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FOXO1 stably knockout transfectants, including UMUC3 (KO FOXO1) (A) and T24T (KO FOXO1) (B), as well as Vector control transfectants were used for determination of their cell migration and invasion in presence of either vehicle or the indicated concentration of ISO treatment for 24 hours.

FOXO1 stably knockdown transfectants, including UMUC3 (shFOXO1-4), UMUC3 (shFOXO1-6) (A), T24T (shFOXO1-4) and T24T (shFOXO1-6) (B), as well as Nonsense transfectants were used for determination of their cell migration and invasion in presence of either vehicle or the indicated concentration of ISO treatment for 24 hours.

Although our most recent studies have identified Isorhapontigenin (ISO), a novel derivative of stilbene that isolated from a Chinese herb Gnetum cleistostachyum, for its inhibition of human bladder cancer growth, nothing is known whether ISO possesses an inhibitory effect on bladder cancer invasion. Thus, we addressed this important question in current study and discovered that ISO treatment could inhibit mouse-invasive bladder cancer development following bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) exposure in vivo. We also found that ISO suppressed human bladder cancer cell invasion accompanied by upregulation of the forkhead box class O 1 (FOXO1) mRNA transcription in vitro. Accordingly, FOXO1 was profoundly downregulated in human bladder cancer tissues and was negatively correlated with bladder cancer invasion. Forced expression of FOXO1 specifically suppressed high-grade human bladder cancer cell invasion, whereas knockdown of FOXO1 promoted noninvasive bladder cancer cells becoming invasive bladder cancer cells. Moreover, knockout of FOXO1 significantly increased bladder cancer cell invasion and abolished the ISO inhibition of invasion in human bladder cancer cells. Further studies showed that the inhibition of Signal transducer and activator of transcription 1 (STAT1) phosphorylation at Tyr701 was crucial for ISO upregulation of FOXO1 transcription. Furthermore, this study revealed that metalloproteinase-2 (MMP-2) was a FOXO1 downstream effector, which was also supported by data obtained from mouse model of ISO inhibition BBN-induced mouse-invasive bladder cancer formation. These findings not only provide a novel insight into the understanding of mechanism of bladder cancer's propensity to invasion, but also identify a new role and mechanisms underlying the natural compound ISO that specifically suppresses such bladder cancer invasion through targeting the STAT1–FOXO1–MMP-2 axis. Cancer Prev Res; 9(7); 567–80. ©2016 AACR.

The MMP-2 stable transfectant and its vector control transfectant were subjected to cell migration and invasion assay in presence of either vehicle or 20 μM of ISO for 24 hours.

Recently, we characterized blue light–mediated relaxation (photorelaxation) of airway smooth muscle (ASM) and implicated the involvement of opsin 3 (OPN3), an atypical opsin. In the present study, ...

Spontaneous preterm birth (sPTB) remains a worldwide healthcare challenge. Preterm labor (PTL) is thought to be the largest reversible cause of sPTB, but current tocolytic therapies are ineffective and associated with systemic side effects from chronic use. Therefore, identifying novel mechanisms that promote human uterine smooth muscle (hUSM) relaxation is essential to improving clinical management of PTL. Here, we aimed to determine if an extraocular opsin receptor (OPN 3,4,5) system is expressed in pregnant hUSM and to characterize how photo-mediated relaxation of pre-contracting hUSM may be facilitated by external application of light. Translational studies were performed with hUSM from healthy late gestation patients (n = 8) and non-pregnant, similarly aged patients undergoing hysterectomy (n = 4). First, RT-PCR screened for mRNA coding for components of the classical extraocular light receptors (OPN 3,4,5). We found a restricted repertoire of opsin receptors (OPN3) expressed in pregnant hUSM tissue. Immunohistochemistry was performed to confirm protein expression. Pre-contracting late gestation hUSM strips were studied in functional organ bath studies to determine if photo-mediated relaxation is intensity or wavelength dependent. Functional organ bath studies revealed acute photo-mediated relaxation occurring in an intensity- and wavelength-dependent manner. Finally, coimmunoprecipitation of OPN3 with Gs following light activation suggests that a component of photo-relaxation occurs via G protein-coupled receptor machinery. This is the first report of light-mediated relaxation of pre-contracted human myometrium. Activation of endogenous light receptors on human myometrium may become a novel, non-invasive tocolytic strategy.

