The granule-dependent cytotoxic activity of T and natural killer lymphocytes has progressively emerged as an important effector pathway not only for host defence but also for immune regulation. The analysis of an early-onset, severe, primary immune dysregulatory syndrome known as hemophagocytic lymphohistiocytosis (HLH) has been decisive in highlighting this latter role and identifying key effectors on the basis of gene mutation analyses and mediators in the maturation and secretion of cytotoxic granules. Studies of cytotoxicity-deficient murine counterparts have helped to define primary HLH as a syndrome in which uncontrolled T-cell activation in response to lymphocytic choriomeningitis virus infection results in excessive macrophage activation and inflammation-associated cytopenia. Recent recognition of late-onset HLH, which occurs in a variety of settings, in association with hypomorphic, monoallelic mutations in genes encoding components of the granule-dependent cytotoxic pathway or even in the absence of such mutations has broadened our view about the mechanisms that underlie the perturbation of immune homeostasis. These findings have led to the development of a model in which disease occurs when a threshold is reached through the accumulation of genetic and environmental risk factors. Nevertheless, validation of this model will require further investigations. However, only recently have studies of inherited deficiencies of lymphocyte cytotoxic activity in humans highlighted the importance of lymphocyte cytotoxicity in the resolution of inflammation2.The role of cytotoxic lymphocytes in defending the organism against virally infected cells and tumor cells has long been recognized. Through the polarized secretion of granules containing cytotoxic proteins, cytotoxic lymphocytes can rapidly kill their cognate target cells+ T cells; this probably results from the failure of cytotoxic T lymphocyte (CTL) and natural killer (NK) cells to clear antigen-presenting cells (APCs) and therefore terminate an immune response4. Uncontrolled T-cell activation leads to macrophage activation, a pro-inflammatory cytokine storm, cytopenia, coagulopathy, multi-organ cellular infiltration, and organ dysfunction7. The link between cytotoxicity and lymphocyte homeostasis was first demonstrated 15 years ago, following the identification of perforin deficiency in a subgroup of patients with an inherited form of HLH 2. Undoubtedly, this step was decisive in the characterization of the other causes of inherited HLH and in the identification of key effectors that mediate the exocytic machinery in cytotoxic lymphocytes9. Naturally occurring or engineered mice with a similar cytotoxicity defect have proven to be very useful tools for further understanding the underlying pathophysiological mechanism11. The observation that a relatively mild cytotoxic defect can be associated with defective immune surveillance or atypical HLH onset or both has now raised the question of the underlying individual’s risk factors that are associated with a subtle cytotoxic defect to drive disease onset.Hemophagocytic lymphohistiocytosis (HLH) syndrome is a life-threatening immune dysregulation condition characterized by an excessive inflammatory response and hypercytokinemia. It is generally triggered by an infective agent, such as the members of the human herpes virus family. HLH manifests as the massive expansion and activation of polyclonal CD812. Within the very first minutes of target cell interaction, the actin network, which was previously positioned across the entire contact area, is progressively depleted from the center of the synapse, as recently highlighted by the use of rapid, super-resolution imaging methods15. The microtubule-organizing centre (MTOC) rapidly moves toward the target cell, while the cytotoxic granules (containing the cytotoxic proteins perforin and granzymes) migrate along microtubules and cluster around the MTOC16. At the IS, the centrosome can touch the membrane and then deliver polarized cytotoxic granules17. The granules fuse with the presynaptic membrane and secrete their contents into the synaptic cleft. This accurate, polarized secretion of lytic reagents ensures that cytotoxic cells destroy only the bound target cell and not bystander cells. Within the synaptic cleft, perforin oligomerizes, creates pores in the target cell membrane, and thus enables the pro-apoptotic granzymes to access the target cytosol20. The mechanism of granzyme uptake has long been subject to debate. By using time-lapse microscopy techniques that pinpoint the moment at which perforin permeabilizes the target cell plasma membrane within the IS, researchers observed that the time course of target cell apoptosis after pore formation is very rapid 22. In contrast to what has been proposed in other studies25, this suggests that granzymes cross the plasma membrane and are not taken up in endosomes.The sequence of events by which T/NK cytotoxic lymphocytes kill targets is now fairly well characterized. When cytotoxic lymphocytes recognize their cognate target cells, they form a transient cellular conjugate and an immunological synapse (IS) at the area of cell-cell contact2, which accounts for about one third of the FHL cases, several inherited forms of HLH are characterized by failure to deliver cytotoxic granule contents. The identification of the underlying molecular causes has contributed to our understanding of the key steps in the secretion of cytotoxic granules at the IS8. Biallelic mutations inUNC13D ,STX11 , andSTXBP2  led to the occurrence of HLH in FHL types 3, 4, and 5, respectively29. In about 10% of FHL cases, the molecular defect remains uncharacterized. Biallelic mutations inRAB27A  andLYST  account for the development of HLH in Griscelli syndrome30 and Chédiak-Higashi syndrome31, respectively. Remarkably, each of these molecules mediates a discrete, non-redundant step in cytotoxic granule exocytosis at the IS. Rab27a and Munc13-4 are respectively required for the granule docking and priming steps at the plasma membrane, whereas syntaxin 11 interacts with Munc18-2 to enable granules to fuse with the plasma membrane8. Although the role of LYST is less well understood, it may regulate a late granule maturation step32. The effector molecules’ partners and interconnections have been progressively characterized to reveal the overall picture of granule exocytosis. Notably, interaction between Rab27a and Munc13-4 was shown to be mandatory for tethering the cytotoxic granules at the IS in order to complete the exocytic process33. Munc13-4 interacts with several syntaxin isoforms, among them syntaxin 1134. Furthermore, Rab27a binds to three different members of the synaptotagmin-like (SLP1-3) family expressed in cytotoxic cells that have partially overlapping functions in granule transport and docking37.In addition to perforin deficiency38. However, there is evidence to suggest that alternative mechanisms are involved, such as the observation that very rapid, effective cytotoxic granule secretion can precede MTOC polarization in some CTL-target cell conjugates39. In the latter study, inhibition of MTOC polarization did not prevent cytotoxic granule release. Furthermore, the Slp3/Rab27a complex expressed in cytotoxic cells was shown to interact with a kinesin motor and to mediate the terminal transport of polarized cytotoxic granules toward the IS35. In view of the diversity ofin vivo settings in which cytotoxic cells are triggered, one can legitimately hypothesize that granule delivery may occur via several different routes. Indeed, recent research has shown that cytotoxic cells are heterogeneous and change their killing performance over time and as a function of antigenic stimulation41. Timescale studies of single NK cells or CTLs have revealed a progressive increase in the rapidity and efficiency of killing during serial killing, which also varies according to the avidity of antigen recognition41. It remains not well understood how what has been shownin vitro appliesin vivo, such as the nature of target cells and the strength of triggering signal.It has been proposed that direct contact between polarized centrosomes and the plasma membrane drives cytotoxic granule delivery at the IS4. LCMV’s potent induction of HLH might be due, at least in part, to its ability to infect APCs and thus strongly stimulate a T-cell response without the need for antigen cross-presentation. Likewise, Epstein-Barr virus (EBV), a major trigger of HLH in humans, can directly infect B cells, which also have an antigen presentation function and trigger prolonged antigenic stimulation when not eliminated by cytotoxic lymphocytes. In addition to viral priming, antigen persistence and prolonged presentation were shown to be critical in the development of primary HLH in murine models10. This appears to contrast with the onset of primary HLH in newborn infants or even fetuses with cytotoxicity defects, since a pathogen trigger cannot be identified in many cases43. Although as-yet-unknown microorganisms may act as the trigger, this observation suggests that, in contrast to the situation in mice, the granule-dependent cytotoxic pathway in humans also has a role in T-cell homeostasis in the absence of an external stimulus . It has been shown that the elimination of a rare, antigen-presenting dendritic cell (DC) population by CD8+ T cells in a negative feedback loop is a critical determinant of the magnitude of T-cell responses45. Thus, the elimination of specific APC populations probably determines the activation status and survival of hyper-reactive T cells and acts as a rheostat by limiting T-cell responses. Whether the granule-dependent