Abciximab is a widely used adjunctive therapy for acute coronary syndrome (ACS). However, the effect of intracoronary (IC) administration of abciximab on cardiovascular events remains unclear when compared with intravenous (IV) therapy.We systematically searched the Medline, Embase, and Cochrane Central Register of Controlled Trials databases and reference lists of articles and proceedings of major meetings for obtaining relevant literature. All eligible trials included ACS patients who received either IC administration of abciximab or IV therapy. The primary outcome was major cardiovascular events, and secondary outcomes included total mortality, reinfarction, and any possible adverse events. Of 660 identified studies, we included 9 trials reporting data on 3916 ACS patients. Overall, IC administration of abciximab resulted in 45% reduction in relative risk for major cardiovascular events , 41% reduction in RR for reinfarction , and 44% reduction in RR for congestive heart failure relative to IV therapy ; however, compared to IV therapy, IC administration of abciximab had no effect on total mortality . No other significant differences were identified between the effect of IC abciximab administration and IV therapy.IC administration of abciximab can reduce the risk of major cardiovascular events, reinfarction, and congestive heart failure when compared with IV therapy. Previous trials have indicated that the management of acute coronary syndrome (ACS) is undergoing major changes Several randomized controlled trials argued that rapidly active abciximab should be used in the ambulance, which contributed to faster recovery and increased efficacy in ACS patients Recently, several large-scale randomized controlled trials have investigated IC abciximab administration We systematically searched the English literature to identify all relevant randomized, controlled trials regardless of publication status . Relevant trials were identified using the following procedure:Electronic searches: We searched the Medline, Embase, and Cochrane Central Register of Controlled Trials electronic databases for articles published through May 10, 2012, using “intracoronary” OR “intravenous” AND “abciximab” AND “randomized controlled trials” OR “clinical trials” as the search terms.Other sources: We searched ongoing randomized controlled trials in the metaRegister of Controlled Trials, which lists trials that are registered as completed but not yet published. Furthermore, we reviewed bibliographies of publications for potentially relevant articles. Medical subject headings, methods, patient population, interventions, and outcome variables of these studies were used to identify relevant trials. This review was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) Statement issued in 2009  [13].The literature search was undertaken independently by 2 authors (Hai-Bo Yuan and Yan Zheng) with a standardized approach. Any inconsistencies between these 2 authors were settled by the primary author (Yu-Hao Zhou) until a consensus was reached. We restricted our research to randomized controlled trials, which were less likely to be subject to confounds and bias than observational studies. Studies were identified for inclusion if: (1) the study was a randomized controlled trial; (2) the trial assessed the effects of IC administration of abciximab compared with IV therapy; (3) the duration of follow-up was at least 30 days; and (4) the trial reported at least 1 outcome of major cardiovascular events, total mortality, reinfarction, or 3 of the aforementioned events.Two investigators (Yuan-Jun Tang and Yan Zheng) independently extracted and collected data using a standardized data extraction protocol. Any discrepancy was settled by group discussion, and then, the primary author (Yu-Hao Zhou) made the final decision. The data collected included baseline patient characteristics , cardiac death, congestive heart failure, major bleeding, and stroke. Study quality was assessed using the Jadad scores 2 statistic We allocated the results of each randomized controlled trial as dichotomous frequency data. Individual study relative risks (RRs) and 95% confidence intervals (CIs) were calculated from event numbers extracted from each trial before data pooling. The overall RR and 95% CI of serious vascular events and any possible adverse events were also calculated. Heterogeneity of the treatment effects between studies was investigated visually by scatter plot analysis and statistically using the heterogeneity IWe identified 660 articles from our initial electronic search, of which 644 were excluded during an initial review (title and abstract). We retrieved the full text for the remaining 16 articles, and 9 randomized controlled trials met the inclusion criteria , which cData for the effect of IC abciximab administration on major cardiovascular events were available from 6 trials Data for the effect of IC administration of abciximab on total mortality were available from 9 trials Data for the effect of IC abciximab administration on reinfarction were available from 7 trials Five of the trials The effect of IC administration of abciximab on the risk of cardiac death was reported in 3 trials The risk of congestive heart failure was reported in 2 trials Four trials Of the 9 trials included in our meta-analysis, only 2 provided data about stroke events Subgroup analyses were conducted for major cardiovascular events and total mortality. We noted that IC administration of abciximab was associated with a reduction in the risk of major cardiovascular events when the number of patients was more than 200, the mean age of patients was below 63, the proportion of men was less than 80%, or the Jadad score was less than 4. Similarly, when compared with IV therapy, IC administration of abciximab showed a clear effect on total mortality when the mean age of patients was below 63, the proportion of men was greater than 80%, or the Jadad score was less than 4. No other significant differences were identified between the effect of IC abciximab administration and IV therapy, when based on additional subset factors .We used Egger’s test This comprehensive, quantitative review included 3916 patients from 9 trials with a broad range of baseline characteristics. Although the studies included clinically diverse populations (with STEMI or ACS), there was little heterogeneity among results of different trials, and the results were stable when subjected to various sensitivity analyses.Our study indicated that IC administration of abciximab produced a 45% reduction in major cardiovascular events compared with IV therapy. In addition, IC abciximab administration played an important role in the incidence of reinfarction. However, IC administration of abciximab was not associated with a statistically significant decrease in the risk of total mortality. Finally, we noted that major bleeding events were less markedly reduced in patients who underwent IC administration of abciximab than those given IV therapy during the active study periods.Our main findings are in contrast with the findings of previous research The subgroup analyses indicated that IC administration of abciximab had no effect on major cardiovascular events when the number of patients was less than 200, the mean age of patients was above 63, the proportion of men was greater than 80%, or the study was high-quality (Jadad score 4 or 5) as compared with IV therapy. The reasons for this lack of difference are as follows: (1) a small sample size always contributed to broad confidence intervals; (2) older patients always had a poor prognosis; and (3) several other factors might play an important role in ACS pathogenesis and affect the efficacy of treatment, including smoking, alcohol abuse, and others, which occurred more frequently in male patients. Similarly, the results of the subgroup analyses were also supported when the mean age of the patients was below 63, and the proportion of men was greater than 80%, and low-quality (Jadad score less than 4) trials indicated that the risk of total mortality was significantly reduced by IC administration of abciximab when compared with IV therapy. Several of the reasons for these findings are the same as those mentioned above. In addition, in the patient subset with a proportion of men that was greater than 80%, the high incidence of reinfarction and low mortality rate might play important roles in the total mortality risk.There were significant differences between IC administration of abciximab and IV therapy for major cardiovascular events and reinfarction; moreover, sensitivity analysis indicated that IC administration of abciximab produced a protective effect on total mortality. The reason for this could be that local doses of IC-administered abciximab might facilitate the diffusion of the antibody to platelets inside flow-limiting thrombi, resulting in improved dissolution of thrombi and microemboli at the culprit lesion and in the distal microcirculation IC abciximab administration played an important role in reducing the risk of congestive heart failure, which may be due to the pathophysiological rationale of improved perfusion and the reduction in infarct size by IC administration of abciximab. Furthermore, because information regarding the effects on infarct size, ST-segment resolution, or left ventricular ejection fraction were not available, this difference in congestive heart failure might be attributable to chance No other significant differences were detected between IC administration of abciximab and IV therapy on the risk of TVR, cardiac death, major bleeding, and stroke. The reason for these absences of differences could be that these data provided by relatively few trials led us to be unable to obtain a reliable conclusion.Previous trials indicated that the effect of IC abciximab administration on cardiovascular outcomes was better than that of IV therapy, whereas other outcomes provided a negative conclusion The limitations of our study are as follows: (1). Relatively few trials reported the results of IC administration of abciximab in patients with ACS. Although subgroup analyses suggested that IC abciximab administration was associated with statistically significant reductions in the risk of major cardiovascular events and total mortality in several subsets, these conclusions were variable because the number of trials included was few that restricted us from exploring the intrinsic effect. (2). Different PCI techniques might provide a biased view of the study question. (3). Inherent assumptions are made for any meta-analysis, the analysis used pooled data either published or provided by individual study authors, and individual patient data or original data were unavailable, which restricted us from performing a more detailed relevant analysis and obtaining more comprehensive results.In conclusion, the findings of our study indicated that IC administration of abciximab produced a significant reduction in the risk of major cardiovascular events and reinfarction. Furthermore, it this treatment might also play an important role in the risk of total mortality in ACS patients. In future research, it will be important to focus on the patients’ baseline characteristics to provide patients the most suitable treatments. We suggest that the ongoing trials should be improved as follows: (1) the adverse effect events of trials should be recorded and reported normatively, particularly for serious adverse events, and the side-effects of treatment should be evaluated in any future trial; and (2) more attention should be paid to the role of treatment duration and dosage and to exploring the optimal dose and the duration of treatment.Figure S1PRISMA Flowchart.(DOC)Click here for additional data file.Table S1PRISMA Checklist.(DOC)Click here for additional data file.


