Protein–protein interactions (PPIs) execute many fundamental cellular functions and have served as prime drug targets over the last two decades. Interfering intracellular PPIs with small molecules has been extremely difficult for larger or flat binding sites, as antibodies cannot cross the cell membrane to reach such target sites. In recent years, peptides smaller size and balance of conformational rigidity and flexibility have made them promising candidates for targeting challenging binding interfaces with satisfactory binding affinity and specificity. Deciphering and characterizing peptide–protein recognition mechanisms is thus central for the invention of peptide-based strategies to interfere with endogenous protein interactions, or improvement of the binding affinity and specificity of existing approaches. Importantly, a variety of computation-aided rational designs for peptide therapeutics have been developed, which aim to deliver comprehensive docking for peptide–protein interaction interfaces. Over 60 peptides have been approved and administrated globally in clinics. Despite this, advances in various docking models are only on the merge of making their contribution to peptide drug development. In this review, we provide (i) a holistic overview of peptide drug development and the fundamental technologies utilized to date, and (ii) an updated review on key developments of computational modeling of peptide–protein interactions (PepPIs) with an aim to assist experimental biologists exploit suitable docking methods to advance peptide interfering strategies against PPIs. Delivering drugs specifically to patient neoplasms is a major and ongoing clinical challenge. Function-blocking monoclonal antibodies were first proposed as cancer therapies nearly four decades ago. The large size of these molecules hindered their commercial development so that the first antibody or antibody-fragment therapies were only commercialized for cancer therapeutics and diagnostics 20 years later ,2. A claIn comparison to small molecules, such as proteins and antibodies, peptides indeed represent a unique class of pharmaceutical compounds attributed to their distinct biochemical and therapeutic characteristics. In addition to peptide-based natural hormone analogs, peptides have been developed as drug candidates to disrupt protein–protein interactions (PPIs) and target or inhibit intracellular molecules such as receptor tyrosine kinases ,6. TheseProtein–protein interactions (PPIs) are the foundation of essentially all cellular process. Those biochemical processes are often comprised of activated receptors that indirectly or directly regulate a series of enzymatic activities from ion transportation, transcription of nucleic acids and various post-translational modifications of translated proteins . Drugs tUnfortunately, understanding the molecular recognition mechanism and delineating binding affinity for PPIs is a complex challenge for both computational biologists and protein biochemists. This is largely because small molecules are superior in binding to deep folding pockets of proteins instead of the larger, flat and hydrophobic binding interfaces that are commonly present at PPI complex interfaces . AlthougSince the first isolation and commercialization of insulin, a 51 amino acid peptide in the early 1920s, peptide drugs have greatly reshaped our modern pharmaceutical industry . With adUnlike peptide drugs that are usually synthetically made, natural polypeptides such as hormones, growth factors or neurotransmitters are known to play central roles in normal physiology. Peptide drugs have two major drawbacks: Their in vivo instability and membrane impermeability . ProteolProteolysis can potentially take place from both N- and C-termini of a peptide by up to 500 proteases and peptidases including serum aminopeptidases and carboxypeptidases . It is wFor biologists, the choice of chemical modification often requires expertise in chemistry through collaborations. There are, however, a number of methods that can be approached easily and yet are important for biological study of peptide drug design. First, it is necessary to identify the minimum amino acid residue(s) essential for peptide activity. This can be achieved by repetitive truncating amino acids from either the N- or C-terminus of a lead sequence to ascertain the critical core peptide motif needed for biological activity. Secondly, a classic screening method called alanine scanning can be used to determine the contribution of individual amino acids to biological activity of the peptide . By screThe amino acid scanning techniques discussed above also provide useful information for the design of further modifications, especially on the side chain group of a particular residue. Synthetic enantiomer amino acids, for instance, have been proposed to enhance resistance to proteases as their stereochemically reversed side-chains are not recognized as protease substrates . ArgininAlthough enantiomer amino acid (D-amino acid) substitution has been a common approach to protect peptides from protease degradation, the conformational changes created can compromise the biological activity of the original L-peptide . In addiThe protease resistance of peptides can also be improved by peptide cyclization. There are a number or strategies for generating cyclized peptides. These include head-to-tail cyclization, which generates a peptide bond between the original N- and C- termini. The amino group on lysine side-chains can be reacted with aspartic or glutamic acid side chains or the free C-terminus, forming an amide bond. Alternatively cysteine pairs can be reacted to form a disulfide bond between the side chains. These strategies can protect termini and restrict conformational flexibility of peptides, maintaining them in their bioactive conformation . CyclizaThe generally poor cell membrane permeability of peptides has limited their application against inaccessible intracellular targets. Peptide therapeutic development has largely focused on extracellular targets due to this limitation. Improvement of membrane permeability or development of strategies that facilitate active intracellular uptake will be critical for successful peptide-based targeting of intracellular PPIs. Potential strategies for improving intracellular uptake include modulation of the hydrophobicity and electrostatic charges to improve passive uptake, or conjugation of the active drug peptide to a cell-penetrating peptide (CPP) to facilitate its active transport.Bioavailability of peptide biotherapeutics is usually greatly enhanced when they are more water soluble, as effective serum concentrations can be easily maintained. Optimization of aqueous solubility is however a largely empirical process, with experimental identification of unnecessary hydrophobic amino acids which can be replaced with charged or polar residues to modulate the pI while maintaining bioactivity ,37. ReceG-protein coupled receptors (GPCRs) are a superfamily of transmembrane receptors, which are responsible for transporting diverse molecules across membranes. Although peptides can be natural ligands for GPCRs, only a small subset of extracellular peptides are actively transported across the plasma membrane. These peptides capable of crossing cell membranes are now termed cell permeable peptides (CPPs). They are generally highly hydrophobic, five to 30 amino acid long peptides . The molAs discussed above, cyclic peptides have superior proteolytic resistance and structural stability than linear peptides. Tremendous efforts have been made towards developing of cell-permeable cyclic peptide drugs to block PPIs. Short CPP motifs have been rationally conjugated to otherwise cell-impermeable cyclic peptides to facilitate their intracellular uptake. This delivery strategy has been applied more generally to develop bicyclic peptide drugs comprised of one membrane-crossing CPP moiety and one cyclic peptide PPI inhibitor, while maintaining target specificity and affinity . The oncG12V. While high content combinatorial library screening method has facilitated the rapid identification of PPI inhibitors, peptides with weaker inhibitory activity may not be efficiently detected. Such modest interactions should not necessarily be ignored because sequence optimization or cyclization may be able to drastically improve affinity. The sequential rounds of “biopanning” enrichment in phage display library screening can improve the detection of weaker interactions. In fact the importance of this approach over the past three decades was recently recognized by the Nobel Prize for Chemistry award [The discovery of the Ras-Raf bicyclic peptide inhibitor in fact stemmed from optimization of hits identified from a screen of 5.7 million bicyclic peptides for interaction with oncogenic K-Rasry award ,56. Overry award . These iry award . Traditiry award ,60. Thes2 to 3000 Å2), relative to the binding pocket size of the small molecules (300–1000 Å2) [PPIs are well-recognized potential therapeutic targets, given that dysregulated protein interaction networks underlie a wide range of pathologies. It is estimated that there are at least 140,000 pairwise PPIs in the human interactome . Many ef1000 Å2) . Small m1000 Å2) . As disc1000 Å2) . With reProtein–protein interactions are at the core of normal and pathogenic cell biology and physiology. Abnormal protein–protein interactions drive signaling changes that underpin pathologies including infection, chronic inflammation, neurodegeneration, cancer and cardiovascular disease to name a few. Protein interaction surfaces are therefore attractive therapeutic targets, and as discussed peptides have advantages over small molecules in this area. A number of promising IPs are currently under clinical investigation. A 28-mer peptide drug that blocks the interaction of the ubiquitin-ligase MDM2 with its target p53 is able to block MDM2-dependent p53 ubiquitination, promoting p53 stabilization and tumor suppression ,65. A 17IPs with α-helical structures that bind to protein interacting surfaces have shown particularly promising interaction-blocking activity, probably due to their good stability and protease resistance. . The litProtein–protein interactions have been experimentally determined by a variety of biophysical techniques such as X-ray crystallography, NMR spectroscopy, surface plasma resonance (SPR), bio-layer interferometry (BLI), isothermal titration calorimetry (ITC), radio-ligand binding, spectrophotometric assays and fluorescence spectroscopy. Experimental data generated by these techniques has advanced our knowledge of how secondary and tertiary protein structure and interaction kinetics influence downstream biological events. These techniques, however, are often time-consuming and applied to study one specific PPI at a time. While protein crystal X-ray diffraction is undoubtedly a very powerful structural analysis approach, which is capable of defining structure down to individual atoms, it has major technical challenges. Many proteins do not crystalize well, or do not crystalize except as smaller protein domains. Even if each protein in a complex forms crystals alone, co-crystallization can be especially challenging. NMR spectroscopy can generate protein complex structures, but has a lower resolution compared to X-ray diffraction. Optical or calorimetric approaches can provide information about the energy, affinity and disassociation properties of an interaction, but do not identify a specific interaction surface the way NMR or X-ray diffraction do. The technical challenges and poor scalability of wet-lab experimental approaches has necessitated the development of reliable computational strategies. Computational docking methods have thus been developed to expedite the process of generating accurate predictions of protein structure, surface charge and interaction affinities. Even for proteins without an available crystal structure, these programs can be applied for in silico identification of key protein interaction surfaces, the structural effect of mutations and binding analysis of potential blocking peptides.Computational PPI docking has provided rapid and useful information for drug designs at the atomic level, as some of the docking methods can be executed within the order of minutes. This can be achieved by rigid-body docking strategies, in which two interacting proteins are both considered absolutely rigid in calculation for optimization of chemical and geometric orientation fit. Z-DOCK is a typical rigid-body protein docking program that generally generates accurate predictions of PPI when proper scoring scaffolds are provided . Due to Many computational docking strategies, especially flexible-body docking methods, require structural information including number of hydrogen bonds, buried surface area, mutation hotspots, geometric angles and allosteric effects for calculations of binding free energies to generate more accurate predictions on binding affinities between the interacting proteins . By contAlthough significant progress has been made in scoring functions from both strategies, the lack of high computing power and larger experimental datasets with high quality have restricted the advances in the field. A community-wide experiment CAPRI compared