cytotoxic pathway is also participating to check self-reactive T/B cells is therefore a possibility that needs to be further investigated.Animal models of primary HLH in which cytotoxicity-deficient mice are challenged with a virus have proven to be invaluable for understanding the pathogenesis of HLH under defined conditions. It has been demonstrated that after lymphocytic choriomeningitis virus (LCMV) infection of perforin-deficient mice, hyperactive CTLs and high levels of interferon-gamma (IFN-γ) are the driving forces behind the development of fatal HLH46. The same is true in humans46  cells and CD4+ T-cell subsets, including specific regulatory T (Treg) populations, in this setting. It is also worth noting that in syntaxin 11-deficient mice, which display a milder cytotoxic defect and less severe HLH than perforin-deficient mice, blockade of inhibitory receptors of T-cell exhaustion (such as PD1/PDL1) dramatically increases the severity of HLH and results in fatal disease49. This finding indicates that T-cell exhaustion is another important modulator of HLH severity.In mice, the degree of cytotoxicity impairment appears to be the best predictor of the development and severity of HLH, as shown by studies of the time course of HLH onset in various HLH-prone strains with defects in the granule-dependent cytotoxic pathwaymans46 . In genein vivo failure of cytotoxic cells to eliminate target cells leads to a fatal cytokine storm, a hallmark of HLH. Previous research has shown that the threshold of T-cell activation determines whether a lytic synapse (which is induced at low antigen concentrations and which enables cytotoxic activity) or a stimulatory synapse (induced at high antigen concentrations and which enables both cytotoxic activity and IFN-γ production) is formed50. Remarkably, it was recently shown that cytotoxicity-deficient lymphocytes form longer contacts with their cognate target, thus resulting in many successive rounds of Ca2+ flux into cytotoxic cells and triggering of pro-inflammatory cytokine secretions51. Thus, the cytokine storm as observed in HLH likely depends on both quantity and quality of contacts formed between cytotoxic cells and APCs.The52. A study of perforin-deficient mice has revealed that IFN-γ specifically triggers this process, which can be reproduced in wild-type mice by inducing the sustained elevation of IFN-γ. Direct, IFN-γ-dependent activation of macrophages prompts the development of severe, consumptive anemia and other types of cytopenia, probably through direct changes in the macrophages’ endocytic uptake53. These results indicate that hemophagocytosis is actually an adapted response to sustained or severe inflammation. Further details on the role of macrophages and other inflammatory cells in the pathophysiology of HLH have been provided in recent reviews54.Phagocytosis of blood cells by macrophages (known as hemophagocytosis) is another hallmark of primary HLH, although it can be observed in a variety of infectious or inflammatory disorders55. Moreover, high levels of IL-4 or repeated TLR9 stimulation in wild-type mice can induce the development of an HLH-like syndrome57. Hence, proteins from the cytotoxic exocytic pathway may have additional functions in other immune cell types (such as inflammatory cells), the absence of which modulates the pathogenesis of HLH. More generally, any regulatory molecule involved in an inflammatory pathway might contribute to the development of the manifestations of HLH.Although cytotoxic lymphocytes exert a key role in the development of primary HLH, other immune cells and signaling pathways may also contribute. It has been shown that MyD88, which mediates Toll-like receptor (TLR) and interleukin-1 (IL-1) signaling, is required for HLH development in Unc13d-deficient mice, suggesting that innate immune cells contribute to the development of HLH58. There are two genetic forms: XLP-1 and XLP-2. Firstly, XLP-1 results from a deficiency in the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP)61. SAP-deficient CTLs and NK cells are selectively impaired in their cytotoxic response to infected B cells; the response requires interaction between SLAM family receptors and subsequent SAP-dependent signaling in T lymphocytes but not in other cell types63. Secondly, XLP-2 results from a deficiency in the X-linked inhibitor of apoptosis protein (XIAP) 64. However, XIAP-deficient CTLs and NK cells exhibit apparently normalin vitro cytotoxic responses (regardless of the SLAM-receptor dependency). The cytotoxic activity of iNKT cells is known to be activated by EBV-infected B cells65. Indeed, the exacerbated apoptosis of XIAP-deficient iNKT cells, induced by EBV infection, might be involved in the development of HLH in XLP-2. Alternatively, the mechanisms underlying EBV-driven HLH in XLP-2 may differ completely from those observed in XLP-1 and other inherited forms of HLH. In a setting of XIAP deficiency, the accumulation of apoptotic cells and the persistence of EBV-infected cells might trigger abnormal inflammation and contribute to the development of HLH. This hypothesis is supported by the observation that XIAP deficiency in mice results in excessive DC death and inflammasome activation66.Genetically determined forms of HLH can occur even when a cytotoxicity defect is only partial or apparently absent. This is the case in X-linked lymphoproliferative syndrome (XLP). Patients with XLP are extremely vulnerable to EBV infection and most go on to develop HLH69. Adult patients with HLH have been found to carry a monoallelic mutation in one or more FHL genes73. These findings suggest that the accumulation of heterozygous mutations that partially impair the granule-dependent cytotoxic pathway may have an additional functional impact. This hypothesis could be tested by studying inter-crossed animal models of HLH with monoallelic mutations. The concept whereby a monoallelic mutation in cytotoxicity-related genes can lead to immune disturbance also requires far more investigation, particularly in much larger cohorts of patients with induced HLH versus healthy controls. When the cytotoxicity defect is mild, the relative weight of additional genetic and environmental factors in HLH triggering is probably greater. It is tempting to speculate that (i) “extreme” stimuli may be sufficient to induce sporadic HLH development in any individual and (ii) the overall risk is augmented by the accumulation of genetic variants promoting excessive or poorly regulated immune response  is needed for the development of HLH in a context of residual cytotoxicity. Some cases of HLH do not appear to be directly related to a cytotoxicity defect, indicating that other genes, notably involved in macrophage-related inflammation, regulating the same disease pathway also have a role. Characterizing the synergistic connections between the various risk factors for HLH will be a key challenge in the coming years.APC, antigen-presenting cell; CTL, cytotoxic T lymphocyte; DC, dendritic cell; EBV, Epstein-Barr virus; FHL, familial hemophagocytic lymphohistiocytosis; HLH, hemophagocytic lymphohistiocytosis; IFN-γ, interferon-gamma; IL, interleukin; iNKT, invariant natural killer T; IS, immunological synapse; LCMV, lymphocytic choriomeningitis virus; MTOC, microtubule-organizing centre; NK, natural killer; SAP, signaling lymphocyte activation molecule-associated protein; SLAM, signaling lymphocyte activation molecule; TLR, Toll-like receptor; XIAP, X-linked inhibitor of apoptosis protein; XLP, X-linked lymphoproliferative syndrome. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Background. There is a strong association between crack/cocaine use and increased sexual risk behavior, but little research on the efficacy of HIV education for decreasing such behavior in crack/cocaine-addicted individuals in substance abuse treatment. Method. Datasets from two cocaine dependence trials including either one or three HIV education sessions, respectively, were analyzed for changes over time in the proportion of participants practicing safe sex. A pooled dataset from two earlier trials not offering HIV education was also analyzed. Results. We included 83 participants from the 1-session trial and 65 participants from the 3-session trial. Both sets of participants evidenced a significant increase in the proportion of participants having safe sex with casual partners. Participants in the 3-session HIV education study also evidenced a significant increase in the proportion of participants having safe sex with regular partners. In the trials without HIV education, no change in safe sex practices was found, and change in condom use was observed only among female participants. Conclusions. These findings are consistent with recommendations that HIV education/counseling should be provided to individuals in substance abuse treatment. A randomized controlled trial to confirm these results may be warranted. This trial is registered with NCT00033033, NCT00086255, NCT00015106, and NCT00015132. Approximately 47,800 Americans contract HIV annually, with sexual contact accounting for about 87% of transmissions . MultiplTo investigate before and after changes in SRB, this paper presents data from two placebo-controlled randomized multisite cocaine dependence trials, one evaluating reserpine  and one In the reserpine and tiagabine trials, a master's level clinician provided each participant with an hour of individual CBT on a weekly basis for 12 weeks. Both studies included HIV education, covering the topics outlined in the “General Guidelines” section of In the reserpine and tiagabine trials, SRB was assessed with the interviewer-administered HIV-risk-taking