These as well as more recent studies indicate that Andeans have somewhat larger lung volumes, narrower alveolar to arterial O2 gradients, slightly less hypoxic pulmonary vasoconstrictor response, greater uterine artery blood flow during pregnancy, and increased cardiac O2 utilization, which overall suggests greater efficiency of O2 transfer and utilization. More recent single nucleotide polymorphism and whole-genome sequencing studies indicate that multiple gene regions have undergone recent positive selection in Andeans. These include genes involved in the regulation of vascular control, metabolic hemostasis, and erythropoiesis. However, fundamental questions remain regarding the functional links between these adaptive genomic signals and the unique physiological attributes of highland Andeans. Well-designed physiological and genome association studies are needed to address such questions. It will be especially important to incorporate the role of epigenetic processes  that are vital for transcriptional responses to hypoxia and are potentially heritable across generations. In short, further exploration of the interaction among genetic, epigenetic, and environmental factors in shaping patterns of adaptation to high altitude promises to improve the understanding of the mechanisms underlying human adaptive potential and clarify its implications for human health.Whether Andean populations are genetically adapted to high altitudes has long been of interest. Initial studies focused on physiological changes in the O Some of the first studies took place in the Andes, where approximately 6 million indigenous Aymara and Quechua  populations reside, chiefly in Peru and Bolivia, however also in neighboring countries. Andeans are derived from the early settlers of the Americas who reached South America 15 to 16 thousand years ago (kya) and then split into two branches, one that settled in the Pacific coastal and Andean regions and the other that moved along the Atlantic coast and then eastward [Since the early 1900s, anthropologists and physiologists alike have sought to determine if there has been genetic adaptation to high altitude, conventionally defined as above 2500 m or 8250 ft since that is where O2-sensitive pathways, numerous questions remain regarding the biological processes driving human adaptation to the chronic hypoxia of high altitude and their importance for human health.Initial debate as to whether genetic adaptation to high altitude has taken place was driven by differences in theoretical orientation and the sources of evidence being considered. Theoretical orientation influenced the way in which the term “adaptation” was being employed. Physiologists used the term to refer to any response regardless of whether it was likely to be beneficial or otherwise affect the chance(s) of being able to live or reproduce, whereas evolutionary biologists or geneticists restricted its usage to those responses likely to influence reproductive success . EvidencThis review discusses the kinds of evidence by which adaptation to high altitude has been assessed and which have led to widespread acceptance of the idea that genetic adaptation to high altitude has occurred. Studies in Andean residents of high altitude are summarized with respect to the physiological characteristics distinguishing them from acclimatized newcomers and the genomic or genetic factors that are potentially involved. While further study is needed, such studies offer the opportunity to identify the importance of interactions between genomic and epigenomic processes for human adaptation to limited oxygen availability.2 transport traits when compared with acclimatized newcomers that cannot be attributed to developmental processes. Second, direct evidence comes from genomic studies that show signals of recent positive selection in specific gene regions. However, despite the remarkable progress in recent years for identifying targets of natural selection and the recognition that many are involved in O2-sensitive signaling pathways, few investigations have been able to show how these gene regions affect specific physiological characteristics and how, in turn, such effects influence reproductive success. From an evolutionary point of view, these relationships are essential since, by definition, only genes with effects on reproductive success are acted upon by natural selection.Two kinds of information support the existence of Andean genetic adaptation to high altitude. First, indirect evidence provided by physiologic studies demonstrates that native highland populations exhibit unique O2 homeostasis, there has been a long history of studies of the O2 transport system and its components  in acclimatized newcomers, Andeans, and other long-term residents of high altitude for establishing the physiologic mechanisms underlying human adaptation to high altitude.Since the hypoxia of high altitude challenges O2 in the arterial blood (PaO2) is determined by alveolar ventilation and the alveolar-arterial (A-a) O2 gradient  can serve as a proxy for arterial PCO2 (PaCO2). Additionally, since according to the alveolar air equation PaCO2 is inversely related to alveolar ventilation, PACO2 can serve as an index of alveolar ventilation per unit of CO2 production . At low altitude, PACO2 averages ~40 mmHg, however it falls to ~10 mmHg at elevations above 3000 m and even further at extreme altitudes. PACO2 or alveolar ventilation is lower in Andeans and acclimatized newcomers than sea-level residents, however Andean values are somewhat higher than those of acclimatized newcomers, indicating lower levels of alveolar ventilation , enabling Andeans to achieve an increased efficiency of O2 transfer [2 transfer is likely important for maintaining arterial O2 saturation and thus, blood O2 content during exercise [Acclimatization does not appreciably change the A-a Otransfer ,14,15. Ttransfer  and givetransfer ,18. Genetransfer , and thetransfer . Howevertransfer  and redutransfer . While mexercise ,22.2 dissociation curve determines the level of arterial O2 saturation (SaO2) for a given PaO2. Little change occurs in the in vivo curve position with acclimatization since the left curve-shifting effect of respiratory alkalosis is offset by an increase in red blood cell 2,3-bisphosphoglyceric acid levels. Current evidence does not support differences in Andean versus acclimatized newcomer curve position  [2 in Quechua regardless of whether they were born at high altitude or at low altitude yet were exposed transiently to high altitude [The position of the hemoglobin-Oication) ,24,25  is either measured directly or calculated using hemoglobin values  multiplied by SaO2, using a correction factor for dissolved O2. Acclimatized newcomers re-establish the sea-level value of ~21 vol % due to their hyperventilation, which helps to overcome the initial fall in PaO2, and increased hemoglobin. Andean and acclimatized newcomers achieve similar CaO2 levels due to the Andeans’ narrower alveolar-arterial O2 gradient offsetting their somewhat lower levels of ventilation.Arterial O2 consumption than acclimatized newcomers and less altitude-associated decrement [Acclimatized newcomers and Andeans have similar levels of cardiac output at a given workload, however values are lower at maximal exercise in both groups compared to sea level . Despiteecrement .2 distribution is determined by regional blood flow. There is increased sympathetic nervous system stimulation in acclimatized lowlanders at high altitude which likely reduced blood flow to the periphery [2 extraction during exercise are reduced in Andeans compared with acclimatized lowlanders as a result of blood being diverted to other tissues [2 delivery due to higher hemoglobin levels [2 delivery [Oeriphery ,45. Bloo tissues . Blood fn levels ,47   . Of inter lipids . Carbohyr lipids ; howeverr lipids . Only a wlanders . The aut hypoxia . Residen hypoxia ,62 as we hypoxia ; this wasumption .2 transport characteristics of Andeans compared to acclimatized newcomers that are not due to developmental factors—namely, lower alveolar ventilation, lower pulmonary vasoconstrictor response, larger lung volumes, higher uterine artery and possibly lower middle cerebral blood flow, less altitude decrement in maximal exercise O2 consumption, and more efficient cardiac O2 utilization—suggest a greater efficiency of O2 transfer and utilization and are consistent with the likelihood of Andean genetic adaptation to high altitude.In summary, the several unique O2-dependent negative regulators of HIF called prolyl hydroxylases (PHDs) enable the hydroxylation of proline residues of HIF1/2α subunits [2-sensitive genes contain HREs. Therefore HIF, while certainly central for governing transcriptional responses to hypoxia, is not the only regulator of molecular responses to changes in O2 tension.Direct evidence for Andean genetic adaptation to high altitude comes from single nucleotide polymorphism (SNP) genome scans and sequencing studies that have identified genomic regions with evidence of recent positive selection . Genes tsubunits ; this prsubunits ,69. In asubunits . While tsubunits . FurtherEGLN1, has thus far been identified as being acted upon by natural selection in both Andeans and Tibetans [PRKAA1, NOS2), vascular growth , cerebral blood flow (CBS), and oxidative defense (FAM213A) [BRINP3, NOS2, and TBX5—with just one (NOS2) having been identified previously in a SNP scan [The first genome scan to study high-altitude adaptation was performed in Andeans residing in Bolivia ; howeverTibetans . Among AFAM213A) ,75,77 , adolescent, and adult periods, with the heaviest concentration occurring during perinatal life .2 delivery, resulting in part from a larger pregnancy-associated rise in uterine artery diameter [2, is responsible for raising uteroplacental O2 delivery since CaO2 is similar in Andeans and newcomers [Residence at high altitude reduces birth weight as the result of slowed fetal growth rather than shortened gestation ,81. Andediameter ,51,86,87diameter ,89,90, sewcomers , and Andewcomers .High altitude also increases the incidence of preeclampsia, an effect that contributes to the altitude-associated decline in birth weight. Preeclampsia is a multisystem vascular disease of placental origin that complicates roughly 8.5 million pregnancies worldwide each year. It not only poses a threat to maternal and perinatal survival but also increases the risk of cardiovascular disease in affected mothers and offspring later in life ,95,96,97PRKAA1  and EDNRA (coding the vascular smooth muscle cell endothelin receptor A) were associated with the preservation of birth weight at high altitude; however only PRKAA1 was also associated with larger uterine artery diameters. In addition, the expression of mTOR-pathway genes in circulating peripheral blood mononuclear cells—a pathway known to play a crucial role in mediating the effects of hypoxia, nutrient restriction, and other factors on fetal growth—differed in women with versus without the selected-for maternal PRKAA1 genotype, suggesting that AMPK may play an important role in vascular adaptation to pregnancy [Given that compromised fetal growth raises the risk of perinatal mortality, an outcome of direct relevance for reproductive success, we tested the relationship between birth weight and 63 single nucleotide polymorphisms in 16 genes with evidence of natural selection at high altitude while making corrections for multiple comparisons . Severalregnancy ,104,105.2 values below 90%, with lower proportions of the night being seen in children and adolescents [Limited data suggest that native compared with acclimatized newcomer groups have better neonatal outcomes. Perinatal mortality is generally higher at high than low altitudes in South America, with the altitude-associated increase being least in the regions of Peru where populations have lived the longest . Infantslescents , but sam2 content under conditions of ambient hypoxia. In contrast, however, excessive red blood cell production, as observed in chronic mountain sickness (CMS), increases in blood