Treatment of pulmonary recurrence from colorectal cancer involving the main bronchus usually entails palliation using interventional bronchoscopy, because the prognosis is generally very poor. Surgical experience has clarified that in this setting pneumonectomy should only be performed in carefully selected patients showing favorable prognostic profiles (defined by low carcinoembryonic antigen serum levels pre-thoracotomy), solitary and completely resectable pulmonary metastasis, and long disease-free intervals. In the few long-term survivors after pneumonectomy for late-recurrent colorectal cancer, the disease has a relatively indolent metastatic course and genetic and epigenetic profiling may provide further insight regarding tumor evolution.KRAS, NRAS, BRAF and PIK3CA mutations, a microsatellite stable phenotype, and no tumor protein p53 alterations or recurrent copy number alterations on chromosome 8. High genetic concordances between the paired primary tumor and metastases suggest that the key tumor biological traits remained relatively conserved in the three metastatic sites. Minor differences in gene specific hypermethylation were observed between the primary tumor and lung and nodal metastases. These differences suggest that epigenetic mechanisms may be causally involved in the microenvironmental regulation of cancer metastasis.We describe a rare case of late hilar-endobronchial and lymph nodal recurrence of rectal cancer, sequential to hepatic metastasectomy, that we successfully treated with pneumonectomy and chemotherapy ; the patient achieved 7-year relapse-free survival after lung metastasectomy and 24-year overall survival after primary rectal cancer resection. To our knowledge, this is the longest survival reported after sequential liver resection and pneumonectomy for recurrent colorectal cancer. In our case the primary rectal cancer and its recurrences showed identical immunohistochemical patterns. The primary rectal cancer and the matched metastases  demonstrated no The exceptionally long survival of the patient in our case study involving favorable clinical features was related to an excellent response to surgery and adjuvant chemotherapy; however, genetic or epigenetic factors that remain unidentified cannot be excluded as contributory factors. Our findings support the concept of a common clonal origin of the primary cancer and synchronous and late metastases, and suggest that aberrant DNA methylation may regulate tumor dormancy mechanisms. The occurrence of sequential or concurrent liver and lung metastases after resection of colorectal cancer (CRC), with the exception of carefully selected patients, has traditionally been associated with poor long-term outcome –3. For lHere, we present a rare case of solitary hilar recurrence of rectal cancer with endobronchial extension, diagnosed at 17 years after primary rectal tumor resection and hepatic metastasectomy; the patient was treated with pneumonectomy and chemotherapy and has achieved an overall survival time of 24 years. In this patient, we performed a comprehensive study of the histologic, immunohistochemical, genetic and epigenetic profiles of the primary tumor and of its metastases to the liver, lung and lymph nodes.WT1, DAPK1, CHFR, PAX5 and GATA5, were found in the lung and in supraclavicular lymph node metastases . This analysis demonstrated no gene mutation in the primary rectal adenocarcinoma, and interestingly this genetic profile remained relatively well preserved in the three metastatic sites.In 1990, a 40-year-old male underwent anterior resection of rectal adenocarcinoma  serum level: 35 ng/mL), that was diagnosed synchronously with a 9-cm metastasis in liver segments V-VIII. After postoperative 5-fluorouracil based chemotherapy, the CEA serum level was slightly decreased (22 ng/mL); liver metastasis was markedly downsized and was radically resected. Follow-up revealed no evidence of residual disease until 2002; follow-up was then intentionally interrupted because the patient was considered to be free of disease. On March 2007, the patient developed dyspnea and cough, and a chest computed tomography (CT) scan revealed a 5-cm right hilar mass bulging into the main bronchus and infiltrating the upper lobe Figs. . BronchoThe decision regarding surgical treatment is critical when an isolated lung metastasis can only be radically resected using pneumonectomy, as was true in the present case. Following sequential surgical resection of the hepatic and pulmonary recurrences from CRC, the following factors are predictive of long-term survival and can help in addressing surgical strategies: 1) low CEA serum level pre-thoracotomy ; 2) solip53 (TP53) or recurrent CNAs on chromosome 8 that are the most frequent events in highly aggressive cancers exhibiting copy number genomic instability [APC and CDH13 methylation, suggesting an aberrant activation of the wingless signaling pathway that is considered a nearly ubiquitous event in CRC [Extensive genetic and epigenetic profiling of the primary rectal adenocarcinoma allowed the characterization of the key biological traits of the tumor and comparison with the recent results of CRC molecular characterization . Accorditability . FinallyComparison between the primary cancer and the matched metastases demonstrated high genetic concordances and minor differences among samples; this was in agreement with the current literature, supporting the concept that concordance is dominant during the metastatic process and reflects the common clonal origin of primary cancer and metastases . Many stKRAS, NRAS, BRAF and PIK3CA mutations, a microsatellite stable phenotype; in addition, there were no TP53 alterations and no recurrent CNAs on chromosome 8. Our findings support the concept of the common clonal origin of the primary cancer and of the synchronous and late metastases, suggesting that the key tumor biological traits remained quite conserved and that epigenetic mechanisms may be causally involved in the microenvironmental regulation of cancer metastasis. The exceptionally long survival time of the current case was probably related to an excellent response to surgery and adjuvant chemotherapy; however, it might also be associated with yet to be identified genetic or epigenetic factors involved in the change from dormancy to the activation of metastasis.Cancer survival is in part related to the natural biology of slow-growing tumors. However, here we presented the case of a patient with pulmonary and lymph nodal metastases that occurred many years after primary tumor removal; these metastases exhibited morphological features of aggressive growth, including marked atypia, high mitotic index and tumor necrosis. The extensive genetic and epigenetic profiling of the primary rectal cancer and of the matched metastases demonstrated the absence of Our data are preliminary and need to be confirmed, but they suggest the importance of future genome-scale analyses of methylomes and microRNA expression in comparing primary tumors and matched metastases.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


MIXTA from Antirrhinum majus. Various MML genes have been shown to be important regulators of epidermal cell differentiation in different plant species, including specifying cell shape in petals, vegetative trichome initiation and branching and seed fiber initiation , the silencing or over-expression of two MML genes (GhMYB25 and GhMYB25Like) expressed predominantly in the ovule epidermis during fiber initiation affect the initiation or timing of expansion of fiber initials , an extant species most closely related to the D-genome progenitor of tetraploid cotton, may help draw a more complete picture about the evolution of the MML gene subgroup in cotton and their apparent expansion and recruitment to specialized functions in epidermal seed fiber development.MYBMIXTA-like (MML) transcription factors form the subgroup 9 of R2R3-MYBs  of subgroup 15, involved in trichome and root hair initiation and development, that are often suggested as the likely models for regulators of seed fiber development in cotton. Subgroup 15 in Gr appears to have only one member, Gorai.012G061800/GrMYB109  and GhMYB25Like  as well as eight other novel MML genes, each with a pair of very close homologs in the A- and D-genomes of tetraploid Gh  and three genes , respectively, in tandem arrangements   are also expressed in leaves and/or cotyledons which are rich in either hair or glandular trichomes that are structurally related to seed fibers and share some common regulators like GhHD-1 , two in inner-integument  and two in the nucellus  , low to medium , and very low  expression ; genes peaking at −1 DPA (GhMML3 and 4); genes peaking at 0 to +1 DPA ; and one member with highest expression at +2 DPA (GhMML10)  Figure  with lit) Figure . These eGhMML members their expression levels were assessed in dissected OI from transgenic tetraploid cotton silenced (through RNAi) for GhMML3/GhMYB25Like  and GhHD-1, necessary for fiber development that are similar to the sorts of complexes seen in Arabidopsis trichome development (Ishida et al., MML genes by silencing and over-expression in transgenic cotton and the study of their interactions in both cotton protoplasts and yeast to confirm their roles in generating the fibers of commercially important cotton species.The MYBMIXTA-like subgroup appears to have expanded in cotton compared to non-fiber plants, probably following a cotton specific triplication as reported in Paterson et al. , thus coMMLs (Figure Phylogeny analysis Figure : Frank Bs Figure : Danny Js Figure –F: Franks Figure : Sally As Figure : Frank BThe 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.


