Allosteric modulation and G-protein selectivity of the Ca2+-sensing receptor.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor1 (GPCR) that has a central role in regulating systemic calcium homeostasis2,3. Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional Gi versus Gq proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both Gi and Gq drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein-lipid interactions. Selective Gi and Gq coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR-G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid L-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands.

Asymmetric activation of the calcium-sensing receptor homodimer.

The calcium-sensing receptor (CaSR), a cell-surface sensor for Ca2+, is the master regulator of calcium homeostasis in humans and is the target of calcimimetic drugs for the treatment of parathyroid disorders1. CaSR is a family C G-protein-coupled receptor2 that functions as an obligate homodimer, with each protomer composed of a Ca2+-binding extracellular domain and a seven-transmembrane-helix domain (7TM) that activates heterotrimeric G proteins. Here we present cryo-electron microscopy structures of near-full-length human CaSR in inactive or active states bound to Ca2+ and various calcilytic or calcimimetic drug molecules. We show that, upon activation, the CaSR homodimer adopts an asymmetric 7TM configuration that primes one protomer for G-protein coupling. This asymmetry is stabilized by 7TM-targeting calcimimetic drugs adopting distinctly different poses in the two protomers, whereas the binding of a calcilytic drug locks CaSR 7TMs in an inactive symmetric configuration. These results provide a detailed structural framework for CaSR activation and the rational design of therapeutics targeting this receptor.

Label-Free Analysis with Multiple Parameters Separates G Protein-Coupled Receptor Signaling Pathways.

Real-time label-free techniques are used to profile G protein-coupled receptor (GPCR) signaling pathways in living cells. However, interpreting the label-free signal responses is challenging, and previously reported methods do not reliably separate pathways from each other. In this study, a continuous angular-scanning surface plasmon resonance (SPR) technique is utilized for measuring label-free GPCR signal profiles. We show how the continuous angular-scanning ability, measuring up to nine real-time label-free parameters simultaneously, results in more information-rich label-free signal profiles for different GPCR pathways, providing a more accurate pathway separation. For this, we measured real-time full-angular SPR response curves for Gs, Gq, and Gi signaling pathways in living cells. By selecting two of the most prominent label-free parameters: the full SPR curve angular and intensity shifts, we present how this analysis approach can separate each of the three signaling pathways in a straightforward single-step analysis setup, without concurrent use of signal inhibitors or other response modulating compounds.

Knowledge and attitudes of physicians in Chile toward Do-Not-Attempt-Resuscitation orders: A cross-sectional nation-wide study.

Objective: Do-Not-Attempt-Resuscitation orders originated in the early 1960s with the establishment of advanced cardiopulmonary resuscitation. These orders aim to limit therapeutic efforts in cases where it may be futile. The decision not to resuscitate a patient is a process that involves a series of ethical, legal, and clinical considerations. Still, it also requires a process in which priority is given to the patients and their autonomy. The objective of this study was to describe the knowledge and attitudes of physicians working in Chile toward Do-Not-Attempt-Resuscitation orders. Methods: A cross-sectional study was conducted, in which a digital questionnaire was sent to physicians from different regions of Chile. Quantitative variables were analyzed using measures of central tendency and dispersion (e.g., median and interquartile range), while qualitative variables were evaluated using frequencies and percentages. Results: Four hundred and thirty-one physicians completed the survey. 85.4% were familiar with the ethical and legal guidelines for cardiopulmonary resuscitation and the rights and duties of the patient. 39.2% believed that patients should have the final decision Do-Not-Attempt-Resuscitation orders, especially if they themselves requested not to be resuscitated. 87.7% mentioned that the Do-Not-Attempt-Resuscitation orders should be reassessed if the patient's prognosis improves. In addition, it was found that the decision not to resuscitate was not always discussed with the patient or their family. Conclusions: The study revealed an ethical conflict regarding Do-Not-Attempt-Resuscitation orders and their management by Chilean physicians. Therefore, it is necessary to create recommendations and provide training to guide professionals in this process, which should also involve patients and their families.

Ligand-directed biased agonism at human histamine H3 receptor isoforms across Gαi/o- and ß-arrestin2-mediated pathways.

