Normal ovarian surface epithelial label-retaining cells exhibit stem/progenitor cell characteristics

Ovulation induces cyclic rupture and regenerative repair of the ovarian coelomic epithelium. This process of repeated disruption and repair accompanied by complex remodeling typifies a somatic stem/progenitor cell-mediated process. Using BrdU incorporation and doxycycline inducible histone2B-green fluorescent protein pulse–chase techniques, we identify a label-retaining cell population in the coelomic epithelium of the adult mouse ovary as candidate somatic stem/progenitor cells. The identified population exhibits quiescence with asymmetric label retention, functional response to estrous cycling in vivo by proliferation, enhanced growth characteristics by in vitro colony formation, and cytoprotective mechanisms by enrichment for the side population. Together, these characteristics identify the label-retaining cell population as a candidate for the putative somatic stem/progenitor cells of the coelomic epithelium of the mouse ovary.     

Role of PKA signaling in D2 receptor-expressing neurons in the core of the nucleus accumbens in aversive learning

The nucleus accumbens (NAc) serves as a key neural substrate for aversive learning and consists of two distinct subpopulations of medium-sized spiny neurons (MSNs). The MSNs of the direct pathway (dMSNs) and the indirect pathway (iMSNs) predominantly express dopamine (DA) D1 and D2 receptors, respectively, and are positively and negatively modulated by DA transmitters via Gs- and Gi-coupled cAMP-dependent protein kinase A (PKA) signaling cascades, respectively. In this investigation, we addressed how intracellular PKA signaling is involved in aversive learning in a cell type-specific manner. When the transmission of either dMSNs or iMSNs was unilaterally blocked by pathway-specific expression of transmission-blocking tetanus toxin, infusion of PKA inhibitors into the intact side of the NAc core abolished passive avoidance learning toward an electric shock in the indirect pathway-blocked mice, but not in the direct pathway-blocked mice. We then examined temporal changes in PKA activity in dMSNs and iMSNs in behaving mice by monitoring Förster resonance energy transfer responses of the PKA biosensor with the aid of microendoscopy. PKA activity was increased in iMSNs and decreased in dMSNs in both aversive memory formation and retrieval. Importantly, the increased PKA activity in iMSNs disappeared when aversive memory was prevented by keeping mice in the conditioning apparatus. Furthermore, the increase in PKA activity in iMSNs by aversive stimuli reflected facilitation of aversive memory retention. These results indicate that PKA signaling in iMSNs plays a critical role in both aversive memory formation and retention.Aversive stimuli induce not only rapid avoidance behavior, but also memory formation to escape from uncomfortable environments, and thus strongly influence animal behavior (1–3). The mesolimbic dopaminergic (DA) system plays a critical role in both rapid aversive reaction and memory formation (3–5). The nucleus accumbens (NAc) receives DA inputs from the ventral tegmental area (VTA) and serves as a key neural substrate for the control of aversive learning (6–8). The NAc consists of two subpopulations of medium-sized spiny neurons (MSNs) (9–11). The MSNs of the direct pathway (dMSNs) send their axons to the substantia nigra pars reticulata (SNr) and VTA, and selectively express dopamine D1 receptors, whereas the MSNs of the indirect pathway (iMSNs) indirectly project to the SNr and VTA via the ventral pallidum (VP) and predominantly express D2 receptors (12, 13). D1 receptors stimulate the cAMP-dependent protein kinase A (PKA) signaling cascade via Gs and exhibit a low affinity for DA (14–16). Conversely, D2 receptors inhibit the cAMP-PKA cascade via Gi and show a high affinity for DA (14–16). Thus, these two distinct types of MSNs, constituting two parallel pathways, contribute to the dynamic modulation of neuronal cell excitability and synaptic plasticity in the NAc circuitry (14–16).Although accumulated evidence indicates that DA modulation of the NAc is critical for both reward-based and aversive reactions (3, 5, 6, 17), the response of DA neurons in the VTA to aversive stimuli is not uniform; that is, some DA neurons are stimulated in response to aversive stimuli, whereas most others react by transiently suppressing their firing (18–22). Recent optogenetic studies have revealed that not only activation of iMSNs, but also inactivation of the VTA neurons, which down-regulates DA levels in the NAc, evoke an aversive reaction and learning (23–26); however, how intracellular cAMP-PKA signaling is involved in the induction and retention of aversive memory in a cell type-dependent manner in the NAc circuit remains largely elusive.In the present investigation, we addressed this issue using two approaches. We first used asymmetric reversible neurotransmission blocking (aRNB) techniques (27, 28), in which either the direct or indirect pathway at one side of the NAc was selectively blocked by the pathway-specific expression of transmission-blocking tetanus toxin and the other intact side was manipulated by injection of PKA inhibitors. In the second approach, we examined temporal changes in PKA activities of these two pathways in the formation of aversive memory by monitoring Förster resonance energy transfer (FRET) responses of PKA selective for either dMSNs or iMSNs with the aid of in vivo microendoscopic analysis (29, 30). These two different approaches explicitly demonstrated that the activation of PKA in iMSNs plays a key role in both the formation and the retention of aversive memory.     

