Schematic model for endocytosis and recycling of KCa2.3 based on data herein as well as our previous publication (18).

<p>Schematic model for endocytosis and recycling of KCa2.3 based on data herein as well as our previous publication (18).</p

KCa2.3 enters Rab5 positive endosomes.

<p>BLAP-tagged KCa2.3 was co-expressed with DsRed-tagged WT (<b>A</b>), S34N (<b>B</b>) or Q79L (<b>C</b>) Rab5 in HEK293 cells. KCa2.3 was labeled with streptavidin-Alexa488 and localization evaluated by confocal fluorescence after 0 or 3 hrs at 37°C. KCa2.3 is localized to the plasma membrane at 0 hrs (upper panels) in each case, as expected. After 3 hrs (lower panels), endocytosed KCa2.3 co-localizes with WT Rab5 (A) as is clear in the merge (yellow color), whereas S34N appears to slow endocytosis (B). As is apparent, Q79L Rab5a induces the formation of intracellular vacuoles (<b>C</b>) and endocytosed KCa2.3 accumulates on these vacuoles; confirming co-localization of KCa2.3 and Rab5. <b>D.</b> Co-IP of myc-tagged KCa2.3 with DsRed-tagged Rab5. KCa2.3 was immunoprecipitated using an anti-myc Ab (lanes 1, 2, 4, 5, 6) or an anti-V5 Ab as IgG control (lane 3) and subsequently IB using an anti-Rab5 Ab. Q79L Rab5 was detected by IB (lane 6), confirming an association between KCa2.3 and Rab5. Note that an association was not detected between KCa2.3 and either WT or Q79L Rab5 suggesting these associations are very transient in nature. Bottom Panel confirms equivalent Rab5 expression (20 µg total protein loaded per lane). Data are representative of 3 experiments.</p

Endocytosis of KCa2.3 is dependent upon dynamin.

<p><b>A.</b> GFP-tagged WT dynamin II and BLAP-tagged KCa2.3 were expressed in HEK293 cells. KCa2.3 was labeled at the plasma membrane with streptavidin-Alexa555 for 10 min at 4°C after which the cells were immediately imaged by live-cell confocal microscopy at 37°C (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044150#s2" target="_blank">Methods</a>). <b>Left Panels</b> show individual images from two separate cells during the time course of the experiment. <b>Right Panels</b> show cropped images from these two cells (labeled 1 and 2) as well as cropped images from 2 additional cells. Arrows denote co-localization of KCa2.3 and WT dynamin II. <b>B.</b> Co-IP of myc-tagged KCa2.3 with GFP-tagged dynamin II. KCa2.3 was immunoprecipitated using an anti-myc Ab (lanes 1, 2, 4, 5) or an anti-V5 Ab as IgG control (lane 3) and subsequently IB using an anti-GFP Ab for dynamin II. WT and K44A dynamin II were detected by IB in lanes 4 and 5, respectively, confirming an association between KCa2.3 and dynamin (Top Panel). Bottom Panel confirms expression of GFP-tagged dynamin in total lysate by IB (5 µg total protein loaded per lane). Data are representative of 3 experiments.</p

Cell surface expression of KCa2.3 is dependent on Rab5.

<p><b>A.</b> BLAP-KCa2.3 was co-expressed with either WT, Q79L or S34N Rab5 and cell surface expression of the channel evaluated in duplicate as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044150#s2" target="_blank">Methods</a> (top panel). Total KCa2.3 expression was also evaluated (total lysate) with no apparent effect of Rab5 expression (<b>A,</b> bottom panel). The data for cell surface expression was quantified by densitometry for three separate experiments and plotted as shown in <b>B.</b> Expression of S34N Rab5 resulted in a significant increase in cell surface KCa2.3 expression (*p<0.05). <b>C.</b> BLAP-KCa2.3 was co-expressed with either control or Rab5 siRNA (siRab5) and cell surface expression of the channel evaluated (top panel). Knockdown of Rab5 was confirmed by IB (middle panel). Tubulin was used as a loading control (bottom panel). The data for cell surface expression was quantified by densitometry for three separate experiments and plotted as shown in <b>D.</b> Knockdown of Rab5 resulted in a significant increase in cell surface KCa2.3 expression (*p<0.05). Densitometry is expressed as mean ± SEM.</p

KCa2.3 is located in a caveolin-rich plasma membrane domain.

