaling pathways in selectively vulnerable MNs in ALS. In fact, recent evidence suggests that abnormalities in cellular Ca2+ signaling are common features in the pathogenesis of a range of neurodegenerative disorders, including ALS. It is well known that Ca2+ is one of the most relevant intracellular messengers, being essential in neuronal development, synaptic transmission and plasticity, as well as in the regulation of various metabolic pathways in the brain level. In subtypes of ALS associated with the SOD1 mutation and the sporadic disease, there have been several reports indicating that the involvement of mitochondria in pathogenesis includes the generation of intracellular free radical species, ultrastructral changes in mitochondrial morphology, swollen and vacuolar mitochondria and increased activity of complex I, III and IV in the Upper and lower MNS, frontal cortex and spinal cord . These findings are in good agreement with a quantitative comparison of Ca2+ homeostasis where low cytosolic Ca2+ buffering capacity acts as an important risk factor for degeneration and in contrast an increase in cytosolic Ca2+ buffering capacity could protect vulnerable MNs from degeneration both in-vitro and in-vivo. Several lines of evidence support altered Ca2+ homeostasis leading to MN degeneration in ALS, notably disturbance of glutamate neurotransmission and subsequent glutamate triggered Ca2+ entry, increased extracellular glutamate levels probably due to reduced glial glutamate uptake caused by oxidative damage to EAAT2, study of fALS in cell lines and animal mouse models where potential mechanism for Ca2+disruption is inhibition of glial glutamate transport by mSOD1 similar to those proposed for sALS. Evidence also suggests that ROS generated in MNs can cross the plasma membrane and cause oxidative disruption of glutamate transporters in neighbouring astrocytes. However, in ALS pathology, the nature of dysfunction is highly controversial after recent findings where non-cell autonomous effect of glia on MNs in an embryonic stem cell-based ALS model and astrocytes expressing ALS-linked mSOD1 that release factors selectively toxic to MNs was shown. The data currently available so far leave several important questions unanswered; these include: (1) does the presence of mSOD1 cause morphological abnormalities of mitochondria when expressed at physiological levels? (2) Does the mitochondrial Ca2+ sequestration source specificity and spatiotemporal properties of [Ca2+]i signaling varies at physiological levels? (3) What are the functional consequences of any changes in mitochondrial function on Ca2+ homeostasis in the presence of mutant SOD1 gene?Further evidence for the involvement of disruption of intracellular Ca2+ homeostasis are prominent (Jaiswal and Keller, 2+ homeostasis we believe that MN possess large number of voltage and ligand gated Ca2+ channels that, when activated, cause rapid Ca2+ influx, which, in part because of relatively weak cytosolic Ca2+ buffering, results in mitochondrial Ca2+ overload and strong ROS generation. Alternatively, the extrusion mechanisms, characterized by the plasma membrane calcium ATPase, Na+/Ca2+ exchanger and accumulation by intracellular organelles contribute to the removal of calcium ions. Furthermore, their selective loss goes a long way explaining the oxidative damage, mitochondrial abnormalities and apoptotic contributions observed in ALS MNs. Chronic mitochondrial membrane depolarisation due to Ca2+ entry can cause the release of pro-apoptotic proteins and activate enzymes involved in apoptotic pathways.Combining the lessons from multiple animal models as well as cell culture model used to determine pathogenic mechanisms in ALS, the central insight is that selective vulnerability of MNs likely arises from a combination of several mechanisms; two of them, mitochondrial dysfunction and Ca2+ homeostasis, continuous activity-dependent Ca2+cycling and the predominant role of mitochondria in buffering Ca2+ transients. Definitive evidence for this will have to wait for further studies. Nevertheless, if Ca2+ homeostasis and mitochondrial defects were found to play a role in disease pathogenesis, this would have broad ranging implications for therapy. Further studies will therefore be of considerable interest in attempting to understand the pathogenesis of cell death in ALS.In the last few years, attention was drawn to the role of mitochondria as an efficient regulator of cytosolic calcium signals. Further studies with mitochondria targeted calcium probes indicate a rapid, dramatic increase in free intramitochondrial calcium. This uptake by mitochondria has an immense effect on the metabolic state of the cell as it can up regulate the activity of the enzymes in oxidative metabolism. The malformations in mitochondrial structure and massive vacuoles derived from degenerating mitochondria found in post mortem human ALS samples further strengthen the proposition. Although the exact molecular mechanism is still not known, we hypothesize that vulnerability to ALS is a consequence of specific physiological features, particularly highly specialized Ca


The incidence of heart failure dramatically increases with aging. An important feature of cardiac aging is an increase in susceptibility to stress, such as ischemia and hemodynamic overload . The heaet al. showed that Sirt3 plays an essential role in fighting against cardiac aging and increases the tolerance of the heart against hemodynamic overload [In a recent publication, Hafner overload . Sirt3 bet al., where the authors proposed that Sirt3 deacetylates FoxO3 in the cytosol and induces nuclear translocation of FoxO3, which promotes transcription of anti-oxidants [Cardiac hypertrophy is often accompanied by oxidative stress, which causes oxidation and dysfunction of mitochondrial proteins, leading to further increases in mitochondrial oxidative stress through an amplification process called reactive oxygen species (ROS)-induced ROS release. Transition from hypertrophy to heart failure is almost always accompanied by increased oxidative stress and mitochondrial dysfunction, which trigger another vicious cycle consisting of oxidative stress, mitochondrial dysfunction and cardiac hypertrophy/dysfunction. This often makes it difficult to determine whether a given intervention acts primarily on cardiac hypertrophy, oxidative stress, or mitochondria to improve overall cardiac function, since all are affected concomitantly. In this regard, the fact that Sirt3 localized in mitochondria directly prevents mPTP opening by modulating cyclophilin D strongly suggests that Sirt3's primary action is to suppress mPTP opening and consequent mitochondrial dysfunction, and that the suppression of cardiac hypertrophy could be secondary to the suppression of mitochondrial/cardiac dysfunction . The facJudging from the fundamental involvement of mPTP opening in myocardial cell death and the development of heart disease, modulation of Sirt3 could be an important modality for medical treatment of heart failure. Sinclair and colleagues have shown that the NAD+ biosynthetic enzyme Nampt plays an important role in maintaining NAD+ content in mitochondria, and that its protective effects are mediated through Sirt3 and Sirt4 .It remains to be shown how the activity of endogenous Sirt3 is regulated by aging in the heart. If the activity of Sirt3 goes down with aging, it could itself be a cause of aging, and this process might be reversed by either increasing mitochondrial NAD+ content (with dietary restriction or exercise) or stimulating expression of Sirt3 (for example by inhibiting angiotensin II type I receptors) . HoweverTransgenic expression of catalase in mitochondria extends lifespan, retards cardiac aging, and confers stress resistance to the heart . We have