viscosity and impairs blood flow and O2 delivery to tissues [Slight elevations of red cell mass increase arterial O tissues ; for thi tissues  and can  tissues ,109.CMS prevalence varies between highland resident populations B. For inEGLN1, and EPAS1, variants related to hemoglobin levels in Tibetans are not so related in Andeans [SENP1 and ANP32D, known to play roles in regulating erythropoiesis and cellular metabolism, respectively) in CMS patients, but not in controls. The association between SENP1 and CMS (however not ANP32D) was replicated in a larger sample of CMS and control residents of 4338 m [SENP1 which codes for a protease that rescues HIF1alpha from degradation, have also been suggested to play a role in increasing susceptibility to CMS [With respect to genetic factors in CMS,  Andeans , suggest Andeans . Using af 4338 m . Other gy to CMS .Genomic studies are well positioned to reveal functional links between genetic regions that appear to have been subject to recent positive selection and adaptive phenotypes of highland populations. It is critical, however, to recognize that phenotypes are the objects on which selective pressures act and are seldom the product of genetic factors alone. Complex phenotypes most often arise through gene-gene and gene-environment interactions, as well as the functional interaction of the genome and epigenome. Epigenetic marks are non-sequence-based features of the genome that are vital for coordinating transcriptional responses to environmental stimuli. In this way, the epigenome acts as an interface through which genetic sequence is “translated” to generate physiological responses to shifting biological or environmental conditions. This section presents evidence supporting the possibility that epigenetic processes contribute to human high-altitude adaptation, emphasizing the role of epigenetics for transcriptional and developmental responses to limited oxygen availability, epigenetic inheritance, and genome-epigenome interactions. Existing literature largely focuses on transient epigenetic effects. However, several recent investigations have explored mechanisms for epigenetic inheritance and the importance of genome-epigenome interactions for driving physiologic responses and phenotype. Taken together, this work indicates that epigenetic modifications could provide a mechanism for the rapid acquisition of potentially heritable features. This flexibility, itself, could be viewed as a selective advantage during periods of rapid environmental change or periods of the lifespan, such as the perinatal life or pregnancy which require widespread physiological changes over a short time period .Numerous epigenetic mechanisms exist, such as DNA methylation, histone modification, RNA-based mechanisms, and histone variants. DNA methylation, the most well-studied epigenetic modification in humans, is defined by the addition of a methyl group to cytosine residues within CpG dinucleotides. Research has predominantly focused on DNA methylation because of its central involvement in the regulation of gene transcription, genomic imprinting, and the silencing of repetitive DNA elements ,126. WhiVHL) and EPAS1 [EPAS1 promoter CpG sites by DNA methyltransferase 3a also prohibits HIF2α-mediated gene expression under hypoxic conditions [2 supply, such as at high altitude.Epigenetic processes are essential to the regulation of the HIF transcriptional program by, for instance, silencing HIF-stabilization genes, including von Hippel-Lindau  as adults compared to lowlanders who did not experience hypoxic pulmonary hypertension during perinatal life . Among AFrom an evolutionary point of view, the relevance of epigenetic marks or the capacity for epigenetic modification for human adaptation depends upon the heritability of epigenetic features themselves and/or the capacity for epigenetic modification in particular regions of the genome. Epigenetic heritability, that is the inheritance of epigenetic marks themselves, remains contentious primarily because non-imprinted genes undergo widespread, yet incomplete, epigenetic reprogramming prior to implantation . HoweverIn short, evidence supporting epigenetic heritability raises novel questions about how genetic sequence orchestrates physiological responses and durable adaptations to environmental exposures such as high altitude. Much work remains to be done in this area, particularly with respect to direct epigenetic inheritance. In the context of human adaptation, understanding the impact of putatively adaptive genetic variants that modify CpG motifs on the epigenetic regulation of gene expression should be of particular interest.EPAS1 SNPs in high-altitude populations modified CpG content [EPAS1 SNPs are apparent in high-altitude populations and have been associated with reduced hemoglobin concentrations in some native highland populations  [EPAS1 promoter lies entirely within a CpG island and is epigenetically regulated under hypoxic conditions [EPAS1 encodes HIF-2α and therefore may be of functional importance for adaptation to hypoxia. Finally, given that CpG-modifying SNPs can influence methylation [As discussed above, SNPs that disrupt (or create) CpG sites are important determinants of epigenetic capacity or, in other words, the potential for epigenetic regulation of gene expression. Prior work has speculated that epigenetics may be involved in high-altitude acclimatization and adaptation  and the  content . This obibetans) . Second,nditions . Third, hylation , heritabEGLN1 [EGLN1 would be anticipated to diminish PHD2 expression and thereby enable the transcription of HIF-regulated genes such as erythropoietin. In support of the hypothesis that hypermethylation of EGLN1 contributes to the excessive production of red blood cells in CMS, EGLN1 inactivation in mice results in an overproduction of erythropoietin and polycythemia, and familial polycythemia in humans has been linked to EGLN1 mutations [Epigenomic processes may also contribute to maladaptive phenotypes of high-altitude populations, including CMS in highland Andeans. Julian contrasted peripheral blood mononuclear cell DNA methylation patterns between Andean men living in La Paz-El Alto, Bolivia who presented with a preclinical form of CMS and healthy controls . Of the EGLN1 , a gene EGLN1 , the hyputations ,165,166.2-transport characteristics of Andeans compared to acclimatized or lifelong newcomer residents of high altitude. As reviewed above, these are lower alveolar ventilation, lower hypoxic pulmonary vasoconstrictor response, slightly larger lung volumes, higher uterine artery and possibly lower middle cerebral blood flows, less altitude decrement in maximal exercise O2 consumption, and more efficient cardiac O2 utilization. Collectively, these are suggestive of greater efficiency in O2 transfer and utilization; in turn, such differences between acclimatized or lifelong high-altitude residents support the existence of Andean genetic adaptation to high altitude.In summary, there are several unique OEGLN1, and on others that are not in the HIF-pathway yet are O2 sensitive, underscoring the importance of looking broadly at the range of genetic factors potentially involved. Whereas whole-genome scans are necessarily more complete, they are considerably more expensive and hence difficult to conduct in large numbers of persons. Thus more, especially high-coverage whole-genome sequencing studies, are needed. The one whole-genome scan to date indicates, intriguingly, that selection has acted not only on genes that are involved in O2 sensing, but also on those regulating cardiovascular responses to hypoxia [Direct support for Andean genetic adaptation to high altitude comes from SNP genome scans and whole-genome sequencing studies. Genome scans can be performed at relatively low cost and in large numbers of persons, but only sample a small portion of the genome . They ha hypoxia .Future studies are also required to provide deeper exploration of the associations between selected-for genotypes and phenotypic traits that are likely to influence reproductive success. The inclusion of epigenomic factors in such studies is also vital as the few studies conducted to date indicate potentially key roles for epigenetic regulation of gene transcription in ways that could affect reproductive fitness. While functional studies on the impact of locus-specific methylation status remains challenging, the advent, for example, of genome-editing technologies such as the CRISPR/Cas-9 system permit the induction of targeted CpG methylation and demethylation events in vitro as well as in vivo experimental animal models . Transcr2 related diseases.In short, the singular nature of the hypoxic stress posed by residence at high altitudes together with the central role played by oxygenation for health and disease states during intrauterine and postnatal life continues to provide a unique study environment for advancing our understanding of the mechanisms underlying human adaptive potential and of human evolutionary processes. Ultimately, such studies can also benefit biomedical research with the identification of new therapeutic targets for treating or preventing O


Acinetobacter baumannii.Multilocus sequence typing has been useful for genotyping pathogens in surveillance and epidemiologic studies. However, it cannot reflect the true relationships of isolates for species with very dynamic genomes. Using a robust genome phylogeny, we demonstrated the limitations of this method for typing  Acinetobacter baumannii, a major source of nosocomial infections, is no exception, and 2 MLST schemes (Oxford and Pasteur) have been established for this species  scheme was proposed as a genotyping method . For instance, under the Pasteur scheme, STp2 represents >15 STs in the Oxford scheme and STp1 encompasses 5 STox. Thus, the Pasteur scheme seems to have considerably less resolution than the Oxford scheme to distinguish isolates. The Pasteur scheme’s lack of resolution was not insignificant, however. STp2 comprises 43 isolates  showing considerable levels of genetic variation according to our phylogeny, but according to this MLST scheme, they constitute just 1 genotype.>2 isolates and therefore only in these STs could we detect problems with the clustering within any given ST. Thus, 3 of these 16 STs did not cluster the isolates properly inasmuch as these STs were polyphyletic. In summary, for the Oxford scheme we demonstrated that some STs form polyphyletic groups because 4 of the 7 loci have signals of recombination (Many of the STs in either scheme formed coherent (monophyletic) groups in our phylogeny. However, we recorded some clear exceptions in which isolates from some STs did not form monophyletic groups, that is, isolates with the same ST did not cluster. The most striking case is STox208 (orange tips in the phylogeny), where there are 2 well-defined groups with several isolates each and an extra isolate not close to either of those well-defined groups. We also noted that the 2 STox455 isolates did not cluster and are located far apart on the tree (green tips). Additionally, 1 of the STox369 isolates did not fall within the ST369 group (blue tips). These 3 examples show that the Oxford MLST does not accurately reflect the relationships among the isolates. Also notable is that, although for the Oxford scheme 36 STs are represented in this dataset, only 16 of them have bination  Table 2,bination  Table 2.A. baumannii. In addition, we highlighted the importance of using more powerful genotyping strategies when analyzing bacteria with highly dynamic genomes; in this regard, the ever-decreasing cost of genome sequencing will make this technology the perfect tool for genotyping bacterial species.In conclusion, we showed that the correct relationships among isolates cannot be recovered using either of the MLST schemes for Acinetobacter baumannii genome dataset, measures of genetic diversity and recombination for both MLST schemes, and maximum-likelihood phylogeny depicting the relationships among isolates in study of multilocus sequence typing of Acinetobacter baumannii.


