Toxoplasmosis is mainly acquired by ingestion of food or water contaminated with oocysts or by ingestion of raw or undercooked meat containing tissue cysts [56]. The infection with T. gondii results in a strong and persistent Th1 responses characterized by the production of pro-inflammatory cytokines (IFN-γ, TNF-α, etc.).

The cytokines produced by professional antigen presenting and T cells trigger effector mechanisms mediated by other cells of the immune system. For example, the IL-12 secreted by dendritc cells enhances NK cell expansion, as well as activation of CD4+ T and CD8+ T cell differentiation in Th1 effector U0126 datasheet cells. Both NK and Th1 cells secret IFN-γ, which activates as plethora of antiparasitic mechanisms in different cells [57] and [58]. Such mechanisms include

activation of respiratory burst in macrophages and production of nitrogen and oxygen intermediates that Screening Library price directly kill phagocytosed parasites. [31]. In addition, IFN-γ induces mechanisms of tryptophan starvation in hematopoietic and non-hematopoietic cells, allowing the limitation of intracellular replication of parasites [59]. In addition to secretion of IFN-γ, CD8+ T cells also control the infection by recognizing and killing parasite-infected cells. It was already demonstrated that CTL activity is related to protection during the early acute phase right after infection [37], [60] and [61]. Moreover, CTL appears to be the major mechanism of controlling development of symptomatic disease during later chronic infection. CTLs are believed to limit the number of parasites initially encysted, and thus, to prevent cyst rupture and reactivation of acute infection within tissues of the CNS [49]. The importance of anti-toxoplasma antibodies in the context of the disease is controversial. Some studies have demonstrated that antibodies directed against surface antigens may prevent infection aminophylline of host cells [62]. Some

studies Modulators performed with mice lacking B cells showed that those animals are susceptible to chronic infection and are not protected after vaccination [62] and [63]. Those studies hypothesize that parasite neutralization and opsonization are important for controlling chronic disease and to prevent the infection reactivation. However, direct evidence of development of both mechanisms “in vivo” is still missing. Our results suggest that IFN-γ produced by T cell is a major mechanism controlling T. gondii infection in mice vaccinated with the heterologous combination of FLU-SAG2 and Ad-SAG2. We support such conclusion by observing that only the heterologous protocol, which induced activation of IFN-γ secreting cells (IN FLU-SAG2 followed by SC Ad-SAG2) conferred protection.

Community interviewers were high school graduates, who underwent initial and refresher training in assessment of a few key signs on exam. Videotapes from WHO depicting sick children with danger signs and signs of dehydration were used in training [11]. No stools were collected at home visits. An additional follow-up period for the last 300 patients enrolled in Kenya was conducted from 1 April to 30 September 2009 and included in

the analysis of home visit and safety data [12]. Infants were randomized in a 1:1 ratio to receive selleck chemical three 2-ml oral doses of PRV (RotaTeq®, Merck & Co., Inc., Whitehouse, New Jersey) or placebo, given with other routine pediatric vaccines, including oral poliovirus vaccine (OPV), at approximately 6-, 10-, and 14-weeks of age [7] and [10]. The placebo had the same composition as PRV without the viral antigens. The primary study outcome for the clinic-based catchment surveillance was severe RVGE, regardless of serotype, occurring ≥14 days after the third dose until the end of the study. Epacadostat order Gastroenteritis was defined as three or more watery or looser-than-normal stools within a 24-h period and/or forceful vomiting [13]. At designated medical facilities, stool samples were collected from subjects

with gastroenteritis; history of symptoms of the current illness was collected through interview with the inhibitors parent/guardian; and physical signs were documented by medical staff. These data were used to define severity using the 20-point modified Vesikari Clinical Scoring System, where “severe” was defined as a score of ≥11 [14]. Secondary objectives included efficacy against RVGE of any severity, and all-cause total and severe gastroenteritis. The primary objective of the home visit surveillance analysis was a comparison of the incidence of severe gastroenteritis episodes between groups.

Because all the variables for the Vesikari score Terminal deoxynucleotidyl transferase were not amenable to being collected at home visits, the severity of gastroenteritis was defined according to WHO’s Integrated Management of Childhood Illness (IMCI) criteria for dehydration as the following: severe dehydration having at least two of the following signs – lethargic or unconscious, sunken eyes, not able to drink or drinking poorly, and skin pinch goes back very slowly (>2 s) and moderate dehydration having at least two of the following signs – restless or irritable, sunken eyes, drinks eagerly or very thirsty, and skin pinch goes back slowly (1–2 s) [11]. A secondary analysis of severity of gastroenteritis at the home visit was done using a modification of the 24-point Clark Clinical Scoring System, which takes into account the number of days of diarrhea and/or vomiting, the maximum numbers of stools and/or vomiting episodes, the behavioral symptoms of the child, and the child’s temperature [15].

