Structure and mechanism of the SGLT family of glucose transporters.

Glucose is a primary energy source in living cells. The discovery in 1960s that a sodium gradient powers the active uptake of glucose in the intestine1 heralded the concept of a secondary active transporter that can catalyse the movement of a substrate against an electrochemical gradient by harnessing energy from another coupled substrate. Subsequently, coupled Na+/glucose transport was found to be mediated by sodium-glucose cotransporters2,3 (SGLTs). SGLTs are responsible for active glucose and galactose absorption in the intestine and for glucose reabsorption in the kidney4, and are targeted by multiple drugs to treat diabetes5. Several members within the SGLT family transport key metabolites other than glucose2. Here we report cryo-electron microscopy structures of the prototypic human SGLT1 and a related monocarboxylate transporter SMCT1 from the same family. The structures, together with molecular dynamics simulations and functional studies, define the architecture of SGLTs, uncover the mechanism of substrate binding and selectivity, and shed light on water permeability of SGLT1. These results provide insights into the multifaceted functions of SGLTs.

Structure and mechanism of blood-brain-barrier lipid transporter MFSD2A.

MFSD2A is a sodium-dependent lysophosphatidylcholine symporter that is responsible for the uptake of docosahexaenoic acid into the brain1,2, which is crucial for the development and performance of the brain3. Mutations that affect MFSD2A cause microcephaly syndromes4,5. The ability of MFSD2A to transport lipid is also a key mechanism that underlies its function as an inhibitor of transcytosis to regulate the blood-brain barrier6,7. Thus, MFSD2A represents an attractive target for modulating the permeability of the blood-brain barrier for drug delivery. Here we report the cryo-electron microscopy structure of mouse MFSD2A. Our structure defines the architecture of this important transporter, reveals its unique extracellular domain and uncovers its substrate-binding cavity. The structure-together with our functional studies and molecular dynamics simulations-identifies a conserved sodium-binding site, reveals a potential lipid entry pathway and helps to rationalize MFSD2A mutations that underlie microcephaly syndromes. These results shed light on the critical lipid transport function of MFSD2A and provide a framework to aid in the design of specific modulators for therapeutic purposes.

An Isoprene Lipid-Binding Protein Promotes Eukaryotic Coenzyme Q Biosynthesis.

The biosynthesis of coenzyme Q presents a paradigm for how cells surmount hydrophobic barriers in lipid biology. In eukaryotes, CoQ precursors-among nature's most hydrophobic molecules-must somehow be presented to a series of enzymes peripherally associated with the mitochondrial inner membrane. Here, we reveal that this process relies on custom lipid-binding properties of COQ9. We show that COQ9 repurposes the bacterial TetR fold to bind aromatic isoprenes with high specificity, including CoQ intermediates that likely reside entirely within the bilayer. We reveal a process by which COQ9 associates with cardiolipin-rich membranes and warps the membrane surface to access this cargo. Finally, we identify a molecular interface between COQ9 and the hydroxylase COQ7, motivating a model whereby COQ9 presents intermediates directly to CoQ enzymes. Overall, our results provide a mechanism for how a lipid-binding protein might access, select, and deliver specific cargo from a membrane to promote biosynthesis.

Insights into distinct signaling profiles of the µOR activated by diverse agonists.

Drugs targeting the µ-opioid receptor (µOR) are the most effective analgesics available but are also associated with fatal respiratory depression through a pathway that remains unclear. Here we investigated the mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two µOR agonists with different safety profiles. LFT, one of the most lethal opioids, and MP, a kratom plant derivative with reduced respiratory depression in animal studies, exhibited markedly different efficacy profiles for G protein subtype activation and ß-arrestin recruitment. Cryo-EM structures of µOR-Gi1 complex with MP (2.5 Å) and LFT (3.2 Å) revealed that the two ligands engage distinct subpockets, and molecular dynamics simulations showed additional differences in the binding site that promote distinct active-state conformations on the intracellular side of the receptor where G proteins and ß-arrestins bind. These observations highlight how drugs engaging different parts of the µOR orthosteric pocket can lead to distinct signaling outcomes.

Cerebellar Ataxia and Coenzyme Q Deficiency through Loss of Unorthodox Kinase Activity.

The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.

CLoNe: automated clustering based on local density neighborhoods for application to biomolecular structural ensembles.

