, 2012) The VENs and fork cells are concentrated within a small

, 2012). The VENs and fork cells are concentrated within a small region in the agranular anterior insula that is located, as in humans, just anterior to the limen insula and medial to the superior limiting sulcus (Figure 1F). (In order to avoid the impression that we infer a clear anatomical and functional homology with the human AIC, in the macaque we use the term “agranular anterior insula” [AAI] to designate the agranular portion of the insula that lies anterior to the limen. We ascribe Tyrosine Kinase Inhibitor Library the concentration of VENs to a small region within the ventral AAI.) A preliminary examination of the cytoarchitecture of this

region in Nissl preparations does not reveal major interspecies differences and suggests that it constitutes a distinct selleck compound architectonic area. In this area, the small granule cells of layer 4 are absent; layer 2 is thin but darkly stained; layer 3 is distinctly sublaminated; and layer 5 is wide with a clear sublamination into a thin superficial layer 5a and a large layer 5b, which contains larger neurons including VENs and fork cells (Figure 1C). VENs and fork cells are also found in the ACC, particularly in area 24b (Figures S1B and S1C), but these are scarcer and more dispersed than those

in ventral AAI. Isolated VENs are infrequently found in other regions of the prefrontal cortex including areas 14 and 10. The present study focuses on the concentration of VENs in the ventral AAI, consistent with the overall aim of our research on primate insula. Other than frequency and number, there are no obvious differences in morphology, size, or laminar distribution between the insular and cingulate VENs. Details on the VEN distribution in ACC will be reported separately. A stereological estimate of the number of VENs in ventral AAI with the optical fractionator (West

et al., 1991) indicates an average of ∼1,500 and ∼2,000 VENs per hemisphere in M. fascicularis and almost M. mulatta, respectively ( Figures 1G and 1H; see Table S1 for details). This number is, as expected, much lower than the numbers reported previously in humans and the average number in great apes, but it is within the lower range of VEN numbers in some individual great apes ( Allman et al., 2010). Macaque VENs represent 2%–3% of the total number of neurons in layer 5, which fits the trend for progressively higher VEN/pyramidal ratio from human to phylogenetically more distant primate species ( Allman et al., 2010). Finally, a comparison of the number of VENs in the left and right AAI reveals a significantly higher number of VENs (F1,5 = 100.358; p = 0.0002) and a nonsignificant trend for a higher VEN/pyramidal ratio (F1,6 = 4.213; p = 0.0953) in the right AAI, also in accordance with the asymmetry reported in hominids ( Allman et al., 2010).

8 × 106, was used) C57BL/6 mice were anesthetized with tribromoe

8 × 106, was used). C57BL/6 mice were anesthetized with tribromoethanol (125–250 mg/kg). Viral solution was injected with a glass pipette at a flow rate of 0.15 μl/min. Coordinates used for the hippocampal injection were AP + 1.95 mm,

ML ± 1.25 mm, DV − 1.20 mm (for CA1), and DV − 1.95 mm (for DG). We injected 1 μl of viral solution in CA1 and another 1 μl in DG. The coordinates used for the prefrontal injection were AP − 1.0 mm, ML ± 0.3 mm, DV − 1.0 mm, and DV − 1.5 mm. The sites at DV − 1.0 mm and DV − 1.5 mm both received 1 μl of injection. The coordinates used for the entorhinal injection U0126 supplier were AP + 4.5 mm, ML ± 3.5 mm, and DV − 4.0 mm. The injections were bilateral except otherwise noted. Two-month-old C57BL/6 mice were injected with AAVs and were used for slice physiology 3–4 weeks after the infection. Transverse hippocampal slices or coronal prefrontal slices (250 μm) were cut in ice-cold solution, comprising

75 mM sucrose, 75 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 8 mM MgSO4, 0.5 mM CaCl2, 26.2 mM NaHCO3, and 20 mM D-glucose saturated with 95% O2/ 5% CO2 and transferred to a holding chamber containing artificial cerebrospinal fluid (ACSF) composed of 117.5 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 1.3 mM MgSO4, 2.5 mM CaCl2, 26.2 mM NaHCO3, and 11 mM D-glucose to recover for at least 1 hr at room temperature Obeticholic Acid mw before being transferred to a recording chamber continually perfused (1 ml/min) with oxygenated ACSF (maintained at 27°C–29°C), containing 50 μM of picrotoxin. Whole-cell voltage-clamp recordings were made with 3–5 Sodium butyrate MΩ pipettes filled with internal solution containing 135 mM CsMeSO4, 10 mM HEPES, 8 mM NaCl, 0.25 mM EGTA, 2 mM MgCl2, 4 mM Mg ATP, 0.3 mM NaGTP, and 5 mM phosphocreatine (pH 7.3). Neurons were clamped at −65mV for recording of EPSC in hippocampal slices. In the prefrontal slices, to avoid contamination from AMPAR-mediated polysynaptic EPSCs, we

