The ability to form long-lasting memories is critical to survival and thus is highly conserved across the animal kingdom. By virtue of its complexity, this same ability is vulnerable to disruption by a wide variety of neuronal traumas and pathologies. To identify effective therapies with which to treat memory disorders, it is critical to have a clear understanding of the cellular and molecular mechanisms which subserve normal learning and memory. A significant challenge to achieving this level of understanding is posed by the wide range of distinct temporal and spatial profiles of molecular signaling induced by learning-related stimuli. In this review we propose that a useful framework within which to address this challenge is to view the molecular foundation of long-lasting plasticity as composed of unique spatial and temporal molecular networks that mediate signaling both within neurons  as well as between neurons . We propose that evaluating how cells integrate and interpret these concurrent and interacting molecular networks has the potential to significantly advance our understanding of the mechanisms underlying learning and memory formation. The ability to form long-lasting memories is an evolutionarily conserved phenomenon that is critical to survival, and the dynamic process of acquiring and storing memories is often compromised in psychiatric and neurodegenerative disorders. It is thus important to understand how memory formation occurs in healthy individuals, and how these processes go awry in pathological states.de novo gene expression )GFs are capable of playing different roles in the different cellular and subcellular compartments in which they are engaged. In particular, mammalian neurotrophins, including NGF, BDNF, NT-3, and NT-4/5, have been extensively studied as signaling modulators critically involved in synapses undergoing learning-induced plasticity. Indeed, numerous studies have shown that the mRNAs and proteins of neurotrophins are widely distributed throughout the mammalian CNS in distinct, but largely overlapping expression patterns in regions important for learning and memory including the hippocampal formation, amygdala, and cortex –97. AddiAdding to the complexity, neurotrophins can be secreted from either postsynaptic dendritic spines or from presynaptic terminals, and both their synthesis and release in the adult central nervous system seems to be activity-dependent ,114,115.Once released at a synapse, neurotrophins as well as other GFs bind to their putative receptors and induce intracellular signaling leading to changes in synaptic transmission and connectivity. Multiple studies in hippocampal and cortical cultures and slices ,126 haveActivity-dependent release of GFs, which is common during a learning event, often occurs at the synapse ; howeverGF signaling is also engaged in temporally discrete profiles during learning and memory formation. BDNF signaling, as discussed above, is often engaged very early in response to neuronal activity . InteresAplysia.Despite extensive data reporting very precise spatial and temporal regulation of both intracellular signaling (i.e. kinase activation) as well as their upstream activators (i.e. GFs), a critical issue remains: what is the meaning of this signaling for the cell? Specifically, how are these disparate  molecular networks integrated into a coherent message? And, what is the value of this complexity? One possible answer is that the integration of individual spatiotemporal profiles is capable of relaying vastly more information than each signal considered in isolation. It is thus possible that individual temporal and spatial molecular networks interact to serve “coincidence detection” functions. In the section that follows we will examine possible mechanisms of spatial and temporal integration, specifically highlighting mechanisms of synapse—soma communication as this trans-compartmental communication contains both a temporal and spatial component. We will then review in detail a specific example of the notion of coincidence detection in GF signaling networks: Two-Trial LTM formation in de novo gene expression [Long-term forms of cellular and behavioral plasticity are distinguished from other forms of memory  by their requirement of pression ,53. HoweMicrotubules are dynamic elements of the cell structure that serve as ‘tracks’ upon which molecular motors can transport cargo. Microtubules typically run throughout the cell and are able to integrate indirectly with the actin framework through cross-linking cytoskeletal proteins such as the CLASP family of proteins –141. TheAplysia [Microtubules tend not to extend into dendritic spines, the primary sites of synaptic input, where instead there is a high concentration of actin filaments ,140. MyoAplysia . TogetheAplysia, as a model, which demonstrates the spatial and temporal integration of distinct GF signaling networks to synergistically support LTM formation.We have briefly discussed how the soma and synapse can communicate, and some aspects of the temporal regulation of this communication. However, because molecular cascades are often studied in isolation, it is experimentally difficult to determine if there are instances in which distinct molecular networks are integrated to mediate functional outcomes that neither network, on its own, can support. We will now review a study using the marine mollusk, Aplysia is well-suited for interrogating the molecular mechanisms of learning and memory formation [Aplysia lasts days to weeks and requires both translation and transcription [The simple central nervous system of ormation . The deformation –149. Upocription .Aplysia, like other species, is responsive to the pattern of training. For instance, four or five repeated, spaced training trials [Aplysia: (i) during the training interval and (ii) after training. A single training trial induces an early phase of MAPK activation in SN somata 45 minutes after Trial 1 [Memory formation in g trials  yields sg trials ,152, whig trials . MAPK ac Trial 1 ,43. This Trial 1 ,43. Howe Trial 1 ,42,154.  Trial 1 ,37.Aplysia. Interestingly, Trial 2 must be delivered within a restricted temporal window: if occurring at 15 minutes or 60 minutes after Trial 1, no LTM is induced [c/ebp [Aplysia: neurotrophins signaling via the receptor TrkB [Philips and colleagues ,43,149 d induced ,43. Colld [c/ebp . Trial 2tor TrkB ,156 and tor TrkB ,157–160.tor TrkB  analyzedThe data demonstrated a striking double-dissociation in the temporal and spatial regulation of these GFs during learning-related stimuli. Trial 1 initiated TrkB signaling at the SN-MN synapse, which was required for early-phase MAPK activation in SN cell bodies. Importantly, these data necessitate an intracellularly transported retrograde signaling to exert the somatic effects of synaptic TrkB signaling. Trial 2 initiated TGFβr-II signaling at SN cell bodies, which was required for late-phase MAPK activation in the same compartment. Surprisingly, the later, TGFβr-II-dependent phase of MAPK was not disrupted if Trial 1 TrkB signaling was inhibited, suggesting that these two GF families were signaling independently despite being in temporal sequence.c/ebp mRNA expression in Aplysia [c/ebp expression was examined. In agreement with the MAPK data, Trial 1 TrkB signaling was required for Trial 1 c/ebp expression, and Trial 2 TGFβr-II signaling was required for Trial 2 c/ebp expression. However, unlike the MAPK data, when TrkB signaling was inhibited during Trial 1, Trial 2 c/ebp expression was also disrupted. These data suggest that Trial 1 TrkB signaling increases the expression of c/ebp mRNAs , and Trial 2 TGFβr-II signaling, rather than engaging a new wave of transcription, prolongs the expression of Trial 1 mRNAs. Finally, in a set of experiments to determine if these molecular observations were behaviorally relevant, Kopec and colleagues [Aplysia.Kopec and colleagues  next sou Aplysia ,161. A uc/ebp expression). Conversely, TGFβr-II signaling was initiated somatically to mediate later, Trial-2 dependent molecular outcomes. Moreover, TrkB signaling resulted in synapse-soma communication, while TGFβr-II signaling prolonged the expression of existing mRNAs. Importantly, Trial 1, despite activating TrkB signaling, synapse-soma communication, MAPK, and transcription, cannot by itself produce LTM [de novo gene expression readily occurs in response to stimuli that alone cannot induce LTM is of critical importance: transcription is necessary, but not sufficient, for the formation long-lasting plasticity. Instead, the coincident arrival of another learning-related signaling cascade, at the appropriate time and in the appropriate subcellular compartment, must occur. Thus, the molecular events induced by Trial 2 serve a coincidence detection role in the cell such that the circuit initiates long-lasting information storage.This report exemplifies many of the principles discussed in the present review see . First, duce LTM . Transcrc/ebp expression, which could result in the increased likelihood of its translation, and thus increased activity of its functions as a transcription factor (i.e. more new gene expression). Interestingly, c/ebp mRNAs contain an AU-rich element in their 3’ untranslated regions, which is a motif targeted by the Hu/ELAV family of RNA binding proteins that can stabilize mRNAs [Precisely what signal induced by Trial 2 is most salient at conferring coincidence is at present unclear, and it is likely that an interaction of several events contribute to this process. However, because TGFβr-II signaling during Trial 2 is required for Two-Trial LTM formation, consequences of the signaling via this GF are likely to be involved. These include, but are not limited to (i) an independent phase of MAPK activity, which could have numerous downstream outcomes, and (ii) prolonged ze mRNAs –164. Posze mRNAs , and thuze mRNAs –168. ThuAplysia offer the spatial resolution needed for these mechanistically detailed studies, as exemplified in the case study above. As it stands, five different GFs have been reported to have a role in plasticity in Aplysia [Collectively, the studies reviewed here support the notion depicted in  Aplysia ,171–173, Aplysia .Aplysia, augmented or restored TrkB signaling may not be sufficient to restore LTM if TGFβr-II signaling is not efficiently interacting with TrkB-dependent signaling. Second, GF signaling is highly spatially regulated and this specificity may well be important for the neural circuit to correctly and effectively interpret the molecular signaling. Utilizing a systemic drug, despite having restored GF signaling, may disrupt the spatial signature of a GF family and thus disrupt behavior. Taking the data from Kopec and colleagues [Aplysia, which may well be the case for other forms of plasticity underlying various forms of learning. Without an understanding of when a GF family is recruited to support behavior, therapeutic interventions may miss the time frame in which it could be most effective. For instance, if TGFβr-II signaling was augmented one hour after Trial 1, apc/ebp expression would have returned to baseline levels and TGFβr-II signaling would not have the opportunity to interact with the TrkB-dependent signaling cascade, and thus may not result in the same behavioral outcome.The notion of spatial and temporal molecular networks is particularly important from the perspective of translational research and therapeutic intervention. Many GFs, including BDNF-TrkB and TGFβ-TGFβr signaling, have been suggested to be dysregulated during cognitive dysfunction of various etiologies in humans, including Alzheimer’s disease –176. GF lleagues  as an ilIn conclusion, plasticity underlying memory formation is an exquisitely complex interplay between differential temporal and spatial molecular networks. Different signaling cascades, including kinases and GF signaling reviewed here, could be initiated in (i) an autocrine or paracrine fashion, (ii) simultaneously or in different temporal phases, resulting in (iii) local or long-distance (e.g. synaptic vs. somatic) molecular consequences, all of which increase the likelihood of interactions and synergism between cascades. Moreover, there can be salient information in these interactions and instances of synergy, such that cells can interpret ‘coincidence,’ pattern, and statistical probability in a series of learning-related stimuli. Discovering how cells integrate and interpret these dynamic networks is an extraordinary challenge, but this challenge is essential to tackle, as it is critical not only for understanding how the plasticity underlying learning and memory formation normally occurs, but also for designing effective therapies to intervene when this plasticity goes awry.


