PMC

Phylogenetic tree of O. tsutsugamushi strains constructed based on base-sequence homologies of 56 kDa type-specific genes. The numbers at nodes indicate bootstrap values. Bar shows genetic distance of 0.02. The isolates of from the study are submitted to GenBank under accession numbers as follows: accession numbers of published sequences: OTNARI-1_BLC (OK019092), OT NARI-3 BLC (OK019093), OT NARI-26 BLC (OK019094), OT NARI-1 LBLC (OK019095), OT NARI-2 BLC (OK019096), OT NARI-2 LCSF (OK019097), OT NARI-6 LCSF (OK019098), OT NARI-6 LBLC (OK019099), OT NARI-7 LCSF (OK019100), and OT NARI-33 WB (OK019101).

Changes in oxytocin-induced uterine peristalsis and Ca²⁺ oscillations in neonatal adenomyotic mice. (A) Time courses of uterine peristalsis in response to accumulative oxytocin (OT) stimulation in postnatal day (PND) 14 mice after treatment with the vehicle for tamoxifen (Ctrl) [A(i)] and the same age mice after treatment with tamoxifen (Adeno) [A(ii)]. The concentrations of OT are labeled above the traces. (B) Uterine peristalsis frequency changes caused by OT in uterine slices from Ctrl (O; blue line) and Adeno (Δ; red line) mice at PND14. (C) Changes in the AUC of uterine peristalsis by OT in uterine slices from Ctrl (O; blue line) and Adeno (Δ; red line) mice at PND14. (D) Time courses of Ca²⁺ oscillations in response to accumulative OT stimulation in Ctrl [D(i)] and Adeno mice [D(ii)] at PND14. The concentrations of OT are labeled above the traces. (E) Ca²⁺ oscillation frequency changes caused by OT in uterine slices from Ctrl (O; blue line) and Adeno (Δ; red line) mice at PND14. (F) OT-induced changes in the AUC of Ca²⁺ oscillations in uterine slices from Ctrl (O; blue line) and Adeno (Δ; red line) mice at PND14. N = 5 Ctrl mice (10 slices) and 5 Adeno mice (10 slices) from for panels (B,C,E,F). ****P < 0.0001 by a two-way ANOVA.

Correlational signatures of interaction support blood pressure evoked expiratory neuron modulation of inspiratory chain recurrent loops. A: correlation feature map for Animal 2 gives a graphical summary of responses to blood pressure elevations and detected short-time scale changes in firing probability (peaks and troughs) that support recurrent loop operations within the inspiratory neuron chain and between the chain and a “belt” of expiratory neurons. See text for details. Numbers in small shaded circles correspond to represented cross-correlograms. B–F: cross-correlograms for pairs of neurons in Animal 2 represented in . The detectability index value of each feature is as follows (O, offset; C, central; P, peak; T, trough): 1: OT, 6.6; 2: OT, 31.4; 3: OT, 7.7; 4: OT, 6.6; 5: OT, 5.4; 6: OT, 6.5; 7: OT, 6.9; 8: OT, 6.5; 9: OT, 4.7; 10: CP, 14.3; 11: CP, 5.3; 12: CP, 5.9; 13: CP, 7.0; 14: CP, 20.0; 15: OP, 53.9; OT, 7.3; 16: CP, 11.9; 17: CP, 4.8; 18: OP, 15.4; 19: OP, 7.5; 20: CT, 6.3; 21: OP, 4.4; 22: CP, 10.9; OT, 4.3; 23: OP, 5.7; 24: OP, 6.4; OT, 6.4; 25: OP, 6.6; OT, 7.8; 26: OP, 5.4; OT, 6.2; 27: CP, 7.3; 28: OP, 5.4; OT, 4.3; 29: OP, 27.9; OT, 5.8 (see ); 30: CP, 6.8; 31: OP, 6.0; 32: CP, 5.2; 33: OP, 5.5; 34: OP, 7.0; 35: OP, 8.5; 36: OP, 19.0; OT, 17; 37: CP, 13.4; 38: CP, 24.1; 39: CP, 22.6; 40: CP, 16.7; OT, 8.0; 41: CT, 4.9; 42: CT, 4.1; 43: CT, 6.8; and 44: CT, 7.2. Number of spikes for each neuron represented in used to calculate cross-correlograms: 801: 92,503; 802: 260,467; 808: 254,595; 809: 66,373; 812: 105,528; 813: 168,259; 814: 53,900; 815: 319,904; 818: 85,646; 820: 245,476; 822: 48,453; 826: 80,901; 831: 69,215; 842: 71,5673; 844: 5,539; 847: 38,755; 851: 103,820; and 869: 35,481. G: averages of rectified left (contralateral) phrenic nerve signals are labeled in the feature map with encircled letters: a and b: triggered by rostral pre-I neurons 801 and 831; c and d: triggered by I-Aug neurons 802 and 808; e and f: triggered by expiratory neurons 815 and 820.