clamped neurons at +30mV to record NMDAR-mediated EPSCs in the presence of 10 μM of NBQX. Two-month-old mice were injected with AAVs and were implanted with recording electrodes 2–3 weeks later. Field potential recordings were obtained from the CA1 field of the right dorsal hippocampus. To implant electrodes, we sedated mice with diazepam (10 mg/kg, intraperitoneally), anesthetized them with isoflurane (1%–3%), placed them in a stereotaxic frame, maintained on a heating pad, and prepared them for aseptic surgery. A hole was drilled 2.2 mm posterior and 1.6 mm right of bregma. An insulated, 50 μm diameter stainless steel wire (California Fine Wire) was implanted 1.7 mm below the surface of the brain. The reference electrode was placed in the cerebellum. Two screws were placed in the skull. Electrode leads were connected to pins that were inserted into a strip connector, which was attached to the screws and skull with cranioplastic cement.

Deletion of TSC also leads to HSC

Deletion of TSC also leads to HSC PLX3397 price depletion, partly by increasing mitochondrial mass and oxidative stress ( Chen et al., 2008 and Gan et al., 2008). The Lkb1-AMPK kinases are key regulators of cellular metabolism that coordinate cellular proliferation with energy metabolism by suppressing proliferation when the ATP to AMP ratio is low. Energy stress prompts AMPK signaling to activate

catabolic pathways such as mitochondrial fatty acid oxidation while inhibiting anabolic pathways such as mTORC1-mediated protein synthesis (Figure 3) (Shackelford and Shaw, 2009). Lkb1 is a tumor suppressor that is mutated in Peutz-Jeghers syndrome patients (Hemminki et al., 1998 and Jenne et al., 1998). Lkb1 deficiency increases the proliferation of many tissues ( Contreras et al., 2008, Gurumurthy et al.,

2008, Hezel et al., DAPT mw 2008 and Pearson et al., 2008) and immortalizes mouse embryonic fibroblasts ( Bardeesy et al., 2002). These data suggest that the primary function of Lkb1 in many adult tissues is to negatively regulate cell division, preventing tissue overgrowth. However, conditional deletion of Lkb1 from hematopoietic cells leads to a cell-autonomous defect in HSCs that rapidly increases proliferation and cell death ( Gan et al., 2010, Gurumurthy et al., 2010 and Nakada et al., 2010). HSCs depend more acutely on Lkb1 for cell-cycle regulation and survival as compared to other hematopoietic cells. Lkb1 also has different effects on signaling pathways and on mitochondrial function within Rolziracetam HSCs as compared to restricted progenitors ( Nakada et al., 2010). This demonstrates that even key metabolic regulators have different functions in different kinds of dividing somatic cells. The Lkb1 pathway regulates chromosome stability in HSCs in addition to energy metabolism. Lkb1-deficient HSCs exhibit supernumerary centrosomes and become aneuploid,

whereas myeloid-restricted progenitors appear to divide normally in the absence of Lkb1 ( Nakada et al., 2010). AMPK-deficient HSCs do not become aneuploid, indicating that Lkb1 regulates mitosis in HSCs through AMPK-independent mechanisms. Lkb1 and AMPK homologs in Drosophila also regulate chromosome stability in neuroblasts, suggesting that Lkb1 is an evolutionary-conserved regulator of mitosis in some cell types ( Bonaccorsi et al., 2007 and Lee et al., 2007). Therefore, regulation of mitotic processes including chromosome segregation differs between stem cells and some other progenitors. Stem cells are particularly sensitive to the toxic effects of oxidative damage and are equipped with protective mechanisms that appear to be less active in some other progenitors. FoxO transcription factors regulate stem cell maintenance by regulating the expression of genes involved in cell cycle, apoptosis, oxidative stress, and energy metabolism (Figure 3) (Salih and Brunet, 2008).