Regulation of CXCL12 is unique in that it may regulate its own expression levels via binding to its scavenger receptor CXCR7/ACKR3. In this review, we explore the diverse roles of these and other homeostatic chemokines expressed within the CNS, including the possible implications of their dysfunction as a cause of neurologic disease.In the adult central nervous system (CNS), chemokines and their receptors are involved in developmental, physiological and pathological processes. Although most lines of investigation focus on their ability to induce the migration of cells, recent studies indicate that chemokines also promote cellular interactions and activate signaling pathways that maintain CNS homeostatic functions. Many homeostatic chemokines are expressed on the vasculature of the blood brain barrier (BBB) including CXCL12, CCL19, CCL20, and CCL21. While endothelial cell expression of these chemokines is known to regulate the entry of leukocytes into the CNS during immunosurveillance, new data indicate that CXCL12 is also involved in diverse cellular activities including adult neurogenesis and neuronal survival, having an opposing role to the homeostatic chemokine, CXCL14, which appears to regulate synaptic inputs to neural precursors. Neuronal expression of CX This is especially the case for chemokines categorized as “homeostatic”, based on their constitutive expression in thymic and lymphoid tissues , where they regulate the migration of leukocytes during immune surveillance  barrier and blood brain barrier (BBB), which prevent the exchange of cells and solutes between the blood and CSF or brain parenchyma, respectively. The BBB is comprised of specialized endothelial cells of the cerebral microvasculature, surrounding pericytes, and astrocytic endfeet, while the blood–CSF barrier is largely made up of the fenestrated endothelium of the choroid plexus. In addition to these anatomical barriers, the expression of chemokines and chemokine receptors at the BBB and blood-CSF barrier serves as an immunological checkpoint and prevents (during non-inflammatory/homeostatic conditions) or promotes (during neuroinflammation) the infiltration of circulating leukocytes into the deeper CNS parenchyma and ventricular or subarachnoid CSF spaces Figure .in vitro studies of the movement of leukocytes towards increasing concentrations of solubilized chemokines , is expressed as three alternatively spliced isoforms . Within the CNS, the expression patterns for CXCL12 is widespread and includes the cortex, olfactory bulb, hippocampus, cerebellum, meninges, and the endothelium of the BBB (β and γ isoforms). Further, expression of CXCR4, the receptor for CXCL12, has been detected in numerous cell types in the CNS including astrocytes, microglia, oligodendrocytes, neurons, and endothelial cells of the BBB -1β and IL-17, increased expression of CXCL12 and CXCR7, respectively, with increased localization of CXCL12 with lysosomal markers. Further, these cytokines enhanced uptake of exogenous CXCL12, which was inhibited in a dose-dependent manner by antagonism of CXCR7, suggesting that under inflammatory conditions, CXCR7 facilitates internalization of CXCL12, leading to loss of polarity at the BBB seen in EAE as well as MS , an inflammatory chemokine expressed by immune cells as well as other stromal cell types. In response to inflammatory cues or tissue injury, CCL2 is up-regulated to recruit CCR2The intensity of immune responses in the CNS increases with proximity to the ventricles of the brain  and facilitate antigen-specific interactions within subcompartments of secondary lymphoid tissue . Thus CCL19 and CCL21 serve to generate adaptive immune responses and are critical for developing and maintaining secondary lymphoid tissues in the periphery and have also been implicated in lymphoid neogenesis within the CNS.In peripheral lymphoid tissues, the chemokine CCL19 guides CCR7-expressing B cells, naïve T cells, and DCs into lymphoid tissue under physiological conditions  of the spinal cord via an IL-6-mediated mechanism. Arima et al. have demonstrated that CCL20 is normally expressed by vasculature at this site. IL-6 mediated upregulation of CCL20 was induced by stimulating the soleus muscle, which led to the infiltration of autoreactive T cells across the BBB in an adoptive transfer model of EAE. These experiments provided evidence that sensory stimulation can trigger chemokine-mediated accumulation of T cells within the CNS microvasculature at specific locations within the lumbar spinal cord. These observations led the authors to postulate that expression of CCL20 at the dorsal L5 blood vessels, even in the absence of pathogenic T cells, could represent a “gateway” by which leukocytes expressing CCR6 could cross the BBB and gain entry into the CNS parenchyma  express CXCR4 and respond to CXCL12 as well as other cytokines to augment neovascularization for restoration of homeostasis following CNS injury  is much longer than most chemokines (373 vs. ∼80 AAs) and also exists in two forms: a 95 kDa membrane-bound form with an N-terminal chemokine domain, a glycosylated mucin-like stalk, a hydrophobic transmembrane region and an intracellular C-terminal domain; and a 70 kDa soluble form that contains only the N-terminal chemokine domain. The soluble chemokine domain of CX3CL1, when cleaved, can act as a signaling molecule -α , orchestrating the balance of homeostasis, while providing immune protection, under physiologic conditions in the CNS. While many functions of chemokines during homeostasis have been identified, there is still much to learn.While chemokines have historically been thought of as mediators of cell migration, recent evidence suggests that chemokines have the capacity to regulate a number of cellular functions critical to inflammatory processes as well as maintenance of homeostasis. Though the characterization of homeostatic chemokines stems from their roles in lymphoid tissues, many parallels can be drawn in the CNS, particularly in the context of the vasculature. Several chemokines contribute to immune cell trafficking and activation during immunosurveillance ; regulation of neural progenitor cell migration, proliferation, differentiation, and integration ; and maintenance of quiescence  is largely expressed by keratinocytes, binds to the receptor CCR10, and may serve as an important regulator of homeostatic immune surveillance. CCL27 has been implicated in inflammatory allergic reactions, primarily in homing memory T cells to the skin (Morales et al., 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.


Arabidopsis thaliana normally six stamen primordia arise in the third whorl of floral organs and subsequently differentiate into stamen filaments and anthers, where male meiosis occurs, thus ending the early developmental phase. This early phase is followed by a late developmental phase, which consists of a rapid elongation of stamen filaments coordinated with anther dehiscence and pollen maturation, and terminates with mature pollen grain release at anthesis. Increasing evidence suggests that auxin transport is necessary for both early and late phases of stamen development. It has been shown that different members of PIN (PIN-FORMED) family are involved in the early phase, whereas members of both PIN and P-glycoproteins of the ABCB (PGP) transporter families are required during the late developmental phase. In this review we provide an overview of the increasing knowledge on auxin transporters involved in Arabidopsis stamen formation and development and we discuss their role and functional conservation across plant species.In flowering plants, proper development of stamens, the male reproductive organs, is required for successful sexual reproduction. In  Arabidopsis thaliana, stamen growth should be coordinated to pistil development to allow self-pollination. Alterations in stamen development lead to male sterile plants that can be utilized to generate hybrids useful for agronomic practice.Stamens are the male reproductive structure of flowers and their function is to produce pollen grains, which house male gametes, and to release them at flower opening to allow plant reproduction. Male fertility results from sequential developmental events that involve an early phase of stamen formation and morphogenesis and a late phase that consists of pollen grain maturation, stamen filament elongation and anther dehiscence. In autogamous plants, such as YUC genes in Arabidopsis  family are the main group of efflux carriers with a polar cellular distribution , contributes to all aspects of plant development mainly through its differential distribution within plant tissues. Auxin concentration controls the expression of hundreds of genes by the ubiquitin-mediated pathway based on the interplay of two classes of transcription factors, auxin-response factors (ARFs) and Aux/IAA repressors  Figure .yuc4 mutant flowers -defective in the auxin biosynthetic gene YUC4- show one or two stamens, and few floral organs  genes such as ARF3/ETTIN or ARF5/MONOPTEROS, show flowers with reduced number of stamens, together with increased number of sepals and petals and abnormal gynoecia  protein kinase that controls PIN1 polarity, show flowers with a range of defects that can include a pin-shaped inflorescence or alterations in stamen number , show flowers with a reduced number of stamens  initiation as a reduction in stamen number is observed in different classes of mutants. Single CUC1 and CUC2 genes. The role of these genes is to inhibit the growth of cells at the boundaries between primordia to avoid organ fusion. As PID and CUC expression domains are overlapping in the boundary of cotyledon primordia, it has been suggested that PID, by promoting auxin transport, reduces the level of auxin at the boundary and increases it in the primordia during cotyledon formation . In addition, it has been recently shown that PIN6 is expressed in anthers at stage 8 of flower development/anther stage 4, as detected by GUS staining of proPIN6:GUS flowers. This would suggest that PIN6, in addition to its role in short stamen formation (see above), could also be necessary for the formation of long stamens , did not impair DR5 activity in stamens; similarly, in anthers severed at a premeiotic stage from DR5:GUS flower buds and matured in vitro, GUS staining was not altered and was comparable to control