Throughout the last decades, magnetic nanoparticles (MNP) have gained tremendous interest in different fields of applications like biomedicine , drug delivery, hyperthermia), but also more technical applications  have been pursued. Different surfactants and polymers are extensively used for surface coating of MNP to passivate the surface and avoid or decrease agglomeration, decrease or modulate biomolecule absorption, and in most cases increase dispersion stability. For this purpose, electrostatic or steric repulsion can be exploited and, in that regard, surface charge is the most important (hybrid) particle property. Therefore, polyelectrolytes are of great interest for nanoparticle coating, as they are able to stabilize the particles in dispersion by electrostatic repulsion due to their high charge densities. In this review article, we focus on polyzwitterions as a subclass of polyelectrolytes and their use as coating materials for MNP. In the context of biomedical applications, polyzwitterions are widely used as they exhibit antifouling properties and thus can lead to minimized protein adsorption and also long circulation times. For decades, magnetic nanoparticles (MNP) have been in focus within a range of scientific disciplines as they show high potential in a variety of different application fields, ranging from chemistry, biology, medicine to physics. One unifying aspect herein are surface properties of such nanomaterials. To date, there have been several reviews focusing on surface modifications of nanomaterials with polyelectrolytes, and most of them have focused on biomedical applications of these materials ,4,5,6,7.All chemical elements or compounds of our planet show under certain conditions different magnetic effects. Since we focus herein on magnetic nanoparticles for medical and technical applications, we concentrate on materials with ferro- or ferrimagnetic, superparamagnetic, and superferrimagnetic behavior at room temperature. In that regard, three classes of materials exist.Metals—The only metallic elements showing ferromagnetism at room temperature are iron, cobalt, and nickel. The preparation of nanoparticles hereof is possible and such materials show promising magnetic behavior for medical applications . . 1). The-monomer .In the following section, polyzwitterions, which were adsorbed onto MNP will be discussed, and the shown examples are summarized in 4) [tert-butyl acrylate (poly(2-vinylpyridine-grad-tert-butylacrylate)) were prepared (5). Subsequently, the tert-butylgroups were hydrolyzed to acrylic acid and the 2-vinylpyridine moieties were sultonated to generate a zwitterionic unit (6). This polymer exhibits a high charge to volume ratio as it consists of a polymeric backbone with directly attached amine and carboxylic acid functionalities. As these functional groups would impede direct polymerization, both functionalities had to be protected prior to polymerization. The protective groups were cleaved off afterwards to generate a polyzwitterion and the carboxyl groups were used for immobilization at the surface of sub 10 nm MNPs [7) [tert-butyldimethylsilyl ethers . The initiator was then used for the polymerization of carboxybetainemethacrylate (CBMA). After deprotection of the catecholic hydroxyl groups, the polyzwitterion was used to coat iron oxide MNPs. The resulting hybrids showed increased dispersion stability in solutions of varying ionic strength and blood serum compared to pristine and citrate stabilized MNPs. Furthermore, macrophage uptake was drastically decreased [block-(glycerol monomethacrylate) (PMPC-b-PGMA) block copolymers by ATRP (9). The double-hydrophilic block copolymer was added to a co-precipitation of FeCl2 and FeCl3.The bis-hydroxides of the PGMA block ensured efficient immobilization of the polymer on the surface of the resulting nanoparticles, and the zwitterionic block increased long term-stability [Although we mainly focus on polyzwitterionic coating materials, here we also included one example of a polyampholyte to show that the resulting hybrid materials can show similar properties to the examples discussed before. Xiao et al. coated in a first step iron oxide nanoparticles with poly(acrylic acid) (PAA) and modified them in a second step by esterification with 3-(diethylamino)propylamine, resulting in a polyampholytic shell material. The resulting nanoparticles exhibited low macrophage cell uptake and low cell toxicity (4) . Billingnic unit D. As a r nm MNPs  and mult nm MNPs . Zhu et MNPs [7) . Besidess TBDMS, B (8). Thecreased . Yuan ettability .In this last section, less frequently employed functionalization methods, like pre-functionalization approaches with polyelectrolytes, utilizing hydrophilic/hydrophobic interactions, or the addition of polyzwitterions during MNP preparation are discussed. The discussed examples are summarized in N-ethylsulfonate), attractive electrostatic interactions led to the formation of a second layer (10). It is noteworthy that the direct attachment of the polyzwitterion is also possible without the underlying PAA layer but the resulting surface coating was by far less stable afterwards [alt-1-octadecene) (PMAO) was used as precursor and modified by opening the anhydrous rings in the polymer in a first step with 3-(dimethyl-amino)-1-propylamine (11). In a second step the generated tertiary amines were reacted with β-propiolactone and 1,3-propanesultone resulting in betaine structures. As the polymer backbone has an amphiphilic character these polymers were immobilized at the nanoparticles by using hydrophilic hydrophobic interactions between the polyampholytes and the hydrophobic nanoparticles [12). The 110–320 nm large microspheres are of interest in the context of biolabeling and imaging [alt-1-decene), which was previously substituted with 3-(dimethylamino)propylamine to give a zwitterionic polymer (PMAL) (13). The surface coating was realized by intercalation of decene with previously attached oleic acid [N-carboxyethylchitosan (CECh) (14) and poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) (15) are capable of stabilizing MNPs in aqueous solution. CECh was synthesized by a polymer-analogous reaction with acrylic acid, while PAMPS was synthesized directly by free radical polymerization of 2-acrylamido-2-methylpropanesulfonic acid. Here, both suspension stability and particle size as well as the resulting magnetic properties were investigated and the obtained nanocomposites were further used for electrospinning [The use of non-covalent interactions (electrostatic or hydrophobic-hydrophilic interactions) to immobilize polyelectrolytes at the surface of MNPs leads to systems which allow the detachment of the respective polymeric shell under specific conditions, which can be either a benefit or a drawback. In order to generate a high surface charge at the surface of MNPs, Yeh et al. used poly(acrylic acid) as a first layer. By applying poly are promising with regard to bioseparation approaches as the magnetic nanoparticles enable a facile and fast way of binding and separating biomolecules  from complex biological systems by external magnetic fields. As this allows an enrichment of the respective molecules, rather high detection sensitivities can be achieved. Further analysis of any separated molecules or macromolecules can afterwards be realized by techniques such as mass spectrometry or various spectroscopy methods ,172,174.Besides biomedical applications, the polyzwitterionic magnetic hybrid materials are also constantly discussed with regard to technical applications such as extraction processes  , as the The synthesis and exploitation of magnetic hybrid materials–in our case consisting of a magnetic core and an organic shell—has already arrived in a broad variety of research areas. However, still only a certain number of research groups have reported on the use of polyzwitterions as coating materials, which we mainly attribute to the fact that the synthesis of polyzwitterions can be challenging and that PEG still is the most prominent biocompatible shell material in many applications. Nevertheless, magnetic hybrid materials which are functionalized by polyzwitterions show several benefits compared to the gold standard PEG, like close similarities to biological tissue, multiple ways of immobilization, and, in some cases, pH responsive behavior rendering those examples interesting candidates for drug delivery systems in the near future. The adjustment of charge at the particle surface allows a potential change in solubility of the particles as well as a change in adsorptive behavior towards any suitable guest molecules (or cargo).Further advance in the context of biomedical applications clearly requires progress concerning the understanding of interactions with proteins and biological macromolecules. Along the same line, a closer look at the influence of the actual combination of strong and weak polyelectrolyte building blocks on the resulting interactions with biological tissues has to be taken as well. The qualitative as well as the quantitative binding of different proteins to the surface of the respective materials might give further information on processes which are important in understanding the governing factors in protein corona formation. Furthermore, basic investigations on the suspension stability depending on ionic strength, and the response to the presence of different counter ions or biological fluids are further important aspects. The examples outlined above also suggest that combinations of PEG and polyzwitterions within polymeric shells are definitely an aspect of interest.