computationally predicted protein complex structures with experimentally determined structures to evaluate and rank the best-performing servers based on prediction accuracy . HADDOCKSimilar to protein–protein interactions, structural information (either a sole target protein structure or complex structure with ligand) that is available for a drug target has often limited prediction accuracy for PepPIs. As a result of the lack of protein co-structures, many studies utilize existing information from structural databases such as the Protein Data Bank (PDB) to identify sequence-binding motifs for peptide designs. . AnotherBefore discussing current computational methods for PepPIs, we would like to first introduce recent advances in the selection of initial peptide scaffolds that also play critical roles in peptide drug development. A number of well-characterized natural occurring peptides had been selected from natural proteins and demonstrated to preserve original functions including structural scaffolds or the ability of recognizing target molecule. Repeated Arg-Gly-Asp (RGD) motifs, for instance, were first derived from cell attachment domain of fibronectin that binds to membrane-bound receptor protein integrins and activates cellular growth, differentiation, adhesion and migration . The capSuccessful docking of the structural pose of a PepPI has been largely dependent on the extent of structural scaffolds available about the interaction complex. The dramatic increases in numbers of peptide–protein structures available in PDB have greatly facilitated the development of more powerful docking and refinement methods in predicting accurate PepPIs. Peptide–protein docking strategies are usually categorized into local or global docking based on the extent of structural information provided as inputs.Local docking is the mostly commonly used strategy as it searches for a potential binding pose for peptide at a user-defined binding site in a resolved structure of its target receptor. A number of methods have the capability to improve initial model quality at atomic resolution within 1 Å to 2 Å RMSD of the experimental peptide conformation. DynaRock, Rosetta FlexPepDock and PepCrawler are the most popular methods that provide different approaches of defining peptide-binding sites. DynaDock uses soft-core potential combined with Molecular Dynamics as refinement approach for conformational sampling and receptor side-chain flexibility determination . As van However, not every query peptide has available information of backbone conformation. Sampling methods that allow acquisition of near-native peptide conformation become essential prior to performing local docking. Rosetta FlexPepDock ab initio, for instance, is a protocol that combines ab initio peptide folding with local docking by placing the query peptide into a user-defined binding site from any arbitrary backbone conformation . The binUnlike local docking that searches for the peptide-binding pose, global docking methods also search for the peptide-binding site at the target protein. Global docking is often the method of choice when no prior information is available on binding sites. A spatial position specific scoring matrix (PSSM) was used in developing the PepSite method to identify potential binding sites with estimated position for each residue ,103. TheIn addition to highly accurate predictions made by PIPER-FlexPepDock, another recently developed method HPEPDOCK used an ensemble of peptide conformations for blind global docking and obtained significantly higher success rates as well as lower simulation time required than pepATTRACT ,109.Template-based docking methods are also known as comparative docking strategies. They use known structures as template scaffolds by threading the sequence of the query peptide and/or target protein to build a model of the interacting complex . TemplatPeptides have attracted a lot of attention and the number of approved peptide biotherapeutics has been on the incline over the recent decades. This has been an attractive approach due to their ability to bind with larger interfacial pockets than small molecules, and has been driven by major advances in computational structural prediction and the expansion of available chemical modifications to improve stability, affinity and specificity. The publicly available computational binding prediction tools have led to effective rational designs of new peptide drugs of higher therapeutic potency. Our recent study utilized both biological and computational methods to develop a cancer-specific targeting peptide that facilitated significantly greater in vitro and in vivo and therapeutic efficacy .Despite recent advances in computational modeling of protein–protein and peptide–protein structures, major challenges remain. For instance, it remains challenging to simultaneously consider the backbone and side-chain flexibility of the peptide and its target protein to accurately predict the bound structure. Secondly, interpretation of experimental data from high-resolution NMR spectroscopy and cryo-electromicroscopy or small-angle X-ray scattering (SAXS) to obtain accurate experimental structures is often ambiguous, which makes integration of such experimental data into computational prediction software very difficult. There are computational servers that have attempted to translate ambiguous experimental data into algorithmic constrains that can be applied as an option for docking ,97. In f


Proteinuria is a common clinical presentation, the diagnostic workup for which involves many non-invasive and invasive investigations. We report on two siblings that highlight the clinically relevant functional role of cubulin for albumin resorption in the proximal tubule and supports the use of genomic sequencing early in the diagnostic work up of patients who present with proteinuria.CUBN (10p12.31).An 8-year-old boy was referred with an incidental finding of proteinuria. All preliminary investigations were unremarkable. Further assessment revealed consanguineous family history and a brother with isolated proteinuria. Renal biopsy demonstrated normal light microscopy and global glomerular basement membrane thinning on electron microscopy. Chromosomal microarray revealed long continuous stretches of homozygosity (LCSH) representing ~ 4.5% of the genome. Shared regions of LCSH between the brothers were identified and their further research genomic analysis implicated a homozygous stop-gain variant in CUBN mutations have been implicated as a hereditary cause of megaloblastic anaemia and variable proteinuria. This is the second reported family with isolated proteinuria due to biallelic CUBN variants in the absence of megaloblastic anaemia, demonstrating the ability of genomic testing to identify genetic causes of nephropathy within expanding associated phenotypic spectra. Genomic sequencing, undertaken earlier in the diagnostic trajectory, may reduce the need for invasive investigations and the time to definitive diagnosis for patients and families. Proteinuria is a common clinical presentation that is associated with poor renal outcomes, especially when severe . HistoriAn 8-year-old boy was referred to the paediatric nephrology service with an incidental finding of persistent proteinuria during investigation for abdominal pain. He had no hematuria or glycosuria. He had normal serum albumin (42 g/L). Haematological, biochemical and immunological parameters were unremarkable. Specifically, C3, C4, ANCA and anti-GBM were negative and ANA was moderately positive 1:320). Renal function was normal (serum creatinine 0.52 mg/dL). Urinary albumin creatinine ratio was raised at 51.4 mg/mmol  and protein creatinine ratio 92 mg/mmol . Urine electrophoresis was 1.47 g/L with non-selective proteinuria pattern detected . Assessment for glycosuria, urinary light chains and urinary amino acids was negative. Urinary electrolytes were normal, and haematuria was absent on serial testing. Renal ultrasound, ophthalmology and audiology assessment did not demonstrate any abnormal findings. Renal biopsy demonstrated normal light microscopy and there was no evidence of immune complex mediated disease or other pathology. Podocyte foot processes were generally well preserved on electron microscopy, however there was global uniform thinning of the glomerular basement membrane ) and one reported case of isolated proteinuria [Family history assessment revealed that his parents were first cousins, of Middle Eastern descent, with no known family history of renal disease or other comorbidity. Further investigation of the family revealed isolated proteinuria  in his 4-year old brother but not in either parent. His brother’s haematological parameters were unremarkable and remaining urinary investigations were within normal range apart from an elevated urinary protein electrophoresis (1.32 g/L). Chromosomal microarray in the older sibling revealed long continuous stretches of homozygosity (LCSH) representing ~ 4.5% of the genome, consistent with parental consanguinity. Shared regions of LCSH between the brothers were interrogated using the Genomic Oligoarray and single nucleotide polymorphism (SNP) evaluation tool© [CUBN mutation in our patients may have led to predominant albuminuria due to a deficiency in tubular reabsorption of albumin.The causes of isolated proteinuria can be divided into glomerular, tubular and overflow proteinuria. Glomerular proteinuria occurs in nephrotic syndrome due to increased permeability of the glomerular capillary wall, resulting in the abnormal passage of proteins from the glomerulus . Tubularcubn heterozygous mice [The role of megalin and cubulin in proximal proteinuria has been postulated in several studies, with the most recent study demonstrating that while megalin/cubulin mouse knockouts result in blocked proximal tubular albumin uptake, this did not affect plasma albumin levels in nephrotic mice  . This sous mice . Future CUBN mutations have previously been described as causative in Imerslund-Grasbeck Syndrome [CUBN mutations [isolated proteinuria as their only phenotypic feature. The previous family was described by Ovunc et al as having a novel biallelic 1-bp homozygous deletion in CUBN resulting in a phenotype of fluctuating proteinuria in otherwise well individuals [CUBN associated with albuminuria in a genome wide association study [CUBN single-nucleotide polymorphisms (SNP) (rs1801239) and ESKD has been demonstrated in African Americans [Syndrome , with ovutations , 7. Our ividuals . Of noteon study . Furthermericans .Persistent proteinuria is an important marker of chronic kidney disease and associated with inferior cardiovascular and renal outcomes , and serCurrently treatment strategies are focussed on glomerular proteinuria, largely by reducing glomerular filtration pressure with renin-angiotensin system inhibition and blood pressure control. Although this has been shown to be effective in reducing decline in kidney function , future CUBN secondary to deficiency of albumin uptake by the proximal tubule, resulting in a renal limited form of Imerslund-Grasbeck Syndrome. This may represent a phenocopy of other isolated proteinuric kidney diseases. Although the patient in our case also demonstrated thinning of the GBM on biopsy, we do not believe that this was the cause of his proteinuria due to the absence of haematuria and other structural anomalies on light and electron microscopy. This family highlights the need to include CUBN in the genomic diagnostic approach to proteinuria. As the availability of next-generation-sequencing (NGS) increases and costs decrease, genomic testing is now being increasingly considered a part of the standard workup of chronic kidney disease. We suggest genomic testing should be performed in patients with proteinuria with a strong suspicion of genetic disease, antecedent to or concurrent with renal biopsy depending upon clinical scenario and urgency, particularly where markers of proximal tubular proteinuria are present [This family, in addition to the one previously described in the literature provides further evidence for gene-disease validity association, which will be important in guiding the interrogation and interpretation of genomic data in renal patients presenting with proteinuria . We prop present .CUBN mutations have been implicated as a hereditary cause of megaloblastic anaemia and variable proteinuria. This family is the second described in the literature to have a monogenic CUBN-mediated cause of isolated proteinuria, approximating a renal-limited form of Imerslund-Brasbeck Syndrome and phenocopying other forms of isolated proteinuria. Therefore, we describe how genomic sequencing can successfully identify a single gene cause of nephropathy in a clinically relevant and applicable manner. This report strengthens the gene-disease validity association for CUBN, with implications for the practice of diagnostic laboratories. Furthermore, this case supports the argument that genomic sequencing should be undertaken earlier in the diagnostic workup in order to reduce the need for invasive investigations, such as renal biopsy. Future cases with long term follow up of isolated proteinuria due to CUBN mutations will help to inform the management and prognosis of these patients.