To the Editor: The reemergence of tuberculosis (TB) as a global health problem over the past 2 decades, accompanied by increased drug resistance, which represents a serious problem both in terms of TB control and clinical management , or resistance to three or more drugs. Confidence intervals were also calculated. Participating laboratories were allowed to use the WHO-recommended drug susceptibility method with which they were most familiar: absolute concentration method, resistance ratio method, proportion method and its variants, or BACTEC 460 radiometric method  (The prevalence of drug resistance detected during the period 1998–2001 is summarized in the Drug-resistant TB in countries with good national control programs, such as in Western Europe, is not commonly a major health problem, although increasing immigration prompts public health authorities to maintain vigilant surveillance systems. The results of our study indicate that throughout Italy, prevalence of resistance to firstline drugs and multidrug resistance among isolates from new cases was consistently low over the 4-year survey period. Prevalence of multidrug resistance among isolates from previously treated patients was high, although a downward trend could be demonstrated during the last 2 years. Since almost 2 out of 10 isolates resistant to rifampicin were multidrug resistant, using rapid molecular methods to identify rifampicin resistance in questionable cases appears cost-effective to facilitate early detection and control of multidrug-resistant TB (


ABCC6 mutations, whereas the mineralization associated with β-thalassemia patients derives from a liver-specific change in ABCC6 expression. DCC is an acquired phenotype resulting from cardiovascular insults (ischemic injury or hyperlipidemia) and secondary to ABCC6 insufficiency. Abcc6-deficient mice develop ectopic calcifications similar to both the human PXE and mouse DCC phenotypes. The precise molecular and cellular mechanism linking deficient hepatic ABCC6 function to distal ectopic mineral deposition is not understood and has captured the attention of many research groups. Our previously published work along with that of others show that ABCC6 influences other modulators of calcification and that it plays a much greater physiological role than originally thought.Abnormal mineralization occurs in the context of several common conditions, including advanced age, diabetes, hypercholesterolemia, chronic renal failure, and certain genetic conditions. Metabolic, mechanical, infectious, and inflammatory injuries promote ectopic mineralization through overlapping yet distinct molecular mechanisms of initiation and progression. The ABCC6 protein is an ATP-dependent transporter primarily found in the plasma membrane of hepatocytes. ABCC6 exports unknown substrates from the liver presumably for systemic circulation. ABCC6 deficiency is the primary cause for chronic and acute forms of ectopic mineralization described in diseases such as pseudoxanthoma elasticum (PXE), β-thalassemia, and generalized arterial calcification of infancy (GACI) in humans and dystrophic cardiac calcification (DCC) in mice. These pathologies are characterized by mineralization of cardiovascular, ocular, and dermal tissues. PXE and to an extent GACI are caused by inactivating  In the absence of any systemic mineral imbalance, the calcification of soft tissues is defined as ectopic or dystrophic calcification. The latter term specifically distinguishes ectopic calcification that occurs in injured, damaged, and/or necrotic tissues. In contrast, elevated levels of calcium and/or phosphate due to abnormal absorption and/or secretion lead to metastatic mineralization. Whether ectopic or metastatic, abnormal calcification is typically composed of calcium phosphate salts, such as hydroxyapatite and can affect most soft tissues. However, the skin, kidneys, tendons, and cardiovascular tissues are particularly prone to this pathology.in vivo , chronic renal insufficiency, and diabetes, as well as certain infrequent genetic conditions. Calcification was long thought to result from passive precipitation of calcium and phosphate but it is now recognized as a complex tightly regulated development involving the osteoblastic differentiation of resident cells such as smooth muscle cells (SMCs), pericytes or adventitial myofibroblasts. It was also viewed as harmless, but calcification is in fact causative in the precipitation of cardiovascular events and mediating chronic damages to these tissues independently of the disease context that brings it about. More importantly, a proliferation of recent data has brought into light the many factors and the complex mechanisms that initiate and promote calcification ABCC6 gene cause PXE  transporter ABCC6 has joined the list of calcification regulators as a new member. Indeed, reduced levels of ABCC6 protein or loss of ABCC6 function in the liver has been linked to four separate ectopic mineralization pathologies in humans and mice. (1) Pseudoxanthoma elasticum (PXE: MIM 264800) is an autosomal recessive disease characterized by a slow and progressive ectopic calcification primarily affecting elastic fibers in dermal, ocular, and vascular tissues. Inactivating mutations in the The aim of this review is to summarize present knowledge on ABCC6 function and its possible molecular and physiological roles in various calcification pathologies where there clearly is more than meets the eye with this unique ABC transporter.in vitro observations demonstrated that ABCC6 has a defined perhaps restricted, substrate specificity. Though the actual endogenous substrate(s) for this transporter and hence the contributing factor to ectopic calcification in PXE, β-thalassemia, GACI, and DCC remains unknown.ABCC6 is a member of the large ABC gene subfamily C. In this group of transmembrane proteins, in addition to active transporters such as ABCC1, −2, −3, −4, and −5, there are also ion channel-forming proteins and ion channel regulators like ABCC7 (CFTR), ABCC8 and −9 (SUR1 and −2). The work of Ilias et al.  and BeliStaphylococcus aureus Sav1866 export pump  and menaquinones (vitamin K2). Three independent studies were carried out and all obtained negative results   expression as a consequence of the lack of adenosine signaling. The partial overlap between the PXE and ACDC phenotypes prompted Markello et al. to suggest that adenosine might be an ABCC6 substrate , a rare condition that has phenotypic similarities with PXE  insect cells overproducing ABCC6 and liquid chromatography/mass spectroscopy-based metabolomics to determine the compounds transported into the vesicles . The third group of laboratories actively seeking the ABCC6 substrate(s) uses the methodology reported by Van et al. . Though no publications or public reports has been made thus far, two of these groups have based their technical approaches on the principle described by Krumpochova et al.  which usn et al. : NMR speAbcc6−/− mice that promoted elastic fibers structural alterations and ectopic calcification  toward a variety of tissues, including but not limited to, the connective tissue. For example, the observations that Fetuin-A, MGP  and encodes a transmembrane protein that uses ATP hydrolysis to export organic anion transport across cellular membranes. Work to characterize the physiological role of ABCC6 has first relied upon tissue expression profiles. ABCC6 is found primarily expressed in liver and the kidneys but is also in the intestine, the retina and to a much lesser extent in most other tissues including the lung, skin, and vasculature  is a recessive disease affecting the connective tissue and is defined by the calcification and fragmentation of elastic fibers . We found a progressive liver-specific downregulation in the Abcc6 gene expression and the corresponding protein levels. This downregulation became significant at 6 months of age and stabilized at older ages at ~25% of the wild type protein levels. Studying the transcriptional regulation of the Abcc6 gene revealed that the main cause of the downregulation resided with the absence of a single transcription factor, the erythroid-specific NF-E2 from the Abcc6 promoter. Coincidentally, NF-E2 is a major transcription factor for the expression of several hemoglobin-related genes  is a monogenic disorder caused by mutations in the β-globin gene that leads to the underproduction of β-globin chains. The stoichiometric excess of α-chains unbound to β-globin is unstable and precipitate in red blood cell precursors forming inclusion bodies. These are responsible for the intramedullary destruction of the erythroid precursors and the ineffective erythropoiesis that characterize the β-thalassemias. Ineffective erythropoeisis in thalassemia major and certain intermedia patients results in considerable marrow expansion causing bone deformities and iron overload that is further exacerbated by frequent blood transfusions Thein, . The β-tAndrews, . Hbbth3/ENPP1 and affected patients suffer from hypertension, severe myocardial ischemia and congestive heart failure. Most affected patients die within the first 6 months of life. The obvious overlapping mineralization phenotype between GACI and PXE led to a recent study that correlated the phenotype to genotype in GACI and PXE patients  and the classic inherited PXE  and MGP but not osteocalcin (OC) clearly indicating that ABCC6 regulates cardiac calcification in conjunction with the local regulators of mineralization  and coronary artery ligation. We first confirmed the propensity to cardiac mineralization of Abcc6−/− mice were structurally altered and presented decreased respiratory capacities  over a period of time counted in years for humans and in months for mice. In contrast, the DCC phenotype is acute when induced and develops over a very short period of no more than 72 h, seemingly affecting only non-elastic muscular tissues. Moreover, the induced DCC calcification is intracellular, occurring within mitochondria. Both chronic and acute molecular pathways leading to calcification share the same molecular origin, i.e., ABCC6 deficiency. However, their mechanism of initiation and progression are clearly different which indicate that ABCC6 signaling (from the liver) has much broader ramifications toward a variety of cellular and molecular processes than we originally thought. Of note, the DCC phenotype has not (yet) been described in human PXE patients, though cardiac calcification occurring within cardiomyocytes mitochondria is not uncommon after severe myocardial damages following an ischemic event  status of the calcification inhibitor MGP and the susceptibility to chronic calcification in PXE  calcification in PXE, it also contributes to and aggravates the pathology of a significant fraction of GACI and β-thalassemia patients. This protein is the root-cause of an acute mineralization phenotype that when triggered dramatically affects the intracellular calcium homeostasis in muscle tissues. ABCC6 is now a fully fledged inhibitor of calcification that works at a systemic level (through the circulation) as part of a larger ensemble of local and general regulators of calcification. But, the loss of ABCC6 function also leads to various physiological changes other than calcification that we have only begun to describe and undoubtedly, more is yet to come.For many years, 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.


