Structural basis of Irgb6 inactivation by Toxoplasma gondii through the phosphorylation of switch I.

Irgb6 is a priming immune-related GTPase (IRG) that counteracts Toxoplasma gondii. It is known to be recruited to the low virulent type II T. gondii parasitophorous vacuole (PV), initiating cell-autonomous immunity. However, the molecular mechanism by which immunity-related GTPases become inactivated after the parasite infection remains obscure. Here, we found that Thr95 of Irgb6 is prominently phosphorylated in response to low virulent type II T. gondii infection. We observed that a phosphomimetic T95D mutation in Irgb6 impaired its localization to the PV and exhibited reduced GTPase activity in vitro. Structural analysis unveiled an atypical conformation of nucleotide-free Irgb6-T95D, resulting from a conformational change in the G-domain that allosterically modified the PV membrane-binding interface. In silico docking corroborated the disruption of the physiological membrane binding site. These findings provide novel insights into a T. gondii-induced allosteric inactivation mechanism of Irgb6.

Structural insights into catalytic promiscuity of chalcone synthase from Glycine max (L.) Merr.: Coenzyme A-induced alteration of product specificity.

Catalytic promiscuity of enzymes plays a pivotal role in driving the evolution of plant specialized metabolism. Chalcone synthase (CHS) catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC), a common precursor of plant flavonoids, from p-coumaroyl-coenzyme A (-CoA) and three malonyl-CoA molecules. CHS has promiscuous product specificity, producing a significant amount of p-coumaroyltriacetic lactone (CTAL) in vitro. However, mechanistic aspects of this CHS promiscuity remain to be clarified. Here, we show that the product specificity of soybean CHS (GmCHS1) is altered by CoA, a reaction product, which selectively inhibits THC production (IC50, 67 µM) and enhances CTAL production. We determined the structure of a ternary GmCHS1/CoA/naringenin complex, in which CoA is bound to the CoA-binding tunnel via interactions with Lys55, Arg58, and Lys268. Replacement of these residues by alanine resulted in an enhanced THC/CTAL production ratio, suggesting the role of these residues in the CoA-mediated alteration of product specificity. In the ternary complex, a mobile loop ("the K-loop"), which contains Lys268, was in a "closed conformation" placing over the CoA-binding tunnel, whereas in the apo and binary complex structures, the K-loop was in an "open conformation" and remote from the tunnel. We propose that the production of THC involves a transition of the K-loop conformation between the open and closed states, whereas synthesis of CTAL is independent of it. In the presence of CoA, an enzyme conformer with the closed K-loop conformation becomes increasingly dominant, hampering the transition of K-loop conformations to result in decreased THC production and increased CTAL production.

Structural-Functional Correlations between Unique N-terminal Region and C-terminal Conserved Motif in Short-chain cis-Prenyltransferase from Tomato.

Neryl diphosphate (C10) synthase (NDPS1), a homodimeric soluble cis-prenyltransferase from tomato, contains four disulfide bonds, including two inter-subunit S-S bonds in the N-terminal region. Mutagenesis studies demonstrated that the S-S bond formation affects not only the stability of the dimer but also the catalytic efficiency of NDPS1. Structural polymorphs in the crystal structures of NDPS1 complexed with its substrate and substrate analog were identified by employing massive data collections and hierarchical clustering analysis. Heterogeneity of the C-terminal region, including the conserved RXG motifs, was observed in addition to the polymorphs of the binding mode of the ligands. One of the RXG motifs covers the active site with an elongated random coil when the ligands are well-ordered. Conversely, the other RXG motif was located away from the active site with a helical structure. The heterogeneous C-terminal regions suggest alternating structural transitions of the RXG motifs that result in closed and open states of the active sites. Site-directed mutagenesis studies demonstrated that the conserved glycine residue cannot be replaced. We propose that the putative structural transitions of the order/disorder of N-terminal regions and the closed/open states of C-terminal regions may cooperate and be important for the catalytic mechanism of NDPS1.

POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos.

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

Impact of Rainfall on the Detection Performance of Non-Contact Safety Sensors for UAVs/UGVs.

