, 2010). Conversely, the ventromedial ATL has strong connections with visual processing regions in ventral posterior temporal cortex (Binney et al., 2012) and shows greater activation when participants make semantic decisions to pictures relative to words (Visser et al., 2012). This visual semantic bias suggests that a C > A effect might be expected in this area, since concrete words are more strongly associated with visual experiences. It is also important to note that other parts of the ATL are equally responsive to all meaningful stimuli, no matter which modality they are presented in. PET and recent distortion-corrected

fMRI studies have identified an area in the inferior temporal and fusiform gyri (which we

term here the ventral ATL) that responds equivalently to spoken words, written words, pictures and non-verbal sounds Ixazomib cell line (Spitsyna et al., 2006, Vandenberghe et al., 1996 and Visser et al., 2012). Hypometabolism in this region has been linked to multi-modal semantic deficits in patients with semantic dementia (Butler et al., 2009 and Mion et al., 2010) and it has been proposed that the ventral ATL acts as a multi-modal convergence Smad inhibitor “hub” that integrates information from modality-specific sites across the brain to form conceptual representations (Binney et al., 2012 and Patterson et al., 2007). While a number of recent neuroimaging studies have demonstrated activation in ventral ATL for concrete concepts (Peelen and Caramazza, 2012, Robson et al., 2014, Visser et al., 2012 and Visser and Lambon Ralph, 2011), we

are not aware of any studies reporting activation in this area for abstract words. This may be RANTES in part due to susceptibility artefacts that make it difficult to obtain reliable signal in this area with standard, gradient-echo fMRI (Devlin et al., 2000 and Visser et al., 2010). While special steps can be taken in image acquisition and processing to combat this problem (e.g., Embleton et al., 2010 and Halai et al., 2014), the vast majority of fMRI studies do not do so and have reduced sensitivity to activation in the ventral ATLs. It is important to address the question of ventral ATL involvement in abstract concepts because, in common with much of the literature on semantic cognition, implemented computational models of the hub theory have focused exclusively on concrete concepts (Lambon Ralph et al., 2007 and Rogers et al., 2004). As a consequence, Shallice and Cooper (2013) have recently proposed that a separate system is required to meet the different challenges of representing abstract concepts. Furthermore, some researchers have proposed that ATL atrophy in semantic dementia primarily affects visual feature knowledge and, as a consequence, has a disproportionate effect on understanding of concrete words (Bonner et al., 2009 and Libon et al., 2013).

Primary

analysis showed that the mean change in distance walked at 12 weeks Bcl-2 inhibitor was an increase of 8.05 ± 55.48 m in the immediate intervention group and a decrease of 11.45 ± 49.46 m in the wait list control group (p = 0.443) (Table 4, Fig. 2). Sensitivity analyses with imputation of data for the one subject in the wait list control group who had missing data on the 6MWT at 12 weeks were based on the best and worst scenarios, and yielded similar results (data not shown). The per-protocol analysis excluded one female subject who was incorrectly randomized but ineligible by hemoglobin criteria (with initial screening hemoglobin of 10.8 g/dL and a subsequent hemoglobin value drawn 12 days later of 11.5 g/dL, thus rendering her ineligible) and showed similar results (data not shown). There was a small but statistically significant increase in hemoglobin of 0.39 ± 0.46 g/dL at 12 weeks in the immediate intervention group compared to a decline in hemoglobin of 0.39 ± 0.85 g/dL in the wait list control group

(p = 0.026, Table 4). One patient in each group had an increase in hemoglobin of at least 1 g/dL 12 weeks after receiving the first dose of IVIS (received immediately after screening in the immediate intervention see more group and after 12 weeks in the wait list control group). Over time, mean hemoglobin levels rose in the immediate intervention group but decreased in the wait list control group (Fig. 3). Mean hemoglobin levels rose slightly in each study group 12 weeks after initiation of treatment with IVIS (from 11.19 g/dL to 11.58 g/dL in the immediate intervention group, and from 10.91 g/dL to 12.01 g/dL in the wait list control group). There were no significant differences in the two groups in change at 12 weeks for other secondary outcomes of physical, cognitive function, quality of life, and frailty (Table 4). There were no statistically significant correlations