Despite its high prevalence, discovery of pathophysiological mechanisms underlying ASD has lagged due to a lack of appropriate model systems. Recent advances in induced pluripotent stem cell (iPSC) technology and neural differentiation techniques allow for detailed functional analyses of neurons generated from living individuals with ASD. Refinement of cortical neuron differentiation methods from iPSCs will enable mechanistic studies of specific neuronal subpopulations that may be preferentially impaired in ASD. In this review, we summarize recent accomplishments in differentiation of cortical neurons from human pluripotent stems cells and efforts to establish  Autism spectrum disorder (ASD) is a debilitating neurodevelopmental disorder characterized by impaired communication and social interactions, as well as restricted interests and repetitive behaviors  recent findings from human iPSC (hiPSC)-based models of RTT, TS, and PMDS, and (3) future directions for optimization of cortical differentiation and modeling of ASD, as well as potential applications of this exciting technology.The generation of iPSCs has become commonplace. However, efficient differentiation of these cells into specific neuronal subtypes remains challenging. As discussed above, one of the prevailing hypotheses suggest that ASD arises due to dysfunctional synaptic communication in the neocortex. Successful generation of ASD-specific cortical neurons will improve our understanding of how ASD develops and may allow for identification of novel therapeutics. In this review, we discuss (1) recent advances in technology of cortical differentiation from human pluripotent stem cells (hPSCs) based on the knowledge of A thorough understanding of neocortical development can inform methodology for cortical neuron differentiation from hPSCs and define neuronal characteristics that should be considered in validating the identity and functionality of resultant neurons. This is especially important for hPSC-based ASD modeling, as abnormal neocortical development has been directly associated with the etiology of some ASDs Kwan, . Thus, wThe mammalian neocortex has a well-organized six-layered structure. Each cortical layer contains a characteristic distribution of neuronal cells with distinctive shape, size, and neurochemical and electrophysiological properties, which make local or long distance connections with other cortical region or subcortical compartments  or orthodenticle homoebox 1/2 (Otx1/2)  Stern, . As neur) Stern, . Once th) Stern, .Pax6), empty spiracles homolog 1/2 (Emx1/2) in mice . Through asymmetric cell division, RGCs give rise to (1) self-renewed RGCs that remain in the ventricular zone (VZ) throughout corticogenesis, and (2) committed daughter cells that can migrate out  Figure  and FoxgA remarkable feature in the development of neocortical interneurons is that they—unlike projection neurons—undergo tangential migration from their place of origin to their cortical destination. Several genetic studies in humans and mice have implicated dysfunctional development or migration of GABAergic interneurons with many psychiatric and neurodevelopmental disorders  compared to the number in hESC differentiation  to generate neurons from mouse ESCs (mESCs). Quantitative analysis revealed that around 80% of total cells were Sox1-positive neural lineage in 5 days. Substantial numbers of cells derived by SFEB culture express forebrain markers such as Another approach that has been used to induce neural progenitors from ESCs was co-culturing with mouse stromal feeder cells that are known to have neural inducing activity  and Emx1 (~89% of Foxg1-positive cells) . This remarkably improved the differentiation efficiency of mESCs to dorsal telencephalic neural precursors, evidenced by expression of in vitro differentiation method adapting the SFEBq system by culturing matrigel-embedded EBs in a spinning bioreactor  that drive alternative fates. Since the cerebral cortex develops in the dorsal telencephalic region of the embryonic brain, blockade of intrinsic ventralizing and/or caudalizing signals during neural induction of ESCs may lead to neural precursors with dorsal telencephalic fate. Vanderhaeghen's group was the first to test this hypothesis in mESCs -type interneurons that arise mostly in the CGE was not well-known. Rodríguez and colleagues illustrated that activation of Activin signaling facilitated the induction of CGE identity during neural differentiation of mouse and human ESCs, and enriched for CR-expressing GABAergic neurons    could be partially reversed by insulin-like growth factor 1 (IGF1) or gentamycin treatment, providing proof-of-principle evidence for the application of RTT-patient derived neurons for drug discovery  gene present with clinical features similar to RTT  and PSD95  gene, which leads to silencing of FMR1 expression. While Benvenisty and colleagues were the first to report the establishment of iPSC lines from FXS patients  is the most commonly inherited mental impairment, and is caused by expansion of CGG-repeats in the 5′ untranslated region of the fragile X mental retardation 1 , by generating iPSC lines from individual with heterozygous deletion of chromosomal locus 22q13.3  and neuronal differentiation pathway (13 genes) with impairment of astrocyte differentiation, suggesting its functional impact on human neurodevelopment  connectivity has been implicated in ASD, and many ASD-associated genes are involved in CStr synapses (reviewed by Shepherd, Differentiation of functionally mature neurons from hPSCs is a long process with multiple steps requiring a few months. This may increase heterogeneity of the final neuronal population, even if the protocol was intended to enrich for a specific neuronal subtype. One way to overcome these difficulties is to convert patient-derived somatic cells directly into neurons, skipping cellular reprogramming and differentiation. A recently introduced method for direct conversion of fibroblasts to functional cortical neurons relies on forced expression of neural-lineage specific transcription factors (Vierbuchen et al., Significant line-to-line variability has been observed in the neuronal differentiation of hPSCs (Wu et al., in vitro may not fully recapitulate neuronal development as it happens in vivo. Proliferation and differentiation of cortical progenitors occurs within specific niche environments characterized by signaling from the VZ, differentiated daughter cells, as well as signaling from non-neural sources, such as astrocytes, blood vessels, meninges (reviewed by Johansson et al., in vitro differentiation culture may obscure disease-relevant phenotypes using neurons generated from hPSCs. In line with this idea, it would be quite informative to determine whether co-culturing healthy cortical neurons with endothelial cells or microglia derived from individuals with ASD impairs neuronal function. Astrocytes are often supplied during the maturation of hPSC-derived neurons, as they promote synaptogenesis (Johnson et al., in vitro conditions for cortical differentiation, and provide clues for therapeutic approaches.Finally, neuronal differentiation With rapid progress in our ability to precisely manipulate hiPSCs, the tremendous knowledge gap between ASD genetics and our understanding of its pathophysiology is beginning to close. Using iPSC technology, it is possible to generate limitless supplies of human ASD-specific cortical neurons, which can revolutionize experimental analyses of ASD. Already, studies of the neurodevelopmental disorders RTT, TS, AS, and PMDS have shown that neuronal phenotypes can be identified using iPSC-derived neurons, and that these phenotypes can be corrected. Given the genetic heterogeneity of idiopathic ASD and the diversity in its clinical presentation, robust and highly reproducible methods for hiPSC manipulation is essential for linking genotype to phenotype. With the advent of facile mammalian genome engineering methods (reviewed in Hsu and Zhang, 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.


Faithful preservation of genome integrity is the critical mission of stem cells as well as of germ cells. Reviewed are the following mechanisms involved in protecting DNA in these cells: (a) The efflux machinery that can pump out variety of genotoxins in ATP-dependent manner; (b) the mechanisms maintaining minimal metabolic activity which reduces generation of reactive oxidants, by-products of aerobic respiration; (c) the role of hypoxic niche of stem cells providing a gradient of variable oxygen tension; (d) (e) the presence of hyaluronan (HA) and HA receptors on stem cells and in the niche; (f) the role of HA in protecting DNA from oxidative damage; (g) the specific function of HA in protecting DNA in stem cells; (h) the interactions of HA with sperm cells and oocytes that also may shield their DNA from oxidative damage, and (e) mechanisms by which HA exerts the anti-oxidant activity. While HA has multitude of functions its anti-oxidant capabilities are often overlooked but may be of significance in preservation of integrity of stem and germ cells genome. MDR1)-encoded adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (P-gp). This efflux machinery can pump out variety of genotoxins in ATP-dependent manner , its percentage in most organs and tissues is reduced to 2% – 9% (15 – 68 mm Hg)  and in the environment of stem cell niche is between 1% and 6% (7.6 – 45 mm Hg) and the intracellular p(O2) is 3 – 4 mm Hg [One of the factors reducing oxidative DNA damage in stem cells is their location. Stem cells reside in their respective niches , the dis 4 mm Hg , 36.2) such damage is expected to be nearly 20-fold lower in the stem cell niche than in tissue culture maintained at ambient air.Interestingly, at the very low oxygen tension (1%) the stem cells remain in the non-proliferating compartment and maintain pluripotency while at the increased tension (3% – 5%) they proliferate and start differentiation , 37. TheSeveral mechanisms are being used by stem cells to mitigate the hazards conveyed by the endogenous reactive oxidants. First of all, for most of their life stem cells remain in quiescent state having minimal metabolic rate and thereby low level of ROS production -47. Thei−Sca+Kit+; LSK) which continuously reside in the LSK compartment, during division segregate their mitochondria unequally delegating more of these organelles to that of the daughter cell which exits the LSK compartment and enters differentiation pathway. The sister cell remaining in the LSK compartment inherits the very low level of energized mitochondria and thus remains with minimal level of ROS production [Further mechanism designed to maintain minimal metabolic rate may involve asymmetric cell division. It was recently shown that the most primitive, long-term reconstituting, hematopoietic stem cells , the ubiquitous component of intercellular matrix, carries out numerous functions that are essential for survival, differentiation, proliferation, motility and intercellular communication of variety of cell types. HA is also of crucial importance for tissue and organ development and architecture [reviews in −) stem cells, and a progressive decline in its expression is seen to be concurrent with differentiation. The mechanistic in vitro studies indicate that HA functions in modulating cell proclivity to differentiation and proliferation by enforcing continuance of the dormant state of the most primitive Lin− cells [0 dormant state when cultured with the mitogen in the presence of high molecular weight HA [The level of expression of HA on the cells surface correlates with the differentiation status of the stem and progenitor cells. The highest HA expression is observed on the surface of the most primitive (Linn− cells -61. Thiseight HA .