Rho guanosine triphosphatases (GTPases) are key regulators in a number of cellular functions, including actin cytoskeleton remodeling and vesicle traffic. Traditionally, Rho GTPases are studied because of their function in cell migration and cancer, while their roles in metabolism are less documented. However, emerging evidence implicates Rho GTPases as regulators of processes of crucial importance for maintaining metabolic homeostasis. Thus, the time is now ripe for reviewing Rho GTPases in the context of metabolic health. Rho GTPase-mediated key processes include the release of insulin from pancreatic β cells, glucose uptake into skeletal muscle and adipose tissue, and muscle mass regulation. Through the current review, we cast light on the important roles of Rho GTPases in skeletal muscle, adipose tissue, and the pancreas and discuss the proposed mechanisms by which Rho GTPases act to regulate glucose metabolism in health and disease. We also describe challenges and goals for future research. The Rho family of small guanosine triphosphatases (GTPases) is a distinct branch within the superfamily of Ras-related small GTPases. Twenty mammalian genes encoding Rho GTPases have been identified of which Rac1, Cdc42, and RhoA are the prototypes and therefore the best characterized. Rho GTPases are crucial organizers of the actin cytoskeleton with essential functions in cell migration, cell–cell contacts, proliferation, differentiation, and many other fundamental cellular processes . In the Today metabolic diseases significantly contribute to early death in Western society. More than 422 million people worldwide are estimated to have diabetes causing 3% of global deaths  and cancWide interest in small GTPases was triggered by the discovery of the Ras oncogenes in 1982 . Those dIn addition to the regulation of the GTP–GDP cycling, post-translational modifications  of Rho GTPases appear to be additionally required for optimal regulation of Rho GTPase function ,19. The As outlined above, the regulation of Rho GTPases is highly complex making it challenging to determine the exact regulatory mechanisms operating under any one circumstance. Active Rho GTPases recruit downstream effector molecules to the plasma membrane and trigger their activation . ConservActin remodeling is controlled through parallel actin polymerization and depolymerization. Generally, Rac1 and Cdc42 regulate comparable downstream events via activation of PAKs and activation of the Wiskott–Aldrich syndrome family of proteins including Wiskott-Aldrich Syndrome protein (WASP), neural-WASP (N-WASP), and WASP family verprolin homologous protein 1 or 2 (WAVE1/2). WASP and N-WASP are critical downstream effectors of Cdc42 that mediate the formation of filopodia through activation of the actin polymerization factor Arp2/3 .Among other downstream effectors, PAKs activate LIM domain kinases (LIMKs) promoting phosphorylation and inactivation of cofilin . Active Critical downstream effectors of RhoA are Rho-associated protein kinase (ROCK) and the Formin family of proteins that promotes the formation of straight, unbranched actin fibers. ROCK phosphorylates a large cohort of actin-binding proteins and intermediate filament proteins to modulate their functions. For example, ROCK phosphorylates LIMK leading to the phosphorylation and inactivation of the actin severing protein cofilin ,51. FormWhereas both downstream pathways of Rac1 and Cdc42 seem to be involved in metabolic control, RhoA may via downstream effectors be involved in muscle mass regulation and thereby play an important role in conditions of muscle mass loss. This will be reviewed below with emphasis on knowledge gained from in vivo studies and humans.Rho GTPases have important functions during cell development and differentiation  so that hyperglycemia ensues . Before Thus, while the Rho GTPases Cdc42 and Rac1 are critical for insulin secretion, pathological hyperactivation of Rac1 and RhoA negatively affects β cell function during times of stress and may thus be targets for preventing β cell failure in diabetes.The Rho GTPases Rac1, RhoA, and Cdc42 are highly expressed in skeletal muscle and adipose tissue and emerging evidence suggests that they have key functions in the regulation of glucose uptake and the maintenance of whole body glucose homeostasis.In response to insulin, the majority of dietary glucose is taken up by skeletal muscle ,80. CompRac1, TC10, and RhoA are activated by insulin in skeletal muscle ,85,86,87The involvement of Rac1 in insulin-stimulated GLUT4 translocation has been ascribed to a Rac1-mediated insulin-stimulated actin remodeling in skeletal muscle cells ,88,89. HIt is largely unknown how Rho GTPases are activated by insulin but it seems to occur downstream of phosphatidylinositol 3-kinase (PI3K). Skeletal muscle evidently expresses the following GEFs for Rac1: Tiam1 , Trio 9, Def6 9, son of RhoGDIα and RhoGDIβ are expressed in skeletal muscle ,103 and Downstream of Rac1, PAK1 and PAK2  are activated in response to insulin ,87,107 aAn input by RhoA in the regulation of insulin-stimulated glucose transport, has also been proposed, although the results are few and rather inconsistent . FurtherChallenging a positive input of RhoA in the regulation of glucose uptake, membrane-bound (active) RhoA is elevated in skeletal muscles of obese Zucker rats . In thatCollectively, there is ample support that Rac1, and possibly RhoA, but seemingly not Cdc42, participate in the insulin-dependent regulation of glucose uptake in skeletal muscle.Canonical insulin signaling via Akt and TBC1D4 is a well-described signaling mechanism by which insulin increases muscle and adipose tissue glucose uptake ,119. In It has been convincingly documented that regular exercise improves glycemic control and insulin action among healthy, as well as obese and type 2 diabetic subjects ,128,129,Rac1 is activated during exercise in mouse and human skeletal muscle  and Rac1Very recent results shed light onto the downstream events by which Rac1 may induce glucose uptake during exercise. Henriquez-Olguin et al. showed that Rac1 is implicated in the increase in ROS-production that occurs during exercise in muscle . As descIn addition to the acute stimulation of glucose uptake by a single bout of exercise, exercise training also induces remarkable skeletal muscle adaptations that benefit metabolic regulation, including the enhancement of insulin sensitivity (recently reviewed in ). RecentInterestingly, exercise results in phosphorylation of RhoGDIα on serine 34 in human skeletal muscle . WhetherIn summary, exercise elicits metabolic benefits, several of which involve regulation of Rho GTPases. Future studies should delineate the mechanisms by which Rho GTPases may be influencing health with exercise and importantly if they can be explored pharmaceutically to harness some of the metabolic benefits of exercise in individuals that cannot adhere to an exercise program.Although being mainly a fat storage tissue, adipose tissue is also essential for maintaining metabolic homeostasis and health. Especially in conditions of obesity, adipose tissue takes up a substantial amount of glucose following a meal, thereby significantly contributing to the clearance of glucose from the blood. Like in skeletal muscle, adipocyte glucose uptake relies on the translocation of GLUT4 to the plasma membrane for glucose to diffuse across the membrane lipid bilayer. Insulin activates TC10 ,145, CdcAn early investigation showed that transfection of a constitutively-active RhoA increased GLUT4 translocation in response to insulin, while dominant-negative RhoA significantly decreased it in 3T3-L1 and rat adipocytes . AnotherThe Rho GTPase TC10 also seems relevant for regulating insulin-stimulated glucose uptake in adipose tissue as depicted in Taken together, Rho GTPases could play roles in adipocyte glucose uptake by regulating GLUT4 vesicle dynamics. Indeed, the fusion of GLUT4 vesicles with the adipocyte membrane requires dynamic insulin-induced actin polymerization, evincing the contribution of the actin cytoskeleton at different steps in the process of GLUT4 translocation . HoweverIn insulin resistant human subjects that are obese or have type 2 diabetes, insulin-stimulated glucose uptake by skeletal muscle is a primary site of dysfunction . Due to RhoA might also regulate glucose homeostasis, as pharmacological inhibition of RhoA’s target, ROCK, using Fasudil prevented high-fat diet-induced hypercholesterolemia and glucose intolerance in mice . FurtherTaken together, Rho GTPases, in particular Rac1 and RhoA, may be highly implicated in muscular insulin resistance and thereby contribute to metabolic dysregulation in metabolic diseases, such as type 2 diabetes.Muscle mass is important for metabolic health because it increases the amount of tissue available for storing glucose. Although studies are few, there is emerging evidence that Rho GTPases may play hitherto unrecognized roles in muscle mass regulation. Indeed, Rho GTPases are highly regulated during conditions of muscle atrophy. For example, a marked reduction in levels of RhoA was noted in the muscles of unweighted hindlimbs  in mice  and in dDrosophila severely disrupted muscle formation. Thus, Rac1 is involved in the regulation of myoblast proliferation and segregation during adult myogenesis [Rho GTPases other than RhoA may also be involved in muscle mass regulation as proteomic analysis of mouse muscles following denervation-induced atrophy identified significant changes in RhoGDIα, PAK1-2, and Cdc42 expression . Dominanogenesis . Downstrogenesis ,169. In ogenesis , suggestogenesis  and PAK1ogenesis ,172 wereA role for Rho GTPases in muscle mass regulation is perhaps not surprising given their well-known requirement for tumor growth. Looking ahead, lessons from the tumor literature may help to understand the mechanisms by which Rho GTPases may be involved in muscle mass regulation in connection with metabolic regulation.Skeletal muscle atrophy is a severe consequence of ageing and many chronic diseases, including cancers. Muscle strength is inversely related to death from all causes  and is oMany cancers are associated with cachexia, a condition of involuntary body weight loss including severe muscle atrophy that is not due to anorexia . InteresAs muscle is the largest organ of the body, completely necessary for mobility and also responsible for the majority of glucose disposal, future studies should investigate the role for Rho GTPases in muscle wasting diseases. Unresolved issues are showed in Box 1Lack of in vivo experiments to support the in vitro literature on Rho GTPase regulation and in particular their role in metabolism.Evidence on the regulatory functions of Rho GTPases in humans is limited.Molecularly, the upstream activators and downstream effectors of Rho GTPases in different tissues and in response to different stimuli are poorly defined.Cross-talk between Rho GTPases is poorly defined but important to delineate, as they challenge all interpretation of data using knockdown or overexpression of a single Rho GTPase.High throughput methodological advances to directly measure in vivo GTP binding (fast hydrolysis) warranted.Whether Rho GTPases can be targeted pharmacologically to benefit metabolic diseases remains to be determined.In this review, we summarize evidence for the role of Rho GTPases in metabolic regulation in health and disease. We demonstrate that Rho GTPases may be hitherto overlooked players in glucose homeostasis by contributing to metabolically essential functions in skeletal muscle, adipose tissue, and the pancreas. However, this area of research is at its early stages and mechanistic in vivo insights are lacking. This will be an exciting area for future discoveries.