The clinical administration of GABAergic medications leads to hypotension which has classically been attributed to the modulation of neuronal activity in the central and peripheral nervous systems. However, certain types of peripheral smooth muscle cells have been shown to express GABAA receptors, which modulate smooth muscle tone, by the activation of these chloride channels on smooth muscle cell plasma membranes. Limited prior studies demonstrate that non-human large-caliber capacitance blood vessels mounted on a wire myograph are responsive to GABAA ligands. We questioned whether GABAA receptors are expressed in human resistance arteries and whether they modulate myogenic tone. We demonstrate the novel expression of GABAA subunits on vascular smooth muscle from small-caliber human omental and mouse tail resistance arteries. We show that GABAA receptors modulate both plasma membrane potential and calcium responses in primary cultured cells from human resistance arteries. Lastly, we demonstrate functional physiologic modulation of myogenic tone via GABAA receptor activation in human and mouse arteries. Together, these studies demonstrate a previously unrecognized role for GABAA receptors in the modulation of myogenic tone in mouse and human resistance arteries.

Background Currently available tocolytic agents are not effective treatment for preterm labor beyond 48 h. A major reason is the development of maternal side effects which preclude the maintenance of an effective steady-state drug concentration. One strategy that can mitigate these side effects is utilizing synergistic drug combinations to reduce the drug concentrations necessary to elicit a clinical effect. We have previously shown that three anoctamin 1 (ANO1) antagonists mediate potent relaxation of precontracted human uterine smooth muscle (USM). In this study, we aimed to determine whether a combination of sub-relaxatory doses of tocolytic drugs in current clinical use [the L-type voltage-gated calcium channel (VGCC) blocker, nifedipine (NIF); and the β2-adrenergic (β2AR) agonist, terbutaline (TRB)] will potentiate USM relaxation with two ANO1 antagonists [benzbromarone (BB) and MONNA (MN)]. Objective This study sought to examine the synergistic potency and mechanistic basis of two ANO1 antagonists with currently available tocolytic drugs. Functional endpoints assessed included relaxation of pre-contracting pregnant human USM tissue, inhibition of intracellular calcium release, and reduction of spontaneous transient inward current (STIC) recordings in human uterine smooth muscle cells. Methods Human myometrial strips and primary human USM cells were used in organ bath and calcium flux experiments with different combinations of sub-threshold doses of ANO1 antagonists and terbutaline or nifedipine to determine if ANO1 antagonists potentiate tocolytic drugs. Results The combination of sub-threshold doses of two ANO1 antagonists and current tocolytic drugs demonstrate a significant degree of synergy to relax human pregnant USM compared to the effects achieved when these drugs are administered individually. Conclusion A combination of sub-threshold doses of VGCC blocker and β2AR agonist with ANO1 antagonists potentiates relaxation of oxytocin-induced contractility and calcium flux in human USM ex vivo. Our findings may serve as a foundation for novel tocolytic drug combinations.

Recently, we characterized blue light-mediated relaxation (photorelaxation) of airway smooth muscle (ASM) and implicated the involvement of opsin 3 (OPN3), an atypical opsin. In the present study, we characterized the cellular signaling mechanisms of photorelaxation. We confirmed the functional role of OPN3 in blue light photorelaxation using trachea from OPN3 null mice (maximal relaxation 52 ± 13% compared with wild-type mice 90 ± 4.3%

The clinical administration of GABAergic medications leads to hypotension which has classically been attributed to the modulation of neuronal activity in the central and peripheral nervous system. However, certain types of peripheral smooth muscle cells have been shown to express GABA(A) receptors, which modulate smooth muscle tone, by the activation of these chloride channels on smooth muscle cell plasma membrane. Limited prior studies demonstrate that non-human large caliber capacitance blood vessels mounted on a wire myograph are responsive to GABA(A) ligands. We questioned whether GABA(A) receptors are expressed in human resistance arteries and whether they modulate myogenic tone. We demonstrate the novel expression of GABA(A) subunits on vascular smooth muscle from small caliber human omental and mouse tail resistance arteries. We demonstrated that GABA(A) receptors modulate both plasma membrane potential and calcium responses in primary cultured cells from human resistance arteries. Lastly, we demonstrate functional physiologic modulation of myogenic tone via GABA(A) receptor activation in human and mouse arteries. Together these studies demonstrate a previously unrecognized role for GABA(A) receptors in the modulation of myogenic tone in mouse and human resistance arteries.