Infectious complications have been known to be a major cause of morbidity and mortality in Chronic Lymphocytic Leukemia (CLL) patients who are prone to infections because of both the humoral immunodepression inherent to the hematologic disease and to the immunosuppression related to the therapy. The majority of infections in CLL patients treated with alkilating agents is of bacterial origin. The immunodeficiency and natural infectious history of alkylator-resistant, corticosteroid-treated patients appears to have changed with the administration of purine analogs, which has been complicated by very severe and unusual infections and also more viral infections due to sustained reduction of CD4-positive T lymphocytes. The subsequent introduction of monoclonal antibodies in therapies, in particular alemtuzumab, further increased the immunodepression, increasing also infections which appeared more often in patients with recurrent neutropenia due to chemotherapy cycles.Epidemiological data regarding fungal infections in lymphoproliferative disorders are scarce. Italian SEIFEM group in a retrospective multicentre study regarding CLL patients reported an incidence of mycoses 0.5%; however, chronic lymphoproliferative disorders emerged as second haematological underlying disease after acute leukemia in a French study on aspergillosis; in particular CLL with aspergillosis accounted for a third of these chronic lymphoproliferative diseases presenting mould infection. Infectious complications have been known for many years to be a major cause of morbidity and mortality in Chronic Lymphocytic Leukemia (CLL) patients. Indeed, they account for the leading cause of death in most series.2Patients with CLL are predisposed to infections because of both the humoral immunodepression related to stage and duration of CLL, and to a further immunosuppression related to therapy with steroids, cytotoxic drugs and monoclonal antibodies.Streptococcus pneumoniae, Haemophilus influenzae) are the predominant pathogens in patients with this disease, but in neutropenic post-chemotherapy phase also Staphilococcus aureus and various Gram-negative enteric patogens such as Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae can be responsible for bacteraemia and septicemia, especially in patients with hypogammaglobulinemia.3Encapsulated bacteria  have been reported rarely, especially in patients with a previous history of infection, because in CLL patients the use of steroids scheduled in chemotherapy cycles or prescribed for autoimmune episodes ( immune haemolytic anemia and/or thrombocytopenia) is an important risk factor for this infection. Mycobacterial infection prevalence in CLL patients was 88 cases/ 10.000 patients.15Mycobacterial infections . Also defects in natural killer (NK) cell activity have been reported.The absolute neutrophil count and neutrophil function are usually normal in untreated CLL patients; neutropenia becomes pathogenetically more important in advanced disease and with more intensive chemotherapy utilization, predisposing these patients to bacterial and fungal infections. Neutrophils and monocytes are defective in their phagocytic function as well as in migration and chemotaxis.21Enzyme deficiency  was found in neutrophils of some, but not all, patients.Heath et al.The risk of infection usually increases with the length of disease, in relation to the natural history of the same and to previous therapies undertaken; both these factors can reduce immunoglobulin level. The risk of a severe infection is 26% at 5 years; the risk increases to 57% in patients with profound hypogammaglobulinemia, and to 68% in patients with profound hypogammaglobulinemia and Binet C stage of disease.2There is a significant correlation between the stage of disease and infections ; infectious episodes are not only more frequent, but also more severe in patients in stage C (82%) rather than stage A (33%).16Patients with stage C are a selected group predisposed to infections, in which the severe defect of immune response plays a critical role for infectious morbidity and mortality.Infections are the major cause of death in CLL patients. In a large 24-years observational study of M.D. Anderson Cancer Center 30% of patients died because of infection;In a review of 518 patients treated with chlorambucil alone, fludarabine alone or with both drugs, chlorambucil-treated patients had fewer major bacterial infections and viral infections than fludarabine treated patients.25In more recent randomized trials,28Opportunistic infections are very rare in patients treated with chlorambucil, and only if multiple lines of chemotherapy had been previously administered. A sporadical report of disseminated atypical mycobacteriosis was reported in a CLL patient chronically treated with both prednisone and chlorambucil.29Bendamustine is an alkilating agent recently introduced in the treatment of CLL and able to induce a high number of remissions. In a trial of first-line therapy regarding 319 CLL patients, bendamustine was shown to be more effective than chlorambucil.The discovery of the purine analogs heralded a new era of CLL therapy. Fludarabine, cladribine and pentostatin have all been shown to be active in CLL, inducing up to 20% of complete response in a pivotal trial;6Fludarabine is very active against indolent lymphoid neoplasms; its efficacy is due to its ability to reduce the number of lymphoid cells rapidly, originating also some cases of tumor lysis syndrome.Profound and prolonged suppression of the CD4 cell count occurs, with median CD4 cell counts decreasing <200/mL in 2–3 months of therapy. Although the CD4 cell count improves in the first 3 months from the end of treatment, quantitative abnormalities may persist for 1–2 years. This immunosuppression and neutropenia secondary to therapy determined an increased number of infections, particularly by opportunistics, also in the absence of neutropenia or steroid therapy. Despite the reduced CD4 cell count, the haematologic and clinical response to purine analogues increases macrophage cell activity and often also hypogammaglobulinemia; for this reason infections are more frequent at the beginning of the disease and decrease with improvement of CLL.Pneumocystis jiroveciii (33%), other mycoses (30%), Listeria monocytogenes (14%), also after many months from completion of therapy, mycobacteria (9%), CMV (7%), Herpes (6%). The majority of these infections doesn’t appear to be related to neutropenia or low levels of immunoglobulins, even if neutropenia in our experience seems to increase the risk of mould infections.In a review of the literaturePneumocystis jirovecii pneumonia, one cryptococcal bronchitis, one cryptococcal meningitis, one Vibrio sepsis, one Strongyloides, and one CMV infection.In more recent years three randomized trials in previously untreated CLL patients 33–35) showed that combined chemotherapy with fludarabine and cyclophosphamide (FC) was superior to fludarabine alone. However, hematologic toxicity was higher than with the monotherapy, but no difference was observed in severe infections by fludarabine alone compared with FC.–35 showeCladribine, another purine analog, was used alone or combined with other agents in CLL patients with efficacy similar to fludarabine. In a recent trial comparing cladribine plus cyclophosphamide with fludarabine plus cyclophosphamideRituximab is an anti-CD20 monoclonal antibody directed against CD20 antigen present on CLL cells. After rituximab all patients show a rapid decrease in B-lymphocytes, while T-cells remain stable; B-lymphocytes return to normal levels usually within 12 months. Addition of the CD20 monoclonal antibody rituximab to chemotherapy for CLL has improved outcomes, particularly in early lines of therapy. The activity of single-agent rituximab in CLL is modest at standard doses with ORR from 15% to 25%. The major impact of targeting the CD20 has been shown in combination with conventional chemotherapy. When rituximab was added to chemotherapy regimens in chronic lymphoproliferative disorders, infectious complications were comparable to chemotherapy-alone regimens. In particular when this monoclonal antibody was added to standard fludarabine regimen at usual dosage of 375 mg/mq/day, infection did not show an increase.In the current use of rituximab unusual infections, in particular viral infections, have been rarely seen, such as some cases of progressive multifocal encephalitis,The humanized monoclonal antibody alemtuzumab binds to CD52 antigen, which is present in B and T lymphocytes, monocytes, macrophages and eosinophils, but not in progenitor cells.Single-agent alemtuzumab showed good efficacy both in first-line therapy and in relapsed/refractory patients and also in combination therapy. However this monoclonal antibody is frequently accompanied by serious complications, in particular cytopenia and subsequent infections. The profound and long-lasting lymphocytolytic activity determined by alemtuzumab is responsible for severe and prolonged immunodepression which produces a major predisposition also to opportunistic infections. In fact, the risk and the type of infection could be different according to indications and doses; its association with chemotherapy, the concomitant administration of immunosuppressive drugs, the characteristics of CLL itself can greatly change the severity of immunodepression, favouring those particular opportunistic infections associated with a low CD4 cell level.In a first-line study comparing alemtuzumab with chlorambucil,erpes simplex virus reactivation was noted in 11 patients and oral candidiasis in five. Other infections in this trial, which did not incorporate antimicrobial or antiviral prophylaxis, included Pneumocystis jirovecii pneumonia (n=2), bacterial pneumonia (n=4) and septicemia (n=3). All patients recovered. A higher percentage of infectious episodes were confirmed also in the pivotal trial using alemtuzumab, which included 93 patients