Cryo-EM and Solid State NMR Together Provide a More Comprehensive Structural Investigation of Protein Fibrils.

The Tropomyosin 1 isoform I/C C-terminal domain (Tm1-LC) fibril structure is studied jointly with cryogenic electron microscopy (cryo-EM) and solid state nuclear magnetic resonance (NMR). This study demonstrates the complementary nature of these two structural biology techniques. Chemical shift assignments from solid state NMR are used to determine the secondary structure at the level of individual amino acids, which is faithfully seen in cryo-EM reconstructions. Additionally, solid state NMR demonstrates that the region not observed in the reconstructed cryo-EM density is primarily in a highly mobile random coil conformation rather than adopting multiple rigid conformations. Overall, this study illustrates the benefit of investigations combining cryo-EM and solid state NMR to investigate protein fibril structure.

Olanzapine Plus Triple Antiemetic Therapy for the Prevention of Carboplatin-Induced Nausea and Vomiting: A Randomized, Double-Blind, Placebo-Controlled Phase III Trial.

PURPOSE: We evaluated the efficacy and safety of antiemetic therapy with olanzapine, a neurokinin-1 receptor antagonist (RA), a 5-hydroxytryptamine-3 (5-HT3) RA, and dexamethasone for preventing chemotherapy-induced nausea and vomiting in patients receiving carboplatin-containing chemotherapy. PATIENTS AND METHODS: Chemotherapy-naïve patients scheduled to receive carboplatin (AUC ≥5) were randomly assigned to receive either olanzapine 5 mg once daily (olanzapine group) or placebo (placebo group) in combination with aprepitant, a 5-HT3 RA, and dexamethasone. The primary end point was the complete response (CR; no vomiting and no rescue therapy) rate in the overall phase (0-120 hours). Secondary end points included the proportion of patients free of nausea and safety. RESULTS: In total, 355 patients (78.6% male, median age 72 years, 100% thoracic cancer), including 175 and 180 patients in the olanzapine and placebo groups, respectively, were evaluated. The overall CR rate was 86.9% in the olanzapine group versus 80.6% in the placebo group. The intergroup difference in the overall CR rate was 6.3% (95% CI, -1.3 to 13.9). The proportions of patients free of chemotherapy-induced nausea in the overall (88.6% in the olanzapine group v 75.0% in the placebo group) and delayed (89.7% v 75.6%, respectively) phases were significantly higher in the olanzapine group than in the placebo group (both P < .001). Somnolence was observed in 43 (24.6%) and 41 (22.9%) patients in the olanzapine and placebo groups, respectively, and no events were grade ≥3 in severity. CONCLUSION: The addition of olanzapine was not associated with a significant increase in the overall CR rate. Regarding the prevention of nausea, adding olanzapine provided better control in patients receiving carboplatin-containing chemotherapy, which needs further exploration.

How do disordered head domains assist in the assembly of intermediate filaments?

The dominant structural feature of intermediate filament (IF) proteins is a centrally located α-helix. These long α-helical segments become paired in a parallel orientation to form coiled-coil dimers. Pairs of dimers further coalesce in an anti-parallel orientation to form tetramers. These early stages of intermediate filament assembly can be accomplished solely by the central α-helices. By contrast, the assembly of tetramers into mature intermediate filaments is reliant upon an N-terminal head domain. IF head domains measure roughly 100 amino acids in length and have long been understood to exist in a state of structural disorder. Here, we describe experiments favoring the unexpected idea that head domains self-associate to form transient structural order in the form of labile cross-ß interactions. We propose that this weak form of protein structure allows for dynamic regulation of IF assembly and disassembly. We further offer that what we have learned from studies of IF head domains may represent a simple, unifying template for understanding how thousands of other intrinsically disordered proteins help to establish dynamic morphological order within eukaryotic cells.

Oxidative regulation of TDP-43 self-association by a ß-to-α conformational switch.