Basophil activation by mosquito extracts in patients with hypersensitivity to mosquito bites

Hypersensitivity to mosquito bites (HMB) is a cutaneous disorder belonging to the group of Epstein–Barr virus (EBV)‐associated T/natural killer (NK)‐cell lymphoproliferative diseases, and is primarily mediated by EBV‐infected NK cells. It is characterized by intense local skin reactions accompanied by general symptoms after mosquito bites, and infiltration of EBV‐infected NK cells into the bite sites. However, the mechanisms underlying these reactions have not been fully examined. We recently described the activation of circulating basophils by mosquito extracts in vitro in a patient with HMB. To further investigate this finding, we studied four additional patients with HMB. All patients showed typical clinical features of HMB after mosquito bites and they had NK lymphocytosis and high peripheral blood EBV DNA loads. We found evidence of EBV infection in NK cells through in situ hybridization that detected EBV‐encoded small RNA‐1, and flow cytometry showed HLA‐DR expression on almost all NK cells. Basophil activation tests with the extracts of epidemic mosquitoes Culex pipiens pallens and Aedes albopictus showed positive responses to one or both extracts in all samples from patients with HMB, suggesting the presence of mosquito antigen‐specific IgE and its binding to basophils. In particular, the extract of Aedes albopictus was able to activate basophils in all available patient samples. These results indicate that basophils and/or mast cells activated by mosquito bites may be involved in initiation and development of severe skin reactions to mosquito bites in HMB.     

Mandibular bone healing after advancement or setback surgery using sagittal split ramus osteotomy

Purpose

The purpose of this study was to compare mandibular bone healing after advancement or setback surgery using sagittal split ramus osteotomy (SSRO).

Disproportionation-induced solid-state fluorescence in 6,13-dihydropentacenes

The thermally and photolytically induced disproportionation of 6,13-dihydropentacene derivatives into tetrahydropentacenes and pentacenes results in unique solid-state fluorescence. The fluorescence thereby depends on the molecular structure and the molecular arrangement in the solid state.

The thermally and photolytically induced disproportionation of 6,13-dihydropentacene derivatives into tetrahydropentacenes and pentacenes results in unique solid-state fluorescence.

