Change in microbiota profile after vaginal estriol cream in postmenopausal women with stress incontinence.

Introduction: Vaginal estrogen is a treatment for genitourinary symptoms of menopause (GSM), which comprises vaginal atrophy and urinary dysfunction, including incontinence. Previous studies show that estrogen therapy promotes lactobacilli abundance and is associated with reduced GSM symptoms, including reduction of stress incontinence. However, detailed longitudinal studies that characterize how the microbiome changes in response to estrogen are scarce. We aimed to compare the vaginal microbiota of postmenopausal women, before and 12 weeks after vaginal estrogen cream. Methods: A total of 44 paired samples from 22 postmenopausal women with vaginal atrophy and stress incontinence were collected pre-vaginal estrogens and were compared to 12 weeks post-vaginal estrogen. Microbiota was characterized by 16S rRNA amplicon sequencing and biodiversity was investigated by comparing the alpha- and beta-diversity and potential markers were identified using differential abundance analysis. Results: Vaginal estrogen treatment was associated with a reduction in vaginal pH and corresponded with a significant reduction in alpha diversity of the microbiota. Healthy vaginal community state type was associated with lower mean pH 4.89 (SD = 0.6), in contrast to dysbiotic state which had a higher mean pH 6.4 (SD = 0.74). Women with lactobacilli dominant community pre-treatment, showed stable microbiota and minimal change in their pH. Women with lactobacilli deficient microbiome pre-treatment improved markedly (p = 0.004) with decrease in pH -1.31 and change to heathier community state types. Conclusion: In postmenopausal women with stress incontinence, vaginal estrogen promotes Lactobacillus and Bifidobacterium growth and lowers vaginal pH. Maximum response is seen in those with a dysbiotic vaginal microbiota pre-treatment.

GPCR kinases differentially modulate biased signaling downstream of CXCR3 depending on their subcellular localization.

Some G protein-coupled receptors (GPCRs) demonstrate biased signaling such that ligands of the same receptor exclusively or preferentially activate certain downstream signaling pathways over others. This phenomenon may result from ligand-specific receptor phosphorylation by GPCR kinases (GRKs). GPCR signaling can also exhibit location bias because GPCRs traffic to and signal from subcellular compartments in addition to the plasma membrane. Here, we investigated whether GRKs contributed to location bias in GPCR signaling. GRKs translocated to endosomes after stimulation of the chemokine receptor CXCR3 or other GPCRs in cultured cells. GRK2, GRK3, GRK5, and GRK6 showed distinct patterns of recruitment to the plasma membrane and to endosomes depending on the identity of the biased ligand used to activate CXCR3. Analysis of engineered forms of GRKs that localized to either the plasma membrane or endosomes demonstrated that biased CXCR3 ligands elicited different signaling profiles that depended on the subcellular location of the GRK. Each GRK exerted a distinct effect on the regulation of CXCR3 engagement of ß-arrestin, internalization, and activation of the downstream effector kinase ERK. Our work highlights a role for GRKs in location-biased GPCR signaling and demonstrates the complex interactions between ligands, GRKs, and cellular location that contribute to biased signaling.

ACKR3 Proximity Labeling Identifies Novel G protein- and ß-arrestin-independent GPCR Interacting Proteins.

The canonical paradigm of GPCR signaling recognizes G proteins and ß-arrestins as the two primary transducers that promote GPCR signaling. Recent evidence suggests the atypical chemokine receptor 3 (ACKR3) does not couple to G proteins, and ß-arrestins are dispensable for some of its functions. Here, we employed proximity labeling to identify proteins that interact with ACKR3 in cells devoid of ß-arrestin. We identified proteins involved in the endocytic machinery and evaluated a subset of proteins conserved across several GPCR-based proximity labeling experiments. We discovered that the bone morphogenic protein 2-inducible kinase (BMP2K) interacts with many different GPCRs with varying dependency on ß-arrestin. Together, our work highlights the existence of modulators that can act independently of G proteins and ß-arrestins to regulate GPCR signaling and provides important evidence for other targets that may regulate GPCR signaling.