Although our most recent studies have identified Isorhapontigenin (ISO), a novel derivative of stilbene that isolated from a Chinese herb Gnetum cleistostachyum, for its inhibition of human bladder cancer growth, nothing is known whether ISO possesses an inhibitory effect on bladder cancer invasion. Thus, we addressed this important question in current study and discovered that ISO treatment could inhibit mouse-invasive bladder cancer development following bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) exposure in vivo. We also found that ISO suppressed human bladder cancer cell invasion accompanied by upregulation of the forkhead box class O 1 (FOXO1) mRNA transcription in vitro. Accordingly, FOXO1 was profoundly downregulated in human bladder cancer tissues and was negatively correlated with bladder cancer invasion. Forced expression of FOXO1 specifically suppressed high-grade human bladder cancer cell invasion, whereas knockdown of FOXO1 promoted noninvasive bladder cancer cells becoming invasive bladder cancer cells. Moreover, knockout of FOXO1 significantly increased bladder cancer cell invasion and abolished the ISO inhibition of invasion in human bladder cancer cells. Further studies showed that the inhibition of Signal transducer and activator of transcription 1 (STAT1) phosphorylation at Tyr701 was crucial for ISO upregulation of FOXO1 transcription. Furthermore, this study revealed that metalloproteinase-2 (MMP-2) was a FOXO1 downstream effector, which was also supported by data obtained from mouse model of ISO inhibition BBN-induced mouse-invasive bladder cancer formation. These findings not only provide a novel insight into the understanding of mechanism of bladder cancer's propensity to invasion, but also identify a new role and mechanisms underlying the natural compound ISO that specifically suppresses such bladder cancer invasion through targeting the STAT1–FOXO1–MMP-2 axis. Cancer Prev Res; 9(7); 567–80. ©2016 AACR.

Spontaneous preterm birth (sPTB) remains a worldwide healthcare challenge. Pre-term labor (PTL) is thought to be the largest reversible cause of sPTB, but current tocolytic therapies are ineffective and associated with systemic side effects from chronic use. Therefore, identifying novel mechanisms that promote human uterine smooth muscle (hUSM) relaxation is essential to improving clinical management of PTL. Here, we aimed to determine if an extraocular opsin receptor (OPN 3,4,5) system is expressed in pregnant hUSM and to characterize how photo-mediated relaxation of pre-contracting hUSM may be facilitated by external application of light. Translational studies were performed with hUSM from healthy late gestation patients (n=8) and non-pregnant, similarly-aged patients undergoing hysterectomy (n=4). First, RT-PCR screened for mRNA coding for components of the classical extraocular light receptors (OPN 3,4,5). We found a restricted repertoire of opsin receptors (OPN3) expressed in pregnant hUSM tissue. Immunohistochemistry was performed to confirm protein expression. Pre-contracting late gestation hUSM strips were studied in functional organ bath studies to determine if photo-mediated relaxation is intensity or wavelength dependent. Functional organ bath studies revealed acute photo-mediated relaxation occurring in an intensity and wavelength-dependent manner. Finally, coimmunoprecipitation of OPN3 with Gs following light activation suggests that a component of photo-relaxation occurs via GPCR machinery. This is the first report of light-mediated relaxation of pre-contracted human myometrium. Activation of endogenous light receptors on human myometrium may become a novel, non-invasive tocolytic strategy.

Nonvisual opsin (OPN) receptors have recently been implicated in blue light-mediated photorelaxation of smooth muscle in various organs. Since photorelaxation has not yet been demonstrated in airway smooth muscle (ASM) or in human tissues, we questioned whether functional OPN receptors are expressed in mouse and human ASM. mRNA, encoding the OPN 3 receptor, was detected in both human and mouse ASM. To demonstrate the functionality of the OPN receptors, we performed wire myography of ex vivo ASM from mouse and human upper airways. Blue light-mediated relaxation of ACh-preconstricted airways was intensity and wavelength dependent (maximum relaxation at 430-nm blue light) and was inhibited by blockade of the large-conductance calcium-activated potassium channels with iberiotoxin. We further implicated OPN receptors as key mediators in functional photorelaxation by demonstrating increased relaxation in the presence of a G protein receptor kinase 2 inhibitor or an OPN chromophore (9- cis retinal). We corroborated these responses in peripheral airways of murine precision-cut lung slices. This is the first demonstration of photorelaxation in ASM via an OPN receptor-mediated pathway.

The reliability of psychiatric diagnosis has a direct effect on the validity of post-mortem analyses of neuropathological data, yet little is known about the reliability of retrospective diagnostic procedures which rely on review of medical records. In this paper, we report on the reliability of DSM-III-R psychiatric diagnoses assigned by a pool of 8 raters to a set of 106 state hospital charts of elderly, chronic patients who had died while institutionalized and were autopsied. Diagnoses were grouped by general diagnostic class, and Kappa coefficients computed for agreement among raters, as well as for agreement between ultimate consensus diagnoses and those made while subjects were living. Interrater agreement for those diagnoses that occurred most frequently in this sample (e.g. Schizophrenia and Dementia) was excellent, and comparable to the the agreement observed for ratings of live patients. Interrater agreement for less frequently occurring diagnoses (e.g. Mental Retardation, Mood Disorders, other non-Schizophrenic Psychoses) ranged from excellent to poor. We found high agreement between our raters diagnoses and those assigned by state hospital personnel while patients were living, although post-mortem review produced lower rates of diagnosis of both schizophrenia and Alzheimer-type dementias. Overall, results suggest that the reliability of chart review diagnosis is comparable to that obtained from interviews of live patients when experienced raters are used and diagnostic base rates are high enough to produce stable estimates of reliability.