In this review, we focus on the zebrafish (Danio rerio), a vertebrate model widely used in biomedical research since its overall nervous system organization is similar to that of humans. A critical analysis of the literature suggests that zebrafish could serve as a powerful experimental tool for molecular and genetic dissection of both HA and HSP. The zebrafish, found to be very useful for demonstrating the causal relationship between defect and mutation, also offers a useful platform to exploit for the development of therapies.Hereditary spastic paraplegia (HSP) and hereditary ataxia (HA) are two groups of disorders characterized, respectively, by progressive dysfunction or degeneration of the pyramidal tracts (HSP) and of the Purkinje cells and spinocerebellar tracts (HA). Although HSP and HA are generally shown to have distinct clinical-genetic profiles, in several cases the clinical presentation, the causative genes, and the cellular pathways and mechanisms involved overlap between the two forms. Genetic analyses in humans in combination with  The term hereditary spastic paraplegia (HSP) refers to a large group of clinically and genetically heterogeneous neurodegenerative diseases, essentially characterized by progressive spasticity and weakness, mainly affecting the lower limbs  assays in mammals, and also in order to develop possible new therapeutic avenues.Using zebrafish, it is easy to induce gain or loss of function of the HSP-related genes. By exploiting strategies designed to obtain transient or stable genetic changes, it is possible to characterize and define patients . The majpatients , in ordeWith reference, mainly, to the mode of inheritance-based genetic classification of HSP, we here review the HSP genes studied in zebrafish, recalling the original contributions and highlighting the recent discoveries that might help to elucidate the functional effects of human HSP-related mutations. In addition, to further expand our review, we also consider genes classified as part of the ASS spectrum and not routinely included in the formal HSP classifications.The PubMed database was queried using the following three search strings: <<zebrafish [All Fields]>> AND <<hereditary spastic paraplegia [All Fields]>>, OR <<zebrafish [All Fields]>> AND <<spasticity [All Fields]>>, OR <<zebrafish>> AND <<paraplegia [All Fields]>>. Articles retrieved by the literature search had to be full-text articles written in English, and they had to have been published by May 31, 2019. Application of the three strings yielded 33, 14, and 39 publications, respectively. We then performed a manual search of the references listed in publications found to discuss HSP in relation to the use of zebrafish. After excluding all articles not reporting direct research on zebrafish and HSP, 42 articles remained for inclusion in the review  morphant zebrafish model (mutations in ATL1/SPG3A are the second most common cause of HSP in humans). Atlastin-1 is a protein implicated in the vesicle trafficking and neurite outgrowth  signaling leading to complete loss of ventral structures. This idea was also supported by the co-localization of Alt1 and type I BMP receptor in endosomal structures along neurites. In addition, the phenotypes observed in atl1 morphant embryos were recovered pharmacologically, by inhibiting the BMP pathway at the receptor level during the late embryonic stage. The recovery of the motility and axonal phenotype observed in this atl1 knockdown model suggested that BMP plays a key role in atl1-dependent HSP  encodes spastin, a microtubule-severing protein that belongs to the AAA (ATPase associated with various cellular activities) family of ATPase and regulates the number and mobility of microtubules -dependent way. In fact, ZFYVE27  is another gene whose mutations cause autosomal dominant HSP  showed impaired head and yolk sac extension with a curly tail phenotype that was more severe than in fish injected with MOs specific for single transcripts. These phenotypes were partially rescued by wild-type human SPAST or PROTRUDIN overexpression  were identified in zebrafish. Knockdown of each of these isoforms led to different motor neuron and locomotion defects, not rescued by the selective expression of the other isoform. Indeed, the expression and the distribution of the two isoforms were found to differ in zebrafish embryos. Isoform-specific knockdown led to morphants with distinctive characteristics: MO-DrM1 morphants displayed a curved tail, whereas MO-DrM61 morphants showed smaller eyes and yolk tube agenesis. In both cases, the embryos failed to hatch from their chorion and at t 72 hpf the larvae showed locomotion defects and secondary motor neuron axon pathfinding defects   lead to larval lethality and sensorimotor deficits similar to those observed in human patients; moreover, zebrafish kif5Aa mutants were found to exhibit hyperexcitability and peripheral polyneuropathy as defective mitochondrial transport resulted in degeneration of peripheral sensory axons. At 6 dpf (days-post-fertilization), kif5Aa mutants had an inflated swim bladder, increased lateral and dorsal pigmentation, and a diminished touch response . VCP is involved in protein aggregation in several neurodegenerative syndromes  membrane acetyl-CoA transporter, which is responsible for carrying acetyl-carboxylase (acetyl-CoA) into the ER lumen , a drug that inhibits BMP receptor-1 activity  gene. UBAP1 is a member of the UBA domain family, which includes proteins that have a role in the ubiquitin and ubiquitination pathways  resulted in misshapen axons and shorter motor neuron length compared with what was observed in controls  HSP, all acting through loss-of-function and dominant-negative molecular mechanisms. Such mechanisms can be easily modeled at genetic level in zebrafish. There follows a brief overview of the AR-HSP genes studied in zebrafish.ALS2/alsin are associated with different neurodegenerative disorders, such as juvenile-onset amyotrophic or primary lateral sclerosis and AR hereditary spastic paraplegia. ALS2 encodes for a protein  with multiple homology motifs similar to guanine-nucleotide exchange factor, indicating Rho and Rab5 activity. Members of the Rho and Rab5 family of GTPase proteins have been implicated in numerous cellular functions, the best characterized being regulation of the actin cytoskeleton (Rho) and protein trafficking through early endosomes (Rab5)  Otomo, . Als2 knype Cai, . Thus, imalities , which wKIAA1840/SPG11 gene encodes spatacsin  led to motor axon outgrowth failure and motor impairment  have been implicated in a broad spectrum of neurodegenerative conditions including the complicated form of spastic paraplegia referred to as type 39 (SPG39). The SPG39 protein is a conservative protein found in many species ranging from yeast to mammals  is another neurodegenerative disorder characterized by onset in childhood of slowly progressive spastic paraplegia and cerebellar signs. Martin et al.  identifiVPS37A in an AR complicated HSP form (SPG53). VPS37A encodes a protein member of the endosomal sorting complex required for transport (ESCRT) system. The ESCRT system is involved in intracellular trafficking, in the maturation of multivesicular bodies, and in the sorting of ubiquitinated membrane proteins into internal luminal vesicles  but only the MO against capn1a led to a phenotype, because capn1a is largely expressed in the brain starting at 24 hpf (hours-post-fertilization)  zebrafish line expressing the green fluorescent protein (GFP) in the motor neurons, including the branchiomotor neurons. Capn1a deficiency resulted in abnormal branchiomotor neuron migration and disorganized axonal networks in the brain, supporting a neuroprotective role of calpain 1 , is KLC2) overexpression. The zebrafish has proved to be an indispensable model for studying in vivo the effects of klc2 knockdown and overexpression. Loss of function was obtained using two different klc2 MOs (a translation-blocking MO and a splice MO); the effects of both MOs were evident at 48 hpf and the phenotypes observed were rescued by injection of WT kcl2 mRNA. Moreover, the kcl2 overexpression was associated with phenotypes similar to those displayed by morphants  were identified in early 2000  and the other targeting the final exon 13 (pcyt2_13)  in zebrafish showed high mortality whereas pcyt2_13 strain with some residual protein function revealed a higher survival compared to pcyt2_03 line. Moreover, at 6 weeks of age, the pcyt2_13 line showed smaller size and abnormal tail-fin morphology compared with the WT. This model highlighted that alterations in lipid metabolism may lead to complex HSP  using a zinc-finger nuclease strategy. Zebrafish mutants showed neurological and behavioral alterations reminiscent of those seen in the human disease. Using several experimental approaches, the authors found abnormalities in the expression of myelin-related genes, defects in neural circuit assembly, a reduction of locomotor activity, and a decreased response to external stimuli. They suggested that these impaired neurological phenotypes and behaviors could be conserved between zebrafish and AHDS patients. In addition, the fact that treatment with TH analogs was able to recover a portion of the neurological phenotypes seems to pave the way for the use of zebrafish for large-scale testing of possible therapeutic treatments for AHDS pathology , a rare disorder of brain development with neuromuscular involvement, is characterized by a combination of severe intellectual disability, spastic paraplegia, and disturbed thyroid hormone (TH) parameters   that encodes a primary constituent of myelin in the central nervous system. Few data are available on plp1 in zebrafish  , also known as spinocerebellar ataxia type 3 (MJD/SCA3)  and these authors also characterized a mutant model from the Zebrafish Mutation Project with a point mutation in abcd1 exon 9 (abcd1sa509)  is another X-linked neurodegenerative disease. Caused by mutations in the n et al.  through  deficit  also see deficit .EXOSC3), a gene associated with pontocerebellar hypoplasia 1 (PCH1) and distal hereditary motor neuronopathy, both of which resemble HSP. EXOSC3 is a component of the RNA exosome complex, and mutations in the relative gene were found in 13 families with signs of PCH1 . Mutations in this gene are responsible for 95% of cases of Niemann-Pick disease, a rare AR lysosomal storage disorder  , have brin vivo, as well as to functionally validate new mutations identified in patients. Even though the main publications in this field focus on loss-of function experiments performed using the MO strategy, research has now started to exploit the possibility of creating stable mutant lines to be used to better identify the mechanisms of complicated forms of HSP, and as a tool for drug screening. Taken together, these approaches have resulted in the development of gene-specific of HSP and ASS zebrafish models.In this review, we have highlighted some of the key features that make the zebrafish a compelling organism for modeling complicated forms of HSP and ASS. Zebrafish has been used to study molecular and functional aspects of HSP and ASS This literature review highlights several common phenotypes among the different disease models thus far described: almost all showed locomotor impairment, motor neuron defects, and body malformations. However, these phenotypes can also be relevant to other forms of neurodegeneration impacting on upper and lower motor neurons and long neuronal tracts. As outlined in FS, VN, and MM: conceptualization. VN and MM: methodology. GF, SM, and VN: investigation. GF and VN: resources and writing original draft preparation. FS, MM, AO, AD, FG, and VN: writing review and editing. SM: visualization. FS and MM: supervision and funding acquisition. VN: project administration.