GPER expression prevents estrogen-induced urinary retention in obese mice.

Long-term administration of exogenous estrogen is known to cause urinary retention and marked, often fatal, bladder distention in both male and female mice. Estrogen-treated mice have increased bladder pressure and decreased urine flow, suggesting that urinary retention in estrogen-treated mice is due to infravesicular obstruction to urine outflow. Thus, the condition is commonly referred to as bladder outlet obstruction (BOO). Obesity can also lead to urinary retention. As the effects of estrogen are mediated by multiple receptors, including estrogen receptors ERα and ERß and the G protein-coupled estrogen receptor (GPER), we sought to determine whether GPER plays a role in estrogen-induced BOO, particularly in the context of obesity. Wild type and GPER knockout (KO) mice fed a high-fat diet were ovariectomized or left ovary-intact (sham surgery) and supplemented with slow-release estrogen or vehicle-only pellets. Supplementing both GPER KO and wild type obese mice with estrogen for 8 weeks resulted in weight loss, splenic enlargement, and thymic atrophy, as expected. However, estrogen-treated obese GPER KO mice developed abdominal distension, debilitation, and ulceration of the skin surrounding the urogenital opening. At necropsy, these mice had prominently distended bladders and hydronephrosis. In contrast, estrogen-treated obese wild type mice only rarely displayed these signs. Our results suggest that, under conditions of obesity, estrogen induces BOO as a result of ERα-driven pathways and that GPER expression is protective against BOO.

GPER deficiency impedes murine myocutaneous revascularization and wound healing.

Estrogens regulate numerous physiological and pathological processes, including wide-ranging effects in wound healing. The effects of estrogens are mediated through multiple estrogen receptors (ERs), including the classical nuclear ERs (ERα and ER ß ), that typically regulate gene expression, and the 7-transmembrane G protein-coupled estrogen receptor (GPER), that predominantly mediates rapid "non-genomic" signaling. Estrogen modulates the expression of various genes involved in epidermal function and regeneration, inflammation, matrix production, and protease inhibition, all critical to wound healing. Our previous work demonstrated improved myocutaneous wound healing in female mice compared to male mice. In the current study, we employed male and female GPER knockout mice to investigate the role of this estrogen receptor in wound revascularization and tissue viability. Using a murine myocutaneous flap model of graded ischemia, we measured real-time flap perfusion via laser speckle perfusion imaging. We conducted histologic and immunohistochemical analyses to assess skin and muscle viability, microvascular density and vessel morphology. Our results demonstrate that GPER is crucial in wound healing, mediating effects that are both dependent and independent of sex. Lack of GPER expression is associated with increased skin necrosis, reduced flap perfusion and altered vessel morphology. These findings contribute to understanding GPER signaling in wound healing and suggest possible therapeutic opportunities by targeting GPER.

Seroprevalence of hepatitis A and hepatitis E in patients at a teaching hospital of northern India over a period of 8 years.

Background: Considering the diverse socio-economic and demographic factors in a vast country like India, it is important to study the long-term trends of hepatitis A (HAV) and hepatitis E (HEV) viruses. This study describes their seroprevalence and long-term trends in a tertiary care center of North India. Methods: The present retrospective observational study was conducted over a period of 8 years (January 2011-December 2018). Serological testing was done for detecting IgM antibodies against HAV and HEV using enzyme-linked immunosorbent assay. Results: A total of 5319 samples were received during the study period, of which 903 (16.9%) were reactive for anti-HAV IgM antibodies and 795 (14.9%) were reactive for anti-HEV IgM antibodies. Majority of the cases occurred from June to October while HEV cases had a constant presence during the later years. Among HAV group, 534 (59.1%) were children, 336 (37.2%) were adults, and 33 (3.7%) were pregnant females. In HEV group, 632 (79.5%) were adults, 114 (14.3%) were pregnant females, whereas only 49 (6.2%) were children. Among those who were co-infected (n = 87), 48 (55.2%) were adults, 22 (25.3%) were pregnant females, and the rest 17 (19.5%) were children. Conclusions: The shift in seroprevalence toward adults, along with an increasing trend of the number of cases reporting to the hospital, warrants active surveillance of HAV. Similarly, screening protocols for HEV should be set up as part of the antenatal management for early detection of the cases among pregnant females.