The histamine H3 receptor (H3R) is a neurotransmitter receptor that is primarily found in the brain, where it controls the release and synthesis of histamine, as well as the release of other neurotransmitters (e.g. dopamine, serotonin). Notably, 20 H3R isoforms are differentially expressed in the human brain as a consequence of alternative gene splicing. The hH3R-445, -415, -365 and -329 isoforms contain the prototypical GPCR (7TM) structure, yet exhibit deletions in the third intracellular loop, a structural domain that is pivotal for G protein-coupling, signaling and regulation. To date, the physiological relevance underlying the individual and combinatorial function of hH3R isoforms remains poorly understood. Nevertheless, given their significant implication in physiological processes (e.g. cognition, homeostasis) and neurological disorders (e.g. Alzheimer's and Parkinson's disease, schizophrenia), widespread targeting of hH3R isoforms by drugs may lead to on-target side effects in brain regions that are unaffected by disease. To this end, isoform- and/or pathway-selective targeting of hH3R isoforms by biased agonists could be of therapeutic relevance for the development of region- and disease-specific drugs. Hence, we have evaluated ligand biased signaling at the hH3R-445, -415, -365 and -329 isoforms across various Gαi/o-mediated (i.e. [35S]GTPγS accumulation, cAMP inhibition, pERK1/2 activation, pAKT T308/S473 activation) and non Gαi/o-mediated (i.e. ß-arrestin2 recruitment) endpoints that are relevant to neurological diseases. Our findings indicate that H3R agonists display significantly altered patterns in their degree of ligand bias, in a pathway- and isoform-dependent manner, underlining the significance to investigate GPCRs with multiple isoforms to improve development of selective drugs. SUBJECT CATEGORY: Neuropharmacology.

Mechanistic insights into G-protein coupling with an agonist-bound G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by promoting guanine nucleotide exchange. Here, we investigate the coupling of G proteins with GPCRs and describe the events that ultimately lead to the ejection of GDP from its binding pocket in the Gα subunit, the rate-limiting step during G-protein activation. Using molecular dynamics simulations, we investigate the temporal progression of structural rearrangements of GDP-bound Gs protein (Gs·GDP; hereafter GsGDP) upon coupling to the ß2-adrenergic receptor (ß2AR) in atomic detail. The binding of GsGDP to the ß2AR is followed by long-range allosteric effects that significantly reduce the energy needed for GDP release: the opening of α1-αF helices, the displacement of the αG helix and the opening of the α-helical domain. Signal propagation to the Gs occurs through an extended receptor interface, including a lysine-rich motif at the intracellular end of a kinked transmembrane helix 6, which was confirmed by site-directed mutagenesis and functional assays. From this ß2AR-GsGDP intermediate, Gs undergoes an in-plane rotation along the receptor axis to approach the ß2AR-Gsempty state. The simulations shed light on how the structural elements at the receptor-G-protein interface may interact to transmit the signal over 30 Å to the nucleotide-binding site. Our analysis extends the current limited view of nucleotide-free snapshots to include additional states and structural features responsible for signaling and G-protein coupling specificity.

Cheating the Rules of Admission With "Observation".

When physicians admit patients to a hospital, their decisions about where-and to whose professional stewardship and services-those patients belong are influenced by federal policies, of which many clinicians are not aware. The distinction between observation and admission has clinical and ethical implications for patients and practices. The evolution of "observation status" from a clinical tool to a catchall of vague and imprecise meaning has been driven by changes to physician payment and compensation structures, particularly Current Procedural Terminology codes and Centers for Medicare and Medicaid Services regulations, and its current value to clinicians and patients is questionable. This article contextualizes clinicians' admission and observation practices and considers how metrics influence patient costs and how clinicians and organizations are compensated.

Differences in parental vaccine confidence and attitudes by health system in Guatemala and their impact on immunization timeliness.

OBJECTIVE: The objectives of this study were to evaluate parental confidence and attitudes towards immunization in urban Guatemala between private versus public health systems and their impact on vaccination timeliness in their children. METHODS: A cross-sectional survey was conducted in parents 6-18-month-old children who attended well-child outpatient clinics from two health systems (public employee-based insurance and private health care) in Guatemala City from November 2017 through August 2018. Parental demographics, household characteristics, food insecurity, vaccine hesitancy using the WHO SAGE Vaccine Hesitancy Scale, and information on parental use of social media platforms and vaccine information sources were collected. RESULTS: Five hundred-three parents were surveyed, most of them mothers. Only 9 parents reported they had previously refused a vaccine for their child: 8 (3.2 %) from private clinics and 1 (0.4 %) from the public clinic (p = 0.02). Significantly more children attending private clinics (226, 90.4 %) were shown to have a delay in any of their vaccines scheduled for the first 2 years of life compared to those in the public clinic (169, 66.8 %; p < 0.01). Children of parents having a college degree (84.5 vs 70.1 %; p < 0.001), earning more than US$ 1,000 per month (81.5 vs 70.7 %; p < 0.001), and having a computer at home (81.4 vs 70.2; p = 0.007) were more likely to have any delays in the scheduled vaccines. Parents seeking care at private clinics were 1.14 times more at risk of delaying a vaccine compared to those at the public clinic, adjusted for other covariates (p = 0.03, 95 % CI: 1.01, 1.28). CONCLUSIONS: In Guatemala, children receiving immunizations at private clinics were significantly more likely than those attending public clinics to be delayed in their immunization schedule and to remain more days without the recommended protection, especially for third doses of the primary vaccine series.