treated at 21 centres in USA and EuropeHerpes simplex was present in 6 cases. CMV reactivation in 7 patients caused concern. Eleven patients, all of whom with advanced disease, developed opportunistic infections during treatment and further 7 in the follow-up period . Six of 9 deaths were due to infections. Responders seemed to experience fewer infections than non-responders. Similar results were obtained in another studyInfectious complications were more frequent in patients who were heavily pretreated or fludarabine-refractory and who did not respond to the same alemtuzumab therapy.Various chemoimmunotherapy combination regimens including alemtuzumab are currently becoming more common therapeutic choices for patients with CLL. Translation of single-agent alemtuzumab into combination regimens has led to variable efficacy but also alteration of the anticipated infectious toxicity profile. Among the combination regimens fludarabine in combination with alemtuzumab (FluCam regimen) was first given to refractory CLL patients,Alemtuzumab is also administered as consolidation treatment to clear minimal residual disease. The study of Montillo and colleaguesListeria meningitis, Legionella, CMV and Pneumocystis jirovecii pneumonia), which occurred up to 7 months after the last therapy. The study was amended to exclude CR patients to receive alemtuzumab.However, a recent CALGB study reported severe infections in patients treated with alemtuzumab as consolidation therapy after induction with fludarabine and rituximab.In all studies the problem of CMV reactivation related to alemtuzumab administration is recurrent, due to loss of CD8 cytotoxic T cells after lymphocyte-depleting treatment regimens. While CMV reactivation is relatively high in CLL patients treated with alemtuzumab (30–40%), symptomatic CMV infection is rarer and appears in 15% in first–line therapyIn summary, the characteristics and the stage of hematologic disease, in particular refractory disease, play an important role for determining an increase of alemtuzumab-related infections, in particular opportunistic infections. Instead a good response of malignancy to induction therapy such as a less aggressive administration schedules of consolidation seem to determine a lower risk of infection.9/L and stable during the course of treatment. Among 37 grade 3 and 4 infections, pneumonia (14 cases) and other respiratory tract diseases (6 cases) were the most common. Thirteen patients died due to infections, including sepsis (6), pneumonia (5), Fusarium mycosis (1), and progressive multifocal encephalopathy (1), which occurred in a patient with low CD4 count at baseline. Finally the incidence of major infections compared favourably with that of similar patients treated with other salvage regimens (56), which showed up to 60% of major infections and 13% of early deaths and emphasizing the need for prospective evaluation of optimal infection prophylaxis in heavily pretreated patients.Ofatumumab is a fully humanized antibody targeting a distinct epitope on the CD20 molecules of CLL cells. It shows more potent complement-dependent cytotoxicity than rituximab, also in cells with low CD20 expression levels, including CLL cells. In an international multicentre studyOfatumumab was also used as front-line therapy in untreated CLL patients in a randomized, 2-doses phase 2 trialThe prevention of infections is very important in CLL patients undergoing innovative therapies.Pneumocystis jiroveci pneumonia in pretreated patients undergoing fludarabine and/or alemtuzumab is of current use, because the incidence of pneumocystosis can be as high as 7% in this patient population.Prevention of The administration of prophylactic intravenous immunoglobulin is not recommended to be used as routine. HD Ig should be performed only in selected cases of severe, recurrent bacterial infectious episodes due to encapsulated bacteria60Considering that only small series or sporadic cases of mycobacterial infections have been described,Herpes viruses and is usually performed as prophylaxis in patients treated with fludarabine and /or monoclonal antibodies (400 mg twice daily), particularly in patients with previous herpetic infections (800 mg twice daily).Acyclovir is active against CMV should be suspected and carefully monitored with antigenemia and/or PCR during and after alemtuzumab treatment, particularly in patients who are CMV seropositive at baseline; only symptomatic infection should be treated. Patients presenting with FUO should be tested promptly, also if negative at baseline. In symptomatic cases ganciclovir (5 mg/kg BID) should be performed as first-line treatment for 14–21 days and alemtuzumab suspended until negative CMV status is obtained.Reactivation of Aspergillus spp have been reported only sporadically in patients treated with both fludarabine and alemtuzumab,General antifungal prophylaxis is not recommended. However, antifungal prophylaxis with fluconazole should be considered, only if mucositis and fungal colonization are present, or during prolonged neutropenia episodes. The suboptimal response to vaccination in CLL patients may be related to impaired antibody production. Anti-pneumococcal vaccination should be recommended, also in presence of a reduced response. The response of CLL patients to influenza vaccine has also been studied;Knowledge of the range of infections  and incr


Examination of the cellular components of BAL and lung revealed neutrophilic infiltrate early that resolves with a dominant macrophage component during the resolution phase of inflammation. Further, genechip analysis of lungs from mice exposed to acute smoke demonstrated that 58 unique genes were significantly elevated in response to tobacco smoke and that many are macrophage associated genes observed in respiratory disease.Steroid insensitive inflammation is implicated with respiratory diseases such as Chronic Obstructive Pulmonary Disease (COPD), and asthma. One of the barriers to effective target screening in this area is the lack of in vivo models that are both relevant and of short duration. We present here our work developing an in-house model for steroid insensitive inflammation that involves the use of a major causative agent of neutrophilic inflammation and respiratory disease, tobacco smoke. We examined the effects of 4 days of cigarette smoke exposure in both Balb/c and C57BL/6 mice. Our investigations of bronchoalveolar lavage (BAL) fluid, lung tissue, and cellular analysis in our murine model indicate strain specific effects in response to acute tobacco smoke and that the neutrophil and macrophage associated cytokines such as KC/GRO, IL-1b, MCP-1, and IL-17A are significantly elevated in the model. Further, acute smoke induced inflammation while unresponsive to steroids such as dexamethasone, is also unresponsive to modulation by known neutrophilic chemotactic mechanisms such as LTB


Lung cancer is the leading cause of cancer deaths worldwide among both men and women, with more than 1 million deaths annually. Non–small cell lung cancer (NSCLC) accounts for about 80% of all lung cancers.Although recent advances have been made in diagnosis and treatment strategies, the prognosis of NSCLC patients is poor and it is basically due to a lack of early diagnostic tools.However, in the last years genetic and biochemical studies have provided more information about the protein and gene’s mutations involved in lung tumors. Additionally, recent proteomic and microRNA’s approaches have been introduced to help biomarker discovery.Here we would like to discuss the most recent discoveries in lung cancer pathways, focusing on the genetic and epigenetic factors that play a crucial role in malignant cell proliferation, and how they could be helpful in diagnosis and targeted therapy. Lung cancer is the leading cause of cancer-related deaths over the world, among both men and women, with an incidence of over 200000 new cases per year and a very high mortality rate. It is responsible for more deaths than breast, colon and prostate tumors combined .Lung tumors can be divided into two histological groups: non-small cell lung cancer (NSCLC) (80.4%) and small cell lung cancer (SCLC) (16.8%) . NSCLC, Many factors potentially contribute to lung cancer formation, e.g. tobacco smoke, ionizing radiation and viral infection, although the mechanisms involved in lung carcinogenesis remain largely unknown.Lung cancer is often suspected on the basis of abnormal chest imaging and/or non-specific symptoms. Bronchoscopy is generally used as an initial diagnostic tool, consisting of cytopathologic examination of bronchoalveolar lavage, endobronchial brushings and biopsies from the suspect area. Despite this procedure being specific for lung cancer, its sensitivity is low . TherefoThe overall five-year survival rate for patients with NSCLC is less than 15% and has remained largely unchanged