Assessment of Metabolic Regulation by Estrogen Receptors.

Estrogens, predominantly 17ß-estradiol (E2), are a class of steroid hormones critical for diverse functions in the body both during normal physiology and disease. Primary actions of E2 include reproduction and development of secondary sexual characteristics. In addition, E2 action is involved in the nervous, immune, vascular, muscular, skeletal, and endocrine systems, all of which contribute to multiple aspects of metabolism. The actions of E2 have traditionally been attributed to the classical nuclear estrogen receptors (ERα and ERß) that largely mediate transcriptional/genomic activities. However, over the last decade, the G protein-coupled estrogen receptor (GPER/GPR30) has become recognized as a mediator of rapid as well as transcriptional actions of E2, employing both in vitro and in vivo approaches. Recent evidence strongly supports the role of GPER in metabolic regulation. Murine genetic knockout (KO) models and pharmacological tools (agonists and antagonists) represent important approaches to understand the mechanisms of E2 action in physiology and disease via GPER. Studies in cells and GPER KO mice have revealed functions for GPER in the regulation of body weight and metabolism. This chapter focuses on methods relevant for the evaluation of metabolic parameters in vivo, ex vivo, and in vitro. We have emphasized glucose homeostasis through the determination of glucose and insulin tolerance, pancreatic islet function, and glucose uptake. In addition, we describe methods of adipocyte isolation, differentiation of preadipocytes, and evaluation of mitochondrial function.

Enhanced Field-Based Detection of Potato Blight in Complex Backgrounds Using Deep Learning.

Rapid and automated identification of blight disease in potato will help farmers to apply timely remedies to protect their produce. Manual detection of blight disease can be cumbersome and may require trained experts. To overcome these issues, we present an automated system using the Mask Region-based convolutional neural network (Mask R-CNN) architecture, with residual network as the backbone network for detecting blight disease patches on potato leaves in field conditions. The approach uses transfer learning, which can generate good results even with small datasets. The model was trained on a dataset of 1423 images of potato leaves obtained from fields in different geographical locations and at different times of the day. The images were manually annotated to create over 6200 labeled patches covering diseased and healthy portions of the leaf. The Mask R-CNN model was able to correctly differentiate between the diseased patch on the potato leaf and the similar-looking background soil patches, which can confound the outcome of binary classification. To improve the detection performance, the original RGB dataset was then converted to HSL, HSV, LAB, XYZ, and YCrCb color spaces. A separate model was created for each color space and tested on 417 field-based test images. This yielded 81.4% mean average precision on the LAB model and 56.9% mean average recall on the HSL model, slightly outperforming the original RGB color space model. Manual analysis of the detection performance indicates an overall precision of 98% on leaf images in a field environment containing complex backgrounds.

G Protein-Coupled Estrogen Receptor in Cancer and Stromal Cells: Functions and Novel Therapeutic Perspectives.

Estrogen is involved in numerous physiological and pathophysiological systems. Its role in driving estrogen receptor-expressing breast cancers is well established, but it also has important roles in a number of other cancers, acting both on tumor cells directly as well as in the function of multiple cells of the tumor microenvironment, including fibroblasts, immune cells, and adipocytes, which can greatly impact carcinogenesis. One of its receptors, the G protein-coupled estrogen receptor (GPER), has gained much interest over the last decade in both health and disease. Increasing evidence shows that GPER contributes to clinically observed endocrine therapy resistance in breast cancer while also playing a complex role in a number of other cancers. Recent discoveries regarding the targeting of GPER in combination with immune checkpoint inhibition, particularly in melanoma, have led to the initiation of the first Phase I clinical trial for the GPER-selective agonist G-1. Furthermore, its functions in metabolism and corresponding pathophysiological states, such as obesity and diabetes, are becoming more evident and suggest additional therapeutic value in targeting GPER for both cancer and other diseases. Here, we highlight the roles of GPER in several cancers, as well as in metabolism and immune regulation, and discuss the therapeutic value of targeting this estrogen receptor as a potential treatment for cancer as well as contributing metabolic and inflammatory diseases and conditions.

Targeted Phage Display-based Pulmonary Vaccination in Mice and Non-human Primates.

