Data of Behavioral sensitivity to heat and threshold temperature of TRPA1

The file includes three sheets:(1) Data_behav_exp_Tbody, body temperature that elicits the first attempt at escaping from heat for three Anolis species; (2)Data_bheav_exp_Tplate,hot plate temperature that elicits the first attempt at escaping from heat for three Anolis species; (3)Data_TRPA1_threshold, arrhenius break temperature (ABT) as an index of temperature threshold of TRPA1for three Anolis specie

Autophagy Induced by HIF1α Overexpression Supports Trophoblast Invasion by Supplying Cellular Energy

<div><p>Extravillous trophoblasts (EVTs) characterize the invasion of the maternal decidua under low oxygen and poor nutrition at the early feto-maternal interface to establish a successful pregnancy. We previously reported that autophagy in EVTs was activated under 2% O₂<i>in vitro</i>, and autophagy activation was also observed in EVTs at the early feto-maternal interface <i>in vivo</i>. Here, we show that autophagy is an energy source for the invasion of EVTs. Cobalt chloride (CoCl₂), which induces hypoxia inducible factor 1α (HIF1α) overexpression, activated autophagy in HTR8/SVneo cells, an EVT cell line. The number of invading HTR8-ATG4B^C74A cells, an autophagy-deficient EVT cell line, was markedly reduced by 81 percent with the CoCl₂ treatment through the suppression of MMP9 level, although CoCl₂ did not affect the cellular invasion of HTR8-mStrawberry cells, a control cell line. HTR8-ATG4B^C74A cells treated with CoCl₂ showed a decrease in cellular adenosine triphosphate (ATP) levels and a compensatory increase in the expression of purinergic receptor P2X ligand-gated ion channel 7 (P2RX7), which is stimulated with ATP, whereas HTR8-mStrawberry cells maintained cellular ATP levels and did not affect P2RX7 expression. Furthermore, the decreased invasiveness of HTR8-ATG4B^C74A cells treated with CoCl₂ was neutralized by ATP supplementation to the level of HTR8-ATG4B^C74A cells treated without CoCl₂. These results suggest that autophagy plays a role in maintaining homeostasis by countervailing HIF1α-mediated cellular energy consumption in EVTs.</p></div

Correlation chart between autophagy and HIF1α for the invasion of EVTs.

<p>(a) In an intact invasion of EVTs, HIF1α activates autophagy via the PI3K pathway. Autophagy then supplies cellular energy to enhance the invasion of EVTs. (b) In an invasion failure, severe hypoxia or long term hypoxia may accelerate HIF1α overexpression in EVTs. EVTs with an impaired autophagy status by soluble endoglin did not produce energy for the invasion of EVTs with HIF1α overexpression, resulting in the inhibition of EVT invasion. Compensatory P2RX7 expression was enhanced by reacting to the decrease in cellular energy in EVTs observed with preeclampsia.</p

Estimation of autophagy in HTR8/SVneo cells, when CoCl₂ induced HIF1α expression.

<p>a) Western blots in HTR8/SVneo cells under 250 µM CoCl₂, 2% oxygen tension, or DMSO (control) for 24 h were shown as follows: HIF1α and α-tubulin. b) The expression of MAP1LC3B-I and -II (LC3-I and LC3-II) in HTR8/SVneo cells was examined in the presence of E64d and pepstatin under 250 µM CoCl₂ for the indicated times. The expression of α-tubulin was used as an internal control. c) Representative panels show anti-LC3 staining in HTR8/SVneo cells under DMSO (control) or 250 µM CoCl₂ for 24 h. Negative control (Nega cont) was treated with rabbit serum instead of anti-LC3 antibody and stained with DAPI (4′, 6-diamidino-2-phenylindole). Scale bar: 30 µm. d) The graph indicates the number of LC3 puncta in HTR8/SVneo cells under DMSO (control), rapamycin (500 nM), 250 µM CoCl₂, or 250 µM CoCl₂ with 5 mM 3-MA for 24 h. These experiments were independently performed at least three times.</p

CoCl₂ decreased cellular ATP levels, but increased P2RX7 expression in autophagy-deficient HTR8/SVneo cells.

