Mechanisms of SARS-CoV-2 entry into cells.

The unprecedented public health and economic impact of the COVID-19 pandemic caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been met with an equally unprecedented scientific response. Much of this response has focused, appropriately, on the mechanisms of SARS-CoV-2 entry into host cells, and in particular the binding of the spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2), and subsequent membrane fusion. This Review provides the structural and cellular foundations for understanding the multistep SARS-CoV-2 entry process, including S protein synthesis, S protein structure, conformational transitions necessary for association of the S protein with ACE2, engagement of the receptor-binding domain of the S protein with ACE2, proteolytic activation of the S protein, endocytosis and membrane fusion. We define the roles of furin-like proteases, transmembrane protease, serine 2 (TMPRSS2) and cathepsin L in these processes, and delineate the features of ACE2 orthologues in reservoir animal species and S protein adaptations that facilitate efficient human transmission. We also examine the utility of vaccines, antibodies and other potential therapeutics targeting SARS-CoV-2 entry mechanisms. Finally, we present key outstanding questions associated with this critical process.

The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids.

CD4+ T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4+ T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1-/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1-/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.

Refphase: Multi-sample phasing reveals haplotype-specific copy number heterogeneity.

Most computational methods that infer somatic copy number alterations (SCNAs) from bulk sequencing of DNA analyse tumour samples individually. However, the sequencing of multiple tumour samples from a patient's disease is an increasingly common practice. We introduce Refphase, an algorithm that leverages this multi-sampling approach to infer haplotype-specific copy numbers through multi-sample phasing. We demonstrate Refphase's ability to infer haplotype-specific SCNAs and characterise their intra-tumour heterogeneity, to uncover previously undetected allelic imbalance in low purity samples, and to identify parallel evolution in the context of whole genome doubling in a pan-cancer cohort of 336 samples from 99 tumours.

Monomer Choice Influences N-Acryloyl Amino Acid Grafter Conversion via Protease Catalysis.

End-capped peptides modified with reactive functional groups on the N-terminus provide a route to prepare peptide-polymer conjugates for a broad range of applications. Unfortunately, current chemical methods to construct modified peptides rely largely on solid-phase peptide synthesis (SPPS), which lacks green preparative characteristics and is costly, thus limiting its applicability to specialty applications such as regenerative medicine. This work evaluates N-terminally modified N-acryloyl-glutamic acid diethyl ester, N-acryloyl-leucine ethyl ester, and N-acryloyl-alanine ethyl ester as grafters and papain as the protease for the direct addition of amino acid ethyl ester (AA-OEt) monomers via protease-catalyzed peptide synthesis (PCPS) and the corresponding formation of N-acryloyl-functionalized oligopeptides in a one-pot aqueous reaction. It was hypothesized that by building N-acryloyl grafters from AA-OEt monomers that are known to be good substrates for papain in PCPS, the corresponding grafters would yield high grafter conversions, high ratio of grafter-oligopeptide to free NH2-oligopeptide, and high overall yield. However, this work demonstrates based on the grafter/monomers studied herein that the dominant factor in N-acryloyl-AA-OEt grafter conversion is the co-monomer used in co-oligomerizations. Computational modeling using Rosetta qualitatively recapitulates the results and provides insight into the structural and energetic bases underlying substrate selectivity. The findings herein expand our knowledge of factors that determine the efficiency of preparing N-acryloyl-terminated oligopeptides by PCPS that could provide practical routes to peptide macromers for conjugation to polymers and surfaces for a broad range of applications.

Utility of Continuous Paravertebral Block After Retroperitoneal Abdominal Aortic Aneurysm Repair.