The MothersBabies Study, an Australian Prospective Cohort Study Analyzing the Microbiome in the Preconception and Perinatal Period to Determine Risk of Adverse Pregnancy, Postpartum, and Child-Related Health Outcomes: Study Protocol.

The microbiome has emerged as a key determinant of human health and reproduction, with recent evidence suggesting a dysbiotic microbiome is implicated in adverse perinatal health outcomes. The existing research has been limited by the sample collection and timing, cohort design, sample design, and lack of data on the preconception microbiome. This prospective, longitudinal cohort study will recruit 2000 Australian women, in order to fully explore the role of the microbiome in the development of adverse perinatal outcomes. Participants are enrolled for a maximum of 7 years, from 1 year preconception, through to 5 years postpartum. Assessment occurs every three months until pregnancy occurs, then during Trimester 1 (5 + 0-12 + 6 weeks gestation), Trimester 2 (20 + 0-24 + 6 weeks gestation), Trimester 3 (32 + 0-36 + 6 weeks gestation), and postpartum at 1 week, 2 months, 6 months, and then annually from 1 to 5 years. At each assessment, maternal participants self-collect oral, skin, vaginal, urine, and stool samples. Oral, skin, urine, and stool samples will be collected from children. Blood samples will be obtained from maternal participants who can access a study collection center. The measurements taken will include anthropometric, blood pressure, heart rate, and serum hormonal and metabolic parameters. Validated self-report questionnaires will be administered to assess diet, physical activity, mental health, and child developmental milestones. Medications, medical, surgical, obstetric history, the impact of COVID-19, living environments, and pregnancy and child health outcomes will be recorded. Multiomic bioinformatic and statistical analyses will assess the association between participants who developed high-risk and low-risk pregnancies, adverse postnatal conditions, and/or childhood disease, and their microbiome for the different sample types.

Location bias: A "Hidden Variable" in GPCR pharmacology.

G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and primarily signal through two main effector proteins: G proteins and ß-arrestins. Many agonists of GPCRs promote "biased" responses, in which different cellular signaling pathways are activated with varying efficacies. The mechanisms underlying biased signaling have not been fully elucidated, with many potential "hidden variables" that regulate this behavior. One contributor is "location bias," which refers to the generation of unique signaling cascades from a given GPCR depending upon the cellular location at which the receptor is signaling. Here, we review evidence that GPCRs are expressed at and traffic to various subcellular locations and discuss how location bias can impact the pharmacologic properties and characterization of GPCR agonists. We also evaluate how differences in subcellular environments can modulate GPCR signaling, highlight the physiological significance of subcellular GPCR signaling, and discuss the therapeutic potential of exploiting GPCR location bias.

Phosphorylation barcodes direct biased chemokine signaling at CXCR3.

G protein-coupled receptor (GPCR)-biased agonism, selective activation of certain signaling pathways relative to others, is thought to be directed by differential GPCR phosphorylation "barcodes." At chemokine receptors, endogenous chemokines can act as "biased agonists", which may contribute to the limited success when pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomics studies. Mutation of CXCR3 phosphosites altered ß-arrestin 2 conformation in cellular assays and was consistent with conformational changes observed in molecular dynamics simulations. T cells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes, leading to distinct physiological processes.

Bacterial extracellular vesicles and their novel therapeutic applications in health and cancer.

Bacterial cells communicate with host cells and other bacteria through the release of membrane vesicles known as bacterial extracellular vesicles (BEV). BEV are established mediators of intracellular signaling, stress tolerance, horizontal gene transfer, immune stimulation and pathogenicity. Both Gram-positive and Gram-negative bacteria produce extracellular vesicles through different mechanisms based on cell structure. BEV contain and transfer different types of cargo such as nucleic acids, proteins and lipids, which are used to interact with and affect host cells such as cytotoxicity and immunomodulation. The role of these membranous microvesicles in host communication, intra- and inter-species cell interaction and signaling, and contribution to various diseases have been well demonstrated. Due to their structure, these vesicles can be easily engineered to be utilized for clinical application, as shown with its role in vaccine therapy, and could be used as a diagnostic and cancer drug delivery tool in the future. However, like other novel therapeutic approaches, further investigation and standardization is imperative for BEV to become a routine vector or a conventional treatment method.