As illustrated in Figure 6 and Table S2, taking TPSM-phase into a

As illustrated in Figure 6 and Table S2, taking TPSM-phase into account to discriminate between IN-PF and OUT-PF firing (IN versus OUT EpF in the wheel) still provided significant

increase in spatial information content when bursts (taken as successive spikes separated by less than 10 ms) were omitted from the original spike train, as well as when only spikes emitted at high frequency (ISI < 10 ms) or on the contrary at frequencies lower than 25 Hz (ISI > 40 ms) were taken into account. These results suggest that a direct relationship between firing rate and TPSM-phase is unlikely to account for the observed TPSM phase-related gain of spatial information. http://www.selleckchem.com/products/EX-527.html Another possibility is that of a location-dependent modulation of theta power itself. For example, if theta amplitude was systematically maximal within a given place field area, the spikes discharged within this place field would likely be biased toward the corresponding phase of TPSM (i.e., π, for maximal theta power). We therefore computed signal-phase histograms corresponding to the TPSM phases expressed in each place field (i.e., distribution of LFP TPSM-phase relative to physical space; see Experimental Procedures). Although in the open field a significant signal modulation relative

to TPSM phase was observed in 41% of TPSM phase-locked place fields (18 place fields among 44 whose IN-PF spikes were significantly phase locked to TPSM; Rayleigh test, p < Sorafenib price 0.05; Figure 7A), it was most often (13 among 18 place fields) significantly different (p < 0.05, Kuiper test) from the distribution of IN-PF spikes relative to TPSM phase. This observation suggests that the TPSM phase-locking of spikes inside a place field cannot be explained by the preferred TPSM phase of the signal within this same place field. A different situation prevailed in the maze in which, as expected from classical track running experiments,

running was accompanied by a highly reproducible sequence of place cells firing, along with the animal’s stereotypical spatial progression (Pastalkova et al., 2008). As observed in Figures 7B–7D and S3, TPSM was remarkably conserved almost from one run to the other, as was the motor behavior of the animal. Accordingly, we observed that TPSM was in fact phase locked to the environment (Figures 7B–7E), in accordance with a recent study reporting a strong correlation between theta power and animal’s position in a maze (Montgomery et al., 2009). As a result, the relationship between IN-PF spikes and TPSM phase in the maze appears to be tightly related to the coincidence of place field position and phase locking of TPSM to space (Figures 6C and 7C–7E). To further investigate the potential relative influences of time and space on hippocampal activity, we examined TPSM during wheel running, in which although the animal is running, its spatial location does not change (Czurkó et al., 1999; Pastalkova et al., 2008).

Our finding of functional heterogeneity in labellar sensilla is c

Our finding of functional heterogeneity in labellar sensilla is consistent with the finding that two taste sensilla on the prothoracic leg responded to this website BER but not quinine, whereas another sensillum responded to quinine but not BER (Meunier et al., 2003). A recent study found that N,N-diethyl-m-toluamide (DEET) elicited different responses from several labellar sensilla tested (Lee et al., 2010). Functionally distinct bitter neurons have also been described in taste organs of caterpillars, and in the case

of the Manduca larva, ARI and salicin activate spike trains that differ in dynamics ( Glendinning et al., 2002 and Glendinning et al., 2006). The functional differences among neurons in the Drosophila labellum suggested underlying molecular differences. In particular, we wondered whether the four classes of bitter taste neurons defined by physiological

analysis could be distinguished by molecular analysis. We constructed a receptor-to-neuron map of the entire IOX1 solubility dmso Gr repertoire and found that four classes of bitter taste neurons emerged on the basis of receptor expression, classes that coincided closely with the four functional classes. Moreover, the neuronal classes that were more broadly tuned expressed more receptors. While the physiological and molecular analyses support each other well, there are limitations to each analysis that raise interesting considerations. Our functional analysis is based on a limited number of taste stimuli. We selected bitter tastants that were structurally diverse, but bitter compounds vary enormously in structure and only a small fraction during of them can be sampled. It is possible that by testing more tastants, by testing them over a greater concentration range, or by analyzing temporal dynamics in greater detail that even more diversity

would become apparent among the bitter-sensing neurons. There are also limitations to our receptor-to-neuron map. First, the map considers exclusively the 68 Grs. There are at least two additional receptors that can mediate bitter taste. DmXR, a G protein-coupled receptor, is expressed in bitter neurons of the labellum and is required for behavioral avoidance of L-canavanine, a naturally occurring insecticide (Mitri et al., 2009); the TRPA1 cation channel, also expressed in a subset of bitter neurons in the labellum, is required for behavioral and electrophysiological responses to ARI (Kim et al., 2010). Second, Gr-GAL4 drivers may not provide a fully accurate representation of Gr gene expression in every case. Genetic analysis has shown that Gr64a is required for the physiological responses of labellar sensilla to some sugars and is therefore expected to be expressed in labellar sugar neurons ( Dahanukar et al., 2007). Our Gr64a-GAL4 driver, however, is not expressed in these neurons, suggesting the lack of a regulatory element.