BACKGROUND: The extensive alveolar capillary network of the lungs is an attractive route for administration of several agents. One key functional attribute is the rapid onset of systemic action due to the absence of first-pass metabolism. METHODS: Here we applied a combinatorial approach for ligand-directed pulmonary delivery as a unique route for systemic targeting in vaccination. FINDINGS: We screened a phage display random peptide library in vivo to select, identify, and validate a ligand (CAKSMGDIVC) that specifically targets and is internalized through its receptor, α3ß1 integrin, on the surface of cells lining the lung airways and alveoli and mediates CAKSMGDIVC-displaying phage binding and systemic delivery without compromising lung homeostasis. As a proof-of-concept, we show that the pulmonary delivery of targeted CAKSMGDIVC-displaying phage particles in mice and non-human primates elicit a systemic and specific humoral response. CONCLUSIONS: This broad methodology blueprint represents a robust and versatile platform tool enabling new ligand-receptor discovery with many potential translational applications. FUNDING: Cancer Center Support Grants to the University of Texas M.D. Anderson Cancer Center (CA016672), University of New Mexico Comprehensive Cancer Center (CA118100), Rutgers Cancer Institute of New Jersey (CA072720), research awards from the Gillson Longenbaugh Foundation, and National Institutes of Health (NIH) grant no. 1R01CA226537.

Targeting the G protein-coupled estrogen receptor (GPER) in obesity and diabetes.

Obesity has become a global epidemic in the modern world with the numbers of obese individuals having risen at alarming rates in the last decades. Obesity represents a serious medical condition that can lead to multiple complications, such as diabetes, dyslipidemia, cardiovascular disease including hypertension and atherosclerosis, stroke and increases in the risk of many types of cancer. Very few effective options exist to treat obesity, with many removed from the market due to associated complications. Obesity and metabolic syndrome display a sexual dichotomy, with (premenopausal) females displaying protection from weight gain and metabolic dysfunction compared to men. These beneficial effects are generally attributed to a class of female ovarian hormone, estrogens, which exert pleiotropic effects in multiple metabolic tissues, such as adipose, skeletal muscle, liver and pancreas. Multiple receptors mediate the actions of estrogens, including the classical nuclear estrogen receptors (ER α and ER ß) and the G protein-coupled estrogen receptor (GPER). While the roles of nuclear ERs are more established, evidence of GPER function in metabolic homeostasis is still emerging. In this review, we will discuss the latest advances concerning the contributions of GPER towards obesity and metabolism utilizing GPER-selective pharmacological (agonists or antagonists) or genetic (GPER knock out mice or cells) tools. We present evidence that GPER regulates body weight, fat distribution, inflammation and glucose and lipid homeostasis via effects on metabolic tissues. Selective agonism of GPER by its agonist G-1 can alleviate symptoms of obesity and metabolic dysfunction in multiple murine models, thereby limiting weight gain, reducing insulin resistance and inflammation and improving glucose and lipid homeostasis in vivo. Thus, GPER represents a novel therapeutic target, with G-1 a first-in-class therapeutic agent, to treat obesity and its associated comorbidities, including diabetes.

Complement Receptor 3 Contributes to the Sexual Dimorphism in Neutrophil Killing of Staphylococcus aureus.

We previously reported sex differences in innate susceptibility to Staphylococcus aureus skin infection and that bone marrow neutrophils (BMN) from female mice have an enhanced ability to kill S. aureus ex vivo compared with those of male mice. However, the mechanism(s) driving this sex bias in neutrophil killing have not been reported. Given the role of opsonins such as complement, as well as their receptors, in S. aureus recognition and clearance, we investigated their contribution to the enhanced bactericidal capacity of female BMN. We found that levels of C3 in the serum and CR3 (CD11b/CD18) on the surface of BMN were higher in female compared with male mice. Consistent with increased CR3 expression following TNF-α priming, production of reactive oxygen species (ROS), an important bactericidal effector, was also increased in female versus male BMN in response to serum-opsonized S. aureus Furthermore, blocking CD11b reduced both ROS levels and S. aureus killing by murine BMN from both sexes. However, at the same concentration of CD11b blocking Ab, S. aureus killing by female BMN was greatly reduced compared with those from male mice, suggesting CR3-dependent differences in bacterial killing between sexes. Overall, this work highlights the contributions of CR3, C3, and ROS to innate sex bias in the neutrophil response to S. aureus Given that neutrophils are crucial for S. aureus clearance, understanding the mechanism(s) driving the innate sex bias in neutrophil bactericidal capacity could identify novel host factors important for host defense against S. aureus.