behavior scale (HRBS) , which an = 60 and n = 67, resp.) and were conducted by the same investigators and in the same geographic region, these datasets were combined for the present analysis.To investigate whether changes in SRB might occur in a clinical trial setting in the absence of HIV education, we analyzed data from two earlier pharmacotherapy cocaine dependence trials in which CBT, but no HIV education, was provided by a master's level therapist. These trials were both ten-week outpatient studies conducted using the Cocaine Rapid Efficacy and Safety Trial (CREST) study design in which three medications and an unmatched placebo are evaluated. The first trial evaluated reserpine, gabapentin, and lamotrigine  while thThe participants and procedures for these trials are described in detail elsewhere , 19. In Participants were included in the data analysis if they completed all of the scheduled SRB assessments . The rationale for including only completers was that we wanted to include only the participants who received all 3 of the HIV education sessions in the tiagabine study. Since the reserpine and tiagabine trials included different assessment time frames, these datasets were analyzed separately. Within each dataset, analysis was completed for two sets of participants: one including all participants who completed the scheduled SRB assessments and one including only participants reporting sexual nonabstinence during the 30 days assessed. The rationale for conducting the analyses for the subset reporting sexual nonabstinence was to determine whether any decrease in the proportion of participants having unsafe sex was due solely to participants being abstinent during a given 30-day period or to individuals who were using condoms consistently during sex.We utilized GENMOD (SAS Institute Inc.) for all analyses. The first step in each analysis was to determine whether there were any significant effects for study site, medication condition, or gender and thus a GEE analysis was conducted including each of these variables. These analyses revealed a significant site effect for the reserpine analyses for regular partners and, thus, site was included as a fixed effect. Otherwise, the GEE analyses regressed the outcome measures only against time. For the tiagabine data, any analysis with a significant time effect was followed up by a GEE analysis treating time as a class variable and including contrasts  to determine the source of the significance.t-tests were conducted for continuous variables.Of the 141 tiagabine participants, 65 (46%) completed the three HRBS assessments. Of the 119 reserpine participants, 83 (69%) completed the two HRBS assessments. The 65 tiagabine participants are referred to as the substance use disorder treatment plus 3-session HIV group (SUD-3-HIV), while the 83 reserpine participants are referred to as the substance use disorder treatment plus 1-session HIV group (SUD-1-HIV). χ2 = 4.84, P < 0.05), with significantly more African Americans in the reserpine completers. SUD-3-HIV and SUD-1-HIV group comparisons revealed several significant differences including a greater proportion of SUD-1-HIV participants being employed . In addition, a greater proportion of the SUD-3-HIV participants engaged in unsafe sex  and inconsistent condom use  with their regular partners at baseline. Finally, a greater proportion of the SUD-3-HIV participants, compared to the SUD-1-HIV participants, used condoms inconsistently with customers at baseline .The comparisons of the tiagabine completers and noncompleters revealed no significant differences. The comparisons of the reserpine completers and noncompleters revealed a significant difference in race , an effect not seen for the SUD-1-HIV participants . The analyses of unsafe sex with casual sexual partners revealed a significant decrease in the proportion of participants having unsafe sex for both the SUD-3-HIV  and SUD-1-HIV  groups as a function of time. The analyses of unsafe sex with customers revealed no significant time effect for either the SUD-3-HIV  or SUD-1-HIV  groups.The unsafe sex analyses included all of the SUD-3-HIV or SUD-1-HIV participants, regardless of their sexual activity. Z = 2.77, P < 0.01), an effect not seen for the SUD-1-HIV participants . The analyses for casual sexual partners revealed a significant decrease in the proportion of the participants using condoms inconsistently for both the SUD-3-HIV  and SUD-1-HIV  participants as a function of time. The analyses for customers revealed no significant time effect for either the SUD-3-HIV  or SUD-1-HIV  participants; this lack of significance is likely due to the very small sample sizes for this comparison, with the sample being as small as five in some cells.Analyses of inconsistent condom use included only participants reporting sexual nonabstinence during the 30 days assessed. The results described above suggest that there were significant increases in safe sex behavior in the tiagabine and reserpine trials, but whether this was due to the provision of HIV education is unclear. To help address this question, we completed an analysis following the plan outlined in X = 39.4, SD = 6.4) than both the SUD-1-HIV  and SUD-3-HIV  participants and had less education  compared to the SUD-1-HIV participants . The CREST participants were more likely to be employed  and had a larger proportion of minority participants  compared to the SUD-3-HIV participants. All other comparisons were nonsignificant.Of the 127 CREST participants, 91 (72%) completed the two RAB assessments and were included in the analysis. The baseline and demographic characteristics of the CREST participants were compared with those of the SUD-1-HIV and SUD-3-HIV participants. The CREST participants were, on average, younger . The analysis of consistent condom use revealed a significant gender by time interaction effect ; a review of the graph (data not shown) revealed that female participants evidenced decreases in the proportion of participants using condoms inconsistently, an effect not seen for the male participants.The unsafe sex analysis revealed no significant change as a function of time , while in the other they were offered three sessions of HIV education (SUD-3-HIV). The results suggest that participants in both trials evidenced significant and clinically meaningful increases in safe sex practices. These findings are consistent with recommendations that HIV education/counseling should be provided to individuals in substance abuse treatment.There are several limitations to the present findings. First, the analyses were conducted post hoc and, thus, should ideally be replicated in a future study in which the analyses are defined a priori. Another potential weakness was the use of self-reported safe sex practices. However, research has found that self-reported SRB correlates highly with corroborator-reported SRB . The lacAnother potential limitation is the inclusion of participants who completed either a 12-week trial (SUD-1-HIV) or a 19-week trial (SUD-3-HIV). While the lack of baseline differences between the completers and noncompleters suggest that the completers were representative of the entire sample, it would have been ideal to include a greater proportion of participants. The lack of a no HIV education control group is another limitation in that the observed reductions in SRB might be due solely to the effects of the substance abuse treatment provided or to other non-treatment related factors. To help address these two issues, we analyzed data from two cocaine dependence trials in which CBT was provided without HIV education. If the improvements in safe sex behavior observed in the SUD-1-HIV and SUD-3-HIV studies were due solely to the inclusion of study completers or CBT, then one would expect to find similar results from the trials that provided CBT without HIV education. The results indicated no change in safe sex practices for the sample as a whole and improved condom use in only the female participants; these finding are consistent with the idea that HIV education is important for increasing safe sex practices.The primary strength of the present evaluation is that it is, to our knowledge, the first to evaluate the efficacy of HIV education in reducing SRB in cocaine dependent participants receiving substance abuse treatment. Both trials found significant decreases in the proportion of participants having unsafe sex and using condoms inconsistently with casual partners. The SUD-3-HIV group, which, compared to the SUD-1-HIV group, had a smaller proportion of participants engaging in safe sex practices with regular partners at baseline, also yielded significant increases in the proportion of participants practicing safe sex and using condoms consistently with regular partners. Previous research with substance abusing populations suggests that increasing safe sex practices with regular partners is significantly more challenging than increasing safe sex practices with casual partners  and so tIn conclusion, the present findings suggest that crack/cocaine addicted individuals receiving substance abuse treatment and HIV education evidenced significant increases on the clinically meaningful measures of safe sex and consistent condom use. It should be noted that even the more intensive 3-session HIV education intervention required less than 1.5 hours of counselor time and, thus, this should be a feasible intervention for substance treatment programs. Given the potential public health significance of these findings, a randomized controlled trial evaluating the effect of HIV education on SRB in crack/cocaine addicted individuals in substance abuse treatment may be warranted.