for the last three decades. Despite new drugs and therapeutic regimens, surgical resection remains the most promising chance for the ~25% of patients who present early-stage disease (stage I-IIIA), although 65% will relapse within two years [These data suggest that strategies for the early detection of lung cancer, aimed at identifying the tumor at a stage in which it is small and locally defined, are urgently needed to increase significantly the chances of a cure . Recent Another major problem linked to lung cancer disease, as for other cancers, is the need to find new and more tailored chemotherapeutical.The variable response to chemotherapy with platinum-based regimens in NSCLC is well recognized clinically. Patients crossing from one regimen to another in clinical trials commonly show responses , suggestIn this short review we summarize what are the main genetic alterations involved in lung cancer disease and what is the prospective for the future development of new biomarker and diagnostic tools to improve the early detection of lung cancer. Different factors contribute to lung cancer formation: tobacco smoke, ionizing radiation and viral infection are among the most well established. However, the mechanisms involved in lung carcinogenesis remain largely unknown to date .As most other cancers, several genes are involved in lung cancer disease , which iKRAS is often mutated and is responsible for 10–30% of lung adenocarcinomas [The proto-oncogene rcinomas , 12.	MYC and Cyclin D1 [yclin D1  are amplBRAF were identified through systematic resequencing of oncogenes and are present in about 2% of adenocarcinoma patients and restricted to tumors that did not show KRAS mutations [ERBB2  or BCL2 over-expression are involved in 25% of cases . Fartherutations , 18. The epidermal growth factor receptor (EGFR) that regulates cell proliferation, apoptosis, angiogenesis and tumor invasion  is over-Basically, mutations involving EGFR, ERBB2 and KRAS are mutually exclusive and are thought to represent early events in the carcinogenesis of lung adenocarcinoma in never (EGFR and ERBB2) and current smokers (KRAS) .MET, NKX2-1, and PIK3CA [Other oncogenes whose expression has been found altered in lung cancer, include d PIK3CA . The tyrosine kinase protein, SRC is also overexpressed and activated in epithelial tumors, and the levels of expression or activation generally correlate with disease progression , althougALK) with the echinoderm microtubule-associated protein-like 4 (EML4) has been identified in a subset of NSCLC. Approximately 5% of all NSCLC cases contain an EML4-ALK translocation. It occurs in mutual exclusion to EGFR and KRAS mutations and is associated with nonsmokers. Since ALK tyrosine kinase activity is oncogenic both in vitro and in vivo, new ALK kinase inhibitors are being evaluated in pre-clinical trials for lung cancer [Recently, the fusion of the anaplastic lymphoma kinase  research-field has been extensively studied and it may also lend insights into the biology of lung cancer, as well as for cancer in general.MiRNAs are small non-coding RNAs about 22 nt long that play key roles in gene expression regulation by modulating the translation and degradation of target mRNAs through base pairing to partially complementary sites -55.MmiRNA microarray analysis for lung cancer were recently examined to investigate miRNA involvement in lung carcinogenesis and the results obtained show that miRNAs could discriminate lung cancers from healthy lung tissues, suggesting that miRNA expression profiles could be diagnostic and prognostic markers of lung cancer  and alsoDNMT 3A and -3B. In particular, it has been shown that expression of miR-29 family members is inversely correlated with DNMT3A and -3B expression in lung cancers and that these miRNAs down-modulate expression levels of both enzymes. Furthermore, enforced expressionof these miRNAs in lung cancer cells leads to reduced global DNA methylation, restores expression of TSGs, and inhibits tumorigenicity both in vitro and in vivo [Recent studies have reported some examples of miRNAs implication in lung cancer. For example miR-29 family members directly target both DNA methyltransferases  in vivo .Another important report shows that miR-107 and miR-185 are down regulated in lung cancer compared with normal lung and their reintroduction in NSCLC cell lines is able to suppress cell growth . SimilarFarthermore, we must remember the role of telomere and telomerases that recently have been demonstrated to be involved in lung cancer.Telomeres are nucleoprotein complexes located at the end of eukaryotic chromosomes, which role is to prevent them from degradation, end to-end fusion and rearrangement.Recently, telomere length has been proposed as prognostic factor in NSCLC, reflecting indirectly chromosomal instability.Telomerase and telomeric complex play a key role in lung tumor progression. As telomere maintenance is essential to tumor cell proliferation, several approaches have been developed to target either telomerase or telomeric complex. Anti telomerase strategies can either target hTERT or hTERC, in addition to modulation of telomerase regulators at the transcriptional and post-transcriptional levels. More recently, the use of specific ligands leading to G quadruplex telomeric structure stabilization and therefore to limitation of telomerase accessibility to its target appears as a promising area of development .Lung cancer is the most prominent cause of cancer-related mortality worldwide. About 60% of those diagnosed with lung cancer die within one year after diagnosis and the five-year survival for all patients with lung cancer is only 16%, a percentage that has not improved significantly in the past 10 years [Thorax computerized tomography (CT) and positron emission tomography (PET) are non-invasive techniques used to detect lymph node involvement and histological sampling of lymph nodes, but often these techniques are not sufficient and thus invasive techniques such as transbronchial needle aspiration (TBNA), needle aspiration by endobronchial ultrasound (EBUS) and mediastinoscopy are frequently used to recover histological samples from lymph nodes . Since pRecent studies provided first evidence for the potential usefulness of a blood-based test for lung cancer diagnosis .For example the mutational status of EGFR can be readily detected in primary tumors and the correlation between EGFR mutations and EGFR TKI (Tyrosine Kinase Inhibitors) sensitivity has been validated in several clinical trials, but it may be difficult to obtain tumor tissues for such analysis. Therefore, since plasma samples of patients with NSCLC often contain circulating DNA derived from tumor tissues, plasma samples have been used for detecting genetic alterations, in particular for EGFR . The research of new biomarkers is important not only for improving diagnosis but also it may offer promise in optimizing treatment. Understanding the genetic mechanisms affecting drug activity and response to treatment is a major challenge for establishing an individualized chemotherapy. To date, cisplatin and platinum-containing drugs are routinely used for treatment of NSCLC and are known to have vital role.DNA adducts, due to cisplatin treatment, are mainly repaired by nucleotide excision repair pathway and proteins such as excision repair cross complementing 1 (ERCC1), ribonucleotide reductase subunit M1 (RRM1) and breast cancer susceptibility gene 1 (BRCA1) have an important role in this process. Recently, ERCC1, RRM1 and BRCA1 and TS (Thymidylate synthase)  have beeBut to date more than two thirds of lung cancer tumors are diagnosed at late stages when the survival rate is low  and thusLung cancer is the leading cause of death over the world and the only chance of cure for patients affected from this kind of cancer is surgical resection.This is mainly due to the fact that several factors are involved in lung cancer develoment and progression and to date the diagnostic methods available for an early and efficient detection are not sufficient.Although lung cancer research data have accumulated dramatically during the past several years, there is no database specifically focusing on lung cancer molecular biology available yet.In this short review we have summarized some of the factors contributing to lung cancer, but it is just an overview of the most important proteins involved in lung cancer disease, which are often mutated or present an unusual pattern of expression compared to the healthy tissue.All together these data are important for understanding the nature behind this type of cancer and also to understand that basic research on proteins, miRNAs and all the other epigenetic modifications could be used to develop more powerful diagnostic tools, as well as prognostic or predictive markers or even for the development of new targeting therapies for lung cancer.