This study comprehensively investigates how rain and drizzle affect the object-detection performance of non-contact safety sensors, which are essential for the operation of unmanned aerial vehicles and ground vehicles in adverse weather conditions. In contrast to conventional sensor-performance evaluation based on the amount of precipitation, this paper proposes spatial transmittance and particle density as more appropriate metrics for rain environments. Through detailed experiments conducted under a variety of precipitation conditions, it is shown that sensor performance is significantly affected by the density of small raindrops rather than the total amount of precipitation. This finding challenges traditional sensor-evaluation metrics in rainfall environments and suggests a paradigm shift toward the use of spatial transmittance as a universal metric for evaluating sensor performance in rain, drizzle, and potentially other adverse weather scenarios.

Guidelines for de novo phasing using multiple small-wedge data collection. Corrigendum.

A figure in the article by Baba et al. [(2021), J. Synchrotron Rad. 28, 1284-1295] is corrected.

Integrated Experimental and Computational Studies on the Organocatalytic Kinetic Resolution of ß-Unfunctionalized Primary Alcohols Using a Chiral 1,2-Diamine: The Importance of Noncovalent Interactions.

The enantioselective kinetic resolution of ß-unfunctionalized primary alcohols with benzoyl chloride was carried out in the presence of a catalytic amount of a novel chiral 1,2-diamine derived from (S)-proline. Several valuable chiral 2-substituted propan-1-ols were obtained with good enantioselectivities. Density functional theory calculations revealed that the noncovalent interaction, such as CH-π interaction, is crucial for the enantioselectivity of the resolution. This study was conducted through an interplay between experiment and computation.

Guidelines for de novo phasing using multiple small-wedge data collection.

Intense micro-focus X-ray beamlines available at synchrotron facilities have achieved high-quality data collection even from the microcrystals of membrane proteins. The automatic data collection system developed at SPring-8, named ZOO, has contributed to many structure determinations of membrane proteins using small-wedge synchrotron crystallography (SWSX) datasets. The `small-wedge' (5-20°) datasets are collected from multiple crystals and then merged to obtain the final structure factors. To our knowledge, no systematic investigation on the dose dependence of data accuracy has so far been reported for SWSX, which is between `serial crystallography' and `rotation crystallography'. Thus, herein, we investigated the optimal dose conditions for experimental phasing with SWSX. Phase determination using anomalous scattering signals was found to be more difficult at higher doses. Furthermore, merging more homogeneous datasets grouped by hierarchical clustering with controlled doses mildly reduced the negative factors in data collection, such as `lack of signal' and `radiation damage'. In turn, as more datasets were merged, more probable phases could be obtained across a wider range of doses. Therefore, our findings show that it is essential to choose a lower dose than 10 MGy for de novo structure determination by SWSX. In particular, data collection using a dose of 5 MGy proved to be optimal in balancing the amount of signal available while reducing the amount of damage as much as possible.

Novel bicyclic pyrazoles as potent ALK2 (R206H) inhibitors for the treatment of fibrodysplasia ossificans progressiva.

Mutant activin receptor-like kinase-2 (ALK2) is associated with the pathogenesis of fibrodysplasia ossificans progressiva, making it an attractive target for therapeutic intervention. We synthesized a new series of bicyclic pyrazoles and evaluated their mutant ALK2 enzyme inhibitory activities, leading to the identification of 8 as the most potent inhibitor. This compound showed moderate microsomal metabolic stability and human ether-a-go-go related gene (hERG) safety. In C2C12 cells carrying mutant ALK2 (R206H), 8 efficiently inhibited the bone morphogenetic protein (BMP)-induced alkaline phosphatase activity.

Crystal structure of chalcone synthase, a key enzyme for isoflavonoid biosynthesis in soybean.

Isoflavonoid is one of the groups of flavonoids that play pivotal roles in the survival of land plants. Chalcone synthase (CHS), the first enzyme of the isoflavonoid biosynthetic pathway, catalyzes the formation of a common isoflavonoid precursor. We have previously reported that an isozyme of soybean CHS (termed GmCHS1) is a key component of the isoflavonoid metabolon, a protein complex to enhance efficiency of isoflavonoid production. Here, we determined the crystal structure of GmCHS1 as a first step of understanding the metabolon structure, as well as to better understand the catalytic mechanism of GmCHS1.

Does screening for prostate cancer improve cancer-specific mortality in Asian men? Real-world data in Yokosuka City 15 years after introducing PSA-based population screening.