DR5:GUS anthers , which regulates polar distribution of PINs. This raises the possibility that auxin levels are also modulated by other PIN proteins in addition to PIN2 in stamens . In particular, GUS staining was observed in the vascular tissues of SAUR63:GUS stamens and reduced expression of SAUR63 causes a short stamen phenotype whereas lines overexpressing SAUR63 showed long stamen filaments. Quite interestingly, when a SAUR63:GUS translational fusion construct was expressed in pgp1pgp19 plants, their flowers had longer stamen filaments than pgp1pgp19 flowers. In contrast, when SAUR63:GUS was expressed in pin3-4pin7-2 double mutant plants, their filament length was comparable to that of pin3-4pin7-2 double mutant flowers  transcription factor genes IDD14, IDD15, and IDD16, exhibits infertile siliques resulted from the asynchronous elongation of stamen filaments and styles. The transcription of AUX1, PIN1, ABCB1, and ABCB4 is severely reduced in the idd mutant, and the IDD proteins could directly bind to the promoter regions of PIN1 to activate its expression  floral system is distinguished from that of Arabidopsis as tomato flowers contain five sepals, alternating with five petals, five anti-sepalous stamens  and two fused carpels are observed. Stamens are fused together to form a cylinder that surrounds the style.The tomato , the putative tomato orthologous gene of AtYUC4, has been identified and it is expressed in immature flowers. In addition tomato genome contains many genes that encode flavin monooxygenases-like proteins, similar to the situation observed for Arabidopsis YUC genes  found in tomato, SlPIN4 is the most expressed, although at a low level, in stamens during flower development up to the anthesis. In agreement P35S:SlPIN4RNAi flowers show alterations in stamen morphogenesis and development together with abnormal sepals and parthenocarpic fruit development   is a model species for the study of flower morphogenesis in legumes. The main difference in Medicago stamen development compared to Arabidopsis is the existence of four common primordia from which petals and stamens differentiate. Medicago flowers are organized in four whorls and show a pentamerous arrangement of sepals and petals, ten stamens and a central carpel. Nine stamen filaments are joined in a staminal tube around the carpel, and the tenth, the vexillary stamen filament at the adaxial position, is free standing.Medicago   primordia formation and separation  family, one of the largest flowering plant families in angiosperms, includes many economically important crops such as maize and rice. The grass inflorescence is composed of different types of branches, including a specialized branch called spikelet. The spikelet is a special unit of the inflorescence and forms one to several florets, depending on the species. In the floret, floral organs such as perianth organs, carpels, and stamens are formed. In grasses, different types of meristem, such as the inflorescence meristem (IM), the branch meristem (BM), the spikelet pair meristem , the spikelet meristem (SM) and the floral meristem (FM), are responsible for the complex development of inflorescences and flowers.Zea Mays) is monoecious and the male inflorescence, the tassel, situated at the shoot apex, consists of a main spike with several long lateral branches at the base. Normal tassels produce florets whose meristems give rise to a lemma and palea , two lodicules , and three stamens.Maize , are clearly associated with the initiation of floral organ primordia, including stamen primordia in tassel  mutants are also seen in plants treated with auxin transport inhibitors , one gene homologous to AtPIN2 (ZmPIN2), three orthologs of PIN5 (ZmPIN5a–c), one gene paired with AtPIN8 (ZmPIN8), and three monocot-specific PINs , 2 ABCB-like genes, ZmABCB1/BR2 and three orthologs of AtABCB19  have recently been identified  inflorescence architecture is quite different from that of maize, as flowers are hermaphrodite, and one rice spikelet has only one floret surrounded by a pair of empty glumes . In addition, rice florets have an asymmetric structure with five types of floral organs: one lemma and one palea in the first outer whorl, two lodicules in the second whorl, six stamens in the third whorl and one pistil with two stigmas in the fourth innermost whorl.Rice . Functional analysis has been reported only for OsPIN1 and revealed that OsPIN1 knockdown plants do not have the characteristic pin phenotype. However, OsPIN1 is highly expressed in flowers, and in particular in stamen filaments and at the junction region between anther and filament during late development (Xu et al., OsPIN3t, which shows high aminoacid sequence identity to AtPIN3, is expressed in mature anthers (Zhang et al., OsPIN1 mutant flowers could shed light on the role of these transporters in stamen development.At least seven Taken together, this data suggest, but are far from being conclusive, a role for auxin transport in stamen primordia formation such as in maize, and also in stamen filament elongation during late flower development.The data reported in this review, (summarized in Table Auxin transport also appears to regulate later developmental processes that require coordination with each other and with gynoecium development. Basipetal auxin transport—exerted by different members of large-loop PINs and ABCB/PGP families—through epidermal and vascular cells regulates stamen filament elongation is described in the model presented in Figure AtPIN1 and other dicot-specific orthologs, with monocot members of the PIN1 family indicates that all PIN1 genes share a common overall structure. However the monocot PIN family is wider and more divergent than the dicot one, with three or four genes homologous to one single Arabidopsis PIN gene. In addition, the overall expression profile of the PIN genes is different between Arabidopsis and maize: while in Arabidopsis each PIN has a specific expression domain, many ZmPIN genes are simultaneously expressed in the same organ or tissue (Forestan et al., PID-like genes have been also identified in maize and rice (McSteen and Hake, AtABCB1/PGP1 and AtABCB19/PGP19 have also been identified in maize (Knöller et al., The mechanisms of auxin transport, together with those of auxin biosynthesis and signal transduction, appear conserved in dicots and monocots. Alignment of In summary, the available data suggests that auxin transport-dependent mechanisms are required for proper stamen development and in particular for stamen primordia formation in dicots as well as in monocots. However to propose a model of more general validity all actors need to be identified to gain a full understanding of how the auxin gradient during stamen development is established. Furthermore more work directly focused on stamen growth is necessary to understand the involvement of PAT in different processes of early and late stamen development.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.


The developing central nervous system (CNS) is vascularised through the angiogenic invasion of blood vessels from a perineural vascular plexus, followed by continued sprouting and remodelling until a hierarchical vascular network is formed. Remarkably, vascularisation occurs without perturbing the intricate architecture of the neurogenic niches or the emerging neural networks. We discuss the mouse hindbrain, forebrain and retina as widely used models to study developmental angiogenesis in the mammalian CNS and provide an overview of key cellular and molecular mechanisms regulating the vascularisation of these organs.  •CNS vascularisation is initiated during embryonic development.•CNS vascularisation is studied in the mouse forebrain, hindbrain and retina models.•Neuroglial cells interact with endothelial cells to promote angiogenesis.•Neuroglial cells produce growth factors and matrix cues to pattern vessels. These organs are vascularised early in their formation to ensure adequate delivery of oxygen and nutrients to neural progenitors, newly born neurons and their associated glia A,B. VascMaturing CNS endothelial cells establish a blood–brain barrier (BBB) to protect the neural tissue from variations in blood composition, to exclude toxins and to maintain ionic homeostasis  9.5, when vascular sprouts begin to emerge from the PNVP and grow in a radial fashion towards the ventricular zone, where neural progenitors are thought to release vascular growth factors A,C e.g.. From arCre–LoxP technology, can even be extended to the study of genes whose constitutive loss causes pre-midgestation lethality. Finally, and typically of mouse models, robust methods for wholemount labelling of endothelial cells and interacting cell types are available and can be combined with high-resolution imaging for reliable quantitation of angiogenic sprouting, network density and vessel calibre.The hindbrain model has several key advantages to study sprouting angiogenesis . Most noAn emerging model of CNS vascularisation is the zebrafish hindbrain, which is particularly amenable to longitudinal live imaging with fluorescent genetic reporters . Using t2.2For mouse embryo forebrain vascularisation, blood vessels begin to sprout at E9.5 from the PNVP at the level of the presumptive ganglionic eminence into the ventrolateral brain. Vascularisation of the forebrain then progresses in a ventrolateral to dorsomedial direction across the entire rostrocaudal axis. By E10, an SVP has formed in the ventral portion of the forebrain, whilst the dorsal part is still largely avascular. This peculiarity was recently explained by the observation that the vasculature in the dorsal forebrain does not sprout from the dorsal PNVP, but instead derives from the SVP of the ventral compartment . Thus, eRecently, angiogenesis has been successfully studied also in the postnatal forebrain one week after birth, when angiogenesis is associated with brain growth. In this system, the distinct angiogenic steps of tip cell selection, vascular sprout migration and lumen formation, as previously studied extensively in the embryonic brain and postnatal retina, could be readily detected and quantified .2.3Anatomically, the retina lies outside the brain, but it originates as an outgrowth of the developing forebrain and is therefore considered part of the CNS. Being the most accessible part of the CNS, it has become a popular