POM archetypes in a mixed ball-and-stick and polyhedral representation with a focus on structures relevant for oxo-replacement in POMs: (A) Lindqvist anion [Mo^VI₆O₁₉]^2– containing six O_t, one μ₆-O, and 12 μ₂-O; (B) Keggin anion γ-[Si^IVW^VI₁₂O₄₀]^4– containing 12 O_t, four μ₄-O, four μ₃-O, and 20 μ₂-O; (C) Wells–Dawson anion α-[{W^VIO₆}(H₂)₂W^VI₁₈O₅₆]^6– containing 18 O_t, eight μ₃-O and 36 μ₂-O; (D) Anderson–Evans anion [Te^VIW^VI₆O₂₄]^6– 12 O_t, six μ₃-O and six μ₂-O; (E) heptamolybdate [Mo^VI₇O₂₄]^6– containing 12 O_t, six μ₃-O, and six μ₂-O; (F) octamolybdate β-[Mo^VI₈O₂₆]^4– containing 14 O_t, two μ₅-O, four μ₃-O, and six μ₂-O; (G) paratungstate [H₂W^VI₁₂O₄₂]^10– containing 18 O_t, six μ₃-O, and 18 μ₂-O; (H) type I-derived lacunary anion A-β-[Si^IVW^VI₉O₃₄]^10– containing 15 O_t, four μ₃-O, and 15 μ₂-O. Hydrogen atoms are omitted. Color code: dark green, Mo^VI; dark blue, W^VI; brown, Te^VI; light gray, Si^IV; orange, V^V; red, O.

a-f, Optogenetic inhibition of Fezf2^BLa→NAc and Fezf2^BLa→OT neurons. a, A schematic of the approach to selectively inhibit Fezf2^BLa→NAc neurons with optogenetics. b, Left: a confocal image showing Fezf2^BLa→NAc neurons expressing stGtACR2. Locations of optical fibers for optogenetics are indicated. Right: a confocal image showing injection location of AAVrg-fDIO-Cre in the NAc, as indicated by fluorescent beads (arrow). c, A schematic of the approach to selectively inhibit Fezf2^BLa→OT neurons with optogenetics. d, Left: a confocal image showing Fezf2^BLa→OT neurons expressing stGtACR2. Locations of optical fibers for optogenetics are indicated. Right: a confocal image showing injection location of AAVrg-fDIO-Cre in the OT, as indicated by fluorescent beads (arrow). e, f, Schematics of the AAA task (e; blue bar on time axis indicates laser delivery) and experimental procedure (f). g-p, Optogenetic activation of Fezf2^BLa→NAc and Fezf2^BLa→OT neurons increases pupil size. g, A schematic of the approach to selectively activate Fezf2^BLa→NAc neurons with optogenetics. h, Left: a confocal image showing Fezf2^BLa→NAc neurons expressing ChR2. Locations of optical fibers for optogenetics are indicated. Right: a confocal image showing injection location of AAVrg-fDIO-Cre in the NAc, as indicated by fluorescent beads (arrow). i, A schematic of the approach to selectively activate Fezf2^BLa→OT neurons with optogenetics. j, Left: a confocal image showing Fezf2^BLa→OT neurons expressing ChR2. Locations of optical fibers for optogenetics are indicated. Right: confocal image showing injection location of AAVrg-fDIO-Cre in the OT, as indicated by fluorescent beads (arrow). k, Images of the pupil in a representative mouse, before (left) and after (right) photoactivation of Fezf2^BLa→NAc neurons. l, Trial-by-trial pupil size changes in response to photoactivation (blue bar, 2 s) of Fezf2^BLa→NAc neurons in an example mouse. m, Quantification of pupil size change in response to laser stimulation in the BLa in all the mice in which ChR2 (n = 9) or GFP (n = 7) was expressed in Fezf2^BLa→NAc neurons (Kruskal-Wallis test (K-stat 18.88) with Dunn’s post-hoc test: P = 0.0003; ChR2: **P = 0.002; GFP: P = 0.99). n, o, same as (k, l), respectively, except that Fezf2^BLa→OT neurons were photoactivated. p, Quantification of pupil size change in response to laser stimulation in the BLa in all the mice in which ChR2 (n = 10) or GFP (n = 7) was expressed in Fezf2^BLa→OT neurons (Kruskal-Wallis test (K-stat 21.9) with Dunn’s post-hoc test: P < 0.0001; ChR2: ****P < 0.0001; GFP: P = 0.99).