However, genetic inactivations of the murine homologs of genes mu

However, genetic inactivations of the murine homologs of genes mutated in human neuronal

migration disorders so far have failed to reproduce these malformations, prompting the suggestion that mutations at other as-yet-unrecognized loci may result in SBH (Bilasy et al., 2009, Croquelois et al., 2009 and Lee et al., 1997). These discrepancies highlight the complexity of human cerebral cortex in comparison to rodents. Not only do migrating neurons have to cover a much longer distance to their final destination, they also need to change radial guides more often due to the increase in pial surface compared to the ventricular surface with additional radial glia (RG) in the outer SVZ (Fietz et al., 2010, Hansen et al., 2010 and Reillo et al., 2010). Thus, migrating neurons in human cerebral cortex SCH772984 clinical trial may require other pathways as they face additional challenges during their journey. Moreover, radial glial cells may need specific pathways, which are yet ill-understood in the mouse model. Indeed, so far only mutation of MEKK4 has been suggested to affect migrating neurons and radial glial cells, causing disruption of the ventricular surface and protrusions

of neuronal ectopia into the ventricle (Sarkisian et al., 2006). Here we set out to examine the role of the small GTPase RhoA for neuronal migration in the developing cerebral cortex, as RhoA had been suggested to play key roles in directed cell migration in various tissues and organs (Govek et al., 2005 and Heasman and Ridley, 2008). By using pharmacological means and dominant-negative or constitutively active constructs, click here several studies suggested that RhoA is crucial for neuronal migration (Besson et al., 2004, Heng et al., 2008, Kholmanskikh et al., 2003, Nguyen et al., 2006 and Pacary et al., 2011). However, the direct role of RhoA in neuronal migration has never been tested in the developing nervous system in vivo. Recently,

conditional deletion of RhoA has revealed insights into its role at early stages of central nervous system (CNS) development Thymidine kinase in the spinal cord and midbrain, highlighting common functions in the maintenance of adherens junction coupling, as previously shown for other members of this family, such as Cdc42 and Rac1 (Cappello et al., 2006, Chen et al., 2009 and Leone et al., 2010), but an opposite role in regulating cell proliferation in spinal cord versus midbrain (Herzog et al., 2011 and Katayama et al., 2011). Moreover, neuronal migration or positioning of neurons was not examined in these mice at later embryonic or postnatal stages. We therefore set out here to delete RhoA in the brain region mostly affected by migrational disorders, namely, the cerebral cortex. In order to determine the role of RhoA in neuronal migration during the development of the cerebral cortex, the Emx1::Cre mouse line driving recombination at early stages selectively in this region (Cappello et al., 2006 and Iwasato et al.

If D112 interacted with R3, as deduced above, then one would pred

If D112 interacted with R3, as deduced above, then one would predict that a substitution of the aspartate at D112 with glutamate would keep a relatively normal G-V because it retained the negative charge. This was indeed observed in the single mutant D112E (Figure 5A). If our inferences so far are correct, and R3 and D112 interact in the open conformation, with R3 lining the pore, then Selleck Natural Product Library D112 would also be expected to line the

open pore. We tested this expectation by considering our observation that substitution of R3 with any of a variety of different amino acids leads to outward current in the high Gu+ solution, i.e., loss of ion selectivity (Figure S1). This led us to predict that a mutation at R3 that has lost ion selectivity should have the selectivity