BACKGROUND: Studies of prostate-specific antigen (PSA)-based population screening have been conducted in western countries, but there is little data in Asian populations. The objective of this study was to determine the efficacy of PSA screening in Asian men using real-world data over a period of 15 years after introducing population screening in Yokosuka City, Japan. METHODS: We investigated patients with pathologically diagnosed prostate cancer at four hospitals and two clinics across the Yokosuka area (Miura peninsula) between April 2001 and March 2015. Patients were divided into two groups; the S group consisted of those diagnosed by PSA-based population screening in Yokosuka City and the NS group consisted of those diagnosed by methods other than screening. Cancer-specific survival (CSS) and overall survival (OS) rates were calculated using the Kaplan-Meier method with the log-rank test to compare survival between the two groups. Clinical and pathological factors for cancer-specific mortality were assessed with Cox regression analyses to calculate the hazard ratio (HR) and 95% confidence interval (CI). RESULTS: A total of 3094 patients had been diagnosed with prostate cancer over the 15-year period. The median follow-up period was 77 months. The S group and the NS group consisted of 977 and 2117 patients, respectively. Patients in the S group were younger (age: 71 years vs 73 years, P < .001) and had a lower Charlson comorbidity index (CCI) with favorable oncological factors, such as lower initial PSA, Gleason score (GS), and risk category. Kaplan-Meier curves for OS and CSS revealed significant differences between the two groups (OS: P < .001, CSS: P < .001). Analysis with Cox proportional hazards model indicated the NS group (HR: 1.584, 95% CI, 1.065-2.356, P = .023), a CCI > 4 (HR: 1.552, 95% CI, 1.136-2.120, P = .006), a GS ≥ 8 (HR: 4.869, 95% CI, 2.631-9.001, P < .001), and nonlocalized cancer (locally advanced; HR: 2.632, 95% CI, 1.676-4.133, P < .001, advanced; HR: 9.468, 95% CI, 6.279-14.278, P < .001) as independent risk factors for cancer-specific mortality. CONCLUSIONS: PSA-based population screening of prostate cancer might be useful in the Japanese population.

Novel method of intraoperative ocular movement monitoring using a piezoelectric device: experimental study of ocular motor nerve activating piezoelectric potentials (OMNAPP) and clinical application for skull base surgeries.

Intraoperative monitoring systems that utilize various evoked potentials for the detection and/or preservation of cranial nerves have become increasingly common due to recent technical and commercial developments, particularly during skull base surgeries. We established a novel system for the intraoperative monitoring of the extraocular motor nerves (eOMNs) using a piezoelectric device capable of detecting imperceptible vibrations induced by ocular movement, with sensors placed on the eyelids alone. We first evaluated the efficacy and reliability of this device for the intraoperative monitoring of eOMNs in two Beagle dogs. Based on the results, we then determined the appropriate stimulation parameters for use in human surgical cases involving removal of various skull base tumors. Animal experiments revealed that a 0.4 mA monopolar electrical stimulation was required to elicit significant responses and that these responses were not inferior to those obtained via the electrooculogram/electromyogram. Significant responses were also detected in preliminary clinical investigations in human patients, following both direct and indirect monopolar electrical stimulation of the oculomotor and abducens nerves, although obtaining responses from the trochlear nerve was difficult. Intraoperative monitoring using a piezoelectric device provides a simple and reliable method for detecting eOMNs, especially the oculomotor and abducens nerves. This monitoring system can be adapted to various surgeries for skull base tumor.

ALK2: A Therapeutic Target for Fibrodysplasia Ossificans Progressiva and Diffuse Intrinsic Pontine Glioma.

Fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG) are diseases that typically manifest in childhood and are associated with severely reduced life expectancy. However, there are currently no effective therapies for these diseases, which remain incurable. Activin receptor-like kinase-2 (ALK2), encoded by the ACVR1 gene, is a bone morphogenetic protein (BMP) type-I receptor subtype that plays an important physiological role in the development of bones, muscles, brain, and other organs. Constitutively active mutants of ALK2 have been identified as causative of FOP and involved in the tumorigenesis of DIPG owing to abnormal activation of BMP signaling, and therefore have emerged as promising treatment targets. Here, we describe these two diseases, along with the link to ALK2 signal transduction, and highlight potential ALK2 inhibitors that are under development to offer new hope for patients with FOP and DIPG.

Bis-Heteroaryl Pyrazoles: Identification of Orally Bioavailable Inhibitors of Activin Receptor-Like Kinase-2 (R206H).