Disproportionation reactions are arguably one of the most important classes of organic reactions, especially in the context of coal liquefaction and aromatization, as well as Cannizzaro and biochemical reactions. However, reports on disproportionation reactions of aromatic hydrocarbons remain scarce. Stein and co-workers have reported the disproportionation of 9,10-dihydroanthracene (>350 °C; liquid phase pyrolysis) into tetrahydroanthracene and anthracene.¹ Pentacene has also been reported to undergo disproportionation at 320 °C in a horizontal vapor phase deposition furnace to produce 6,13-dihydropentacene and polycondensed aromatic hydrocarbons.² It has been postulated that H-atom-transfer processes play a crucial role in such disproportion reactions. Indeed, H-atom transfer from 9,10-dihydroantharacene,^3,4 xanthene,⁴ fluorene,⁵ acridan,⁶ and other H donors (D–H) with weak C–H bonds⁷ to unsaturated H acceptors has been widely explored.On the other hand, the molecular design of π-conjugated systems is crucial for the development of new functional materials. Nevertheless, reports on π-conjugated systems exhibiting disproportionation-induced photoluminescence changes are relatively rare. Against this background, we decided to explore the utility of disproportionation reactions for the development of fluorescent chromic materials. Herein, we report the synthesis and disproportion of 6,13-dihydropentacene derivatives, as well as a detailed investigation into the fluorescence properties of the obtained compounds in the solid state.The synthesis of N,N′-bisalkyl-6,13-dihydropentacene[2,3:9,10]biscarboxyimides 3a–c is outlined in Scheme 1. We have previously reported the synthesis of key starting material 6,13-dihydropentacene-2,3,9,10-tetracarboxylic acid (1).⁸ Tetracarboxylic acid 1 was converted into the corresponding anhydride (2) in good yield using a modification of the procedure reported by Qian and co-workers.⁹N,N′-Bisalkyl-6,13-dihydropentacene[2,3:9,10]biscarboxyimides 3a–c were then obtained in 20–64% yield by stirring anhydride 2 under reflux in the presence of the corresponding amines. The structure of the obtained dihydropentacene bisimides was confirmed by ¹H and ¹³C NMR spectroscopy, IR spectroscopy, FAB mass spectrometry, and elemental analysis. The ¹H NMR spectrum of 3a in CDCl₃ shows a singlet at 4.36 ppm, which was assigned to the central ring of dihydropentacene. In the aromatic region, two singlets appear at 8.03 and 8.23 ppm, which were attributed to the naphthalene ring. The ¹³C NMR of 3a displays 13 signals, including five aromatic carbon atoms (123.5, 128.0 × 2, 134.4, 138.4, and 168.3 ppm) for the naphthalene ring, one carbonyl carbon atom (186.3 ppm), and one sp³-hybridized carbon atom of the central ring (38.5 ppm). In general, the spectra of 3a–c are very similar.Open in a separate windowScheme 1Synthesis of N,N′-bisalkyl-6,13-dihydropentacene[2,3:9,10]bis-carboxyimides 3a–c.Subsequently, we examined the disproportionation reaction of dihydropentacene 3a in the solid state (Scheme 2). Interestingly, upon heating solid 3a from room temperature to 230 °C, the color of the solid changed from colorless to green. To elucidate the origin of this green material, we investigated the absorption features of a thin film of 3a before and after heating (250 °C; 3 h). In the difference spectrum of 3a (Fig. 1), the positive absorption bands at 523, 560, and 611 nm were attributed to an expansion of the π-conjugated system. A comparison with the absorption spectrum of independently synthesized N,N′-bispentylpentacene[2,3:9,10]biscarboxyimide (4a) revealed very similar features. The formation of pentacene derivative 4a was also confirmed by ¹H NMR spectroscopy (Fig. S1^†). After heating (250 °C; 40 min), the main signals were assigned to the starting material (3a), and the new signals suggested a conversion <7%. The ¹H NMR spectrum in CDCl₃ showed three singlets at 8.50, 8.94, and 9.12 ppm, which were assigned to the protons attached to the pentacene core, respectively. Moreover, three singlets for aromatic protons (8.06, 8.22, and 8.39 ppm) and two singlets for methylene protons (4.27 and 4.81 ppm) were observed for tetrahydropentacene derivative 5a. Upon exposure to air, the signals of 4a disappeared and two singlets at 8.56 and 9.14 ppm appeared, which were assigned to pentacene quinone 7a, i.e., the oxidation product of pentacene 4a (Fig. S2^†).^8a,10 An integral 5a : 7a ratio of 1 : 1 was estimated. These results strongly indicate that the thermal reaction of dihydropentacene 3a affords pentacene 4a and tetrahydropentacene 5a (Scheme 2).Open in a separate windowScheme 2Disproportionation reaction of dihydropentacene 3a and decomposition of pentacene derivative 4a.Open in a separate windowFig. 1The absorbance difference spectrum of a thin film of 3a before and after the heating.Moreover, we discovered that N,N′-bisalky-6,13-dihydropentacene[2,3:9,10]bis-carboxyimide 3a exhibits solid-state fluorescence (Fig. 2); under illumination with UV light (λ_ex = 365 nm), bright blue fluorescence was observed. Interestingly, upon heating 3a in solid state from room temperature to 230 °C, the color of the apparent fluorescence gradually changed from bright blue to yellow and then red (Fig. 2a). Such phosphorchromaticity was observed for both film and crystalline samples (Fig. 2b), and the original color was not recovered even after cooling to room temperature. The differential scanning calorimetry (DSC) cooling and heating curves of 3a showed no peaks between room temperature and 270 °C (Fig. S3^†). In the case of 3b and 3c, similar fluorescence color changes were observed, while the crystal appearance changed from transparent to opaque. Polarized optical microscopy images revealed rough and cracked surfaces of the crystals of 3b and 3c after heating (Fig. S4^†).Open in a separate windowFig. 