The 3' UTR of vigR is required for virulence in Staphylococcus aureus and has expanded through STAR sequence repeat insertions.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteremia, and treatment failure is often associated with vancomycin-intermediate S. aureus isolates. The regulatory 3' UTR of the vigR mRNA contributes to vancomycin tolerance and upregulates the autolysin IsaA. Using MS2-affinity purification coupled with RNA sequencing, we find that the vigR 3' UTR also regulates dapE, a succinyl-diaminopimelate desuccinylase required for lysine and peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the 3' UTR increased virulence, while the isaA mutant is completely attenuated in a wax moth larvae model. Sequence and structural analyses of vigR indicated that the 3' UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions that contribute sequence for the isaA interaction seed and may functionalize the 3' UTR.

Diminished androgen levels are linked to irritable bowel syndrome and cause bowel dysfunction in mice.

Functional gastrointestinal disorders (FGIDs) have prominent sex differences in incidence, symptoms, and treatment response that are not well understood. Androgens are steroid hormones present at much higher levels in males than females and could be involved in these differences. In adults with irritable bowel syndrome (IBS), a FGID that affects 5% to 10% of the population worldwide, we found that free testosterone levels were lower than those in healthy controls and inversely correlated with symptom severity. To determine how this diminished androgen signaling could contribute to bowel dysfunction, we depleted gonadal androgens in adult mice and found that this caused a profound deficit in gastrointestinal transit. Restoring a single androgen hormone was sufficient to rescue this deficit, suggesting that circulating androgens are essential for normal bowel motility in vivo. To determine the site of action, we probed androgen receptor expression in the intestine and discovered, unexpectedly, that a large subset of enteric neurons became androgen-responsive upon puberty. Androgen signaling to these neurons was required for normal colonic motility in adult mice. Taken together, these observations establish a role for gonadal androgens in the neural regulation of bowel function and link altered androgen levels with a common digestive disorder.

Anoctamin 1 antagonism potentiates conventional tocolytic-mediated relaxation of pregnant human uterine smooth muscle.

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 ß2-adrenergic (ß2AR) 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 ß2AR 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.

Opsin 3-Gαs Promotes Airway Smooth Muscle Relaxation Modulated by G Protein Receptor Kinase 2.

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%, P < 0.05). We then demonstrated colocalization of OPN3 and Gαs using co-IP and proximity ligation assays in primary human ASM cells, which was further supported by an increase in cAMP in mouse trachea treated with blue light compared with dark controls (23 ± 3.6 vs. 14 ± 2.6 pmol cAMP/ring, P < 0.05). Downstream PKA (protein kinase A) involvement was shown by inhibiting photorelaxation using Rp-cAMPS (P < 0.0001). Moreover, we observed converging mechanisms of desensitization by chronic ß2-agonist exposure in mouse trachea and correlated this finding with colocalization of OPN3 and GRK2 (G protein receptor kinase) in primary human ASM cells. Finally, an overexpression model of OPN1LW (a red light photoreceptor in the same opsin family) in human ASM cells showed an increase in intracellular cAMP levels following red light exposure compared with nontransfected cells (48 ± 13 vs. 13 ± 2.1 pmol cAMP/mg protein, P < 0.01), suggesting a conserved photorelaxation mechanism for wavelengths of light that are more tissue penetrant. Together, these results demonstrate that blue light photorelaxation in ASM is mediated by the OPN3 receptor interacting with Gαs, which increases cAMP levels, activating PKA and modulated by GRK2.

Light-Mediated Inhibition of Colonic Smooth Muscle Constriction and Colonic Motility via Opsin 3.

Opsin photoreceptors outside of the central nervous system have been shown to mediate smooth muscle photorelaxation in several organs. We hypothesized that opsin receptor activation in the colon would have a similar effect and influence colonic motility. We detected Opsin 3 (OPN3) protein expression in the colonic wall and demonstrated that OPN3 was present in enteric neurons in the muscularis propria of the murine colon. Precontracted murine colon segments demonstrated blue light (BL) -mediated relaxation ex vivo. This photorelaxation was wavelength specific and was increased with the administration of the chromophore 9-cis retinal and a G protein receptor kinase 2 (GRK2) inhibitor. Light-mediated relaxation of the colon was not inhibited by L-NAME or tetrodotoxin (TTX). Furthermore, BL exposure in the presence of 9-cis retinal decreased the frequency of colonic migrating motor complexes (CMMC) in spontaneously contracting mouse colons ex vivo. These results demonstrate for the first time a receptor-mediated photorelaxation of colonic smooth muscle and implicate opsins as possible new targets in the treatment of spasmodic gastrointestinal dysmotility.

Correction to: Activation of an Endogenous Opsin 3 Light Receptor Mediates Photo-Relaxation of Pre-Contracting Late Gestation Human Uterine Smooth Muscle Ex Vivo.

This article was updated to correct Joy Y. Vink's name in the author listing.

Activation of an Endogenous Opsin 3 Light Receptor Mediates Photo-Relaxation of Pre-Contracting Late Gestation Human Uterine Smooth Muscle Ex Vivo.

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.

Novel Expression of GABAA Receptors on Resistance Arteries That Modulate Myogenic Tone.

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.