MOTIVATION: Proteins are intrinsically dynamic entities. Flexibility sampling methods, such as molecular dynamics or those arising from integrative modeling strategies, are now commonplace and enable the study of molecular conformational landscapes in many contexts. Resulting structural ensembles increase in size as technological and algorithmic advancements take place, making their analysis increasingly demanding. In this regard, cluster analysis remains a go-to approach for their classification. However, many state-of-the-art algorithms are restricted to specific cluster properties. Combined with tedious parameter fine-tuning, cluster analysis of protein structural ensembles suffers from the lack of a generally applicable and easy to use clustering scheme. RESULTS: We present CLoNe, an original Python-based clustering scheme that builds on the Density Peaks algorithm of Rodriguez and Laio. CLoNe relies on a probabilistic analysis of local density distributions derived from nearest neighbors to find relevant clusters regardless of cluster shape, size, distribution and amount. We show its capabilities on many toy datasets with properties otherwise dividing state-of-the-art approaches and improves on the original algorithm in key aspects. Applied to structural ensembles, CLoNe was able to extract meaningful conformations from membrane binding events and ligand-binding pocket opening as well as identify dominant dimerization motifs or inter-domain organization. CLoNe additionally saves clusters as individual trajectories for further analysis and provides scripts for automated use with molecular visualization software. AVAILABILITY AND IMPLEMENTATION: www.epfl.ch/labs/lbm/resources, github.com/LBM-EPFL/CLoNe. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Delineating the Ligand-Receptor Interactions That Lead to Biased Signaling at the µ-Opioid Receptor.

Biased agonists, which selectively stimulate certain signaling pathways controlled by a G protein-coupled receptor (GPCR), hold great promise as drugs that maximize efficacy while minimizing dangerous side effects. Biased agonists of the µ-opioid receptor (µOR) are of particular interest as a means to achieve analgesia through G protein signaling without dose-limiting side effects such as respiratory depression and constipation. Rational structure-based design of biased agonists remains highly challenging, however, because the ligand-mediated interactions that are key to activation of each signaling pathway remain unclear. We identify several compounds for which the R- and S-enantiomers have distinct bias profiles at the µOR. These compounds serve as excellent comparative tools to study bias because the identical physicochemical properties of enantiomer pairs ensure that differences in bias profiles are due to differences in interactions with the µOR binding pocket. Atomic-level simulations of compounds at µOR indicate that R- and S-enantiomers adopt different poses that form distinct interactions with the binding pocket. A handful of specific interactions with highly conserved binding pocket residues appear to be responsible for substantial differences in arrestin recruitment between enantiomers. Our results offer guidance for rational design of biased agonists at µOR and possibly at related GPCRs.

Extra Ossification Center at the Tip of the Medial Malleolus Suspected as Fracture: A Clinical Clue.

The appearance of displaced bone inferior (distal) to the medial malleolus, present on radiographs in an adolescent patient, can be confused with a fracture, when, in fact, it is the radiographic appearance of a secondary center of ossification. Foot and ankle surgeons should be aware of extra ossification centers and accessory bones, including one at the tip of the medial malleolus, to avoid misdiagnosis and overtreatment. In the present report, I describe the case of a 9-year-old male with bilateral extra ossification centers at the tip of each medial malleolus.

Os subtibiale displacement secondary to pronation-external rotation ankle injury: A case report.

Os subtibiale is a rare accessory ossicle of the ankle, considered not of clinical significance. But its presence in trauma patients may cause misdiagnosis as malleolar fracture and over-treatment. Because the ossicle is attached to the deltoid ligament, ankle trauma that cause medial compartment injury may detach os subtibiale from medial malleolus. In these situations, MR imaging may help to diagnose the deltoid injury, demonstrate the features of the ossicle, and guide treatment.

Signaling Modulation Mediated by Ligand Water Interactions with the Sodium Site at µOR.

The mu opioid receptor (µOR) is a target for clinically used analgesics. However, adverse effects, such as respiratory depression and physical dependence, necessitate the development of alternative treatments. Recently we reported a novel strategy to design functionally selective opioids by targeting the sodium binding allosteric site in µOR with a supraspinally active analgesic named C6guano. Presently, to improve systemic activity of this ligand, we used structure-based design, identifying a new ligand named RO76 where the flexible alkyl linker and polar guanidine guano group is swapped with a benzyl alcohol, and the sodium site is targeted indirectly through waters. A cryoEM structure of RO76 bound to the µOR-Gi complex confirmed that RO76 interacts with the sodium site residues through a water molecule, unlike C6guano which engages the sodium site directly. Signaling assays coupled with APEX based proximity labeling show binding in the sodium pocket modulates receptor efficacy and trafficking. In mice, RO76 was systemically active in tail withdrawal assays and showed reduced liabilities compared to those of morphine. In summary, we show that targeting water molecules in the sodium binding pocket may be an avenue to modulate signaling properties of opioids, and which may potentially be extended to other G-protein coupled receptors where this site is conserved.