−/−) leads to aberrant distribution of the grafted cells in bone marrow compared to the wild type recipients [−/− mice. The presence of HA CD44 receptors on surface of stem cells mediates their adhesion and rolling movement on HA surfaces [HA plays also essential role in homing and lodgment of the transplanted stem cells. The transplanted hematopoietic stem and progenitor cells preferentially home to the trabecule-rich metaphysic of femur where they become lodged in endosteal niche, being associated with blood vessels. The presence of HA, which is highly expressed in endothelium of blood vessels, provides the homing and lodgment mechanism for the stem cells having strong expression of the HA receptors CD44 and RHAMM , 63, 64.cipients . Apparensurfaces .One of the mechanisms by which HA facilitates homing and engraftment of transplanted hematopoietic stem cells  involves its effect in promoting synthesis of membrane type 1 (MT1) of matrix metalloproteinase 2 (MMP-2) . Since MInterestingly, HA can substitute hypoxia for the long-term maintenance of embryonic stem cells in culture, preserving their pluripotency and thereby providing a useful alternative that enhances their viability . A combiThe importance and protective effect of HA on stem cells are well recognized in the field of transplantation of these cells in regenerative medicine. Variety of hydrogel scaffolds providing a niche-like environment, are being used as stem cell vehicles for their transplantation -73. HA, It should be noted that the HA receptor CD44 is frequently used as a marker to identify and sort out drug-resistant stem-like cells from different tumors -76. InteThe increased production of ROS takes place during inflammation and the oxidants generated in the inflamed tissue mediate and further amplify many inflammatory reactions -87. It h2O2 was markedly attenuated by HA; especially effective was HA of high molecular weight [Ataxia Telangiectasia Mutated (ATM) and phosphorylation of histone H2AX on Ser139 and reporting persistent DNA damage induced by endogenous oxidants, the by-products of aerobic respiration in mitochondria [We have recently reported that DNA damage signaling evoked by exposure of cells to exogenous oxidants such as Hr weight . In anotr weight  was alsor weight . Moreovechondria , 115, wachondria . Here agchondria . These fProtection of DNA by HA through its antioxidant activity has been also observed in fibroblast cultures in which oxidative stress was induced by treatment with iron and ascorbate . In addiInterestingly, HA has found wide utility in assessment of integrity of sperm cells DNA. Specifically one of the methods to separate fertile sperm cells that have undamaged DNA is based on the ability of sperm cells attach to HA -121. TheOocytes were shown also to synthesize and secrete HA -133. TheSimilar to stem cells there are several mechanisms protecting DNA in germ cells, including the blood-testis barrier and efflux pumps -137. TheAs discussed, strong evidence points out that the antioxidant properties of HA provide protection of genomic DNA against the damage by ROS as well as are involved in attenuation of inflammatory processes. Two different mechanisms may contribute to the antioxidant properties of HA. One mechanism involves the ability of HA to chelate Fe++ and Cu++ , 139. ThThe second mechanism by which HA may attenuate damage induced by oxidants involves the direct scavenging of oxidant molecules, particularly the reactive products of Fenton's reaction OH* radicals, by HA , 116. Ofin vitro upon HA exposure to free radicals and in vivo, in the inflamed tissues [2O2 while leads to HA breakage it also enhances HA synthesis [The ROS scavenging by HA while it depletes the pool of reactive oxidants potentially damaging DNA at the same time results in a breakdown of HA molecules , 104. Th tissues . It was ynthesis . Likely,ynthesis . BecauseWhen DNA damage occurs the highly effective and as much as possible error-free DNA repair is the subsequent mechanism by which stem cells protect genome integrity. Robustness of DNA repair in stem cells, and in particular of embryonic stem cells, is greater compared to other somatic cells -148. Forin vitro experiments in which the cells were growing at standard tissue culture conditions in an atmosphere of air and CO2. As mentioned, the partial pressure of O2 (pO2) of 159 mm Hg (21%) in ambient air is much higher than in tissues, especially in the microenvironment of the stem cells niche [in vitro findings on cells growing under standard atmosphere conditions may be hampered by an experimental bias, i.e., the consequence of exposure to non-physiological pO2.Most observations regarding modulation of cell growth and proliferation of stem cells by HA came from the ls niche , 36. Becls niche  as well ls niche , are mucin vitro studies were also carried on stem cells growing attached to glass or plastic surfaces and exposed to HA that was included in the culture media. Such cells when attached to solid, dried out surfaces treated with various chemicals have very different migration properties, proliferation capability, and cell-to-cell interactions compared to in vivo conditions. The cells growing on solid surfaces exhibit also a very different behavior than that of the same cells grown on highly viscoelastic HA.Most of the When cells from the lymphomyeloid system, tumor cells, fibrocytes or stem cells are placed on highly hydrated elastoviscous solutions of HA, they remain round, divide and move toward each other and on top of each other, forming a multilayered assembly. These assemblies are separated from each other. Depending on the cell lines, they survive or die slowly , 152. Thin vitro results of cells growing on solid surfaces with the in vivo conditions when the cells are embedded and exposed in a highly hydrated intercellular matrix. The cell-to-cell interactions may have an entirely different character.Since in the vertebrate intercellular matrix, especially in the connective tissues, the cells are surrounded by liquid hyaluronan of various molecular weights and concentrations, it is logical to argue that the cells must have different behavior and activities – even gene expression – when surrounded with hyaluronan and not growing on plastic or glass surfaces. A caution, therefore, should be exercised in interpolating the The mechanisms associated with maintenance of genome integrity of stem cells, particularly focused on elucidation of the role of ROS and DNA damage response, have been recently reviewed . In thisAs yet there is no direct experimental evidence that unequivocally demonstrates that HA protects stem cells from oxidative DNA damage. However, the wealth of indirect evidence presented in this review strongly advances the notion that one of the functions of HA associated with stem cells is DNA protection against oxidative damage. The most supporting evidence to this conception provide the findings that DNA damage response triggered by exogenous as well endogenous oxidants in several cell types, most likely reporting persistent oxidative damage, is strongly attenuated by HA , 113. ThOf interest and of further support for this notion are observations that the level of expression of HA on surface of stem cells correlates with their differentiation status and the mechanistic studies showing that HA modulates cell proclivity to differentiation and proliferation -61. On t


Generalist herbivores are challenged not only by the low nitrogen and high indigestibility of their plant foods, but also by physical and chemical defenses of plants. This study investigated the foods of wild parrots in the Peruvian Amazon and asked whether these foods contain dietary components that are limiting for generalist herbivores  and in what quantity; whether parrots chose foods based on nutrient content; and whether parrots avoid plants that are chemically defended.We made 224 field observations of free-ranging parrots of 17 species in 8 genera foraging on 102 species of trees in an undisturbed tropical rainforest, in two dry seasons (July-August 1992–1993) and one wet season (January-February1994). We performed laboratory analyses of parts of plants eaten and not eaten by parrots and brine shrimp assays of toxicity as a proxy for vertebrates. Parrots ate seeds, fruits, flowers, leaves, bark, and insect larvae, but up to 70% of their diet comprised seeds of many species of tropical trees, in various stages of ripeness. Plant parts eaten by parrots were rich in protein, lipid, and essential minerals, as well as potentially toxic chemicals. Seeds were higher than other plant materials in protein and lipid and lower in fiber. Large macaws of three species ate foods higher in protein and lipids and lower in fiber compared to plant parts available but not eaten. Macaws ate foods that were lower in phenolic compounds than foods they avoided. Nevertheless, foods eaten by macaws contained measurable levels of toxicity. Macaws did not appear to make dietary selections based on mineral content.Parrots represent a remarkable example of a generalist herbivore that consumes seeds destructively despite plant chemical defenses. With the ability to eat toxic foods, rainforest-dwelling parrots exploited a diversity of nutritious foods, even in the dry season when food was scarce for other frugivores and granivores. Psittrichus fulgidus, is a specialized frugivore, feeding exclusively on the pulp of figs The ecological role of parrots in tropical forests may yet be underestimated, but a growing number of recent studies have described the feeding ecology and diets of wild parrots  and plant secondary compounds. As part of a broader study of parrots in the Peruvian Amazon of Manu National Park Ara ; Amazona ; Pionus (P. menstruus); Aratinga ; Orthopsittaca (O. manilata); Pionites (P. leucogaster); Pyrilia (P. barrabandi); Pyrrhura ; Brotogeris ; and Forpus (F. modestus).We observed foraging in 17 species of parrots at two field sites, Manu National Park  and in the Tambopata-Candamo Reserved Zone , described in detail elsewhere We observed parrots foraging by walking census routes in Manu during the dry seasons (July and August) of 1992 and 1993. We made all Tambopata observations during the wet season (January and February) of 1994. We located foraging birds by direct observation from emergent trees, by hearing vocalizations, or most often, by hearing falling fruit. Observers walked the same pre-established paths through the forest for 1 to 3 hours each day (0630–0930 hours) during these months. We conducted 10–13 morning censuses in each habitat and stratified observations so that each habitat type received equal censusing effort.When we located foraging parrots, we noted: the species identity of both tree and parrot(s); which plant parts were consumed and the stage of ripeness of fruits and seeds. Because of concern for statistical independence, for this study, we noted only each first observation of parrot species and exploited tree species. Sample size was therefore highly conservative; we noted 224 total unique observations of members of a given species of parrot feeding on a given species of tree. We collected plant samples from the ground below a foraging event, or we climbed the tree and collected fruit directly from the canopy at the same stage of maturity as those being consumed. We defined seed predation as the destructive use of seeds, consumption of whole fruits (such as figs) or of unripe fruits within which seeds might be consumed whole but were not ready for dispersal when consumed. Therefore to estimate granivory (digestion of seeds) in contrast to frugivory (digestion of fruit pulp), we counted as granivory any instance in which we observed whole fruits, unripe fruits, or seeds taken by parrots. We estimated use of unripe fruit or seed based on color and state of plant parts falling to the ground. Plants were identified to species by botanists working in the two reserves . We determined crude protein with standard methods using a nitrogen gas analyzer . In laboratories on the UC Davis campus, we re-dried the samples in a vacuum oven overnight (40°C) and ground them for passage through a 1 mm sieve using either a Wiley Mill or a coffee grinder. We determined lipid content by extracting 0.5–1.0 g dried plant