Modulation of Treg function has been proposed as a means of treating autoimmune conditions and preventing rejection of organ transplants, although achieving this goal will require a detailed understanding of Treg signaling pathways. Signaling within Tregs is known to differ considerably from that observed in other T cell subsets. Of note, Tregs are the only cell type known to constitutively express CD25, the main ligand-binding subunit of the IL-2 receptor. The PI(3)K/Akt/mTOR cascade constitutes a major signaling pathway downstream of IL-2 and is closely tied to cellular metabolism. Due to increasing recognition of the links between cellular fuel usage and immune cell function, the interplay between IL-2 signaling and Treg metabolism represents an important space for exploration and a potential approach for immunomodulation. Here, we discuss how IL-2 may affect Treg metabolism via PI(3)K signaling, as well as the effects of altered metabolism on Treg lineage stability and suppressor function.CD4 Modulatpulation  which ispulation . Althougpulation  or humanpulation  lacking pulation  or humanpulation  lacking pulation , 17.It is now appreciated that cell-intrinsic metabolic pathways directly impact cellular fate and function . Broadlyc  subunits K, and MAPK/ERK pathways. STAT5 is particularly important for Treg development, as it is necessary to initiate Foxp3 expression  (−/−(−/− (−/− (+ cells and do not develop autoimmunity.The fact that IL-2 and CD25 knockout mice maintain appreciable numbers of Foxp3d spleen  indicateubunits) , 36. In its) K signaling and subsequent glycolysis, with PD-1 also actively promoting FAO by increasing expression of CPT1A , which is needed for optimal Treg suppressor function. Engaging glycolysis forces Enolase-1 into the cytoplasm, thereby allowing transcription of Foxp3-E2 , or with dichloroacetate (an inhibitor of PDHK isoforms) reduced the severity of experimental autoimmune encephalomyelitis with associated decreases in the percentage of Th17, but not Treg, cells , 84. SimConversely, interventions that promote glycolysis enhance immune function, presumably by increasing the proliferation and function of effector T cells while inhibiting Treg function. Pharmacological blockade or genetic loss of PTEN leads to Akt-dependent inhibition of Foxo3a and subsequent loss of Foxp3 and tumor regression . FurtherThe metabolic state of Tregs defies simple categorical explanations with regard to glycolysis and FAO. Although elevated glycolysis is generally associated with immune activation and can be detrimental to Treg lineage stability and function, controlled levels of glycolysis are necessary to sustain the same processes. The list of known links between metabolism and Treg function is far from complete, and the coming years will likely reveal other metabolic enzymes with moonlighting roles in Treg biology. In particular, the “futile cycle” approach of tTregs to FAO, and its preference for cholesterol synthesis may be a promising area of discovery.Metabolic interventions offer a promising new approach to modulating Treg function and may be used to fine-tune therapies targeting other signaling pathways or used as a primary therapy in their own right. Of note, while there is clear potential for interplay between IL-2 signaling and immunometabolism through the PI(3)K/Akt/mTOR signaling cascade, to date no studies have specifically evaluated the effects of IL-2 signaling on Treg metabolism. In part, this is due to the inadequacies of germline knockout models to address this question. As mentioned before, such knockouts experience an autoimmune environment in which immune cells are already highly active and presumably glycolytic. It would be more appropriate to use a model in which Tregs can be inducibly made to lose IL-2 signaling while maintaining immune homeostasis. A tamoxifen-inducible CD25 knockout, with tamoxifen dosage adjusted to leave enough CD25-competent cells to prevent autoimmunity, would be well suited for this approach. Such studies would lay the framework for combination treatments in which metabolic interventions would be used with existing therapies such as CD25 blockade.MF and LT conceived of and wrote the review.LT discloses personal or family financial interests with Third Rock Ventures, Tango Therapeutics, and Neon Therapeutics. LT has served as a consultant to Lycera, Solid Bio, UCB, and Intellia. M.F 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.


Danio rerio (zebrafish). At least part of that role is achieved by its interaction with the mTORC2 component SIN1, but the mechanistic details of this interaction and/or additional functions that CCDC28B might play in the context of cilia remain poorly understood. Here we uncover a novel interaction between CCDC28B and the kinesin 1 molecular motor that is relevant to cilia. CCDC28B interacts with kinesin light chain 1 (KLC1) and the heavy chain KIF5B. Notably, depletion of these kinesin 1 components results in abnormally elongated cilia. Furthermore, through genetic interaction studies we demonstrate that kinesin 1 regulates ciliogenesis through CCDC28B. We show that kinesin 1 regulates the subcellular distribution of CCDC28B, unexpectedly, inhibiting its nuclear accumulation, and a ccdc28b mutant missing a nuclear localization motif fails to rescue the phenotype in zebrafish morphant embryos. Therefore, we uncover a previously unknown role of kinesin 1 in cilia length regulation that relies on the BBS related protein CCDC28B.Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, polydactyly, renal disease and mental retardation. CCDC28B is a BBS-associated protein that we have previously shown plays a role in cilia length regulation whereby its depletion results in shortened cilia both in cells and  The physiological relevance of primary cilia is best supported by the fact that its dysfunction results in a number of clinical manifestations, including retinal degeneration, obesity, cystic kidney disease, central nervous system malformations and skeletal defects, while its complete absence is incompatible with life. Furthermore, several of these phenotypes are now considered hallmarks of an underlying ciliary defect and characterize the group of human disorders known as ciliopathies8.Primary cilia play a critical role in the regulation of cellular and tissue homeostasis acting in mechano- and chemo-sensation and paracrine signaling. Several important cascades have been shown to operate through the cilium or depend on the integrity of the organelle for their correct sensing and transduction including Sonic Hedgehog (Shh), transforming growth factor β (TGFβ), platelet-derived growth factor receptor α (PDGFRα) and Wnt12. CCDC28B was shown to interact with a number of BBS proteins and reduced levels of CCDC28B due to a splicing mutation correlated with a more severe presentation of the disease in some families10. More recently we have shown that CCDC28B plays a role in cilia length regulation both in cells and in vivo in Danio rerio (zebrafish)14. We have shown that CCDC28B modulates cilia length at least in part through its interaction with SIN1, a member of the mTOR complex 2 (mTORC2), but independently of the mTORC2 pathway in a process not completely understood13.CCDC28B was originally identified as a second site modifier of the ciliopathy Bardet-Biedl syndrome  although its contribution is likely variable among cohorts16. Therefore, dissecting the mechanism by which CCDC28B affects cilia length is critical to understand its role in human disease. Here we demonstrate that CCDC28B associates with the kinesin 1 molecular motor via an interaction with kinesin light chain 1 (KLC1). In the kinesin 1 motor complex the light chains associate with KIF5 heavy chains which are encoded by three genes in mammals: KIF5A, KIF5B and KIF5C. These proteins present a similar motor domain located on the N-ter region (N-kinesins) but distinct cargo binding motifs on their C-ter region18. We show here that CCDC28B interacts not only with KLC1 but also with KIF5B.Ciliogenesis and ciliary length are tightly regulated and the later varies between different cell types suggesting that it can influence or determine specific ciliary functions. Moreover, both short and abnormally elongated cilia have been associated with cilia dysfunction and the ciliopathies19. Importantly, different kinesin motors have been shown to play an important role in cilia formation and cilia length regulation. Heterotrimeric kinesin 2 in mammals and other kinesins in other organisms are a key component of the process of intraflagellar transport (IFT). This motor-based machinery moves cargo, IFT particles, important for the formation and function of the cilium both into and out of the organelle. Kinesin 2 is responsible for anterograde movement (from base to tip) while cytoplasmic dynein mediates the retrograde (tip to base) transport21. Not surprisingly, affecting either structural or regulatory components of IFT has a direct impact on cilia length25.The kinesins are members of a superfamily of proteins (KIFs) that is composed of 15 families (kinesin 1 to kinesin 14B)27. Furthermore, KIF2A is activated by the cell cycle regulated Polo-like kinase 1 (PLK1), thus promoting cilia disassembly in proliferating cells27. Other kinesins localize inside the cilium and their depletion results in abnormally elongated cilia. For example, KIF7 (kinesin 4) and KIF19A (kinesin 8) organize intra-ciliary compartments and affect the stability of the axoneme and tubulin levels through microtubule depolymerizing activities29. In addition, and depending on the context and organism, kinesin 13 members have been shown to regulate cilia length either entering the cilium and affecting microtubules in the ciliary compartment or by controlling cytoplasmic microtubule depolymerization and hence the availability of tubulin for cilia formation and maintenance33. Thus, we reasoned that the CCDC28B-Kinesin 1 interaction could be relevant to further understand the role of the BBS-associated protein in ciliogenesis.In addition to those involved in IFT, other kinesins are known to play a role in cilia biology and cilia length regulation, linking assembly/disassembly of the organelle with the cell cycle. Some kinesins have been shown to promote cilia shortening by affecting microtubule dynamics either in the intraciliary compartment (axoneme) or at the basal body. The kinesin 13 family members KIF24 and KIF2A both localize to the centrosome/basal bodies and remodel cilia through depolymerization of microtubulesHere we show that both KLC1 and KIF5B are found at the basal body region of cilia and play an inhibitory role on cilia extension whereby their depletion results in abnormally elongated cilia. Thus, our results uncover a previously unknown role for kinesin 1 in cilia length regulation. Importantly, through genetic interaction experiments in cells we show that this activity of kinesin 1 is mediated by CCDC28B. Furthermore, our data indicate that kinesin 1 regulates the subcellular localization of CCDC28B, unexpectedly, affecting the nuclear/cytoplasmic distribution of the protein whereby targeting kinesin 1 results in nuclear accumulation of CCDC28B. Importantly, we show that eliminating residues encompassing a NLS domain in the zebrafish ccdc28b ortholog impairs its capacity to rescue the depletion of ccdc28b in zebrafish. Altogether, our results underscore a novel role for a main molecular motor in cilia length regulation and provide further insight into the function of the Bardet-Biedl associated protein CCDC28B, a critical step to understand its contribution to the pathogenesis of BBS.13. Temperature sensitive cdc25h yeasts were able to grow at the non-permissive temperature of 37 °C only when pMyr-KLC1 was co-expressed with pSOS-CCDC28B but not with the pSOS empty vector (EV)  as bait and identified an interaction with kinesin light chain 1 (KLC1) cloned in the pMyr library vector as reportedEV) Fig. . To confEV) Fig. . In addiEV) Fig.  to immunEV) Fig. . Overall14 and hypothesized that the CCDC28B-kinesin 1 interaction could be relevant to this function. There are three isoforms of KLC1, which only differ in their C-termini . In contrast, the single KD of the other KIF5s was not sufficient to affect cilia length , targeting either exon 2 (first coding exon) or exon 3 of CCDC28B, and the Cas9 nuclease36 and transduced hTERT-RPE cells. We selected transduced cells with puromycin and assessed levels of CCDC28B 3 and 10 days after transduction. While we did observe a reduction in CCDC28B levels at day 10 for