<p><b>A.</b> BLAP-tagged KCa2.3 was expressed in HEK293 cells and labeled at the plasma membrane with streptavidin-Alexa555 (left panel), while lipid rafts were localized using the cholera toxin B-subunit (CTX-B) labeled with Alexa488 (middle panel). <b>Top Panels.</b> Single confocal section at the interface where the cells are attached to the glass coverslip showing extensive co-localization between KCa2.3 and CTX-B (merge) at T = 0 min. <b>Bottom Panels.</b> Single mid-plane confocal section after 30 min at 37°C showing endocytosed KCa2.3 co-localized with CTX-B (arrowheads in merge), indicating KCa2.3 is endocytosed in caveolae with CTX-B. <b>B.</b> Transmission Electron Micrograph (TEM) of Quantum Dot-labeled KCa2.3 following endocytosis for 30 min at 37°C. Electron-dense Quantum Dots are observed in structures consistent with caveolae (Cav). A clathrin coated vesicle (CCV) is indicated for comparison. <b>C.</b> Co-IP of KCa2.3 with GFP-tagged caveolin-1 was carried out in HEK293 cells as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044150#s2" target="_blank">Methods</a>. Caveolin-1 was immunoprecipitated using an anti-GFP Ab (lanes 1, 2, 4) or an anti-V5 Ab as IgG control (lane 3) and subsequently IB using an anti-KCa2.3 Ab. When only a single construct was transfected the empty vector (vec) was included to maintain the plasmid at the same final concentration. KCa2.3 was detected by IB in lane 4, confirming an association between KCa2.3 and caveolin-1 (Top Panel). Bottom Panel confirms expression of KCa2.3 in total lysate by IB (5 µg total protein loaded per lane). Data are representative of 3 experiments.</p

Absence of caveolin-1 decreases KCa2.3 endocytosis.

<p><b>A.</b> Wild-type (WT) and caveolin-1-deficient [Cav-1(−/−)] mouse embryonic fibroblasts (MEFs) were infected with BLAP-KCa2.3-Ad, labeled at the plasma membrane with streptavidin-Alexa555 at 4°C after which the cells were imaged by confocal microscopy (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044150#s2" target="_blank">Methods</a>). In Cav-1(−/−) MEFs, KCa2.3 cell surface expression appears to be significantly increased compared to WT MEF cells. <b>B.</b> Plasma membrane proteins were biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044150#s2" target="_blank">Methods</a>) after which the cells were incubated at 37°C for 20 min. Initial plasma membrane expression was determined by omitting the 37°C incubation step (T = 0 min, lane 2). The cell surface biotin which remained following endocytosis was stripped (MESNA) after which the endocytosed, biotinylated protein was pulled down using streptavidin-agarose, the proteins separated by SDS-PAGE and blotted for KCa2.3. The densitometries were determined and plotted in <b>C.</b> In the absence of caveolin-1, plasma membrane KCa2.3 was increased 1.44±0.16-fold (<i>n</i> = 3; p<0.05). Subsequently, the cell surface biotin which remained following endocytosis was stripped (MESNA) and KCa2.3 expression evaluated as above. The efficiency of stripping was determined by subjecting cells to MESNA in the absence of a 37°C endocytosis step (control, lane 1). Lane 3 demonstrates the amount of KCa2.3 endocytosed in 20 min (T = 20 min). The ratio between the amount of KCa2.3 detected following endocytosis (lane 3) to that at time 0 (lane 2) was determined by densitometry and plotted as % endocytosis in <b>D.</b> Knockout of caveolin-1 (Cav-1(−/−)) resulted in a significant decrease of KCa2.3 endocytosis, relative to WT MEFs (n = 3; *p<0.05). Caveolin-1 knockout was confirmed by IB. Densitometry is expressed as mean ± SEM. Tubulin was used as a loading control.</p

DN dynamin II (K44A) slows down endocytosis of KCa2.3.