material in diethyl ether using a Soxtec extractor, evaporating the ether from the extract, and comparing dried extract to sample proportion by weight 3 and 2 ml 30 percent H2O2 in a teflon vessel and heated under pressure in a microwave oven . We diluted this to 15 ml with H20, and then analyzed the extracts using inductively coupled plasma optical emission spectroscopy (Thermo Jarrell Ash Atomscan 25) for the following minerals: Na, Mg, P, S, K, Ca, Fe, Zn. We converted raw results to mg/kg using a three point standard curves based on reference solutions for the appropriate element (Fisher).We tested all plant samples for mineral content by digesting 500 mg dry material in 0.5 ml concentrated HNOWe tested all plant materials for total phenolic compounds using the Folin-Ciocalteau method in vacuo. After adding 5 ml of artificial seawater  to each cell, we transferred 10 to 25 individual 24-hour old brine shrimp nauplii to each cell. We covered the plates and counted live versus moribund or dead shrimp 24 hours later. We analyzed counts using a probit analysis, which estimates the amount of extract that caused 50 percent mortality (LD50) using POLO software 50 values, we tested several pure phenolics, saponins, and other toxins with the following LD50 results in mg/g: α-amanitin 0.001, digitonin 0.07, digitoxin 0.02, quercetin >5.0, quinidine 0.32, quinine 0.55, rutin >5, ß-escin 0.05, strychnine 0.11, and tannic acid 0.26.We tested plant materials for toxicity using a standard brine shrimp bioassay versus selected plants, we collected samples of plant parts that fit into two broad categories: plants that we had evidence for at least one parrot species consuming (eaten); and parts of those same plants discarded or ignored by foraging parrots  or similar and abundant reproductive parts of other plants to which parrots had clear access but did not eat (non-eaten). These collections are neither complete, nor are they necessarily representative. Nevertheless, by sampling foods that a given species of parrot does consume and comparing with those that it does not, we can generate a rough approximation of parrot food choices, particularly in macaws where our data are best, and estimate the criteria by which parrots select or reject potential foods. Our results therefore represent an inventory of known food plants for the parrots at this site. Because of our sampling methods, we do not claim to estimate diet or foraging niche of these species.Determining how nutritional components affect dietary selection in wild animals is notoriously difficult To assess how the quality of available foods varied with plant structure, we compared mean values of different plant parts: seeds; whole fruit; fruit pulp; and other parts , using a series of one-way ANOVAs on the various components. For the three largest macaws, we compared plant parts that were eaten by parrots of a given species against plant parts that were available to them but not eaten, using a series of one-way ANOVAs. Where differences among means were significant in one-way ANOVAs, we employed Bonferronni post-hoc analysis for multiple comparisons, including both standard Bonferroni estimates on the 14 univariate ANOVAs, as well as sequential Bonferroni estimates on means of different plant parts within each univariate analysis of components. Although use of Bonferroni or other corrections are appropriate where multiple comparisons are made, a number of researchers question the overly conservative estimates of statistical significance provided by the Bonferroni tests, particularly in disciplines in which data are difficult to come by (such as in this study) Ara macaws, which used 43 percent of the 102 plant species exploited by parrots. Our data herein represent an inventory of tree species exploited by parrots in this region of southern Perú and are not presented as an estimate of diet or niches of the parrot species involved.Parrots of the 17 species observed in this study fed from a total of 102 species of plants, for a total of 224 unique observations of parrots of a given species exploiting trees of a given species . This vaParrots fed from both reproductive and non-reproductive parts of these plants, but by far most plant species (92%) were exploited for their reproductive parts . For theOf the plant parts used by parrots, protein and lipid levels were relatively high, as were fiber levels , Table 350, which we assayed with brine shrimp and calibrated against pure known standards of pure phenolics, saponins, and other toxins  than is fruit pulp from the same species of plants. Likewise, seeds and fruit analyzed in this study were much higher in protein and lipids and much lower in fiber than were vegetative parts of the same plants. Thus our study corroborated the general findings of studies on digestive physiology reviewed in Karasov & Martinez del Rio cf.HuraHeveaIn our study, virtually all plant parts chosen by parrots contained phenolics and other substances known to be toxic to vertebrates; a significant proportion of their observed foods contained levels that are considered to be highly toxic. Although direct testing of toxicity of each of these food items in a bird or even a vertebrate was beyond the scope of this study, as well as ethically problematic, the brine-shrimp bioassay was useful in providing a rough measure of toxicity, partly because the toxicities of these parrot foods varied over three orders of magnitude cf.. Indeed,Plant secondary compounds are known to deter foraging in herbivorous birds and mammals, yet toxic foods were nevertheless consumed by these herbivores, presumably as a trade-off in obtaining higher nutrition or abundant available food in vivo in food detoxification in parrots No one has yet studied the physiological effects of toxins or costs of detoxification in parrots. Studies of other herbivores reveal that these costs may be high, depending on the environmental demands. For example, processing of secondary plant compounds that serve as toxic deterrents to vertebrate herbivores may have significant effects on sodium and water balance or require high energy or other costs in producing enzymes or carrier proteins such as P-glycoprotein Our study was inconclusive on whether minerals are potentially limiting in the diet and whether parrots chose foods based upon their mineral content. Only one mineral, phosphorus, was higher in foods eaten than not eaten by macaws. Certainly, natural foods of these parrots contain measurable amounts of eight nutritionally important minerals; future studies should compare these levels with those in formulated captive diets. Others have argued that consumption of clay may play a role in mineral acquisition Strigops habroptilus)Understanding and providing adequate nutrition may be critical to recovery programs for endangered and threatened species of granivorous parrots, as it has been for the folivorous Kakapo Click here for additional data file.Dataset S1Plants eaten by parrots in southeastern Peru, noting part consumed and stage of maturity.(XLS)Click here for additional data file.


HIV-associated nephropathy (HIVAN) is the leading cause of end-stage renal disease in patients with HIV-1 infection. While HIVAN involves all portions of the kidney, marked changes in podocyte proliferation and dedifferentiation are central to the pathogenesis of this disease. This review focuses on identifying the genes and signaling pathways that trigger terminally differentiated quiescent podocytes to lose their normal markers of maturity and reenter the cell cycle. The parallels between these alterations to malignant transformation are quite striking.HIV-associated nephropathy (HIVAN) is the leading cause of chronic kidney failure in patients infected with HIV . The majThe HIV virus directly infects tubular cells and podocytes leading to the development of HIVAN. Not surprisingly, the HIV-1 transgenic mouse (Tg26 mice) model in which renal cells express viral proteins has greatly advanced our knowledge of the pathogenesis of HIVAN. The Tg26 mice develop proteinuria, progressive renal failure, and renal pathologic findings identical to HIVAN patients . Reciprogag, pol, and env leads to the development of massive proteinuria, severe collapsing FSGS, and microcystic tubular dilatation , synaptopodin, podocalyxin, common acute lymphoblastic leukemia antigen (CALLA), and ezrin . The losin vitro . This isgag, pol, env), two regulatory genes (tat and rev), and four accessory genes . In HIV-1 infected hosts, the 27-kDa protein Nef is at an early phase in the viral replication cycle and performs several functions  which normally prevent mature podocytes from reentering the cell cycle  . PhosphoActivation and signaling of the MAPK family plays a role in mitogenesis and cell differentiation. Nef can activate this downstream signaling pathway via Raf and Src activation . MAPK sinef and downstream signaling pathways in vitro  are binding sites for the SH3 domain of the Src kinase family and PAK2. Using immortalized murine podocytes, mutations of Nef in the PxxP region have been shown to have the following effects in vitro: inhibition of Stat3 and MAPK1, 2 phosphorylation, reduction of cyclin E expression, restoration of synaptopodin expression, and reversal of Nef-induced cell proliferation (105R106 domain did increase synaptopodin expression, mutation at this site failed to change phosphorylation of Stat3 or MAPK1, 2 and only minimally decreased cell proliferation (105R106 motif may exert some of Nef’s effects leading to the HIVAN phenotype in podocytes, it is likely through a signaling cascade independent of Stat3 and MAPK1, 2 activation. A summary of known pathways of nef signaling and its downstream effectors is presented in Two Nef domains (PxxP and Rferation . While mferation . The datin vitro. Immunostaining for phospho-Stat3 is markedly elevated in the glomeruli of transgenic mice with podocyte-specific nef expression compared to control littermates (nef generated mice that no longer exhibited the HIVAN phenotype (in vivo evidence that nef induces the myriad of renal cortical abnormalities that characterize the HIVAN phenotype via activation of the Src family of tyro-sine kinases.We confirmed these signaling pathways are critical to mediating Nef’s effect on the HIVAN phenotype termates . Also, itermates . Additiohenotype . This stnef interaction and activation of PAK2 in the murine model contributes to the HIVAN phenotype (nef alleles but lacking the Nef—PAK2 association do not exhibit kidney disease. This identifies Nef binding and activating PAK2 as critical to the development of HIVAN. Activation of PAK2 alone, however, is not sufficient to develop kidney disease. Transgenic mice expressing nef mutants that were able to bind and activate PAK2 failed to exhibit kidney disease (Vincent et al. have proposed that henotype . Activathenotype . HIV tra disease .In vitro and in vivo evidence indicates that Nef induces these changes in the podocyte via Src kinase-dependent activation of Stat3 and MAPK1,2 signaling pathways. Additionally, Nef interaction with PAK2 may contribute to some of the features of the HIVAN phenotype. There are still several downstream targets that have yet to be identified which potentiate the development of podocyte proliferation and dedifferentiation. Evidence also indicates that Nef may activate other pathways, in addition to Stat3 and MAPK1, 2, as part of the pathogenesis of HIVAN. Continued studies are needed to identify downstream effectors of Nef and therapeutic interventions that may abrogate the activity of these signaling pathways.HIVAN is characterized by marked proteinuria, renal insufficiency and characteristic pathologic changes on kidney biopsy. Marked podocyte proliferation and dedifferentiation occurs within the collapsing glomerulopathy found in HIVAN. These podocyte changes are not seen in other glomerular diseases. HIV-1 Nef is the critical protein mediating reentry of podocytes into the cell cycle and down-regulation of mature podocyte differentiation markers.