An evolutionarily conserved region of the TDP-43 low-complexity domain (LCD) twenty residues in length can adopt either an α-helical or ß-strand conformation. When in the latter conformation, TDP-43 self-associates via the formation of a labile, cross-ß structure. Self-association can be monitored via the formation of phase-separated protein droplets. Exposure of droplets to hydrogen peroxide leads to oxidation of conserved methionine residues distributed throughout the LCD. Oxidation disassembles the cross-ß structure, thus eliminating both self-association and phase separation. Here, we demonstrate that this process reciprocally enables formation of α-helical structure in precisely the same region formerly functioning to facilitate ß-strand-mediated self-association. We further observe that the α-helical conformation allows interaction with a lipid-like detergent and that exposure to lipids enhances the ß-to-α conformational switch. We hypothesize that regulation of this oxidative switch will prove to be important to the control of localized translation within vertebrate cells. The experimental observations reported herein were heavily reliant on studies of 1,6-hexanediol, a chemical agent that selectively dissolves labile structures formed via the self-association of protein domains of low sequence complexity. This aliphatic alcohol is shown to exert its dissociative activity primarily via hydrogen-bonding interactions with carbonyl oxygen atoms of the polypeptide backbone. Such observations underscore the central importance of backbone-mediated protein:protein interactions that facilitate the self-association and phase separation of LCDs.

Oxidative regulation of TDP-43 self-association by a ß-to-α conformational switch.

An evolutionarily conserved region of the TDP-43 low complexity domain twenty residues in length can adopt either an α-helical or ß-strand conformation. When in the latter conformation, TDP-43 self-associates via the formation of a labile, cross-ß structure. Self-association can be monitored via the formation of phase separated protein droplets. Exposure of droplets to hydrogen peroxide leads to oxidation of conserved methionine residues distributed throughout the low complexity domain. Oxidation disassembles the cross-ß structure, thus eliminating both self-association and phase separation. Here we demonstrate that this process reciprocally enables formation of α-helical structure in precisely the same region formerly functioning to facilitate ß-strand mediated self-association. We further observe that the α-helical conformation allows interaction with a lipid-like detergent, and that exposure to lipids enhances the ß-to-α conformational switch. We hypothesize that regulation of this oxidative switch will prove to be important to the control of localized translation within vertebrate cells. The experimental observations reported herein were heavily reliant on studies of 1,6-hexanediol, a chemical agent that selectively dissolves labile structures formed via the self-association of protein domains of low sequence complexity. This aliphatic alcohol is shown to exert its dissociative activity primarily via hydrogen bonding interactions with carbonyl oxygen atoms of the polypeptide backbone. Such observations underscore the central importance of backbone-mediated protein:protein interactions that facilitate the self-association and phase separation of low complexity domains. Significance Statement: The TDP-43 protein is a constituent of RNA granules involved in regulated translation. TDP-43 contains a C-terminal domain of 150 amino acids of low sequence complexity conspicuously decorated with ten methionine residues. An evolutionarily conserved region (ECR) of 20 residues within this domain can adopt either of two forms of labile secondary structure. Under normal conditions wherein methionine residues are reduced, the ECR forms a labile cross-ß structure that enables RNA granule condensation. Upon methionine oxidation, the ECR undergoes a conformational switch to become an α-helix incompatible with self-association and granule integrity. Oxidation of the TDP-43 low complexity domain is hypothesized to occur proximal to mitochondria, thus facilitating dissolution of RNA granules and activation of localized translation.

A 41-Year-Old Woman with Spontaneous Hemoperitoneum in Pregnancy at 28 Weeks.