2(a) Apparent fluorescence color change of a 3a film with the increasing temperature. (b) Apparent fluorescence color change of crystals of 3a.We also observed significant changes in the fluorescence spectra of a thin film of 3a with increasing temperature (Fig. S5^†). The intensity of the fluorescence of the naphthaleneimide chromophore (300–550 nm) decreased with increasing temperature, while the intensity of the new fluorescence band at 638 nm, which was assigned to a pentacene chromophore, increased. The decaying emission of the naphthaleneimide skeleton may be explained in terms of an energy transfer from 3a to 4a.¹¹Fig. S6^† shows the concentration-dependent emission spectra of 3a in CHCl₃. At a concentration of 1.9 × 10⁻⁴ M, a monomer emission band from the naphthaleneimide moiety was observed at 390 and 400 nm (λ_ex = 280 nm). Upon increasing the concentration of 3a beyond 7.7 × 10⁻⁴ M, the fluorescence intensity of the naphthaleneimide moieties decreased and two new excimer emission bands emerged at approximately 440 and 460 nm with growing intensity. These results indicate that the solid-state fluorescence at 440 nm does not correspond to monomer emission, but to aggregate emission.In order to determine the molecular conformation in the crystals and rationalize the observed reactivity, a single-crystal X-ray diffraction analysis was carried out (Fig. 3). Depending on the solvent, two different crystalline structures were identified in the case of 3a. A structural analysis of single crystals of 3a–c grown from benzonitrile solutions revealed a planar central six-membered ring of the dihydropentacenes (Fig. 3a, and S8^†). The bond angle sums of the central six-membered rings are close to 720°. Numerous hitherto reported crystal structures are characterized by flattened boat conformations for dihydroanthracene and dihydropentacene derivatives, whereas the preferred conformation of dihydroaromatics should be a planar according to a theoretical study.¹² In fact, there are a few crystallographic reports on dihydroanthacene¹³ and dihydropentacene¹⁴ skeletons with a planar geometry. In contrast, the structural analysis for crystals of 3a grown from a THF solution revealed a V-shaped dihydropentacene skeleton with a bent central six-membered ring 3a_(v).Open in a separate windowFig. 3ORTEP drawings of (a) 3a_(planer) and (b) 3a_(v) (thermal ellipsoids at 50% probability).The energy difference between the planar and V-shaped conformers was examined using density functional theory (DFT) calculations on model compound 3′, which bears H atoms instead of alkyl chains. The V-shaped conformer 3′_(v) is by 4.0 kcal mol⁻¹ thermodynamically more stable than the planar conformer 3′_(planar). These results suggest a small energy difference between the two conformers. Consequently, the solid-state structure of 3a is most likely determined by packing forces in the crystal.Interestingly, the phosphorchromaticity changes in the solid state were also triggered by photoirradiation (>300 nm); however, the reaction was limited to the surface of the specimen. The phosphorchromaticity switching rate was sensitive to the substituents on the dihydropentacene. Upon photoirradiation of solids 3a_(planar) and 3b (λ_ex > 300 nm; 1 h), the apparent fluorescence color gradually changed from bright blue to red. In the case of 3_(v) and 3c, the apparent fluorescence color did not change, not even after two days of photoirradiation. To clarify the different reactivity of the planar and V-shape conformers of 3a, further DFT calculations were carried out. Fig. 4 shows the relevant Kohn–Sham molecular orbitals for the optimized structures of 3′_(planar) and 3′_(v). Both HOMOs are clearly localized on the C–H bonds of the central six-membered ring and the two naphthalene units. Interestingly, the LUMO of 3′_(planar) is notably less localized around the C–H bond of the central six-membered ring than the LUMO of 3′_(v). These results indicate that the excited state of 3a_(planar) favors the elimination of the C–H protons of the central six-membered ring compared to the case of 3a_(v).Open in a separate windowFig. 4Molecular Kohn–Sham orbitals of 3′_(v) and 3′_(planer), calculated at the M06-2X/6-31G* level of theory.To evaluate the differences in photoreactivity depending on the substituents, the molecular arrangements in the crystals were investigated. None of the crystals contained any solvent in the lattice. The distances between the H (from the C–H bonds of the central six-membered ring) and C (from the naphthalene ring of the nearest-neighboring molecule atom) atoms were ca. 3.0 Å (3a), 2.8 Å (3b), and 3.3 Å (3c). The long distance in the crystals of 3c should thus be unfavorable for hydrogen-atom transfer from the C–H bond of the central six-membered ring to the naphthalene ring of the neighboring molecule.     