most gRNAs, in some cases there appeared to be a recovery of CCDC28B when compared to the knockdown at day 3  mutations leading to premature stop codons (PTCs) due to insertions of 2 and 1 bases respectively (not shown). In the ones that did grow, we checked CCDC28B levels by western blot and unexpectedly detected the protein in all cases Fig. . AltogetCCDC28B mRNA and at lower levels than the wild type mRNA from control cells  and the first 127 bases of intron 2 , which normally elongates cilia, resulted in a subtle rescue in the proportion of cilia-positive cells in CCDC28B clone B1 Fig. . Since tent Fig. . Thus, o13 we observed signal at the pericentriolar region at the base of cilia, as was previously reported10, but also in a diffuse cytoplasmic pattern and in the nuclear compartment 38, is functional. This NLS is conserved between human and zebrafish and therefore we deleted residues 4 to 10 (Δ4–10) in both orthologs to generate NLS mutants. We determine that the human CCDC28B NLS mutant retains its capacity to interact with KLC1 , encompassing residues 4 to 10 in CCDC28B  zebrafish ccdc28b mRNA14. To further confirm their specificity however, we performed genome editing in zebrafish embryos targeting exon 2 of ccdc28b by CRISPR/Cas9. We designed and tested four different gRNAs  classified morphant embryos into three different categories according to their external phenotype  mutant ccdc28b could rescue the phenotype of ype Fig. : class Aype Fig. , similarype Fig. .To assess rescue we scored embryonic phenotypic classes in five independent experiments. For each one we injected embryos obtained from one female fish to generate all the experimental conditions , and all the controls needed for validation . By doing this we eliminated the variability that could be introduced by using different batches of embryos but resulted in low number of embryos per condition in each individual injection.ccdc28b MO alone resulted in 41% class A, 46% class B and 13% class C. Co-injecting the wt ccdc28b mRNA increased the percentage of mildly affected class A embryos (67%) and reduced the more severe Class B to 19% .Therefore, we first analyzed the individual experiments Fig.  and thenccdc28b could rescue cilia defects specifically. We processed age-matched 12-somite embryos to visualize cilia in the Kuppfer’s vesicle (KV) by confocal microscopy using antibodies against acetylated- and γ-tubulin . Co-injecting the wt mRNA rescue the phenotype of the morphants restoring cilia length to control values with a median of 3.8 μm (P = 0.339). In contrast however, the cilia length of morphant embryos co-injected with ccdc28b Δ4–10 was significantly different from controls  and embryos co-injected with wt ccdc28b mRNA (P = 0.0051)  Fig. . These r14. Therefore, we have hypothesized that the main role of this BBS associated protein is to mediate the function of proteins that physically interact with it, for example the BBS proteins and SIN113. In this context we reasoned that it was vital to continue with the identification of proteins interacting with CCDC28B, as this would represent a critical step to understand its cellular function. Here we uncovered a link between CCDC28B and kinesin 1, providing novel insights to understand the function of CCDC28B and importantly, linking a major kinesin motor with cilia length regulation.CCDC28B is a 200 amino acid protein characterized primarily by a coiled coil domain located at its C-terminal region, a motif involved in protein-protein interactionsAs mentioned, kinesin 1 is composed of both heavy and light chains. There are three different KIF5 heavy chains that can be part of the kinesin 1 molecular motor: KIF5A, KIF5B and KIF5C. By knocking down each one individually or in combination, our data show that KIF5B is the main heavy chain involved in this novel cilia-related function in hTERT-RPE cells. In contrast, we found that KIF5A is not heavily expressed in this cell line while KIF5C KD alone was not sufficient to affect cilia. However, KIF5C KD in combination with KIF5B KD consistently resulted in a mild increase in ciliary length compared to KIF5B KD alone, although not statistically significant. Thus, KIF5C could play at least a minor role regulating cilia. It will be interesting to test whether the contribution of different kinesin heavy chains to ciliogenesis varies between different cell types.33. Although it is difficult to conclude based on negative findings, a role of kinesin 1 modulating axonemal microtubules is unlikely since we were not able to detect KLC1 or KIF5B inside cilia. Supporting this notion, KLC1 KD or KIF5B KD did not produce gross changes in cilia ultrastructure or axonemal tubulin acetylation levels, features that have been reported upon KIF7 KD, a kinesin that enters the cilium and has microtubule depolymerizing activity28. We did detect KLC1 and KIF5B at the pericentriolar region at the base of cilia, which could be due to the increased density of microtubules in the region of the microtubule organizing center. At this location, microtubule depolymerization by KIF2A has been shown to promote cilia disassembly27. However, kinesin 1 has not been reported to present a microtubule depolymerizing activity but rather to stimulate microtubule elongation in the cytoplasm through the modulation of the c-Jun N-terminal kinase (JNK) pathway39. Furthermore, our biochemical and genetic studies underscore a different role for kinesin 1 on cilia linked to CCDC28B.Different kinesins have been shown to participate in ciliogenesis. For example kinesin 13 , kinesin 4 (KIF7), and kinesin 8 (KIF19A) affect cilia by modulating microtubule dynamics at either the basal body, the ciliary axoneme or in the cytoplasmWe show that CCDC28B can physically interact with the kinesin 1 motor and our genetic interaction studies strongly suggest that kinesin 1 and CCDC28B regulate cilia length through a common pathway. Using our hTERT-RPE CRISPR CCDC28B cell line we were able to show that KLC1 KD or KIF5B KD require CCDC28B to regulate cilia length. In contrast, KIF7 KD, which is known to affect axonemal microtubule dynamics, resulted in cilia elongation and rescue independently of CCDC28B. Of note, we were unable to isolate a complete CCDC28B knockout cell line suggesting that at least a minimal activity of this protein is required for hTERT-RPE cells to grow. Furthermore, clone B1, albeit carrying only one CCDC28B coding allele presented levels of CCDC28B comparable to controls. This may indicate the presence of compensatory mechanisms maintaining CCDC28B levels. Our RT-PCR data suggest that if this is the case it would be a post-transcriptional event for example increasing mRNA occupancy and translational rates or modulating protein turnover. Further studies will require to test these possibilities.34. In this context, KLC1 could be in charge of loading CCDC28B onto KIF5B-based motors at the pericentriolar region. This type of effect has been reported for other kinesin 1 cargoes such as disrupted in schizophrenia (DISC1), a centrosomal protein that requires kinesin 1 to translocate away from the pericentriolar region and into the axon in neurons42.Therefore, we report here a previously unknown role for kinesin 1 in cilia length regulation, an activity that it achieves, at least in part, through the Bardet-Biedl associated protein CCDC28B. In addition, finding the link between CCDC28B and a microtubule based motor such as kinesin 1 provided a new entry-point to continue dissecting the still largely unknown mechanism by which CCDC28B regulates cilia length. Altogether, our data support a model where the function of kinesin 1 is to inhibit the pro-ciliogenic role of CCDC28B, which lies downstream in the pathway. Given the known function of kinesin 1, we reasoned that it could affect the function of CCDC28B by modulating its subcellular distribution. Interestingly, our results suggest this could be the case since depletion of KLC1 and KIF5B resulted in changes in CCDC28B localization: we observed nuclear accumulation of CCDC28B upon kinesin 1 KD, although other changes in localization cannot be discarded. Nuclear entry requires directing the target protein towards the nuclear envelope, often depending on the microtubule network and the tight association between the pericentriolar region/centrosome and the nucleus. Thus, kinesin 1, a microtubule plus end molecular motor, could be transporting CCDC28B away from the pericentriolar region and nucleus. In ciliated cells, kinesin light chains were shown to interact with rootletin, the main component of the ciliary rootlet, a structure that has been proposed to function as a site for loading cargo onto kinesin 1 motorsin vivo rescue assays. Eliminating amino acids 4–10 comprising the NLS could result in a null ccdc28b because it impairs nuclear translocation of the protein but also by other mechanisms that we cannot discard at this point. For example, it could impact protein folding and hence function although, at least for human CCDC28B, the fact that the NLS mutant protein retains its ability to interact with KLC1 suggests that is not the case. Also, deletion of residues 4–10 could impact the ability of CCDC28B to localize to the base of cilia, a possibility that we could not assess due to difficulties assessing localization upon overexpression of the mutant protein. Finally, the deletion could affect a yet unknown functional motif in the protein that is relevant to cilia length regulation. Further studies will be required to fully dissect the contribution of this NLS motif.Driven by the observed changes in cellular localization we analyzed CCDC28B further and show that both human and zebrafish CCDC28B likely possess a functional NLS domain at their N-ter. Moreover, deleting this motif in the zebrafish ortholog resulted in a protein that behaves as a null in our 10, enters the nucleus and modulates gene transcription through an interaction with RNF2, a member of the polycomb chromatin remodeling complex43. This finding was reinforced recently by a report showing that BBS6 actively translocates between the cytoplasm and nucleus and interacts with the SWI/SNF chromatin remodeling protein SMARCC1 thus affecting gene transcription44. In the case of SIN1, the other reported CCDC28B interacting protein linked to cilia length regulation13, there are at least five isoforms produced by alternative splicing that present a dynamic sub-cellular localization being found in the plasma membrane, cytoplasm, nucleus, and in the case of the shorter SIN1γ, the basal body47. Thus, it is tempting to speculate that CCDC28B, together with kinesin 1, could play a role modulating the subcellular localization and function of BBS proteins or SIN1.Our results therefore raised the intriguing possibility of CCDC28B playing a cilia-associated role in the nuclear compartment. Thus, one relevant question is whether the CCDC28B interactors, BBS proteins or SIN1, could also play a relevant function in the nucleus. Interestingly, we have shown that BBS7, which interacts with CCDC28B48. Interestingly, it has been postulated that modulating the levels of BBS4 at the pericentriolar region could represent a major regulatory point for the formation of the BBSome, a complex of BBS proteins with a critical role in cilia formation, maintenance and function51.In addition to nuclear enrichment of CCDC28B we could have expected an accumulation of CCDC28B in the pericentriolar region upon depletion of KLC1/KIF5B. However, this was not evident although this could be expected if CCDC28B rapidly translocates into the nucleus. Therefore, it is tempting to speculate that kinesin 1 could also regulate CCDC28B levels in the pericentriolar region. CCDC28B interacts with BBS4, a protein mainly localized at the pericentriolar regionFuture research will have to address the question of whether CCDC28B, via kinesin 1, can in fact regulate the subcellular distribution and function of its interactors, both in the nucleus and in the cytoplasm, and whether this could impact their function, in particular, regarding ciliogenesis. Fully determining the function of CCDC28B will allow us to understand its role in the pathogenesis of BBS thus providing insight to fully dissect the cellular and molecular basis of the syndrome.We performed the Cytotrap yeast two-hybrid screen following the manufacturer’s instructions (Stratagene) using CCDC28B (NM_024296) cloned into the pSOS bait vector and a human fetal brain library as prey (Stratagene).2 and hTERT-RPE cells in a 1:1 mix of Dulbecco’s modified Eagle medium (DMEM) and F12 (Invitrogen) supplemented