Corelease of OT and adenosine inhibits presynaptic GABA release. A, In the presence of the OT receptor antagonist vasotocin (1 μm), the effect of electrical stimulation on GABAergic sIPSC frequency was partly, although significantly, blocked (paired t test;p < 0.05; n = 8). ○, Control; ▪, vasotocin. B, Summary of the effect of postsynaptic stimulation after block of the OT receptor. Note that after blockade, the effect of postsynaptic OT release was reduced but still present, indicating potential release of an additional retrograde messenger. C, Bath application of OT (1 μm) mimicked the effect of postsynaptic depolarization on sIPSC frequency, inducing an increase in sIPSC interval (paired t test; p < 0.01;n = 6). D, Application of saturating concentrations of OT (5 μm) induced an increase in sIPSC interval. The dotted line indicates the level of electrical stimulation in the absence of OT application. Electrical stimulation (E.S.) in the presence of OT induced an additional increase in sIPSC interval (paired t test;p < 0.01; n = 3).E, Under control conditions, electrical stimulation resulted in increase in the interval of GABAergic sIPSCs. In the presence of the specific adenosine antagonist CPT (10 μm), the effect of electrical stimulation was significantly reduced (Mann–Whitney; p < 0.05;n = 12). Asterisks indicate significant difference from control, and in D, OT + E.S. from OT.

(A, B, D) BMDMs were primed with LPS (10 ng/ml) for 16 h and infected with OT. IL-1β release was assessed by ELISA. (A) LPS-primed BMDMs were treated with ATP (5 mM) for 3 h or infected with OT (ICU/cell=50) for the indicated time periods. (B) LPS-primed BMDMs were infected with OT of the indicated ICU/cell for 6 h. (C) BMDMs were primed with LPS (10 ng/ml), live OT, heat-inactivated (HOT) or UV-inactivated (UVOT) for 16 h and then treated with ATP (5 mM) for 3 h. (D) LPS-primed BMDMs were challenged with ATP, live OT, heat-inactivated OT (HOT), or UV-inactivated OT (UVOT) for 6 h. (A-D) Error bars represent SD of triplicate samples. N.D.; not detected. (E) LPS-primed BMDMs were challenged with the vehicle (-), ATP (5 mM, for 3 h), Salmonella enteritidis (Sal., MOI=25) or OT (ICU/cell=50) for the indicated time periods. The caspase-1 activation was analyzed by western blotting using rabbit polyclonal antibodies specific for the p10 subunits of caspase-1. Data are representative of three independent experiments in A-E.