The leads were placed to monitor standard bipolar derivations (F3-C3, C3-O1, C4-O2 and/or Cz-Oz). These animals were used for the caffeine challenge (as detailed subsequently in 2.3 Experimental methods) with qEEG spectral analysis (see below). A telemetry transmitter (TL11M2-C50-PXT or F40-EET, Data Science International, St.-Paul, Erastin cost MN, USA) for EEG monitoring was used with one standard bipolar derivation (Fz-Oz) in forty nine (49) adult rats. Animals were aged 9 to 14 weeks old. The animal room environment was controlled (temperature 21 ± 3 °C, humidity 30%–70%, 12 h light, 12 h dark, 10–15 air changes per hour) and temperature and relative humidity

were monitored continuously. Penicillin G procaine (Vetoquinol, Lavaltrie, QC, Canada, 1.0 mL, 300 000 IU/mL) was administered SC once daily for three days beginning on the day of surgery. Buprenorphine (Champion Alstoe, Whitby, ON, Canada, 0.04 mL, 0.3 mg/mL) was administered twice daily for three days. Local anesthetics (Bupivacaine, Hospira, Montreal, QC, Canada, 0.25%, 0.1 mL; Lidocaine, Vetoquinol, Lavaltrie, QC, Canada, 20 mg/mL, 0.1 mL) were injected in 4 SC sites distributed over the skull surgical site. The animal was placed on a heating pad and inhaled a mixture of O2 and isoflurane. A longitudinal incision was performed on the linea alba, and a telemetry

transmitter was secured in the abdominal cavity. Both EEG and EMG electrodes were LBH589 tunneled subcutaneously to a small skin incision in the neck. The abdominal skin incision was closed with interrupted buried sutures and the animal was placed in sternal recumbency to expose the skull for the remainder of the surgery. The EEG leads were secured on the cranial bone to monitor one bipolar derivation while EMG leads were sutured to longitudinal muscles of the neck. A linear groove was done in the cranial cortical all bone to secure the electrodes with surgical glue (Vetbond, 3M, St.-Paul, MN, USA) and acrylic. A period of three weeks was allowed between surgery and the start of experimental procedures. An additional twenty-four (24) Sprague–Dawley rats were used to illustrate the qEEG response

to PTZ infusion as described subsequently in Experimental Modulators methods. Electroencephalographic data were obtained from animals using telemetry transmitter leads using bipolar derivations (Monkey: F3-C3, C3-O1, C4-O2 and/or Cz-Oz; Dog: Cz-Oz and C4-O2; Rat: Fz-Oz). The EEG, and EMG, were recorded continuously from at least 24 h prior to dosing to at least 24 h post-dosing completion (Dataquest ART, Data Science International, St.-Paul, MN, USA). The EEGs were subjected to computer analysis from at least one hour pre-dosing to at least 24 h post-dosing (NeuroScore, Data Science International, St.-Paul, MN, USA). Digital color cameras (Geovision, Irvine, CA, USA), with daylight and infrared night vision connected to a computerized system (IBM Intellistation Z pro, Xeon 3.8 Ghz, 3.

We followed up the child till 14 days after enrollment and there was daily record of symptoms by the parents. Probably this makes the study highly sensitive and obtained the detailed

information of the duration and frequency of symptoms of AGE. Finding of more severe cases by Vesikari scale as compared to Clark scale is similar to earlier studies that have used both scales. The Vesikari scale more frequently scores gastroenteritis episodes as severe as compared to the Clark scale [8], [9] and [21]. All severe cases were not hospitalized in our study. The decision to hospitalize a child is based Akt inhibitor mainly on requirement of supervised rehydration as determined by the treating physician. In addition, SB203580 factors like economic condition of parents and distance between home and healthcare setting influence decision