BACKGROUND: Open abdominal aortic aneurysm (AAA) repairs can be associated with significant pain and morbidity. Previous studies have demonstrated utility of adjunctive epidural analgesia (EA) in addition to general anesthesia (GA) to reduce pain and blunt the maladaptive surgical stress response. However, EA may be complicated by epidural hematomas and severe hypotension. Recently, we started using continuous paravertebral block (PVB) for perioperative analgesia after retroperitoneal AAA repair. PVB has some distinct advantages over EA such as unilateral localization, reduced risk of hypotension, and minimal risk of epidural hematoma in the setting of systemic heparinization. This study aimed to examine the utility of PVB by comparing total opioid consumption in the postoperative period among patients who received GA + PVB and those who received GA alone. METHODS: This retrospective matched cohort study included 62 patients who underwent elective retroperitoneal AAA repair between January 2019 and August 2022. Thirty-one subjects managed with GA + PVB were compared with 31 control subjects treated with GA alone, matched on following criteria: age, sex, and cross-clamp location. Outcome measures included total opioid analgesics administered during their inhospital postoperative course, time to extubation, time to return to baseline activity, time to normal bowel function, and length of stay. Opioid doses were converted to morphine milligram equivalents (MMEs). RESULTS: The GA + PVB group required significantly less opioid analgesics (81 ± 53 MME) than the GA group (171 ± 121 MME) (P < 0.001). Compared to GA alone, GA + PVB was superior in every clinical metric examined: time to extubation (3 vs. 1 hr, P < 0.001), recovery of bowel function (3 vs. 2 days, P = 0.002), recovery of baseline physical activity (4 vs. 2 days, P = 0.019), and length of stay (5 vs. 3 days, P < 0.001). CONCLUSIONS: Continuous paravertebral block provides better pain management with significantly decreased opioid requirements in the postoperative period compared to GA-alone for patients undergoing elective retroperitoneal AAA repair.

Extensor Mechanism Disruption Remains a Challenging Problem.

BACKGROUND: Extensor mechanism disruption (EMD) following total knee arthroplasty (TKA) is a devastating problem commonly treated with allograft or synthetic reconstruction. Understanding of reconstruction success rates and patient recorded outcomes is lacking. METHODS: Patients who have an EMD after TKA undergoing mesh or whole-extensor allograft reconstruction between 2011 and 2019, with minimum 2-year follow-up were reviewed at two tertiary care centers. Functional failure was defined as extensor lag >30 degrees, amputation, or fusion, as well as revision extensor mechanism reconstruction (EMR). Survivorship was assessed using Kaplan-Meier curves, and factors for success were determined with logistic regressions. RESULTS: Of fifty-six EMRs (49 patients), 50.0% (28/56) were functionally successful at 3.2 years of mean follow-up (range, 0.2 to 7.4). In situ survivorship of the reconstructions at 36 months was 75.0% (42 of 58). There were 50.0% (14 of 28) of functionally failed EMRs that retained their reconstruction at last follow-up. Mean extensor lag among successes and failures was 5.4 and 71.0° (P = .01), respectively. Mean Knee Injury and Osteoarthritis Outcome Score, Joint Replacement scores were 67.1 and 48.8 among successes and failures (P = .01). There were 64.0% (16 of 25) of successes and 1 of 19 failures that obtained a Knee Injury and Osteoarthritis Outcome Score, Joint Replacement score above the minimum patient-acceptable symptom state for TKA. Survivorship and success rates were similar between reconstruction methods (P = .86; P = .76). All-cause mortality was 8.2% (4 of 49), each with EMR failure prior to death. All-cause reoperation rate was 42.9% (24 of 56), with a 14.3% (8 of 56) rate of revision EMR and 10.7% (6 of 56) rate of above-knee-amputation or modular fusion. CONCLUSIONS: This multicenter investigation of mesh or allograft EMR demonstrated modest functional success at 3.2 years. Complication and reoperation rates were high, regardless of EMR technique. Therefore, EMD after TKA remains problematic.

E-selective Semi-hydrogenation of Alkynes under Mild Conditions by a Diruthenium Hydride Complex.