between the week 12 change in hemoglobin from baseline and any of the iron indices at baseline in either the immediate intervention Hydroxychloroquine in vitro group or the wait list control group (data not shown). This is the first exploratory intervention study aimed solely at treating older adults with UAE utilizing intravenous iron. We treated subjects with UAE and serum ferritin levels between 20 and 200 ng/mL (inclusive) with five weekly 200-mg doses of IVIS. Unfortunately, because of early termination of the study due to poor recruitment, the study is substantially underpowered to detect differences in the primary outcome. Thus, although the direction of changes in 6MWT results were as hypothesized—that is, the 6MWT improved in the immediate intervention group compared to the wait list control group—the differences between the groups were not significant. These results are compatible with a trial in older adults with heart failure and similar ferritin levels treated with intravenous ferric carboxymaltose [19].

In a naïve representation, as the split-beam passes by a single scatterer, the measured alongship angle will

suffer a monotonous variation from positive to negative values, while the athwartship angle detected EX 527 order will show a more uniform value. In the case of a shellfish patch, the multiple scatterings will cause the angles (determined from the phase differences detected) to spread around the actual positions, but the time evolution of the angles will be retained. Although their backscattered intensity is superimposed in the same way on the rest of the bottom backscatters, making them indistinguishable in the energy echogram, their angular information will compete with the interface returns and sediment volume backscatter, drawing a complex picture. The split-beam angular information was processed to provide a textural characterisation AZD0530 price of the echogram. First-order statistics do not offer information about variations in the angular echograms that would denote the presence of razor shells. Thus, a second-order statistical procedure, aimed at detecting correlations between neighbouring acoustic samples, should be applied in the form of a textural analysis

(Haralick et al., 1973 and Zaragozá et al., 2010). The most used second-order statistic is the co-occurrence matrix, whose cell pij contains the fraction of pairs of the neighbouring signal samples (echo bins) having quantised levels i and j respectively in a preset window and after signal quantisation in N levels ( Haralick et al. 1973). The neighbouring samples of a bin can be defined in two natural ways: along the pings (being neighbours, the previous and the next bin in the same ping) or along depths (being neighbours, the bins of consecutive pings corresponding to the same depth below the detected sea bottom). We will refer to the first neighbour definition as Type 1

(or along pings) and the second one as Type 2 (or across pings). The CYTH4 resulting co-occurrence matrix will be symmetric as if i is followed by j, then both (i, j) and (j, i) bin pairs are counted. Based on the co-occurrence matrices, Haralick et al. (1973) introduced the so-called textural features. Thirteen Haralick textural features (denoted as H1 to H13) have been calculated for both the alongship and athwartship angles. Another textural feature (lacunarity, Lac), describing the relationship between the co-occurrence standard deviation and the mean value, was also calculated. These variables are mathematically defined in the Appendix. We have restricted the textural analysis to those bins contained between the bottom surface and the equivalent to 30 cm of sediment depth. This depth corresponds to the main insonified region of the echogram and also to the corer sample depth range.

We apologize to all scientists whose work could not be properly discussed and cited here due to limited space. “
“Current Opinion in Genetics & Development 2014, 27:14–19 This review comes from a themed issue on Developmental mechanisms, patterning and evolution Edited by Lee A Niswander and Lori Sussel For a

complete overview see the Issue and the Editorial Available online 8th May 2014 0959-437X/$ – see front matter, © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.gde.2014.03.006 The homeostasis of all multicellular organisms requires active cell–cell communication, which can be achieved through direct contact or via secreted factors that travel Ibrutinib clinical trial and signal at a distance. For example, proper embryonic patterning during fetal development needs accurate

signaling orchestrated by a multitude of factors termed morphogens [1]. Furthermore, malignant cancer cells often hijack normal intercellular signaling pathways to communicate with each other and the microenvironment that serves to promote tumorigenesis and metastasis [1]. During transport, signaling molecules must face the challenges of stability and solubility in the extracellular milieu. Therefore cells have evolved a number of mechanisms to overcome these issues and to ensure that secreted factors can successfully Osimertinib price transmit information. One such mechanism involves tethering signaling molecules to membranous extracellular vesicles (EVs), which can be classified based on criteria that include cellular origin, biological function, or pathways of biogenesis [2 and 3]. First identified three decades ago in reticulocytes, exosomes are typically cup-shaped EVs with 40–100 nm diameter [4]. It is generally agreed that exosomes originate from multivesicular bodies (MVB) within the endocytic system and are released into the extracellular milieu upon the docking and fusion of the MVB with the plasma membrane [5 and 6]. The composition of exosomes