Mutant activin receptor-like kinase-2 (ALK2) was reported to be closely associated with the pathogenesis of fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG), and therefore presents an attractive target for therapeutic intervention. Through in silico virtual screenings and structure-activity relationship studies assisted by X-ray crystallographic analyses, a novel series of bis-heteroaryl pyrazole was identified as potent inhibitors of ALK2 (R206H). Derived from in silico hit compound RK-59638 (6a), compound 18p was identified as a potent inhibitor of ALK2 (R206H) with good aqueous solubility, liver microsomal stability, and oral bioavailability.

Perspectives on End-of-Life Treatment among Patients with COPD: A Multicenter, Cross-sectional Study in Japan.

Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality. Since patients with severe COPD may experience exacerbations and eventually face mortality, advanced care planning (ACP) has been increasingly emphasized in the recent COPD guidelines. We conducted a multicenter, cross-sectional study to survey the current perspectives of Japanese COPD patients toward ACP. "High-risk" COPD patients and their attending physicians were consecutively recruited. The patients' family configurations, understanding of COPD pathophysiology, current end-of-life care communication with physicians and family members, and preferences for invasive life-sustaining treatments including mechanical ventilation (MV) and cardiopulmonary resuscitation (CPR) were evaluated using a custom-made, structured, self-administered questionnaire. Attending physicians were also interviewed, and we evaluated the patient-physician agreement. Among the 224 eligible "high-risk" patients, 162 participated. Half of the physicians (54.4%) thought they had communicated detailed information; however, only 19.4% of the COPD patients thought the physicians did so (κ score = 0.16). Less than 10% of patients wanted to receive invasive treatment (MV, 6.3% and CPR, 9.4%); interestingly, more than half marked their decision as "refer to the physician" (MV 42.5% and CPR 44.4%) or "refer to family" (MV, 13.8% and CPR, 14.4%). Patients with less knowledge of COPD were less likely to indicate that they had already made a decision. Although ACP is necessary to cope with severe COPD, Japanese "high-risk" COPD patients were unable to make a decision on their preferences for invasive treatments. Lack of disease knowledge and communication gaps between patients and physicians should be addressed as part of these patients' care.

Structural Insights into the Altering Function of CRMP2 by Phosphorylation.

Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity by controlling microtubule dynamics. CRMP2 activity is regulated by semaphorin-induced phosphorylation at the C-terminal tail domain. Unphosphorylated CRMP2 induces effective axonal microtubule formation to give the axonal characteristics to a neurite, whereas phosphorylated CRMP2 leads to the apparently opposite effect, growth cone collapse. We have recently characterized the structural detail of CRMP2-induced axonal microtubule formation (Niwa et al. (2017) Sci. Rep., 7: 10681). CRMP2 forms the hetero-trimer with GTP-tubulin to induce effective axonal microtubule formation in the future axon. Phosphorylation of CRMP2 has been reported to decrease the affinity between CRMP2 and the microtubule, albeit the molecular mechanisms of how the phosphorylation of CRMP2 changes the structure to achieve distinct effects from unphosphorylated CRMP2 is not well understood. Here we performed a series of biochemical and structural analyses of phospho-mimic CRMP2. Phosphorylation of CRMP2 undergoes small conformational changes at the C-terminal tail with shifting the surface charge, which not only alters the interactions within the CRMP2 tetramer but also alters the interactions with GTP-tubulin. Consequently, phospho-mimic CRMP2 fails to form a hetero-trimer with GTP-tubulin, thus losing the ability to establish and maintain the axonal microtubules.Key words: CRMP2, phosphorylation, microtubule, axon, crystal structure.

[A Case of Synchronous Malignant Pheochromocytomas in Bilateral Adrenal Glands].

We present a case of synchronous malignant pheochromocytoma in bilateral adrenal glands. A 73- year-old man presented to our hospital with bilateral adrenal masses incidentally found during abdominal ultrasonography examination for an unrelated issue. The patient had a 30-year history of hypertension and paroxysmal atrial fibrillation. Computed tomography and magnetic resonance imaging showed heterogeneous tumors in bilateral adrenal glands and an enlarged para-aortic lymph node. Hormonal examinations revealed a high value of urinary catecholamines. Metaiodobenzylguanidine (MIBG) scintigraphy showed increased uptake in bilateral adrenal glands and the lymph node. Both adrenal tumors and the node were surgically removed. Pathological examination revealed histologically distinct tissue between the two adrenal tumors. The patient received five cycles of adjuvant chemotherapy, consisting of cyclophosphamide, vincristine, and dacarbazine. The patient has been in remission for 32 months following surgical treatment.