Arabidopsis thaliana. We show that B-GATAs can be subdivided based on structural features and their biological function into family members with a C-terminal LLM- (leucine–leucine–methionine) domain or an N-terminal HAN- (HANABA TARANU) domain. The paralogous GNC  and CGA1/GNL (CYTOKININ-INDUCED GATA1/GNC-LIKE) are introduced as LLM-domain containing B-GATAs from Arabidopsis that control germination, greening, senescence, and flowering time downstream from several growth regulatory signals. Arabidopsis HAN and its monocot-specific paralogs from rice (NECK LEAF1), maize (TASSEL SHEATH1), and barley (THIRD OUTER GLUME) are HAN-domain-containing B-GATAs with a predominant role in embryo development and floral development. We also review GATA23, a regulator of lateral root initiation from Arabidopsis that is closely related to GNC and GNL but has a degenerate LLM-domain that is seemingly specific for the Brassicaceae family. The Brassicaceae-specific GATA23 and the monocot-specific HAN-domain GATAs provide evidence that neofunctionalization of B-GATAs was used during plant evolution to expand the functional repertoire of these transcription factors.GATA transcription factors are evolutionarily conserved transcriptional regulators that recognize promoter elements with a G-A-T-A core sequence. In comparison to animal genomes, the GATA transcription factor family in plants is comparatively large with approximately 30 members. Here, we review the current knowledge on B-GATAs, one of four GATA factor subfamilies from  Arabidopsis have a type IV zinc finger with the consensus C-X2-C-X17-20-C-X2-C  followed by a highly basic amino acid stretch  or an adenosine (A); R, guanidine (G) or adenosine (A);  stretch . While tc finger .Aspergillus nidulans is a key regulator of nitrogen signaling, which suggested that studies of plant GATAs may also lead to advances in understanding nitrogen signaling in plants . LLM- and HAN-domain containing B-GATAs can be identified in all sequenced dicot and monocot species suggesting that they existed before the monocot-dicot divergence  and CGA1/GNL , representative B-GATAs with an LLM-domain; second, HAN and HANL (HAN-LIKE) proteins from Arabidopsis and monocots, B-GATAs with a HAN-domain. Furthermore, there are Brassicaceae-specific as well as monocot-specific B-GATAs that together provide evidence that the neofunctionalization of B-GATAs was used during plant evolution to expand their functional repertoire . In this review, we will summarize the current knowledge about B-GATAs, their structure, their regulation, and their role in plant development.The knowledge about the identity of GATA factors from  class D . This cl class D  others cGNC and its paralog GNL (GNC-LIKE) had first been noted based on their transcriptionally regulation by nitrate . GNL had initially been designated CGA1 based on its strong transcriptional regulation by cytokinin (CK) and light . Subsequent studies could then show that both GATAs, GNC, and GNL, contribute to the control of greening and also play a role in the regulation of plant development downstream of the hormones gibberellin (GA) and auxin . Thus, these B-GATAs are under the control of multiple signaling pathways including nitrogen availability, several phytohormones as well as light . Common to at least some of these input pathways is that they modulate the greening of the plant, which is the most prominent phenotype not only in the loss-of-function mutant but also in the overexpressors of GNC and GNL . GNC and GNL were also identified as direct targets of the floral homeotic regulatory APETALA3 and PISTILLATA but the functional significance of this regulation remains to be explored  or related complexes .Gibberellin signaling is mediated by interactions between GA and the GIBBERELLIN INSENSITIVE DWARF1 (GID1) GA receptors . GA-bindomplexes . DELLAs omplexes . StudiesGNC and GNL have a number of phenotypes that can be explained by defects in GA signaling in that they promote greening and hypocotyl elongation but repress germination and flowering . When compared to mutants with a strong GA pathway defect, the contribution of GNC and GNL to plant growth regulation is comparatively subtle. For example, the strong flowering time delay of the ga1 mutant is only partially suppressed in ga1 gnc gnl . Interestingly, these arf2 phenotypes are partially or fully suppressed in the presence of gnc and gnl loss-of-function mutants  are phenotypically similar to GNC and GNL overexpressors. In line with a direct activity of ARF7 on the B-GATA promoters, an auxin-modulated binding of ARF7 to the GNC and GNL promoters could be demonstrated. Thus, GNC and GNL transcription is under the control of auxin- and AUX/IAA-independent (ARF2) as well as auxin- and AUX/IAA-dependent (ARF7) transcription factors .Although ARF2 is an auxin regulation-independent ARF-, the transcriptional repression of GNC and GNL suggested that modulation of the expression of the two GATAs would allow for a transcriptional cross-talk between these two pathways. In fact, several phenotypes of the arf2 mutant could be suppressed by GA treatments or in the presence of a spy (spindly) mutation, which phenotypically mimics the phenotypes of plants with constitutively active GA signaling .The observation that the two phytohormones, GA and auxin, repress the transcription of ignaling . Thus, GGNC and GNL expression . CK induces chloroplast division by activating the expression of the chloroplast division regulators PDV1 and PDV2 in a manner that is dependent on the CK-induced regulator CRF2 . Although the number of chloroplasts is not reduced, gnc gnl mutants have smaller chloroplasts in the hypocotyls and reduced chlorophyll levels in seedlings  and ARR12 are important for this regulation since CK-induced gene expression of GNL is strongly compromised in arr1 arr12 mutants where CK effects on chloroplast division are also compromised . Furthermore, gnc gnl mutants exhibit reduced CK sensitivity in chloroplast division . In turn, when SOC1 expression is uncoupled from GNC and GNL control in a SOC1 overexpression line, the flowering repressive effects of GNC and GNL overexpression are suppressed. Thus, GNC and GNL act upstream of SOC1 in flowering time control. a month . A dedicnditions . In shoron by GA . Based oSOC1 and the GATAs in the control of other B-GATA-regulated responses that are not directly related to flowering time control . In fact, the genetic interaction experiments between SOC1, GNC, and GNL indicated that two other phenotypes of soc1 mutants, enhanced greening and decreased cold tolerance, are suppressed in the absence of the GNC and GNL regulators  was independently identified in genetic screens as a mutant with altered floral organ identity . The HAN-domain, which is specific for this family of B-GATAs was first noted in HAN and its HAN-LIKE paralogs from Arabidopsis and later used to classify further B-GATAs as monocot-specific HAN-paralogs . han mutant embryos have several developmental defects, including a vacuolation of the lower tier cells of the embryo and a decrease in suspensor cell divisions  are further indications for defects in proembryo boundary positioning in these mutants. Since lower tier cells normally give rise to the root, root formation is impaired in han embryos. han mutants fail to form an embryonic root and they are unable to undergo an essential cell division of the uppermost suspensor cell, the hypophysis that produces the quiescent center (QC). Interestingly, most han mutants can later recover from this defect and produce a root independently of the hypophysis at a later stage of embryo development  and PIN7 . In the 1 by HAN . Thus, than mutants with altered shoot meristem morphology . When compared to the wild type, han loss-of-function mutants have small flat shoot meristems, reduced numbers of floral organs in all four whorls as well as fused sepals. HAN is expressed between the meristem and between newly initiated floral organ primordia and in the boundaries between the different floral organ whorls. HAN overexpression, on the other side, results in delayed plant growth, disturbed cell divisions, and a loss of meristem activity. Taken together these findings suggest that HAN acts as a repressor of cell proliferation and that loss of this repressive function could lead to the reduced meristem size, which may be the cause for the reduced floral organ numbers and fused floral organs seen in its mutants  pathway  and CLV3 expression overlies the expression domain of WUS suggesting, in combination with evidence from mutant analyses, that WUS controls meristem size by restricting CLV3 expression as an essential ligand for the CLV1 and CLV2 receptor proteins. A han mutation combined with clv gene mutations resulted in increased inflorescence fasciation and increased floral abnormalities . Alternatively, it may be envisioned that defects in nutrient or signal transport hinder meristem growth and floral organ development since HAN is also expressed early in provascular cells  after glucocorticoid treatment were used for the sampling of the material and that many transcription factor genes were found to be regulated downstream from HAN in this experiment argues that the majority of these downstream genes could represent indirect rather than direct targets of HAN.Molecular analysis for transcription factor targets identifies f JAGGED . Genes af JAGGED . This anGNC. Furthermore, genetic interaction studies using mutants of these B-GATAs found that mutant combinations of han with hanl2, gnc, and gnl mutations resulted in a strong decrease in the number of petals formed in these mutants, sepal fusion defects, fertility defects, as well as carpel abnormalities  from rice, TSH1 (TASSEL SHEATH1) from maize and TRD (THIRD OUTER GLUME) from barley . In line with the mutant phenotype, it could be shown that the expression of these HAN-domain B-GATAs is restricted to a cryptic bract in the zone where the suppression of bract formation is observed in the wild type. Interestingly, these B-GATAs form a monocot-specific subclade of HAN-domain B-GATAs indicating that these B-GATAs were recruited for the suppression of bract outgrowth specifically during monocot evolution. The apparent role as a repressor of bract growth also fits to the proposed function for HAN as a repressor of growth and cell cycle activities in the shoot meristem.Whereas the formation of bract leaves is blocked in flowers of cultivated rice, maize, or barley, mutants