BACKGROUND Spontaneous hemoperitoneum in pregnancy (SHiP), defined as nontraumatic, acute intra-abdominal bleeding during pregnancy or the postpartum period, is a serious life-threatening complication to mother and child. Endometriosis is a major risk factor for SHiP. This study presents the case of a 41-year-old woman with adenomyosis who developed hemoperitoneum due to endometriosis at 28 weeks of pregnancy. CASE REPORT The patient was a 41-year-old woman (gravida 1, para 0) who conceived via artificial insemination. She had diffuse adenomyosis in the posterior uterine wall and was admitted to our hospital at 12 weeks of gestation with persistent lower abdominal pain. She had started treatment with hydroxyprogesterone caproate to reduce the focal inflammation of adenomyosis. At 28 weeks of gestation, she developed severe lower abdominal pain, and ultrasonography revealed prolonged fetal heart rate deceleration. An emergency cesarean delivery was performed, and a 907 g female infant with an Apgar score of 2/3 was delivered. Umbilical artery blood pH was 7.15. Bleeding from the veins surrounding an endometriotic lesion on the posterior wall of the uterus was observed, and SHiP was diagnosed. CONCLUSIONS Pregnancies complicated by endometriosis or adenomyosis require perinatal management, considering the possibility of SHiP complication. If acute abdominal pain and fetal heart rate deceleration occur during pregnancy, a search for intra-abdominal bleeding should be performed and emergent open hemostasis or cesarean delivery should be considered.

Dosimetric effect of six degrees of freedom couch top with rotational setup error corrections in proton therapy.

PURPOSE: To investigate the dosimetric effect of six degrees of freedom (6DoF) couch top with rotational corrections in proton therapy (PT). METHODS: The water equivalent thickness (WET) was measured using a proton beam with a 6DoF couch top and patient immobilization base plate (PIBP) placed in front of a motorized water phantom. The accuracy verification was performed with the beam axis set perpendicular to the 6DoF couch top and tilted in 10° steps from 10° to 30°. Up to 3° rotational correction may be added during the actual treatment to correct the rotational setup error on our system. The measured and calculated values using the treatment planning system were compared. Additionally, the effect of the 3° difference was evaluated using actual measurements concerning each angle on the proton beam range. RESULTS: The WET of the 6DoF couch top and PIBP were 8.5 ± 0.1 mm and 6.8 ± 0.1 mm, respectively. The calculation and the actual measurement at each angle agreed within 0.2 mm at the maximum. A maximum difference of approximately 0.6 mm was confirmed when tilted at 3° following 30° with the 6DoF couch top plus PIBP. CONCLUSIONS: The dosimetric effect of the 6DoF couch top with rotational corrections in PT differs depending on the incidence angle on the couch top, and it increased with the increased oblique angle of incidence. However, the effect on the range was as small as 0.6 mm at the maximum. The amount of rotational correction, the angle of incidence of the beam, and the effect of rotational corrections on the proton beam range may differ depending on the structure of the couch top. Therefore, sufficient prior confirmation, and subsequent periodical quality assurance management are important.

High-flow nasal cannula oxygen therapy for respiratory management after postoperative re-intubation/re-extubation in patients with trisomy 18 and trisomy 13: Two case reports.

We present two cases of general anesthesia in children with 18, 13 trisomy. One patient had difficulty with intubation and had to be reintubated postoperatively, another developed postoperative acute respiratory distress syndrome. The use of postoperative high-flow nasal cannula oxygen therapy to avoid reintubation is considered a feasible strategy.

Breaking the hard-sphere model with fluorite and antifluorite solid solutions.

Using the hard-sphere model with the existing tabulated values of ionic radii to calculate the lattice parameters of minerals does not always match experimental data. An adaptation of this crystallographic model is proposed by considering the cations and anions as hard and soft close-packed spheres, respectively. We demonstrate the relevance of this "hybrid model" by combining Pauling's first rule with experimental unit-cell parameters of fluorite and antifluorite-structured systems to revise the ionic radii of their constitutive species.

Mutations linked to neurological disease enhance self-association of low-complexity protein sequences.

Protein domains of low sequence complexity do not fold into stable, three-dimensional structures. Nevertheless, proteins with these sequences assist in many aspects of cell organization, including assembly of nuclear and cytoplasmic structures not surrounded by membranes. The dynamic nature of these cellular assemblies is caused by the ability of low-complexity domains (LCDs) to transiently self-associate through labile, cross-ß structures. Mechanistic studies useful for the study of LCD self-association have evolved over the past decade in the form of simple assays of phase separation. Here, we have used such assays to demonstrate that the interactions responsible for LCD self-association can be dictated by labile protein structures poised close to equilibrium between the folded and unfolded states. Furthermore, missense mutations causing Charcot-Marie-Tooth disease, frontotemporal dementia, and Alzheimer's disease manifest their pathophysiology in vitro and in cultured cell systems by enhancing the stability of otherwise labile molecular structures formed upon LCD self-association.