Location bias contributes to functionally selective responses of biased CXCR3 agonists.

Some G protein-coupled receptor (GPCR) ligands act as "biased agonists" that preferentially activate specific signaling transducers over others. Although GPCRs are primarily found at the plasma membrane, GPCRs can traffic to and signal from many subcellular compartments. Here, we determine that differential subcellular signaling contributes to the biased signaling generated by three endogenous ligands of the GPCR CXC chemokine receptor 3 (CXCR3). The signaling profile of CXCR3 changes as it traffics from the plasma membrane to endosomes in a ligand-specific manner. Endosomal signaling is critical for biased activation of G proteins, ß-arrestins, and extracellular-signal-regulated kinase (ERK). In CD8 + T cells, the chemokines promote unique transcriptional responses predicted to regulate inflammatory pathways. In a mouse model of contact hypersensitivity, ß-arrestin-biased CXCR3-mediated inflammation is dependent on receptor internalization. Our work demonstrates that differential subcellular signaling is critical to the overall biased response observed at CXCR3, which has important implications for drugs targeting chemokine receptors and other GPCRs.

Fecal DNA Virome Is Associated with the Development of Colorectal Neoplasia in a Murine Model of Colorectal Cancer.

Alteration of the gut virome has been associated with colorectal cancer (CRC); however, when and how the alteration takes place has not been studied. Here, we employ a longitudinal study in mice to characterize the gut virome alteration in azoxymethane (AOM)-induced colorectal neoplasia and identify important viruses associated with tumor growth. The number and size of the tumors increased as the mice aged in the AOM treated group, as compared to the control group. Tumors were first observed in the AOM group at week 12. We observed a significantly lower alpha diversity and shift in viral profile when tumors first appeared. In addition, we identified novel viruses from the genera Brunovirus, Hpunavirus that are positively associated with tumor growth and enriched at a late time point in AOM group, whereas members from Lubbockvirus show a negative correlation with tumor growth. Moreover, network analysis revealed two clusters of viruses in the AOM virome, a group that is positively correlated with tumor growth and another that is negatively correlated with tumor growth, all of which are bacteriophages. Our findings suggest that the gut virome changes along with tumor formation and provides strong evidence of a potential role for bacteriophage in the development of colorectal neoplasia.

GPCR systems pharmacology: a different perspective on the development of biased therapeutics.

G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and are the target of approximately one-third of all Food and Drug Administration (FDA)-approved pharmaceutical drugs. GPCRs interact with many transducers, such as heterotrimeric G proteins, GPCR kinases (GRKs), and ß-arrestins. Recent experiments have demonstrated that some ligands can activate distinct effector proteins over others, a phenomenon termed "biased agonism." These discoveries have raised the potential of developing drugs which preferentially activate therapeutic signaling pathways over those that lead to deleterious side effects. However, to date, only one biased GPCR therapeutic has received FDA approval and many others have either failed to meet their specified primary end points and or demonstrate superiority over currently available treatments. In addition, there is a lack of understanding regarding how biased agonism measured at a GPCR leads to specific downstream physiological responses. Here, we briefly summarize the history and current status of biased agonism at GPCRs and suggest adoption of a "systems pharmacology" approach upon which to develop GPCR-targeted drugs that demonstrate heightened therapeutic efficacy with improved side effect profiles.

Implementation of a Surgical Intensivist Model Increases Palliative Care Consultation in the Care of Trauma ICU Patients.