Finally, our molecular dynamics computer simulations indicate that the local breathing dynamics of the PPARG promoter DNA coincides with the gene specific response to the THz radiation. We propose that THz radiation is a potential tool for cellular reprogramming.We report that extended exposure to broad-spectrum terahertz radiation results in specific changes in cellular functions that are closely related to DNA-directed gene transcription. Our gene chip survey of gene expression shows that whereas 89% of the protein coding genes in mouse stem cells do not respond to the applied terahertz radiation, certain genes are activated, while other are repressed. RT-PCR experiments with selected gene probes corresponding to transcripts in the three groups of genes detail the gene specific effect. The response was not only gene specific but also irradiation conditions dependent. Our findings suggest that the applied terahertz irradiation accelerates cell differentiation toward adipose phenotype by activating the transcription factor peroxisome proliferator-activated receptor gamma  Terahertz (THz) radiation occurs ubiquitously in our environment, as part of the solar spectrum and through the natural black-body radiation within the earth's atmosphere. Despite this abundance, the non-ionizing character of this radiation, and the lack of practical and powerful THz emitters, has left the biological significance of this region of the electromagnetic spectrum relatively unexplored. Interestingly, the energy scale of THz radiation is within the range of hydrogen bonds, van-der-Waals interactions, and charge-transfer reactions. This energy overlap is associated with the unique sensitivity of the emerging THz techniques in vitro and in vivo studies were mostly conducted at frequencies below 0.15 THz, at low power, and with short exposure times (10–30 min), and did not provide conclusive evidence regarding the influence of THz radiation on mammalian cells. Further, results from the multi-national “THz-bridge” project aimed at investigating the interaction of THz radiation with biological systems in vitro to powerful THz radiation (over 30 mW/cm2), at a specific frequency, develop infringements on the morphology of the cellular membranes and intracellular structures Unfortunately, the available data related to the influence of THz radiation on biological systems, and the understanding of the precise mechanisms governing this influence, are limited and the subject of debate Here we report that extended exposure to broad-spectrum THz radiation (centered at ∼10 THz) results in specific  changes in the functionality of cellular DNA. Certain genes in irradiated mouse stem cell (MSC) cultures are activated, while other genes are repressed. Many of the MSC genes do not respond to the selected radiation conditions at all, showing that the effect is specific. Additionally, 9 hours of exposure causes significant changes in the MSC gene expression, while the response to shorter duration (2 and 4 hours) is appreciably less pronounced. Hence, we argue that the effect of THz radiation is gene and exposure specific and most likely is at the level of DNA transcription. In this context, our EPBD-based 2. To exclude any external variations in the growth conditions, unrelated to the THz radiation, two MSC culture dishes, control and experimental, were placed next to each other during the THz treatment  have been reported in an experiment with the eukaryotic microorganism To validate the array data, namely that the response to the radiation is gene specific, we measured quantitatively the messenger RNA (mRNA) levels using RT-PCR. With selected available gene probes, corresponding to transcripts from the three groups of genes identified in the array experiment, we confirmed the observed gene array results . Four ofThe observed accumulation of mRNA depends on the duration of exposure. The accumulation of adiponectin, GLUT 4, FABP4 and PPARG mRNA transcripts was evaluated by RT-PCR after 2, 4, and 9 hours irradiation. The results for the selected time frames revealed clear difference in the accumulation of specific mRNA transcripts (panel d). Significant changes in specific mRNA transcripts levels were measured only after 9 hours of exposure. The level of TBP transcripts did not change detectably and was used as an internal control for equal sample loading.PPARG and PPARA belong to the same group of nuclear receptor proteins, with overlapping activity as transcription factors, and a well-documented difference of transcriptional regulation of their DNA gene promoters THz radiation, with a fundamental period in the pico-second range, is uniquely suited to control functions in molecular systems of central importance for living organisms. Biologically important collective modes of proteins vibrate at THz frequencies and are therefore THz radiation sensitive Here we report that extended exposure to a broad-spectrum THz radiation results in specific rather than global changes in cellular functions that are closely related to the DNA-directed gene transcription. Our gene chip and RT-PCR survey on gene expression reveals that some genes in irradiated MSC cultures are activated, while other genes are repressed. Yet other genes do not respond to the selected radiation conditions at all, demonstrating the gene specific, rather than general, effect on gene transcription. Our experimental data further argues that the response is not only gene specific but also depends on the conditions of irradiation, as cell morphology and gene specific accumulation of RNA transcripts was dependent on exposure duration. Further, it is known that THz radiation affects biomolecules via interaction with the internal dipole moments Exposure of cells to THz radiation for 9 hours causes changes in gene expression, while in response to shorter duration of exposure the changes are significantly less pronounced. The appearance of lipid droplet-like inclusions that is a characteristic sign of MSC differentiation into adipocytes, was clearly visible after 9 hours exposure rather than in the typical time frame of several days. This may be explained by the observed significant elevation of the PPARG expression. This transcription factor is known to be required for transcriptional activation and expression of adipocyte genes including adiponectin and GLUT4 that produce the differentiated phenotype The observations of the variations of DNA transcription due to THz radiation support a correlation between specific THz radiation effects, the level of accumulation of gene specific RNA transcripts, and the natural nonlinear process of DNA transcription initiation. One particularly pronounced variation in gene transcription is between PPARA and PPARG. Our EPBD based Langevin molecular dynamic simulations show that the DNA propensity for local breathing at the PPARG promoter is significantly higher, and focused at the TSS compared to the noticeably dynamically silent PPARA. The correlation with the THz induced PPARG promoter activation is striking. This difference indicates that the selected irradiation parameters, and the breathing promoter signature, may together direct the functional response to THz irradiation. On the other hand a PPARA response may require an extended exposure, since DNA breathing with higher amplitudes is needed for the THz effect to take place via nonlinear resonances In addition to effects pertaining to the formation of DNA transient openings, it is also possible that DNA-protein interactions and transcription factor conformational changes could be induced by THz irradiation Further investigations involving a large number of genes and variations in the THz radiation characteristics as well as longer time exposure are needed to generalize these striking correlations between promoter-specific breathing dynamics, probability for THz induced nonlinear resonances, transcriptional response, and cellular behavior.Mouse mesenchymal stem cells  were cultured on tissue-culture treated plastic T-75 flasks (Corning). Once the cells reached 80–90% confluence, they were sub-cultured in supplemented medium  for THz irradiation treatment. Roziglitazone (1 µM), insulin (5 µg/ml), 3-Isobutyl-1-methylxanthine (100 µM), and dexamethasone (1 µM) were added to the medium 24 hours before irradiation.2, broadband THz radiation (∼10 THz) at a high repetition rate (1 kHz). Cells were irradiated in parafilm-sealed tissue culture plates in the presence of culture medium and at ambient temperature. Cells were irradiated for 2, 4, 6, and 9 hours in two replicates. Nonirradiated identical plates were used as a control in all experiments. Cell viability and morphology were monitored by light microscopy, 100x magnification (Nikon). Temperature of the cells during irradiation was monitored using an IR detector  this technique is capable of producing electromagnetic radiation at THz frequencies detector , and sepOur EPBD model is an extension of the classical Peyrard-Bishop-Dauxois nonlinear model Cellular RNA was extracted using the RNeasy Mini kit (Qiagen) following the manufacturer's instructions. For RT–PCR experiments, 600 ng total RNA was reverse transcribed using a cDNA Synthesis kit (Invitrogen) with random-oligo primers. For cellular qRT–PCR, 500 ng total RNA was reverse transcribed using the same conditions as above; 1/10 dilutions were used in triplicates with 0.2 µM gene specific primers and 5 µl LightCycler 480 SYBR Green I Master kit (Stratagene) in 20 µl reactions. qPCR was executed in a 96-well block Stratagene RTQPCR Mx3000 instrument. Gene specific primers were selected , synthesized, HPLC, and gel purified. The sequences of the primers have been published elsewhere A gene chip analysis assay was performed with a mixture of total RNA from cells that were irradiated for 9 hours in two independent experiments and the same procedure was performed separately for the control cells. The Affymetrix gene chip (GeneChip Mouse Genome 430 2.0 Array) hybridization procedure was conducted as recommended by the supplier (Affymetrix). The microarray data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus http://www.bioconductor.org) and the Significance Score (S-Score) algorithm The comparative CT method (Applied Biosystem) was used to analyze the data resulting from the RT-PCR experiments and the gene chip analysis. Statistically significant differentially expressed genes in the Affymetrix GeneChip® microarray data were identified via the Bioconductor software framework (