Congenital hypothyroidism (CH) is one of the most common endocrine disorders in the infant population. It is also one of the most important causes of foreseeable brain damage and severe mental impairment. Current views on CH pathogenesis, screening schema and treatment standards have been presented. Also outlined have been the newest reports on the impact on CH epidemiology and classification generated by the introduction of low TSH cut-off (6 mU/l).The use of low TSH cut-off allowed the detection of an unsuspected number of children with neonatal hypothyroidism, evolving in mild permanent thyroid dysfunction later in life. Constantly growing, especially in developed countries, number of preterm infants is a separate group of children, mainly with transient fluctuations in thyroid function. Thyroid gland function develops and matures during fetal life, with production of serum thyroxine (T4) beginning around 12 weeks gestation and increasing to term. Infants born prior to term have lower cord serum T4 concentrations which correlate with gestational age or/and birth weight. This is partially a result of lower thyroxine-binding globulin (TBG) concentrations. The cord serum free thyroxine (FT4) concentrations also correlate with gestational age, but they are not proportionately as low as cord T4 concentration. Preterm infants have a postnatal thyrotropin (TSH) surge and rise in serum T4 and triiodothyronine (T3), which is qualitatively similar to, but quantitatively smaller than term infants. In contrast to term infants, preterm infants often experience a fall in serum T4 and T3 in the first week of life to below birth levels. This drop appears to be the result of many factors, including nutritional problems and decreased hepatic TBG production, immaturity of hypothalamic-pituitary control of the thyroid gland, immaturity of the thyroid gland itself, and increased tissue utilization of T4. Several studies have correlated different measures of morbidity and mortality in the preterm infant with lower serum T4 concentrations. This has led to speculation that T4 treatment might be beneficial in improving these complications of prematurity, in particular the neurological outcome.Latest studies do not confirm that hypothyroidism in preterm infants is for them harmless. Many factors, however, influence mental development of those children. Thus, treatment strategies, including thyroid hormone substitution, should consider both the etiology of the hypothyroidism and the impact of aforementioned factors. There is no “hard evidence” to support introducing thyroid hormone supplementation in preterm infants as a rule.


Autoantibodies directed to chromatin components date back to the discovery of the LE cell and the LE cell phenomenon circa 1950, and subsequent evidence that major components of that reaction were chromatin components and histones in particular. Over time, immunoassays ranging from ELISA and line immunoassays to more modern bead-based assays incorporated histone and DNA mixtures, purified histones, and purified nucleosomes leading to a more thorough understanding of the genesis and pathogenetic relationships of antibodies to chromatin components in systemic lupus erythematosus and other autoimmune conditions. More recently, interest has focussed on other components of chromatin such as high mobility group (HMG) proteins both as targets of B cell responses and pro-inflammatory mediators. This review will focus on immunoassays that utilize chromatin components, their clinical relationships, and newer evidence implicating HMG proteins and DNA neutrophil extracellular traps (NETs) as important players in systemic autoimmune rheumatic diseases. Eukaryotic chromatin is comprised of approximately 40% DNA, 40% histones, 20% nonhistone proteins , RNA, and other macromolecules. The fundamental subunit of chromatin is the mononucleosome, which is composed of ~180 base pairs of DNA and two molecules each of the core histones H2A, H2B, H3, and H4 and one of the linker histone H1. The core histones are organized as a histone octamer (containing two H2A-H2B dimers and one H3-H4 tetramer) around which 146 base pairs of DNA are wrapped, thus constituting the “core particle.” This structure is stabilized by histone H1 which binds across the surface of the nucleosome [Human autoantibodies that bind to chromatin targets can be divided into those that recognize dsDNA, protein components of chromatin , mononucleosomes, or macromolecular components of nucleosomes as represented by low salt extracted nucleosomes (core particle) , 5–7. ScAnti-dsDNA are quite specific for SLE, although they have been found in normal individuals where they are mostly the IgM isotype as encoded by germline DNA with few or no somatic mutations , 10. TheAutoantibodies to dsDNA were first recognized as an important serological marker for the diagnosis of idiopathic SLE, and eventually both the American College of Rheumatology and Systemic Lupus International Cooperating Clinics (SLICC) criteria for classification of the disease included the presence of these autoantibodies as a formal criterion , 18. AntBy comparison, ANuA are a more sensitive biomarker of SLE than anti-dsDNA and are almost exclusively found in SLE and in much lower frequency in systemic sclerosis (SSc), mixed connective tissue disease, and other systemic autoimmune rheumatic diseases (SARD) . SeveralSLE is characterized by the production of both antigen driven autoantibodies such as anti-dsDNA and anti-histone antibodies and polyclonal, apparently nonspecific, autoantibodies. Precise mechanisms leading to production of these autoantibodies are still unclear, but several data suggest that the nucleosome plays a key role –25. SincAn understanding of the sources of the nucleosomes that drive the anti-chromatin response is becoming more and more complex. It has been held for almost two decades that aberrant apoptosis and a reduced clearance of apoptotic cells by phagocytes may lead to an increased exposure of apoptotic nucleosomes to the immune system  lpr mutation in murine lupus and found that the Toll-like receptor 9 (TLR9) is absolutely required for the ANuA response but not for anti-dsDNA antibody production. As a general paradigm, apoptosis results in modified chromatin components appearing at the surface of apoptotic cells, the removal of apoptotic debris is defective, and the massive release of these nucleosomes into the circulation incites their recognition by the immune system (T and B cells) and the production of ANuA [Nevertheless, a prevailing paradigm is that nucleosomal constituents, which have been cleaved and modified  during the process of apoptosis , 43, esc of ANuA . in vitro that also displayed DNA and other nucleosomal structures in an antigenic form [A number of reports have discussed the role of circulating microparticles generatednic form , 41. Thenic form . It was nic form .The notion that one of the primary B cell targets in SLE is the nucleosome is supported by a number of observations. Autoantibodies directed against histones are found in 95–100% of DIL sera and also in 70% of SLE , 51. Ear Crithidia luciliae immunofluorescence test (CLIFT) and various enzyme-linked immunoassays (ELISA). More recently, other methods include the use of strips blotted with the DNA molecules (immunoblotting (IB) and line immunoassays (LIA)) and the use of microarrays and addressable laser bead immunoassays (ALBIA) [ Crithidia luciliae kinetoplast and purified dsDNA in an ELISA [Over the last five decades since their first description, several methods for anti-dsDNA detection have been developed . Th (ALBIA) –68. Theran ELISA , 65.In a multicenter study of four different anti-dsDNA immunoassays, autoantibody titres detected by EliA and FARR assay were correlated with SLE disease activity . It has A more recently developed high avidity anti-dsDNA IgG ELISA is reported to have highly specific performance characteristics for the SLE although it is less sensitive than certain other dsDNA IgG assays . In a muELISA techniques for ANuA detection differ in terms of the antigen preparations used. Some methods use purified nucleosomal particles obtained by reconstituting histone core proteins or histone dimers onto DNA; others use purified chromatin generally obtained by digestion with micrococcal nuclease and subsequent removal of histone H1 and HMG proteins with 0.5 M NaCl at a neutral pH . BecauseBardin et al.  found thP < 0.0005) and anti-histone antibodies . Ghillani-Dalbin et al. [At the outset, it must be made clear that ANuA represents a very complex autoantibody system comprising a variety of potential protein targets, epitopes, and B cell responses leading some to take a rather nihilist view of their clinical value . Neverthn et al.  studied n et al.  reportedn et al. , 72  SLE patients as compared to healthy individuals . SimilarIn children, anti-dsDNA antibody testing was slightly more sensitive than ANuA (66.5% versus 64.1%), while specificity slightly favored ANuA (98.8% versus 97.1%). However, ANuA displayed a more relevant predictive value than anti-dsDNA antibodies  and extracellular microvesicles may provide other vehicles by which inflammatory and immunogenic components of chromatin can be released into the extracellular environment. Although not discussed in detail here, recent evidence suggests that the use of nucleosomal peptides as tolerogens may be a successful approach to suppressing certain aspects of lupus pathogenesis and could lead to the design of novel therapeutics .High mobility group (HMG) proteins are operationally defined by their extractability from chromatin in 0.35 M NaCl and their solubility in 5% perchloric acid (PCA) and 2% trichloroacetic acid (TCA) , 115. AsDating to the seminal studies of Bustin et al. in 1982 , HMG antIn a study of sera from 42 DIA patients, we found reactivity with HMG proteins 14 and/or 17 in 67% of the sera by immunoblotting assays and in 58% by an ELISA . The sliUntil the last decade, one of the HMG proteins, HMGB1, was primarily regarded as a DNA-binding protein that participated in chromatin structure and transcriptional regulation , 144. HoHMGB1 is also secreted from various cell types during activation and/or cell death and may act as a proinflammatory mediator, alone or as part of DNA-containing immune complexes in SLE , 169. A Last, a few reports have implicated autoantibodies to sex related Y HMG box (SOX) proteins in type I diabetes  and antiObservations that there is a high frequency of antibodies to the HMG proteins associated with nucleosomes add further evidence implicating the nucleosome or nucleosomal subunits as immunogens. Furthermore, observations that the most common autoantibody targets in human diseases are HMGN1/HMG-14 and HMGN2/HMG-17, which are preferentially associated with the nucleosomes of transcriptionally active chromatin, suggests that functionally and structurally distinct forms of nucleosomes are the inciting immunogens in autoimmune disease.Neutrophils kill certain extracellular pathogens by releasing their highly decondensed chromatin as extracellular traps (NETs) . The impANuA and anti-dsDNA are independent and complementary biomarkers that have value in the diagnosis and disease activity assessment of SLE. ANuA are specially a useful marker in the diagnosis of SLE patients who are anti-dsDNA negative. ANuA and anti-dsDNA both are associated with disease flare and lupus nephritis.In general, ANuA have equal specificity but higher sensitivity, positive likelihood ratio, and diagnostic odds ratio than anti-dsDNA antibodies for the diagnosis of SLE.The emergence of newer immunoassays for the detection of antibodies to chromatin components requires ongoing pre- and postmarketing evaluation. Standardization of nomenclature and assay performance is a desirable goal.The renaissance of autoantibodies to HMG proteins and their relationship to other chromatin components including nucleosomes and DNA NETs are beginning to weave interesting paradigms in autoimmunity that requires further investigation. Circulating anti-HMGB1 and HMGB1 levels may be useful when assessing the protective effects of autoantibodies.