Relationship between Radiological Measurement of Subcoracoid Impingement and Subscapularis Tendon Lesions.

Background: The value of radiological measurements of subcoracoid impingement such as the coracohumeral interval in predicting subscapularis tendon injuries is controversial. We aimed to assess the relationship between radiological measurements of subcoracoid impingement and subscapularis tendon lesions in young and middle-aged adults. Methods: This study was designed as a retrospective cohort study. Patients between the ages of 18-55 years without a history of shoulder surgery or major trauma were included and patients with arthritis, instability, or retracted rotator cuff tears were excluded from the study. Magnetic resonance images were evaluated and patients were grouped into two according to the subscapularis tendon condition: normal or pathologic. Glenoid version, axial coracohumeral distance, coracoglenoid angle, coracoid index, sagittal coracoid-glenoid tubercule distance, and axial coracoacromial inclination-glenoid version difference were measured for all patients. Measurement findings were compared between the groups. Correlation analysis was performed for age and radiologic measurements. A p < 0.05 was considered statistically significant for all tests. Results: A total of 298 patients, 107 women (35.1%) and 191 men (64.9%), with a mean age of 34.46 ± 10.10 years (range, 18-55 years) were examined in the study. Subscapularis tendon pathology was noted in 85 patients (28.5%). The diagnosed pathologies were tendinosis in 48 patients (56.5%), partial tears in 28 (32.9%), and full thickness tears in 9 (10.6%). A significant relationship was observed between increasing age and subscapularis tendon lesions (p = 0.001). There was no statistically significant relationship between subscapularis pathology and calculated measurements. Axial coracohumeral distance and coracoglenoid angle measurements showed a statistically significantly negative correlation with age. A positive correlation was found between axial coracohumeral distance and coracoglenoid angle measurements (p < 0.001) and also between glenoid version and coracoid index measurements (p = 0.004). Axial coracohumeral distance and coracoglenoid angle measurements showed a negative correlation with glenoid version and coracoid index measurements (p < 0.05). Conclusions: In this study, the coracohumeral distance and coracoglenoid angle decreased and the incidence of subscapularis tendon lesions increased as the age progressed. However, no relationship was found between radiological measurements and subscapularis tendon lesions.

Metal chelating and anti-radical activity of Salvia officinalis in the ameliorative effects against uranium toxicity.

Uranium is a highly radioactive heavy metal that is toxic to living things. In this study, physiological, cytogenetic, biochemical and anatomical toxicity caused by uranium and the protective role of sage (Salvia officinalis L.) leaf extract against this toxicity were investigated with the help of Allium test. Germination percentage, root length, weight gain, mitotic index (MI), micronucleus (MN) formation, chromosomal aberrations (CAs), superoxide dismutase (SOD) and catalase (CAT) enzyme activities, malondialdehyde (MDA) levels and changes in root meristem cells were used as indicators of toxicity. In the experimental stage, a total of six groups, one of which was the control, were formed. Group I was treated with tap water, while group II and III were treated only with sage (190 mg/L and 380 mg/L). Groups IV, V and VI were germinated with uranyl acetate dihydrate (0.1 mg/mL), uranyl acetate dihydrate + 190 mg/L sage and uranyl acetate dihydrate + 380 mg/L sage, respectively. Allium cepa L. bulbs of each group were germinated for 72 h, and at the end of the period, routine preparation techniques were applied and physiological, cytogenetic, biochemical and anatomical analyzes were performed. As a result, uranium application caused a significant decrease (p < 0.05) in all physiological parameters and MI values. MN, CAs numbers, SOD and CAT enzyme activities and MDA levels increased significantly (p < 0.05) with uranium application. Uranium promoted CAs in the root tip cells in the form of fragment, vagrant chromosome, sticky chromosome, bridge and unequal distribution of chromatin. In addition, it caused anatomical damages such as epidermis cell damage, cortex cell damage and flattened cell nucleus in root tip meristem cells. Sage application together with uranium caused significant (p < 0.05) increases in physiological parameters and MI values and significant decreases in MN, CAs, SOD and CAT activities and MDA levels. In addition, the application of sage resulted in improvement in the severity of anatomical damages induced by uranium. It was determined that the protective role of sage observed for all parameters investigated was even more pronounced at dose of 380 mg/L. The protective role of sage against uranium toxicity is related to its antioxidant activity, and sage has 82.8% metal chelating and 72.9% DPPH removal activity. As a result, uranyl acetate exhibited versatile toxicity in A. cepa, caused cytotoxicity by decreasing the MI rate, and genotoxicity by increasing the frequencies of MN and CAs. And also, Sage acted as a toxicity-reducing agent by displaying a dose-dependent protective role against the toxic effects induced by uranyl acetate.