The potential applications of nanomaterials as drug delivery systems and in other products continue to expand. Upon introduction into physiological environments and driven by energetics, nanomaterials readily associate proteins forming a protein corona (PC) on their surface. This PC influences the nanomaterial’s surface characteristics and may impact their interaction with cells. To determine the biological impact of nanomaterial exposure as well as nanotherapeutic applications, it is necessary to understand PC formation. Utilizing a label-free mass spectrometry-based proteomics approach, we examined the composition of the PC for a set of four silver nanoparticles (AgNPs) including citrate-stabilized and polyvinlypyrrolidone-stabilized (PVP) colloidal silver (20 or 110 nm diameter). To simulate cell culture conditions, AgNPs were incubated for 1 h in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, washed, coronal proteins solubilized, and proteins identified and quantified by label-free LC-MS/MS. To determine which attributes influence PC formation, the AgNPs were characterized in both water and cell culture media with 10% FBS. All AgNPs associated a common subset of 11 proteins including albumin, apolipoproteins, keratins, and other serum proteins. 110 nm citrate- and PVP-stabilized AgNPs were found to bind the greatest number of proteins (79 and 85 respectively) compared to 20 nm citrate- and PVP-stabilized AgNPs (45 and 48 respectively), suggesting a difference in PC formation based on surface curvature. While no relationships were found for other protein parameters , the PC on 20 nm AgNPs (PVP and citrate) consisted of more hydrophobic proteins compared to 110 nm AgNPs implying that this class of proteins are more receptive to curvature-induced folding and crowding in exchange for an increased hydration in the aqueous environment. These observations demonstrate the significance of electrostatic and hydrophobic interactions in the formation of the PC which may have broad biological and toxicological implications. In biological environments, it has been recently established that nanomaterials interact readily with proteins, peptides, and lipids thus forming a protein corona (PC) on their surface In vitro toxicity studies have demonstrated that exposure to AgNPs can result in mitochondrial damage, reactive oxygen species production, cell-cycle arrest, apoptosis, and cytotoxicity Silver nanoparticles (AgNPs) have been incorporated as antibacterial/antifungal agents in medical devices, textile engineering, water treatment, and have been integrated into the surfaces of many household appliances and food storage containers in vitro techniques, which have often yielded inconsistent results. It is generally accepted that physicochemical characterization of the nanomaterial is needed to understand and predict toxicity, yet less attention has been given to the disparities between cell culture media and the subsequent effect on PC formation in the interpretation of in vitro results. An understanding of protein-nanoparticle interactions resulting in PC formation would not only benefit toxicity assessment by in vitro approaches, but also their usefulness in human health applications. Recently we demonstrated through label-free quantitative mass spectrometry that carbon nanotubes with different surface functionalization acquire different PCs following incubation in typical cell culture media containing fetal bovine serum proteins Screening the biocompatibility of nanoparticles typically utilizes a variety of In the current study, through the use of label-free quantitative mass spectrometry, we have characterized the formation of the PC on AgNPs of different sizes, 20 or 110 nm, and with different coatings, PVP or citrate. We utilized a comprehensive proteomics approach to determine the identities and abundance of proteins forming the PC on these AgNPs following incubation in commonly used cell culture media (DMEM) with 10% fetal bovine serum. Through the use of particles that share the same chemical composition, but vary based upon size and surface coating, insight can be gained regarding the effects of these characteristics on PC formation and, ultimately, its effects on AgNP-induced biological responses. Specifically, we examined differences in constituent or overall proteins found to associate with AgNPs (Section 2.2), and unique proteins which were found to associate only with a particular AgNP (Section 2.3).Citrate- and PVP-stabilized AgNPs were characterized for hydrodynamic size and zeta potential in suspensions of water or DMEM cell culture media without serum . DiameteNanoparticle bioactivity and toxicity is influenced through the addition of the PC in biological fluids by modifying the surface characteristics and thus the interface by which the cell interacts. The PC is a complex and dynamic entity which consists of two layers in general. The first layer is a hard corona which forms rapidly by the association of strongly bound proteins, which do not readily disassociate Proteomic analysis by label-free quantitative mass spectroscopy identified and quantified 133 different protein components of the various AgNP-PCs. A complete list of these proteins, along with mass spectral and quantitation data, can be found in Recently we identified 2,507 individual proteins, polypeptides, or protein fragments/isoforms in the 10% FBS-DMEM used in this study and determined individual peptide sequences and abundance data Many of the 133 proteins found to associate with AgNPs were shared; however, all AgNP-PC were found to share 11 common proteins . These pnd most for 20 nm AgNPs was serum albumin  whereas 60 were found to only bind to 110 nm AgNPs (PVP and citrate) . These hTo assess how characteristics of the nanomaterial govern the formation of the PC, we analyzed the correlation between the number of unique proteins found to bind with the hydrodynamic sizes and zeta potentials of each AgNP . We deteRecently, we examined the physiochemical interactions of a simplified PC by examining the association of an individual protein, bovine serum albumin, with 110 nm PVP- and citrate-stabilized AgNPs In a recent study, we described the PC of a variety of carbon nanotubes utilizing the same label-free quantitative mass spectroscopy approach used in our current study in vitro conditions and in vivo. These variations in conditions resulting in differential PC formation are likely to cause variability in the formation of the PC in individuals suffering from diseases treated with nanomedicines. Specifically these disease states may modify both serum chemistry and concentrations of individual proteins thus altering the PC and the therapeutic applications of the nanomedicine.It is likely that the PC on AgNPs and other nanomaterials may be significantly different based on the media they are suspended in. In the current study we utilized an extremely common cell culture media (DMEM) with 10% FBS. Other medias with different pH, salt concentrations, protein components, FBS concentrations, and other various characteristics may modify the individual constituents of the PC and their abundance within the PC. These alterations in the PC have been shown to alter the cellular uptake and activity of the nanomaterials in vitro toxicity testing are immense. In our current study, we have described the differences in the diverse protein composition between particles of identical chemical composition that differ based on size (20 and 110 nm) and coating (PVP and citrate). The interactions and association of proteins with the surface of the nanomaterials may also induce conformational changes to the proteins resulting in toxicity through modified immune responses. It is likely that these interactions with protein may also modify AgNP dissolution and thereby affect toxicity + leading to cell death. Ultimately, through a thorough understanding of the formation and composition of these PCs we may be able to mitigate toxicity and selectively utilize the PC to increase the therapeutic applications of nanomaterials.The implications of the PC on the eventual activity and fate of intravenously injected nanomaterials and the assessment of nanomaterials via dl-Dithiothreitol (DTT), urea, triethylphosphine, iodoethanol, and ammonium bicarbonate were purchased from Sigma-Aldrich . LC-MS grade 0.1% formic acid in acetonitrile and 0.1% formic acid in water were purchased from Burdick & Jackson . Modified sequencing grade porcine trypsin was obtained from Princeton Separations . Dulbecco’s Modified Eagle’s Medium (DMEM) with glutamax and 10% heat inactivated fetal bovine serum were purchased from Invitrogen (Carlsbad CA). AgNPs were obtained from nanoComposix  by the NIH Nanocharacterization Laboratory.The hydrodynamic size  and zeta potential  of all AgNPs were characterized in DI water and DMEM culture media without fetal bovine serum (FBS). Scanning electron microscopy was utilized to verify the diameters of the AgNPs.in vitro exposure protocols) in situ using a lysis buffer specific for label-free quantitative mass spectrometry (LFQMS) . For comparative reference purposes, 100 µg of FBS supplemented culture media proteins were also solubilized for LC-MS/MS analysis. Briefly, protein samples were reduced and alkylated by triethylphosphine and iodoethanol and proteolyzed using porcine trypsin Bos taurus (Bovine) database using SEQUEST (v. 28 rev. 12) algorithms in Bioworks (v. 3.3). Peptide and protein identifications were validated by PeptideProphet and ProteinProphet in the Trans-Proteomic Pipeline  Using a modification of Tenzer’s method, 1 mg of each AgNP type was suspended in 10 mL of DMEM culture media supplemented with 10% FBS, briefly sonicated in a bath sonicator, diluted 1∶10 in FBS/media, and incubated for 1 h at 37°C (to simulate Protein abundance was determined using IdentiQuantXL™ http://web.expasy.org/protparam/) . The GRAVY score for a peptide or protein is calculated as the sum of hydropathy values of all the amino acids, divided by the number of residues in the sequence. The aliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains  and may be regarded as a positive factor for increased thermostability of globular proteins.Grand average of hydropathicity (GRAVY) scores, aliphatic indices, and cysteine content for all identified proteins and peptides were calculated using the Protein Identification and Analysis Tools (ProtParam) on the ExPASy Server Click here for additional data file.Table S2Quantification of all proteins and peptides found to associate in the corona of each AgNP by label-free mass spectroscopy.(PDF)Click here for additional data file.File S1This file contains Figures S1 through S3. Figure S1, Evaluation of characteristics of constituent protein components found to associate with each AgNP after incubation in DMEM containing 10% fetal bovine serum. Average GRAVY score, isoelectric point, and aliphatic index for constituent proteins and peptides (1A). Average GRAVY score, isoelectric point, and aliphatic index for unique proteins and peptides (1B). Evaluation of cysteine residues present in constituent and unique proteins and peptides (1C). Figure S2, Correlation of the characteristics of individual protein components to protein quantity in the protein corona formed on 20 nm citrate and PVP suspended AgNPs. Characteristics correlated include GRAVY score, isoelectric point, and aliphatic index. Figure S3, Correlation of the characteristics of individual protein components to protein quantity in the protein corona formed on 110 nm citrate and PVP suspended AgNPs. Characteristics correlated include GRAVY score, isoelectric point, and aliphatic index.(PPTX)Click here for additional data file.