OBJECTIVE: This study investigated patient outcomes of care before and after transitioning to a surgical intensivist-led trauma-intensive care unit (ICU) team. The intensivist team provided daily multidisciplinary rounds and continuity of care. Prior to an intensivist model, general surgeons cared for trauma patients admitted to the unit. METHODS: Outcomes of 1,078 trauma patients, admitted to the ICU at a Level II trauma center, under care of general surgeons (1/2011-8/2012, n = 449) were retrospectively compared with care managed by a surgical intensivist team (1/2013-5/2015, n = 629) by Pearson Chi-squared and Wilcoxon tests. A multivariable logistic regression technique was used to control for covariates. Demographics and injury severity were analyzed. The primary outcome was ICU mortality. The secondary outcomes were length of stay (LOS), ventilator-free and ICU-free days, and ICU readmission rate. Other data collected included palliative care consultation. Results: There were no statistically significant differences in ICU mortality (P = 0.055), hospital LOS (P = 0.481), ventilator-free days (P = 0.174), or ICU readmission rate (P = 0.587). The surgical intensivist team consulted palliative care more frequently (4.0% vs 13.5%, P < 0.001), while managing more trauma patients who were older than 65 years (P < 0.001) with lower Glasgow Coma Scale (P = 0.048) and higher injury severity (P = 0.025) and abbreviated injury scale (P < 0.001) scores. DISCUSSION: There were no differences in outcomes. However, incorporating palliative care consultation in the ICU is essential in the support of critically ill patients and their families. These data demonstrate that a surgical intensivist team utilized palliative care more often in the management of trauma patients admitted to the ICU.

Fractalkine is a "find-me" signal released by neurons undergoing ethanol-induced apoptosis.

Apoptotic neurons generated during normal brain development or secondary to pathologic insults are efficiently cleared from the central nervous system. Several soluble factors, including nucleotides, cytokines, and chemokines are released from injured neurons, signaling microglia to find and clear debris. One such chemokine that serves as a neuronal-microglial communication factor is fractalkine, with roles demonstrated in several models of adult neurological disorders. Lacking, however, are studies investigating roles for fractalkine in perinatal brain injury, an important clinical problem with no effective therapies. We used a well-characterized mouse model of ethanol-induced apoptosis to assess the role of fractalkine in neuronal-microglial signaling. Quantification of apoptotic debris in fractalkine-knockout (KO) and CX3CR1-KO mice following ethanol treatment revealed increased apoptotic bodies compared to wild type mice. Ethanol-induced injury led to release of soluble, extracellular fractalkine. The extracellular media harvested from apoptotic brains induces microglial migration in a fractalkine-dependent manner that is prevented by neutralization of fractalkine with a blocking antibody or by deficiency in the receptor, CX3CR1. This suggests fractalkine acts as a "find-me" signal, recruiting microglial processes toward apoptotic cells to promote their clearance. Next, we aimed to determine whether there are downstream alterations in cytokine gene expression due to fractalkine signaling. We examined mRNA expression in fractalkine-KO and CX3CR1-KO mice after alcohol-induced apoptosis and found differences in cytokine production in the brains of these KOs by 6 h after ethanol treatment. Collectively, this suggests that fractalkine acts as a "find me" signal released by apoptotic neurons, and subsequently plays a critical role in modulating both clearance and inflammatory cytokine gene expression after ethanol-induced apoptosis.

The effect of ratio and interval training on Pavlovian-instrumental transfer in mice.

Conditional stimuli (CS) that are paired with reward can be used to motivate instrumental responses. This process is called Pavlovian-instrumental transfer (PIT). A recent study in rats suggested that habitual responses are particularly sensitive to the motivational effects of reward cues. The current experiments examined this idea using ratio and interval training in mice. Two groups of animals were trained to lever press for food pellets that were delivered on random ratio or random interval schedules. Devaluation tests revealed that interval training led to habitual responding while ratio training produced goal-directed actions. The presentation of CSs paired with reward led to positive transfer in both groups, however, the size of this effect was much larger in mice that were trained on interval schedules. This result suggests that habitual responses are more sensitive to the motivational influence of reward cues than goal-directed actions. The implications for neurobiological models of motivation and drug seeking behaviors are discussed.