Phyto-endoparasitic nematodes induce specialized feeding cells (NFCs) in their hosts, termed syncytia and giant cells (GCs) for cyst and root-knot nematodes (RKNs), respectively. They differ in their ontogeny and global transcriptional signatures, but both develop cell wall ingrowths (CIs) to facilitate high rates of apoplastic/symplastic solute exchange showing transfer cell (TC) characteristics. Regulatory signals for TC differentiation are not still well-known. The two-component signaling system (2CS) and reactive oxygen species are proposed as inductors of TC identity, while, 2CSs-related genes are not major contributors to differential gene expression in early developing NFCs. Transcriptomic and functional studies have assigned a major role to auxin and ethylene as regulatory signals on early developing TCs. Genes encoding proteins with similar functions expressed in both early developing NFCs and typical TCs are putatively involved in upstream or downstream responses mediated by auxin and ethylene. Yet, no function directly associated to the TCs identity of NFCs, such as the formation of CIs is described for most of them. Thus, we reviewed similarities between transcriptional changes observed during the early stages of NFCs formation and those described during differentiation of TCs to hypothesize about putative signals leading to TC-like differentiation of NFCs with particular emphasis on auxin an ethylene. Vicia faba , syncytia, and giant cells (GCs), the nourishing cells for cyst and root-knot nematodes (RKNs), respectively  where GCs are embedded surrounded by heterogeneous tissues. Although gall microscopy sections were not examined, the confined GUS activity observed suggests that the promoter is probably active in GCs  or ARF19 . This suggests that auxin concentration is very high at early infection stages in GCs as compared to the rest of the gall tissues. There are several hypotheses to explain this local increase in auxin levels, i.e., the presence of auxins in the nematode secretions . Ethylene responsive genes were over-represented among the 310 up-regulated genes in GCs, relative to the total number of hormone responsive genes in Nemhauser et al. (ETR2 induction coincides with the presence of other ethylene receptors (as ETR1) in developing TCs from barley and rice endosperm , were less susceptible to H. schachtii , with a peak at medium infection stages  or syncytia (PDK). However, most HKs and HKLs are down-regulated in NFCs. ETR2, HK2, HK3, ERS1, and PHYA are down-regulated in syncytia and HK1 and CKI1 in GCs  that act as signaling intermediates between HKs and response regulators. From them, AHP3 is up-regulated in syncytia while AHP1 is down-regulated, and the rest of the genes are not differentially expressed in the transcriptome of micro-dissected GCs and syncytia  activity was suggested , ethylene (AAP3), or both , all induced by auxin, were also induced in NFCs (Table UGD2 and UGD3 are necessary for the production of CIs in syncytia and loss of function in double mutants severely affected nematode development (Siddique et al., It is interesting to point that many genes induced in early differentiating NFCs correspond to typical categories of downstream regulated genes that might participate in CIs formation, as those involved in vesicle trafficking, cell wall biogenesis, cell shape control and expansion, or nutrient transport, also induced in cells undergoing differentiation into TCs (Thiel et al., 8G Table  or AtFH6both ABCG; Table 1In conclusion, although genes encoding proteins with similar functions are differentially expressed in differentiating NFCs and typical TCs, a clear knowledge of their implication, either upstream or downstream, in the signaling cascades leading to TCs characteristics of NFCs is still lacking. Further research will probably elucidate the contribution of signals such as hormones to those differentiation events in NFCs.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.


Our understanding of immunity has historically been informed by studying heritable mutations in both the adaptive and innate immune responses, including primary immunodeficiency and autoimmune diseases. Recent advances achieved through the application of genomic and epigenomic approaches are reshaping the study of immune dysfunction and opening up new avenues for therapeutic interventions. Moreover, applying genomic techniques to resolve functionally important genetic variation between individuals is providing new insights into immune function in health. This review describes progress in the study of rare variants and primary immunodeficiency diseases arising from whole-exome sequencing (WES), and discusses the application, success, and challenges of applying genome-wide association studies (GWAS) to disorders of immune function and how they may inform more rational use of therapeutics. In addition, the application of expression quantitative-trait mapping to immune phenotypes, progress in understanding MHC disease associations, and insights into epigenetic mechanisms at the interface of immunity and the environment are reviewed. Effective immune function is critical to health, and dysregulation underlies a large proportion of human diseases. Elucidation of the genetic basis of rare heritable diseases involving deficiencies in the innate and adaptive immune response have been highly informative in advancing our understanding of both disease mechanisms and of the fundamental processes underpinning immunity  [1–3]. CGenomic approaches such as GWAS have allowed previously unrecognised genomic loci, genes, proteins, and pathways involved in a particular process to be identified. In this context GWAS have been a notable success, particularly in terms of autoimmune and other immune-related conditions , as exemAnother layer of complexity is added by the fact that, in most instances, non-coding sequence variants have been causally implicated by GWAS, and immune dysfunction is no exception to this trend. In these cases, expression quantitative-trait mapping offers an important complementary approach for resolving regulatory genetic variants and the genes affected. Interestingly, these studies have revealed significant overlap between disease and expression-associated genetic markers In addition to understanding genetic variation in both coding and regulatory regions, progress in understanding disease pathogenesis and opportunities for treatment of immune diseases will require the integration of genetic and environmental risk in a much more holistic way than has been achieved to date, taking into account epigenetic processes and context-specific effects in immune function Historically, genetic investigation of rare Mendelian primary immunodeficiency disorders has been highly informative in understanding immune function Primary immunodeficiencies characterised by significant autoimmune dysfunction have provided important insights into central tolerance from study of the rare disease autoimmune polyendocrine syndrome 1 (APS1) and the identification of mutations in the autoimmune regulator (AIRE), a transcription factor regulating expression of self antigens in the thymus Candida albicans involving the nails, skin, oral, or genital mucosa Despite these successes, the identification of causative genes for some primary immunodeficiencies through classical approaches has been hampered by the small numbers of informative cases. Recent developments in genomic technology such as whole-exome sequencing (WES) have opened the door to resolving these rare instances and promise to advance this field significantly. This approach has highlighted how both loss-of-function and gain-of-function alleles of STAT1  may perturb normal immune function, leading to clear disease phenotypes ORC4L, a gene important for initiation of DNA replication that was previously implicated in B cell lymphoproliferative disorders Although the cost of WES and whole-genome sequencing is decreasing, it remains a less common genomic approach than GWAS. The rarity of primary immunodeficiency disorders makes it challenging, and in some instances impossible, to identify sufficient cases for the GWAS to be adequately powered because this approach requires large numbers of cases and controls to test for association using a genome-wide panel of genetic markers. Nevertheless, for the most common immune-deficiency disorders, common variable immunodeficiency (CVID) and selective IgA deficiency, such studies have recently been performed. In the case of CVID, a GWAS involving 363 patients confirmed the role of the MHC, but also implicated other loci such as the disintegrin and metalloprotease (ADAM) genes as well as many rare structural variants (insertions and deletions) involving for example HLA-DQB1*02IFIH1, which encodes an interferon-inducible RNA helicase important in response to viral infections and implicated by GWAS in psoriasis IFIH1 were also subsequently found to show independent association to T1D CLEC16A, which encodes a member of the C-type lectin domain family, is also associated with T1D, multiple sclerosis (MS), systemic lupus erythematosus, and rheumatoid arthritis (RA) A recent similarly sized GWAS investigating IgA deficiency also confirmed the previous MHC class II association with this disease PTPN22 R620W; it is associated with increased risk of RA, thyroid disease and T1D, protection for Crohn's disease, and no effect on MS The associated loci shared between IgA deficiency and other immune diseases reflects a general trend that has been uncovered through GWAS, particularly of autoimmune diseases , namely TNFRSF11One outcome of identifying these shared associations is the potential to treat diseases with drugs previously approved for seemingly unrelated diseases (drug repositioning). Indeed, it has been found that genes currently implicated by GWAS are significantly more likely to be ‘druggable’ by small molecules (21%), or potentially modulated by ‘biopharmaceuticals’ (therapeutic antibodies/protein therapeutics) (47%), than those derived from considering the whole genome (17% and 38% respectively) IL12A, IL18RAP, and SH2B3 in the context of coeliac disease de novo sequence assembly When considering the role of genetic variation in the immune system, the highly polymorphic MHC locus on chromosome 6p21 is sometimes viewed as being too complex and difficult to resolve, but this region of the genome is undoubtedly the elephant in the room that refuses to go away. GWAS have underlined what was already known from associations based on serological testing in the early 1970s, namely that the predominant genetic risk for autoimmune disease resides in the MHC, together with associations for infectious and inflammatory diseases as well as traits such as cancer and drug hypersensitivity The majority of autoimmune-disease associations are thought to arise completely or in part from structural variants involving HLA class I and II molecules, acting in conjunction with a variety of known and unknown germline and somatic genetic variants, epigenetic mechanisms, and specific environmental triggers to modulate tolerance  Catalog of Published Genome-Wide Association Studies (17.3% of reported GWAS SNPs) were associated with one or more eSNPs in this dataset, with 4.6% of GWAS SNPs showing association with expression of different genes in a cell type-specific manner In terms of immune phenotypes, recent expression quantitative-trait mapping studies have proved to be highly informative, notably for autoimmune diseases DARC, a variant that confers a strong selective advantage in malaria CSF3 that show association with neutrophil and total white blood cell count CSF3) in treating neutropenia. Other striking associations  and PTPN2 (Crohn's disease and T1D) were bound by VDR HLA-DRB1, which illustrates how environmental and genetic risk factors may be related Epigenetic mechanisms modulating gene expression, such as DNA methylation, histone modifications, and non-coding RNAs, play a critical role in the immune system trans-acting variants. The use of RNA sequencing should further increase the informativeness of such studies, identifying alternatively spliced transcripts and non-coding RNAs as well as allele-specific expression involved in immune dysfunction.Genomic approaches such as GWAS and WES have proven very powerful in defining the extent and nature of genetic variation in the context of immune function and disease. However, considerable challenges remain, not least in establishing causal functional variants . The desGenetic variation plays a fundamental role in modulating the development and effective functioning of the immune system. Recent work has illustrated how we can use genomic and epigenomic approaches to define such variation and understand the immune response in health and disease. This offers the promise of improved insights into disease pathogenesis, better diagnostic accuracy, new or repositioned therapeutics targeted to maximise benefit for the individual, and opportunities to improve outcome. To deliver on this will require continued excellence in basic science and translational research spanning genetics and immunology which takes advantage of current technological advances and our ability to resolve ‘experiments of nature’ seen in immune dysfunction.