model for studies of both physiological and pathological angiogenesis. Whilst the human retinal vasculature develops before birth, the mouse retinal vasculature develops postnatally and therefore offers unique advantage to experimental manipulation e.g. . The posRetinal vascularisation in the mouse begins on the day of birth, when vessel sprouts emerge from the optic nerve head and spread radially over the retina, guided by a template of fibronectin (FN)-expressing astrocytes B Frutti. During op/opCsf1 mutants with defective macrophage recruitment. In these mutants, the primary plexus undergoes radial expansion, but has reduced vascular network complexity compared to wild type littermates  model, which recapitulates hallmarks of neovascularisation in the human conditions retinopathy of prematurity and proliferative diabetic retinopathy  and low levels of VEGFR1 in tip cells relative to neighbouring stalk cells promote tip cell-mediated vessel sprouting in chimeric embryoid bodies . Recent GLUT1 gene cause a rare autosomal dominant disorder termed GLUT1 deficiency syndrome, which is characterised by a low cerebrospinal fluid glucose concentration, due to reduced transport across the BBB , VEGFR2  and VEGFR3 (FLT4) . VEGFR2 VEGFR3, best known as a VEGF-C receptor in lymphangiogenesis, is highly expressed in angiogenic sprouts , and theVegfr1 gene into a short secreted form termed soluble VEGFR1 (sVEGFR1 or sFLT1) and a long transmembrane form with a tyrosine kinase domain  and VEGF . A role 4.3NRP1 is a non-catalytic transmembrane protein shown to be essential for the vascularisation of the mouse spinal cord , hindbraInitially, NRP1 was initially studied in the nervous system as an adhesion molecule and a receptor for a member of the class 3 semaphorin family, termed SEMA3A, and later discovered to also act as a VEGF165 receptor  receptor of the frizzled (FZ) family, often referred to as the G protein-coupled receptor (GPCR) family. FZ receptors function together with a co-receptor of the low-density lipoprotein receptor-related protein (LRP) family, LRP5 or LRP6, to activate transcription via the cadherin-associated protein beta 1 . Specifically, the activated receptor complex recruits dishevelled (DSH), which inhibits a destruction complex that otherwise ubiquitinates cytoplasmic beta-catenin. When its degradation is prevented, beta-catenin translocates to and accumulates in the nucleus to regulate the transcription of WNT target genes. In the CNS of developing mice, the genetic loss of WNT7B, in particular in the context of additional WNT7A loss, or the vascular-specific loss of beta-catenin reduce neural tube vascularisation  Daneman. Moreove−/−Fz4 endothelial cells, but the Fz4–/– endothelial cells are progressively eliminated ; moreover, Ang2 deficient mice showed delayed and incomplete development of the superficial vascular bed of the retina  is abundantly expressed in motor neurons at a time when its main receptor TIE2 is expressed in endothelial cells within the neural tube  Nagase . Further embryos , and PNVnockouts . In the nockouts . The altnockouts . In contnockouts   impairs vessel ingression by blocking induction of motor neurons in the neural tube . As neurSoluble epoxide hydrolase in Muller glia converts docosahexenoic acid into 19,20-dihydroxydocosapentaenoic acid, which inhibits γ-secretase to suppress endothelial notch signalling  Hu et a. This in5The cellular interactions and molecular signals critical for CNS vascularisation and the formation of the neurovascular unit are being elucidated at an accelerating speed, and we are beginning to appreciate the significance of defective vascular development in the emergence of CNS pathologies. Yet, it is likely that additional signalling mechanisms and interactions remain to be identified before we will fully understand how the cross-talk of neural and vascular cells regulates blood vessel ingression into the CNS and the formation of a fully functional BBB. Ultimately, understanding these signalling pathways will reveal how a defective neurovascular unit impacts on CNS function during aging and in neurological disease. It will also benefit the development of new therapeutic strategies aimed at delivering drugs through the BBB, as well as restoring or improving vascular supply to ischemic retina and brain in diseases such as diabetic retinopathy, age-related neurodegeneration and stroke. In the mature brain, neuronal activity stimulates changes in blood flow that can be measured by fMRI, but how this flow information is sensed and leads to structural changes in blood vessels is largely unknown and therefore requires further research. Finally, the specific features of the tumour microenvironment that promote the differentiation of tumour cells into endothelium, and the functional consequence of this transdifferentiation for glioblastoma remain to be determined.


It is required for survival and serves as an important catalytic cofactor in redox chemistry for proteins that carry out fundamental biological functions, important in growth and development. The deficit of copper can result in impaired energy production, abnormal glucose and cholesterol metabolism, increased oxidative damage, increased tissue iron (Fe) accrual, altered structure and function of circulating blood and immune cells, abnormal neuropeptides synthesis and processing, aberrant cardiac electrophysiology, impaired myocardial contractility, and persistent effects on the neurobehavioral and the immune system. Increased copper level has been found in several disorders like e.g.: Wilson’s disease or Menke’s disease. New findings with the great potential for impact in medicine include the use of copper-lowering therapy for antiangiogenesis, antifibrotic and anti-inflammatory purposes. The role of copper in formation of amyloid plaques in Alzheimer’s disease, and successful treatment of this disorder in rodent model by copper chelating are also of interest. In this work we will try to describe essential aspects of copper in chosen diseases. We will represent the evidence available on adverse effect derived from copper deficiency and copper excess. We will try to review also the copper biomarkers (chosen enzymes) that help reflect the level of copper in the body.Copper (Cu) is an essential microelement found in all living organisms with the unique ability to adopt two different redox states—in the oxidized (Cu Copper is absorbed from diet, mainly in duodenum, although it is thought that some absorption takes place in the stomach and in the distal part of the small intestine. Copper uptake into enterocyte is managed mainly by human copper transport protein 1 (hCTR1). The processes is mediated by action of reductases in the apical membrane, which reduce dietary Cu(II) to Cu(I)—the acceptable state of copper for this receptor. Recent studies show that transport of Cu(I) ions can be carried out using the divalent metal transporter 1—it is an iron transport protein located on the membrane of enterocytes. Following absorption by the gut, copper is secreted into the circulation and bound as Cu(II) to albumin, transcuprein and low molecular weight copper-histidine complexes. Upon gaining the liver, copper is rapidly taken up by hepatocytes also via hCTR1. Inside the cytoplasm of cells, copper is either chelated by metallothionein (MT) for storage or it binds to a copper chaperone for delivery to specific proteins or it binds to reduced glutathione. Three copper chaperones have been identified: CCS (copper chaperone for superoxide dismutase 1), COX17 (chaperone for cytochrome c oxygenase) and ATOX1 (chaperone for ATPases—ATP7A and B). ATP7A delivers copper to tyrosinase and ATP7B to lysyl oxidase and ceruloplasmin (Cp)  and Wilson disease (WD). Wilson’s disease  and Menke’s disease (X-linked) represent the most well recognized and understood disorders of copper homeostasis , bone fractures, and aortic aneurysms , high levels of zinc supplements (40–50 mg three times per day) and low Cu diet (Uriu-Adams and Keen Wilson’s disease (WD) is an autosomal recessive genetic disorder, and is caused by disabling mutations in both copies of the ATP7B gene. This gene is responsible for copper’s transfer into the secretory pathway for both binding into Cp and excretion into the bile Brewer . ClinicaSyndrome called idiopathic copper toxicosis (ICT) occurring in infants and young children has been seen in many parts of the world, and is usually associated with high levels of hepatic copper, caused by high concentrations of copper in drinking water or food Brewer , b. In wAceruloplasminemia—an autosomal recessive disease—is caused by mutations in the Cp allele on chromosome 3q, and results in a total absence of Cp in the blood. Therefore an iron accumulation, causing clinical problems in the brain and liver is observed Brewer , b. The Alzheimer disease (AD) is a multifactorial disorder in which many altered cellular processes are involved, such as oxidative stress, neuroinflammation, impairments in energy metabolism, and others. Major histopathological features associated with AD are senile plaques and neurofibrillary tangles, related to aberrant processing of amyloid precursor protein (APP) leading to the deposition of amyloid β and hyperphosphorylation of tau protein, respectively , and redox-based neurochemical reactions occur  and superoxide dismutase (involved in the detoxification of reactive oxygen species). An excess of copper could result in oxidative stress related health disorders, many of which can be linked partially to its redox reactivity. Copper has been suggested to facilitate oxidative tissue injury through a free radical mediated pathway analogous to the Fenton reaction. Copper deficiency also affects directly or indirectly the components of the oxidant defense system and as a result increased ROS and oxidative damage to lipid, DNA and proteins have been observed in human cell culture models. Copper on Cu(I) state can bind directly to free thiols of cysteine or methionine, resulting in oxidation and subsequent cross-links between proteins leading to impaired activity. Copper on Cu(II) state prefers nitrogen donors, such as histidine, or oxygen donors, such as glutamate or aspartate.Copper deficiency has been described mostly in infants on total parenteral nutrition without adequate mineral supplementation or in persons with persistent