of hospitalization [21]. It is evident from our study that in diarrheal disease and especially in RVGE, taking early treatment from health care setting would be of utmost importance to prevent complications of disease. Our study suggests that RVGE places a considerable financial and emotional burden on parents of the affected children and they lost up to 7 days of work. The RVGE cases had higher healthcare cost and difference between RVGE and non RVGE cases was significant in OPD managed cases. Our results show that pediatric RVGE caused considerable Modulators stress for parents. This is consistent with results of a study conducted across European countries where stress scores of >5 on 10-point scale were reported irrespective of settings under which the child was treated [22]. Though study provides substantial data on RVGE in specified setting and overall proportion of RVGE is in concurrence with earlier studies, the results of this study need to be interpreted with caution because of certain important limitations. Study was conducted only in private outpatient clinics in urban areas of India and is not representative of rural and non-private healthcare settings such as government healthcare facilities or non-profit hospitals/clinics. These settings

might have different rotavirus disease profile and economic impact on subjects who utilize these Phosphatidylinositol diacylglycerol-lyase services may be different. It is noteworthy however that in our study, the private and urban setting has shown RVGE as important health problem, reaffirming the universal occurrence of RVGE. IRSN data has shown that though rotavirus disease occurs throughout the year, higher proportion is observed in winter season (December–February) particularly in northern India. It has also been shown that proportion of rotavirus disease is higher in younger age and more severe cases [4]. Even in our study, when total PP population was considered, we did find that RVGE is associated with younger age, multiple symptoms, more severity of the disease as per Clark and Vesikari scale and higher proportion in the months of January–March.

Both enzyme-linked find protocol immunospot (ELISpot) and intracellular cytokine staining (ICS) assays

have been identified for harmonization on this basis. In the blood-stage field there are two functional assays of note: growth inhibition (GIA) and antibody-dependent cellular inhibition (ADCI) assays. Investigators proficient in GIA have participated in several harmonization efforts resulting in conformity in some aspects of the assay procedure, and selection and support of one intramural NIAID laboratory as a PATH MVI Reference center [3], [4] and [5]. ADCI is more difficult to standardize, but has the advantage of requiring far lower IgG concentrations for activity [6] and has therefore been identified for harmonization, with the anticipation that this will be challenging. A PATH MVI ELISA Reference laboratory is funded

for the performance of both blood-stage and pre-erythrocytic stage ELISAs at the Walter Reed Army Institute of Research (WRAIR). In the EGFR inhibitor spirit of growing coordination and collaboration between groups of funders and scientists, the OPTIMALVAC assay harmonization activity has been initiated (www.optimalvac.eu). This is a European Union funded project whereby funds have been allocated to harmonize the following assays: ICS, ELISpot, ADCI and blood-stage IFA. The European Vaccine Initiative provides project management and coordination expertise. The PATH Malaria Vaccine Initiative is closely involved with the project both through its steering committee and through targeted, complementary funding of certain components. PATH MVI also supports the NIAID GIA Reference Center as well as the WRAIR ELISA Reference Center along with USAID support. WHO Initiative for Vaccine Research (IVR) acts to identify and synergize other malaria vaccine assay harmonization activities with OPTIMALVAC

and to link with other disease areas where appropriate. PATH MVI is, in parallel, conducting comparisons of alternate pre-erythrocytic functional assays and assays of infectivity for sexual stage and mosquito antigen vaccine research. Thus, Farnesyltransferase though choice of immunological outcomes is complex in malaria vaccination, a great deal of progress is being made. In the medium term, consensus harmonized SOPs should be available for the community and identification of laboratories with an interest in serving as additional central testing centers may be facilitated. There are currently no WHO designated reference centers. Ultimately a particular assay may progress to the stage where it has met the requirements of a WHO reference center and where establishment of such a center is appropriate and feasible in the malaria vaccine field. To conclude, many different Libraries approaches to malaria vaccination are under clinical or advanced pre-clinical evaluation.

While in the vast majority of scenarios explored vaccination reduced the risk of unvaccinated individuals by 50–80% (due to indirect effects), direct effects of vaccination (i.e. reductions in the number of cases in vaccinated individuals as compared to unvaccinated RG7420 research buy individuals) were smaller ( Fig. 4). Interestingly, in scenarios that included high heterogeneity in the transmission intensity and very low vaccine efficacy against DENV-2, direct effects of vaccination were negative. However, even under these scenarios, there was an absolute reduction in the cumulative Libraries incidence among vaccinated individuals, as compared to themselves had no vaccination

program been implemented (counterfactual effect). This reduction reflects the cumulative effects of both direct and indirect protection that vaccinees experience. We assessed the impact of vaccination on the yearly incidence of clinically apparent dengue, across all serotypes, for 50 years after vaccine introduction (Fig. 5). While significant decreases were observed in all scenarios (relative to the average incidence prior to vaccination), several short-term increases over pre-vaccine levels occur within thirty years of vaccine introduction. These increases result from the build up of susceptible individuals in certain