Arabidopsis to draw an emerging picture of overlapping and specific roles of plant actin isotypes. Finally, we examine hypotheses explaining the mechanisms of isotype-specific functions.Plant actins are encoded by a gene family. Despite the crucial significance of the actin cytoskeleton for plant structure and function, the importance of individual actin isotypes and their specific roles in various plant tissues or even single cells is rather poorly understood. This review summarizes our current knowledge about the plant actin gene family including its evolution, gene and protein structure, and the expression profiles and regulation. Based on this background information, we review mutant and complementation analyses in  The structure, regulation of expression and expression patterns of actin genes, and the roles of the proteins have been extensively researched, yet fundamental questions remain. For example, what are the factors that account for the occurrence of different actin isotypes and what are their functions? Although the issue of diverse roles of actin isotypes has been partly addressed at the tissue level, information at the cellular level is scarce. To better understand the existence and role of multiple actin isotypes in plants, this review first summarizes what is known about the structure of actin genes, evolution of the gene family, and expression regulation. Further, we examine the current evidence for diverse and overlapping roles of actin isotypes and the molecular mechanisms determining the diverse roles.Actin is an essential protein that is expressed throughout the plant body in many distinct isotypes , although some of them may represent pseudogenes. Similar situation may occur in other species as well, so the actual number of actin isotypes can differ from the values identified so far , eight of which encode functional proteins and two likely pseudogenes (ACT5 and ACT9). According to protein sequences, the actin family is divided into two major classes: vegetative and reproductive. The eight protein isotypes can be further divided into five subclasses: two single-member subclasses ACT7 and ACT11, and three pairs of closely related proteins, namely, ACT2/8 in the vegetative class and ACT1/3 and ACT4/12 in the reproductive class  is inserted 4 bp upstream from the exon-intron 2 junction so that the intron 2 is located at amino acid residue 153 instead of the usual residue 152, which is common in other cotton actin genes  ago  led to the preservation of the ACT1 expression, but only in vegetative tissues and not in mature pollen  tend to be expressed in reproductive tissues, whereas the vegetative actins (ACT2/8 and ACT7) are expressed in vegetative ones. Specifically, the ACT1/3 pair is expressed in developing floral meristem, the emerging carpel and ovules, and in mature pollen (An et al., Arabidopsis actin isotypes are summarized in Table As mentioned above, individual plant actins show different expression patterns, congruent with their evolutionary relationships. Generally, the Picea abies actin isotype Pa1 was expressed in the whole somatic embryo, while isotypes Pa2-4 were expressed predominantly in the suspensor and their expression decreased as the suspensor degenerated later in development (Schwarzerova et al., A. thaliana, although no detailed data are available on differential expression of actin isotypes during the embryogenesis. Therefore, results from experiments in various plants are difficult to compare and more plant models are urgently needed to better understand the diversity, regulation, and the role of actin isotypes in plants.Developmental changes in the dynamics of actin isotypes certainly play an important role in the complex process of embryogenesis. We found that In animal cells, the localization of muscle and cytoplasmic actin isotypes in muscle and non-muscle cells was described in several studies, even though with somewhat conflicting results (Perrin and Ervasti, In the preceding sections we attempted to summarize existing knowledge about plant actin genes and proteins. The differences among plant actin isotypes at the protein level are higher and amino acid substitutions are more frequent than those in animal actins, suggesting that individual actin isotypes may have specific roles in plant tissues. The expression analysis likewise suggested the existence of vegetative and reproductive actins. However, is there direct experimental evidence that plant actin isotypes have specific, non-overlapping functions? What is the molecular mechanism of diverse actin functions in the cell? And importantly, is there biochemical evidence for diverse roles of actin isotypes?Drosophila results in a flightless phenotype because the cytoplasmic actin is not able to substitute for muscle actin (Brault et al., Studies in animals suggest that actin isotypes fulfill specific roles in these organisms, with some overlapping functions. Substitution of a muscle actin isotype with cytoplasmic actin in Arabidopsis. Aberrant root hairs were exhibited in several mutants, namely, missense act2 mutants (Ringli et al., act2-1 T-DNA insertional line null for ACT2, and act8-2 T-DNA insertional mutant (Kandasamy et al., act2-1 mutants or ACT2 was overexpressed in act8-2 mutants, suggesting the two isotypes are indeed mutually interchangeable in their roles in root hair development (Kandasamy et al., In plants, the specific roles of individual actin isotypes were mostly studied by mutation analyses. In shoots, the vegetative actin isotype ACT2 comprises approximately 50% of the total actin protein and closely related ACT8 is much more weakly expressed, comprising 10–15% of the total actin protein. ACT7, a more divergent isotype, represents 40–45% of the total actins (Kandasamy et al., ACT7 using native regulatory sequences in transgenic plants, or ectopic expression of the ACT1 coding region under the regulatory regions of ACT2, efficiently complemented act2-1 loss-of-function mutant root hair phenotype. Kandasamy et al. (act7-4 had 20-fold reduced levels of ACT7 protein and the total actin protein was reduced to 60–80% of the wild-type levels. In contrast to the act2-1 loss-of-function mutant, which exhibited root hair phenotypes, act7-4 mutants showed specific defects in germination, root growth control, and polarity control (Gilliland et al., act7-4 phenotype (Kandasamy et al., act2-1 act7-4 mutant plants could be fully rescued by overexpressing ACT8, only a partial rescue of act2-1 act8-2 phenotype was achieved in plants overexpressing ACT7 despite the fact that the total actin protein level in the rescued plants was elevated twofold compared with wild-type plants (Kandasamy et al., Gilliland et al.  reportedy et al. , howevery et al. . Indeed,y et al.  experimeOther studies showing functional non-equivalence of plant actin isotypes are also available. Ectopic expression of reproductive actin ACT1 in vegetative tissues of transgenic plants caused severe phenotypes including dwarfism and morphological abnormalities in most organs (Kandasamy et al., Monosiga brevicollis, Acanthamoeba castellanii, and Coleochaete scutata, which are close to the common ancestor of plant and animal actins, completely suppressed root phenotypes in Arabidopsisact8-2 act7-4 double mutants. Interestingly, human cytoplasmic actin, but not human α-skeletal muscle actin, partially suppressed the phenotype as well (Kandasamy et al., As mentioned above, phylogenetic analysis showed that plant vegetative actins represent a more primitive group of actins, whereas reproductive actins are more diverged. An exciting study of Kandasamy et al.  suggestsWhat is the molecular basis of the functional specificity between actin isotypes? As mentioned above, actins are highly conserved and differences between isotypes are very small. Nevertheless, actin–actin or actin–ABP interactions may still be affected by these differences, resulting in different phenotypes. Similarly to the family of actin isotypes, plant profilins likewise constitute a family where the isotypes are divided into vegetative and reproductive classes (Huang et al., ACT2 gene was isolated in which a missense mutation resulted in an amino acid substitution at the surface of the protein molecule (Nishimura et al., act2 mutant did not show most of the root developmental defects (Gilliland et al., A dominant-negative mutation in the Unfortunately, only limited information is available about the biochemical properties of individual actin isotypes. Recently, Bergeron et al.  purifiedAvailable data suggest that plant actin isotypes have both overlapping and distinct functions. Complementation studies suggest a modified view of actins’ evolution. As suggested by Kandasamy et al. , in spitBesides the generally high functional inter-changeability, some functional specialization can be detected even within the group of plant vegetative actins. Indeed, the relative proportions of actin isotypes with different biochemical properties may determine the characteristics of actin polymers. Theoretically it may be advantageous for a cell to express multiple isotypes with overlapping expression patterns, capable of mutual interactions, and cooperation with specific isotypes of ABPs. Using the analogy of steel as an alloy composed of multiple metals, an actin filament can be considered as an alloy composed of multiple actin isotypes (Perrin and Ervasti, 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.