Sex differences in murine myocutaneous flap revascularization.

Sex differences in susceptibility to ischemia/reperfusion injury have been documented in humans. Premenopausal women have a lower risk of ischemic heart disease than age-matched men, whereas after menopause, the risk is similar or even higher in women. However, little is known about the effects of sex on myocutaneous ischemia/reperfusion. To explore sex differences in wound revascularization, we utilized a murine myocutaneous flap model of graded ischemia. A cranial-based, peninsular-shaped, myocutaneous flap was surgically created on the dorsum of male and female mice. Physiological, pathological, immunohistochemical, and molecular parameters were analyzed. Flaps created on female mice were re-attached to the recipient site resulting in nearly complete viability at post-operative day 10. In contrast, distal full-thickness myocutaneous necrosis was evident at 10 days post-surgery in male mice. Over the 10 day study interval, laser speckle imaging documented functional revascularization in all flap regions in female mice, but minimal distal flap reperfusion in male mice. Day 10 immunostained histologic sections confirmed significant increases in distal flap vessel count and vascular surface area in female compared to male mice. RT-PCR demonstrated significant differences in growth factor and metabolic gene expression between female and male mice at day 10. In conclusion, in a graded-ischemia wound healing model, flap revascularization was more effective in female mice. The recognition and identification of sex-specific wound healing differences may lead to a better understanding of the underlying mechanisms of myocutaneous revascularization and drive novel discovery to improve soft tissue wound healing following tissue transfer for traumatic injury and cancer resection.

Management of progressive facial asymmetry owing to a rare temporomandibular joint tumour: A case report.

Osteochondroma of the condyle is a rare, slow-growing, benign tumour of the temporomandibular joint that can result in facial asymmetry, limited mouth opening, temporomandibular joint dysfunction and malocclusion. The large majority of osteochondromas occur at the distal metaphysis of the femur and the proximal metaphysis of the tibia, whereas only 0.6% of osteochondromas have been reported as occurring in the craniofacial region. We discuss the diagnosis and treatment of a 56-year-old fit and well male patient who presented to the Orthodontic Department at Wexham Park Hospital with a four-year history of progressive facial asymmetry and functional concerns, owing to a rare osteochondroma of the condyle.

Preclinical efficacy of the GPER-selective agonist G-1 in mouse models of obesity and diabetes.

Human obesity has become a global health epidemic, with few safe and effective pharmacological therapies currently available. The systemic loss of ovarian estradiol (E2) in women after menopause greatly increases the risk of obesity and metabolic dysfunction, revealing the critical role of E2 in this setting. The salutary effects of E2 are traditionally attributed to the classical estrogen receptors ERα and ERß, with the contribution of the G protein-coupled estrogen receptor (GPER) still largely unknown. Here, we used ovariectomy- and diet-induced obesity (DIO) mouse models to evaluate the preclinical activity of GPER-selective small-molecule agonist G-1 (also called Tespria) against obesity and metabolic dysfunction. G-1 treatment of ovariectomized female mice (a model of postmenopausal obesity) reduced body weight and improved glucose homeostasis without changes in food intake, fuel source usage, or locomotor activity. G-1-treated female mice also exhibited increased energy expenditure, lower body fat content, and reduced fasting cholesterol, glucose, insulin, and inflammatory markers but did not display feminizing effects on the uterus (imbibition) or beneficial effects on bone health. G-1 treatment of DIO male mice did not elicit weight loss but prevented further weight gain and improved glucose tolerance, indicating that G-1 improved glucose homeostasis independently of its antiobesity effects. However, in ovariectomized DIO female mice, G-1 continued to elicit weight loss, reflecting possible sex differences in the mechanisms of G-1 action. In conclusion, this work demonstrates that GPER-selective agonism is a viable therapeutic approach against obesity, diabetes, and associated metabolic abnormalities in multiple preclinical male and female models.