with 10% fetal bovine serum and 0.01 mg/ml hygromicin B, at 37 °C in 5% CO2. We transfected cells using either FuGene (Promega) for plasmids or LipofectamineRNAiMax for stealth double stranded RNA oligos following the manufacturers recommendations (Invitrogen). Control cells were transfected with the recommended Stealth RNAi Low GC Negative Control duplex (Invitrogen).We maintained Hek293 cells in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum at 37 °C in 5% COin vivo biotinylated VHH as described52. All activities were performed in accordance with international guidelines with special care to establish high standards of biosafety and animal welfare. All protocols were authorized by the ethics committee for animal experimentation of the Universidad de la República (UdelaR), Montevideo, Uruguay, and the animal ethics committee of the Lecocq Municipal Zoo of Montevideo, Uruguay.We immunized a llama with recombinant CCDC28B and prepared a VHH phage display library from peripheral blood leukocytes. We selected the VHH to CCDC28B by panning on the polystyrene-adsorbed antigen and confirmed its reactivity by ELISA and western-blot. For immunodetection of CCDC28B and pull-down experiments, we produced the 3VO4. The lysates were cleared by centrifugation at 17000 g for 15 minutes at 4 °C. The supernatants were incubated with the appropriate antibody immobilized onto protein A/G Sepharose beads (Invitrogen) or with biotinylated-VHH against CCDC28B immobilized onto Streptavidin-Agarose resin (Pierce), overnight at 4 °C under rotation. After extensive washing of the beads, proteins were recovered with Laemmli’s sample buffer.We transfected Hek 293 cells in 10 cm dishes and harvested them 48 hours post-transfection to obtain cell lysates using CoIP buffer  supplemented with a protease inhibitor cocktail (Sigma) and 100 mM NaWe prepared nuclear and cytoplasmic fractions from hTERT-RPE cells using a hypotonic buffer  to swell cells prior to homogenization and addition of buffer T (3% Triton X-100 and 10 mM Hepes pH 7.9). We then separated intact nuclei and the cytoplasmic fraction by centrifugation at 800 g for 5 minutes. RIPA buffer  was used to solubilize proteins of the nucleoplasmic fraction. We performed western blots with the corresponding primary antibodies: anti-KIF5B and anti-KLC1 from Abcam, anti-Histone H3 from Cell Signaling, anti-HA and anti-Myc from Sigma, and rabbit polyclonal anti-CCDC28B custom made by Genscript using a human peptide. We quantified western blot bands by densitometry using the ImageJ software .We cultured hTERT-RPE cells on glass coverslips. 24 hours post-transfection with Stealth RNAi we serum starved cells for 48 hours in order to induce ciliation. We fixed cells with cold methanol at −20 °C or 4% PFA at 4 °C for 10 minutes, permeabilized for 10 minutes with 0.1% Triton X100 and blocked with 5.5% FBS for 1 hour. We incubated with the appropriate primary antibodies for 1–2 hours at room temperature and used secondary antibodies coupled to either Tetramethylrhodamine or Alexa Fluor 488 (Invitrogen). We stained basal bodies and cilia (in cells and zebrafish) using anti-γ-tubulin and anti-acetylated α-tubulin antibodies (Sigma) respectively and isotype specific secondary antibodies (Invitrogen). ARL13B was detected using a specific antibody from Abcam. 4,6-diamidino-2-phenylindole (DAPI) was used to stain DNA. Images were obtained in a Leica TCS-SP5 confocal microscope using a 63x oil 1.4 NA objective.53 and data was analyzed using Pyhton scripts. For each cilium, the 3D distal and proximal points were manually defined by the user using the ARL13B (red) channel. Based in these two 3D points, the Simple Neurite Tracer (SNT) plugin54 was used in FIJI, using default parameters, to build a simple path (curve) between them. This curve in the volume was find using two directional searches based on a cost defined by the selected points and considering the intensities distribution of the image and a measure of ‘tubeness’ at the points. This curve defined in the red channel was then manually validated in the green (acetylated α-tubulin) channel where its end points were eventually adjusted and the curve updated. The 3D curves for every cilia were projected to the 2D along with the transformation of the Z-stack in an image using FIJI’s maximum intensity Z-projection tool. 2D curves were divided in ten segments of similar distances, and each segment was thickened to about three pixels wide defining ten regions of interest covering each cilium.To evaluate levels of acetylated tubulin Z-stack images of individual cilia were semi-automatically processed in the image processing package FIJI2 in a DPC-1 Denton Vacuum Critical Point Drying apparatus. Dried samples were affixed to aluminum stubs with carbon tape and sputter-coated with gold in DeskII Denton Vacuum for 120 seconds. Samples were analyzed in high vacuum in a Jeol-5900 LV SEM operated at an accelerating voltage of 20 kV.For scanning electron microscopy (SEM) analysis, cells were grown on glass coverslips, fixed overnight in a mixture of 4% paraformaldehyde and 3,5% glutaraldheyde in 0.1 M phosphate buffer saline pH 7,2 (PBS) at 4 °C. After that, cells were washed in PBS, dehydrated through a series of ethanol solutions of increasing concentration and submitted to critical point drying with COhttp://crispr.mit.edu and cloned them into the pLentiCRISPRv2 lentiviral vector  following Zhang’s laboratory protocol36. To obtain lentivirus we co-transfected Hek293 cells plated in 10 cm dishes with the pLentiCRISPRv2 construct and the packaging vectors pCMV-VSV-G (Addgene #8454) and psPAX2 (Addgene #12260). The supernatant containing the virus was collected 48 hours after transfection, centrifuged a 4000 RPM for 15 min, passed through a 0.45μm filter and stored at −80 °C until used. hTERT-RPE cells were transduced at 60% confluency in 6-well dishes with virus-containing supernatant diluted 1/2 with fresh medium in the presence of 8 μg/ml Polybrene (Sigma). Selection was started 24 hours after transduction supplementing media with 10 μg/ml puromycin. For cell sorting and single cell deposition, hTERT-RPE cells were grown and transduced in a 10 cm dish. After 5 days under puromycin selection (6 days post-transduction), cells were resuspended and sorted into a 96-well plate using a BD FACSAriaTM Fusion (BD Biosciences). To identify the mutations in CCDC28B, genomic DNA was obtained by standard methods, exon 2 was amplified by PCR, PCR products were cloned into pGEM-T-easy following the manufacturer’s instructions (Promega) and individual constructs were purified and sequenced. For clone B1, cDNA was generated using SuperScript® III First-Strand Synthesis kit (Invitrogen) with oligodT primers. CCDC28B was amplified with specific primers, PCR products were cloned into pGEM-T-Easy and sequenced. The sequences of all primers and gRNAs used in this study are available upon request.We designed five gRNAs using the online design tool at ccdc28b in zebrafish we used our previously validated splice-blocking morpholino14. MOs were obtained from Gene Tools. We injected TAB5 wild-type zebrafish embryos at a one to two-cell stage with 0,5 nanoliters of MO solutions prepared to deliver 5 ng of MO. mRNA for rescue experiments was prepared using the ccdc28b wt and ccdc28bΔ4–10 ORFs cloned into pCS2+ and the Ambion mMessage mMachine SP6 mRNA kit. Experiments in zebrafish were performed following protocol 010–13 approved by the Institut Pasteur de Montevideo ethics committee for the use of animal models.To target ccdc28b following the methods described in55. Four gRNAs were designed using CRISPRscan tool. gRNAs were produced as described55. In short, a DNA template containing T7 promoter, the sgRNA targeting sequence and sgRNA invariable 3′end was generated by fill in PCR. This template was purified and used for in vitro transcription using Ambion MAXIscript SP6/T7 In Vitro Transcription Kit (Thermo Fisher Scientific). After DNAse treatment, sgRNAs were purified through precipitation with sodium acetate and ethanol. All gRNAs were tested and then two of them targeting exon 2 were selected for the study. All sequences and estimated efficiencies are available upon request. Cas9 mRNA was synthetized in vitro from plasmid pT3TS-nCas9n56 which codifies zebrafish codon-optimized Cas9 fused to nuclear localization signals, using the mMESSAGE mMACHINE Tanscription Kit (Thermo Fisher Scientific). zf-nCas9n mRNA was purified using MicroSpin G-50 columns . Zebrafish embryos in one cell-stage were injected in the cell cytoplasm with a combination of 50 pg of each sgRNA and 100 pg of zf-nCas9n mRNA. General embryo morphology and the aspect of cilia in different organs were analyzed at 48 hpf.CRISPR/Cas9 was used to generate mutations in the coding region of zebrafish 57, using a test of hypothesis specific for comparison of two proportions (hypothesis test for proportions). For the analysis of cilia length, we first tested the sets of data for normal distribution, using the Shapiro-Wilk test, and variance homogeneity using the Levene test. When necessary, data were Ln transformed in order to meet the distributional assumptions required by the statistical tests. To compare three or more groups with normal distribution we used one-way ANOVA. In the case of datasets without normal distribution we used non-parametric tests, the two-tailed Mann Whitney in order to compare two sets of data or the Kruskal Wallis to compare more than two sets. To compare the distribution of classes in the zebrafish experiments we used Chi-squared test (χ2) when data was pooled and both Wilcoxon rank test and paired t-test for the analysis of percentage of embryos per class and condition. The tests used are appropriately indicated in the corresponding figure legends.The proportion of cilia positive cells was calculated by counting the number of cilia with clear acetylated-tubulin signal over the total number of cells and results are expressed as a proportion with a 95% confidence interval (CI). The comparison between the different samples was performed as describedSupplementary Information


The mean serum UA levels were lowest in the severe group (0.281 ± 0.074 mmol/l) followed by moderate (0.282 ± 0.080 mmol/l) and mild (0.297 ± 0.090 mmol/l) with significant differences among the three groups (p = 0.032). In multivariate regression analysis, there was a significant negative correlation between UA level and vertical cup-disc ratio . Significantly lower serum UA concentration in PACG and its negative association with disease severity presented it as an important candidate in reaction to oxidative stress in glaucoma pathogenesis.Uric acid (UA) is a major antioxidant molecule and has been hypothesized to have a protective effect on the central nervous system against oxidative damage. We prospectively investigated the serum concentration of UA in primary angle closure glaucoma (PACG), and explored the association between serum concentration of UA and the severity of PACG. Using a retrospective case-control study design, 886 PACG subjects and 994 control subjects who attended the Eye & ENT Hospital of Fudan University, were eligible for this study. Glaucoma severity was classified as mild (MD ≤ 6.00 dB), moderate (12 dB ≥ MD > 6 dB) and severe (MD > 12 dB) based on the MD (mean deviation). The levels of UA were significantly lower ( Primary glaucoma is a progressive optic neuropathy and one of the leading causes of global irreversible blindness, and the number of people (aged 40–80 years) with glaucoma worldwide is projected to increase from 76.0 million in 2020 and 111.8 million in 2040 . AlthougAs the final product of the common pathway of purine metabolism, UA is a major antioxidant with metal-chelating properties  as well The objective of this study was to measure serum UA and creatinine concentration and to further explore the relationships between UA and UA/Cr ratio and glaucoma severity in PACG.p > 0.05). The mean serum levels of UA and UA/Cr ratio were significantly lower in the PACG compared with the control group (p < 0.001). PACG group had a higher serum creatinine concentration compared with the control group (p < 0.001). The demographic, Cr, UA, UA/Cr ratio of the PACG and control groups were summarized in Table A total of 886 PACG subjects and 994 control subjects from the Eye & ENT Hospital of Fudan University were eligible for the study from January 2010 to December 2015. Only one eye was selected randomly if both eyes suffered from PACG. There was no statistical difference in the mean age and gender