nephritic syndrome that increases copper losses. Low copper status has been associated with bone malformation during development, increased osteoporosis, impaired melanin synthesis, poor immune response, therefore increasing frequency of infections, cardiovascular disease, alterations with cholesterol metabolism, and disturbance of metabolism of other trace elements, such as iron mobilization.Copper toxicity is usually an acute episode resulting from copper contamination of drinking water or food  to water. In addition to O2 reduction, cytochrome C oxidase pumps protons from the inside to the outside of the membrane  is bound with inner mitochondrial membrane. It is a component of the mitochondrial respiratory chain so it takes part in energy production. It has four redox active metal sites, two hem sites and two copper sites. It is the terminal oxidase in most aerobic organisms and reduces molecular oxygen  are cysteine-rich metal binding proteins at low molecular weight. They come at least in two isoforms encoded by several genes. The biological roles of metallothioneins are: detoxification of metals ions, including non-essential and excess of essential metals ions, storage of essential microelements, sequestration of ROS and nitrogen species  and is found largely in cytosol. Mutations of Cu/Zn-SOD have been recently of interest in connection with amyotrophic lateral sclerosis, where a gain of function is responsible for the underlying neurological symptomatology . It may oxidize iron ions for binding to apotransferrin, allowing its release into the blood as holotransferrin , dopamine-β-hyroxylase (important in catecholamine production), peptidylglycine α-amidating monooxygenase (required for neuropeptide and peptide hormone processing), monoamine oxidase (needed for pigment and neurotransmiter production and metabolism)  has been recently studied. Numerous Cu(II) complexes of NSAIDs, showing enhanced anti-inflammatory and antiulcerogenic activity as well as reduced gastrointestinal toxicity compared to the un-complexed drug have been prepared and structurally characterized  complexes showed antifungial activity against Aspergillus niger, Rhizopus stolonifer, Aspergillus flavus, Rhizoctonia bataticola and Candida albicans. Antifungal activity of clomiphene citrate copper compounds has been determined against two fungi: Aspergillus flavus and Aspergillus niger  complexes showed antibacterial activity against


Microcell-mediated chromosome transfer (MMCT) is a technique to transfer a chromosome from defined donor cells into recipient cells and to manipulate chromosomes as gene delivery vectors and open a new avenue in somatic cell genetics. However, it is difficult to uncover the function of a single specific gene via the transfer of an entire chromosome or fragment, because each chromosome or fragment contains a set of numerous genes. Thus, alternative tools are human artificial chromosome (HAC) and mouse artificial chromosome (MAC) vectors, which can carry a gene or genes of interest. HACs/MACs have been generated mainly by either a “top-down approach” (engineered creation) or a “bottom-up approach” (de novo creation). HACs/MACs with one or more acceptor sites exhibit several characteristics required by an ideal gene delivery vector, including stable episomal maintenance and the capacity to carry large genomic loci plus their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. The MMCT technique is also applied for manipulating HACs and MACs in donor cells and delivering them to recipient cells. This review describes the lessons learned and prospects identified from studies on the construction of HACs and MACs, and their ability to drive exogenous gene expression in cultured cells and transgenic animals via MMCT. New avenues for a variety of applications to bio-medical challenges are also proposed. Fournier and Ruddle performed for the first time microcell-mediated chromosome transfer (MMCT)  cells are induced to multinucleate their chromosomes (referred to as micronucleus formation). Micronuclei are then forced through the cell membrane to create microcells by centrifugation in the presence of cytochalasin B which disrupts the cytoskeleton  cells. Improvement of the efficiency has emerged as an issue to be resolved.Since the dawn of MMCT trials, established cell lines such as mouse ES cells have been preferably used as recipient cells. Efficiency of MMCT has been described by the ratio of drug-resistant colony number to recipient cells. Depending on the type of recipient cells, the efficiency is generally 10As mentioned in the previous section, MMCT comprises multiple steps, viz., (1) induction of micronuclei in donor cells, (2) isolation of microcells from micronucleated donor cells, (3) fusion of microcells with recipient cells, (4) integration of the transferred chromosome into host cell nucleus, and (5) selection of microcell hybrid cells by drug selection. Eventual efficiency is determined by the summation of contribution from each step. Of these steps, we will discuss microcell fusion and provide a perspective for effective isolation of microcells.−5∼10−6. The mixture of microcells and recipient cells is transiently exposed to PEG, followed by dilution and removal by washing. Theoretically, one-to-one fusion between a pair of microcell and recipient cell produces a seed of microcell hybrid. But the PEG treatment is injurious to cell membrane, and overexposure to PEG reduces the viability of hybrid cells  a chromosome (HAC or MAC) to be transferred, (2) chromosomes derived from host cells, or (3) no choromosomal DNA. Only the first subpopulation contributes to intended MMCT, but it is minor among all the components. In the current protocol, total mixed population is used for fusion with recipient cells, and intended microcell hybrids are chosen by selection culture with antibiotics by utilizing the drug-resistant gene tagged onto the HAC/MAC. Preferential fractionation of required microcell subpopulation from the total mixture, if possible, might aid in achieving more intentional fusion reaction and in needing a smaller number of recipient cells.The conventional MMCT method is usually performed immediately after the purification of microcells. The timing of the isolation of microcells and the preparation of recipient cells is very important. A cryopreservation method to store microcells at −80 °C was performed and compared the efficiency of MMCT with conventionally (immediately) method. There was no significant difference between the two methods regarding chromosome transfer efficiency. Thus, cryopreservation of ready-to-use microcells is useful for the MMCT , transactivator-like effector (TALE), and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 system , which they can carry is not limited Fig. . The de Another HAC with a conditional centromere that includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array has been generated  was created via amplicon-dependent de novo chromosome formation induced by the integration of exogenous DNA sequences into centromeric DNA regions near the pericentric heterochromatic or acrocentric chromosome , since multiple lox71 sites were integrated into the HAC, a lox66 sequence in the donor vector containing desired gene is useful for the sequential insertion of multiple desired genes into the HAC A9 mini-chromosome), iterative site-specific integration (ISSI) system on the 49B(A)A9 mini-chromosome derived from human Y chromosome were developed  system, was reported  vectors, where often the gene cannot be expressed at a physiological level because the gene copy number is not regulated and a BAC/YAC transgene randomly integrates into the host genome. The HACs and MACs described above provide a way to overcome these problems  cDNA, was constructed and inserted into CHO cells  gene was inserted into the 21HAC and then transferred into hiMSC. The EGFP was specifically expressed in the hiMSCs that differentiated into osteocytes in coordination with the transcription of the endogenous OPN gene but was not expressed after adipogenic differentiation induction or in non-inducing culture conditions, indicating that the use of HAC vectors is suitable for regulated expression of transgenes not only in stem-cell-mediated gene therapy, but also in promoter analyses. Another application of tissue specific promoter is that erythropoietin (EPO) gene expression driven by the 5′ untranslated region of the human ubiquitin C gene on the newly developed chromosome 14-derived HAC greatly increased (over 1000-fold) the EPO production in hPFs  high transfection efficiency; (2) long-term stable maintenance in host cells without integration into the host genome; (3) appropriate levels of spatial and temporal expression of therapeutic genes in specifically desired cells; (4) no risk of cellular transformation or stimulation of the host’s immune system; and (5) a system to safeguard against tumor formation. Although a number of different approaches have been attempted to achieve efficient gene transfer and long-term gene expression, this challenging task remains unfulfilled because all current methods have certain limitations, including transient expression, consequent toxicity, undesired immunological response, integration of target genes into the host cell genome, and transcriptional silencing. An alternative solution to these problems could be the use of HAC vectors. For example, the advantages of HAC vectors have been demonstrated for reprogramming mouse embryonic fibroblasts (MEFs) into iPS cells . GALC gene was microinjected into the model mouse-derived blastocysts. However, the life-span extension is possibly attributable to the genomic copy of the GALC gene present in the wild-type ES cells.The other example is Globoid cell leukodystrophy , which is an autosomal recessively inherited disease caused by a deficiency of galactocerebrosidase (GALC), a lysosomal enzyme that degrades galactosylceramide, a major glycolipid component of myelin and myelin-forming cells (Katona et al. Finally, advances in the efficiency of methods used for the differentiation and purification of stem cells, including ES and iPS cells, are anticipated, and the application of these methods to ES/iPS cells combined with HAC vector systems may enable the development of more sophisticated gene therapies. Thus, stem cells, potentially derived from multiple sources, combined with HAC-mediated gene delivery, should permit safe treatment of various genetic defects. The next step in the future of gene therapy is to demonstrate functional restoration and safety in vivo using large animal models such as dogs and monkeys.The chromosomal vector systems offer complementary and desirable characteristics for use as gene delivery vectors to overcome various problems in existing viral and non-viral vector systems. The most important property of HAC and MAC vectors is that they can express entire complex signaling pathways under their normal physiological regulation, which is of great potential benefit. Stem cells possess two characteristic features: the ability for self-renewal and the ability for multi-lineage differentiation. A number of other applications of HACs/MACs in addition to gene therapy and animal models are possible Fig. . For exaA combination of chromosome engineering technologies and genome-editing technologies should facilitate the applications to bio-medical challenges Fig. . Genome