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aggressive maintenance of a target hematocrit level lower than 45% with phlebotomy (PHL), either alone or associated with cytoreductive drugs, and low-dose aspirin have been shown to reduce the thrombosis rate in the randomized controlled CYTO-PV2 and European Collaborative Low-dose Aspirin Polycythemia Vera (ECLAP)3 clinical trials, respectively, and are recommended therapies in the clinical practice5.Clonal proliferation of hematopoietic precursors leading to progressive expansion of myeloid cells with a predominant increase of red cells characterizes the hematological phenotype of Polycythemia Vera (PV). The resulting blood hyperviscosity is a major determinant of vascular complications which severely impact on morbidity and mortality of these patients6.In patients at high-risk because of age and/or history of thrombosis, hydroxyurea (HU) is the recommended front-line cytoreductive drug based on a small observational study (PVSG protocol 08), in which 51 HU-treated patients experienced a lower incidence of thrombosis compared with historical controls managed with PHL7.More recently, interferon (IFN)-α and ruxolitinib (a JAK1 and JAK2 inhibitor) have been introduced into the therapeutic armamentarium for patients with PV, yet in the absence of controlled evidence of superiority over HU as regards prevention of thrombosis, and a relative lack of information about safety over long-term use for ruxolitinib7. Moreover, the concern that HU may increase the risk of leukemic transformation led to suggest therapeutic PHL as the only first line therapy, irrespective of patient risk category8. Recently, we documented an advantage of HU over PHL in a cohort of 1042 patients with PV included in the ECLAP trial consistently significant with respect to the proportion of fatal/non-fatal CV events  and myelofibrosis transformation, that was more frequent in patients treated with PHL only9.The recommendation that HU should be a first line therapy has been criticized since no solid demonstration of its efficacy to prevent thrombosis or prolong survival has been produced so far10. (ii) HU may predominantly exert its antithrombotic property by reducing the cellular components of thrombi formation, including leukocytes, and (iii) leukocytosis is associated more with arterial than venous thrombosis in PV1.In the present work we investigated more thoroughly the same cohort of PV patients to determine whether cytoreduction therapy with HU, in comparison with therapeutic PHL, was differently effective in protecting from arterial and venous thrombosis. The rationale of this analysis, approved by our IRB, is supported by the following considerations: (i) the two vascular complications are biologically different processes with distinct physiopathology supporting that arterial and venous events are separate entities with specific risk factors that require careful evaluation and management11 by forming matched sets of 1 PHL and up to 2 randomly sampled HU-treated subjects who shared a similar values of PS  were well balanced for the parameters included in the PS . Over a comparable treatment period with PHL (median: 25.8 months) and HU (median: 24.0 months), we documented a significant advantage of HU over PHL with respect to the proportion of non-fatal arterial thrombosis , while HU did not result more effective than PHL in reduction of unprovoked venous thromboembolism (p = 0.574) (Table p = 0.001).We selected patients who, during the follow-up, had received only PHL or HU to maintain the HCT level < 45%. To assure comparability, we conducted a 1:2 Propensity Score (PS) matching analysisThe cumulative incidence curves for arterial thrombosis Fig. a, b showThe results of this study comparing two well-balanced cohorts of PV patients by using a propensity score matching, should be interpreted as a semi-experimental prospective trial and although the findings of current analysis are possibly exposed to intrinsic limitations, the rigorous application of 1:2 matching  generation12. These processes are more involved in arterial thrombosis, while the changes in blood flow due to hyperviscosity are predominantly associated with venous thrombosis, providing an explanation for the comparable efficacy of PHL and HU on the background of similar control of HCT levels < 45%. However, it should be underscored that the rate of both arterial and venous thrombosis remains high, also in low-risk PV (rate 2.5 per 100 PY2) compared to the normal population 13, suggesting the need of further therapeutic proposals.To our knowledge, this is the first study documenting the greater antithrombotic protection of HU over PHL against arterial thrombosis while the two treatments produce similar results in the protection from venous thrombosis. This observation deserves to be confirmed with studies that have a larger number of venous events including splanchnic thrombosis separately from the common deep vein thrombosis. One possible explanation for these findings is that HU reduces the abnormalities of MPN clone-derived blood cells, such as leukocytosis and consequently the interaction with platelets and vascular endothelium which lead to an inflammatory state reduces the inflammatory state we know involved in the pathogenesis of thrombosis


Graft vs. host disease (GVHD) is the major non-relapse complication associated with allogeneic hematopoietic stem cell transplantation (HSCT). Damage to the gastrointestinal (GI) tract from acute GVHD is a particularly serious event that can result in significant morbidity and mortality. Proinflammatory cytokines play a critical role in the pathophysiology of intestinal GVHD, in part by activating donor T cell populations which subsequently induce tissue damage. In this review, we summarize pre-clinical data derived from experimental murine models that have examined the role of inflammatory cytokine pathways that play critical roles in the pathophysiology of GVHD of the GI tract. Specific areas of focus are on STAT 3-dependent cytokines , and members of the IL-1 cytokine family, both of which have been shown to induce pathological damage within the GI tract during this disease. We also review established and ongoing efforts to translate these pre-clinical findings into the clinic in an effort to reduce morbidity and mortality due to this complication. Graft-vs.-host disease (GVHD) is the major complication that occurs after allogeneic hematopoietic stem cell transplantation (HSCT) and is the leading cause of transplant-related mortality , 2. MatuGVHD has been divided into two phases, termed acute and chronic, which are distinguishable based on the timing of onset as well as unique clinical and pathological manifestations –12. Duriin vitro using human cells where small molecule inhibition of STAT3 signaling suppressed alloreactive T cell proliferation while enhancing expansion of induced Tregs (iTregs)  proteins are responsible for much of these gene expression changes as a result of signaling through cytokine receptors. Upon ligation of a cytokine to its receptor, Janus kinases (JAKs) bind to the cytoplasmic domain of the receptor and become active, phosphorylating the appropriate STAT proteins, which then dimerize and translocate to the nucleus where they execute their function in driving transcriptional changes . Of thes(iTregs) . In addipatients .+ and CD8+ T cells in the spleen and an elevation in serum IFN-γ and IL-17 in LysM-Cre STAT3fl/− recipients compared to WT recipients, suggesting that this subset of recipient myeloid cells might indirectly regulate donor T cell responses. Interestingly, deficiency in donor myeloid cells had no impact on overall GVHD severity  that have received FDA approval for the therapy of psoriasis , 49. How+ T cells, CD8+ T cells, and NKT cells, while the receptor for IL-21 is expressed on T cells, B cells, NK cells, dendritic cells macrophages, and epithelial cells  was one of the first cytokines described and was named for the soluble product of macrophages during inflammation . Since tA role for the IL-1 family in the biology of GVHD was first postulated in 1991 when increased levels of IL-1α mRNA was detected in the skin of GVHD recipients in an MHC-matched, minor antigen mismatched murine transplant model . In addiWith the discovery of the inflammasome  there haJankovic and colleagues provided the first evidence that the NLRP3 inflammasome is important in the pathophysiology of gastrointestinal GVHD . This re+ subepithelial myelofibroblasts which have also been termed pericryptal fibroblasts   . Notablylations) .+ regulatory T cells, a cell population that is able to survive after total body irradiation. A reduction in GVHD was also observed in mice that received peritranspant sST2 blockade, which potentiates the effects of IL-33 due to an inhibition of its decoy receptor  and cytokine pathways  that are operative in the pathophysiology of GVHD of the GI tract. Notably, many of these cytokines or specific pathways can be targeted with existing, clinically available antibodies or small molecules designed to inhibit their activity in human transplant recipients. Thus, there is now optimism that the further evolution of this work will lead to the rational development of new strategies designed to reduce the severity of this complication in man and ultimately result in improved overall survival.CP and WD wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Congenital anomalies are significant causes of mortality and morbidity in infancy and childhood. Embryogenesis requires specific signaling pathways to regulate cell proliferation and differentiation. These signaling pathways are sensitive to endogenous and exogenous agents able to produce several structural changes of the developing fetus. Oxidative stress, due to an imbalance between the production of reactive oxygen species and antioxidant defenses, disrupts signaling pathways with a causative role in birth defects. This review provides a basis for understanding the role of oxidative stress in the pathomechanism of congenital malformations, discussing the mechanisms related to some congenital malformations. New insights in the knowledge of pathomechanism of oxidative stress-related congenital malformations, according to experimental and human studies, represent the basis of possible clinical applications in screening, prevention, and therapies. Embryogenesis represents a complex process requiring temporal and spatial regulatory mechanisms . These mEnvironmental drugs, chemicals, and physical agents can produce congenital malformations and reproductive effects. The most common known cause is genetic, but the largest group, unfortunately, is unknown.It is important to remember that a teratogenic exposure includes not only the agent but also the dose and the time in pregnancy when the exposure has to occur. The dose is a crucial component in determining the risk, since those teratogenic agents follow a toxicologic dose-response curve .Known agents that have been demonstrated to result in malformations cannot produce every type of malformation. So, it is easier to exclude an agent as a cause of birth defects than to conclude definitively that it was responsible for birth defects .Oxidation–reduction (redox) homeostasis, like pH control, is central to life. Redox processes pervade practically all fundamental processes of life from bioenergetics to metabolism and life functions . BiologiOxidative stress is an imbalance between oxidants and antioxidants in favour of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage . OxidatiBiological redox equilibria do not denote, as a matter of fact, true thermodynamic equilibria but instead are “nonequilibria” as defined by steady state . ImportaEmbryonic development requires specific signaling events that regulate cell proliferation and differentiation to occur at the correct place and the correct time in order to build a healthy embryo. Signaling pathways are sensitive to perturbations of the endogenous redox state and are also susceptible to modulation by reactive species and antioxidant defenses, contributing to a spectrum of passive versus active effects that can affect redox signaling and redox stress .Redox signaling plays a pivotal role in developmental processes, and it is largely regulated during embryogenesis. Disruption of redox signaling pathway alters the control of intracellular redox potential and causes redox stress through the generation of reactive oxygen species (ROS) . These dIn addition to ROS, further important reactive species have notable impacts on redox biology and, consequently, on oxidative stress: reactive nitrogen species (RNS) , reactivEnzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) are important in scavenging ROS and have been shown to increase 150% during the last 15% of gestation. There are three forms of SOD that have been identified: copper-zinc superoxide dismutase (Cu/ZnSOD) that is present primarily in the cytoplasm, manganese superoxide dismutase (MnSOD) in the mitochondria, and extracellular superoxide dismutase (EC-SOD) located in the extracellular spaces in adults but primarily intracellular in newborns. The only known function of SOD is to convert extremely reactive superoxide radicals to hydrogen peroxide and water. Catalase, GPx, and glutathione reductase then convert hydrogen peroxide to water .Antioxidant enzyme expression generally increases in most fetal compartments throughout the progression of pregnancy. Qanungo and Mukherjea found that SOD, catalase, GPx, and glutathione reductase activities increased with gestational age, as evidence of lipid peroxidation decreased in human placental and fetal tissues .Development of the antioxidant system during fetal life must also include redox signaling in the maintenance of pregnancy through uterine-placental-fetal interactions .There is evidence of regulation of antioxidant enzymes in the context of local nitric oxide (NO) generation via nitric oxide syntheses and downstream NO-dependent signaling in the placenta, critically important to normal vascular development.In this review, we present data concerning redox signaling in developmental processes and discuss the role of oxidative stress during pregnancy and embryogenesis. Models of congenital malformations in which redox modulations