The synthesis, characterization and catalytic activity of a new class of diruthenium hydrido carbonyl complexes bound to the tBu PNNP expanded pincer ligand is described. Reacting tBu PNNP with two equiv of RuHCl(PPh3 )3 (CO) at 140 °C produces an insoluble air-stable complex, which was structurally characterized as [Ru2 (tBu PNNP)H(µ-H)Cl(µ-Cl)(CO)2 ] (1) using solid-state NMR, IR and X-ray absorption spectroscopies and follow-up reactivity. A reaction with KOtBu results in deprotonation of a methylene linker to produce [Ru2 (tBu PNNP* )H(µ-H)(µ-OtBu)(CO)2 ] (3) featuring a partially dearomatized naphthyridine core. This enables metal-ligand cooperative activation of H2 analogous to the mononuclear analogue, [Ru(tBu PNP*)H(CO)]. In contrast to the mononuclear system, the bimetallic analogue 3 catalyzes the E-selective semi-hydrogenation of alkynes at ambient temperature and atmospheric H2 pressure with good functional group tolerance. Monitoring the semi-hydrogenation of diphenylacetylene by 1 H NMR spectroscopy shows the intermediacy of Z-stilbene, which is subsequently isomerized to the E-isomer. Initial findings into the mode of action of this system are provided, including the spectroscopic characterization of a polyhydride intermediate and the isolation of a deactivated species with a partially hydrogenated naphthyridine backbone.

Functional importance of the D614G mutation in the SARS-CoV-2 spike protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus which binds its cellular receptor angiotensin-converting enzyme 2 (ACE2) and enters hosts cells through the action of its spike (S) glycoprotein displayed on the surface of the virion. Compared to the reference strain of SARS-CoV-2, the majority of currently circulating isolates possess an S protein variant characterized by an aspartic acid-to-glycine substitution at amino acid position 614 (D614G). Residue 614 lies outside the receptor binding domain (RBD) and the mutation does not alter the affinity of monomeric S protein for ACE2. However, S(G614), compared to S(D614), mediates more efficient ACE2-mediated transduction of cells by S-pseudotyped vectors and more efficient infection of cells and animals by live SARS-CoV-2. This review summarizes and synthesizes the epidemiological and functional observations of the D614G spike mutation, with focus on the biochemical and cell-biological impact of this mutation and its consequences for S protein function. We further discuss the significance of these recent findings in the context of the current global pandemic.

Phosphatidylethanolamine and Phosphatidylserine Synergize To Enhance GAS6/AXL-Mediated Virus Infection and Efferocytosis.

Phosphatidylserine (PS) receptors mediate clearance of apoptotic cells-efferocytosis-by recognizing the PS exposed on those cells. They also mediate the entry of enveloped viruses by binding PS in the virion membrane. Here, we show that phosphatidylethanolamine (PE) synergizes with PS to enhance PS receptor-mediated efferocytosis and virus entry. The presence of PE on the same surface as PS dramatically enhances recognition of PS by PS-binding proteins such as GAS6, PROS, and TIM1. Liposomes containing both PE and PS bound to GAS6 and were engulfed by AXL-expressing cells much more efficiently than those containing PS alone. Further, infection of AXL-expressing cells by infectious Zika virus or Ebola, Chikungunya, or eastern equine encephalitis pseudoviruses was inhibited with greater efficiency by the liposomes containing both PS and PE compared to a mixture of liposomes separately composed of PS and PE. These data demonstrate that simultaneous recognition of PE and PS maximizes PS receptor-mediated virus entry and efferocytosis and underscore the important contribution of PE in these major biological processes.IMPORTANCE Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are usually sequestered to the inner leaflet of the plasma membrane of the healthy eukaryotic cells. During apoptosis, these phospholipids move to the cell's outer leaflet where they are recognized by so-called PS receptors on surveilling phagocytes. Several pathogenic families of enveloped viruses hijack these PS receptors to gain entry into their target cells. Here, we show that efficiency of these processes is enhanced, namely, PE synergizes with PS to promote PS receptor-mediated virus infection and clearance of apoptotic cells. These findings deepen our understanding of how these fundamental biological processes are executed.

Recommendations to Increase Neuromuscular Electrical Stimulation Training Intensity During Quadriceps Treatments for Orthopedic Knee Conditions.