Heterosubtypic antibody recognition of the influenza virus hemagglutinin receptor binding site enhanced by avidity.

Continual and rapid mutation of seasonal influenza viruses by antigenic drift necessitates the almost annual reformulation of flu vaccines, which may offer little protection if the match to the dominant circulating strain is poor. S139/1 is a cross-reactive antibody that neutralizes multiple HA strains and subtypes, including those from H1N1 and H3N2 viruses that currently infect humans. The crystal structure of the S139/1 Fab in complex with the HA from the A/Victoria/3/1975 (H3N2) virus reveals that the antibody targets highly conserved residues in the receptor binding site and contacts antigenic sites A, B, and D. Binding and plaque reduction assays show that the monovalent Fab alone can protect against H3 strains, but the enhanced avidity from binding of bivalent IgG increases the breadth of neutralization to additional strains from the H1, H2, H13, and H16 subtypes. Thus, antibodies making relatively low affinity Fab interactions with the receptor binding site can have significant antiviral activity when enhanced by avidity through bivalent interactions of the IgG, thereby extending the breadth of binding and neutralization to highly divergent influenza virus strains and subtypes.

Clinical outcomes of prostate cancer patients in Yokosuka City, Japan: A comparative study between cases detected by prostate-specific antigen-based screening in Yokosuka and those detected by other means.

OBJECTIVES: To investigate whether prostate-specific antigen-based screening reduced the prostate cancer mortality rate in Yokosuka, Japan. METHODS: We carried out a cohort study, in which we compared clinical outcomes between patients detected by prostate-specific antigen-based screening (S group n = 524) versus those detected by other means (NS group n = 1044). Clinical and pathological factors were evaluated using Cox regression analyses and the Kaplan-Meier method. RESULTS: A total of 1.5% (8/524) of patients in the S group and 6.7% (70/1044) of those in the NS group died from prostate cancer during follow up. A total of 8.0% (42/524) of patients in the S group and 11.4% (119/1044) in the NS group died from other causes. The 10-year cancer specific survival rates of the S and NS groups were 97% and 86%, respectively (P < 0.001). The median age was significantly lower in the S group than the NS group: 71 and 73 years, respectively (P < 0.001). The rate of Gleason score 8-10 was significantly lower in the S group than the NS group: 9.7% and 16.7%, respectively (P < 0.001). The rate of patients with metastasis or prostate-specific antigen 100 ng/mL or more was significantly lower in the S group than the NS group: 7.8% and 23.0%, respectively (P < 0.001). On multivariate analysis, Gleason score 8-10 compared with Gleason score 6 was independently associated with cancer-specific survival (hazard ratio 4.808, 95% confidence interval 1.044-22.14, P = 0.044). CONCLUSIONS: Prostate-specific antigen-based population screening in Yokosuka City might help to reduce the prostate cancer mortality rate.

Primary Central Nervous System Vasculitis Mimicking Susac Syndrome and Multiple Sclerosis With Long-Term Remission and Spontaneous Resolution of Lesions: A Case Report.

Primary central nervous system vasculitis (PCNSV) is an angiitis localized to the central nervous system (CNS), with various manifestations and no specific biomarkers. Herein, we report a case of PCNSV that presented with an unusual course. A 40-year-old Japanese male developed inner ear symptoms and visual field disturbances. Later, at 42 years of age, the patient developed right hemiparesis and was diagnosed with multiple sclerosis (MS). He received methylprednisolone pulse therapy, which improved his symptoms and resolved most brain lesions. Subsequently, he did not visit the hospital for 13 years, during which time he experienced no relapse. At 55 years of age, he presented to our hospital with fatigue and dizziness. Susac syndrome was suspected because of sensorineural hearing loss and snowball lesions in the corpus callosum. Some of the brain lesions resolved spontaneously. A biopsy was performed on a right frontal lobe lesion, which revealed vasculitis with fibrinoid necrosis, no demyelinating lesions, no amyloid positivity, and no infiltration of atypical lymphocytes. With no evidence of vasculitis in other organs, the patient was diagnosed with PCNSV. The patient was treated with methylprednisolone pulse therapy, followed by oral prednisolone (1 mg/kg/day). The prednisolone was tapered off, and no relapse of symptoms or new lesions on magnetic resonance imaging (MRI) were noted. As observed in this case, even in a scenario suggestive of Susac syndrome or multiple sclerosis, PCNSV should be considered a differential diagnosis and confirmed via brain biopsy.