Actin cytoskeleton remodeling, which drives changes in cell shape and motility, is orchestrated by a coordinated control of polarized assembly of actin filaments. Signal responsive, membrane-bound protein machineries initiate and regulate polarized growth of actin filaments by mediating transient links with their barbed ends, which elongate from polymerizable actin monomers. The barbed end of an actin filament thus stands out as a hotspot of regulation of filament assembly. It is the target of both soluble and membrane-bound agonists as well as antagonists of filament assembly. Here, we review the molecular mechanisms by which various regulators of actin dynamics bind, synergize or compete at filament barbed ends. Two proteins can compete for the barbed end via a mutually exclusive binding scheme. Alternatively, two regulators acting individually at barbed ends may be bound together transiently to terminal actin subunits at barbed ends, leading to the displacement of one by the other. The kinetics of these reactions is a key in understanding how filament length and membrane-filament linkage are controlled. It is also essential for understanding how force is produced to shape membranes by mechano-sensitive, processive barbed end tracking machineries like formins and by WASP-Arp2/3 branched filament arrays. A combination of biochemical and biophysical approaches, including bulk solution assembly measurements using pyrenyl-actin fluorescence, single filament dynamics, single molecule fluorescence imaging and reconstituted self-organized filament assemblies, have provided mechanistic insight into the role of actin polymerization in motile processes. The actin cytoskeleton is a dynamic architecture of the living cell, made of several structurally and functionally distinct arrays of actin filaments that define modular motile activities. Nucleation and polarized assembly of actin filaments are driven locally in a stimulus-responsive fashion in each module by specific protein machineries. These reactions develop forces of compression or traction against the membranes to elicit protrusive, adhesive and contractile activities , fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), and fluorescence speckle microscopy (FSM) , 4. To gIn a living cell, actin is in a dynamic equilibrium (or rather steady-state) between two states—the globular monomeric state (G-actin) and the polymeric filament state (F-actin). At physiological ionic strength, actin is essentially polymerized in filaments (F-actin). Movement is intimately linked to actin exchanges between these two states. These exchanges are dissipative due to ATP hydrolysis that is associated with actin assembly  for revia, b. TheThe relative sizes of the assembled and unassembled actin pools are regulated in several ways, which are interconnected. The mechanically simplest regulators are G-actin sequestering proteins. Proteins such as β-thymosins bind G-actin in a complex that does not assemble in filaments. This pool of sequestered actin is in rapid equilibrium with only G-actin, not with F-actin, and cannot be invoked to support actin assembly directly. It can only amplify the changes in amount of F-actin that result from changes in stationary concentration of free monomers, elicited by regulators of assembly dynamics at filament barbed ends. In contrast with G-actin sequestering proteins, profilin binds G-actin in a complex that participates in barbed end assembly specifically Fig. b, thus eListeria monocytogenes, which exploit actin-based motility, move in the cytoplasm at constant rates for long periods of time [Live-cell imaging of actin filament dynamics in lamellipodia of migrating cells indicates that filaments treadmill at constant rate as the leading edge moves forward , 4. Simi of time , 7. Thes of time , which wIn the cellular context, fundamental questions regarding the mechanism of production of force and movement by actin polymerization remain unanswered. For polarized growth of filaments to be maintained at defined sites in the cell, e.g., at the protein–membrane interface, some transient attachment of the growing barbed ends appears to be required. What structural and functional mechanisms are able to locally restrain and control filament growth? How are these chemical reactions at the interface of filaments and membranes transduced into mechanical properties, at the molecular and supramolecular scale? What laws of physical chemistry of protein self-assembly account for coordinated filament turnover in various arrays and for actin homeostasis? If dendritic arrays are assembled using the same basic molecules in different motile functions, how does the cell avoid indirect effects resulting from one motile activity (e.g. endocytosis) on the efficiency of other modules? Answering these questions requires a detailed understanding of the underlying molecular mechanisms at high spatial and temporal resolution. Integrated interdisciplinary approaches have shown promise of providing answers at different scales to these burning issues.An actin filament is a chiral helical polymer in which all actin subunits share the same polarity, defining a “barbed end” and a “pointed end” Figs. b, 2. A lFirst, the two terminal subunits are subject to weaker bonding constraints compared to the actin subunits further in the core of the filament, which are connected via two lateral and two longitudinal contacts. The acknowledged structural plasticity of the filament core  is potenSecond, barbed end binding proteins may associate either with only one or both actins at the barbed ends Fig. . OccupanA frequently used binding site, targetted by actin-binding proteins as well as by drugs like macrolides, is the hydrophobic pocket in the shear zone between subdomains 1 and 3 also called target binding cleft, TBC Figs. a, 3. AmpMost of these effectors have been characterized, using bulk solution assays and single filament assembly dynamics. Importantly, in cellular conditions, some barbed end regulators are soluble and free in the cytoplasm; others act in a membrane-bound state. The interplay between soluble and immobilized regulators of filament barbed end assembly drives the dynamic coupling between the actin cytoskeleton and membranes (or vesicles), and is pivotal in force production and shape definition. The functional competition between agonists and antagonists of barbed end growth raises structural and mechanistic issues.The most important agonist of barbed end assembly is profilin. Profilin is a ubiquitous protein, present at concentrations in the range of 10–100 µM in cells. As a result of its binding to the barbed face of actin, it displays a dual interaction with G-actin and terminal subunits at filament barbed ends , its mu−11 M , twinfil−11 M , IQGAP1 −11 M , or Ca2+molecule , displaymolecule . CappingThe maintenance of a stationary concentration of monomeric polymerizable actin, which supports sustained local nucleation and polarized growth of filaments is made possible if a large enough fraction of the population of filament barbed ends are capped, thus setting the high concentration of monomeric actin imposed by pointed end dynamics . The avaCapping proteins thus act in synergy with actin depolymerizing factor, which causes an increase in the critical concentration at the pointed ends by enhancing the rate of pointed end disassembly. The resulting higher stationary concentration of actin monomers facilitates spontaneous nucleation. Spontaneously formed nuclei abort in the cytoplasm by their association with capping proteins, but are stabilized locally by association with membrane-bound nucleators Fig. . Thus, iAlong the same line of logic, overexpression of soluble, constitutively active protein machineries that promote filament barbed end assembly, like the catalytic domain VCA of WASP proteins, or constitutively active formin fragments, should functionally antagonize capping proteins by imposing filament barbed end dynamics in the cytoplasm and causing a massive assembly of actin filaments similar to the phenotype of capping protein deletion. The resulting imposed lower concentration of polymerizable monomeric actin impairs migration and all motile processes. A different phenotype is generated by overexpressing the CA fragment of WASP proteins or any CA-related protein. These do not activate Arp2/3 complex but sequester it, hence they impose a lower limit of filament branching by insufficient amount of substrate. In conclusion, cells understand and use the intrinsic physico chemical aspects of actin self-assembly.The rate of elongation in propulsive processes displays a bell-shaped dependence on CP concentration . As discDirect competition for barbed ends takes place between soluble CP and other barbed-end binding proteins like membrane-activated formins. This competition, which develops through so far unexplored mechanisms, is important in regulating the number of formin-initiated filaments in filopodial bundles, and the length and morphology of these bundles. The presence of a punctate pattern of CP along the filopodia and the rod-like appearance of filopodia in CP-depleted cells, contrasting with the tapered shape of filopodia in control cells , altogetThe interplay between cappers and positive regulators of barbed end growth has drastic effects when the extent of barbed