ABSTRACT: Neuromuscular electrical stimulation (NMES) is often used by clinicians as a therapeutic adjunct to improve quadriceps strength deficits following orthopedic knee conditions. The efficacy of NMES treatments is primarily dependent on the NMES training intensity, which is a direct result of NMES-induced torque production. The importance of NMES training intensity is well known, yet adequate NMES training intensities are often difficult to achieve due to a variety of limitations associated with NMES (eg, fatigue and patient discomfort). This article provides recommendations that a clinician can use to increase NMES training intensity when strengthening the quadriceps with NMES for orthopedic knee conditions. These recommendations should allow forceful contractions that can be sustained over a treatment with multiple repetitions without the rapid decline in force that is typically seen when NMES is used.

Emergent Approaches to Efficient and Sustainable Polyhydroxyalkanoate Production.

Petroleum-derived plastics dominate currently used plastic materials. These plastics are derived from finite fossil carbon sources and were not designed for recycling or biodegradation. With the ever-increasing quantities of plastic wastes entering landfills and polluting our environment, there is an urgent need for fundamental change. One component to that change is developing cost-effective plastics derived from readily renewable resources that offer chemical or biological recycling and can be designed to have properties that not only allow the replacement of current plastics but also offer new application opportunities. Polyhydroxyalkanoates (PHAs) remain a promising candidate for commodity bioplastic production, despite the many decades of efforts by academicians and industrial scientists that have not yet achieved that goal. This article focuses on defining obstacles and solutions to overcome cost-performance metrics that are not sufficiently competitive with current commodity thermoplastics. To that end, this review describes various process innovations that build on fed-batch and semi-continuous modes of operation as well as methods that lead to high cell density cultivations. Also, we discuss work to move from costly to lower cost substrates such as lignocellulose-derived hydrolysates, metabolic engineering of organisms that provide higher substrate conversion rates, the potential of halophiles to provide low-cost platforms in non-sterile environments for PHA formation, and work that uses mixed culture strategies to overcome obstacles of using waste substrates. We also describe historical problems and potential solutions to downstream processing for PHA isolation that, along with feedstock costs, have been an Achilles heel towards the realization of cost-efficient processes. Finally, future directions for efficient PHA production and relevant structural variations are discussed.

Myxoma virus M013 protein antagonizes NF-κB and inflammasome pathways via distinct structural motifs.

Among the repertoire of immunoregulatory proteins encoded by myxoma virus, M013 is a viral homologue of the viral pyrin domain-only protein (vPOP) family. In myeloid cells, M013 protein has been shown to inhibit both the inflammasome and NF-κB signaling pathways by direct binding to ASC1 and NF-κB1, respectively. In this study, a three-dimensional homology model of the M013 pyrin domain (PYD) was built based on similarities to known PYD structures. A distinctive feature of the deduced surface electrostatic map of the M013 PYD is the presence of a negatively region consisting of numerous aspartate and glutamate residues in close proximity. Single-site mutations of aspartate and glutamate residues reveal their role in interactions with ASC-1. The biological significance of charge complementarity in the M013-ASC-1 interaction was further confirmed by functional assays of caspase-1 activation and subsequent secretion of cytokines. M013 also has a unique 33-residue C-terminal tail that follows the N-terminal PYD, and it is enriched in positively charged residues. Deletion of the tail of M013 significantly inhibited the interactions between M013 and NF-κB1, thus compromising the ability of the viral protein to suppress the secretion of pro-inflammatory cytokines. These results demonstrate that vPOP M013 exploits distinct structural motifs to regulate both the inflammasome and NF-κB pathways.

Whole-Genome Approach to Understanding the Mechanism of Action of a Histatin 5-Derived Peptide.