Delayed osteochondral fracture fixation of the knee in a pediatric patient.

The frequency of osteochondral fractures in the knee joint in the pediatric population is not clearly known. Although fragment fixation is generally considered to be the ideal treatment method in acute injuries, the data of the results of late fixation in neglected and/or late-diagnosed cases are very limited. In this paper, we report our findings regarding the fixation of a delayed large osteochondral fracture in lateral femoral condyle in a pediatric patient.

Computerised Tomography Analysis of Pelvic Inlet and Outlet Fluoroscopic View Angles.

BACKGROUND: Pelvic inlet and outlet fluoroscopy views are routinely used in operative treatment of posterior pelvic ring injuries. In this study, we aimed to evaluate the angles of pelvic inlet and outlet fluoroscopic view, their differences with hip flexion and the correlation of these differences with sacral slope changes. MATERIALS AND METHODS: Sagittal reconstructions of 100 lumbopelvic CT were used to measure sacral slope, pelvic inlet and outlet view angles. The range of pelvic inlet-outlet view angles and their relation with age, sex and sacral slope were analyzed. In ten of these 100 patients, who were undergone a second CT imaging, hips were passively flexed to 60° to change pelvic tilt. The difference in sacral slope and pelvic inlet-outlet view angles in different positions were compared. RESULTS: Mean angles for inlet view, outlet view and sacral slope were 28.9, 41.4 and 37.0, respectively. There was no difference between males and females (p > 0.05). Pelvic outlet angles had a negative correlation with age (p < 0.05). Sacral slope changes with hip flexion showed a negative correlation with inlet angles and positive correlation with outlet angles (p < 0.05). The differences in sacral slope, pelvic inlet and outlet view angles between two measurements were equal. CONCLUSIONS: The pelvic inlet and outlet view angles shows a wide range without a standard so we suggest preoperative CT scan to plan the optimal angles before pelvic ring surgery. The difference in these angles due to pelvic tilt during the surgery may be corrected by measuring the sacral slope difference.

Evaluation of the Knees of Asymptomatic Kangoo Jumpers with MR Imaging.

PURPOSE: The aim of this study is to determine the prevalence of pathologic findings in asymptomatic knees of Kangoo Jumpers by using a 3T MRI and to compare them with age and sex-matched controls who do not regularly participate in any impact sports. METHODS: Both knees of 18 Kangoo Jumpers were examined by 3T MRIs in a total of 36 MRI scans. The control group was comprised of 20 volunteers from the same age group and with similar weights who did not participate in any competitive sports, in a total of 40 MRI scans. Two orthopedists and one radiologist independently assessed all images for the presence or absence of any abnormalities. RESULTS: In 32 (88.9%) of the 36 Kangoo Jumpers' knees, one or more abnormalities were observed. The most prevalent abnormality was bone marrow edema, which was detected in 32 knees (88.9%). The other significant findings were quadriceps tendinopathy (80.6%), patellar tendinopathy (63.9%), gastrocnemius tendinopathy (63.9%), infrapatellar fat pad edema (75%), suprapatellar fat pad edema (63.9%), meniscal signal change (72.2%) and cartilage damage in the patellofemoral joint (72.2%). There were no statistically significant differences in terms of joint effusion (8.3%), ganglion cysts (8.3%) or tibiofemoral joint cartilage injury (0%). CONCLUSION: This study reveals many types of knee MRI findings of asymptomatic Kangoo Jumpers compared to the control group. These MRI findings may be associated with acute knee injuries or chronic joint problems such as osteoarthritis, which may develop in long-term follow-up studies.