Structural and Molecular Determinants for Isoform Bias at Human Histamine H3 Receptor Isoforms.

The human histamine H3 receptor (hH3R) is predominantly expressed in the CNS, where it regulates the synthesis and release of histamine and other neurotransmitters. Due to its neuromodulatory role, the hH3R has been associated with various CNS disorders, including Alzheimer's and Parkinson's disease. Markedly, the hH3R gene undergoes extensive splicing, resulting in 20 isoforms, of which 7TM isoforms exhibit variations in the intracellular loop 3 (IL3) and/or C-terminal tail. Particularly, hH3R isoforms that display variations in IL3 (e.g., hH3R-365) are shown to differentially signal via Gαi-dependent pathways upon binding of biased agonists (e.g., immepip, proxifan, imetit). Nevertheless, the mechanisms underlying biased agonism at hH3R isoforms remain unknown. Using a structure-function relationship study with a broad range of H3R agonists, we thereby explored determinants underlying isoform bias at hH3R isoforms that exhibit variations in IL3 (i.e., hH3R-445, -415, -365, and -329) in a Gαi-dependent pathway (cAMP inhibition). Hence, we systematically characterized hH3R isoforms on isoform bias by comparing various ligand properties (i.e., structural and molecular) to the degree of isoform bias. Importantly, our study provides novel insights into the structural and molecular basis of receptor isoform bias, highlighting the importance to study GPCRs with multiple isoforms to better tailor drugs.

Participation of Latin American surgeons in Twitter using the hashtag #SoMe4Surgery and #SoMe4IQLatAm.

Background: In medicine, social networks contribute to the professional training because it is a way to improve the knowledge and skills of students, residents and specialists; additionally, these networks allow the dissemination of evidence. However, Latin American surgeons' influence within this social network is not highlighted. In this study, using the hashtags #SoMe4Surgery and #SoMe4IQLatAm, the participation of Latin American surgeons in Twitter is established. Study Design: This is a prospective cross-sectional study of the academic tweets published in the period between October 13 and October 19, 2020, on Twitter by the academic accounts @Cirbosque and @MISIRG1 who are users of the social network Twitter in Latin America who participate in the network with the hashtags #SoMe4Surgery and #SoMe4IQLatAm with academic use. Results: A total of 56 tweets and 665 retweets were analyzed. Male sex presents greater interaction, as well as Mondays and Tuesdays week days. Geolocation was recorded as 37.1% in Latin America and 17.6% in Europe. #SoMe4Surgery was mentioned in 31 tweets and generated 211,700 impressions and 25,557 interactions, and #SoMe4IQLatam was mentioned in 25 tweets and achieved 57,585 impressions and 21,901 interactions. A growth of the participation rate of 6.5% in @Cirbosque and 10.5% in @ MISIRG1 was estimated for 2021. Conclusion: The use of social networks, particularly Twitter, in the surgeon community has proven to be a valuable tool during the last decade. The tweet that needs to be shared among more surgeons should be linked to the hashtag #SoMe4IQLatAm and #SoMe4Surgery and Twitter surgeon leaders mentions.

Spinal paraganglioma at the conus medullaris mimicking schwannoma: A case report.

BACKGROUND: Paragangliomas of the spine are extremely rare, and they should be considered in the differential diagnosis of spinal tumors due to its overlapping clinical and radiological features with many spinal tumors. CASE REPORT: In this article, we present a 30-year-old lady who presented with low back pain and radicular neuropathic pain at L1 dermatome which was intractable to medical surgery. Her magnetic resonance imaging (MRI) of the lumbosacral spine revealed a T1 isointense, T2 heterogeneously hyperintense intradural extramedullary lesion at the conus medullaris with strong homogenous enhancement on contrast administration. The lesion was surgically excised completely with L1 laminectomy, and the histopathological picture was suggestive of paraganglioma. The patient's complaints resolved fully postoperatively, and there was no evidence of recurrence on long-term follow-up. CONCLUSION: Due to the absence of pathognomonic clinical or radiological features of paragangliomas, they should be taken into consideration in the differential diagnosis of spinal tumors. They share similar clinical and radiological features of schwannomas, ependymomas, and hemangioblastomas. The diagnosis is usually made postoperatively based on histopathological examination.