Pressure ulcer induced by discontinuation of levodopa: Case report of an older patient with Parkinson's disease.

We report the case of a pressure ulcer that developed consequent to the discontinuation of levodopa (L-3,4-dihydroxyphenylalanine) administration. The 86-year-old female patient had a 5-year history of Parkinson's disease treated with levodopa. She developed a sacral pressure ulcer due to unanticipated immobilization induced by the discontinuation of levodopa. Discontinuation of mandatory drugs is therefore a risk factor for the development of pressure ulcers in patients with Parkinson's disease.

How do protein domains of low sequence complexity work?

This review covers research findings reported over the past decade concerning the ability of low complexity (LC) domains to self-associate in a manner leading to their phase separation from aqueous solution. We focus our message upon the reductionist use of two forms of phase separation as biochemical assays to study how LC domains might function in living cells. Cells and their varied compartments represent extreme examples of material condensates. Over the past half century, biochemists, structural biologists, and molecular biologists have resolved the mechanisms driving innumerable forms of macromolecular condensation. In contrast, we remain largely ignorant as to how 10%-20% of our proteins actually work to assist in cell organization. This enigmatic 10%-20% of the proteome corresponds to gibberish-like LC sequences. We contend that many of these LC sequences move in and out of a structurally ordered, self-associated state as a means of offering a combination of organizational specificity and dynamic pliability to living cells. Finally, we speculate that ancient proteins may have behaved similarly, helping to condense, organize, and protect RNA early during evolution.

Dosimetric Effects of the Supine and Prone Positions in Proton Therapy for Prostate Cancer.

Purpose: To quantitatively evaluate how much the doses to organs at risk are affected in the prone position compared to the supine position in the proton therapy (PT) for prostate cancer. Materials and Methods: Fifteen consecutive patients with clinically localized prostate cancer underwent treatment planning computed tomography scans in both the supine and prone positions. The clinical target volume (CTV) consisted of the prostate gland plus the seminal vesicles. The PT plans were designed using the standard lateral opposed fields with passively scattered proton beams for both treatment positions. The prescribed dose for each plan was set to 78 Gy (Relative biological effectiveness)/39 fractions to 50% of the planning target volume. Dose-volume metrics of the rectum and bladder in the two treatment positions were analyzed. Results: It was confirmed that all the parameters of D05, D10, D20, D30, Dmean, and V90 examined in the rectum were significantly reduced in the prone position. There was no significant difference between the two positions in the bladder dose except for Dmean. The distance between the CTV and the rectum tended to increase with the patient in the prone position; at the prostate level, however, the maximum change was approximately 5 mm, and there was significant variation between cases. Conclusions: We confirmed that the rectal doses were significantly lower in the prone compared with the supine position in PT. Although uncertain, the prone position could be an effective method to reduce the rectal dose in PT.

The low-complexity domain of the FUS RNA binding protein self-assembles via the mutually exclusive use of two distinct cross-ß cores.

The low-complexity (LC) domain of the fused in sarcoma (FUS) RNA binding protein self-associates in a manner causing phase separation from an aqueous environment. Incubation of the FUS LC domain under physiologically normal conditions of salt and pH leads to rapid formation of liquid-like droplets that mature into a gel-like state. Both examples of phase separation have enabled reductionist biochemical assays allowing discovery of an N-terminal region of 57 residues that assembles into a labile, cross-ß structure. Here we provide evidence of a nonoverlapping, C-terminal region of the FUS LC domain that also forms specific cross-ß interactions. We propose that biologic function of the FUS LC domain may operate via the mutually exclusive use of these N- and C-terminal cross-ß cores. Neurodegenerative disease-causing mutations in the FUS LC domain are shown to imbalance the two cross-ß cores, offering an unanticipated concept of LC domain function and dysfunction.