Survivin, an antiapoptotic molecule from inhibitor of apoptosis protein (IAP) family, is most known for its implication in cancer as there are some efforts to apply it for diagnostic as well as therapeutic purposes in oncology. On the other hand, it could be a useful molecule to be positively targeted when trying to save tissue and promote cells viability. Since protecting the allograft from ischemia reperfusion injury and inflammation-induced damage is a considerable objective in transplantation, it is reasonable to take advantage from antiapoptotic agents like survivin in order to achieve this goal. However, survivin's potential ability to induce malignancies makes some concerns about its use in clinic. The other barrier is this molecule's involvement in lymphocytes development and proliferation which might increase the risk of graft rejection due to adaptive immune system overactivation. In this review we summarize the few studies carried out about survivin's effect on graft survival and probable advantages and disadvantages of its overexpression in transplantation. To keep up with the rapid surgical and pharmacological advancements in transplantation and the rising number of organ recipients, there is a need for designing fast non-invasive graft monitoring techniques to take proper actions in order to prevent irreversible tissue damage. In addition, long-term adverse effects and the financial burden of immunosuppressor (IS) agents have resulted in a trend towards finding practical ways to replace these drugs with tolerance induction methods and to improve graft survival with molecular manipulation techniques. Therefore, finding molecules which play both diagnostic and therapeutic roles would be of great interest in this field.The role of antiapoptotic molecules has already been recognized as both safe biomarkers in diagnosis of certain malignancies and potential targets in cancer therapy; however, manipulating antiapoptotic agents to improve graft survival is a novel topic to study in transplantation. There are also some efforts to produce IS drugs in combination with antiapoptotic agents in order to reduce these drugs toxic effects; for instance, Wu et al. demonstrated that JP-3-110 induces significantly less activated caspase-3 and apoptotic death of transplanted islet cells than mycophenolic acid, while exerting the same immunosuppressive effect [Survivin was first described in 1997 by Ambrosini et al. as an antiapoptotic molecule involved in fetal development which is overexpressed in cancerous tissues. This small protein of 16.5 kDa is a member of inhibitor of apoptosis protein (IAP) family, responsible for apoptosis inhibition and cell cycle regulation . The BIRA specific feature of IAP family is the Baculoviral IAP Repeat (BIR) domain with conserved residues rendering a death-preventing activity to N-terminal by direct inhibition of caspase proteins. Survivin has a single BIR domain, which discriminates it from other IAPs. In addition, it has been found that the interaction between two hydrophobic positions in survivin structure forms a bow tie-shaped dimer in protein product  Figure .Zwerts et al. showed that lack of survivin causes mice to die at the early stages of embryogenesis due to the abnormal generation of heart. These mice also suffered from diffuse hemorrhage and neural tube defects .After birth, survivin plays a significant role in viability and appropriate function of normal tissues, particularly those with high regeneration rates such as bone marrow , colonicSurvivin overexpression in cancer has made it an appealing topic in molecular research because it has been shown that survivin overexpression is associated with high grade tumors and poor prognosis in a range of malignancies. Moreover, a link has been drawn between certain survivin gene polymorphisms  and susceptibility for cancer, as well as responsiveness to the chemotherapy . In addiApoptosis can occur in various types of tissue injury such as ischemia, inflammation, drugs cytotoxicity, and infections , most ofContrary to cancer, tissue survival and apoptosis inhibition are considerable advantages in transplantation. To overcome ischemia reperfusion injury (IRI) and inflammation-induced tissue damages, has always been desired by clinicians; therefore, any means which makes the cells resistant to apoptosis would be welcomed in this area.Since kidney is the most transplanted organ, molecular studies are mainly directed to the renal transplantation. According to an investigation of Lechler and colleagues, survivin is expressed in adult renal epithelial cells, particularly in proximal tubules, and plays an essential role in tubular maintenance. They have even warned about targeting this molecule in cancer therapy because of its harmful effects on tubular integrity . Musiał Chen et al. demonstrated a protective role for survivin in mouse model of acute kidney injury (AKI). They compared ischemia reperfusion injury between the selective renal proximal tubule survivin knockout mice and a control group. The study showed a significant upregulation of survivin expression within 48 hours of IRI insult in control mice lasting for six days which was absent in knockout group; in addition, a progressive tubular damage was evident after two weeks of follow-up in the knockout mice comparing to the control group which exhibited only minimal injuries; therefore, they attributed delayed recovery and more tissue damage to the survivin deficiency in knockout mice . Such a Survivin involvement in tissue repair has also been investigated in other tissues. One such study used mesenchymal stem cells (MSCs) expressing survivin in mouse model of cardiac infarction. MSCs were infected with lentiviral vectors carrying green fluorescent protein (GFP) together with survivin genes . After inducing heart permanent hypoxia, Fan and colleagues applied intramyocardial injection of phosphate-buffered saline (PBS), MSC-GFP, and MSC-GFP-SURVIVIN to three groups of mice. Cell survival was assessed one and four weeks later; accordingly, more cell viability, upregulated expression of VEGF, increased capillary density, reduced infarct size, less collagen deposition, and improved cardiac function were observed in MSC-GFP-SURVIVIN recipients compared to the other groups . We coulβ-cells affected neither insulin release nor glucose levels; moreover, there was no difference between wild-type and transgenic mice in islets size and number. Nevertheless, the latter group was protected against staurosporine (STS)-induced cell death comparing to the other.Regarding antiapoptotic effects of survivin, there have been some interventional experiments to apply such a property in transplantation for the purpose of promoting engraftment and allograft survival. In an experiment by Dohi et al. survivin expression in mice transgenic (full-length mouse survivin complementary DNA was cloned and microinjected into C57Bl/6 embryos) β-cells while there has not been any curative effect using normal cells. So they demonstrated survivin molecule's effectiveness in protecting tissue against STS toxicity and transplantation insults [Besides, they transplanted wild-type and survivin-expressing islet cells (infected with a replication-deficient adenovirus encoding haemagglutinin-tagged wild-type survivin) to the streptozotocin (STZ)-induced diabetic mice. After one and two months of follow-up corrected hyperglycemia and appropriate glucose tolerance were observed in all transplanted diabetic mice receiving transgenic  insults .P.Cassis et al. have also generated mice with 50% of survivin gene expression (haplo-insufficient) and investigated different syngenic as well as allogenic renal transplant models. They reported three main findings: first, survivin haplo-insufficient mice would reject syngenic kidney grafts due to the sustained inflammation and deficient tissue repair after a period of ischemia; the second finding was that survivin gene delivery (using plasmid vectors) could improve tissue survival in both syngenic and allograft models; finally, in fully mismatch transplantation models survivin overexpression resulted in better graft survival and less chronic allograft nephropathy. Besides, considering the oncogenic potential of survivin, they commented that no malignancy was detected in overexpressing group during a long-term follow-up period .Quillard et al. has shown that Notch2 gene silencing which regulates survivin gene expression could protect endothelial cells from TNF-induced apoptosis ; this fiFurthermore, it has been shown that survivin inhibition in mouse and human bone marrow results in fewer MSC and clonogenic colony forming unit fibroblasts, whereas survivin overexpression in MSC promotes their proliferation; moreover, survivin inhibition in MSCs reduces their hematopoiesis-supporting capacity. According to the considerable role of MSCs in engraftment and maintenance of hematopoietic stem cell transplantation (HSCT), survivin-expressing MSCs seem to promote HSCT outcome . In addiIn spite of positive effect of survivin overexpression on allograft survival, this antiapoptotic molecule's involvement in cancer arises some concerns about its use in clinic. Decision-making would get even more complicated considering the increased risk of posttransplant lymphoproliferative disorders due to the intensive immunosuppression.A notable study in this regard was carried out by Michele Bernasconi et al. to investigate early molecular events leading to B-cell transformation after Epstein-Barr virus (EBV) infection. They used tonsils as the source of B lymphocytes. After induction of EBV infection, gene expression profile assay was performed using Affymetrix microarray. Three genes whose expression had been significantly elevated by EBV were cyclin-dependent kinase 1 (CDK1), cyclin B1 (CCNB1), and survivin ; this fiAnother concern about survivin overexpression is its role in T lymphocytes homeostasis. Zheng Xing et al. inactivated survivin gene in mice and performed an apoptosis assay on primary thymocytes and peripheral lymphocytes. The survivin-deficient mice at early stages of thymic development showed impaired pre T cell proliferation. Although later stages passed normally at thymus, peripheral blood T cells displayed immature phenotypes and reduced number. These findings propose an essential function for survivin at both early and late phases of T cells development . SurviviL/LCD21Cre mice [B cells life cycle is also markedly affected by survivin. Miletic et al. studying mice bone marrow showed survivin high expression in proliferating CD43+B220+IgM−IgD− cells which is maintained through small pre-B cell stage but it was undetectable in immature B cells. Subsequent to mitotic stimulation as well as in germinal centers, survivin expression is restored significantly in mature B cells. In addition, the mice lacking survivin expression failed to develop sufficient B lymphocytes in BM. Although survivin seems not to be required for mature B cells maintenance, it is necessary for their proper function as it was demonstrated that IgM and IgG serum levels along with peritoneal B cell populations were reduced in survivinCre mice . AccordiIn brief, to protect allograft from apoptosis induced by various insults such as ischemia reperfusion injury, toxic effect of IS drugs, infection, and sustained allo-responses leading to chronic rejection, it might be useful to apply synthetic antiapoptotic agents or induce overexpression of the biologic ones like survivin. Regarding the findings of gene deletion and overexpression studies, there is some evidence in favor of survivin's beneficial effect on tissue repair, ischemia resistance, and engraftment; nonetheless, the risk of developing malignancies and lymphocytes overactivation should be taken into account. To overcome these risks, it is suggested to apply tissue restricted survivin expression for a limited time such as reperfusion period posttransplant .