Effects of temperature (OT: 25/15 °C: daytime maximum/nighttime minimum temperature and HT: 35/25 °C) during booting stage on leaf surface morphology. Abaxial surface of OT leaf (a, c, e) and HT stressed leaf (b, d, f) showing the disintegration of wax and closure of stomata. Similarly, the adaxial surface of OT (g, i, k) and HT stressed leaf (h, j, l) showing the integration of wax and closure of stomata. Arrows indicate disintegrated wax and closed stomata under HT stress. The corresponding information in OT is indicated by *. The decreased mesophyll thickness under HT stress is shown in (n) and its corresponding OT was shown as (m). The vascular bundle size and morphology of OT and HT was shown in (o) and (p), respectively. OT, optimum temperature; HT, high temperature

M. algicola OT MT023716 growth under different experimental conditions. (A) M. algicola OT MT023716 monoculture growth in ESAW-F/2 and marine broth media; (B) O. tauri RCC4221 and M. algicola OT MT023716 coculture growth in ESAW minus vitamin B₁₂, showing that a sufficient number of O. tauri cells in stationary phase induces M. algicola OT MT023716 growth.

Responses and cross-correlograms features support pontine neuron circuit operations for tuning of an inspiratory drive by blood pressure. A: cross-correlograms for pairs of neurons in Animal 1 represented in . Each correlogram was scaled to facilitate compact visual representation; minimum and maximum firing rates, normalized to spikes/s/trigger event, are indicated. Numbers in yellow circles correspond to represented cross-correlograms. Arrowheads in correlograms 1 and 2 note a positive-lag offset peak superimposed upon a broader peak. The detectability index values for troughs or peaks in each histogram are as follows (O, offset; C, central; P, peak; T, trough): 1: OP, 8.5; 2: OP, 23.6; 3: OP, 10.4; 4: OP, 8.2; 5: OT, 5.1; 6: OT, 9.4; 7: OT, 8.2; 8: OT, 5.4; 9: OT, 3.5; 10: OT, 4.1; 11: CP, 47.4; 12: OT, 4.1; 13: OP, 6.9; 14: OT, 6.3; 15: OT, 6.9; 16: OP, 3.5; 17: OP, 5.9; 18: OP, 3.9; 19: OT, 3.6; 20: OT, 3.5; 21: OP, 4.2; 22: CP, 5.4; 23: OP, 4.0; 24: OT, 4.8; 25: OP, 4.3; and 26: OT, 3.0. Number of spikes for each neuron used to calculate cross-correlograms: 511: 5,347; 524: 19,393; 533: 28,002; 534: 38,613; 803: 298,388; 805: 462,009; 807: 200,008; 813: 44,427; 815: 203,049; 837: 248,893; 842: 138,279; 852: 40,619; 853: 77,355; 881: 71,915; 909: 55,815; 913: 6,577; 914: 8,883; 915: 24,060; 919: 5,232; 931: 37,612; and 933: 18,051. B: correlation feature map represents simultaneously monitored pairs of neurons as circles linked by lines with symbols near target neurons that correspond to the direction of change in their firing probability (+, increased firing probability is characterized by a peak in the CCH; –, decreased probability, trough), following spikes in the indicated reference or trigger neurons. Such maps and neuron responses permit inferences about intra and interregional relationships and directed functional excitation and inhibition. Red lines indicate putative inhibitory influences of pontine neurons on raphe and VRC cells. C: features in spike-triggered averages of rectified nerve recordings support links between responsive neurons and phrenic and lumbar motor neurons shown in B. VRC, ventral respiratory column.

Changes in oxytocin-induced uterine peristalsis and Ca²⁺ oscillations in adult adenomyotic mice. (A) Time courses of uterine peristalsis in response to accumulative oxytocin (OT) stimulation in postnatal day (PND) 55 mice after treatment with the vehicle for tamoxifen (Ctrl) [A(i)] and the same age mice after treatment with tamoxifen (Adeno) [A(ii)]. The concentrations of oxytocin are labeled above the traces. (B) Uterine peristalsis frequency changes caused by oxytocin in uterine slices from Ctrl (O; blue line) and Adeno (Δ; red line) mice at PND55. (C) Changes in the AUC of uterine peristalsis caused by OT in uterine slices from Ctrl (O) and Adeno (Δ) mice at PND55. (D) Time courses of Ca²⁺ oscillations in response to accumulative OT stimulation in Ctrl [D(i)] and Adeno mice [D(ii)] at PND55. The concentrations of OT are labeled above the traces. (E) Ca²⁺ oscillation frequency changes caused by OT in uterine slices from Ctrl (O; blue line) and adeno (Δ; red line) mice at PND55. (F) Change in the AUC of Ca²⁺ oscillations by OT in uterine slices from Ctrl (O; blue line) and Adeno (Δ; red line) mice at PND55. Note that a burst or a spike was counted as an event in this Figure (and ). N = 5 Ctrl mice (10 slices) and 5 Adeno mice (10 slices). A two-way ANOVA to compare individual groups shows no significant difference in panels (B,C,E,F).