restored if a complementary mutation could be made at D112 that would reestablish an interaction. If the interaction between D112 and R3 were electrostatic, then a charge reversal would provide a good test. Since, as seen with the 14 other substitutions made initially at R3, a mutation of R3 to aspartate (R3D) also compromises ion selectivity, giving rise to outward current at 100 mM Gu+ at pH 8 (Figures S1 and 6), we tested the effect of a charge reversal. Strikingly, the addition of the mutation D112R to the selectivity-compromised R3D to generate the double mutant find more D112R-R3D (thus swapping the charges between D112 and R3) yielded a channel with an outward current at pHi = 6, pHo = 8, but not with 100 mM Gu+ pH 8 as the internal solution (Figure 6). In other words, the introduction of the charge reversal by the D112R mutation complemented the effect of the R3D charge reversal until and restored proton selectivity (Gu+ exclusion), as predicted for an interaction between D112 and R3 in the open state of the channel. Based on what we have seen so far, the contribution of D112 to ion selectivity could be explained by an indirect effect of D112 on the role of R3 in selectivity. We next set out to determine if D112 has a direct effect on selectivity. To do this we extended our analysis to metal cations and compared the

conductance of WT channels and channels mutated at either R3 alone or both R3 and D112. Our first approach was to test outward currents elicited by voltage steps (to +60 mV) in patches where the pipette was filled (external solution) with 100 mM NaCl solution and to sequentially test 100 mM internal Na+, Li+, K+, Cs+, and Gu+. The experiment was carried out in symmetric pH 8 to minimize contribution by proton current. WT channels supported outward current in the presence of the metal cations, with modestly larger currents in Na+, K+, and Cs+ than in Li+ (Figure 7A). In Gu+, current was almost entirely abolished (reduction to 8.6 ± 1.4%, n = 8) (Figure 7A), consistent with pore block, as shown above (Figure 2A and S1).

LED illumination was restricted to a spot (∼150 μm diameter) and

LED illumination was restricted to a spot (∼150 μm diameter) and we compared the amplitude of IPSCs elicited when the photostimulus was over the GC layer versus when the illumination surrounded the glomerulus containing the dendritic tuft of the recorded mitral cell (filled with fluorescent indicator). Shifting the location of the photostimulus from the GC layer to the glomerular layer largely abolished light-evoked mitral cell IPSCs (Figure 3C; 4.0 ± 1.6% of GC layer

response, n = 6), indicating that cortically-evoked mitral cell inhibition 3 Methyladenine arises primarily from the GC layer. Taken together, these results are consistent with the idea that activation of cortical fibers is sufficient to elicit disynaptic inhibition onto mitral cells

that results from click here AMPAR-mediated excitation of GCs. Intriguingly, activation of cortical feedback projections also elicited feedforward IPSCs in GCs. GABAAR-mediated IPSCs (recorded at the reversal potential for excitation) followed light-evoked EPSCs with a disynaptic delay (3.5 ± 0.5 ms, n = 14; Figures 4A1 and 4A2) and were abolished following application of glutamate receptor antagonists (Figure 4A3). Short-latency feedforward inhibition plays an important role in regulating time windows for excitation (Pouille and Scanziani, 2001). Indeed, in current clamp recordings (Vm = −60 mV) of cells with a mixed EPSP-IPSP, blocking the disynaptic IPSP greatly prolonged the duration of cortically-evoked EPSPs (½ width = 6.5 ± 1.7 ms versus 58.4 ± 18.7 before and after gabazine, respectively) without effecting peak EPSP amplitude (110.4 ± 7.7% of control, n = 5, Figure 4B). Although the amplitudes PDK4 of light-evoked excitatory and inhibitory conductances were similar across the population

of recorded GCs (average excitation [GE] = 1.1 ± 0.3 nS, inhibition [GI] = 1.4 ± 0.3 nS, n = 42), the relative contribution of inhibition to the total conductance (GI/(GE + GI)) varied widely within individual cells (Figure 4C). Anatomical reconstruction of dye-filled GCs did not reveal an obvious correlation between cell morphology and the excitation/inhibition ratio (n = 7, data not shown). Heterogeneity in the relative amount of excitation versus inhibition received by individual GCs suggests that cortical feedback inputs could have diverse effects: activation of the same cortical fibers could cause a net increase in the excitability of some GCs while neighboring GCs are suppressed. We tested this idea by giving nearby (within 100 μm) GCs depolarizing current steps sufficient to elicit APs and interleaving trials with and without trains of light flashes. Indeed, we found that cortical fiber activation in the same region could either enhance or suppress AP firing in GCs (Figure 4D1).