from each of these species are known where the formation of such bract leaves is derepressed. In each case, the respective locus was identified and found to correspond to the Figures 9). Whereas there is no information about the role of GATA29, GATA23 has been proposed to act in the root following its identification in a search for genes that are induced during the early steps of lateral root initiation (GATA23 is specifically expressed in xylem pole pericycle cells before their first asymmetric division. Auxin accumulation is the first marker for lateral root founder cells and in line with an early role for GATA23 during lateral root initiation, GATA23 is auxin-induced. Since the expression of GATA23 is impaired in gain-of-function mutants of the AUX/IAA gene IAA28, which is defective in lateral root formation, and since IAA28 interacts with several ARFs including the previously introduced ARF+ ARF7 and ARF19, a model was proposed, according to which auxin promotes lateral root initiation through degradation of the AUX/IAA IAA28 and subsequent ARF+-mediated GATA23 expression. This model is supported by observations that lateral root initiation is partially suppressed in plants expressing an GATA23 RNAi construct. The cell type specific expression of GATA23, in turn, correlates with increased lateral root initiations and uncoupling GATA23 expression from auxin control also interferes with the normally regular spacing of lateral roots.Within both B-GATA subfamilies, there is one family member with a degenerate HAN- or LLM-domain, GATA29, and GATA23, respectively (itiation . GATA23 GATA23 is important for lateral root initiation across species remains to be seen. Phylogenetic analyses have revealed that GATA23 from Arabidopsis belongs to a specific clade of B-GATAs with a degenerate LLM-domain that is closely related to GNC and GNL but functionally distinct (Figures 9; Brassicaceae and thus its function in non-Brassicaceae in lateral root initiation cannot be conserved outside of this family. Future research will have to elucidate the apparent functional diversification of these specific B-GATAs.To what degree Important advances have been made in understanding the role of B-GATA transcription factors in plant growth and development. Although there is now a comprehensive understanding of how the expression of these B-GATA genes is regulated at the transcriptional level, the knowledge about the identity of their target genes and cell type-specific activities is scarce. Candidate target genes of B-GATAs were genetically validated in a few exceptional cases only and, as yet, high quality transcription target analyses remain to be performed. Such experiments will be key to understand to what extent B-GATAs have overlapping and distinct transcription targets and should permit to delineate further to what extent differences in their expression domains or differences at the protein level contribute to their functional diversification.Research on the LLM-domain containing B-GATAs has to date largely focused on the signaling events regulating their expression as well as on their role in the control of physiological processes such as greening and flowering. In turn, research on HAN-domain B-GATAs mainly focused on their role in the control of development. It can be anticipated that this apparent separation in the biological functions of B-GATAs between physiology and development will become more and more blurred in the future when developmental biologists will start studying LLM-domain B-GATAs and physiologists will study HAN-domain B-GATAs.The fact that B-GATAs are unstable proteins that are turned-over by the 26S proteasome with a half-life of about 30 min is one interesting observation regarding all B-GATAs that requires further exploration . It implArabidopsis GNC was isolated as an interactor of the transcriptional co-regulator SIN3-LIKE1 (The observation that different members of the plant B-GATA family, namely HAN, GNC, and GNL proteins, can interact with each other, could suggest that also other B-GATAs may act as homo- or heterodimers and may thus engage in interactions that could modulate their DNA-binding specificity or their function as transcriptional activators or repressors . MammaliN3-LIKE1  and secoN3-LIKE1 . Future The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A common finding in time psychophysics is that temporal acuity is much better for auditory than for visual stimuli. The present study aimed to examine modality-specific differences in duration discrimination within the conceptual framework of the Distinct Timing Hypothesis. This theoretical account proposes that durations in the lower milliseconds range are processed automatically while longer durations are processed by a cognitive mechanism. A sample of 46 participants performed two auditory and visual duration discrimination tasks with extremely brief (50-ms standard duration) and longer (1000-ms standard duration) intervals. Better discrimination performance for auditory compared to visual intervals could be established for extremely brief and longer intervals. However, when performance on duration discrimination of longer intervals in the 1-s range was controlled for modality-specific input from the sensory-automatic timing mechanism, the visual-auditory difference disappeared completely as indicated by virtually identical Weber fractions for both sensory modalities. These findings support the idea of a sensory-automatic mechanism underlying the observed visual-auditory differences in duration discrimination of extremely brief intervals in the millisecond range and longer intervals in the 1-s range. Our data are consistent with the notion of a gradual transition from a purely modality-specific, sensory-automatic to a more cognitive, amodal timing mechanism. Within this transition zone, both mechanisms appear to operate simultaneously but the influence of the sensory-automatic timing mechanism is expected to continuously decrease with increasing interval duration. A common finding in time psychophysics is that temporal acuity is much better for auditorily than for visually presented stimuli . This alCommon Timing Hypothesis and the Distinct Timing Hypothesis  approach. In their study on visual-auditory differences in temporal information processing, In another, more recent CFA study on the internal structure of auditory interval timing in the subsecond and 1-s range, In the present study, we transferred these conclusions to visual-auditory differences in duration discrimination of extremely brief and longer intervals. By doing so, we arrived at two predictions. First, if the timing of longer durations involves not only cognitive processes but also depends, at least to some degree, on input from the sensory-automatic timing system, then visual-auditory differences in duration discrimination observed with extremely brief intervals should also become evident for longer intervals. Our second prediction, therefore, was that visual-auditory differences observed with longer intervals in the 1-s range can be explained by modality-specific differences in initial sensory-automatic processing. More precisely, if performance on duration discrimination of longer intervals, in fact, depends on sensory-automatic as well as cognitive processes, then the relative contribution of the sensory-automatic mechanism should become evident when performance scores on auditory  duration discrimination with longer intervals are statistically controlled for performance on auditory  duration discrimination obtained for extremely brief intervals. With such a methodological approach, the visual-auditory difference should decrease, or even disappear, in the adjusted performance scores for longer intervals, if modality-specific differences in duration discrimination indeed originate from the sensory-automatic level of temporal information processing. This line of reasoning, underlying Predictions 1 and 2, implies the following two assumptions: (1) It is possible to dissociate the contribution of the temporal processing of extremely brief intervals from that associated with cognitive processing of longer intervals and (2) the sensory-automatic mechanism is independent of the cognitive timing mechanism.To test our predictions, participants performed auditory and visual two-alternative forced-choice duration discrimination tasks with extremely brief intervals in the subsecond range and longer intervals in the 1-s range. The durations of the standard intervals were 50 ms for the extremely brief and 1000 ms for the longer intervals. These standard durations were chosen because the hypothetical shift from one timing mechanism to the other is supposed to occur somewhere between 100 and 500 ms . FurtherTwenty male and 26 female undergraduate students participated in the present study. Participants’ age ranged from 18 to 28 years (mean age ± standard deviation: 22.7 ± 2.5 years). All participants were naïve with regard to the purpose of the study and reported normal hearing and normal or corrected-to-normal vision. The study was approved by the ethics committee of the Faculty of Human Sciences, University of Bern, and all participants gave their written informed consent.2) positioned at eye level of the participant. Testing took place in a sound-attenuated room with constant ambient light.Temporal stimuli were auditory and visual intervals. Auditory stimuli were white-noise signals presented through headphones (Vivanco SR85) at an intensity of 63 dB(A) SPL. Visual stimuli were generated by a red LED  was computed. Following Table 1. For both extremely brief and longer intervals, smaller DL values and, thus, better performance on duration discrimination, were observed for auditory compared to visual stimuli. Subsequent t-tests revealed that these visual-auditory differences in DL values were statistically significant (see Table 1). In Figure 1, these visual-auditory differences are displayed graphically. For enhancing the presentation of results and to facilitate a comparison across the two ranges of interval duration, Weber fractions  are diagramed instead of absolute DL values  = 1.77, p = 0.19. Performance on auditory and visual duration discrimination of longer intervals was virtually identical as indicated by adjusted mean Weber fractions of 0.175 and 0.173 for auditory and visual intervals, respectively. The adjusted means of longer intervals after controlling for the influence of the sensory-automatic timing system are depicted