Equipotency of the behavioural impact of the blood‐brain barrier‐impenetrable oxytocin (OT)‐vitamin B₁₂ (B₁₂) and native OT. Equimolar doses of OT and OT‐B₁₂ are equally potent at reducing food intake at 30 min post‐injection in (A) male and (B) female rats. (C) Both OT‐B₁₂ and OT remain equally effective at food intake suppression in male rats at 1 h. (A‐D) In female rats, only OT‐B₁₂ reduced feeding behaviour at 1 h n = 7 for male rats and n = 18‐19 for female rats. In an open field test, (E) male rats reduce the time spent in the centre, and (F) latency to enter central part of the field after both OT and OT‐B₁₂. (G) Male rats also reduce the distance travelled in the centre in response to either OT or OT‐B₁₂. (I) In females time spent in the centre is reduced by OT but not OT‐B₁₂. (J) Both compounds reduce the latency to enter the central area. (K) However, only OT reduces the distance travelled in the centre. Track visualizations display a representative open field locomotion in (H) males and (L) females during each drug condition. Data are expressed as mean ± SEM. (E‐K) Individual data points are shown as black dots, squares, or triangles. n = 12 for OT and vehicle (Veh) and n = 4 for OT‐B₁₂ in male rats and female rats. The acoustic startle response test indicated an anxiolytic response to both OT and OT‐B₁₂ in (M) males and (N) females when challenged with a 90 dB stimulus. An anxiolytic response was also noted at the 120 dB intensity, with both OT and OT‐B₁₂ showing an anxiolytic response in (O) males but not (P) females. n = 11‐12 for OT and vehicle, and 8‐9 for OT‐B₁₂. *p < .05, **p < .005, ***p < .0005, ****p < .00005, compared with vehicle (saline)

Effect of PDTC (50 μM) or TPCK (100 μM) on O. tsutsugamushi-induced MCP-1 mRNA level and NF-κB in HUVEC. (A) MCP-1 mRNA level was analyzed using an RNase protection assay with total RNA samples that were prepared from uninfected cells (Mock), cells infected with O. tsutsugamushi for 3 h (OT), and infected cells in the presence of PDTC (OT + PDTC) or TPCK (OT + TPCK). (B) EMSA was performed to analyze the activation of NF-κB using nuclear extracts prepared from HUVEC treated for 2 h with medium (Mock), L-929 cell lysate (Lysate), or O. tsutsugamushi. Nuclear extracts from cells pretreated with PDTC (OT + PDTC) or TPCK (OT + TPCK) for 1 h before infection with O. tsutsugamushi were also analyzed. (C) In the supershift assay, nuclear extracts from HUVEC were preincubated with antibodies against the p65 subunit of NF-κB.

Dung removal of single dung beetle species and their combinations. T1: just soil, T2: soil + dung, T3: soil + dung + O. taurus (OT), T4: soil + dung + D. gazella (DG), T5: soil + dung + P. vindex (PV), T6: soil + dung + OT + DG, T7: soil + dung + OT + DG + PV.

Mean amplitude and phase of the vector sum of OT for Y-Supine and Z-RED (○) compared with Y-Upright OT (•) as a function of frequency (OT averaged across both eyes). Standard errors of the mean are shown (N = 4).