affect the development and function of the system are also shown.Intracellular ROS are important factors in signaling mechanism, as they modulate physiological processes during embryogenesis. Besides, cellular proliferation, differentiation, and apoptosis are often driven by specific redox signals.2-) is mainly produced by the oxidation of NADPH by NAPH oxidase enzymes (NOXs) or by electron leak from aerobic respiration in the mitochondria. Superoxide is then quickly converted into hydrogen peroxide (H2O2) by superoxide dismutases (SODs). H2O2 may either oxidize cysteine residues on proteins to initiate redox biology, or it may be converted to H2O by cellular antioxidant proteins, such as peroxiredoxins (PRx), glutathione peroxidase (GPx), and catalase (CAT). When H2O2 levels raise significantly, hydroxyl radicals (OH) form via reactions with metal cations (Fe2+) and irreversibly damage cellular macromolecules [Intracellular superoxide  causes oxidative stress.An increased production of ROS during organogenesis, period in which cells continue to differentiate, disrupts critical signaling events causing structural abnormalities, loss of cellular function, or spontaneous abortion of the developing fetus .Different conditions may produce abundant ROS in human tissues, resulting in a state of oxidative stress for both the mother and the developing fetus. Although the relationship between oxidative stress and congenital malformations is not clear and need further investigations, experimental studies in animal models have shown that oxidative stress might play a significant causal role in birth defects.For instance, Long and colleagues investigated the relationship between the toxicological effects of bacterial component LPS via oxidative stress and pulmonary dysplasia in chick embryos. The FGF and Wnt signaling pathways are considered to control lung development. GATA binding protein 6 (GATA-6) is a member of the zinc finger GATA protein family that presumably plays a key role in maintaining the balance between the proliferation and differentiation of pulmonary epithelial progenitor cells during lung development through modulating Wnt signaling. Long and colleagues demonstrated that LPS could induce the oxidative stress, which subsequently led to altered embryonic lung development. Specifically, LPS induced an intracellular ROS production enhancement, which was partially blunted by the addition of vitamin C to the culture medium. LPS significantly inhibited GATA-6 expression. However, GATA-6 was partially restored by vitamin C. Moreover, LPS induced downregulation of SP-C, ABCA3, and GATA-6 expressions, which again could be restored by vitamin C .Over the course of development, the delicate balance between oxidants and antioxidants can be disrupted by various factors  that induce ROS production and lead to oxidative stress. Many investigators have evaluated the effects of antioxidants on embryonic development. In general, antioxidants reduce the abundance of highly reactive ROS by becoming radicalized themselves. The most important, glutathione, exists in a couple of its oxidized (GSSG) and reduced (GSH) forms. Another significant group of antioxidants are selenium and selenoproteins, such as thioredoxin, GSH peroxidase (GPX4), and selenoprotein W. Lipoic acid is a potent natural antioxidant. Mice deficient in lipoic acid are retarded in their development and die early with a lack of organization and smaller size. The heterozygotes have significantly reduced erythrocyte GSH levels and lower antioxidant capacity.The enzyme G6PD is also very important to oxidative stress. It is a developmentally critical enzyme that protects the embryo from endogenous and xenobiotic-initiated oxidative stress and DNA damage. In fact, G6PD-deficient dams have higher embryonic oxidation and more fetal death and birth defects than their wild-type counterparts.Other antioxidants, including vitamin C, are vital to the fetoplacental unit, so that in cryopreserved embryos, addition of ascorbate reduced the levels of hydrogen peroxide, increased the rate of metabolism, and enhanced inner cell mass development .A better understanding of the mechanisms behind the relation between oxidative stress and congenital malformations may be important from both diagnostic and therapeutic perspective, providing both early prenatal diagnostic tools and new possible preventive treatments with antioxidant administration during pregnancy to reduce any oxidative damage of abundant ROS during organogenesis.Oxidative stability can be assessed using different markers such as antioxidant enzymes: glutathione peroxidase (PxG), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), vitamin C, vitamin A, vitamin E, glutathione (GSH), or by determining the total antioxidant capacity (TAC) and nitrite levels .β-HCG, already used to assess the risk of chromosomal aberration in the first trimester [Oxidative stress may also affect the levels of PAPP-A and rimester .Plentiful evidence both from in vivo and in vitro studies and animal models have suggested that the pathogenesis of Down syndrome (DS), due to complete or partial trisomy of chromosome 21, might be linked to the effects of oxidative stress on early embryonic development .Trisomy of chromosome 21 causes deregulation of gene/protein expression either in dosage-sensitive genes (gene dosage hypothesis) or in several other genes  . FurtherPrevious studies have been supposed that a chronic oxidative injury in the brain would act as a risk factor for abnormal brain development, the higher incidence of hyperactivity with attention deficits and Alzheimer disease clinical features of DS patients , 22.Genes located on chromosome 21, overexpressed due to triplication of the chromosome, include copper-zinc superoxide dismutase (SOD1), amyloid precursor protein (APP), transcription repressor BACH1 genes and other several genes with a role in ROS metabolism .2·−) into the reactive oxygen species (ROS) hydrogen peroxide (H2O2) converted into water (H2O) and molecular oxygen (O2) by catalase (CAT) and glutathione peroxidase (GPX). The triplication of chromosome 21 causes an excessive activity of SOD1 and an altered ratio of SOD-1/CAT and GPX, resulting in the accumulation of endogenous H2O2 and/or its conversion products  with cellular oxidative damage [SOD1 gene encodes for the enzyme catalyzing the conversion of superoxide anion  and oxidative damage antioxidants (protein carbonyls levels and lipoperoxidation), all measured spectrophotometrically in whole blood of 20 DS patients and 18 healthy controls, showed an increase in the SOD and CAT activities and a decrease in protein carbonyls levels in DS individuals, revealing a systemic prooxidant status in the blood of DS patients. Finally, in a cross-sectional study, total SOD activity in plasma from 36 DS children measured by spectrophotometric methods was found increased when compared with 40 healthy controls .Trisomy 21 also causes overexpression of BACH1 gene, a basic leucine zipper protein belonging to the cap'n'collar (CNC) family. BACH1 is a transcription repressor that binds the antioxidant response elements of DNA (AREs) and suppresses the expression of specific genes/proteins controlled by ARE such as quinone oxidoreductase-1 (NQO1), glutathione S-transferase (GST), glutamate-cysteine ligase (GCL), and heme oxygenase-1 (HO-1). In oxidative stress conditions, the function of BACH1 is suppressed thus promoting the expression of these genes involved in the cell stress response. It was supposed that BACH1 overexpression might promote oxidative stress blocking the expression of oxidative stress-responsive and antioxidant genes. Increased total BACH1 protein levels were found into frontal cortex tissue from 16 DS individuals coupled with reduced induction of brain HO-1 compared to healthy individuals, suggesting that BACH1 overexpression in DS leads to the repression of HO-1 transcription and may contribute to the increased OS found in DS .β) that is considered to be the most important pathogenic molecule in Alzheimer's disease representing the core protein of neuritic plaques. Aβ accumulates in the brains of DS individuals as early as 8–12 years of age, and this accumulation increases during the lifespan resulting in Alzheimer's disease-like neuropathology found in all DS individuals over 40 years of age. Soluble forms of Aβ generated from APP commonly end at C-terminal residue 40 (Aβ40) or 42 (Aβ42). Plasma concentrations of both Aβ40 and Aβ42, quantitated by sandwich ELISA from 35 DS children and adolescents, were reported significantly higher in DS patients than in controls and the ratio of Aβ42/Aβ40 was lower in DS than in controls [β-peptide overexpression leads to accumulation into neuritic plaque inducing neuronal loss and cognitive dysfunction and could be associated with ROS production and oxidative stress [In addition, the overexpression of the amyloid precursor protein (APP) gene, located on chromosome 21, in DS patients causes an increased production of amyloid beta-peptide  that normally detoxifies the cytotoxic metabolic intermediates carbonyls catalyzing the reduction of free carbonyls compounds to their corresponding alcohols. Carbonyls are cytotoxic metabolic intermediates that are detoxified by either oxidation catalyzed by aldehyde dehydrogenase (ALDH) or by reduction to alcohols by CBR and/or alcohol dehydrogenase (ADH). Of note, increased levels of CBR protein have been shown in different brain regions of DS patients . Thus, iA systemic prooxidant status in DS individuals has been confirmed in various studies that demonstrated an increased activity of some important antioxidant enzymes  together with decreased glutathione (GSH) levels in DS whole blood and higher levels of biomarkers of oxidative damage, such as protein carbonyls, malondialdehyde (MDA), allantoin, or 8-hydroxydeoxyguanosine than in controls .Finally, the prooxidant condition in DS patients may be linked to reduced activity of complex I in the respiratory electron transport chain in the mitochondria associated with an increase in cellular ROS . The oliIn an effort to better understand the role of oxidative stress in DS, a set of oxidative biomarkers were evaluated in amniotic fluid collected from ten women undergoing amniocentesis and carrying confirmed DS fetuses compared with ten women carrying normal fetuses in a retrospective matched case control study . IncreasIt is clear from these data that DS fetuses are exposed to oxidative stress early in pregnancy with consequent damage of many fetal organs and tissues .β also affects redox imbalance and could exacerbate oxidative damage into the brain.In conclusion, it has been suggested that trisomy of chromosome 21 causes stress oxidative conditions and oxidative injury early in embryogenesis altering gene/protein expression and particularly inducing overexpression of SOD1 and also reduction of antioxidant enzymes. Moreover, the overproduction of ABased on these findings, the administration of antioxidant nutrients could have a role in ameliorating the clinical pattern of DS patients. In previous studies on the effects of antioxidant elements, controversial results were obtained. In a 2-year randomized, double-blind, and placebo-controlled trial with daily oral antioxidant supplementation in DS patients and dementia , it was demonstrated that the supplementation was safe and well tolerated but not associated with any stabilization or improvement in the cognitive function .Further studies are needed in order to elucidate the relationship between oxidative stress and DS clinical expression to identify clinical biomarkers of early oxidative stress and damage and to find any possible therapeutic agents.The incidence of congenital heart defects (CHDs) varies from 4/1000 to 50/1000 live births .The embryonal heart tube is composed of myocardium and an inner lining of endocardial cells separated by an extensive extracellular matrix the so-called cardiac jelly. The formation of cardiac cushions is a complex event under the direction of specific signaling pathways.Nowadays, despite there are some progresses in understanding the genetics of heart defects, only 15% of CHDs can be attributed to a genetic cause. All other cases result from a complex interaction between genetic susceptibility and environmental factors  whose common embryotoxic effect might be related to excessive production of reactive oxygen species and to reduced antioxidant-defense mechanisms .Despite the role of ROS in cardiovascular diseases (CVD) is well documented , 44, theIn a study by Ercan and colleagues, the relationship between congenital heart diseases and oxidative stress in children with cyanotic and acyanotic congenital heart diseases was investigated. The authors concluded that the oxidant and antioxidant values of the cyanotic patients were significantly higher than the acyanotic and control groups. So, they have speculated that due to the underlying anatomical defect, hypoxia develops and increases both the free oxygen radicals and the antioxidant substances for compensation