in Figure 1. As the adjusted means represent the predicted means for the visual and auditory sensory modality, respectively, there is no individual variability and, thus, no standard deviations can be reported. The outcome of the analysis of covariance provided clear evidence for the notion of a modality-specific sensory-automatic timing system. When the influence of this system was statistically controlled for, a visual-auditory difference in duration discrimination of longer intervals could no longer be established.Following these considerations, a repeated-measures analysis of covariance was conducted with performance on duration discrimination with auditory and visual intervals in the 1-s range as dependent variables using BMDP 2V statistical software . Again, The aim of the present study was to systematically investigate modality-specific differences in duration discrimination within the conceptual framework of the Distinct Timing Hypothesis. For this purpose, performance on duration discrimination of extremely brief and longer intervals in the auditory and visual modality was assessed by means of a within-subjects design. Proceeding from a modified version of the Distinct Timing Hypothesis, introduced by This pattern of results is consistent with the general notion that a ‘hard’ boundary between the sensory-automatic and the cognitive mechanism is rather unlikely to exist cf. . InsteadTo date, the mechanism underlying the observed visual-auditory difference in duration discrimination of extremely brief intervals in the 10s-of-ms range still remains unclear. One notion refers to a finer temporal resolution due to more neural pulses accumulated with auditory intervals than with visual ones e.g., . It is damodal, cognitive mechanism for temporal processing of longer intervals. In a most recent series of experiments, however, A large number of studies on interval timing applying a dual-task approach support the view that processing of temporal information in the range of seconds occurs in working memory e.g., . Within Taken together, our findings are consistent with the general notion of two dissociable timing mechanisms underlying the obtained pattern of visual-auditory differences in duration discrimination of extremely brief intervals in the 10s-of-s range and longer intervals in the 1-s range: a modality-specific, sensory-automatic and an amodal, cognitive mechanism. Most importantly, however, the marked visual-auditory differences observed for duration discrimination of extremely brief intervals appeared to depend on the predominating sensory-automatic temporal processing system. Only with increasing interval duration, the amodal, cognitive timing mechanism progressively contributes to the timing process. The present study also showed that it is possible to dissociate the contribution of the sensory-automatic timing system from that of the amodal, cognitive timing system. Finally, unlike the Distinct Timing Hypothesis in its strict sense, our findings argue for a transition zone characterized by a sensory-automatic and cognitive processing overlap. From this perspective, temporal processing of longer intervals in the 1-s range seems to be controlled by and functionally related to both sensory-automatic and cognitive timing mechanisms. As the evidence that the amodal, cognitive mechanism is impacted by the modality-specific, sensory-automatic timing mechanism is based on a null result, future studies are needed to provide additional converging evidence for this notion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N = 76, range 49–80 years at inclusion) at two time points, 3 years apart. A subset of the participants (N = 24) was included at a third time-point. In addition to the frontal-subcortical fibers, the anterior callosal fiber (ACF) and the corticospinal tract (CST) was investigated by its mean FA together with tract parameterization analysis. Our results demonstrated fronto-striatal structural connectivity decline (reduced FA) in normal aging with substantial inter-individual differences. The tract parameterization analysis showed that the along tract FA profiles were characterized by piece-wise differential changes along their extension rather than being uniformly affected. To the best of our knowledge, this is the first longitudinal study detecting age-related changes in frontal-subcortical WM connections in normal aging.Motivated by the frontal- and white matter (WM) retrogenesis hypotheses and the assumptions that fronto-striatal circuits are especially vulnerable in normal aging, the goal of the present study was to identify fiber bundles connecting subcortical nuclei and frontal areas and obtain site-specific information about age related fractional anisotropy (FA) changes. Multimodal magnetic resonance image acquisitions [3D T1-weighted and diffusion weighted imaging (DWI)] were obtained from healthy older adults ( It is well-established that normal aging is associated with changes in brain morphometry and white matter (WM) integrity   MRI-based tractography to extract frontal-subcortical fiber bundles  across tracts, subjects, and age, where results are likely influenced by both methodological variations and biological processes. The study is thus an important supplement to cross-sectional overall mean FA analyses, and provides more comprehensive spatio-temporal information about the aging process in frontal WM  who took part in a longitudinal study on cognitive aging. Demographic data are summarized in Table Twenty-four of the subjects  did also participate in a third wave, 3–4 years (mean 3.6) after the second one. Their scores on the MMSE were equal to or greater than 25 (range 25–30). The neuropsychological and MRI examination protocols were similar to the ones used in the two earlier waves.Three participants were excluded from the longitudinal analyses: two because they were diagnosed with Parkinson's Disease, and one due to a low MMSE score (23) in the third wave. All participants signed an informed consent form, and the Regional Committees for Medical and Health Research Ethics of southern (wave 1) and western (wave 2 and 3) Norway approved the study.b = 1000 s/mm2; 5 images with b = 0; for all three waves. Details about the MRI pulse sequences are given in Table Multimodal MRI examinations, both at baseline and at the follow-ups, were performed on the same 1.5 T GE Signa Echospeed scanner  using a standard 8-channel head coil. No major scanner upgrades were done between wave 1 and wave 3. The 3D T1-weighted and the DWI data were acquired during the same imaging session, without repositioning the subject, allowing the positional information in the DICOM file headers to be used in subsequent multimodal image registration. At each wave, two consecutive T1-weighted 3D volumes were recorded using a fast spoiled gradient echo (FSPGR) sequence. DWI were collected using a spin echo echo planar (SE-EPI) sequence including 25 directions with http://surfer.nmr.mgh.harvard.edu) was used for structural image analysis at all three waves. The two 3D T1 weighted volumes recorded in succession were first coregistered and averaged to improve signal to noise ratio (SNR). The following semi-automated FreeSurfer processing pipeline included Talairach registration . The Diffusion Toolkit software package  was then used for tensor reconstruction from the DWI images and voxel-wise calculation of the eigenvalues and eigenvectors of the tensor  was used for visualizing the tracts. Figure The algorithm used tracts from the entire volume and selected those specific tracts fulfilling the criteria of running between or crossing one or more of the desired ROIs . The mean percentage change was first calculated for each subject and then averaged for the total sample; the latter one is presented in the Tables 7. In the tract parameterization analysis we segment-wise calculated mean FA ± standard error (SE) and percentage rate of change in FA per year, and performed paired t-test analysis . For the tract parameterization analyses, corrected alpha level was set to 0.0005 after correcting for 100 multiple comparisons (the number of segments in one tract).Our combined segmentation and fiber reconstruction methods provided identification, reconstruction, and information about the FA properties of several frontal-subcortical fiber connections. Examples of the reconstructed frontal-subcortical fibers are visualized in Figure Striatum, including caudate and putamen, exhibited the highest number of frontal connections. Connectivity between the caudate nucleus extended especially to the superior frontal, the lateral, and medial orbitofrontal, and the rostral middle frontal WM areas. Putamen had projections to the same frontal areas as the caudate nucleus, with the addition of the pars triangularis. Thalamus had fewer connections to orbitofrontal areas compared to the striatum. With the thalamus as seed region, superior frontal, and rostral middle frontal areas seemed to be those target areas exhibiting the highest numbers of connections. Fiber bundles arising from nucleus accumbens as the seed region had distinct connections to the orbitofrontal areas (mainly found in between 80 and 90% of the subjects), leaving other frontal areas without detectable connections. Still, only the connection to the left medial orbitofrontal area fulfilled the threshold criterion. Sparse connections were seen between pallidum and the frontal WM areas, not fulfilling the threshold criteria.Ten of the 19 fiber connections being included according to the selection criteria revealed significant mean FA decrease from Wave-1 to Wave-2 Table , exhibitWhile the 10 frontal-subcortical connections revealed significant decline in tract mean FA with age  and a maximum decrease of 1.71% , with a mean of 0.82% decrease in tract mean FA each year Table .As illustrated in Figure Highly significant age related longitudinal decrease in mean FA was found bilaterally in the ACF. In the full cohort, the annual mean FA reduction in this fiber bundle was −1.50% across Wave-1 to Wave-2 Table , and 1.3The longitudinal tract parameterization analysis of FA along the ACF bundle is depicted in Figure To the best of our knowledge, this is the first longitudinal investigations of FA time courses obtained for a large set of frontal-subcortical fiber tracts in healthy aging across an observation time of at least 7 years. Using multimodal image registration, image segmentation, and tractography, we were able to identify