Biological nitrogen fixation (BNF) is controlled by intricate regulatory mechanisms to ensure that fixed nitrogen is readily assimilated into biomass and not released to the environment. Understanding the complex regulatory circuits that couple nitrogen fixation to ammonium assimilation is a prerequisite for engineering diazotrophic strains that can potentially supply fixed nitrogen to non-legume crops. In this review, we explore how the current knowledge of nitrogen metabolism and BNF regulation may allow strategies for genetic manipulation of diazotrophs for ammonia excretion and provide a contribution towards solving the nitrogen crisis. Only a selected group of Bacteria and Archaea, called diazotrophs, have evolved the ability to reduce N2 to generate NH3 in a process known as biological nitrogen fixation (BNF) catalysed by nitrogenase, an oxygen-sensitive enzyme complex. The reaction catalysed by the nitrogenase enzyme is an extremely energy-demanding process since reduction of 1 mol of N2 requires 16 mol of ATP in vitro [in vivo can increase up to 40 mol of ATP per mol of N2 in aerobic diazotrophs particularly at high oxygen tensions when respiratory activity has to be diverted to protect nitrogenase from oxygen damage [Nitrogen is an essential element for all living organisms as it is part of the building blocks that constitute nucleic acids and proteins. Although gaseous nitrogen  genes , additional enzymes that carry out reversible post-translational modification of proteins in response to metabolite control (GlnD and GlnE), and a two-component regulatory system (NtrB or NRII and NtrC or NRI) that controls the expression of many genes involved in nitrogen metabolism –18. In dg family ) to ensug family . Gaseousg family ,22.54-dependent genes important for nitrogen metabolism [54 factor controls not only genes related to nitrogen metabolism but also other physiological traits such as motility, virulence, and biofilm formation [Azotobacter vinelandii, two in Escherichia coli and Klebsiella oxytoca M5a1  to threH72, see ) and Rhom rubrum . The preith NtrB ,40. In aKm (∼1 mM) for ammonium [Km for ammonium [Nitrogen assimilation primarily occurs via the synthesis of glutamate from ammonium and 2-oxoglutarate and is catalysed by two alternative pathways, involving either the enzyme glutamate dehydrogenase (GDH) or the GS-GOGAT pathway which combines the activities of the enzyme glutamine synthetase (GS) and glutamate synthase (GOGAT)  enzyme [In ) enzyme . Adenyly) enzyme ).54-dependent activator NifA is controlled by the two-component NtrB–NtrC nitrogen regulatory system in response to covalent modification and the ligand-binding state of the PII proteins [A. brasilense, Herbaspirillum seropedicae and R. rubrum, uridylylated PII proteins are required to activate NifA when nitrogen is limiting, whereas in Rhodobacter capsulatus, Azorhizobium caulinodans and Gluconacetobacter diazotrophicus, the non-modified form of PII seems to be required to inhibit NifA activity when nitrogen is abundant [K. oxytoca, A. vinelandii and Pseudomonas stutzeri A1501) and Beta (A. olearius) proteobacteria, NifA activity is controlled by a partner protein NifL, which senses environmental cues and interacts with PII proteins, to modulate NifA activity in response to the nitrogen status. In A. vinelandii, the non-modified form of GlnK is required for the formation of the inhibitory complex between NifL and NifA under nitrogen excess conditions [K. oxytoca, where GlnK (irrespective of its modification state) is required to relieve inhibition of NifA activity by NifL under nitrogen-limiting conditions [The PII proteins also play a pivotal role in coupling nitrogen fixation with ammonium assimilation by signalling the nitrogen status to the nitrogen fixation machinery at both the transcriptional and post-translational levels. In many diazotrophic Proteobacteria, expression of the σproteins ,44–46 . Nif reviews –53. TherR. rubrum and A. brasilense , mediates formation of a ternary complex between AmtB, GlnZ and DraG, that sequesters DraG to the membrane, inhibiting its ability to remove the covalent modification from nitrogenase [rnfABCDE), which potentially provides a dedicated electron transfer pathway to nitrogenase in A. vinelandii [nifLA operon in diverse diazotrophs (see below). The interaction between the PII signal transduction GlnK and the Rnf1 complex has been proposed to control electron transfer to nitrogenase in A. olearius BH72. Under conditions of ammonium shock, the non-modified form of GlnK interacts with the RnfC component of the membrane-bound Rnf1 complex. This is likely to decrease electron transport and potentially disrupt the electron flow to nitrogenase [H. seropedicae, for example, rapid nitrogenase switch-off during ammonium upshift requires both the amtB and glnK genes, suggesting that this elusive mechanism may require formation of an AmtB-GlnK complex in the membrane [In addition to the complex signal transduction cascades that couple nitrogen fixation to ammonium assimilation, the activity of the nitrogenase enzyme itself is regulated by fixed nitrogen availability. At least three different mechanisms, involving PII proteins, are known to switch-off nitrogenase activity under excess nitrogen conditions in Proteobacteria. The first mechanism involves inactivation of nitrogenase Fe protein by ADP ribosylation, catalysed by the DraT (ADP-ribosyltransferase) enzyme and has been extensively characterised in iew, see ). This piew, see –57. In arogenase ,58,59. Unelandii . Genes erogenase   compared with A. vinelandii (G2 clade), which might reflect physiological lifestyles and a requirement for the Rnf1 complex to support electron flux to nitrogenase activity under aerobic conditions. The absence of co-location between rnf and nifLA in group G3, represented by the methanotrophs , is more difficult to rationalise, given that these organisms can fix nitrogen under microaerobic conditions [rnf1 and nifLA operons in diazotrophic genomes may reflect physiological traits within particular phylogenetic groups and can possibly shed light into signal transduction mechanisms controlling nitrogen fixation in these organisms. Although it is difficult to speculate at this stage, one potential hypothesis for the neighbourhood co-location of rnf1 with nifLA, is that it provides integration of the regulation of nif gene transcription with the expression of electron transfer components required for nitrogenase activity, in addition to the possibility of conferring physiological switch-off of nitrogenase in response to ammonia.Although the NifL–NifA system was originally considered to be restricted to diazotrophic Gammaproteobacteria and a single representative of the Betaproteobacteria , the incnscribed . Notablyandii DJ , P. stutri A1501  and A. oius BH72 , the rnfrogenase . Whetherobserved . Organisnditions . Overallnif gene expression , (c) nitrogenase switch-off mechanisms , or (d) coupling between the PII uridylylation and GS adenylation states in response to nitrogen . Ideally, these metabolic perturbations should result in the excretion of a major proportion of the ammonia produced by nitrogenase while maintaining assimilation rates at sufficient levels to support bacterial fitness.The complex regulatory circuits described above ensure that components required for the biosynthesis and activity of nitrogenase are expressed only under demanding physiological conditions. They also ensure that regulation of nitrogen fixation and ammonia assimilation are intertwined, so that fixed nitrogen becomes readily available to support bacterial growth instead of being altruistic released to the environment. Knowledge of the control hierarchies and feedback regulation in response to the nitrogen status provide the potential for metabolic engineering of plant-associated diazotrophs to excrete surplus ammonium to benefit plant growth. The obvious targets for this metabolic engineering approach include perturbation of (a) ammonium assimilation , (b) nifL in A. vinelandii, which removes all regulatory inputs controlling NifA activity, results in the excretion of millimolar levels of ammonia [glnA substitution that decreases GS activity results in increased ammonium release as anticipated [A. brasilense, mutant strains selected for resistance to ethylenediamine (EDA) have been shown to excrete ammonia [A. brasilense strain HM053, the combination of low GS activity  with imr levels .A. brasilense provides substantial growth benefit to wheat plants [Setaria viridis with A. brasilense strain HM053 has been demonstrated to provide sufficient nitrogen to support plant growth [P. stutzeri A1501 nif gene cluster when engineered into Pseudomonas protegens Pf-5 leads to ammonia excretion and plant growth promotion [In some cases, ammonia-excreting strains have provided evidence that BNF can contribute to substantial plant growth promotion in model experiments in which single bacterial strains are used to inoculate plant varieties grown on sterile substrates. For example, inoculation with ammonium-excreting strains of t plants ,73 and it growth . Mis-regromotion . In someromotion –79. Howeromotion ,15.The use of BNF to tackle the nitrogen problem in agriculture has been attracting research efforts from many laboratories across the globe. The two major synthetic biology approaches to this problem: (a) engineering the nodule symbiosis in non-legumes ,80 and (


Following the failure of acute neuroprotection therapies, major efforts are currently made worldwide to promote neurological recovery and brain plasticity in the subacute and post-acute phases of stroke. Currently, there is hope that stroke recovery might be promoted by cell-based therapies. The field of stem cell therapy for cerebral ischemia has made significant progress in the last five years. A variety of stem cells have been tested in animal models and humans including adipose stem cells, human umbilical cord blood-derived mesenchymal stem cells, human amnion epithelial cells, human placenta amniotic membrane-derived mesenchymal stem cells, adult human pluripotent-like olfactory stem cells, human bone marrow endothelial progenitor cells, electrically-stimulated human neuronal progenitor cells, or induced pluripotent stem cells (iPSCs) of human origin. Combination therapies in animal models include a mix of two or more therapeutic factors consisting of bone marrow stromal cells, exercise and thyroid hormones, endothelial progenitor cells overexpressing the chemokine CXCL12. Mechanisms underlying the beneficial effects of transplanted cells include the “bystander” effects, paracrine mechanisms, or extracellular vesicles-mediated restorative effects. Mitochondria transfer also appears to be a powerful strategy for regenerative processes. Studies in humans are currently limited to a small number of studies using autologous stem cells mainly aimed to assess tolerability and side-effects of human stem cells in the clinic. Cerebral ischemia is a metabolic disease of the blood vessels affecting mostly the older population for which no rehabilitative therapy exists. Therefore, worldwide there is a huge investment in research aimed at alleviating the sequelae after stroke and improving restorative recovery. Unlike other species, the human brain is extremely sensitive to lack of oxygen which causes within minutes neuronal death ,2. In adHuman placenta amniotic membrane-derived mesenchymal stem cells (AMSC) regulate the immune response. Under hypoxic conditions, the gene expression of several factors involved in the regulation of immunity is shifted. CD200, an anti-inflammatory