between the PACG and control group  among the three groups. The mean serum levels of UA was lowest in the severe PACG group, followed by moderate PACG and mild PACG, and the differences among groups were significant (p = 0.032). The mean serum level of creatinine was not significantly different among the three groups (p > 0.05). A similar trend was observed when UA were compared among the 3 groups with respect to gender. The ocular parameters were significantly different among the three groups (p < 0.05). The level of UA, creatinine, UA/Cr ratio, and ocular characteristics of the three groups were shown in Table Based on the MD, the PACG subjects were categorized into 3 subgroups of different severity level of which 286 were classified as mild, 198 as moderate and 402 as severe. There was no statistical difference in the mean age and gender , and positive correlation between UA and AL   were observed. In the mild PACG group, a negative correlation between UA and VCDR , UA and MD , and positive correlation between UA and AL , UA and MS (mean sensitivity)  were observed. In the moderate PACG group, the correlations between UA and AL, UA and MD, UA and MS were also statistically significant. A similar trend was observed when correlation among UA and ocular parameters were performed with respect to gender.Table p = 0.035) in the overall PACG patients. In the mild PACG group, a negative correlation between UA and VCDR , and positive correlation between UA and MS  were observed. In the severe PACG group, a positive correlation between UA and VCDR  was observed. A similar trend was observed when association among UA and ocular parameters were performed by multiple linear regressions with respect to gender.Table To the best of our knowledge, this study has been the first to focus on the investigation of serum UA and UA/Cr ratios in PACG and further evaluate their relationship with disease severity. The principal finding was that serum UA levels and UA/Cr ratios in PACG patients were lower compared with sex and age-matched control group subjects, in addition, UA levels were found to be associated with the severity of PACG. The serum UA/Cr ratio was used to reduce the possible interference caused by sex and differences in renal function . These dThe levels of peripheral blood UA have been reported to be associated with hypertension  and AlzhIn this study, we found significantly lower serum UA levels in PACG patients compared with control group in both male and female subjects. However, Yuki K et al.  reportedThere have been reports suggesting that glaucomatous damage is associated with episodes of systemic inflammation which are likely to cause a systemic decrease in UA . OxidatiMoreover, the decreased serum levels of UA in PACG patients were an outcome of an oxidative stress-related process in the optic nerve system, eye tissue and possibly in other tissues which meant that this decrease was likely to be a result of the direct reaction between UA and oxidizing agents , 38. SacIn vitro studies showed that oxidative stress accelerated cell apoptosis and led to accumulation of extra cellular matrix in the trabecular meshwork, which could both increase the resistance of the aqueous humor outflow and result in IOP elevation and later visual field loss [eld loss , 39, 40.eld loss . Hooper eld loss  reportedeld loss  reportedeld loss . Therefoeld loss , 44. TheMoreover, based on the MD, the PACG subjects were categorized into 3 subgroups of different severity level . The mean serum levels of UA were lowest in the severe PACG group, followed by moderate PACG and mild PACG. However, the mean serum level of UA was not statistically different between moderate and severe PACG group Figure . This suMoreover, Table Although this is the first study which focused on the evaluation of serum level of UA, UA/Cr ratios and its relationship between PACG and disease severity, we acknowledged that our present study has some limitations. (1) We did not consider treatment with brinzolamide eye drops, carbonic anhydrase inhibitor, and mannitol and how it might affect renal function. In this study, there was no statistical difference of creatinine among the mild, moderate, and severe PACG subgroups. The serum UA/Cr ratio was analyzed considering the existence of possible interference such as medication and difference in renal function. UA/Cr ratios in PACG patients were also lower compared with the control group. (2) Owing to this study was a retrospective case-control study, information on ocular parameters in controls subjects was lacking.The present study demonstrated that the mean serum level of UA was decreased and negatively associated with PACG severity which suggested the possible association between UA with the development of glaucoma and the involvement of oxidative stress in the pathogenesis of PACG.This was a retrospective case-control study design. The study was approved by the Ethics Committee of the Eye-ENT Hospital of Fudan University, Shanghai, China and was conducted according to the Declaration of Helsinki. Written informed consent for the use of any clinical data in research was obtained for all patients at the time of admission to the Eye-ENT Hospital of Fudan University. PACG subjects and control subjects who attended the Eye-ENT Hospital of Fudan University from January 2010 to December 2015 were selected according to the inclusion criteria listed below.n = 1350) during January 2010 through December 2015 and control subjects (n = 1645) were collected from the inpatient electronic database. 446 PACG subjects and 595 control subjects were excluded according to the inclusion criteria. The exclusion rate of PACG and control group was 33.04% and 36.17% respectively. A further 56 control subjects were randomly excluded because they were not age and sex matched to the PACG group. At last, a total of 886 PACG subjects and 994 control subjects from the Eye & ENT Hospital of Fudan University were eligible for the study.The data of all the PACG subjects ((1) PACG subjects were selected from inpatients. Each subject underwent a standardized ophthalmic examination, which included refractive status, slit-lamp biomicroscopy, fundus examination, IOP, CCT , AL, ACD (anterior chamber depth), visual field examination, and gonioscopy, performed by glaucoma specialists. MD  and MS (mean sensitivity) were measured by Octopus automated perimetry . IOP was measured using Goldmanapplanation tonometry. Fundus photography was performed with a retinal camera . A-scan ultrasound  was used to measure AL, ACD, and CCT.(2) PACG was diagnosed on the basis of narrow angles with glaucomatous optic neuropathy with corresponding visual field loss. This was defined as glaucomahemifield test outside normal limits including a cluster of three or more, non-edge, contiguous points on the pattern deviation plot, not crossing the horizontal meridian with a probability of less than 5% of being present in the age-matched normal (one of which was less than1%), an abnormal pattern standard deviation with P less than 5% occurring in the normal population, and fulfilling the test reliability criteria . PACG was diagnosed in eyes with narrow angles, with elevated IOP (IOP >21 mm Hg); at least 180 of angle-closure obliterating the pigmented part of the trabecular meshwork, whether synechial or appositional, segmented or continuous; and eyes in which the degree of peripheralanteriorsynechial is too extensive to be managed by laser peripheral iridotomy. , 47, 48 (3) PACG subjects had no other ocular diseases and systemic diseases such as hyperuricemia, diabetes, cardiac, autoimmune disease and cancer.n = 168), amblyopia (n = 146), nystagmus (n = 111), epicophosis (n = 144), vocal cords leukoplakia (n = 163), lacrimal ducts obstruction (n = 155), and nasal septal deviation (n = 107). They were selected from the inpatients record at the Eye-ENT Hospital of Fudan University from January 2010 to December 2015. Each subject underwent a standardized physical examination and preliminary ophthalmic examinations, which included refractive status, slit-lamp, and IOP, performed by glaucoma specialists. IOP was measured using Goldmanapplanation tonometry. All the control subjects were excluded from any family history of glaucoma and systemic diseases which included but not restricted to hyperuricemia, diabetes, cardiac, autoimmune disease and cancer.Control subjects were composed of patients with strabismus . Internal controls were analyzed daily over the 6-year period, with typical monthly CVs of 2%–4% and no significant changes in the values. The reference ranges for UA between males and females were different , 52. Thet-test and chi-square test were used for comparison of characteristics of patients between the PACG group and control group. The one-way ANOVA test was used to compare the levels of serum UA, serum creatinine, UA/Cr, and ocular parameters among the three groups with different severity. The associations between UA and ocular parameters in PACG were analyzed by Spearman correlation. After Spearman correlation, multivariate linear regression analysis  to prevent bias) was performed to evaluate the association between UA and disease severity, namely visual field indices , VCDR, and other ocular parameters. A P value of less than 0.05 was considered statistically significant.The data was analyzed by SPSS13.0 . UA and creatinine concentrations were presented as mean ± standard deviation (SD). UA/Cr ratio = (UA*1000)/Cr. Normality was assessed with the Kolmogorov-Smirnoff test. The independent student's


Ever since its initial discovery in Drosophila, hedgehog signaling has been linked to foregut development, The mammalian genome expresses three Hedgehog paralogues, sonic hedgehog (Shh), Indian Hedgehog, and desert hedgehog. In the mucosa of the embryonic and adult foregut, Shh expression is the highest. It has now become clear that hedgehog signaling is of pivotal importance in gastric homeostasis. Aberrant activation of hedgehog signaling is associated with a range of pathological consequences including various cancers. Also in gastric cancer, clinical and preclinical data support a role of Hedgehog signaling in neoplastic transformation, and gastrointestinal cancer development, also through cancer stroma interaction. Technological advance are facilitating monitoring Hedgehog signaling broadening options for the more efficient screening of individuals predisposed to eventually developing gastric cancer and targeting Hedgehog signaling may provide opportunities for prophylactic therapy once atrophic gastritis develops. Nevertheless, convincing evidence that Hedgehog antagonists are of clinically useful in the context of gastric cancer is still conspicuously lacking. Here we analyze review the role of Hedgehog in gastric physiology and the potential usefulness of targeting Hedgehog signaling in gastric cancer. Hedgehog proteins are fundamental regulators of embryological development, and tissue homeostasis in adult organisms. Disturbed hedgehog signaling is associated, amongst others, with a range of congenital disabilities, oncological malignancies and immunological defects . HedgehoThe physiological importance of Hedgehog signaling in the physiology of the proximal tract is illustrated by the phenotypes observed in mice with genetic loss of Hedgehog paralogues. Genetic knockout of both Shh and Dhh provoke by malrotation of the gastrointestinal tract, oesophageal atresia, gastric overgrowth and other gross abnormalities , 18. TheHedgehog signaling in general is unusual and complicated, and an immense scientific effort has been necessary to unravel its general principles , 22-24. Following binding of Hedgehog to Patched. Smo translocates to the primary cilium in the cell membrane. The subsequent signaling culminates in altered transcription through Gli transcription factors. The molecular details of Smoothened signaling to Gli is still partly obscure but involves the microtubule transport proteins , 27. TheEver since its initial detection in Drosophila, Hedgehog has long been associated with foregut development. Of the three mammalian Hedgehogs (3) Shh levels are most highly expressed h, in the mucosa of the embryonic foregut, . Also inwww.proteinatlas.org). Pepsin A prefers to cleave proteins at hydrophobic and aromatic residues, particularly at phenylalanine (F) when the pH is less than 2. By contrast, pepsin C recognizes a broader consensus site and uses more comprehensive pH spectrum than pepsin A[During progression from the inflamed stomach to gastric cancer, the epithelium goes through