frontal-subcortical fiber connections between 11 cortical regions and five subcortical structures in each hemisphere, and further address changes in tract-wise mean FA and local segment-wise FA. A major finding was age-related reduction in tract-wise FA, confirming previous reports from cross-sectional studies  and faulty registration  are called upon. One important class of such LDA methods are linear and non-linear mixed effects models (e.g., Long, AV: Statistical analyses and interpretation of the results; writing the manuscript. EH: Design and implementation of workflow and algorithms for image registration, estimation of diffusion tensor, FA, and the along-tract analysis; commenting on the manuscript. MY and JH: Interpretation of the results and critical comments on the manuscript. AJL: PI of the project; design of study and inclusion of participants; writing and commenting on the manuscript. AL: Design of the MRI-protocol and the image analysis approach; writing and commenting on the manuscript. All authors have approved the final version of the manuscript and agreed on all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mindfulness meditation has gained momentum in medical circles for bolstering wellbeing and other facets of professionalism. This study evaluated the feasibility and benefits of a peer-led mindfulness meditation program (MMP) on medical student wellness and professionalism.Pre-clerkship students were recruited and randomized to the 8-week MMP or wait-list. Feasibility outcomes included ease of recruitment, program attendance and homework compliance. Other outcomes included self-reported psychological distress, empathy, self-compassion, mindfulness, altruism and program satisfaction.The MMP decreased levels of stress and enhanced mindfulness, self-compassion and altruism from baseline to post-study. Changes were not significant for the wait-list condition. Although satisfaction with the MMP was high compliance was suboptimal.A peer-led MMP is feasible and may be a promising approach to enhance medical student wellbeing. Further research is needed to explore strategies to improve program compliance in this student population. Medical school is a stressful and challenging time for students. Studies have demonstrated that rates of psychological distress are high among students.2Student wellness has become a priority in many medical schools. Although a number of initiatives have been developed to foster positive mental health in medical students, the effectiveness of these programs has not been well researched. One intervention that has gained momentum in medical circles for its effectiveness in bolstering wellbeing and other facets of professionalism is mindfulness meditation.While mindfulness-based programs have been evaluated in medical student populations, no previous investigations have incorporated medical students as part of the core research development team. Medical students, a group with an otherwise vibrant and powerful voice, have been silent regarding the domain of their own wellbeing. Previous research has also documented that medical students are reluctant to seek mental health support from professionals.The primary aim of this pilot study was to assess the overall interest and ability of pre-clerkship medical students to participate in an 8-week peer-led mindfulness meditation program (MMP). A secondary aim was to obtain preliminary data on the efficacy of the program in improving medical student wellness, mindfulness and professionalism skills.The study used a randomized waitlist (WL) control design. Participants assigned to the WL received the intervention following the 8-week wait period.Participants were pre-clerkship medical students at the University of Ottawa who were recruited at the beginning of September 2012. All first and second year medical students (n=300) received an email inviting them to participate in the study. The first 30 students who responded to the recruitment announcement were included in the pilot study. Students who consented to participate received medical school performance record (MSPR) credit through the Wellness Student Interest Group. The study received ethics approval from the Ottawa Health Science Network Research Ethics Board.The MMP was an adapted version of the mindfulness based stress reduction (MBSR) program developed by Kabat-Zinn.Our feasibility outcomes included ease of recruitment, program retention and homework compliance. We considered the study feasible if 75% of participants completed four or more sessions and if compliance with homework completion was 70% or higher. Efficacy outcomes included the following self-report scales: the Depression Anxiety and Stress Scale,A five-point Likert scale, ranging from Strongly Disagree (1) to Strongly Agree (5), was used to assess student satisfaction with the MMP and perceived benefit on levels of academic and clinical performance. All participants who completed the mindfulness program, including the WL participants who received the intervention after the wait period, completed the satisfaction questionnaire.Data were analyzed with SPSS Version 21. Main analyses for this study included calculation of feasibility outcomes using descriptive statistics. Efficacy analysis was performed on the intent-to-treat sample. For students with missing post-study assessments, we carried forward their baseline scores as an approach to imputing missing data (i.e. last observation carried forward - LOCF). Paired sample t-tests were used to evaluate pre- to post-study changes in outcome within each group. Between-group comparisons were assessed with analysis of covariance (ANCOVA), with the baseline score of each measure used as covariate. Within- and between-group effect sizes (Cohen’s d) were calculated to examine the magnitude of effect of the MMP. Calculation of within-group effect sizes accounted for correlations between repeated measures. According to convention, a small effect is considered to be .20, a medium effect .50 and a large effect .80. Significance was set at p<0.05, two-tailed tests.n=22, 73.3%) and equally composed of first (n=14) and second (n=16) year students. The average number of sessions attended was 3.9 out of 8 sessions. Nine (60%) participants completed four or more sessions. Among participants who dropped out early  one did not start the program, 4 dropped out after session 2 and one dropped out after session 3. Regarding homework compliance, only 4 participants reported a regular meditation practice. Post-study assessments were completed by 80% (n=13) of MMP and 60% (n=9) of WL participants.We recruited our target sample of 30 pre-clerkship students within the first two weeks of active recruitment. Our sample was predominately female , self-compassion (p=.024), altruism (p=.033) and two facets of mindfulness: describe (p=.05) and non-react (p=.034), with the magnitude (effect size) of change ranging from small to medium. Pre- to post-test changes were not significant for the WL. Between group comparisons revealed that post-test scores on the FFMQ-observe subscale were significantly higher for the MMP versus WL (p=.026), with a moderate effect size.All participants who received the MMP reported a high level of satisfaction with the program. 88% reported that the program had a positive impact on their academic performance and 69% reported a positive impact of the program on their clinical performance.To our knowledge this is one of the first studies to evaluate the feasibility and potential benefits of a peer-led MMP for medical students. Although the sample size is small and compliance with the program was suboptimal, results are nevertheless encouraging and suggest that a peer-led MMP may have a positive impact on student wellbeing, mindfulness, self-compassion and aspects of professionalism.There has been a shift in medical education over the recent past from top-down teaching models towards peer teaching.Training in mindfulness demands a considerable time-investment from participants and, as noted above, the rate of program completion in this study was lower than expected with 40% of participants failing to complete at least 4 of the 8 weekly lunch-time sessions. Lamothe and colleaguesSimilarly, participants reported difficulty engaging in daily mindfulness home practice, although they were more likely to practice yoga or informal meditation practices . The level of homework compliance observed in this study is consistent with other reports that mindfulness home practice among participants in mindfulness-based interventions is low.21Because sessions were held during the day, third and fourth year medical students were automatically excluded from participating in this study. An interesting avenue for future research would be to examine the impact of an online mindfulness program that would allow greater participation of third and fourth year students, and allow greater ease for first and second year medical students to engage in ongoing mindfulness training at their own pace. Online mindfulness and psychological interventions have been shown to be similarly effective as face-to-face equivalents, while also being a more feasible option given the reduced cost and logistical requirements.There are study limitations to note. First, the small sample size limits the generalizability of findings to other medical students at the University of Ottawa, and reduces the power to detect differences between the MMP and WL condition on most outcomes. Second, the completion rate of post-study questionnaires was lower in the WL than MMP condition, and the use of the last observation carried forward method to impute missing data may be problematic here. Although a linear mixed model would have been ideal as this approach uses all time points without the need to use imputation procedures, our sample size was too small for this analytic approach. Third, the sample comprised predominantly female medical students, although this is in keeping with gender ratios reported in the literature. In a review of 39 mindfulness studies involving health care provider populations 81% of participants were female.Although these preliminary findings must be viewed with caution, results suggest an 8-week peer-led mindfulness based intervention is acceptable to pre-clerkship medical students and may improve well-being and aspects of professionalism for those who express interest in such an intervention. Further research on the benefits of a peer-led mindfulness program is warranted.