factor and a TGF-β positive regulator, was more strongly expressed under hypoxic conditions than under normotoxic conditions. AMSC transplanted in the rat ischemic stroke model inhibited the expression of proinflammatory cytokines and increased CD200 levels compared to control group. In addition, rats treated with AMSC have shown low activation of microglia in the penumbra and improved behaviour. The results suggest that CD200 strongly expressed from AMSC in a hypoxic environment modulates inflammatory cytokine levels and microglia activation, thus increasing the therapeutic recovery potential after hypoxic-ischemic brain lesions, demonstrating the immunomodulatory function of AMSC in a stroke model .In another study, human pluripotent stem cells with the potential to differentiate into cortical neurons, were evaluated upon transplantation in an animal model of ischemia in rats. Grafting at the lesion demonstrated reactivity to the margin of ischaemia, indicated by preferential axonal growth and cell migration, without influencing cell survival. Behaviourally, limb asymmetry was improved in the treatment group as compared to the control group .Human amnion epithelial cells (hAECs) are non-immunogenic, non-tumorigenic, anti-inflammatory cells normally discarded with the placenta. These biological features, broad availability and lack of ethical barriers in their use could make them useful as a therapy for ischemic stroke. The effectiveness of intravenous injection in the acute phase (1.5 h) or post-stroke (1–3 days) of the cells was tested in 4 animal models of cerebral ischemia, in young and old mice of both sexes, 7–14 weeks and 20–22 months as well as adult marmosets of either sex. hAECs administered at 1.5 h after stroke in mice migrated to the ischemic area via a CXC chemokine receptor type 4 dependent mechanism. The beneficial effects included reduced inflammation, infarct volume and functional deficits. Improved functional recovery was reported in young and elderly mice of both sexes even if hAEC administration was delayed up to 1 or 3 days after stroke. Intravenous injection of hAECs during the acute phase in marmosets led to a reduction in the infarct size. Systemic administration of hAECs determines marked neuroprotection and facilitates repair and recovery mechanisms .The protective effect of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) has been tested by administering hUCB-MSCs  intravenously, through the tail vein, in male Sprague–Dawley rats that were subjected to a transient cerebral artery occlusion. Interestingly, hUCB-MSCs treatment prevented the induction of matrix metalloproteinases (MMPs), which were overexpressed in the control group, at both dosages. HUCB-MSCs treatment also prevented the induction of tissue inhibitors of metalloproteinases .Conductive polymers have been recently used for cell delivery, drug release, and electrical stimulation to improve post-stroke recovery. Thus, an electrically conductive polymer scaffold was used to induce changes in the expression of genes involved in cell survival, inflammatory response, and synaptic remodelling in human neuronal progenitor cells (hNPCs). hNPCs are a promising alternative for stroke treatment, but optimum delivery conditions are not known. Electrical stimulation of hNPC using conductive polymers was performed the day before implantation, and the cells were transplanted in a MCAo rat model. Electrochemically manipulated pre-conditioned hNPCs reportedly improved functional results as compared to unstimulated hNPCs ,9. HowevBlood brain barrier (BBB) impairment is a pathological manifestation secondary to ischemic stroke, being one of the targets of new stroke recovery therapies. Using electron microscopy, the ability of bone marrow endothelial progenitor cells (hBMEPCs) to repair BBB in adult Sprague–Dawley rats subjected to transient MCAo) was evaluated. hBMEPCs pre-labelled with β-galactosidase were injected intravenously 48 h after MCAo. Ultrastructural analysis of microvessels in rats with stroke in the control group revealed the typical pathology of BBB at 5 days post-transplantation. In contrast, in the hBMEPCs treated group the animals showed vigorous repair of the network of the blood vessels the striatum and motor cortex without detectable perivascular edema. The transplanted endothelial cells displayed a normal morphology with intact mitochondria .Adult human olfactory mucosa, a highly regenerative tissue, is a mosaic of stem cell population that can be used for therapeutic purposes. Adult pluripotent-like olfactory stem cells (APOSCs) have the ability of self-renewal and differentiate into all three germinal cell lines. A subpopulation of adult pluripotent-like olfactory stem cells are modulated by an epigenetic repressor of CBX7, which is essential for the maintenance of embryonic stem cells by preventing cellular senescence. Unlike APOSCs from CBX7 −/− mice, the CBX7 +/+ mice APOSCs respond to induced tissue regeneration or induced lesions. Quite interestingly, in vitro cultured CBX7 −/− APOSCs suffered from premature senescence, while CBX7 +/+ APOSC is actively dividing, indicating that CBX7 is required for self-renewal of APOSCs. Intracerebral implantation of APOSCs, obtained from biopsy specimens of human olfactory mucosa and tranduced with the Luc gene; migrated to the injury site; differentiated into neuronal, glial, endothelial cell types; and improved neurological stroke-mediated function in mice subjected to MCAo .Induced pluripotent stem cells (iPSCs) are derived from differentiated cells by transfection with transcription factors which can induce pluripotency. iPSCs are a good alternative as replacement for neural stem/progenitor cells (NSCs/NPs). In addition, iPS of human origin can be easily tracked in animal studies. Thus, human induced pluripotent neural stem cells (hiNSC BC19) transplanted into a pig model of stroke correlated with good recovery of brain integrity and perfusion, as evaluated by magnetic resonance imaging (MRI). In vitro hiNSCs expressed Nestin and Sox1 and differentiated into neurons, astrocytes, and oligodendrocytes. In vivo, iNSCs had a 12-week survival time and differentiated into oligodendrocytes and neurons, conferring neuroprotection by decreasing microglial activation and stimulating neurogenesis [Quite recently, intra-parenchymal injection of human neuronal progenitor cells (hNPCs) in a model of stroke in pig, was associated with better functional recovery. The animals were divided into a treatment and a control group; the treatment group received intra-parenchymal stem cell injections on day 5 post stroke while the control group received injections of phosphate buffer saline (PBS). Animals were followed on weeks 1–12 post injection. Animals in the treatment group showed a quicker functional recovery and better preservation of appetite and posture as compared to the control group .MSCs exposed to extracts from ischemic brains seem to secrete a wide range of neuroprotective proteins including brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) . TherefoEndothelial progenitor cells (EPCs) along with the chemokine CXCL12 have the advantages of both promoting angiogenesis and would promote neurogenesis brain in part by secreting trophic factors. Much like G-CSF, the chemokine CXCL12 has been shown to attract EPCs, neural progenitor cells, and oligodendrocyte progenitor cells (OPCs) to the injury site. By using a lentivirus overexpressing CXCL12 in human endothelial progenitor cells (hEPC-cxcl12), it was reported on synergistic effects of the combination therapy over monotherapies by stimulating angiogenesis, neurogenesis, and migration of oligodendrocyte progenitor cells to the lesion site. Increased vessel density and preservation of the myelin sheath integrity in the treatment groups were also reported .In a recent study, the authors hypothesized that restoration of the interhemispheric connectivity of the motor cortex might be an additional functional recovery mechanism. Using imaging methods (MRI diffusion tensor imaging) and neuroanatomical tracing techniques by injecting a GFP-expressing adeno-associated (AAV) virus, an anatomical restoration of interhemispheric connections through the corpus callosum could be demonstrated following intravenous MSC administration of autologous cells in a rat model of stroke (MCAo). Moreover, the degree of connectivity was higher in the MSC treated group than in the vehicle-treated group. Accordingly, both the thickness of corpus callosum and synaptic puncta in the contralateral motor cortex were greater in the MSC treated group than in the control group .6 ASC as compared to controls or the group treated with 5 injections of 104 ASCs. At 9 days after MCAo, injection of 3 × 105 ASCs resulted in a reduced infarct volume. It was concluded that intra-arterial administration is an efficient method, but its usefulness greatly depends on the proper dose of delivered stem cells, as this factor has a strong influence on the migration rate and infarct volume, with better outcomes at the lower doses [Recently, adipose tissue-derived mesenchymal stem cells (ADMSCs) have demonstrated their therapeutic potential in stroke models by improving functional recovery, with an increase in markers related to brain repair in animal models of stroke. Thus, adipose stem cells (ASCs) labelled with superparamagnetic iron oxide particles (VSOP), which allow cell tracking by MRI, were given intra-arterially to a rat model of stroke, and an MRI analysis was performed at 48 h and 9 days after MCAo. An increased ASCs signal strength in the affected hemisphere at 48 h post MCAo was indicative of an active migration of ASCs to the infarction site which was proportional with the number of injected cells. However, the infarct volume was increased after injection of 1 × 10er doses .Quite recently, an adipose tissue stem cell line RESSTORE01, has been used to improve functional recovery in adult male Sprague–Dawley rats subjected to permanent occlusion of the MCA. After the intravenous infusion of vehicle or 2 × 106 ADMSCs behavioural recovery was assessed by Rogers’ functional evaluation scale, which assess reflexes, sensory responses, and simple motor functions and performance in the cylinder and sticky label tests during a 42-day behavioural follow-up. Tissue recovery was assessed using histological markers of angiogenesis (RECA-1), gliosis (GFAP), and glial scar formation .Bone marrow stromal cells (BMSC) give rise to various non-hematopoietic cells including osteoblasts and adipocytes and are normally used for treating bone-related abnormalities. Hypoxia is essential in the mobilization and homing of MSCs, by its ability to induce stromal cell-derived factor-1 expression along with its receptor, CXCR4. For this reason, transplantation of BMSC holds the promise for ischemic stroke therapy. Within the hypoxic environment, BMSC secrete vascular endothelial growth factor and interleukin-6, thereby promoting wound healing and diabetic fracture healing. A recent study investigated the hypothesis that hypoxic preconditioning could increase the effectiveness of BMSC transplantation in a rat model of ischemic stroke. Post-stroke rats received normally cultured bone marrow stromal cells (N-BMSCs), BMSCs exposed to hypoxia (H-BMSCs), or DMEM cell culture medium (control) at 24 h after cerebral ischemia. Compared to the control group, the H-BMSCs-transplanted group exhibited significantly improved functional recovery and diminished infarct volumes over the N-BMSCs- and DMEM-transplanted groups. The increased survival and differentiation of transplanted cells