<p><b>A.</b> BLAP-KCa2.3 was co-expressed with either WT or K44A dynamin II and cell surface expression of the channel evaluated in triplicate as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044150#s2" target="_blank">Methods</a> (top panel). Tubulin was used as a loading control (bottom panel). The data were quantified by densitometry for three separate experiments and plotted as shown in <b>B.</b> Expression of K44A dynamin II resulted in a significant increase in cell surface KCa2.3 expression (*p<0.05). <b>C.</b> BLAP-KCa2.3 was co-expressed with either WT or K44A dynamin II and endocytosis of the channel assessed. Plasma membrane proteins were biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044150#s2" target="_blank">Methods</a>) after which the cells were incubated at 37°C for 20 min. Initial plasma membrane expression was determined by omitting the 37°C incubation step (T = 0 min, lane 2). The cell surface biotin which remained following endocytosis was stripped (MESNA) after which the endocytosed, biotinylated protein was pulled down using streptavidin-agarose, the proteins separated by SDS-PAGE and blotted for KCa2.3. The efficiency of stripping was determined by subjecting cells to MESNA in the absence of a 37°C endocytosis step (control, lane 1). Lane 3 demonstrates the amount of KCa2.3 endocytosed in 20 min (T = 20 min). The ratio between the amount of KCa2.3 detected following endocytosis (lane 3) to that at time 0 (lane 2) was determined by densitometry and plotted as % endocytosis in <b>D.</b> Expression of K44A dynamin resulted in a significant decrease of KCa2.3 endocytosis, relative to WT dynamin (n = 3; *p<0.05). Densitometry is expressed as mean ± SEM.</p

DN Rab5 (S34N) retards endocytosis of KCa2.3.

<p><b>A.</b> BLAP-KCa2.3 was co-expressed with either WT or S34N Rab5 and endocytosis of the channel assessed. Plasma membrane proteins were biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044150#s2" target="_blank">Methods</a>) after which the cells were incubated at 37°C for 20 min. Initial plasma membrane expression was determined by omitting the 37°C incubation step (T = 20 min, lane 2). The cell surface biotin which remained following endocytosis was stripped (MESNA) after which the endocytosed, biotinylated protein was pulled down using streptavidin-agarose, the proteins separated by SDS-PAGE and blotted for KCa2.3. The efficiency of stripping was determined by subjecting cells to MESNA in the absence of a 37°C endocytosis step (control, lane 1). Lanes 3 demonstrates the amount of KCa2.3 endocytosed in 20 min (T = 20 min). The ratio between the amount of KCa2.3 detected following endocytosis (lane 3) to that at time 0 (lane 2) was determined by densitometry and plotted as % endocytosis in <b>B.</b> Expression of S34N Rab5 resulted in a significant decrease of KCa2.3 endocytosis, relative to WT Rab5 (<i>n</i> = 3; *P<0.05). Densitometry is expressed as mean ± SEM.</p

Data_Sheet_1_Relationship between maternal–infant gut microbiota and infant food allergy.docx

The gut microbiota plays a crucial role in food allergies. We sought to identify characteristics of the maternal gut microbiota in the third trimester and the infant gut microbiota in early life and the association of these microbiotas with infant food allergy. A total of 68 healthy pregnant women and their full-term newborns were selected from a cohort of 202 mother–infant pairs; among them, 24 infants had been diagnosed with food allergy within 1 year of age, whereas 44 infants were healthy without allergic symptoms. We collected 65 maternal fecal samples before delivery and 253 infant fecal samples at five time points following birth. Fecal samples were microbiologically analyzed using 16S rRNA gene sequencing. Holdemania abundance in the maternal gut microbiota in the third trimester was significantly higher in the non-allergy group than in the food allergy group (P = 0.036). In the infant gut microbiota, Holdemania was only found in meconium samples; its abundance did not differ significantly between the two groups. The change in the abundance of Actinobacteria over time differed between the non-allergy and food allergy groups (FA, P = 0.013; NA, P = 9.8 × 10−5), and the change in the abundance of Firmicutes over time differed significantly in the non-allergy group (P = 0.023). The abundances of genera Anaerotruncus, Roseburia, Ruminococcus, and Erysipelotricaceae were significantly different between the non-allergy and food allergy groups at different time points. Our results showed that maternal carriage of Holdemania during the third trimester strongly predicted the absence of food allergies in infants; there was no correlation between the presence of food allergies and the abundance of Holdemania in the infant gut microbiota. More dynamic fluctuations in phyla Actinobacteria and Firmicutes early in life protect against food allergy. Thus, the enrichment of the infant gut microbiota early in life with short-chain fatty acid-producing bacteria may be beneficial in preventing the development of food allergies in infants.</p

Additional file 1 of Dose-response associations of triglyceride to high-density lipoprotein cholesterol ratio and triglyceride–glucose index with arterial stiffness risk

Supplementary Material