afterwards .Increased oxidative stress and reduced antioxidant capacity might lead to CHDs, through ROS production, which affect many intra- and intercellular signaling pathways , 48.Furthermore, recent evidences in humans, i.e., mothers of offspring with congenital heart disease, have shown elevated homocysteine level related to low folate and/or vitamin B12 levels thus supporting that folic acid pathway alteration may exert an indirect embryotoxic effect by increasing oxidative stress. The metabolic pathway from homocysteine to glutathione is referred to as the transsulfuration pathway. Approximately 50% of homocysteine generated from methionine is metabolized to cystathionine. This is an irreversible reaction that permanently removes homocysteine from the methionine cycle for the synthesis of cysteine and glutathione. Elevated homocysteine is associated with alterations in the transsulfuration pathway that lead to greater oxidative stress . In a prExperimental models have suggested that, in addition to evidence of a direct teratogenic effect, elevated homocysteine may have an indirect embryotoxic effect by increasing oxidative stress through excessive production of reactive oxygen species and by decreasing the glutathione-dependent antioxidant-defense mechanism. Hobbs and colleagues indicate that higher homocysteine observed among women with CHD-affected pregnancies may extend beyond impairments in remethylation of homocysteine to impairments in the transsulfuration of homocysteine. Specifically, in comparison to controls, cases with CHD-affected pregnancies had significantly lower concentrations of vitamin B-6, glutamylcysteine (GluCy), and reduced glutathione (GSH) and significantly higher concentrations of oxidized glutathione (GSSG).Role of oxidative stress for congenital cardiovascular malformations is well studied in maternal diabetes . Congenird trimester.In fact, diabetic pregnancy is considered an independent risk factor for major embryonic malformations, and cardiac outflow tract defects are among the most frequent alterations observed in epidemiological studies . The higStudies on rats have shown that hyperglycemia in the embryo induces production of reactive oxygen species that, together with a reduced ability of fetal cells to activate antioxidant defense mechanisms, mediate adverse effects on cardiac neural crest migration and cardiac outflow tract septation through proapoptotic signaling , 54.In humans, oxidative stress markers have been evaluated in the cord blood of newborns delivered by mothers with diabetes, suggesting that hyperglycemia induces oxidative stress . HoweverGlucose may also affect gene expression in embryonic development via epigenetic changes (histone acetylation and microRNA expression) .The alternative that offspring CHD reflects maternally inherited genetic or epigenetic variations that confer risk of both diabetes mellitus and cardiac abnormalities is less likely, because the risk of maternal diabetes mellitus subsequent to birth of a child with CHD was only modestly increased. Conotruncal defect risk increased in the offspring of diabetic women consistent with experimental study findings that hyperglycemia in early pregnancy affects regulatory gene expression in the embryo, leading to cardiac neural crest cell death and increased CHD risk, particularly for conotruncal and outflow tract abnormalities.Detailed mechanistic studies will be required to define the role of glucose sensitivity in cells from the neural crest and anterior second heart field during cardiac development. Maternal diabetes mellitus was also associated with the entire spectrum of CHD phenotypes. The nonspecific nature of the association suggests that hyperglycemia in early pregnancy may not only influence specific sequences in cardiac development but affects cardiac development in general or exerts its detrimental effect before formation of the primitive heart tube, with subsequent early and late consequences for fetal cardiac development.Studies in rats have shown that oxidative stress during pregnancy can be reduced by using vitamin E and Vitamin C as antioxidants , 59, theFurthermore, recent evidences in humans, i.e., mothers of offspring with congenital heart disease, have shown elevated homocysteine level related to low folate and/or vitamin B12 levels , 60, thuDespite some human studies have shown that women using multivitamin supplements and folic acid during the periconceptional period had a lower risk of having babies with congenital heart defects, there are still concerns about the role of multivitamin supplements in reducing embryotoxic effect and risk for CHD in humans , 63.Recent studies have also shown that ROS overproduction and/or imbalance in the antioxidant system could lead to pulmonary hypertension in cases of CHD associated with increased pulmonary blood flow in lamb  and rodeFurther studies are needed to identify the key factors in the development of CHDs in order to develop and implement effective primary prevention program with preconceptional screening and development of nutritional intervention.The human brain is particularly vulnerable to the damaging effects of reactive oxygen intermediates due to both its complexity and the long period of development .Embryonic and fetal brain tissues are especially susceptible to peroxidative injury due to the fact that their membranes are rich in easily oxidable polyunsaturated fatty acid side chains .Several studies indicate that antioxidant enzymes and molecules exhibit extremely low activities in fetal tissues, especially the brain , 68. CimHoloprosencephaly (HPE) is one of the most common birth defects and is characterized by midline defects of the brain, facial, and oral structures. In humans, it has been estimated that HPE affects 1 in every 5000–10,000 live births and 1 in every 200–250 miscarried fetuses . Many caShh, produced by the axial mesendoderm, prechordal mesoderm (PME), and notochordal plate, acts as a crucial signal in mammalian brain and facial development, and Shh gene alterations are the most frequent causes of autosomal-dominant inherited HPE in humans and Shh2/2 mouse embryos exhibit severe HPE –76.Ethanol-induced activation of PKA in the anterior PME results in a reduction of Shh expression and enhanced apoptosis of anterior PME cells causing the characteristic severe midline defects of HPE.The inhibition of PME cells apoptosis by antioxidants, i.e., vitamin C and vitamin E, may protect from the teratogenic actions of ethanol.Neural tube defects (NTDs) are a group of common and devastating congenital malformations that arise early in pregnancy due to the disturbance of normal neural tube closure. NTDs occur in about one in every 1000 established pregnancies worldwide , and it The aetiology of NTD is thought to be heterogeneous, including genetic and environmental factors and their interactions , 79. FacPax3) gene plays a major role in the development of neuroepithelium of embryos. In the absence of Pax3, neural tube defects occur [Pax3 expression leads to an increased risk of neural tube defects and diminished gene expression [Moreover, oxidative stress induces DNA damage and defects in DNA repair mechanisms. Single- and double-stranded fractures, base modifications , and nucleoside damage may occur in DNA. There may also be crosslinking between DNA and protein depending on oxidative damage , 93. Earts occur . Oxidatipression .Myelomeningocele (MM) is a common congenital malformation that occurs when the embryonic neural tube fails to close properly during early embryogenesis. Common pathogenetic mechanisms for MM include folate deficiency, genetic susceptibility, environmental factors, in utero drug exposure, and biochemical factors –100.Numerous reports have described free radical-mediated congenital defects –89. Kao In addition, SOD is involved in FR-mediated neurological diseases and acting a fundamental role in modulating reactive oxygen species toxicity . In tissArslan and colleagues found that malondialdehyde (MDA), an oxidative damage marker, and a lower activity of erythrocyte carbonic anhydrase, an antioxidant enzyme which regulates the acid-base homeostasis, differ in newborns with MM and in their mothers compared to healthy newborns and their mothers .This study suggests that the finding of low antioxidant enzyme activities in addition to ultrasound and maternal serum alpha fetoprotein may be an index of suspicion of neural tube defect.In pregnant women with high risk, antioxidant enzymes administration together with folic acid may be an opportunity to reduce the incidence of neural tube defect.Pregnancy is a state of oxidative stress as a consequence of high metabolic activity in the fetoplacental compartment. Fetal tissues are especially sensitive to oxidative damage because of the rapidly growing nature of their cells, which makes them vulnerable to the harmful effects of free radicals.Despite reactive oxygen species and free radicals, in the presence of a good antioxidant capacity, are important for developing embryos, promoting and controlling cellular fate, and playing a crucial role in normal development through cellular signaling, when overproduced, in the absence of a parallel increase in antioxidative activity, resulted in a wide range of biological toxic effects.Due to the rapidly growing nature of their cells, fetal tissues are especially sensitive to oxidative damage that lead to lipid, protein, and polysaccharides oxidation and DNA damage with disruption of apoptosis processes that, during organogenesis, are highly needed in an appropriate location and temporal pattern.Thus, the increase of oxidative stress, together with the impaired antioxidant activity, is clearly related to the induction of fetal malformations.There are still gaps in our knowledge in the role of oxidative injury in the activation of complex array of genes involved in different biological processes of fetal structure such as inflammation, coagulation, fibrinolysis, cell cycle, cell adhesion, and signal transduction. Future studies addressing the role of oxidative stress in this field are encouraged.Moreover, there are few published studies evaluating oxidative stress biomarkers and management of oxidative stress with antioxidants therapeutic approaches.Oxidative stress is widely implicated in failed reproductive performance, including infertility, miscarriage, diabetes-related congenital malformations, and preeclampsia. Poston et al. have focused on the role of free radicals and antioxidant capacity in preeclampsia. By measuring markers of lipid peroxidation and antioxidant capacity, they demonstrated the role of oxidative stress in this disorder .Recent studies suggest that ischemia-reperfusion in the placenta as well as endoplasmic reticulum stress in the placenta may contribute to oxidative stress in trophoblasts. The recognition of oxidative stress in the placenta and the maternal circulation has led to evaluate the potential benefit of prophylactic antioxidant supplementation in women with a known risk of preeclampsia, particularly with an early supplementation with vitamins C and E . HoweverOther potential approaches include the use of supplements in the preconceptual period, selenium supplements, antiperoxynitrite strategies, and statins , 110.In clinical practice, early markers of oxidative stress might reveal that gravidic prophylactic use of antioxidants could help to prevent or at least reduce oxidative stress-related malformations in fetuses. Anyway, maternal antioxidant supplementation during pregnancy is important for protecting newborns against oxidative DNA damage.Potential therapies for ROS-induced disease include both enzymatic and nonenzymatic antioxidant preparations. Supplementation with enzymatic and/or nonenzymatic antioxidants might have beneficial effects in decreasing injury from excess production of ROS, particularly in disorders such as bronchopulmonary dysplasia, retinopathy of prematurity, periventricular leukomalacia, and necrotizing enterocolitis in preterm newborns who are especially susceptible to ROS-induced damage because of inadequate antioxidant stores at birth, as well as impaired upregulation in response to oxidant stress . NonenzyMore research is required to determine the beneficial effects of supplemental antioxidant therapy. There are multiple potential therapeutic antioxidants currently under investigation that could benefit newborns, particularly premature infants. For example, one protein under investigation is Pon3 that was shown in laboratory studies to have antioxidant properties and to be upregulated in rat, sheep, and human cord blood late in gestation . Other cCaution must be taken since ROS are critical second messengers in various cell signaling pathways that control normal cellular functions, but strategies that maintain normal antioxidant balance may be beneficial to the newborns. New studies should more extensively investigate the diagnostic and therapeutic value of various oxidative stress biomarkers and antioxidants to reduce oxidative tissue injury to developing newborns.