and improved neurological function involved the inhibition of caspase-3 activation and an increased expression of HIF-1α, which is indispensable for angiogenesis and neurogenesis ,24.6) of such stem cells alone or in combination with the chemoattractant, Granulocyte Colony Stimulating Factor (G-CSF) at 3 hrs after cerebral ischemia in aged rats led to some improvement of some behavioural indices of recuperation at 48 days after the event [It is unlikely that monotherapies will address the tissue and functional recovery after cerebral ischemia. Rather, a monotherapy might be helpful at a certain stage of post-stroke recovery. Therefore, it is reasonable that combination therapies could be more efficacious at multiple time points of tissue and functional recovery after cerebral ischemia. Our group was interested in the potentially beneficial effect of combination therapies using bone marrow derived mesenchymal stem cells (BM-MSCs) and bone marrow derived mononuclear cells (BM-MNCs). Intrajugular administration of a single dose  with enriched environment was superior to the monotherapy alone .A recent study evaluated the combined effect of bone marrow stromal cells (BMSC) administration with exercise and administration of thyroid hormones in a model of middle cerebral artery occlusion (MCAO) in young mice. The stem cells were injected into the right ventricle at 24 h after MCAO, followed by daily T3 injections and physical training for 6 days. Animals were perfused on day 7 after stroke. Functional recovery, infarct volume, and the number of BrdU and TUNEL-positive cells were evaluated. All combination therapies led to a decrease in the infarct volume: BMSCs and exercise by 51%, BMSCs and T3 by 26%, BMSCs + T3 and exercise by 70%. A significant decrease in the infarct volume (by 26%) was also seen in the BMCS monotherapy as well as the exercise alone group, by 44%. A functional improvement on neurobehavioral tests was observed in the exercise groups, BMSCs and Physical Exercise and the full combination, BMSC + T3 and Physical Exercise. Combination therapy also led to a decrease in the number of TUNEL cells, which suggests a decrease in apoptosis, and an increase in the number of BrdU-positive cells . An overA prospective study involving a small cohort of 20 stroke patients, divided into 2 groups, intervention and control, assessed the safety and clinical outcome after treatment with autologous stem cells in the subacute stage of ischemic stroke. Autologous stem cells were given intra-arterially to the ipsilateral MCA of subacute ischemic stroke patients. Patients in the intervention group received intra-arterial stem cell infusion in the ipsilateral middle cerebral artery on days 8–15 post-stroke. Final analysis at 6 months was performed for primary (safety) and secondary outcomes (efficacy). No complications were reported after intra-arterial injection, and procedure-related mortality was not an issue for both groups. The clinical response using the modified Rankin scale was superior for patients in the treatment group. Intra-arterial infusion of stem cells can be carried out safely in the subacute stage of ischemic stroke. Improved clinical outcomes were observed with intra-arterial stem cell therapy .Another prospective pilot trial involved a small group of 12 patients with a history of stroke (from 3 months to 2 years). Patients were allocated to two groups, those who received intravenous autologous ex vivo cultured mesenchymal stem cells (MSC group) or those who did not (control group), and all were followed for four years from the day of cell transplantation. Patients were followed for 4 years, with regular assessment of muscle strength on the upper limbs. The transplanted cells were well tolerated. There were no cell-related side effects and all patients survived. Moreover, a modified Barthel Index showed a statistically significant improvement at 156 and 208 weeks of transplantation in the MSC group as compared to controls. The results of the study indicated some potential for the use of autologous mesenchymal stem cells for stroke patients to improve functional recovery .Adult pluripotent-like olfactory stem cells (APOSCs) have the ability of self-renewal and differentiate into all three germinal cell lines. In a small clinical trial autologous APOSCs were injected to six patients under stereotactic guidance and were followed up to 3 mo. Patients in the treatment group seemed to present better clinical outcomes than those with low expression of CBX7 in their autologous APOSCs. Overall, despite a low differentiation efficiency into neuronal cells, these findings indicate the safety and feasibility of autologous APOSC implantation in the treatment of stroke .During the early stages of cell therapy, it was hypothesised that MSCs contribute to tissue regeneration by replacement of lost cells. However, MSCs cannot survive for long at the site of transplantation and do not generate functional neurons to replace the lost ones. Therefore, focus has shifted to their paracrine actions or the so called the ‘‘bystander’’ effect. A common hypothesis is that the beneficial effects of transplanted MSCs are mediated either by “chaperones” or through paracrine mechanisms . Cell-baOther mechanisms include the preservation of the blood brain barrier (BBB) integrity. Ischemic stroke leads to increased MMP-9  levels, causing the breakdown the blood brain barrier (BBB). The hypothesis of this study was that mesenchymal stem cells protect the blood-brain barrier by inhibiting MMP-9 through a mechanism that involves intercellular adhesion molecule-1 (ICAM-1). Treatment of post-stroke male adult IRC mice with 2 × 105 MSCs given by intracranial injection, diminished the infarct volume and led to an improvement in behavioural indices of functional recovery. At molecular levels, the authors reported reduced levels of inflammatory markers IL-1β, IL-6, and TNF-α in MSCs-treated animals. Similarly, a reduced MMP-9 activity in polymorphonuclear cells in treated mice was associated with resistance to IgG leakages and loss of tight junction proteins .Administration of stem cells at the lesion site is not necessary to achieve a therapeutic effect. Beneficial effects on tissue and behavioural recovery were reported after the administration of conditioned medium obtained from cultured stem cells. Further, hypoxic pre-conditioning enhances the production of factors, including IGF-1, BDNF, GDNF, VEGF, SDF-1, and HGF, that promote survival and plasticity ,36,37,38For example, to assess whether secreted factors can mimic the neuroprotective and restorative effects of NPC, male C57BL6 mice were exposed to focal cerebral ischaemia and received intravenous treatment with a conditioned medium (CM) containing biologically active substances derived from cell cultures of neuronal progenitor cells from the subventricular zone up to 12 h after focal cerebral ischemia. The reduction in infarct volume was dose-dependent, as was brain leukocyte infiltration at 48 h post-administration, when the treatment. Neuroprotection persisted in the post-acute phase of stroke, producing an improved neurological recovery maintained throughout the observation period of 28 days. Long-term neuroprotection was associated with increased concentration of glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF) at 28 days, resulting in increased neurogenesis and angiogenesis. The observation that conditioned medium derived from the neural progenitor cell induce neuroprotection and neurological recovery suggests that cell transplantation can be dispensable by administering secreted factors .Extracellular vesicles (EVs) are 40–1000 nm nanosized vesicles that are released from many cell types into the extracellular space. Such vesicles are widely distributed in various body fluids and can readily cross body barriers, such as the blood brain barrier (BBB). The biogenesis of EV production starts from the plasma membrane invagination, which then furthers form inward buds forming so-called multivesicular bodies. During this process, several cellar containing molecules, such as mRNAs, miRNAs, proteins and metabolites are selectively packed into the vesicles. Thus, EVs can not only transfer biological messages between the cells in the body but also represent the physiological status of their parent cells, which makes EVs potential biomarker candidates . Indeed,Remodelling of ischemic brain tissue involves interactions between neurons, glial and microvascular cells that create a microenvironment, in which neurological recovery may ensue . The remExosomes are released by virtually all cell types and have previously been shown to have restorative actions in the ischemic brain promoting neurological recovery and brain plasticity. In a head-to-head study, we have demonstrated that EVs obtained from mesenchymal stem cells (MSCs), which were intravenously administered 24 h after intraluminal middle cerebral artery occlusion (MCAO), promoted neurological recovery, long-term neuronal survival, angiogenesis, and neurogenesis very similar to MSCs, at the same time restoring post-ischemic immunodepression in peripheral blood . These dRecent research has focused on the roles of exosomes, acting as bidirectional cargo between neurons, glia, vascular and perivascular cells, in brain homeostasis and plasticity ,47. The Brain recovery after an overt injury requires energy supply normally provided by the mitochondria. However, following brain injury, most of the cellular structures are destroyed, including the mitochondria. Therefore, one strategy to restore the energy supply is mitochondria transplantation which is supported by the ability of mitochondria to undergo mitochondrial fission and remodelling in response to higher metabolic demands. Therefore, mitochondria transfer is essential for a successful regenerative attempt of the damaged brain cellular structures . In partThe mitochondrial transfer is not unidirectional. Available data suggest that cues released by the damaged cells may trigger a bidirectional mitochondrial and mtDNA transfer between transplanted MSCs and the recipient’s cells. Thus, it could be that the transfer of mtDNA from the damaged cells to the transplanted MSCs may trigger mitophagy and as a consequence, more mitochondria are available to rescue the injured cells by providing more energy and a robust immune response within the damaged tissue ,50. The therapeutic potential of mitochondria transfer has been assessed in an experimental model of stroke. One study investigated the effect of the transfer of mitochondria from multipotent mesenchymal stem cells (MMSC) to astrocytes and neuron-like PC12 pheochromocytoma cells after lentivirus-driven increased expression of Miro1 in MMSC. Miro1 , a calcium-sensitive adaptor protein which organize mitochondrial movement along microtubules inside the cells, was given intravenously to MCAo rats. The most important result of the transfer of mitochondria from the MMSC was the restoration of neural functions observed in vivo. More specifically, behavioural data analysis suggests a reduction of the neurological deficit of the upper limbs at 14 days post stroke in animals receiving intravenous injections of multipotent mesenchymal stem cells. Thus, it was concluded that the transfer of mitochondria is a very important component of the neuroprotective effect of MMSC after experimental ischemic stroke .The field of stem cell therapy for cerebral ischemia has made significant progress in the last five years. Combination therapies in animal models include a mix of two or more therapeutic factors consisting of bone marrow stromal cells, exercise and thyroid hormones, endothelial progenitor cells and lentiviruses overexpressing the chemokine CXCL12. Mechanisms underlying the beneficial effects of transplanted cells include the “bystander” effects, paracrine mechanisms, or extracellular vesicles-mediated restorative effects. Mitochondria transfer also appears to be a powerful strategy for regenerative processes. We believe that the most promising stem cells for stroke therapy in animal models are the adipose tissue-derived mesenchymal stem cells (ADMSCs) which have demonstrated their therapeutic potential in stroke models by improving functional recovery, with an increase in markers related to brain repair.We should not forget that there are also risks associated with the transplantation of stem cells into a lesioned brain . Studies