Parametric wavelength conversion with bidirectional utilization of a multiple QPM device.

A configuration for wavelength conversion and optical amplification by parametric interaction using a nonlinear optical device is proposed. It enables pump generation through second harmonic generation (SHG), difference frequency generation (DFG), and optical parametric amplification (OPA) using a multiple-quasi-phase-matched (M-QPM) LiNbO3 waveguide in a bidirectional manner. Wavelength conversion for the 1.4-1.6 µm band is experimentally demonstrated. In addition, it is demonstrated that the parametric gain band can be changed using various detunings between the pump and QPM wavelengths used for the DFG/OPA process. The proposed method would be useful for enabling high-capacity optical transmission outside the 1550-nm band.

The impact of different setup methods on the dose distribution in proton therapy for hepatocellular carcinoma.

PURPOSE: To investigate the impact of different setup methods, vertebral body matching (VM), diaphragm matching (DM), and marker matching (MM), on the dose distribution in proton therapy (PT) for hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Thirty-eight HCC lesions were studied retrospectively to assess changes in the dose distribution on two computed tomography (CT) scans. One was for treatment planning (1st-CT), and the other was for dose confirmation acquired during the course of PT (2nd-CT). The dose coverage of the clinical target volume (CTV-D98 ) and normal liver volume that received 30 Gy relative biological effectiveness (RBE) (liver-V30 ) were evaluated under each condition. Initial treatment planning on the 1st-CT was defined as reference, and three dose distributions recalculated using VM, DM, and MM on the 2nd-CT, were compared to it, respectively. In addition, the relationship between the CTV-D98 of each method and the distance between the center of mass (COM) of the CTV and the right diaphragm top was evaluated. RESULTS: For CTV-D98 , significant differences were observed between the reference and VM and DM, respectively (P = 0.013, P = 0.015). There were also significant differences between MM and VM and DM, respectively (P = 0.018, P = 0.036). Regarding liver-V30 , there was no significant difference in any of the methods, and there were no discernable difference due to the different setup methods. In DM, only two out of 34 cases with a distance from right diaphragm top to COM of CTV of 90 mm or less that CTV-D98 difference was 5% or more and CTV-D98 was worse than VM were confirmed. CONCLUSION: Although MM is obviously the most effective method, it is suggested that DM may be particularly effective in cases where the distance from right diaphragm top to COM of CTV of 90 mm or less.

Transiently structured head domains control intermediate filament assembly.

Low complexity (LC) head domains 92 and 108 residues in length are, respectively, required for assembly of neurofilament light (NFL) and desmin intermediate filaments (IFs). As studied in isolation, these IF head domains interconvert between states of conformational disorder and labile, ß-strand-enriched polymers. Solid-state NMR (ss-NMR) spectroscopic studies of NFL and desmin head domain polymers reveal spectral patterns consistent with structural order. A combination of intein chemistry and segmental isotope labeling allowed preparation of fully assembled NFL and desmin IFs that could also be studied by ss-NMR. Assembled IFs revealed spectra overlapping with those observed for ß-strand-enriched polymers formed from the isolated NFL and desmin head domains. Phosphorylation and disease-causing mutations reciprocally alter NFL and desmin head domain self-association yet commonly impede IF assembly. These observations show how facultative structural assembly of LC domains via labile, ß-strand-enriched self-interactions may broadly influence cell morphology.

Redox-mediated regulation of low complexity domain self-association.

Eukaryotic cells express thousands of protein domains long believed to function in the absence of molecular order. These intrinsically disordered protein (IDP) domains are typified by gibberish-like repeats of only a limited number of amino acids that we refer to as domains of low sequence complexity. A decade ago, it was observed that these low complexity (LC) domains can undergo phase transition out of aqueous solution to form either liquid-like droplets or hydrogels. The self-associative interactions responsible for phase transition involve the formation of specific cross-ß structures that are unusual in being labile to dissociation. Here we give evidence that the LC domains of two RNA binding proteins, ataxin-2 and TDP43, form cross-ß interactions that specify biologically relevant redox sensors.