A Selective Ligand for Estrogen Receptor Proteins Discriminates Rapid and Genomic Signaling.

Estrogen exerts extensive and diverse effects throughout the body of women. In addition to the classical nuclear estrogen receptors (ERα and ERß), the G protein-coupled estrogen receptor GPER is an important mediator of estrogen action. Existing ER-targeted therapeutic agents act as GPER agonists. Here, we report the identification of a small molecule, named AB-1, with the previously unidentified activity of high selectivity for binding classical ERs over GPER. AB-1 also possesses a unique functional activity profile as an agonist of transcriptional activity but an antagonist of rapid signaling through ERα. Our results define a class of small molecules that discriminate between the classical ERs and GPER, as well as between modes of signaling within the classical ERs. Such an activity profile, if developed into an ER antagonist, could represent an opportunity for the development of first-in-class nuclear hormone receptor-targeted therapeutics for breast cancer exhibiting reduced acquired and de novo resistance.

Challenges in the management of late developing malocclusions in adulthood: A case report of acromegaly.

The development of a malocclusion in adulthood can present as a diagnostic and management challenge to an orthodontist. It is prudent to identify the aetiology of changes to the occlusion which will influence the management plan. Uncommon causes include acromegaly. Orthodontists are in a good position to identify certain underlying disorders based on a patient's presenting malocclusion that may otherwise go unnoticed and undiagnosed until other systemic signs and symptoms present themselves at the latter stages of the condition. This case report highlights possible aetiological factors of a developing malocclusion in adulthood and presents the clinical manifestations and joint orthodontic-surgical management of a patient with a developing skeletal III base attributed to acromegaly.

GPER activation protects against epithelial barrier disruption by Staphylococcus aureus α-toxin.

Sex bias in innate defense against Staphylococcus aureus skin and soft tissue infection (SSTI) is dependent on both estrogen production by the host and S. aureus secretion of the virulence factor, α-hemolysin (Hla). The impact of estrogen signaling on the immune system is most often studied in terms of the nuclear estrogen receptors ERα and ERß. However, the potential contribution of the G protein-coupled estrogen receptor (GPER) to innate defense against infectious disease, particularly with respect to skin infection, has not been addressed. Using a murine model of SSTI, we found that GPER activation with the highly selective agonist G-1 limits S. aureus SSTI and Hla-mediated pathogenesis, effects that were absent in GPER knockout mice. Specifically, G-1 reduced Hla-mediated skin lesion formation and pro-inflammatory cytokine production, while increasing bacterial clearance. In vitro, G-1 reduced surface expression of the Hla receptor, ADAM10, in a human keratinocyte cell line and increased resistance to Hla-mediated permeability barrier disruption. This novel role for GPER activation in skin innate defense against infectious disease suggests that G-1 may have clinical utility in patients with epithelial permeability barrier dysfunction or who are otherwise at increased risk of S. aureus infection, including those with atopic dermatitis or cancer.

Mandibular premolar migration: two case reports.

Premolar migration is an infrequent and unusual phenomenon. Although several cases of distal premolar migration have been reported in the literature, the management of these cases is often challenging and to date, there are no agreed guidelines regarding best practice. This report describes two cases of distal migration of a lower second permanent premolar with sequential panoramic imaging mapping the progress of the teeth as they migrated distally. Cone Beam Computerised Tomography was subsequently used in one case to locate the migrated premolars' precise position. We report on one case which was managed with the surgical removal of the migrated premolar and one case which has been managed conservatively to date, largely due to patient wishes. Alternative treatment strategies are also discussed in the following case report.

Effect of immediate periodontal surgical treatment on periodontal healing in combined endodontic-periodontal lesions with communication-A randomized clinical trial.