The incidence of opportunistic fungal infections that threaten immunocompromised patients, along with the limited arsenal of antifungal drugs, calls for renewed efforts to develop novel antifungal therapies. Antimicrobial peptides have garnered interest as potential therapeutics. Among naturally occurring peptides, histatin 5 is a well-characterized 24-amino-acid peptide with strong antifungal activity. Our lab has identified a smaller histatin derivative, KM29, with stronger activity against multiple Candida spp., prompting us to investigate its fungicidal mechanism. A genetic screen was developed to test the Saccharomyces cerevisiae genomewide deletion collection for mutants with increased or decreased peptide sensitivity. The goal was to identify genes that would reveal insights into the mechanism of action of KM29, to be assessed in Candida albicans Several biological processes yielded increased sensitivity, with endosomal transport and vacuolar function appearing at high frequencies. Among the pathways involved in increased resistance, mitochondrial function showed the highest normalized genome frequency; hence, we focused on characterizing this pathway. KM29 localizes to mitochondria, and the killing activity depends on a functional electron transport chain. In addition, KM29 triggered reactive oxygen species (ROS) production, which was responsible for some cell death but insufficient to account for the complete killing activity. In agreement with this finding, we found that KM29 induced mitochondrial fragmentation and a mild loss of mitochondrial membrane potential. Furthermore, respiratory mutants exhibited severely diminished KM29 uptake. We confirmed this behavior in a C. albicans respiratory mutant. Taking our findings together, this work delineates the mitochondrial functions associated with KM29 fungicidal activity and provides additional pathways for further characterization in Candida spp.

Characteristics of Dog Bites in Arkansas.

OBJECTIVE: Dog bite injuries are encountered frequently in emergency departments and can cause significant morbidity. The objective of this study was to explore the associations between the multiple variables at play during these occurrences (eg, the patient's age, the bite location, the bite severity, the dog's relationship with the patient, the breed of dog). METHODS: This two-institution study collected and analyzed dog bite data from Arkansas' only Level I trauma centers. The charts of 740 patients were included in our retrospective chart review. The chart review extracted data, including each individual patient's age, sex, dog bite location, and dog bite severity, as well as the patient's relationship to the dog and the dog's breed. To determine the relation between and among variables, contingency tables were created and analyzed to determine odds ratios (ORs) and confidence intervals (CIs). In addition, standard t tests were used in statistical comparisons of means and proportions. RESULTS: Of the 740 patient charts reviewed, 574 were for patients who presented to Arkansas Children's Hospital and 166 were for patients who presented to the University of Arkansas for Medical Sciences. Of the patients across both institutions, 267 (37.1%) required some form of repair, with 225 (30.4%) receiving closure within the emergency department and 42 (6.7%) requiring an operative intervention. Among children, those younger than age 5 years were >8 times as likely to require an operative repair (OR 8.1, 95% CI 2.77-23.58, P < 0.0001), >4 times as likely to be bitten on the head and neck (OR 4.30, 95% CI 3.00-6.16, P < 0.0001), and ≤3 times as likely to be bitten by a family dog (OR 2.97, 95% CI 2.10-4.20, P < 0.0001). Conversely, children older than age 12 years were >3 times as likely to be bitten on an extremity (OR 3.43, 95% CI 2.08-5.65, P < 0.0001). CONCLUSIONS: The results of this retrospective review are aligned mostly with the general trends found in previous national and global studies, supporting the notion that family dogs represent a more significant threat than often is realized and that, among the breeds identified, pit bulls are proportionally linked with more severe bite injuries. Our data further validate previous studies that note an increased risk of bites and bite severity in children younger than 5 years. In addition, our data show that bites to the head and neck occurred more frequently among children younger than 5 years than among older children, and that boys younger than 5 years were bitten more frequently than girls.

Asymmetric [3 + 2] Cycloaddition of Chiral N-Phosphonyl Imines with Methyl Isocyanoacetate for Accessing 2-Imidazolines with Switchable Stereoselectivity.

Asymmetric [3 + 2] cycloaddition of chiral N-phosphonyl imines with methyl isocyanoacetate has been established, enabling controllable/switchable stereoselectivity access to 21 examples of cycloadducts with good to excellent chemical yields (up to 92%) and high diastereoselectivities (up to 99:1 dr). The cycloaddition reaction promoted by Cs2CO3 resulted in diastereoenriched (4R,5S)-products with >99:1 dr. However, it showed the reverse stereoselectivity as diastereoenriched (4S,5R) products when AgF was employed as the catalyst. The synthesis followed the group-assisted purification (GAP) chemistry/technology in which the pure 2-imidazoline products were readily provided by washing the crude products with cosolvents of hexane and ethyl acetate.