includes a broad range of molecules, such as lipid, protein, carbohydrates, DNA and RNA, reflecting their diverse biological ADAM7 functions [7 and 8]. Proteomic studies have identified a growing list of proteins that are enriched in exosomes, such as the tetraspanin molecules Cd63 and Cd81 [9 and 10]. The mechanism that meditates exosomal biogenesis remains elusive and may vary depending on cell types and functional contexts [4]. Key molecular regulators of MVB/exosomes formation and release include components of the Endosomal Sorting Complex Required for Transport (ESCRT) [11 and 12], as well as members of the Rab GTPase family (e.g. Rab11, Rab27, and Rab35) that are also important for MVB trafficking and exosome secretion [13, 14 and 15]. The stable nature and ability to travel over long distances make exosomes an ideal platform for integrating and transmitting signaling molecules between cells.

It has been suggested that the osteocyte processes might be attached directly to the canalicular wall by β3 integrins at the apex of infrequent, previously

unrecognized canalicular projections [43]. A theoretical model was developed that predicts that the tensile forces acting on these integrins can be as large as 15 pN, Selleckchem AZD1208 and thus provide stable attachment in the range of physiological loading [20]. The model also predicts that axial strains caused by the sliding of actin microfilaments relative to the fixed attachments are two orders of magnitude greater than whole-tissue strains thereby producing local membrane strains in the cell process that can exceed 5%. In vitro experiments indicated that membrane strains of this order are large enough to open stretch-activated cation channels [74]. It is likely that stretch-activated ion channels play a role in the transduction of mechanical stimuli into a chemical response in osteocytes. A well known early response to mechanical stimulation of osteocytes and other mechanosensitive bone cells in vitro is an increase in intracellular calcium concentration [75], which could well be caused by opening of stretch-activated ion channels. Although no direct evidence exists that transient receptor potential (TRP) channels are stretch-activated ion channels, it is an idea that has often been

put forward. In MLO-Y4 osteocytes the Seliciclib price calcium response to mechanical stimulation can be partially blocked by Gd3 + [76], suggesting that some kind of TRP channel is involved in this response. Little is known about TRP channel expression in osteocytes, only TRPV6 is known to be expressed at low levels in murine osteocytes [77]. So far the involvement of specific ion channels in the mechanoresponse of osteocytes has not been elucidated. As described

above, actin microfilaments in the osteocyte cell extensions may slide relative to the fixed attachments. As a result, stretch-activated ion channels may be pulled open, since such channels are connected to the cytoskeleton. On the outside of the osteocyte next process any stretch-activated ion channels may also be connected to the extracellular matrix via tethers, further enabling the opening of ion channels. Such tethering filaments appear to be absent in the pericellular space surrounding the cell body, likely due to the wide pericellular space (~ 1 μm) between the cell membrane and the wall of the lacuna. In contrast, the pericellular space surrounding the cell process on average is 80 nm [32]. You et al. [78] were the first to propose that there were regularly spaced tethering filaments that attached the cell processes to the canalicular wall. They postulated that the flow through the pericellular matrix, which was supported by these tethers, would put them in tension creating a hoop strain on the cell process membrane.

, 2008). Retrogradely labeled MePD cells were mainly located in deeper layers and varied markedly

in number in the different cases. In BSTp 762 and MPN 783 cases, a particularly heavy retrograde labeling was observed in the MePD, especially in its dorsal extent (Figs. 11C1, D1, C2, D2). In contrast, in LA 181 and BMP 737 cases, the MePD contained RG7204 concentration a much smaller number of faintly labeled cells (Figs. 9C1, D1, C2, D2). The present investigation provides the first detailed description of MeAV projections using anterograde and retrograde tract tracing techniques in the rat. The results suggest that the MeAV displays a relatively simple pattern of projections, innervating prominently a few targets it shares with the MeAD and/or the MePV, namely the ventromedial hypothalamic nucleus, especially the dorsomedial and central parts, the amygdalostriatal transition area, the lateral and posterior basomedial amygdaloid nuclei and the intraamygdaloid part of the BST. Overall, they reinforce the view that the MeAD, MeAV and MePV are interrelated and differ markedly from