end capping varies within the narrow range of 95–100 %, in which a large change develops in the steady-state concentration of both polymerizable monomeric actin and sequestered (unpolymerizable) actin , 36–38. As discussed earlier , de novoIn conclusion, the regulation of barbed end capping, both in the bulk cytoplasm and at specific loci, is of crucial importance in motility.As briefly outlined in the introduction, if two regulators share only partial binding subsites on terminal subunits, they may bind the barbed end simultaneously, thus increasing the structural and functional complexity. Transient binding of the two proteins together may also facilitate and lead to the dissociation of one of them by the other. Typical physiologically relevant cases concern the regulation of the dwell time at barbed ends of proteins that by themselves dissociate very slowly, like CP and formins.CP binds barbed ends with high affinity (Kd = 0.1 nM) and by itself dissociates from barbed ends with a half-time of about 30 min . Both thVibrio cholerae/Vibrio parahaemolyticus outer proteins VopF/VopL harbor a unique organization in dimers of three WH2 repeats. This feature is responsible for their ability to uncap actin filaments from barbed end-bound capping protein [Uncapping of CP was also shown to be mediated by dimerized WH2 domains, present in VopF/VopL or in Ena/VASP proteins. The  protein . The bar protein , 43  and spire in mediating the massive assembly of cytoplasmic actin networks, which is required for spindle translocation to the cortex in mammalian asymmetric meiotic division, and for axis patterning at mid-oogenesis of Drosophila [Two other barbed end binding proteins, formin 2 and the four-WH2 repeat containing protein Spire, together regulate barbed ends in cytoplasmic actin assembly in asymmetric meiotic cell division. As compared to other formin FH1–FH2 domains, formin 2 by itself nucleates poorly in the presence of profilin–actin and binds extremely slowly to barbed ends. Spire, on its own,  caps barbed ends via its WH2 domains. Via association of its KIND domain with the C-terminal region (tail) of formin 2, Spire rapidly recruits formin 2 to the barbed end. This local interaction leads to rapid displacement of Spire by formin 2 at the barbed ends and onset of fast processive assembly  by formiosophila –47.Formins and CP are considered as strict competitors for barbed end binding with formins “protecting” the barbed ends from CP , to filaments of up to 20 µm in length before filament detachment from formin occurs. In bundles of filaments initiated by clustered formins, a fraction of formins remains bound to the bundle at any given time, increasing the period of time spent by the bundle in the attached state and the resulting bundle length. In vivo, however, the length of filopodia or of the actin filaments that compose the cytokinetic ring appears to reach only a limited length, suggesting that some down-regulation of processive assembly occurs. Bud14 has been identified as such a displacement factor that kicks off yeast formin Bnr1 from the barbed end . FurtherSimilarly, capping proteins  and CytoAmong the site-directed nucleators of filaments, the WASP family proteins Table  are partSCAR/WAVE proteins generate branched filament arrays to promote extension of lamellipodia  as well Listeria ActA protein, which catalyzes filament branching responsible for Listeria propulsion in the host cytoplasm, harbors motifs structurally and functionally analogous to the WH2, C and A motifs of WASP proteins, but in different order in the sequence [Central to the mechanism of force production is the catalysis of filament branching. Activation of WASP proteins always results in exposure of the C-terminal catalytic domain, called VCA or WCA. This domain consists of a WH2 motif (in one or two repeats), C (connector) and A (acidic) regions stand adjacent to each other in the sequence of most WASP proteins except WASH in which 100 residues separate C from A . Incidensequence , 85.At each catalytic cycle of filament branching, one molecule of Arp2/3 is incorporated at a branch junction from which a “daughter” filament is initiated Fig. . FluoresThe general topology, high resolution structure and dynamics of the large macromolecular complex formed at the membrane between a filament, WASP and Arp2/3 in the catalysis of branching represent a major challenge for future research.The isolated VCA has been extensively used to analyze filament branching both in bulk solution and in single filament TIRF microscopy assays. Polymerization of actin in the presence of VCA and Arp2/3 complex shows constant acceleration due to autocatalytic multiplication of growing filaments by branching. Addition of pre-assembled filaments at time zero shortens the initial acceleration period, in a manner dependent on the number of barbed ends rather than on the mass of added F-actin. This feature is suggestive of barbed end branching by Arp2/3. It is further consistent with the measured length correlation of mother and daughter filaments ; Contrad−1 s−1 per F-actin subunit); growth of a daughter branch occurs extremely infrequently, from only 1 % of the filament-bound Arp2/3 [TIRF microscopy was used to visualize live assembly and branching of single filaments anchored by myosin or maintained in close proximity of the glass surface . In thisd Arp2/3 , and is d Arp2/3 . The remBiochemical and structural analysis of the interactions between VCA, actin and Arp2/3 complex is at the heart of the possible mechanisms of filament branching. Binding of Arp2/3 to VCA induces a structural change in Arp2/3 complex that strengthens binding of ATP to Arp2 . The ArpThe VCA–actin–Arp2/3 complex, considered as the “branching complex” that interacts with the mother filament , displayTechnical difficulties that have slowed our progress include the limited available resolution in the structures of large macromolecular complexes, the limited available amounts of native and recombinant genetically modified Arp2/3 complex, and the 2D constraints imposed by TIRF microscopy in monitoring formation of the 3D structure of branched filaments. Due to these difficulties, the proposed models are dominated by assumptions made ab initio on structural changes in the organization of the Arp2/3 complex during branching.The high density of the intricate array of branched filaments in the actin tail of propelling pathogens precludes the accurate tracking of individual filaments in electron micrographs , 114. ThAssuming that a pool of polymerizable actin monomers is established and feeds site-directed barbed end elongation, an unsolved issue concerns the seemingly homogeneous rate of protrusion and filament turnover in the lamellipodia, where both dendritic filament arrays and formin-mediated filopodia and microspikes co-exist . ForminsThe control of actin filament barbed end dynamics is mediated by a large number of soluble as well as membrane-bound effectors, which may act either directly by binding barbed ends, or indirectly by affecting the on flux of actin monomers at barbed ends.Effectors that bind barbed ends may either exclude each other or bind in synergy together at the barbed ends, or may displace each other from the barbed end via formation of transient ternary complexes. The binding of regulatory proteins may further be affected by the state of the nucleotide bound to actin subunits at the barbed end. Many of these processes also include irreversible ATP hydrolysis. Elucidating such complex binding schemes, will require more extensive biochemical, kinetic, as well as structural studies.High resolution electron microscopy of complexes formed at the barbed ends is anticipated to foster our progress. Rapid kinetics of the changes in reactivity of filament barbed ends using microfluidics-assisted TIRF microscopy of individual filaments, in assembly and disassembly regimes and presence of various regulatory ligands, is clearly an avenue for mechanistic studies.Because most of the regulation of polarized filament assembly is mediated by regulators that are bound both to membranes and to filament barbed ends, the chirality of the growing helical filament plays an important role. A consequence of the chirality of the actin filament is revealed at high scale in the chiral organization of cytoskeletal patterns of radial and transverse fibers in fibroblasts constrained to a circular shape . Future


Understanding the role of acidosis in osteoclast biology may lead to development of novel therapeutic approaches for the treatment of diseases related to low bone mass. In this review, we aim to discuss the recent investigations into the effects of acidosis in osteoclast biology and the acid-sensing molecular mechanism.The adverse effect of acidosis on the skeletal system has been recognized for almost a century. Although the underlying mechanism has not been fully elucidated, it appears that acidosis acts as a general stimulator of osteoclasts derived from bone marrow precursors cells and enhances osteoclastic resorption. Prior work suggests that acidosis plays a significant role in osteoclasts formation and activation  LocalizOsteoclasts are the only cells known to be involved in resorption of large quantities of bone material and mineralized cartilage , (b) Fusion of these TRAP-positive mononuclear preosteoclasts to become non-functional polykaryons, and finally, (c) the activation of these nonfunctional multinucleated cells into fully activated functional osteoclasts  differentiation of monocytic/macrophage lineage precursor cells into preosteoclast cells expressing mononuclear tartrate-resistant acid phosphatase (TRAP) gene  concentration, which further stimulated the nuclear translocation/activation of NFATc1 and promoted osteoclasts bone resorption activity  are located in areas where osteoclasts adhere to bone during osteoclastic resorption  using the intracellular Ca2+ chelator, BAPTA, abolished the ability of acidosis to increase osteoclast survival. Calcium signaling results in activation of protein kinase C (PKC), which either regulates the phosphorylation status of pro- or anti-apoptotic proteins, or promotes ERK1/2 phosphorylation. Activation of the MAPK pathway is known to be important for osteoclast survival. Studies using PKC inhibitor also showed that pharmacological inhibition of PKC completely blocked acidosis-induced