defined series of morphological transitions. First, the acid-producing parietal cells are lost and are replaced by mucus-secreting cells that express spasmolytic polypeptide (SP) or trefoil factor 2.7 . Mostly  pepsin A, 51. Exp pepsin A. This maSeveral studies have examined the impact of blocking Hedgehog signaling in vivo, for instance by employing a transgenic mouse that secretes a natural-inhibitor of Hedgehogs called HHIP employing the parietal cell-specific H+, K+-ATPase β subunit promotor . This apUpregulation of Hedgehog signaling pathway is involved in tumor development . De SauvAs stated, Hedgehog signaling in the stomach plays a significant role in gastric development, homeostasis, and neoplastic transformation . InitialThe Hedgehog signaling pathway is a significant target for cancer therapy. Various molecules that may inhibit the pathway have been evaluated in both preclinical and clinical studies. These inhibitors include: SMO inhibitors, ligand-receptor inhibitors, Gli targeted inhibitors, and these classes of molecules are Illustrated in figure Cyclopamine, originally isolated from the flower veratrum californicum that causes congenital disabilities when eaten by pregnant cattle, was the first compound that was used as a Hedgehog inhibitor and it targets SMO. Consequently, GLIs activation is inhibited. In the clinic, it is side effect-prone and exhibits substantial toxicity . FrustraAlso in view of resistance development against vismodegib through SMO mutation efforts have been made to target Gli. Two inhibitory compounds (GANT 58. And GANT 56) were identified through cellular screens aimed at identifying compounds able to inhibit transcription mediated by GLI. Both compounds at the cellular level have been revealed. Understanding the mechanism of action of these compounds at a molecular level is still incomplete. GLI1 posttranslational modification by GANT 61 impedes binding to DNA or changes the conformational structure of the GLI1-DNA complex. In xenograft mice, models of human prostate cancer cells inhibition of cancer cell growth is observed . A studyA monoclonal antibody 5E1 (ch5E1) from murine-human chimeric has been proven to bind Shh and thereby to inhibit Hedgehog signaling. A smallFor gastric cancer treatment using Hh signaling targeted agents, only two clinical trials have been performed. A phase II multi-centered, randomized, a prospective clinical trial involving 124 participants was performed to determine the vismodegib efficacy, potency, and safety. For adenocarcinoma patients under FOLFOX chemotherapy, vismodegib an SMO inhibitor was administered in conjunction with this regimen. The study did not meet the primary endpoint with a no significantly-improved progression-free survival between the placebo group and vismodegib group , although with a noticeable tendency for prolonged progression-free survival . The lacGastric cancer has long been seen as one of the most difficult gastrointestinal malignancies to treat. Encouragingly, recent progress with targeted therapies offers hope for patients with advanced gastric cancer and is substantially expanding the therapeutic armamentarium with regard to this infaust disease. As these treatments continue to be developed, we must focus on determination of predictive markers, and preferably co-develop drugs with these markers. The mechanisms underlying primary or acquired resistance to targeted agents also should be clarified to help further drug development. DevelopWhile there is good evidence that Smoothened inhibition may be useful for a selection of gastric cancers, we feel that its untargeted application on gastric cancer patients, in general, is likely to prove disappointing. In this sense efforts to select patients characterized by unusually high SMO expression in gastric tumor material with high probability to have cancers that are truly dependent on a functional Hedgehog pathway may likely yield positive results. As both approaches are currently being attempted in clinical trials, it should prove interesting to see whether this notion holds true.


Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The Rosa26AOX mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The Rosa26AOX mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from  Summary: Previous limitations are overcome in this first genetically tractable mouse model expressing invertebrate alternative oxidase, AOX, which can suppress pathological stresses in the mitochondrial respiratory chain. The mitochondrial system for oxidative phosphorylation (OXPHOS) comprises four multisubunit complexes supporting stepwise respiratory electron flow from primary electron acceptors to oxygen, and a fifth complex (ATP synthase) that uses the proton gradient thereby generated across the inner mitochondrial membrane to synthesize ATP. In many lower organism and plants, alternative oxidases (AOXs) are expressed that branch the mitochondrial respiratory chain, thus transferring electrons directly from ubiquinol to oxygen in a non-proton-motive manner. AOXs are absent in mammals  Fig. 1AAFig. 1A.Ciona intestinalis, a sister group to the vertebrates, was shown to be expressible and catalytically active in human cells , which enhances expression several-fold , a chimeric line was established via blastocyst injection, with subsequent elimination of the positive-selectable (neomycin resistance) cassette  by Flp recombination in vivo, following germ-line transmission. Founders were backcrossed over more than seven generations to strain C57Bl/6J, with transgene presence checked at each step by PCR . The rate of transmission of the AOX transgene from heterozygous parents of either sex did not significantly deviate from 50% . Brain expression was highest in newborn mice , but declined substantially by one month of age . As expected, AOX expression was higher in homozygotes compared with heterozygous animals . The enzyme was found to be associated with the membrane fraction of isolated mitochondria after carbonate extraction , albeit less tightly bound than some integral membrane proteins of the OXPHOS complexes, such as subunit 1 of cIV (Mtco1).Northern blotting E confirmFig. S2A). Moreover, the overall structure of the respiratory membrane, specifically its organization into supercomplexes, was similarly unaltered, based on blue-native electrophoresis (BNE) followed by in-gel histochemistry of heart mitochondria . In BNE gels, AOX itself migrated mainly at the size of a dimer and as multimers thereof , rather than associating specifically with any other respiratory complex. In each tissue tested, the mobility of the respiratory chain complexes detected by BNE was identical to that in controls . Principal component analysis of metabolite levels in skeletal muscle  showed no consistent effect of AOX expression, nor did any of 100 individual metabolites analyzed show any significant difference .In each tissue tested, the expression of representative subunits of the five OXPHOS complexes was essentially unaffected by AOX expression  oxygen consumption between 30% and 70% of the uninhibited level driven by succinate . In heart mitochondria from AOX-expressing compared with control mice, antimycin A- and azide-resistant substrate oxidation was evident across a wide range of drug concentrations . Compared with littermate controls, mitochondrial ROS production driven by succinate was greatly decreased . This analysis covered metabolic, behavioural, morphological, immunological, cardiac and neurological parameters, amongst others. None of the parameters tested showed substantial or systematic deviations from littermate controls.The high level of AOX expression, capable of replacing a large fraction of electron flow when cIII/IV is inhibited, raised the question of potentially deleterious consequences under normal physiological conditions. Surprisingly, elopment A. Muscleelopment B, treadmelopment B, cardiaelopment C and lefelopment C, conducRosa26AOX transgenic mice tested survived the treatment, whereas three of six littermate controls succumbed as expected (Despite the absence of any meaningful phenotype under standard (non-stressful) physiological conditions, we reasoned that the ubiquitous expression  of functexpected . AlthougCiona AOX, via a single-copy insertion into the Rosa26 locus, controlled by the synthetic CAG promoter. AOX protein was widely expressed and enzymatically functional when tested in the presence of antimycin A in organello. AOX expression produced negligible phenotypic effects under standard physiological conditions, but seemed able to protect mice from the lethal effects of injected cyanide. The Rosa26AOX mouse provides a genetically tractable tool for analyzing the pathophysiology of a wide spectrum of diseases proposed to be linked to mitochondrial respiratory dysfunction.In this study we successfully engineered mice for stable, ubiquitous expression of Ciona AOX expression in the mouse is not harmful. However, owing to the multi-copy nature of the inserted transgene at different genomic sites, as well as varying expression levels, MitAOX mice were not suitable for studies using genetic disease models. To avoid these problems, we created a revised model containing a single insertion of AOX cDNA at the Rosa26 locus on chromosome 6. We demonstrated . (2) Inefficient mobilization of nutritional resources by AOX-expressing mice should alter metabolic parameters in vivo. However, there were no differences in whole body weight (2 exchange or activity (see GMC Phenomap). (3) No significant differences in heart performance were detected by electro- or echocardiography, or MRI  is somewhat puzzling, given previous reports. The CAG promoter has previously been used to drive transgene expression at a high level in the mouse brain during development , indicating that the transgene can be active, but apparently regulated, in neural cells. At present we do not have a convincing explanation for these anomalies. However, the relatively low expression of AOX in the adult brain seemed sufficient to protect against the lethality of systemically delivered cyanide  other OXPHOS complexes. The electrophoretic mobility of the standard OXPHOS complexes was indistinguishable from that in controls, in all tissues tested . Our findings imply that these multimers are themselves benign, although it remains unknown whether AOX is structurally arrayed in a similar manner in its natural context in Ciona. We reason that, by remaining uncomplexed with other respiratory chain components, the enzyme would be functionally adapted to act as a sink for electrons transferred from diffusible quinols in the inner mitochondrial membrane. Quinone reduction might arise from the operation of diverse dehydrogenases, including, for example, cII, electron transferring flavoprotein dehydrogenase, the mitochondrial isoform of glycerol-3-phosphate dehydrogenase and dihydroorotate dehydrogenase. Under normal physiological conditions, diffusible quinols would be efficiently mopped up by (dimeric) cIII, whether alone or attached to cIV as a supercomplex. As in plants  upstream and downstream of the neomycin selection cassette. Ciona AOX (Escherichiacoli (EL250) with a Rosa26-targeting plasmid (pRosa26-DTA). Positive clones were selected by kanamycin resistance (pRosa26-Aox), verified by PCR and sequencing, and electroporated into v6.5 ESCs following linearization with SalI. After negative  and positive (G418) selection, homologous integration was verified by Southern blotting .Standard cloning and recombineering procedures  were useiona AOX  was inteblotting  using geESC clones positive for integration were injected into blastocysts and transferred to pseudopregnant mice. Chimeric males were then backcrossed onto the C57Bl/6J strain background to generate heterozygous animals, and subsequently bred with mice ubiquitously expressing FLP recombinase , in ordeAOX, and Rosa26_wt s: 5′-GACCTCCATCGCGCACTCCG-3′ plus Rosa26_wt as: 5′-CTCCGAGGCGGATCACAAGC-3′ for the wild-type Rosa26 locus, giving respective products of 317 and 523 bp. PCR reactions of 20 μl contained 4 pmol of each primer, DMSO at 2% and 0.2 μl DyNazyme II (Thermo Fisher Scientific), with cycle parameters of initial denaturation at 95°C for 5 min, then 39 cycles of denaturation at 95°C for 20 s, annealing at 56°C for 30 s and extension at 72°C for 60 s, with final extension step at 72°C for 10 min, followed by 1.5% agarose gel electrophoresis. See Fig. S1D for example gel.Crude DNA for genotyping was extracted from ear punches or tail cuts by standard methods . Multiplex PCR genotyping