Can pretreatment ADC values predict recurrence of bladder cancer after transurethral resection?

Objective

The aim of this retrospective study was to investigate the association between the pretreatment apparent diffusion coefficient (ADC) value and recurrence of bladder cancer after transurethral resection.

Methods

Patients with superficial bladder cancer were identified. Mean ADC values of the tumors were compared between patients with and without recurrence following trans-urethral resection. A receiver–operator characteristic curve was used for determining the optimal cutoff ADC value. Univariate and multivariate analyses were used to determine the effect of ADC values and other factors.

Results

With a mean follow-up period of 25 months, bladder cancer recurred in 14 of 44 patients (32%). The mean ADC value of tumors in patients with recurrence was lower than in those without recurrence (1.08 mm²/s vs. 1.28 × 10⁻³ mm²/s; p = 0.003). The optimal cutoff ADC value for predicting recurrence was determined to be 1.12 × 10⁻³ mm²/s. A modest and significant negative correlation was observed between the ADC values and tumor size (r = −0.436, p = 0.008). After adjustment for size and risk groups, an ADC value equal to or less than the optimal cutoff remained a significant predictor of recurrence (odds ratio 6.3, 95% CI 1.23–32.2, p = 0.027).

Conclusion

Pretreatment ADC values may be an independent predictor of bladder cancer recurrence.     

Hypovascular hepatic nodule showing hypointensity in the hepatobiliary phase of gadoxetic acid-enhanced MRI in patients with chronic liver disease: prediction of malignant transformation

Purpose

To investigate the predictive factors of malignant transformation of hypovascular hepatic nodule showing hypointensity in the hepatobiliary phase images of gadoxetic acid-enhanced MRI (HHN).

Materials and Methods

The clinical data and imaging findings of dynamic contrast-enhanced computed tomography (DCE-CT) and gadoxetic acid-enhanced MRI for a total of 103 HHNs in 24 patients with chronic liver disease were retrospectively investigated. After the results of follow-up examinations were investigated, HHNs were categorized into the three groups for each comparison: (1) nodules with enlargement and/or vascularization and others, (2) nodules with only enlargement and others, (3) nodules with only vascularization and others. Enlargement and/or vascularization during the follow-up period were defined as malignant transformation of HHN. The frequency of each clinical datum and imaging finding in each group was compared to identify the predictive factors for malignant transformation in HHN.

Results

Multivariate analysis showed that a nodule size of 9 mm or more on the initial gadoxetic acid-enhanced MRI was a significant predictive factor for the enlargement and/or vascularization of HHN (P < 0.05). On the other hand, the hypoattenuation on the delayed phase imaging of the initial DCE-CT was a significant predictive factor for the enlargement or vascularization of HHN (P < 0.05).

Conclusion

A nodule size of 9 mm or more on the initial gadoxetic acid-enhanced MRI and hypoattenuation on the delayed phase imaging of initial DCE-CT would be helpful for predicting the outcome of HHN in patients with a risk of hepatocellular carcinoma.     

The interpolated projection data estimation method improves the image quality of myocardial perfusion SPECT with a short acquisition time

Objective  

The interpolated projection data estimation processing (IPDE) method increases the amount of projection data by interpolation of the projection data. We examined the usefulness of the IPDE method for ²⁰¹Tl myocardial perfusion imaging (MPI) single photon emission computed tomography (SPECT) with a short acquisition time.     

Brain penetration of telmisartan,a unique centrally acting angiotensin II type 1 receptor blocker,studied by PET in conscious rhesus macaques

IntroductionTelmisartan is a widely used, long-acting antihypertensive agent. Known to be a selective angiotensin II type 1 (AT₁) receptor (AT₁R) blocker (ARB), telmisartan acts as a partial agonist of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and inhibits centrally mediated effects of angiotensin II in rats following peripheral administration, although the brain penetration of telmisartan remains unclear. We investigated the brain concentration and localization of telmisartan using ¹¹ C-labeled telmisartan and positron emission tomography (PET) in conscious rhesus macaques.MethodsThree male rhesus macaques were bolus intravenously administered [¹¹ C]telmisartan either alone or as a mixture with unlabeled telmisartan (1 mg/kg). Dynamic PET images were acquired for 95 min following administration. Blood samples were collected for the analysis of plasma concentration and metabolites, and brain and plasma concentrations were calculated from detected radioactivity using the specific activity of the administered drug preparation, in which whole blood radioactivity was used for the correction of intravascular blood radioactivity in brain.ResultsTelmisartan penetrated into the brain little but enough to block AT₁R and showed a consistently increasing brain/plasma ratio within the PET scanning period, suggesting slow clearance of the compound from the brain compared to the plasma clearance. Brain/plasma ratios at 30, 60, and 90 min were 0.06, 0.13, and 0.18, respectively. No marked localization according to the AT₁R distribution was noted over the entire brain, even on tracer alone dosing.ConclusionsTelmisartan penetrated into the brain enough to block AT₁R and showed a slow clearance from the brain in conscious rhesus macaques, supporting the long-acting and central responses of telmisartan as a unique property among ARBs.