Staphylococcus aureus is a major human pathogen, and a leading cause of soft tissue and blood stream infections. One of the causes of its success as a pathogen is the peculiar array of immune evasion factors through which the bacterium avoids host defenses, where the staphylococcal protein A (SpA) plays a major role thanks to its IgG binding activities. Moreover, SpA has recently been proposed as a promising vaccine antigen. In this study, we evaluated the expression of SpA in a collection of staphylococcal strains, about 7% of which did not express SpA (SpA- strains), despite the presence of the gene. By a comparative genomic analysis, we identified that a mutation in the spa 5′ UTR sequence affecting the RBS is responsible for the loss of SpA in a subset of SpA- strains. Using a high-throughput qRT-PCR approach on a selected panel of virulence-related genes, we identified that the SpA- phenotype is associated with lower spa transcript levels and increased expression and production of capsule as well as other changes in the transcription of several key virulence factors. Our data suggest that the SpA- phenotype has occurred in geographically distinct strains through different molecular mechanisms including both mutation, leading likely to translation alterations, and transcriptional deregulation. Furthermore, we provide evidence that SpA- strains are highly susceptible to phagocytic uptake mediated by anti-capsule antibodies. These data suggest that S. aureus may alter its virulence factor expression pattern as an adaptation to the host or environment. Vaccination strategies targeting both SpA and capsule could therefore result in broader coverage against staphylococcal isolates than SpA alone. Staphylococcus aureus is a Gram-positive bacterium that colonizes the human nares and skin  are more likely to be expressed in the stationary phase . We investigated whether the absence of SpA is associated with genetic polymorphisms common to the SpA- subset of strains, and whether other changes in the virulence gene expression profile are associated with the SpA- phenotype, highlighting a profound interconnectedness of SpA and other immune evasion factors. Understanding how different staphylococcal immune evasion mechanisms interact and how their relative expression levels are controlled will give a deeper insight on staphylococcal pathogenesis, and support to the design of new therapies against this important human pathogen.Considering the central role of SpA in staphylococcal pathogenesis and the rising interest in SpA as potential vaccine antigen, in the present study we aimed to understand the prevalence of SpA expression in a large collection of staphylococcal isolates. We identified a subset of strains carrying the gene but lacking expression to detectable levels of SpA . For SpA detection, the membrane was incubated with SpA-specific chicken antibodies conjugated with biotin and revealed with HRP-conjugated streptavidin. Alternatively, SpA was detected as previously reported  according to manufacturer’s instructions. Sequencing was performed with HiSeq 2500 Sequencing System from Illumina and Paired-Ends. Paired reads were assembled using CLC genomic work bench (Qiagen). Comparative analysis was performed using BRIGG software and N315 genome as reference. Alignments of specific DNA regions were performed with MUSCLE algorithm in Geneious software (Biomatters).spa gene were fused to mCherry in a pOS1 plasmid backbone . The bacterial pellet was resuspended in 1ml of Trizol reagent (Thermo Fisher Scientific) and lysed in a FastPrep®-24 homogenizer . The RNA was purified using the PureLink kit (Ambion) applying an on-column DNase digestion step using the RNase-free DNase kit (QIAgen) according to the manufacturer’s instructions. Residual DNA was removed by a second DNase treatment using RQ1 DNase (Promega) followed by a second RNA purification using the PureLink kit. cDNA synthesis was performed with SuperScript First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific), using random hexamer primer for reverse transcription (RT).Samples for RNA extraction were collected from gyrB. gyrB was used as housekeeping reference gene, and each assay was normalized to the mean of the gyrB replicates for each Chip. For the transcription kinetic profiles, all the samples were normalized to the early stationary phase using the ΔΔct method. Clusterization of genes or samples was performed using the Multiple array viewer (Mev) application with Hierarchical Clustering, Pearson correlation as distance metric.The virulence factors transcript profile was assessed using the high-throughput qRT-PCR system BIOMARK HD (Fluidigm), with 84 specific TaqMan assays . Two Chi+ and SpA- strains was performed using GraphPad Prism 7 software as follows: the difference between mean Δct for the SpA+ and SpA- strains was calculated for each gene at each time point; two-way ANOVA was used to establish the significance of the difference, comparing values at same time points, and correcting for multiple comparisons by Sidak correction.Comparative transcriptional analysis between SpAgyrB. The statistically significant difference between SpA+ and SpA- strains was determined by unpaired t-test with Welch’s correction using GraphPad Prism 7 software.qRT-PCR was done using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen–Life Technologies) and ROX as internal control on a STRATAGEN Mx3000P QPCR system. Final data were analyzed with the ΔΔct method, normalizing samples to the expression levels of Staphylococcus aureus isolates were grown to OD600 = 12.0, and then pelleted. The bacterial pellets were resuspended in 0.5% SDS (v/v), 5 mM DTT, 100 mM Tris (pH = 8) and treated with proteinase K for 1h at 45°C. Serial dilutions were loaded onto a nitrocellulose membrane. The membrane was blocked with PBS-Milk 10% (w/v) Tween20 0.05% (v/v) and then incubated with CP5-specific rabbit antiserum, followed by goat anti-rabbit HRP. The blots were developed using the Pierce ECL Western Blotting substrate (Thermo Fisher Scientific), the images were acquired using Chemidoc (BIO-RAD) and dot intensities were defined using Imagelab (BIO-RAD). The statistically significant difference between SpA+ and SpA- strains dot intensities was determined by unpaired t-test with Welch’s correction using GraphPad Prism 7 software.7 stained bacteria were incubated with rabbit anti capsule serum, 1% (v/v) guinea pig complement and 106 HL60 differentiated cells for 30 min in the dark. Samples were washed in PBS and fixed in 1% (v/v) paraformaldehyde for 20 min, then washed in PBS and incubated in PBS 1% (w/v) BSA and 1:100 anti-S. aureus polyclonal antibodies (Thermo Fisher) for 1 h. After a wash in PBS 1% (w/v) BSA, the samples were incubated in Rhodamine (TRITC) F(ab′)2 Fragment Goat Anti-Rabbit IgG (Jackson Immunoresearch), for 30 min. After a wash in PBS 1% (w/v) BSA and a second wash in PBS, the samples were resuspended in PBS and read with flow cytometry with BD LSRII (BD Biosciences). Neutrophils with only internalized bacteria were gated selecting for FM-64fx positive and TRITC negative events. For each sample, 10,000 events were analyzed using FlowJo software. In each sample, the delta geometric mean of the positive and negative populations was multiplied by the percentage of positive neutrophils, giving an estimate of the bacteria internalized for each sample. The results were analyzed using a two-way ANOVA implemented in GraphPad prism 7 software, comparing each condition -S -C within each strain to the control, and applying Sidak correction for multiple comparisons.Phagocytic uptake was measured similarly to the protocol already described by spa gene and its expression in a collection of S. aureus strains. The collection was composed of 133 strains, including commensal and clinical isolates from diverse niches and different geographic origin as well as some lab strains, and categorized by Multilocus sequence typing (MLST) , nine of them resulted negative in Western blot analysis (-) strains include members of clonal complex (CC) 5: Mu50  . The colg (MLST) , of whicanalysis . The SpA 5: Mu50 , four st 5: Mu50 . Figure ain N315 , four stain N315 . Their gain N315 . Given t- phenotype in a subset of strains, we investigated the possible reasons for the loss of SpA expression. In particular, we investigated if at the level of genome sequence, we could identify a genetic basis for the SpA- phenotype that could be shared by the different SpA- strains. For this purpose, we analyzed the genomes of the eight United States strains  showed a deletion of eight nucleotides, six nucleotides upstream of the spa start codon  maintained a similar profile of expression of the SpA+ strains, even though at lower steady state levels in comparison with SpA+ strains. Strains MB01 (that harbors the RBS mutation) and S27 maintained this profile only partially, as reflected by the flatter curve. Mu50 shows a completely flat trend, with similar transcript levels across the growth. This means that some of the SpA- strains lost spa regulation during growth and expressed lower steady state spa transcripts.Our genomic analysis showed that the genetic bases for the SpAgulation , therefogulation  shows thand fnbB   and the first gene in the capsular polysaccharide type 5 specific region (cap5H), respectively  produced no detectable capsule at the time point tested, while all SpA- strains expressed capsule at detectable levels.Having identified significant transcriptional differences in the United States subset of strains, we expanded the subset to include the remaining SpA strains . To quanth point . The ana- and SpA+ strains produce different amounts of capsule, we explored whether the capsule produced by the SpA- strains was enough to elicit neutrophil uptake in the presence of capsule-specific antibodies. N315 and Mu50 were used as SpA- and SpA+ reference strains and a phagocytosis experiment was set up using capsule-specific rabbit antisera as described in Section “Materials and Methods.” Phagocytic uptake was visualized by confocal microscopy and quantified by Flow cytometry. - strains express higher quantities of capsule, and that this capsule amount is enough to elicit phagocytosis in presence of capsule specific antibodies.After we gained evidence of the fact that SpA-). The molecular mechanisms that determine the SpA- phenotypes were found to be different among the SpA- strains and involve both RNA translation in the three SpA- strains that lacked a functional spa RBS, and likely alterations in the transcriptional network. The different kinetic profiles of the spa transcript in the SpA- strains, showing lower steady state levels and lower or completely absent growth phase regulation, indicate that there are several independent factors influencing spa transcription