BACKGROUND AND OBJECTIVE: Management of combined endodontic-periodontal lesions needs more clinical investigations. The aim of this prospective randomized clinical trial was to evaluate the effect oftime interval between the non-surgical endodontic treatment (ET) and open flap debridement (OFD) on periodontal healing in combined endodontic periodontal lesions with apical communication. METHODS: Forty patients were randomly allocated to two treatment protocols. Group 1(immediate periodontal surgery): OFD was performed at 21 days of initiation of ET and SRP, and Group 2(delayed periodontal surgery): OFD was performed after 3 months of initiation of ET and SRP. The primary parameters included probing pocket depth (PPD), relative attachment level (RAL) and bleeding on probing (BOP) and tooth mobility (TM). RESULTS: Significantly more reduction in PPD, TM and gain in RAL was observed in Group 1 at 3 months of OFD. (P < 0.05) Whereas at 6 months follow up of OFD (6 and 9 months of ET in Group1 and Group 2, respectively), intergroup analysis showed statistically comparable reduction in BOP (%), PPD, TM and gain in RAL (P > 0.05) in both the groups. CONCLUSION: Immediate periodontal surgery may not affect the outcome of the treatment of combined endo-perio lesions with apical communication.

Selection of phage-displayed accessible recombinant targeted antibodies (SPARTA): methodology and applications.

We developed a potentially novel and robust antibody discovery methodology, termed selection of phage-displayed accessible recombinant targeted antibodies (SPARTA). This combines an in vitro screening step of a naive human antibody library against known tumor targets, with in vivo selections based on tumor-homing capabilities of a preenriched antibody pool. This unique approach overcomes several rate-limiting challenges to generate human antibodies amenable to rapid translation into medical applications. As a proof of concept, we evaluated SPARTA on 2 well-established tumor cell surface targets, EphA5 and GRP78. We evaluated antibodies that showed tumor-targeting selectivity as a representative panel of antibody-drug conjugates (ADCs) and were highly efficacious. Our results validate a discovery platform to identify and validate monoclonal antibodies with favorable tumor-targeting attributes. This approach may also extend to other diseases with known cell surface targets and affected tissues easily isolated for in vivo selection.

Innate Sex Bias of Staphylococcus aureus Skin Infection Is Driven by α-Hemolysin.

Numerous studies have reported sex bias in infectious diseases, with bias direction dependent on pathogen and site of infection. Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs), yet sex bias in susceptibility to S. aureus SSTI has not been described. A search of electronic health records revealed an odds ratio of 2.4 for S. aureus SSTI in males versus females. To investigate the physiological basis of this bias, we compared outcomes between male and female mice in a model of S. aureus dermonecrosis. Consistent with the epidemiological data, female mice were better protected against SSTI, with reduced dermonecrosis followed later by increased bacterial clearance. Protection in females was disrupted by ovariectomy and restored by short-term estrogen administration. Importantly, this sex bias was mediated by a sex-specific response to the S. aureus-secreted virulence factor α-hemolysin (Hla). Infection with wild-type S. aureus suppressed inflammatory cytokine production in the skin of female, but not male, mice when compared with infection with an isogenic hla deletion mutant. This differential response was conserved following injection with Hla alone, demonstrating a direct response to Hla independent of bacterial burden. Additionally, neutrophils, essential for clearing S. aureus, demonstrated sex-specific S. aureus bactericidal capacity ex vivo. This work suggests that sex-specific skin innate responsiveness to Hla and neutrophil bactericidal capacity play important roles in limiting S. aureus SSTI in females. Understanding the molecular mechanisms controlling this sex bias may reveal novel targets to promote host innate defense against S. aureus skin infection.

Roles of G protein-coupled estrogen receptor GPER in metabolic regulation.

Metabolic homeostasis is differentially regulated in males and females. The lower incidence of obesity and associated diseases in pre-menopausal females points towards the beneficial role of the predominant estrogen, 17ß-estradiol (E2). The actions of E2 are elicited by nuclear and extra-nuclear estrogen receptor (ER) α and ERß, as well as the G protein-coupled estrogen receptor (GPER, previously termed GPR30). The roles of GPER in the regulation of metabolism are only beginning to emerge and much remains unclear. The present review highlights recent advances implicating the importance of GPER in metabolic regulation. Assessment of the specific metabolic roles of GPER employing GPER-deficient mice and highly selective GPER-targeted pharmacological agents, agonist G-1 and antagonists G-15 and G36, is also presented. Evidence from in vitro and in vivo studies involving either GPER deficiency or selective activation suggests that GPER is involved in body weight regulation, glucose and lipid homeostasis as well as inflammation. The therapeutic potential of activating GPER signaling through selective ligands for the treatment of obesity and diabetes is also discussed.