the MePD, as proposed by Canteras et al. (1995). Importantly, in contrast to the MeAD and MePV, the MeAV sends only light Talazoparib molecular weight inputs to the medial extended amygdala, main olfactory system and components of the reproductive hypothalamic network. A similar pattern of projections was observed in hamsters after Me injections restricted to the MeAD or involving the MeAD and MeAV (Coolen and Wood, 1998 and Gomez and Newman, 1992) however, in rats, these injections were

found to originate distinctive outputs to the ventromedial hypothalamic nucleus, ending in the shell and core (the dorsomedial and central divisions) regions, respectively (Canteras et al., 1995; present findings). The existence of a massive MeAV projection to the ventromedial hypothalamic nucleus is also supported by retrograde tracing evidence in rats (Berk and Finkelstein, 1981; present data) and mice (Choi et al., 2005). In particular, Choi et al. (2005), using exquisitely localized injections, showed in mice that MeAV neurons projecting Orotidine 5′-phosphate decarboxylase to the ventromedial hypothalamic nucleus express the Lhx5 gene of the LIM homeodomain and target the dorsomedial rather than the ventrolateral part. Moreover, in accord with the present results, Gomez and Newman (1992) noted in a case with a PHA-L injection primarily confined to the hamster MeAV that the projections of the MeAV, although similar to, are not as extensive as those of the MeAD, particularly in view of the absence of fiber labeling in the thalamus and nucleus of the horizontal limb of the diagonal band.

Post-hoc comparison following mixed model analysis was carried out using Bonferroni adjustment. Statistical analysis was performed using SPSS for Windows (version 17.0; SPSS Inc., Chicago, USA) and p < 0.05 was considered to be significant. Initial and final body weight and longitudinal

lengths of the left control and right loaded tibiae are shown in Table 1. There were no significant differences between the body weights or bone lengths of mice treated with vehicle or risedronate at any dose. In trabecular SD-208 bone, treatment with risedronate at a dose of 15 or 150 μg/kg/day resulted in a significantly higher BV/TV of the left non-loaded tibiae than in vehicle-treated controls (Table 2, Fig. 2). This increase was primarily associated with higher trabecular number. In cortical bone, there were no significant differences in bone volume between vehicle-treated Epigenetics inhibitor and risedronate-treated animals at any dose. A dose of 0.15 μg/kg/day induced a lower medullary volume than in vehicle-treated controls, while at a dose of 1.5 μg/kg/day there was a slightly lower periosteally enclosed volume (Table 2, Fig. 2). As has been shown previously [34], [37] and [38], mechanical loading significantly increased both trabecular BV/TV and cortical bone volume (Table 2, Fig. 2). The former effect was primarily due to an increase in

trabecular thickness, while the latter response was mainly associated with an increase in periosteally enclosed volume. Mechanical loading-related increases in trabecular BV/TV and cortical bone volume, as assessed by the difference between the right loaded tibiae and their contra-lateral non-loaded controls, were not significantly influenced by treatment

with risedronate, even when given at a high dose (15 or 150 μg/kg/day) (Fig. 3 and Fig. 4). Consistent with previous reports [34] and [40], the fluorochrome-labeled images supported the inference that such loading-related bone gain was primarily associated with increased osteogenesis all (Fig. 5). The additive effect of risedronate and loading on trabecular BV/TV was found at a dose of 15 or 150 μg/kg/day (Table 2, Fig. 2), while there was no synergistic effect of risedronate and loading on trabecular or cortical bone at any dose (Fig. 3). A slight reduction in the loading-related increase in trabecular thickness was observed with high doses of risedronate, but this only reached statistical significance at a dose of 15 μg/kg/day (Fig. 3). In the present study, vehicle or risedronate at various doses was administered to 17–19 week old female C57BL/6 mice and changes in the structure of the tibiae three-dimensionally analyzed by high-resolution μCT. Although the treatment period was short, high doses of risedronate (15 and 150 μg/kg/day) resulted in higher trabecular BV/TV and trabecular number.