Objective Measurement of Medial Joint Space Widening with Percutaneous "Pie Crust" Release of Medial Collateral Ligament during Knee Arthroscopy.

Knee arthroscopy may be called the most commonly and increasingly performed orthopaedic procedure. Posterior medial compartment visualization may be quite challenging. The aim of the present study is to detect objective measurement of medial joint space widening with percutaneous "pie crust" release of medial collateral ligament (MCL) during knee arthroscopy. We used this technique for all knees that require any intervention in the posteromedial compartment and for tight knees in which adequate visualization of the posteromedial compartment cannot be obtained. Eighteen patients (18 knees) were included in this study. Patients were evaluated clinically with the Lysholm and Tegner scores at the final office visit. Joint balance, valgus instability, pain or tenderness on MCL region, and numbness over the medial side of the joint were also noted. Measurements of medial joint space (mm) were obtained at three different times with perioperative C-arm images: normal, controlled valgus force, and after pie crusting. The median follow-up time was 9 (6-12) months. Final follow-up Lysholm (p < 0.05) and Tegner scores (p < 0.05) increased significantly compared with preoperative scores. At the final follow-up, there was no pain or tenderness over MCL and there were no signs of saphenous nerve or vein injury. Medial joint space values in after pie crusting increased significantly (p < 0.05) compared with neutral position measurements and controlled valgus force application (p < 0.05). Controlled release of the MCL in knees provided ∼2.45 times wider visualization place. Furthermore, pie crusting of MCL is a safe and effective technique that provides enough space for visualization and instrumentation in knees. This is a Level IV study.

Severe central nervous system depression in a patient with acute imidacloprid poisoning.

Imidacloprid belongs to a relatively new class of insecticidal chemistry, the chloronicotinyl neonicotinoid compounds. We report a case of acute ingestion of an insecticide formulation containing imidacloprid. Clinical manifestations included respiratory failure and coma. A previously healthy 67-year-old male patient ingested an unknown quantity of imidacloprid for suicidal purpose. Disorientation, drowsiness, and increased salivation had developed by the time of arrival to a local hospital. On admission, his mental status was decreased (Glasgow Coma Scale score, 3/15). He was intubated and mechanically ventilated. Symptomatic and supportive treatments were given. At the fourth day, the patient was discharged from the hospital. None of the symptoms can be considered characteristic and specific for imidacloprid poisoning as a neonicotinoid compound. Although the symptoms can be resolved with supportive measures, none of them can be considered characteristic and specific for imidacloprid poisoning as a neonicotinoid compound.

Medial Swivel Dislocation of the Talonavicular Joint with Associated Cuboid Fracture.

Medial talonavicular dislocation associated with cuboid fracture is rare. We report an 18-year-old man with this injury who exhibited excellent results after open reduction and stabilization of the joint with temporary Kirshner wires.

Missed Capitellar Fracture Caused by Avoidance of Radiological Evaluation in Pregnancy.

In this paper, we report a pregnant woman with a missed capitellar fracture of the elbow, who was treated successfully with open reduction and internal fixation using two headless screws. A 29-year-old 6-month pregnant woman presented to the emergency department due to a history of falling on her outstretched hand. A long-arm splint was applied without radiological evaluation due to pregnancy. She came to the orthopaedics and traumatology outpatient clinic 6 weeks after trauma and her examination after splint removal revealed pain and restriction in the elbow joint movements. Radiography was taken by using a lead shield in order to protect the fetus. Radiographs showed a displaced osteochondral capitellar fracture. Using the posterolateral approach as described by Kocher, the fracture was fixed using headless canulated compression screws. The follow-up examination showed excellent functional and radiological results. Radiological evaluation should not be avoided in case of obvious fracture findings after trauma even in case of pregnancy. It is also highlighted that good results in terms of union and functional recovery can be achieved with open reduction and headless compression screw fixation followed by early rehabilitation even in delayed treatment of capitellum fractures.

Conserved Lipid and Small-Molecule Modulation of COQ8 Reveals Regulation of the Ancient Kinase-like UbiB Family.

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here, we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by binding to membranes containing cardiolipin and by phenolic compounds that resemble CoQ pathway intermediates. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, this work defines lipid and small-molecule modulators of an ancient family of atypical kinase-like proteins and establishes a chemical genetic system for further exploring the mechanistic role of COQ8 in CoQ biosynthesis.