age groups and, as expected, are less http://www.selleckchem.com/products/SB-203580.html frequent in scenarios with higher efficacy against DENV-2. Despite these periodic increases, the expected cumulative incidence of clinically apparent dengue was significantly lower than the cumulative Sitaxentan incidence without vaccine for the great majority of scenarios explored (Fig. 5, right panel). We also explored the impact of vaccination on the mean-age of clinical cases (Fig. 6). While vaccination with high efficacy across all serotypes led to an increase in the mean age of cases, in certain instances of low vaccine efficacy against DENV-2 we observed decreases

in the mean-age. The largest decreases were observed in scenarios that included heterogeneity in transmission intensity (Fig. 6B), and result mostly from breakthrough infections by DENV-2 in vaccinated children. Sudden increases in the mean-age of cases were also observed at varying times after vaccine introduction and result from susceptibility accumulating in certain age-classes. The impact of any particular vaccine formulation depends on at least four separate effects: (1) direct protection of vaccinees against infection and/or disease, (2) indirect protection of all members of vaccinated communities, (3) an impact on serotype distribution, and (4) the immunopathogenic effects of partial vaccine-induced immunity. Our results from a four-serotype, age-specific compartmental dengue transmission model suggest that partially effective vaccines can have a significant positive impact, on average, in reducing dengue transmission and disease.

We found that FSTL1 mainly appeared in small vesicle fractions that contained synaptoporin, a member of the synaptophysin superfamily and an integral membrane component of synaptic vesicles in afferent terminals (Sun et al., 2006), but not in LDCV fractions labeled by CGRP (Figure 2C). Thus, FSTL1 is localized to small translucent vesicles. The identity of FSTL1 vesicles was further analyzed in the afferent axons of dorsal roots. Double

immunostaining showed that ∼53% of FSTL1 vesicles (n = 2050 in 212 axon profiles) contained vesicular glutamate transporter 2 (VGluT2) in the axons of rats (Figure 2D) and mice (Figure S3A). Also, ∼39% of the vesicles contained vesicle-associated membrane protein 2 (VAMP2) (n = 378 in 35 axon profiles) Cell Cycle inhibitor (Figure 2D). However, only a small number of FSTL1 vesicles contained synaptoporin (∼12%, n = 2624 in 138 axon profiles) or synapsin (∼14%, n = 777 in 64 axon profiles) (Figure 2D). Thus, newly synthesized FSTL1 is mainly transported via VGluT2- and VAMP2-containing vesicles, while only a small amount of FSTL1 is transported via the vesicles carrying synaptoporin and synapsin

(Hannah et al., 1999 and Santos VX770 et al., 2009). We directly examined the potential secretion of FSTL1 in DRG neurons cultured from young rats. Immunoblot analysis showed that the level of FSTL1 in the culture medium was increased in the absence of the stimulus (Figure 2E), indicating spontaneous secretion of FSTL1. Furthermore, FSTL1 secretion was elevated by K+-induced membrane Amisulpride depolarization (55 mM KCl, 1 hr), but only in the presence of extracellular Ca2+ ([Ca2+]o) (Figures 2F and 2G). The FSTL1 level was increased by the TRPV1 channel activator, capsaicin, and that response was abolished by the TRPV1 channel blocker, capsaizepine (Figure 2F). Additionally, high-K+ stimulation for 15 min enhanced FSTL1 secretion from the spinal cord slices of rats (Figure 2H) and mice

(Figure S3B) as well as synaptosomes prepared from the spinal dorsal horn (Figure 2I). As expected, the secretion of CGRP, SP, and glutamate was also increased in the same preparation (Figures 2H and 2I). The stimulus-evoked secretion of FSTL1 from synaptosomes occurred only in the presence of [Ca2+]o (Figure 2I). In contrast, the spontaneous secretion of tenascin-C, an extracellular matrix glycoprotein (Joester and Faissner, 2001), was unaffected by the K+ stimulation (Figure 2I and Figure S3C). Together, these results suggest that FSTL1 is stored in small translucent vesicles and can be secreted either spontaneously or by depolarization in a manner similar to neurotransmitters. To determine whether secreted FSTL1 plays a physiological role in the spinal cord, we performed whole-cell recording of lamina II neurons to monitor afferent synaptic transmission in dorsal root-attached spinal cord slices from rats (Nakatsuka et al., 2000).