Asthma in COVID-19: Mitigating or exacerbating? An interesting case report.

Introduction: Bronchial asthma is an age-old disease whereas COVID-19 is an officially declared pandemic on March 11, 2020 by WHO. Since both are primarily a disease of the respiratory system, researchers across the globe tried to explore the potential relationship between them; to date, there is no convincing data. Here, we tried to present a case to explore potential relationships between these two, if present. Case presentation: A 30-year-old male patient with well-controlled cough variant asthma was diagnosed with a case of covid-19 infection 12 months back. All other sign symptoms subsided except dry cough. The patient is treated with an inhaled bronchodilator, oral and inhaled steroid, Tab montelukast as well as other conservative management like hot water vapor, lozenge, honey, etc but symptoms were not controlled for the last 12 months. The patient could not do his job because of this problem. All examination and investigation findings were normal. After long-term use of inhaled steroids, he is now 50-60% improved and gradually improving. Discussion: Covid can exacerbate cough in an asthmatic patient. Neuronal activation and neuroinflammatory mechanisms may aggravate this cough after covid. Diagnosis confirmed clinically with the relevant improvement of symptoms. Other important differentials were excluded by appropriate history, examinations, and investigation. Cough is improved by steroids in this case. Conclusion: Summary of conclusion: Cough variant asthma may be aggravated with covid 19 infection and meticulous history, treatment, and follow up needed for an asthmatic patient who is infected with covid 19.

Euthanasia and assisted suicide: An in-depth review of relevant historical aspects.

End-of-life care is an increasingly relevant topic due to advances in biomedical research and the establishment of new disciplines in evidence-based medicine and bioethics. Euthanasia and assisted suicide are two terms widely discussed in medicine, which cause displeasure on many occasions and cause relief on others. The evolution of these terms and the events associated with their study have allowed the evaluation of cases that have established useful definitions for the legal regulation of palliative care and public policies in the different health systems. However, there are still many aspects to be elucidated and defined. Based on the above, this review aimed to compile relevant historical aspects on the evolution of euthanasia and assisted suicide, which will allow understanding the use and research of these terms.

Undergraduate research in medicine: A summary of the evidence on problems, solutions and outcomes.

Scientific research plays a fundamental role in current medical practice and it is of great importance that medical students relate to it from the beginning of their professional career, since it generates multiple benefits that will be reflected during the course of their careers as university students and future professionals. However, getting involved in research during the undergraduate years is not always easy, as there are different obstacles and challenges that result in a reduced number of research students. Because of this, it is necessary to adopt appropriate strategies and measures to help facilitate this process, in order to foster the early development of essential skills that will facilitate responsible clinical practice. Knowing the evidence on this issue is fundamental to propose educational solutions depending on each context.

The use of facial nerve fasciculus motor evoked potential (MEP) as intraoperative neurophysiological monitoring modality in a child with a diffuse intrinsic pontine glioma: A case report.

BACKGROUND: Intraoperative neurophysiological monitoring (IONM) has improved the diagnosis and surgical treatment of brainstem and posterior fossa tumors. Several modalities are available for IONM such as electroencephalography, brainstem mapping (BSM), cranial nerves evoked potentials), somatosensory evoked potentials (SEP), motor evoked potentials (MEP), brainstem auditory evoked potentials (BAEPs), nerve conduction, and electromyography (EMG) signals. Though motor evoked potential (MEP) and brainstem mapping are the most common IONM modalities used for surgical management of brainstem gliomas, cranial nerve potentials can also be of great help. CASE DESCRIPTION: This article describes a 10-year-old child with diffuse intrinsic pontine glioma (DIPG) who presented with gradual progressive crossed hemiparesis. His brain images carried a range of potential differential diagnoses. Her underwent a successful brainstem biopsy via using motor evoked potential for facial nerve without injuring nearby structures. CONCLUSION: Motor evoked potential of the facial nerve can be used solely for biopsy taking in cases of DIPG.

Midterm outcomes of the covered endovascular reconstruction of the aortic bifurcation for aortoiliac occlusive disease in a latinoamerican population.