Quantitative and qualitative defects of Tregs have been postulated to contribute to failed immune regulation in multiple sclerosis (MS) and other autoimmune diseases. This paper highlights the potential uses of T regulatory cell epitopes (Tregitopes), natural Treg epitopes found to be contained in human immunoglobulins, as immunomodulating agents in MS. Tregitopes expand Treg cells and induce “adaptive Tregs” resulting in immunosuppression and, therefore, are being considered as a potential therapy for autoimmune diseases. We will compare Tregitopes versus intravenous immunoglobulin (IVIg) in the treatment of EAE with emphasis on the potential applications of Tregitope for the treatment of MS.The induction of immunologic tolerance is an important clinical goal in autoimmunity. CD4 H) cells and their mediators triggering injury of axons and their myelin sheaths through a complex sequence of events + and CD4One explanation that may tie the two observations together is that DC-SIGN may enable the trafficking of sialylated IgG to the antigen processing and presentation pathway. The requirement for sialylation does not explain the induction of natural and inducible Tregs following administration of Tregitope peptides in saline . Our finThe important discovery of Tregitopes has the potential to bring understanding about a number of phenomena related to Ig, including tolerance to antibody (Ab) variable regions, the tolerogenic properties of immunoglobulin-antigen (Ag) conjugates, the weak immunogenicity of Fc fusion proteins, and the therapeutic and regulatory effects of clinical preparations of intravenous immunoglobulin (IVIg) on autoimmune and inflammatory diseases. Immunoglobulin (Ig) has long been known to have tolerogenic properties. Thus, Ags conjugated to Ig elicit tolerance rather than immunity, and intravenous administration of pooled Ig from multiple donors, known as IVIg, is used in clinical practice to treat autoimmune and inflammatory diseases.γ receptors, and others have concluded that the effect is due to Fc-independent mechanisms such as epitope masking [The presence of Tregitopes in IgG may explain the induction of tolerance with intravenous IgG . Even th masking , while s masking . Of note masking .P < 0.002) correlation was found between Tregitope content and lower reported immunogenicity .γR) are required for rapid uptake of IgG and immune complexes into antigen-presenting cells during the initial inflammatory phase, and the inhibitory Fc receptor, FcγRIIb, increases the threshold for cell activation during the refractory phase of immune response. In our model, Tregitope activation of Treg would stimulate the release of cytokines such as IL-4 and IL-10 that are known to shift expression from the activating FcγRI and FcγRIIa to FcγRIIb [This model does not ignore the contribution of Fc receptors to IgG-mediated anti-inflammatory processes. Fc-gamma Receptors  to mice in 1986 , causingThe discovery of Tregitopes inspired a reconsideration of published research on IVIg and Fc-fusions. The Tregitope hypothesis may change the interpretation of work published almost a century ago  as well A study published by Ephrem et al. in Blood also demonstrated that IVIg therapy induced expansion of Tregs and protected against development of EAE induced by active immunization with myelin oligodendrocyte glycoprotein (MOG)35–55 . IVIg haIs there a role for Tregitope in future clinical practice? Emerging approaches to autoimmune disease treatment currently involve induction of Tregs using monoclonal antibodies (mAbs) such as anti-CD3 , which induce Treg cells. Anti-CD3 treatment has shown some efficacy in human studies, but the mechanism of Treg induction is elusive and the effect appears to be short lasting , 50. Ant


The aging process decreases tissue function and regenerative capacity, which has been associated with cellular senescence and a decline in adult or somatic stem cell numbers and self-renewal within multiple tissues. The potential therapeutic application of stem cells to reduce the burden of aging and stimulate tissue regeneration after trauma is very promising. Much research is currently ongoing to identify the factors and molecular mediators of stem cell self-renewal to reach these goals. Over the last two decades, fibroblast growth factors (FGFs) and their receptors (FGFRs) have stood up as major players in both embryonic development and tissue repair. Moreover, many studies point to somatic stem cells as major targets of FGF signaling in both tissue homeostasis and repair. FGFs appear to promote self-renewing proliferation and inhibit cellular senescence in nearly all tissues tested to date. Here we review the role of FGFs and FGFRs in stem cell self-renewal, cellular senescence, and aging. In the 1960's, Hayflick observed that human cells displayed a finite lifespan when cultured in vitro . These oINK4a pathway in human cells and also the p19/ARF pathway in murine cells. In human cells, both intrinsic and extrinsic senescence can thus coalesce to play a role in aging.Cellular senescence can be caused by intrinsic or extrinsic factors and this distinction is important . IntrinsThe process of aging is a systemic degenerative process caused by intrinsic  and extrinsic  factors. It affects multiple organs, mainly those with a high metabolic demand or those which are mitotically active and require constant or frequent regeneration . As suchAdult stem cells provide constant replacement cells for tissue homeostasis and repair while at the same time maintaining a pool of stem cells by the process of self-renewal, where following cell division at least one daughter cell is still a stem cell whereas the other is either a stem cell (symmetric division) or a differentiated progeny (asymmetric division). The stem cell pool only regresses if a symmetric division giving rise to two differentiated progeny occurs, or if the stem cell undergoes cellular senescence (these two processes not being exclusive). In recent years, a number of studies have identified fibroblast growth factors (FGFs) and their receptors (FGFRs) as key regulators of both senescence and self-renewal in a variety of stem cell types.FGFs (23 known members) and FGFRs  have long been known for their important roles in embryonic development , 9. HoweThe purpose of this Research Perspective article is to review the roles of FGFs and FGFRs in different stem cell populations and highlight their roles in stem cell self-renewal, cellular senescence and aging.In early murine embryonic development FGF-4 is the first member to be expressed, from the 4 cell stage onto the blastocyst, egg cylinder and primitive streak . Its del−/− embryos fail to develop because of defects in inner cell mass proliferation and germ layers specification, it has been assumed that FGF signaling in mESCs was required for their differentiation or lineage commitment. Of note, mESCs constitutively express FGF-4 which is thought to act in an autocrine manner. Undifferentiated mESCs were found to express high levels of FGFR1 and 4 which are maintained during differentiation [−/− mESCs do not display defects in proliferation in vitro and are capable of multilineage differentiation, however the survival of those differentiated progeny is severely compromised, although the underlying mechanism for this phenotype is still unclear [−/− mESCs or specific inhibitors of FGFR1 and 3 confirmed that inhibition of FGF signaling through these receptors could maintain mESCs in a self-renewing, pluripotent state [Murine embryonic stem cells (mESCs) have historically been derived from the inner cell mass of the blastocyst , 19 or tntiation . They al unclear . Furthernt state , 22. HowThe study of molecular events in human post-implantation embryogenesis is complicated by ethical and technical limitations. It is however possible to study pre-implantation embryos from which are derived humans ESCs. It must be noted that hESCs might be more related to epiblast-derived (primed) mESCs in terms of properties and functionality. Nevertheless, hESCs have been found to express several molecular-mass isoforms of FGF-2, 11 and 13 (but not FGF-4) as well as the whole repertoire of FGFRs with the following relative abundance (mRNA levels): FGFR1 > FGFR3 > FGFR4 > FGFR2 . These lIn summary, the roles of FGF signaling in murine and human embryogenesis are diverse and appear stage specific. They are probably modulated by the context (presence and type of HSPGs), the differentiation status of target cells, and the repertoire of FGFRs these cells express. However, FGF signaling is important for self-renewal and proliferation of primitive ESCs and for migration, proliferation and lineage commitment of more differentiated cells.