<p>a) Cellular ATP levels in HTR8-Atg4B^C74A mutant cells, the autophagy-deficient EVT cell line, or HTR8-mStrawberry cells, the control cell line, were determined after the treatment with DMSO (control: white bars) or 250 µM CoCl₂ (black bars) for 48 h. b) P2RX7 expression in HTR8-Atg4B^C74A mutant cells (upper panel) or HTR8-mStrawberry cells (lower panel) treated with DMSO (red lines) or 250 µM CoCl₂ (black solids) were determined by flow cytometry for 48 h. Isotype controls are shown as black lines. c) P2RX7 expression was estimated by the mean fluorescent intensity in HTR8-Atg4B^C74A mutant cells or HTR8-mStrawberry cells treated with DMSO (control: white bars) or 250 µM CoCl₂ (black bars) for 48 h. These experiments were independently performed at least three times. N.S.: not significant</p

ATP supplementation recovered the suppression of invasion in autophagy-deficient HTR8/SVneo cells.

<p>a) Invasion assays were performed with HTR8-Atg4B^C74A mutant cells (upper panel), the autophagy-deficient EVT cell line, or HTR8-mStrawberry cells (lower panel), the control cell line, under DMSO (control) or 250 µM CoCl₂ in the presence (black bars) or absence (white bars) of 100 µM ATP for 48 h. The <i>Y</i>-axis indicates the number of invading cells. b) Invasion assays were performed with HTR8-Atg4B^C74A mutant cells under 250 µM CoCl₂ in the presence of increasing concentrations of ATP, as indicated for 48 h. The <i>Y</i>-axis indicates the number of invading cells. These experiments were independently performed at least three times.</p

Total dose of supplemental analgesic rescue.

<p>The efficacy or the dose-response of SRLS was evaluated by comparing the dose of supplemental analgesic rescue across the non-administration, PLGA Control, SRLS 100 mg, 200 mg, and 400 mg groups.</p

Intestinal Microbiota is Different in Women with Preterm Birth: Results from Terminal Restriction Fragment Length Polymorphism Analysis - Figure 1

<p>A. Principal component analysis of fecal microbiota. Principal component analysis scores are plotted based on the relative abundance of OTUs of vaginal microbiota. The percentage of variation explained by the principal coordinates is indicated on the axis. Open circles (○) represent the non-PTL group, open triangles (△) the PTL group, and closed triangles (▴) the PTB group. A dotted line, on the left in Figure 1A, shows ‘cluster 1′, which contains all 10 cases of the PTB group, as well as 2/20 of the non-PTL group and 7/11 of the PTL group. A solid circle, on the right in Figure 1A, shows 90% of the non-PTL group (18/20) and 36.4% of the PTL group (4/11). The PTL group occupied an intermediate position between the non-PTL group and the PTB group. B. Principal component analysis of vaginal microbiota. Principal component analysis scores are plotted based on the relative abundance of OTUs of vaginal microbiota. The percentage of variation explained by the principal coordinates is indicated on the axis. Open circles (○) represent the non-PTL group, open triangles (△) the PTL group, and closed triangles (▴) the PTB group.</p

Exodontia procedure.

<p>a. Incision design for the third molar extraction. The envelope flap design without a vertical releasing incision, with lateral midcrestal incision to protect against lingual nerve damage. b. Bone resection before extraction of impacted wisdom teeth. The mandibular bone around the impacted wisdom teeth is removed with a surgical hand piece and burs to expose the cervical contour of the tooth. c. Crown sectioning with a surgical hand piece and burs. Extraction of the impacted portion usually requires crown sectioning, which prevents damage to the periodontium of the second molar and mandibular alveolar and lingual nerves. d. Wound closure following extraction of the tooth. The alveolar buccal margin is removed, and the defect socket is irrigated. Finally, the operator inserts a study drug into the socket and closes the incision by rough sutures to allow drainage.</p

Average OTU scores in vaginal samples.

<p>OTU: operational taxonomic unit.</p><p>T-RF: terminal restriction fragment.</p><p>non-PTL: non-preterm labor.</p><p>PTL: preterm labor.</p><p>PTB: preterm birth.</p><p>Average OTU scores in vaginal samples.</p