Cell-Free Synthesis of Human Endothelin Receptors and Its Application to Ribosome Display.

Engineering G-protein-coupled receptors (GPCRs) for improved stability or altered function is of great interest, as GPCRs consist of the largest protein family, are involved in many important signaling pathways, and thus, are one of the major drug targets. Here, we report the development of a high-throughput screening method for GPCRs using a reconstituted in vitro transcription-translation (IVTT) system. Human endothelin receptor type-B (ETBR), a class A GPCR that binds endothelin-1 (ET-1), a 21-residue peptide hormone, was synthesized in the presence of nanodisc (ND) composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG). The ET-1 binding of ETBR was significantly reduced or was undetectable when other phospholipids were used for ND preparation. However, when functional ETBR purified from Sf9 cells was reconstituted into NDs, ET-1 binding was observed with two different phospholipids tested, including POPG. These results suggest that POPG likely supports the folding of ETBR into its functional form in the IVTT system. Using the same conditions as ETBR, whose three-dimensional structure has been solved, human endothelin receptor type-A (ETAR), whose three-dimensional structure remains unsolved, was also synthesized in its functional form. By adding POPG-ND to the IVTT system, both ETAR and ETBR were successfully subjected to ribosome display, a method of in vitro directed evolution that facilitates the screening of up to 1012 mutants. Finally, using a mock library, we showed that ribosome display can be applied for gene screening of ETBR, suggesting that high-throughput screening and directed evolution of GPCRs is possible in vitro.

Prevalence of adverse childhood experiences and their association with suicidal ideation and non-suicidal self-injury among incarcerated methamphetamine users in Japan.

BACKGROUND: Adverse childhood experiences (ACEs) are distressing and/or traumatic events that occur during childhood that increase the risk of negative health outcomes in adulthood. OBJECTIVE: This study estimated the prevalence of ACEs in a nationwide sample of Japanese methamphetamine users in prison and examined associations among ACEs, suicidal ideation, and non-suicidal self-injury. PARTICIPANTS AND SETTING: Participants were 636 inmates (418 male and 218 female) who were newly incarcerated in Japan for Stimulants Control Act violations. METHODS: First, 699 participants completed an anonymous self-report questionnaire. Of these, 636 participants who did not have any missing responses were included in the analysis. After calculating descriptive statistics, the associations between ACEs and suicidal behaviors were assessed using binary logistic regression analyses. RESULTS: Results showed that 76.1 % of the participants reported at least one ACE before the age of 18, and female participants reported a significantly higher number of adversities than their male counterparts. The most common ACEs were parental death or divorce, followed by psychological abuse. Logistic regression analyses revealed that ACE scores significantly increased the risk of suicidal ideation (SI; adjusted odd ratio [AOR] = 1.18, p < .001) and non-suicidal self-injury (NSSI; AOR = 1.18, p < .001) after controlling for possible confounding variables. CONCLUSIONS: Findings suggest the importance of early prevention and intervention for traumatic experiences, and have implications for the recommendation of gender-responsive, trauma-focused interventions, especially for female inmates in the criminal justice system, to break the intergenerational chain of abuse. Future research directions and treatment are discussed.

Primary Experiences with Robot-assisted Navigation-based Frameless Stereo-electroencephalography: Higher Accuracy than Neuronavigation-guided Manual Adjustment.

The use of robot-assisted frameless stereotactic electroencephalography (SEEG) is becoming more common. Among available robotic arms, Stealth Autoguide (SA) (Medtronic, Minneapolis, MN, USA) functions as an optional instrument of the neuronavigation system. The aims of this study were to present our primary experiences with SEEG using SA and to compare the accuracy of implantation between SA and navigation-guided manual adjustment (MA). Seventeen electrodes from two patients who underwent SEEG with SA and 18 electrodes from four patients with MA were retrospectively reviewed. We measured the distance between the planned location and the actual location at entry (De) and the target (Dt) in each electrode. The length of the trajectory did not show a strong correlation with Dt in SA (Pearson's correlation coefficient [r] = 0.099, p = 0.706) or MA (r = 0.233, p = 0.351). De and Dt in SA were shorter than those in MA (1.99 ± 0.90 vs 4.29 ± 1.92 mm, p = 0.0002; 3.59 ± 2.22 vs 5.12 ± 1.40 mm, p = 0.0065, respectively). SA offered higher accuracy than MA both at entry and target. Surgical times per electrode were 38.9 and 32 min in the two patients with SA and ranged from 51.6 to 88.5 min in the four patients with MA. During the implantation period of 10.3 ± 3.6 days, no patients experienced any complications.

Endoscopic Transcortical Selective Amygdalohippocampectomy for Mesial Temporal Lobe Epilepsy: 2-Dimensional Operative Video.

This surgical video shows a 19-yr-old woman with focal impaired awareness seizures. Seizure semiology showed no lateralizing signs. Ictal electroencephalography (EEG) failed to determine the seizure origin. Interictal EEG showed bilateral spike-and-waves at the temporal electrodes. Magnetic resonance imaging (MRI) showed suspected hippocampal sclerosis on the right side. To determine the side of the focus, depth electrodes were implanted in both hippocampi. Invasive video EEG identified the seizure origin on the right. The decision was made to perform selective amygdalohippocampectomy (SelAH) via the middle temporal gyrus (MTG). An endoscope was used to minimize the craniotomy and shorten the skin incision. A 5-cm linear skin incision and 2.5-cm craniotomy were made. A thin tube was inserted to the inferior horn of the lateral ventricle (Inf-H) under neuronavigation to guide the route to the Inf-H. The endoscope was introduced. A 1.5-cm corticotomy was made at the MTG, and white matter was aspirated until opening the Inf-H. The hippocampus and parahippocampal gyrus were removed with the usual steps in microsurgical SelAH. The surgical time was 4 h 20 min. The patient was discharged without complications and has remained seizure free. In addition to the preoperative objectives, using an endoscope widens the surgical view in the Inf-H compared with microsurgical procedures. Although seizure and cognitive outcomes are expected to be comparable to those from other methods of SelAH, invasiveness might be reduced. This appears to represent the first video report of endoscopic SelAH. The patient consented to the procedure and publication of her images and surgical video.