“Fibroblast growth factor” was first isolated in 1974 from bovine pituitary gland and shown to have a mitogenic effect on many cells types . The proThe importance of FGF signaling in skeletal tissues was first highlighted by genetic linkage analysis demonstrating that the etiology of achondrodysplasia (one of the most common forms of dwarfism in humans) was due to activating point mutations in FGFR3 , 35. OthIn the early stages of bone development, FGFR1(IIIc) is expressed in limb mesenchyme whereas FGFR2(IIIb) is expressed in overlying ectoderm . There aBecause FGFR1 and 2 knockout murine embryos die before skeletal development, conditional knockout techniques have been used to elucidate the roles of these receptors in bone lineage cells. FGFR1 signaling in osteogenic cells appears to have developmental stage-specific effects. When inactivated at the early condensation phase in brachyury expressing cells, a decreased proliferation of mesenchymal progenitors is observed, along with decreased condensation sizes and numbers and delayed patterning . When inA similar strategy has been used to study the role of FGFR2 in bone lineage cells. Conditional inactivation of this receptor in Dermo1  expressing cells resulted in severe dwarfism accompanied by reduced bone mineral density . At E16.The role of FGF signaling in skeletal cells has also been studied in vitro in mesenchymal stem cells , which are thought to give rise to all skeletal or mesenchymal cells in bones and sustain bone homeostasis and repair throughout life. The mitogenic effect of FGF on mesenchymal stem cells (MSCs) was first described over 20 years ago . SeveralThe observations that FGF acts as a mitogen (probably by ERK1/2 activation), a multipotency factor (through Sox2 induction) and an inhibitor of cellular senescence (through a PI3K-AKT-MDM2 pathway) are significant in that they may explain how MSCs are capable of maintaining a sufficient pool of progenitors during bone development, growth, homeostasis and repair for the lifespan of the organism. These observations also provide potential therapeutic targets as senescence of osteoblasts and their progenitors is an important cause of age-associated bone loss and osteoporosis . Data frIn embryonic development, mesodermal cells arising from the posterior primitive streak are thought to migrate to the yolk sac and to the para-aortic splanchnopleura  and differentiate into haemangioblasts, common progenitors of both endothelial and hematopoietic cells , 64. As −/− ES cells, which under appropriate conditions recapitulate haemangioblasts differentiation in vitro, Magnusson et al.  demonstrated that FGFR1 was indeed not necessary for ECs differentiation and vascular plexus formation, it was however required for hematopoietic development [−/− embryoid bodies contained more blood vessels and ECs derived from them proliferated faster. In a more recent study, Murakami et al. (2008) used soluble FGFRs in vivo to demonstrate the importance of FGF signaling in maintaining vascular integrity, more specifically in maintaining adherens and tight junctions between ECs [The first evidence that the mitogenic effect of FGF-2 on ECs may be indirect was provided when it was shown that FGF-2 upregulated VEGF expression and blocking of VEGF using antibodies completely abolished FGF-2-induced ECs proliferation in vitro and angiogenesis in vivo . Using eelopment , 69. Morween ECs . Howeverween ECs . These aween ECs ) indicatween ECs  whereas ween ECs . Moreoveween ECs .−/− embryoid bodies show no obvious defects in angiogenesis. It could be that FGF signaling serves to protect ECs from cellular senescence during active proliferation or that FGFR1 is only expressed in endothelial progenitors, but this requires more investigations. A fundamental requirement that needs to be addressed before answering some of these unresolved questions is a better understanding of the heterogeneity of ECs in vivo and in vitro, of their phenotypic differences and various physiological roles. As not all ECs are the equivalent, this would enable to test the effects of FGF stimulation on specific subsets of ECs.From what has just been described and contrary to widespread belief, it is obvious that FGFs are not mere mitogens for ECs and in fact they may even exert their effects mostly by indirect means, whether by acting on accessory cells such as pericytes or by modulating the activity of other growth factors. This is supported by the low FGFR1 expression by ECs in vivo and the fact that FGFRAs already mentioned, hematopoietic stem cells (HSCs) arise from haemangioblasts located in the AGM and yolk sac during embryonic development, before undergoing a journey that will take them to the placenta and fetal liver and eventually the bone marrow shortly before birth . In thisFGF-2 was initially shown to be a mitogen for multipotent progenitors from bone marrow, mostly in the myeloid lineage -80. AlthWhile the mitogenic effect of FGF on myeloid cells is obvious, its effect on embryonic and adult HSCs is more controversial. Berardi and colleagues(1995) found noIn mice, it was first shown that FGF-2 signaling through FGFR1 was required for hematopoietic commitment of haemangioblasts derived from ESCs , 88. MorThe wide variety of cell types used, purity of cell populations, culture conditions and endpoint assays to determine the stemness of HSCs in the studies described here probably explain the controversy regarding the role of FGF in hematopoiesis. As our definition of HSCs constantly evolves and better techniques are available to study these cells at near purity , it willThe importance of FGF signaling in central and peripheral nervous system both during development and postnatal life has been long recognized. In addition to the difficulties in studying FGF signaling mentioned above for other tissues, our knowledge of brain development and neural stem cells has greatly evolved in the last two decades rendering previous conclusions obsolete or in any case requiring re-evaluation. The skin is another ectoderm-derived tissue containing various stem cell populations where FGFs and their receptors are widely distributed, yet very little is known about the precise role of FGF signaling in skin homeostasis or repair and it will not be discussed here. This section will review what is known about FGF signaling in neural tissue.During embryonic development, the nervous system arises shortly after gastrulation from neuroepithelium located along the dorsal midline of the embryo  and then folds into the neural tube before undergoing various patterning events and specification. Whereas most neural cells in early embryonic development are multipotent (they can give rise to both neurons and glia), neural stem cells (NSCs) become restricted to specific areas later in development and postnatal life: the cerebellum, the subgranular zone (SGZ) of the dentate gyrus in the hippocampus, and subependymal zone  lining the lateral ventricles , 99. NSCFGF signaling has long been acknowledged for its neural induction role in the developing embryo , 103 as Aging is usually associated with a decline in cognitive functions including memory as well as decreased regenerative capacity. Aging has also been associated with decreased NSCs or progenitors number and self-renewal capacity in the SEZ . Most of the stem cell populations described in this review have enormous therapeutic potential and increasing our capacity to harness their power to address unmet medical needs and reduce human suffering is the holy grail of current biomedical research. FGFs could well be added to the toolbox required to achieve this goal.The roles played by FGFs and FGFRs in aging or age-related disorders are gradually being unveiled. This is exemplified by the accelerated aging-like phenotype of FGF-23 knockout mice  and by t