Chiral GAP catalysts of phosphonylated imidazolidinones and their applications in asymmetric Diels-Alder and Friedel-Crafts reactions.

The design and synthesis of recyclable imidazolidinone catalysts using GAP chemistry/technique was described. Their applications in asymmetric Diels-Alder and Friedel-Crafts reactions with α,ß-unsaturated aldehydes resulted in excellent yields and higher enantioselectivities than previous processes. As recyclable small molecular catalysts, phosphonylated imidazolidinones can be recovered and reused for up to three runs without costing significant decrease in catalytic activity.

Varying age-gender associations between body mass index and urban greenspace.

Urban greenspace benefits urbanites in numerous ways ranging from regulating flooding, air quality, and local climate to providing opportunities for exercise and relaxation. These benefits may influence human health. Greenspace, for example, may facilitate exercise, thereby helping to reduce body mass index (BMI) and combat obesity, a current epidemic of great public health concern. Little evidence exists to support this assertion, however, and we lack a full understanding of the mechanisms whereby this relationship operates, the populations for whom greenspace is linked to weight status, and the aspects of urban greenspace that are linked to weight status. This study seeks to identify relationships among the composition and arrangement of greenspace and BMI for different populations using regression models for eight age and gender groups in Cleveland, Ohio, US. We find that several greenspace variables are related to BMI for women under 65 years and males under 51 years, but not for older groups, and that the aspects and types of greenspace that are significantly related to BMI vary among groups. Relationships between greenspace attributes and BMI are generally stronger for female groups and for younger groups. Providing access to greenspace with particular attributes such as greenspaces with water, canopy cover, or connected greenspaces could support a healthy weight status for some populations, but these attributes are not consistent across age and gender groups. These results could help to inform policy aimed at designing urban greenspace to benefit the health of different population subgroups.

The Effectiveness of Neuromuscular Electrical Stimulation in Improving Voluntary Activation of the Quadriceps: A Critically Appraised Topic.

Clinical Scenario: Orthopedic knee conditions are regularly treated in sports-medicine clinics. Rehabilitation protocols for these conditions are often designed to address the associated quadriceps strength deficits. Despite these efforts, patients with orthopedic knee conditions often fail to completely regain their quadriceps strength. Disinhibitory modalities have recently been suggested as a clinical tool that can be used to counteract the negative effects of arthrogenic muscle inhibition, which is believed to limit the effectiveness of therapeutic exercise. Neuromuscular electrical stimulation (NMES) is commonly accepted as a strengthening modality, but its ability to simultaneously serve as a disinhibitory treatment is not as well established. CLINICAL QUESTION: Does NMES effectively enhance quadriceps voluntary activation in patients with orthopedic knee conditions? Summary of Key Findings: Four randomized controlled trials (RCTs) met the inclusion criteria and were included. Of those, 1 reported statistically significant improvements in quadriceps voluntary activation in the intervention group relative to a comparison group, but the statistical significance was not true for another study consisting of the same sample of participants with a different follow-up period. One study reported a trend in the NMES group, but the between-groups differences were not statistically significant in 3 of the 4 RCTs. Clinical Bottom Line: Current evidence does not support the use of NMES for the purpose of enhancing quadriceps voluntary activation in patients with orthopedic knee conditions. Strength of Recommendation: There is level B evidence that the use of NMES alone or in conjunction with therapeutic exercise does not enhance quadriceps voluntary activation in patients with orthopedic knee conditions (eg, anterior cruciate ligament injuries, osteoarthritis, total knee arthroplasty).

The bile acid receptor TGR5 does not interact with ß-arrestins or traffic to endosomes but transmits sustained signals from plasma membrane rafts.

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of ß-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with ß-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with ß-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-ß-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with ß-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.