Endogenous glutamate release and intracellular calcium increase in response to 4-AP induced depolarization in striatal gliosomes. Modulation by OT. (A) Inhibition by OT 3 nM of the 4-AP-evoked endogenous glutamate efflux. Bars represent the overflow of the glutamate release, expressed as pmol/mg protein in 3 min sample, in the presence of the drugs at the concentrations indicated. 4AP was added (6 min) during superfusion; OT was added together with 4-AP; the OT receptor antagonist L371,257 was added 8 min before the agonist. Other experimental details in Materials and Methods. Data are means ± SEM (bars) of n = 3–6 independent experiments. ** p < 0.01 compared with the effect of 4-AP, according to Kruskal-Wallis two tailed test and multiple comparison analysis. (B–F) CG-loaded gliosomes were treated with 3 nM OT (B), or with 300 µM 4-AP in the absence (●) or presence (○) of 3 nM OT (C,D), or with 300 µM 4-AP + 3 nM OT in the absence (○) or presence (▼) of the OT receptor antagonist L371,257 (E,F) for the indicated time at 37 °C. CG-dependent fluorescence was monitored every 10 s from 0 to 300 s. [Ca²⁺]_i increase is expressed as “Delta Fluorescence”, which is the difference between the CG-dependent fluorescence of the stimulated samples and the ones of the vehicle-treated samples, both measured at each recording time and subtracted by the one measured at the starting time. The areas reported in (D,F) were quantified to estimate the calcium influxes after 300 s. Data are means ± SEM from three (B), nine and eight ((C,D), ● and ○, respectively), and eight and five ((E,F), ○ and ▼, respectively) experiments in duplicate. * p < 0.05 and ** p < 0.01, according to Mann-Whitney test. 4-AP, 4-aminopyridine; CG, Calcium Green™-1 AM; L371,257, 1-[4-[(1-Acetyl-4-piperidinyl)oxy]-2methoxybenzoyl]-4-(2-oxo-2H-3,1-benzoxazin-1(4H)-yl)piperidine; OT, oxytocin.

OT infection requires attachment to the host cell surface (binding), followed by uptake of the bacteria by clathrin-mediated endo/phagocytosis (internalization). After maturation of the endo/phagosome, OT can be released into the cytoplasm (escape), where OT multiplies (replication). OT in the mature endo/phagosome or cytoplasm activates ASC inflammasome, which induce activation of caspase-1. Active caspase-1 processes pro-IL-1β cleavage, which results in the maturation and secretion of IL-1β.

Synteny between the chromosomes of O. tauri (Ot) and O. lucimarinus (Ol). Depicted areas in red show collinear regions (conserved gene order and content) as described in Methods. Blocks of different colors denote different sorts of duplications: blue, an internally duplicated segment; green, a duplicated segment that is collinear with a segment on a different chromosome in both Ot and Ol; yellow, a duplicated segment that is collinear with a segment on a different chromosome in Ol; orange, a duplicated segment that is collinear with a segment on a different chromosome in Ot.

Effect of PDTC and TPCK on the levels of O. tsutsugamushi-induced chemokine mRNAs in J774A.1 cells. (A) Levels of each chemokine mRNA were analyzed in total RNA samples prepared from uninfected cells (C), O. tsutsugamushi-infected cells (OT), and infected cells in the presence of PDTC (PDTC + OT) or TPCK (TPCK + OT) by RT-PCR analysis. (B) The intensities of bands were determined and normalized as specified for Fig. .

OTR and OT in the porcine PVN. Quantification of the immunohistochemical stainings as positive percentage area in the PVN for the OTR (vehicle: n = 5; Na₂S₂O₃: n = 7) and OT (vehicle: n = 5; Na₂S₂O₃: n = 7). Boxes represent the interquartile ranges with the median (black line), whiskers represent minimum and maximum values. Exemplary immunohistochemical pictures of the OTR and OT in the PVN of vehicle and Na₂S₂O₃ treated animals (10X). Higher magnification pictures of the OTR and OT originate from the black box in the respective 10X picture. OT, oxytocin; OTR, oxytocin receptor; Na₂S₂O₃, sodium thiosulfate.