was carried out using primers Aox 317 s: 5′-GCGATGCAAGATGGAGGGTA-3′ plus Aox 317 as: 5′-TGAATCCAACCGTGGTCTCG-3′ for gmax for 15 min at 4°C. The upper (aqueous) phase was decanted and RNA recovered by isopropanol precipitation and centrifugation. Using standard procedures  of Trizol reagent (Sigma). After incubation for 5 min at room temperature, samples were gently extracted with 0.2 volumes of chloroform and centrifuged at 12,000 ocedures , air-drigmax for 5 min at 4°C. Supernatants were saved and protein concentration was measured using Bradford reagent (Bio-Rad) before dilution into SDS-PAGE sample buffer for electrophoresis on SDS 12% polyacrylamide gels. After semi-dry transfer to PROTRAN nitrocellulose membranes (PerkinElmer), western blots were probed using primary antibodies for AOX , using a glass-teflon homogenizer (tight-fitting pestle). Hard tissues , chopped to a similar size, were pre-treated with 3 ml (∼10 volumes) ice-cold trypsin-EDTA  for 10 min, followed by blocking with 300 μl foetal bovine serum (Gibco/Life Technologies) and recovery by low-speed centrifugation  before homogenization. Homogenates were centrifuged at 1300 gmax for 5 min at 4°C, after which supernatants were collected and re-centrifuged at 17,000 gmax for 15 min at 4°C. The mitochondrial pellet was resuspended, according to its size, in 75-250 μl ice-cold MiR05 buffer  and stored on ice until respirometry. Mitochondrial protein content was assayed using Bradford reagent (Bio-Rad). Respirometry, using an O2K oxygraph (Oroboros), was conducted in MiR05 buffer in a 2 ml chamber, to which was added 50 or 100 µg of mitochondria according to the tissue. Substrates and inhibitors were added in the following order: (1) 5 mM sodium pyruvate+5 mM sodium glutamate+5 mM sodium malate, (2) 4 mM ADP, (3) 150 nM rotenone (Sigma), (4) 17 mM sodium succinate, (5) 22.5 ng/ml antimycin A (Sigma), (6) 200 µM n-propyl gallate , (7) 0.5 mM N,N,N′,N′-tetramethyl-p-phenylenediamine +2 mM sodium L-ascorbate, (8) 100 mM NaN3 or 1 mM KCN. The flux values [pmol/(s×ml)] obtained from the trace were normalized to the amount of mitochondrial protein. For measurements of ROS production, mouse heart mitochondria were isolated essentially as described . Comprehensive phenotyping by the German Mouse Clinic (GMC) was conducted using the protocols described and referenced at https://www.mouseclinic.de (search ‘phenomap’). In all tests, mouse genotypes were blinded to the experimenter and verified subsequently.Mouse body weight was measured using a small electronic balance suitable for rodents. Grip strength was measured using the BIO-GS3 apparatus (Bioseb). Mice were placed on the platform until all four limbs were engaged on the grid, and then pulled to measure the force generated. The mean of three measurements was normalised to body weight (g/g) for each animal tested. All animals were trained for three successive days before the actual experiment. Endurance running was measured as previously , as the The procedure was implemented under contract by Luria Scientific Industries, Herzliya, Israel (responsible scientist Dr Iris Maimon). Mice, whose genotypes were blinded to the experimenter, were anesthetized with 3% isoflurane in an induction chamber, after which anaesthesia was maintained by 2% isoflurane using a nose cone. Core temperature was kept at 36.5°C using a heating pad. KCN was dissolved in distilled water at 10 mg/ml and delivered by IP injection to the mice at 8.5 mg/kg. Animals were observed for 48 h for the onset of death, defined as apnea without further respiratory effort or movement or palpable cardiac pulsation.All mouse breeding and experiments were approved by the national ethical committee in Finland, under permits ESAVI/8766/04.10.07/2015 and ESAVI/2954/04.10.07/2015. Mouse experiments conducted under contract by Luria Scientific Industries were approved by IACUC under assurance 7433J45, 07/22/2015. Maintenance of mice in Magdeburg was in accord with procedures specified by the Animal Health and Care Committees of the Otto-von-Guericke University, Magdeburg, and of the State of Sachsen-Anhalt, Germany.Images were optimized for brightness and contrast and cropped for clarity. No other manipulations such as gamma corrections were made, nor was any relevant information excluded by cropping. Full, original gel images are available on request.


Mycobacterium tuberculosis (Mtb) infection, suggesting that immunological defect are associated with HTLV-1 infection. Evidence suggests an important role for Mucosal-associated invariant T (MAIT) cells in the early control of Mtb infection. Chronic viral infections like HIV and HCV have been associated with decreased frequency and functionality of MAIT cells. We hypothesized that HTLV-1 infection is associated with similar perturbations in MAIT cells. We investigated MAIT cell frequency, phenotype, and function by flow cytometry in a cohort of 10 asymptomatic and 10 HAM/TSP HTLV-1 infected patients. We found that MAIT cells from HTLV-1-infected subjects were reduced and showed high co-expression of the activation markers CD38 and HLA-DR but normal levels of CCR6 and CD127. MAIT cells had a lower expression of the transcription factor PLZF in HAM/TSP patients. Unlike Tax-specific CD8+T cells, which are hyperfunctional, MAIT cells from HTLV-1-infected subjects had a poor IFNγ response following antigen stimulation. MAIT cell perturbations in HTLV-1 infection were not associated with HTLV-1 proviral load and MAIT cells were not infected by HTLV-1 in vivo. Rather, MAIT cells loss was associated with immune activation. Overall, our results do not support a role for MAIT cells in HAM/TSP pathogenesis but reduced numbers of MAIT cells, together with their poor functionality, could contribute to the increased susceptibility of HTLV-1-infected individuals to other infectious agents.HTLV-1 infection is associated with several inflammatory disorders, including the neurodegenerative condition HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is unclear why a minority of infected subjects develop HAM/TSP. The cellular immune response has been implicated in the development of inflammatory alterations in these patients; however the pathogenic mechanisms for disease progression remain unclear. Furthermore, HTLV-1-infected individuals have an increase incidence of  Mycobacterium tuberculosis (Mtb) infection[Human T-lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus estimated to infect around 10 million people worldwide. Other iinfection, 9, suggin vivo[CD4+ T cells are the primary targets of HTLV-1 in vivo. The immin vivo, 13. Thein vivo–16. Othein vivo, but manin vitro and in vivo[Mucosal-associated invariant T (MAIT) cells are a population of innate T cells recently identified. In human, they express a semi-invariant TCR using Vα7.2 coupled with Jα33 and have a limited Vβ repertoire. They cad in vivo, 21. MAId in vivo. MAIT ced in vivo, 24 and d in vivo, 26 and d in vivo and MS[2d in vivo, 29. TheIn this study, we determined the frequency, phenotype, and functionality of MAIT cells in a cohort of HTLV-1-infected subjects. Here, we describe for the first time phenotypic and functional alterations in MAIT cells of HTLV-1-infected individuals and discuss the implications such alterations might have in HAM/TSP and in the immune response of HTLV-1-infected subjects to other pathogens, specifically Mtb.We selected 20 HTLV-1 patients from the HTLV-1 outpatient clinic at the University of Sao Paulo, Brazil. All participants were enrolled in a longitudinal cohort of HTLV-1-infected subjects after signing a written consent form approved by the University of São Paulo’s Institutional Review Board (#0855/08). Patients were recruited and peripheral blood was collected between October 2014 and October 2015. Patients were invited to participate in the study based on the following criteria: capacity to understand and sign consent form, age > 18, HIV negative, no therapy with corticosteroids or immunomodulators in the last 3 months and absence of active co-infection by Tuberculosis or Syphilis. We randomly selected 10 patients diagnosed with neurological complications associated to HTLV-1 infection (HAM/TSP), 10 HTLV-1 asymptomatic carriers and 12 uninfected controls age-and-gender-matched . ClinicaCryopreserved PBMC specimens were thawed and washed, and counts and viability were assessed using the Countless Automated Cell Counter system . Cells were washed and stained in Brilliant Violet Stain Buffer  at room temperature for 15 min in 96-well V-bottom plates in the dark. Samples were then washed and fixed using Cytofix/Cytoperm (BD Biosciences) before flow cytometry data acquisition. Intracellular staining for IFNγ and promyelocytic leukaemia zinc finger (PLZF) were performed in Perm/Wash (BD Biosciences) at room temperature for 15 min in the dark. mAbs used in flow cytometry: CD3 AF700, CD3 PerCP-Cy5.5 (both clone UCHT1), CD4 BV605 (clone RPA-T4), CD8 BV711 (clone RPA-T8), CD38 APC-H7 (clone HB7), CD69 AF00 (clone FN50de), CD161 BV421 (clone DX12), CCR6 BV786 (clone 11A9), HLA-DR APC (clone L243), IFNγ APC (clone B27), and PD-1 PE-Cy7 (clone EH12.1) were all from BD Biosciences, TCR Vα7.2 PE, TCR Vα7.2 PerCP-Cy5.5 (clone 3C10) were from Biolegend , and PLZF APC was from R&D Systems . Live/dead aqua fixable cell stain was from Life Technologies  and added to the cells together with the cell surface antibodies. Data were acquired on a BD LSRFortessa instrument (BD Biosciences) and analyzed using FlowJo Version 9.8.5 software .in vitro using paraformaldehyde-fixed E. coli stimulation  in the presence of 1.25 μg/ml anti-CD28 mAb [2 in RPMI medium supplemented with 10% fetal bovin serum (FBS). Monensin  was added during the last 6 hours of the stimulation.MAIT cell function was determined sciences). PBMCs wPBMC samples were thawed as previously described and stained with CD3 PerCP-Cy5.5, CD4 APC , CD161 FITC , Vα7.2 PE, and DAPI (BD Bioscience) for 10 minutes at RT and washed with PBS 2% FBS. CD4+ T cells and CD4- MAIT cells were then sorted on a SH800Z . Purity was typically over 90%. Sorted cells were then used for pro-viral quantification.10 dilutions (from 105 to 100 copies). For quantitation of HTLV-1 and the human housekeeping gene albumin in genomic DNA, previously described primers combined with TaqMan® Universal Master Mix II (ThermoFisher Scientific) were used[Total DNA was extracted from PBMCs using a commercial kit  following the manufacturer's instructions. For HTLV-1 proviral load absolute quantification, a double standard plasmid pcHTLV-ALB previously described was used to generate standard curves. For eacwere used. Each sa≤ 0.05 were considered statistically significant.All statistical analysis was performed using Graph Pad Prism version 6.0 for Mac OSX . The comparison between healthy controls and HTLV-1 patients and between asymptomatic and HAM/TSP patients were analyzed using Mann Whitney U-test. Associations between groups were determined by Spearman's rank correlation. P values First, we evaluated the frequency of MAIT cells by flow cytometry in cryopreserved PBMCs from 20 HTLV-1-infected individuals (10 asymptomatic carriers and 10 with HAM/TSP) and in 12 matched healthy controls . MAIT ceNext, we studied the expression of the activation markers CD38 and HLA-DR by CD4 T cells, CD8 T cells, and MAIT cells . CD4 T cWe then looked for associations between immune activation and HTLV-1 proviral load. As expected, we found a positive association between HTLV-1 proviral load and CD4 T cell activation Click here for additional data file.S2 FigRepresentative flow plots showing PD-1 expression by MAIT cells  from healthy controls and HTLV-1-infected subjects (A). PD-1 expression by CD4 T cells (B), CD8 T cells (C), and MAIT cells (D) from healthy controls (n = 12), asymptomatic carriers (n = 10), and HAM/TSP patients (n = 10). Association between co expression of CD38 and HLA-DR by MAIT cells and PD-1 expression by MAIT cells (E).(TIFF)Click here for additional data file.S3 FigRepresentative flow plots showing CCR6 expression by MAIT cells  from healthy controls and HTLV-1-infected subjects (A). Representative flow plots showing PLZF expression in healthy controls, asymptomatic carriers, and HAM/TSP patients (B).(TIFF)Click here for additional data file.S4 FigIFNγ production by unstimulated MAIT cells from healthy controls (n = 11) and HTLV-1-infected subjects (n = 17) (A). IFNγ production by unstimulated MAIT cells from asymptomatic carriers (n = 8), and HAM/TSP patients (n = 9) (B).(TIFF)Click here for additional data file.