Non-Hodgkin lymphoma (NHL) is the most common AIDS-related malignancy in developed countries. An elevated risk of developing NHL persists among HIV-infected individuals in comparison to the general population despite the advent of effective antiretroviral therapy. The mechanisms underlying the development of AIDS-related NHL (A-NHL) are not fully understood, but likely involve persistent B-cell activation and inflammation.This was a nested case-control study within the ongoing prospective Multicenter AIDS Cohort Study (MACS). Cases included 47 HIV-positive male subjects diagnosed with high-grade B-cell NHL. Controls were matched to each case from among participating HIV-positive males who did not develop any malignancy. Matching criteria included time HIV+ or since AIDS diagnosis, age, race and CD4+ cell count. Sera were tested for 161 serum biomarkers using multiplexed bead-based immunoassays.A subset of 17 biomarkers, including cytokines, chemokines, acute phase proteins, tissue remodeling agents and bone metabolic mediators was identified to be significantly altered in A-NHL cases in comparison to controls. Many of the biomarkers included in this subset were positively correlated with HIV viral load. A pathway analysis of our results revealed an extensive network of interactions between current and previously identified biomarkers.These findings support the current hypothesis that A-NHL develops in the context of persistent immune stimulation and inflammation. Further analysis of the biomarkers identified in this report should enhance our ability to diagnose, monitor and treat this disease. Non-Hodgkin lymphoma (NHL) is the most common AIDS-related malignancy in developed countries The precise pathogenic mechanism underlying A-NHL development is a current focus of intense scrutiny, and several basic themes have emerged. Immune deficiency appears to contribute to the development of A-NHL through the loss of T-cell mediated control over B-cell proliferation. This mechanism appears to be particularly important in the development of Epstein-Barr virus (EBV)-positive A-NHL  at each collection site: Johns Hopkins Medicine IRB, Northwestern University IRB, University of California, Los Angeles IRB, University of Pittsburgh IRB. The current study was approved by the University of Pittsburgh IRB.3) at the time of blood collection in the case or matched visit in the control, and race (white or black). Following matching, a total of 49 cases and 49 controls were selected. Sera obtained for three subjects  were not evaluable due to insufficient volume or hemolysis. In the resulting groups of 47 cases and 48 controls (The Multicenter AIDS Cohort Study (MACS) is an ongoing prospective study of the natural and treated histories of HIV-1 infection in homosexual and bisexual men conducted at sites located in Baltimore, Chicago, Pittsburgh and Los Angeles controls , age, raA combination of 161 bead-based immunoassays for a diverse set of serum biomarkers available on the Luminex platform was utilized in this study . This liEach serum sample was analyzed for each multiplex immunoassay using the Bio-Plex suspension array system and the Bio-Plex Manager software (version 4.1) . For each analyte, 100 beads were analyzed and the median fluorescence intensity was determined. Analysis of serum sample biomarker data was performed using five-parameter logistic curve fitting to standard analyte values. A number of samples fell below lower limit of extrapolation of the Bio-Plex Manager software for certain analytes and were therefore reported as out of range low (OOR <). The percentages of cases and controls classified in this manner for each analyte are listed in Biomarker distributions among the A-NHL and control groups were analyzed using both parametric and non-parametric statistical tests. In each analysis the minimum level of significance was p<0.05. For the parametric analysis, exact conditional logistic regression was performed on the biomarker data obtained for the A-NHL and control groups (n = 46 matched case-control pairs). The results were further analyzed using the Bonferroni Step-Down (Holm) correction for multiple comparisons. For the non-parametric analysis, the Mann-Whitney U test was utilized on data from 47 cases and 48 controls without regards to matching. Here, the False Discovery Rate (FDR) was controlled at 5% according to the method described by Benjamini and HochbergThe Ingenuity Pathway Analysis (IPA) software package  was used to examine pathways and interactions associated with the most promising biomarkers identified in our analysis described above. Additional serum biomarkers demonstrating associations with A-NHL in previous reports, which were not measured in the current investigation, were also included in this analysis. The list of additional biomarkers included IL-6 The non-parametric analysis of biomarker distributions among all cases and controls revealed that 19 out of the 161 biomarkers tested were significantly altered in the comparison of A-NHL cases and controls . Among tBiomarker measurements from the A-NHL and control groups were combined in order to identify correlations between specific biomarkers and HIV viral load. Among the complete panel of 161 biomarkers, a total of 17 biomarkers exhibited significant correlations with HIV viral load (data not shown). Most notably, 11 of the significantly correlated biomarkers were among the consensus biomarkers described above, and are presented in Each of the 17 consensus biomarkers were evaluated in case subsets including subjects diagnosed with B-cell diffuse, CNS, or Burkitt's Lymphoma (BL)/BL-like NHL using pair-wise one-way ANOVA. As this analysis included cases only, biomarker levels were normalized to CD4 counts prior to ANOVA to account for variations in disease severity. In a comparison of BL/BL-like and CNS NHL cases, OC and Timp-1 were significantly different with p<0.05. In the comparison of CNS and B-cell diffuse NHL cases, three significant alterations were observed including OC, SAP and Timp-1 (data not shown). In each comparison, significantly elevated levels of each biomarker were observed in CNS NHL relative to the other subtypes.−14–1.9×10−3), Cell-to-Cell Signaling and Interaction , Hematological System Development and Function , and Immune Cell Trafficking . Interactions between molecules were then examined for each biomarker included in the consensus list and several biomarkers examined in association with A-NHL in previous studies including IL-6, sCD30, IL-10, sCD23, sCD44, sCD27, CRP, CXCL13, and IgE. An extensive array of interactions within and among the two lists of biomarkers was identified  have been reported to have an association with various forms of leukemia and lymphoma Biological roles related to the development of lymphoma have not been conclusively described for MIP-1δ, M-CSF, sIL-1R1, HPN, SAP, and OC and, to the best of our knowledge, we are the first to report on these associations. Each of these proteins does possess functions relating them to inflammation and were linked mechanistically to other potential biomarkers in our pathway analysis. Further investigation into the precise activity of each of these proteins in lymphoma would be of great interest.Many of the biomarkers found to be significantly altered in A-NHL subjects appear to be positively associated with HIV disease progression, as indicated by their observed correlation with HIV viral load . Each ofWhen A-NHL subjects of differing disease subtypes were evaluated separately, relatively few significant alterations were found. Several biomarkers including OC, SAP, and Timp-1 were found to be significantly elevated in CNS NHL in comparison to other subtypes. To the best of our knowledge, we are the first to report on such associations. For the vast majority of the biomarkers examined, we were not able to detect any significant differences between NHL subtypes, although this may be due to the relatively small sample size of each subset and the amount of variability within both case and control groups. Our results are similar to the recent findings of Breen et al. in which no significant differences were observed when comparing biomarker levels of diffuse large B cell cases to BL cases; however, in that study all significantly increased biomarker levels in cases compared to controls were associated with the systemic form of NHL but not CNS NHL The results of the pathway analysis indicate the existence of a complex network of interactions among the collective list of biomarkers identified in the current and previous investigations. An examination of this network suggests central roles for two previously identified biomarkers, IL-6 and IL-10 . SeveralIn the current study, an analysis of a broad array of circulating proteins yielded a relatively small but robust collection of inflammatory mediators associated with the development of A-NHL in comparison to controls. Many of the identified biomarkers were positively correlated to HIV viral load and indicate heightened immune responses in A-NHL with respect to HIV infection and/or AIDS in the absence of lymphoma. A minority of the subjects included in the current study were former or current recipients of HAART. Although the distribution of HAART recipients was similar among cases and controls, we cannot rule out some effect of treatment on our biomarker findings. Our findings further support the hypothesis that A-NHL develops in response to persistent immune activation and inflammation and adds new points of focus for further investigation. Ongoing analysis into the precise role of each of the biomarkers identified in our report should enhance our ability to diagnose, monitor and treat this disease.Table S1Description of Biomarker assays utilized in the current study.(DOCX)Click here for additional data file.Table S2Percentage of samples classified as out of range low according to the Bioplex Manager software.(DOCX)Click here for additional data file.


The development of oncolytic viruses has led to an emerging new class of cancer therapeutics. Although the safety profile has been encouraging, the transition of oncolytic viruses to the clinical setting has been a slow process due to modifications. Therefore, a new generation of more potent oncolytic viruses needs to be exploited, following our better understanding of the complex interactions between the tumor, its microenvironment, the virus, and the host immune response. The conventional method for creation of tumor-targeted oncolytic viruses is based on homologous recombination. However, the creation of new mutant oncolytic viruses with large genomes remains a challenge due to the multi-step process and low efficiency of homologous recombination. The CRISPR-associated endonuclease Cas9 has hugely advanced the potential to edit the genomes of various organisms due to the ability of Cas9 to target a specific genomic site by a single guide RNA. In this review, we discuss the CRISPR-Cas9 system as an efficient viral editing method for the creation of new oncolytic viruses, as well as its potential future applications in the development of oncolytic viruses. Further, this review discusses the potential of off-target effects as well as CRISPR-Cas9 as a tool for basic research into viral biology. Oncolytic viruses (OVs) are anti-tumor viruses that selectively infect and kill cancer cells without damaging normal tissues . They caModifications of viral genomes have been made to render OVs more selective to cancer cells. The deletion of thymidine kinase (TK) in OncoVEX and JX594 increases their selectivity to cancer cells, while H101 lacking the E3B-55kDa gene replicates selectively in cancer cells. Further, minimizing the host immune response represents an important target for cancer therapeutics ,11,12.Thus, viruses have become important potential vectors for cancer therapy. The conventional method for generating tumor-targeted OVs with large genomes  is based on homologous recombination using a shuttle vector, either in bacteria or in mammalian cells. Currently, there are three major established methods which are used to edit the adenovirus genome: a bacteria-based homologous recombination system ,14, a baStreptococcus pyogenes, a single guide RNA (sgRNA) and the trans-activating crRNA (tracrRNA), the type II CRISPR-Cas9 system has dramatically altered the way genomes are being edited in eukaryotic cells .,37.18,37The CRISPR-Cas9 system has been used successfully in creating mutations with high efficiency in the target region of AdV and HSV-1 , which iAnother potential use of the CRISPR-Cas9 system is to elucidate as yet unknown functions of specific viral genes, particularly within the VV as almost every gene of VV can be targeted by gRNA guided Cas9 . A mutan