Where insufficient data were reported, first authors were contact

Where insufficient data were reported, first authors were contacted by email to request data. The PEDro scale was used to assess trial quality and it is a reliable JNJ-26481585 solubility dmso tool for the assessment of risk of bias of randomised controlled trials in systematic reviews.14 The PEDro scale consists of 11 items, 10 of which contribute to a total score.12 In the

present review, PEDro scores of 9 to 10 were interpreted as ‘excellent’ methodological quality, 6 to 8 as ‘good’, 4 to 5 as ‘fair’, and < 4 as ‘poor’ quality.15 Two reviewers (DS and ES) independently assigned PEDro scores and any disagreements were adjudicated by a third reviewer (TH). The number of participants, their ages and genders, and the types of cardiac surgery were extracted for each trial. The country in which each trial was performed was also extracted. To characterise the preoperative interventions, the content of the intervention, its duration and the health professional(s) who Sotrastaurin concentration administered it were extracted for each trial. The data required for meta-analysis of the outcome measures presented in Box 2 were also extracted

wherever available. Meta-analysis aimed to quantify the effect of preoperative intervention on the relative risk of developing postoperative pulmonary complications, on time to Modulators extubation (in days), and on the length of stay in ICU and in hospital (also in days). An iterative analysis plan was used to partition out possible heterogeneity in study results by sub-grouping studies according to independent variables of relevance, eg, age, type of

intervention or type of outcome. Due to the differences in clinical populations and therapies being investigated across the studies, random effects meta-analysis and meta-regression models were used. The principal summary measures used were the pooled mean difference (95% CI) and the pooled relative risk (95% CI). Where trials included multiple intervention groups, the meta-analyses were performed using the outcome data of the most-detailed intervention group. Sensitivity new analyses were conducted for length of stay using meta-regression to examine: the influence of population differences (age as a continuous variable); study design (randomised versus quasi-randomised); global geographical region (Western versus Eastern); intensity of education (intensive, defined as anything more than an educational booklet, versus non-intensive, defined as a booklet only); and type of intervention (breathing exercises versus other). Thresholds for sensitivity analyses were defined according to median values (eg, age) or defined using investigator judgment and clinical expertise. Two studies could only be included in analyses for outcomes assessable until time to extubation, as they provided postoperative physiotherapy intervention following extubation in ICU.16 and 17 To aid interpretation of the effect on postoperative pulmonary complications, the relative risk reduction and number needed to treat were also calculated.

Finally, while CTC implementation does not require the use of col

Finally, while CTC implementation does not require the use of cold boxes, their use during this study allowed us to protect vaccines from high temperatures

(reported ambient temperatures reached 39 °C) and direct sunlight, and they remain a known ‘signal’ of vaccination activities within the community. Although MenAfriVac is not the only vaccine to be kept outside the 2–8 °C range, it is the first vaccine approved with this type of variation by WHO, and this study marks the first demonstration of potential benefits from this type of use in low income setting. This landmark decision opens the door for the development of Galunisertib new immunization strategies and approaches to ensure the vaccine reaches all those who are at risk, not just those reached by a cold chain. However in order to achieve CTC vaccine labels, close collaboration with manufacturers, regulatory experts and WHO technical staff is essential. The data that is necessary for these types of variations is not yet systematically generated, and collaboration to define the parameters for which additional testing

should follow in order to apply for a variation is essential [13]. As the current CTC work aims to take advantage of existing stability without requiring reformulation, the length of time available in a CTC is likely to be constrained by the limited stability of today’s vaccines. This means CTC will likely provide benefits in the very Sodium butyrate last mile, rather than alleviate cold chain inhibitors capacity issues higher up in the supply chain. However, NSC 683864 further work to assess full impact on health care workers, coverage and potential cost savings from the approach is needed. In the longer term, combining the CTC workstream with other more upstream efforts on vaccine development and thermostability, and generating the data necessary to achieve a CTC license systematically, have the potential to enable routine EPI services without cold chain for longer periods of time and should be explored. The operational costs of the campaign were covered

within the standard new vaccine introduction support window to the Government of Benin by the Global Alliance for Vaccine and Immunization; project Optimize, a WHO/PATH collaboration funded by the Bill & Melinda Gates Foundation, provided additional specific funding for training, supervision and the evaluation. The authors wish to extend their sincere thanks to the following: For operational and planning support, the Ministry of Health in Benin, the WHO country office in Benin, especially Dr. Aristide Sousou and Dr. Jose Biey; AMP Benin, in particular Philippe Jaillard. Regulatory support and expertise from Maria Baca-Estrada, Tong Wu, Dean Smith and their colleagues at Health Canada; and from Carmen Rodriguez and Nora Dellepiane at WHO, Quality Safety and Standards team.