The coherence Cxy(f) of two signals x check details and y is defined as their normalized cross-spectral density. For a given frequency f, the coherence is higher the more stable the phase difference of the signals: Cxy=|Pxy|2PxPy.

Coherence was computed using PSDs by the Welch periodogram method with 256 FFT points. Slope-triggered averages (Figures 4D and 9D) were computed by averaging windows of 40 ms LFP data centered to the times of strongest 10% slopes of each cPSC. Ripple modulation of the resulting averages indicates a consistent phase of the putative EPSC onsets (or putative IPSC onsets, Figure 9D) in the oscillation defined by the extracellular ripple. The presence of the bulk of the signal after the onset (0 ms offset) indicates a tendency for stronger slopes to concentrate at the beginning of the cPSC. cPSCs

with only one strong slope were removed from the analysis. For the analysis shown in Figure 4, a total of 1,085 cPSCs were represented with 5,161 onsets, each cell having an average of 4 to 5 onsets per cPSC. For the complementary analysis presented in Figure 9, a total of 849 cPSCs from 6 cells were represented with 3,037 onsets, Antidiabetic Compound Library cell assay each cell showing an average of 3 to 5 onsets per cPSC. To assign phases with respect to the ripple component of the LFP, we applied a Hilbert transform on the 120–300 Hz-filtered extracellular potential. Each putative EPSC event (or putative IPSC event, Figure 9D) detected by its extremal slope was assigned a Hilbert phase, ϕj. As an indicator for locking quality

of these collected EPSC ADAMTS5 (or IPSC) phases, we use the vector strength (VS) or mean resultant length, which is normalized between 0 and 1: VS(ϕ)=1N|∑j=1Ne−iϕj|. Figures 4E and 9C illustrate the dependence of vector strength and phase (polar plots in insets) as a function of the slope threshold used for EPSC (or IPSC) detection (in the range of 1% to 25%). All vector strengths and phases plotted are significant according to a Rayleigh test for the uniformity of a phase distribution with p < 0.05. Data are presented as means ± standard error of the mean (SEM), unless otherwise stated. Statistical significance was assessed using Wilcoxon’s rank-sum test, the two-sample Kolmogorov-Smirnov test, or the Rayleigh test for circular statistics. Statistical significance was indicated at the given level (p) with α = 0.05 regarded significant, unless otherwise designated. We wish to thank Sarah Shoichet, Anja Gundlfinger, Alexey Ponomarenko, Christian Wozny, José R. Donoso, Nikolai Axmacher, and Robert Schmidt for constructive comments on earlier versions of the manuscript; Peter Barry (UNSW) for advice on estimation of LJPs; Roger D. Traub for valuable discussions; as well as Serena Dudek and Robert J. Bridges for help on establishing recordings with DNDS. We highly appreciate the technical assistance of Susanne Rieckmann and Anke Schönherr.

To test the anatomical this website requirements for insomniac function, we used the Gal4/UAS system ( Brand and Perrimon, 1993) to direct RNAi against insomniac. More than thirty Gal4 driver lines were tested, including those driving expression in muscle, the eye, glia, and various regions of nervous system ( Supplemental Experimental

Procedures and data not shown). Among the lines tested, only the panneuronal elavC155-Gal4 driver ( Lin and Goodman, 1994) was able to recapitulate the sleep defect of insomniac null mutants. Both male and female animals bearing elavC155-Gal4 and a UAS-inc-RNAi transgene integrated at either of two independent sites exhibited sharply reduced sleep ( Figure 4A and data not shown). Control animals lacking either elavC155-Gal4 or the UAS-inc-RNAi transgene exhibited wild-type sleep patterns ( Figure 4A). Furthermore, the RNAi phenotype induced by elavC155-Gal4 is suppressed by the coexpression of insomniac from a UAS-inc

transgene ( Figure 4B), indicating that the sleep defects elicited by neuronally restricted RNAi arise from the specific depletion of insomniac and not from off-target effects ( Scacheri et al., 2004 and Ma et al., 2006). Thus, we conclude that insomniac is required in neurons for the normal regulation of sleep and wakefulness. In a second series of experiments, we employed the Gal4/UAS system 5-Fluoracil to restore insomniac expression to inc1 and Edoxaban inc2 mutants. The inc2 transposon ( Figure 2A) contains a UAS/TATA element within its downstream-facing terminus, juxtaposed in the correct orientation for Gal4 to drive transcription through the insomniac locus. Introduction of one copy of actin-Gal4 or tubulin-Gal4