A sentence is omitted after the first sentence in the first paragraph under the subheading “Flow cytometry” in the Materials and Methods section.The missing sentence should read: For Glut-1 staining, MDDCs (1 10e5 cells) were incubated with 2.5 µl of Glut1-RBD-GFP construct (Metafora biosystems) for 30 min and fixed with 4% PFA before FACS analysis.In addition, a reference is omitted from the second sentence of the first paragraph under the subheading “HTLV-1 viral entry is more efficient in DCs that restrict HTLV-1 infection” in the Results section.The sentence should read: Two proteins, the binding receptor NRP-1 [25] and the fusion receptor Glut-1  have been involved in HTLV-1 entry in CD4+ T lymphocytes.The reference is: Manel N, Kim FJ, Kinet S, Taylor N, Sitbon M, et al. (2003) The ubiquitous glucose transporter GLUT-1 is a receptor for HTLV. Cell 115: 449–459.


Abnormalities in GABA signaling in the forebrain at all stages - prenatal, postnatal and adult have been implicated with a wide range of neuropsychiatric illnesses ,2. AdditA receptors and GABA function with defective behaviors have been vital for understanding the pathobiology of neuropsychiatric illnesses, but all of these models were systemic or region-specific knockouts that could not serve to establish a cause - effect relationship between neuronal versus endothelial cells. Our observation of expression of GABAA receptor subunits and GAD65/67 in forebrain endothelial cells signified that forebrain angiogenesis has its own intrinsic GABA signaling mechanisms. To discover the functional significance of this endothelial GABA signaling pathway in vivo, we designed strategies to specifically turn off GABA release from endothelial cells or render endothelial GABAA receptors dysfunctional using cre-lox technology during early embryonic development. Both approaches first affected forebrain angiogenesis and in turn impaired neuronal migration. As endothelial GABA’s roles were delineated individually for angiogenesis, neurogenesis, long distance GABAergic neuronal migration and radial migration of projection neurons, so too was the realization that neuronal GABA could not serve to compensate these roles. The data laid the foundation for a novel positive feedback signaling pathway in endothelial cells that functions via GABAA receptor mediated GABA release [Though there have been reports of GAD immunoreactivities in endothelial cells of cerebral arteries  and GAD6A receptors, NKCC1-KCC2 expression, and chloride concentration in forebrain endothelial cells and alterations in disease scenarios. And most importantly, additional questions begin to emerge about the significance of this vascular GABA pathway and novel mechanisms specifically in the postnatal, adult and aging brain (As we now know that a common GABA pathway operates in both endothelial cells and GABAergic neurons of the embryonic telencephalon, it is essential to gain further mechanistic insights by segregating this pathway in individual cell types. It is important for future work to understand how the endothelial GABA signaling pathway specifically influences angiogenesis related genes and specific processes like tight junction formation, tip cell functions, vascular sprouting and migration. Also, it is important to see how components of the neuronal GABA pathway are affected in the absence of endothelial GABA signaling. For instance, how does endothelial GABA modulate interneuron migration - does it affect receptor-mediated signaling that transduces extracellular information or directly the transcription factors that provide interneurons with their intrinsic migratory ability or both? It also brings up several new questions with respect to roles of GABAng brain . New und


The purpose of this review is to discuss the possibility of the treatment of spinal cord injury (SCI) with curcumin via regulating the mTOR signaling pathway, which may provide another strong support for curcumin to be a promising medicine applied to the treatment of SCI. Curcumin is termed as a multifunctional targeting therapy drug that regulates the mTOR signaling pathway in the treatment of numerous diseases. Previous research has already revealed that mTOR signaling pathway plays a vital role in prognosis, which involves the axon regeneration and autophagy. This review discusses a potential mechanism that curcumin suppresses the activation of this pathway and ameliorates the microenvironment of axons regeneration which would provide a new way that induces autophagy appropriately. There after SCI  , interleukin- (IL-) 1α, IL-1β, and IL-6, and lead to the disorder of the microenvironment of the spinal cord. Besides, infiltrating macrophages, neutrophils, and activated microglia also join the oxidative stress with providing extra sources of free radicals. Free radicals react with the polyunsaturated fatty acid of the cellular membrane yielding to the peroxidation and disorder of the normal phospholipid structure of cellular and subcellular organelle membranes, which significantly contributes to neural tissue damage. In particular, extracellular amino acids, glutamate and aspartate, are increasingly released from disorder cells. The excessive activation of relevant amino acid receptors produces excitotoxicity and leads to the loss of neurons both in necrosis and apoptosis. Apoptosis occurs in neurons, oligodendrocytes, microglia, and astrocytes. Oligodendrocytes are easily subjected to apoptotic loss that contributes to postinjury demyelination [The secondary phase is a delayed and progressive tissue damage following the initial onset of SCI, which occurs over the time course of hours to weeks and is caused by a cascade of biological events. Following the mechanical insult, there is an unavoidable structural destroy and vascular dysfunction that end up with edema, necrosis and ischemia. Vascular dysfunction includes hemorrhage, vasospasm, thrombosis, loss of autoregulation, and disruption of blood spinal cord barrier. These impairments lead to more inflammatory cells across the defective barrier and expansion of the initial damage. The inflammatory response is regarded as an important contribution to secondary damage; inflammatory cells such as macrophages, microglia, T cells, and neutrophils infiltrate the lesion area, which is releasing inflammatory factors such as tumor necrosis factor-lination , 11.Eventually, all of these processes bring neuron death, axon demyelination, reactive gliosis, and syringomyelia around the lesion. Considering the pathophysiology of SCI, alleviating secondary damage is a key point to the treatment .mTOR signaling pathway is a promising pharmacological option that is well known for its roles in the process of growing cells, metabolism, and cancer. mTOR, is a core of the signaling networks, which belongs to the phosphoinositide 3-kinase- (PI3K-) related protein kinase (PIKK) family. It assembles into two complexes, mTORC1 and mTORC2, which were composed of its subunit raptor and rictor. Respectively, both contained mLST8 subunit, with distinct inputs and downstream effects –15. Likeβ (TGF-β) are upregulated after SCI through non-Smad-dependent activation of the PI3K/Akt/mTOR signaling pathway, rather than the Smad2/3 signaling pathway [Glial scar, an extrinsic environment for axon regeneration, acts like physical and chemical barriers and is mainly composed of reactive astrocytes, with the upregulation of glial fibrillary acidic protein (GFAP) and vimentin, the hallmarks of the gliosis process, and extracellular matrix molecules particularly interact with the chondroitin sulfate proteoglycans (CSPGs), which is degenerated by Chondroitinase ABC (ChABC) allowing axon regeneration and function recovery in SCI . Neverth pathway , but the pathway   are directly phosphorylated and suppressed by mTORC1 . LC-3 an Curcuma longa, was first found to have antibacterial action in 1949 [Curcumin, a pleiotropic molecule from in 1949 . Extensi in 1949 –55, diab in 1949 , 57, dia in 1949 –60 and d in 1949 –63, card in 1949 , CNS dis in 1949 , and oth in 1949 .At present, curcumin, a well known anticancer medicine entering phase III clinical trials approved by FDA, aims at several signaling pathways and demonstrated the suppression of mTOR signaling pathway , 68. WhiPrevious researches have already shown the positive influence of curcumin on the treatment of SCI, and a meta-analysis revealed the neurological recovery effect of curcumin . The expThe mTOR signaling pathway is involved in the axon regeneration and autophagy in the pathological process of SCI. While curcumin is also well known as a multiple targets medicine that can inhibit the mTOR signaling pathway in numerous diseases, likewise, the mentioned evidence above indicates that mTOR signaling pathway is a potential therapeutic target of curcumin in the treatment of SCI by ameliorating the microenvironment of axons regeneration which induced autophagy appropriately as well as having little impact on the intrinsic mechanism of the axon regeneration. However, there is still no investigations referring to the curcumin's influence involved in mTOR signaling pathway; thus another research should be done to make the related mechanisms about it clear, and it will provide another strong evidence for the potential utility of curcumin through the treatment of SCI.