2A) and crypt proliferative activity (Fig. 2C) were decreased in carcinogenic FLX-treated rats (P < 0.007 and 0.001; Fig. 2B and D), despite its activity in the promotion of proliferation in non-carcinogen treated

rats (P < 0.01). As previously shown (Liang et al., 2004 and Waldner et al., 2010), dysplastic ACF development is also selleck screening library related to microvessels enlargement. Therefore, crypt surrounding microvessels (Fig. 2E) were reduced in carcinogenic FLX-treated rats (P < 0.05; Fig. 2F). Also, it decreased VEGF expression within PCCS ( Fig. 3A) in DMH-treated rats (P < 0.001; Fig. 3B) and, reduced COX-2 expression ( Fig. 3C) in non-DMH and DMH-treated groups (P < 0.01; Fig. 3D). In this study we demonstrated that FLX and its metabolite are present in the colon tissue and this treatment possibly increased 5-HT levels by decreasing SERT activity resulting in the suppression of 5-HIAA release. Thus, FLX was quickly diffused into multiples body-sites, as colon, due to its high lipophilicity (Lefebvre et

al., 1999) and possibly blocked SERT-function I BET 762 (Gill et al., 2008), resulting in the imbalance of 5-HT metabolism (Bertrand et al., 2010). Despite the current knowledge that high 5-HT levels are implicated in the induction of cell proliferation and tumor growth (Arends et al., 1986), 5-HT selectively inhibited the colon adenocarcinoma growth by constricting tumor arterioles (Lubbe and Huhnt, 1994). Furthermore, FLX has been revealed as a great apoptosis inducer inhibiting tumor

development (Arimochi and Morita, 2006 and Lee et al., 2010). Our analysis is driven by the hypothesis that besides FLX effect on the upregulation of 5-HT levels, Ribonuclease T1 their co-related activity possibly promoted the blockade of 5-HT2C receptors. On the other hand, endogenous upregulation in this amine levels seemed not to be correlated to the promotion of malignant crypt changes, as noticed by its metabolism and recognition. FLX and N-FLX have been shown to enhance the rate of desensitization in 5-HT-receptors (Brink et al., 2004 and Choi et al., 2003), reducing both Na+ and Ca2+ currents as a noncompetitive antagonism activity (Eisensamer et al., 2003). Also, 5-HT potentially desensitized 5-HT2C receptors after a short cell exposition to this amine (Briddon et al., 1998), and the blockade of 5-HT1 and 5-HT2-receptors subtypes inhibited tumor cell proliferation (Tutton and Barkla, 1980 and Tutton and Barkla, 1986). Additionally, 5-HT treatment promoted tumor but not crypt cell proliferation (Tutton and Barkla, 1980), whereas colon tumor cells treated with sulforaphane revealed decreased 5HT1A, 5-HT2C, and SERT levels, suggesting a lower tumor progression (Mastrangelo et al., 2008). Although FLX greatly controlled dysplastic ACF development, the results regarding epithelia proliferation seemed to be conflicting between non-DMH and DMH treated rats that received FLX.

Fluorescent-stained areas of vessel walls were selected and the fluorescence intensity was quantified using image analyzer software (Axio Vision® 4.8 version, Carl-Zeiss, Germany). The same procedures were carried out in sections

of lung tissue incubated without antibody or using goat anti-mouse immunoglobulin G to evaluate the background reaction. Leucocytes collected from blood of the abdominal aorta of vehicle or HQ exposed mice were employed to quantify L-selectin, β2-integrin, β3-integrin and PECAM-1 expression. Briefly, erythrocytes were lysed by the Alectinib mw addition of ammonium chloride solution (0.13 M) to the samples and leukocytes were recovered after washing with Hank’s balanced salt solution (HBSS). To quantify the expression of adhesion molecules, leukocytes (1 × 105) were incubated for 20–60 min in the dark at 4 °C with 10 μl of monoclonal antibody (L-selectin conjugated with FITC; β2 or β3-integrin Selleck ZD1839 conjugated with FITC or PECAM-1 conjugated with PE). After that, the cells were analyzed in

a FACS Calibur flow cytometer (Becton & Dickinson, San Jose, CA, USA). Data from 10,000 events were obtained and only the morphologically viable leukocytes were considered for analysis. Results are presented as arbitrary units of fluorescence. In order to study the ability of HQ to induce peroxidation of fatty acids in cell membranes, plasma levels of MDA