restores insomniac expression from the inc2 allele ( Figure 4C), but not from inc1 (data not shown), indicating that inc2 functions as a null allele that can be reverted in the presence of Gal4. The restoration of insomniac expression by these ubiquitous drivers rescues the sleep defect of inc2 animals completely ( Figure 4D). To further map the anatomical requirements for insomniac, we used a panel of isogenic Gal4 drivers to restore insomniac expression in restricted patterns, particularly within the brain. We were unable to assess rescue using elavC155-Gal4, as this driver is closely linked to insomniac and recombinants containing both alleles could not be isolated (data not shown). Drivers expressed broadly within the nervous system, including nsyb-Gal4, sss-Gal4, and D42-Gal4, restored the sleep of inc2 animals to 70% of wild-type levels or greater ( Figure 4D). A similarly strong rescue was observed with the Cha-Gal4 driver ( Figure 4D) expressed in abundant cholinergic neurons. Two drivers with relatively broad patterns of neuronal expression, glutamatergic neuron-specific VGlut-Gal4, and c309-Gal4, rescued the sleep defect of inc2 animals weakly, as did the pars intercerebralis-specific driver Mai301-Gal4 ( Figure 4D).

We stained the eye discs of the third-instar eyFLP;Rab6 mutant larvae with anti-24B10, anti-Elav, anti-Senseless (labels R8), and anti-Prospero (labels R7). The staining patterns all appear normal, indicating that

there are no differentiation defects in Rab6 mutant eyes (data not shown). We then labeled R7 cells with Pan-R7 Gal4 and assessed the R7 targeting see more phenotypes of Rab6 mutants. 18.8% ± 3% (n = 265) of the R7 cells in Rab6 mutants (versus 19.7% ± 3% [n = 268] for rich1 mutants) fail to target to the correct layers in the medulla ( Figures 7C, 7C′, and 7D). The similarity in phenotype between Rab6 and rich mutant PR suggests that they function in the same pathway. To test this hypothesis, we removed one copy of Rab6 in the

eyFLP; rich1 or eyFLP; rich2 mutant animals and quantified the R7 targeting defects in the adult medulla. Rab6 heterozygous animals do not have R7 targeting defects, while the hypomorphic allele rich2 only has weak R7 targeting defects (8.5% ± 2%, n = 329; Figures 7F and 7M). We reasoned that loss of one copy of Rab6 in the partial loss of function mutant rich2 would enhance the phenotype. Indeed, loss of a copy of Rab6 enhanced the R7 targeting defects in eyFLP; rich2 animals (17.0% ± 3%, n = 483) similar to the rich1 mutant phenotype (compare Figures 7E and 7F to 7G; Figure 7M). However, loss of a copy of Rab6 does not modify the R7 targeting defects of the null allele rich1 (19.0% ± 3%; n = 308; compare Figures 7E to 7H; Figure 7M). Hence, the phenotypic and genetic data strongly argue Talazoparib solubility dmso that rich and Rab6 indeed function in a common pathway. To provide additional evidence that Rich and Rab6 function together,

we overexpressed a constitutively active form of Rab6 (Rab6CA) in rich1 mutant cells using the MARCM system. This greatly suppressed the R7 targeting GPX6 defects caused by loss of rich. Moreover, overexpression of a dominant negative form of Rab6 (Rab6DN) did not suppress or enhance the rich1 phenotype, indicating that Rich positively regulates Rab6 activity, possibly as a GEF ( Figures 7I–7L and 7N). To determine if Rab6 physically interacts with Rich, we overexpressed the WD40 domain (AA8–AA521) and RIC1 domain (AA711–AA995) of Rich in S2 cells and performed GST pull-down experiments using GST fusion proteins containing either wild-type Rab6 (Rab6WT), Rab6CA, or Rab6DN. A typical GEF protein tends to bind to the nucleotide-free form (e.g., Rab6WT) or GDP-bound form (e.g., Rab6DN). Consistent with this, both the WD40 domain and the RIC1 domain have a strong affinity for Rab6WT and Rab6DN but not Rab6CA, suggesting that Rich directly regulates Rab6 (Figure 7O). To directly assess the GEF activity of Rich, we overexpressed Rich in S2 cells and performed GTP exchange assay with the cell lysate. In this experimental setting, a positive control, Tiam1, showed proper GTP exchange activity for Rac1.