INTRODUCTION: Surgical approach of aortoiliac occlusive disease (AOD) with aorto-bi-femoral graft or endarterectomy, has been the first line treatment with patency rates up to 90%. Nevertheless, this procedure has an early mortality rate of 4%. Vascular complications of aorto-bi-femoral graft have an average incidence of 5-10% and development of incisional hernia in 10% of the cases. The Covered Endovascular Reconstruction of Aortic Bifurcation or CERAB technique, as a new approach is shaping up to be a promising approach. However, there are few studies in Latin America and the Caribbean. MATERIALS AND METHODS: Retrospective multicenter study. All patients treated with the CERAB technique between February 2015 and June 2021 in three hospitals. RESULTS: A total of 9 patients (5 male and 4 female) were treated with the CERAB technique. Only one patient died. Of the total number of patients, 41.2% had a TASC II - C classification, and 58.8% had a TASC II - D classification. Complications included dissection in only 2 patients, massive bleeding in 1 patient and hematoma in 3 patients. The average number of days in critical care was 1.2 days and 2.6 in hospitalization. Two patients required endovascular reintervention. Primary patency was present in 66.7% of the patients. DISCUSSION: The CERAB technique presents a low morbidity and mortality with an 88.9% of technical success rate. None of our patients needed Chimney CERAB procedure. Our results are similar to those reported in the literature, where they report primary patency rates between 82% and 97%.

Bilateral congenital inguinal hernia with right-sided Amyand's hernia in a premature twin: Case report and a summary of clinical presentations, management and outcomes in neonates and infants with Amyand's hernia.

INTRODUCTION AND IMPORTANCE: Congenital hernias occur 70% on the right side, 25% on the left side, and approximately 5% bilaterally. The finding of a congenital Amyand's hernia is of interest, especially in patients who do not present risk factors associated with connective tissue disorders, ascitic conditions, fetal developmental disorders or any condition that increases abdominal pressure. CASE PRESENTATION: Male patient, 6 months old, was brought to the pediatric surgery department due to a visible mass in the bilateral inguinal region, which protruded with crying. The parents report that he was a 36-week preterm, low birth weight, monochorionic monoamniotic twin with bilateral congenital inguinal hernia. An open herniorrhaphy was performed, showing a left communicating hydrocele with an indirect left inguinal hernia and right communicating hydrocele with indirect inguinal hernia containing cecal appendix with no signs of inflammation. CLINICAL DISCUSSION: The most common clinical presentation is the presence of a reducible or irreducible mass, erythema and/or inguino-scrotal edema, irritability manifested by crying and recurrent pain in older infants. This condition may be associated with cryptorchidism, intrauterine structural developmental disorder, and the presence of fistulas. Appendectomy and traditional hernia reduction are the most common surgical approach. The evolution of this condition is favorable with extremely low complication rates. CONCLUSION: Amyand's hernia in the neonate is a rare presenting condition, which frequently involves nearby structures with risk of inflammation, incarceration and perforation, so repair should be performed early.

Robotic surgery in Asia.

The percentage of robot-assisted radical cystectomy (RARC) is comparatively low in Asia compared to other continents [11]. Few studies showed, RARC with intracorporeal urinary diversion causes less bleeding and hospital stay compared with extracorporeal urinary diversion.

Broken beer bottle as a cause of sigmoid perforation: A summary of causes and predictors in the management of traumatic and non-traumatic colorectal perforation.

INTRODUCTION AND IMPORTANCE: The presence of foreign bodies at the colorectal level and associated complications is a problem that has become increasingly common in emergency departments. This condition carries high health costs, in addition to having high morbidity and mortality rates, due to the large number of complications such as perforation. CASE PRESENTATION: 46-year-old male patient, who came to the emergency department of a low-level hospital with a clinical picture of approximately one day of evolution consisting of the violent introduction of a foreign body (bottle) at the anorectal level secondary to aggression in a fight, with subsequent endoluminal rupture. CLINICAL DISCUSSION: The most recent evidence indicates that the incidence of perforation as a complication of colorectal foreign body introduction is low. However, the presence of profuse bleeding, advanced age, presence of comorbidities and sepsis are predictors of poor prognosis in these cases. In general, perforation secondary to non-traumatic causes is more frequent, being predominantly due to colorectal cancer, ischemia, diverticulitis, inflammatory bowel disease, inadequate use of enema, iatrogenic endoscopy or anorectal manometry or fecal impaction. The presence of unfavorable factors prolongs hospital stay, the risk of reoperation, perianal infection, peritonitis, sepsis and wound infection, generating mortality rates of up to 38%. CONCLUSION: Colorectal perforation is more frequent in non-traumatic situations and carries health costs and risk of mortality. Its management depends on hospital aspects, clinical context of the patient and training of health personnel. However, most of the outcomes are favorable.