were determined in mice exposed to vehicle or 25 ppm HQ. For this purpose, 250 μl of plasma were added to 36 μl of 0.2% butylated hydroxytoluene (BHT) in ethanol and 12.5 μl of 10 M NaOH followed by incubation at 60 °C for 30 min. Afterward, 1500 μl of 7.2% trichloroacetic acid with 1% potassium iodide were added to the sample and placed on ice for 10 min. The sample was centrifuged (1000 × g for 10 min), and 1000 μl of the supernatant were removed and mixed with 500 μl of 0.6% thiobarbituric acid (TBA). The solution was incubated at 90 °C for 45 min. Next, 250 μl of n-butanol were added to the sample, and the mixture was vortexed and centrifuged (600 × g for 5 min). The n-butanol phase was collected and injected in the HLPC-DAD system, using the following chromatographic conditions. A 150 mm × 4.6 mm ID, 5 μm C18 column (Phenomenex, Torrance, DNA Methyltransferas inhibitor CA) with a C18 security guard cartridge, 4.0 mm × 3.0 mm (Phenomenex, Torrance, CA), was eluted in isocratic mode with a mobile phase consisting of 35% MeOH and 65% potassium phosphate buffer (50 mM, pH 7.0), at a flow rate of 1 ml/min and 30 °C. The diode array detector was set at 532 nm and calibration curves were constructed in the range of 0.5–5.0 μM of MDA standard dissolved in PBS. Leukocytes collected from blood from the abdominal aorta of vehicle or HQ exposed mice were employed to quantify oxidative burst.

After washing three times with PBS-T, 100 μl of goat anti-rabbit antiserum conjugated

with horseradish peroxidase (diluted 1:3000 in PBS-T) was added as secondary antibody and plates were incubated for 40 min at 37 °C. After three PBS-T buy Cabozantinib washes, 100 μl of substrate solution (25 mg O-phenylenediamine, 25 μl H2O2 in 25 ml 0.1 M citrate buffer, pH 5.0) was added to each well and incubated for 40 min at 37 °C. The reaction was stopped by addition of 50 μl 1.0 N H2SO4 to each well and optical density was read at 492 nm on a Labsystems Multiskan MCC/340 (ThermoQuest SEG Ltd., Basingstoke, UK). MAGs and SVs of male mosquitoes were dissected into ice-cold PBS and fixed in 4% (w/v) paraformaldehyde in PBS at 4 °C overnight. Fixed tissues were washed four times in PBS before incubation for 2 days at 4 °C with anti-FMRFamide primary antibody diluted 1:500 in 0.3% v/v Triton X-100 in PBS (TX-PBS) containing 2% v/v goat serum). A control was performed by incubating fixed tissue with 2% (v/v) goat serum in TX-PBS without the primary antibody. Excess reagent was washed away

with TX-PBS (4 × 15 min) before incubating samples for 2 days at 4 °C with secondary antibody (Alexa Fluor 546 goat anti-rabbit IgG, Invitrogen, Paisley, UK). Secondary antibody was diluted 1:500 in TX-PBS containing MEK inhibitor clinical trial 2% v/v goat serum. A further control was performed by pre-incubating 250 μl of secondary antibody (diluted 1:500 in TX-PBS containing 2% v/v goat serum) with 25 μl of 1 mM Aea-HP-1 prior to incubation with tissue. Excess reagent was washed away with TX-PBS (4 × 15 min) before mounting tissue on slides for confocal microscopy. Mounting was performed in 4,6′-diamidino-2-phenylindole (DAPI) Loperamide diluted 1:1000 in Vectashield® Mounting Medium (Vector Laboratories Ltd., Peterborough, UK). Slides were stored

in the dark at 4 °C overnight before microscopic examination. Images were captured using an inverted LSM510 META laser scanning confocal (Carl Zeiss) microscope. Pinholes were set to 1 Airy Unit which gave a 1 μm optical section with a 40× oil immersion objective. Alexa Fluor 546 was excited with the 543 nm HeNe laser and emission was collected through a long pass LP560 emission filter. DAPI was excited with a 405 nm laser diode and emission was collected through a LP420 emission filter. For determining the volume of the MAG, the gland surface was non-specifically coated with Alexa Fluor 546 goat anti-rabbit IgG and serial optical z-sections were collected using confocal microscopy as described above (omitting the collection of the DAPI channel) through the full depth of the gland with z-steps of 0.5 μm. Approximately 60–80 images were required to image the full volume of the MAG. Image stacks were then imported into Imaris software (version 5.7, Bitplane AG, Zurich, Switzerland).