prolongation of osteoclasts survival, suggesting that acidification-increased osteoclast lifespan was dependent on PKC activation. Although activation and nuclear translocation of NFATc1 have been suggested to be critical for osteoclast differentiation and activity under acidic conditions  fusion of mononuclear pre-fusion osteoclasts into multinucleated osteoclasts; (2) attachment of osteoclasts to the bone surface; (3) polarization [characterized by ruffled border and clear zone (actin ring)], increased secretion of acid and lysosomal enzymes into the space beneath the ruffled border; and (4) apoptosis  motif on the bone matrix components, osteopontin and bone sialic protein  and non-GPCR sensors  gene expression and subsequent prostaglandin E2 (PGE2) production. This regulation was sensitive to Gq/11 inhibitor and OGR1-specific siRNA, which stimulate bone resorption activity of osteoclasts by inducing the RANKL/RANK system. It has also been reported that extracellular acidic pH, mimics RANKL and induces the Ca2+/calcineurin/NFATc1 pathway, which is critical for osteoclast differentiation and function, possibly by activated OGR1 in the osteoclasts (Komarova et al., in vivo and in vitro, and is crucial for osteoclast differentiation. Iwai et al. (2+]i levels (Pereverzev et al., 2+]i levels in primary calvarial cells and Ca2+ efflux from calvariae (Figure 2+ signaling pathway. Further evidence for a positive role of OGR1 in osteoclastogenesis was detected in OGR1 knockout mice (Li et al., in vivo X-ray scans did not show overall abnormality in the bones of these animals (Li et al., in vivo loss of OGR1 increased bone mineral density probably by increasing the bone formation and directly contributing to the decreased bone resorption that was observed in rapidly growing mice (Krieger et al., More specifically, OGR1 has been shown to be activated by acidosis to increase i levels in osteoclasts and also involved in extracellular acidification-stimulated NFATc1 signaling in osteoclastogenesis (Figure ASICs are proton-gated channels that are distributed throughout the central and peripheral nervous systems. Currently, there are six isoforms of ASICs (Xiong et al., s Figure .Recent evidences based on animal model studies, implicate several acid sensing receptors in various diseases. However, going forward it will be important to determine how our current knowledge of acid sensing receptors can be translated to human patients. One way would be to first identify if there are any existing genetic associations between acid sensing receptors and human bone resorption-related diseases. Another path would be to directly test if inhibitor of acid sensing receptors can produce beneficial effects in human patients. In addition, a better understanding of the molecular mechanisms involved in action of acid sensing receptors will help to clarify how inhibiting or potentiating these receptors could affect the pathophysiology and behavior of human patients. In this review, we have tried to highlight the critical role of acidosis in osteoclast fusion, differentiation, activity, survival, adhesion, migration and acid sensing mechanism. This effort will help to provide further insight into the molecular and cellular understanding of bone resorptive disorders in an acidic environment.All authors wrote, edited and approved the final submission of the manuscript. FY, JD, HX, and WF conceived the project and designed experiments. XL, MX, HX, and JD performed experiments and collected and analyzed data. All authors developed analytical tools.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.


On posttreatment radioiodine scintigraphy, mediastinal 131I uptake was noted that finally was histologically/anatomically diagnosed as thymus gland uptake. It should be borne in mind as a potential cause of false-positive whole-body 131I scintigraphy.Radioiodine uptake is not commonly seen by the thymus gland. On the contrary, the gland is slowly replaced by fat after puberty. Herein, we present 2 patients with papillary thyroid carcinoma, follicular variant, and cervical lymph node involvement. After total/near-total thyroidectomy, the patients received  Radioiodine (131I) therapy is often used as an adjunct to surgery because it has been reported to improve survival rate.3 The whole body scan (WBS) with 123I or 131I is the gold standard imaging for the detection of thyroid remnant and regional or distant metastases, thus being routinely used for evaluation before and after radioiodine therapy.7 However, false-positive WBS may occur because of radioiodine uptake by normal or pathological tissues that are not related to the thyroid tissue. This could lead to unsuitable therapy, such as unnecessary surgery or administration of 131I. There are many normal tissues that commonly accumulate iodine, such as breasts, salivary glands, gastrointestinal, and urinary systems.5 In addition, pathological uptake has been reported in neoplastic diseases or pulmonary inflammatory, sinusitis, pericardial effusions, esophagus pathologies, urinary tract diseases, ovarian cysts or tumors, and traumatic lesions.8 Radioiodine uptake by normal thymus is an uncommon reason of false-positive WBS that might be considered as lung or mediastinal nodes metastases.12 It should be borne in mind to recognize this condition in order to facilitate distinction between this normal variant and metastases. We present two patients with DTC, who underwent total thyroidectomy and 131I therapy that had 131I concentrate in the mediastinum during the follow-up, and provide a review of the latest insights.Differentiated thyroid cancer (DTC) is an unusual tumor with a good prognosis when adequately treated, but can recur locally or with distant metastases even decades after initial therapy.131I for ablation (first 131I therapy). First posttreatment scan was only positive in the superior mediastinum with no evidence of cervical region uptake  of 50 mCi of131I 1 year ago. In first posttreatment WBS, thyroid remnant tissue with no evidence of regional or distant metastasis was noted. Diagnostic WBS was performed 12 months after ablative therapy and was in favor of lymph node metastasis in the left-side of the neck. The patient submitted to neck dissection and then received 4440 MBq (120 mCi) of 131I. Second posttreatment WBS was only positive in the superior mediastinum with no evidence of cervical region uptake and the uptake in the mediastinum appeared to increase from 2 to 14 days  of 2 Cognizance of false-positive findings in the diagnostic or posttreatment WBS in patients with DTC is essential to avoid unnecessary treatment. False-positive findings in the chest may be because of inflammatory processes, ectopic thyroid tissue, neoplasms of nonthyroidal origin, body secretions, and pathological transudates.13 Thymic uptake of radioiodine is one of the possible causes that can be either because of the misinterpretation of metastatic mediastinal spread or because of an intrathymic ectopic thyroid tissue,13 thyroidal metastases to the thymus,14 residual normal thymus,12 or thymic hyperplasia.11 The mechanism of iodine uptake by the thymus is not yet fully understood. Vermiglio et al15 considered the possibility of iodine accumulation in the Hassal's bodies present in the thymic tissue because of the similarity to the follicular cells of the thyroid. Spitzweg et al16 showed the presence of the human Na+/I symporter (hNIS) in extra thyroidal tissue, including the thymus gland, with diminished capacity to transport and concentrate iodine, compared with the thyroid gland, which could explain why higher activities, late scanning, after the ablation of the thyroid remnant, and absence of metastatic spread (loss of tissue with higher hNIS expression than the thymus), increase the possibility of visualization of the thymic tissue after administration of radioiodine.13 In the present case report, two patients presented thymic uptake in the posttherapy WBS. Surgical removal of mediastinal mass in 1 patient was done with histopathologic verification of thymic tissue, without any evidence of thyroidal or neoplastic cells. Another patient, without histological confirmation, had a CT scan indicative of thymus tissue and is in periodic monitoring. The association between thymic hyperplasia and benign thyroidal disease, especially Graves disease, is well known. The identity of Thyroid Stimulating Hormone (TSH) receptors in the human thymic tissue has also been reported.20 Niendorf et al21 for the first time showed relationship between thymic hyperplasia and DTC. The mechanism of this association is not thought to be similar to the thymic hyperplasia demonstrated after chemotherapy or after stress conditions or acute infections, when hyperplasia happen because of an immunological reaction.22 It seems that l-thyroxin hormone therapy in supraphysiological doses is responsible for the thymic hyperplasia in patients with thyroid carcinoma.21 Heath et al,23 with the technique of reverse transcription and polymerase chain reaction from analyses of mRNA, showed the thymic expression of thyroglobulin (Tg) in embryonic mice and adult rats. Sospedra et al24 reported transcription of Tg in 4 of 12 specimens of human thymus, and Gotter et al25 found the existence of Tg expression in medullary epithelial cells of human thymus. Mello et al4 considered the possibility of the thymic tissue as a source of benign production of Tg, and suggested randomized studies to evaluate whether the Tg generation by the thymus is actually efficient and whether it could be stimulated by TSH. Michigishi et al9 considered reliable criteria to distinguish malignant from benign mediastinal uptake. Thymic uptake that visualized only by higher activities on posttreatment scintigraphy becomes more intense with repeated 131I treatment, low serum Tg levels, younger age (<45 years) and a hyperplastic thymus indicative of nonmetastatic radioiodine accumulation. It should be born in mind that beside thymic uptake, radioiodine accumulation in the midline of the thorax may be because of various causes, for example tracer accumulation in the esophagus, hiatal hernia, and aspiration of radioactive saliva in the tracheobronchial tree.26Radioiodine is very valuable for the diagnosis and treatment of both benign and malignant residual thyroid tissue in patients who have submitted to thyroidectomy for DTC.