and causing the loss of SpA expression in these strains.This study aimed to investigate the expression of SpA, both virulence factor and recently proposed vaccine antigen, and its interplay with other virulence determinants. Despite the important role of SpA in staphylococcal pathogenesis, the screening of a library of strains allowed us to identify a subset of strains lacking SpA expression  are known to effect the same regulatory activity on those three genes, confirming that they are part of a similar regulatory network. Conversely, the analysis of the factors influencing spa and capsule transcription showed that several regulators  determine opposite effects on these two virulence factors, indicating that multiple systems closely control inverse regulation to balance the expression of capsule and SpA. Alterations in any one of these factors involved in maintaining this balance would lead to the inverse shift that we observed between the SpA- strains.The regulators shared by - phenotype has occurred repeatedly through diverse mechanisms in geographically distinct locations, suggests that the loss of SpA expression may represent the response to a selective pressure under specific conditions. Moreover, multiple examples have been reported of adaptive responses where SpA and capsule expression are inversely altered. For example, lower SpA expression and higher capsule production are common phenotypic features in Vancomycin Intermediate S. aureus (VISA) strains , the overexpression of capsule in strains lacking SpA is a feature shared with that of the VISA phenotype.This inverse regulation could be necessary due to the common localization of SpA and capsule on the cell surface and the somewhat redundant nature of their functions, especially in relation to evading opsonophagocytosis.  strains . VISA st strains . Through strains . While tspa RNA in the SCV clones compared to parental strains is evident in their dataset . The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objectives: Patients with systemic lupus erythematosus (SLE) are characterized by increased cardiovascular disease (CVD) risk as well as heightened rates of psychological distress. Since a link between psychological issues and CV morbidity has been previously suggested, the influence of psychological burden on subclinical atherosclerosis in SLE patients was investigated.Methods: 71 SLE patients were assessed for the presence of subclinical atherosclerosis—defined either as carotid and/or femoral plaque formation or arterial wall thickening [Intima Media Thickness (IMT) levels > 0.90 mm by Doppler ultrasound]; personality traits, anxiety and depression, sleeping habits and fatigue levels were also evaluated by specific questionnaires including Eysenck Personality Questionnaire Scale, State-Trait Anxiety Inventory (STAI), Zung Depression Scale, Athens Insomnia Scale and Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F). Disease related clinical and laboratory features and traditional risk factors for atherosclerosis were documented. Univariate and multivariate analysis were performed.Results: SLE patients with arterial wall thickening displayed higher STAI anxiety scores  compared to those without . In a multivariate model, trait anxiety and extraversion personality scores were found to be independently associated with arterial wall thickening and plaque formation, respectively , following adjustment for potential confounders. No other associations were detected.Conclusions: Anxiety and extraversion personality traits have been independently associated with subclinical atherosclerosis in lupus patients, implying psychoneuroimmunological interactions as contributors in SLE related atherosclerosis. Cardiovascular disease (CVD) is a well-established comorbidity of systemic lupus erythematosus (SLE), severely affecting both prognosis and mortality. Traditional CVD risk factors are present or overrepresented in SLE patients. However, the high rates of ischemic events observed are not entirely explained by Framingham scores, implemented to calculate the 10-year CV risk of an individual based on traditional CV risk factors .Depression and anxiety have been long recognized as risk factors to CVD in the general population. Chronic activation of the hypothalamic-pituitary axis, imbalance between sympathetic and parasympathetic effects on cardiac function and promotion of a pro- inflammatory milieu with higher C-reactive protein (CRP) levels as a result of imbalance between proinflammatory  and anti-inflammatory cytokines IL-10) have been all proposed to underlie this link –6. Moreo have beeIn addition, exposure to major life stressing events during childhood has been also linked to CVD, mortality and autoimmune diseases in adult life . Other pper se appears to influence anxiety; active disease in SLE patients [higher Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores] has been independently associated with anxiety, irrespectively of the presence of depression  scores –19.Thus, in view of the associations observed between psychological stressors and atherogenesis in general populations, we aimed to explore whether psychopathological issues, personality traits and sleep habits are associated with subclinical atherosclerosis in a cohort of SLE patients.Seventy-one consecutive SLE patients according to the American College of Rheumatology Classification Criteria were enrolled in the study. All patients were followed at the Department of Rheumatology, General Hospital of Athens “G.Gennimatas” . Exclusion criteria were pregnancy, age <18 years old and renal dysfunction . The study has been approved by the Ethics Committee of the General Hospital of Athens “G.Gennimatas.” All patients provided informed consent prior to their entry to the study. The Institutional Review Board waived the requirement for written, informed consent.All patients underwent evaluation for subclinical atherosclerosis [ultrasound measurement of intima-media thickness scores (IMT) and detection of carotid and/or femoral (C/F) plaque] at the General Hospital of Athens “G.Gennimatas.” Arterial wall thickening was defined as IMT score>0.90 mm. Demographic data, clinical features, therapeutic regimens and classical risk factors for atherosclerosis were recorded in detail, as previously described .Psychological features were assessed using self-administered psychometric questionnaires: (i) Zung Depression Scale, a validated tool for assessing depressive illness  ; (ii) Stp < 0.05 for univariate analyses and of < 0.1 for multivariate analyses, respectively, were considered statistically significant. Data were stored in the SPSS statistical package.Two-sided Fisher's exact/chi-square and Mann-Whitney tests were implemented to compare qualitative and quantitative characteristics, respectively, between patients with and without arterial wall thickening or plaque. Correlations between IMT and anxiety scores were detected by Spearman's rho test. Univariate analyses were performed to test whether the presence of plaque formation or arterial wall thickening [high IMT (>0.9 mm)] was associated with traditional risk factors for CVD, as well as disease-related and psychological/personality features. Multivariate models including variables found to be significant in univariate analysis were constructed to detect independent associations between subclinical atherosclerosis  and psychometric variables. A Plaque formation and arterial thickening  were detected in 40/71 (56.3%) and 26/71 (36.6%) of SLE patients, respectively. As shown in Table We next wished to evaluate whether psychological/personality features were associated with subclinical atherosclerosis in patients with SLE. While in univariate analysis, no association between plaque formation and various psychometric scores was detected Figure , multivap-value: 0.03 and 49.2 ± 4.4 vs. 45.7 ± 6.8, p-value: 0.009, respectively). Following adjustment for variables found to be significant in univariate analysis . No associations were detected between high IMT values and other psychometric parameters studied .As shown in Figure is Table , arteriaThe aim of the present study was to investigate whether psychological and personality features contribute to the development of subclinical atherosclerosis in the setting of lupus. To the best of our knowledge this is the first study in SLE patients to detect an independent association between markers of subclinical atherosclerosis and personality characteristics, with anxiety playing an aggravating while extraversion a protective role.Among the psychological and personality features under investigation, only depression has been linked to subclinical atherosclerosis in lupus populations to date. Depression has been associated with a 2-fold increase in coronary artery calcification risk; notably, this association was influenced by body mass index (BMI) . A subseThe lack of association with depression in the present study could be attributed to differences in the methodology implemented, as well as environmental influences. It should be noted that in all studies- including ours- the diagnosis of depression was based on single-assessment questionnaires rather than psychiatric evaluation or taking into consideration prolonged time periods.In general population, anxiety disorders have been long related to CVD development and unfavorable CVD related prognosis, being a more potent predictor for heart disease than depression. Patients with acute myocardial infarction exhibit more often type A personality behavior , which was found to be linked to higher degrees of anxiety as state and as a personality trait .Mechanisms of atherogenesis are poorly understood in anxiety disorders; daily stressors and prolonged worry (anxious apprehension) have been shown to influence the activity of the autonomic nervous system (increasing sympathetic and decreasing parasympathetic activity) leading to high heart rate and low heart rate variability  . FurtherThe finding that personality traits such as anxiety or extraversion are associated with subclinical atherosclerosis in lupus patients is in accord with general population studies. Since these factors can be modified by cognitive-behavioral stress reducing techniques, medication or psychotherapy sessions, these interventions could provide a complementary tool in the management of lupus related CV risk.There are several limitations to our study. The lack of a healthy control group and the relatively small number of SLE patients enrolled warrant the confirmation of our results in larger studies. Moreover, the cross sectional design of the current study does not allow the identification of direct causal relationship between psychological burden and atherogenesis in lupus patients. On the other hand, the strengths of this study derive from the concomitant analysis of a wide range of parameters related to atherosclerosis including traditional CVD risk factors and SLE related factors, together with psychological and personality characteristics of the patients enrolled using validated psychometric tools.In conclusion, in the present report an independent link between personality traits and CV risk among SLE individuals is revealed, pointing out the importance of a holistic approach in the management of SLE related CV comorbidities.MG and DT recorded clinical, laboratory and psychometric data from all study participants and drafted the manuscript. EA performed all ultrasonographic measurements and revised the manuscript. CM conceived, designed and coordinated the study, performed the statistical analysis and revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.