Structures and mechanisms of actin ATP hydrolysis.

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG-O3B bond cleavage; 3) four concomitant events: W1-PO3- formation, OH- and proton cleavage, nucleophilic attack by the OH- against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi-bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Usefulness of cardiac fusion imaging with computed tomography and Doppler echocardiography in the assessment of conduit stenosis in complex adult congenital heart disease.

BACKGROUND: Noninvasive assessment of stenotic lesions in patients with complex adult congenital heart disease (ACHD) is challenging due to its complex morphology. The simultaneous two-screen display of multidetector-computed tomography (MDCT) and real-time echogram (STDME) technology can display a virtual multi-planar reconstruction from MDCT corresponding to the same cross-sectional image from transthoracic echocardiography (TTE). We investigated the usefulness of the STDME technology for stenosis severity assessment in complex ACHD patients. METHODS: Twenty-four complex ACHD patients with stenotic lesions were enrolled in this study. All patients underwent TTE and the STDME technology within a week after MDCT. Peak velocity and pressure gradient (PG) across the stenotic site were measured using continuous wave Doppler. Cardiac catheterization was performed in 17 patients. RESULTS: Nine out of the twenty-four patients had undergone repair with a conduit. Peak velocity and PG from the STDME technology were higher than those from TTE (peak velocity: 3.1 ± 1.1 vs. 2.8 ± 1.0 m/s; peak PG: 43 ± 28 vs. 34 ± 21 mmHg; both p < 0.01). Peak PG from the STDME technology showed significant correlations with those from catheterization in patients with a conduit (n=7) and those without a conduit (n=10) (r = 0.795 and 0.880, respectively; both p < 0.05), while peak PG from TTE was correlated with catheterization measurements only in patients without a conduit (r = 0.850, p < 0.05). CONCLUSIONS: The STDME technology enables more accurate assessment of conduit stenosis severity than does TTE in complex ACHD patients.

Multiple Late Complications After Takeuchi Repair of Anomalous Left Coronary Artery From the Pulmonary Artery.

Takeuchi repair is a unique surgical approach in anomalous left coronary artery from the pulmonary artery. We present an adult patient with anomalous left coronary artery from the pulmonary artery with multiple late structural complications after Takeuchi repair who was evaluated using multimodality imaging, including newly developed cardiac fusion imaging with computed tomography and echocardiography. (Level of Difficulty: Advanced.).

Efficacy and safety of atrial septal defect closure using Occlutech Figulla Flex II compared with Amplatzer Septal Occluder.

Few studies have reported the efficacy of Occlutech Figulla Flex II (FFII) device compared with Amplatzer Septal Occluder (ASO) device. The aim of this study was to examine the efficacy and safety of FFII compared with ASO for transcatheter atrial septal defect (ASD) closure. We retrospectively evaluated 190 patients using FFII and 190 patients using ASO who underwent transcatheter ASD closure. ASD characteristics were evaluated by transesophageal echocardiography. The prevalence of procedural complications, including erosion, device embolization, stroke, and new-onset atrial arrhythmia, and the presence of a residual shunt were evaluated between the two groups during 12-month follow-up. FFII was used more frequently than ASO in patients with a deficient aortic rim or septal malalignment (P = 0.02, P < 0.01, respectively). The procedural complications of erosion, device embolization, and stroke did not occur in any patients. New-onset atrial arrhythmia occurred in 3 patients of the FFII group and 4 patients of the ASO group, and the difference between the two groups was not significant (P = 0.70). A large residual shunt (≥ 3 mm) was observed in 6 patients of the FFII group and 5 patients of the ASO group, and the difference between the two groups was not significant (P = 0.76). FFII was used frequently in patients with high-risk ASD morphology; however, there was no difference in the prevalence of procedural complications or efficacy between patients using FFII and those using ASO.

An anaerobic membrane bioreactor using a hollow fiber membrane and biogas agitation.

Many biomass disposal demonstration projects are based on anaerobic digestion. However, the excessively slow anaerobic microorganism growth rate is a drawback because a decreased anaerobic microorganism population limits methane fermentation's efficiency. To ensure operation at higher loads, this study used an anaerobic membrane bioreactor (AnMBR) for maintaining anaerobic microorganisms' growth, and this article introduces a series of improvements to address the reactor's shortcomings. Finally, we chose to mix-the internal biogas and conducted the experiment using a hollow fiber AnMBR.•Introducing the design of a highly efficient and compact anaerobic membrane bioreactor (AnMBR).•Introducing the initial OLR and the changes of HRT, SRT, TS, and flux of the permeate in the AnMBR after gradually increasing the load.•Monitoring decomposition characteristics in the gas meter connection.

A common allosteric mechanism regulates homeostatic inactivation of auxin and gibberellin.

Allosteric regulation is protein activation by effector binding at a site other than the active site. Here, we show via X-ray structural analysis of gibberellin 2-oxidase 3 (GA2ox3), and auxin dioxygenase (DAO), that such a mechanism maintains hormonal homeostasis in plants. Both enzymes form multimers by interacting via GA4 and indole-3-acetic acid (IAA) at their binding interface. Via further functional analyses we reveal that multimerization of these enzymes gradually proceeds with increasing GA4 and IAA concentrations; multimerized enzymes have higher specific activities than monomer forms, a system that should favour the maintenance of homeostasis for these phytohormones. Molecular dynamic analysis suggests a possible mechanism underlying increased GA2ox3 activity by multimerization-GA4 in the interface of oligomerized GA2ox3s may be able to enter the active site with a low energy barrier. In summary, homeostatic systems for maintaining GA and IAA levels, based on a common allosteric mechanism, appear to have developed independently.

Impact of Right Ventricular Dilatation in Patients with Atrial Septal Defect.

OBJECTIVE: The aim of this study was to examine the relationship between right ventricular (RV) volume and exercise capacity in adult patients with atrial septal defect (ASD) and to determine the degree of RV dilatation for transcatheter ASD closure. BACKGROUND: RV dilatation is an indication of transcatheter ASD closure; however, few studies have reported the clinical significance of RV dilatation. METHODS: We enrolled 82 consecutive patients (mean age, 49 ± 18 years; female, 68%) who underwent cardiac magnetic resonance imaging and symptom-limited cardiopulmonary exercise test before ASD closure. The relationship between RV volume and peak oxygen uptake (VO2) was evaluated. RESULTS: The mean RV end-diastolic volume index was 108 ± 27 ml/m2 (range, 46 to 180 ml/m2). The mean peak VO2 was 24 ± 7 ml/min/kg (range, 14 to 48 ml/min/kg), and the mean predicted peak VO2 was 90 ± 23%. There were significant negative relationships of RV end-diastolic volume index with peak VO2 (r = -0.28, p < 0.01) and predicted peak VO2 (r = -0.29, p < 0.01). The cutoff value of RV end-diastolic volume index <80% of predicted peak VO2 was 120 ml/m2, with the sensitivity of 49% and the specificity of 89%. CONCLUSIONS: There was a relationship between RV dilatation and exercise capacity in adult patients with ASD. RV end-diastolic volume index ≥120 ml/m2 was related to the reduction in peak VO2. This criterion of RV dilatation may be valuable for the indication of transcatheter ASD closure.

Clinical Significance of Septal Malalignment for Transcatheter Closure of Atrial Septal Defect.

BACKGROUND: Septal malalignment is related to erosion and device embolization in transcatheter closure of atrial septal defect (ASD), but limited information is available. OBJECTIVES: This study aimed to assess clinical significance of septal malalignment and to determine appropriate evaluation of ASD diameter, including the selection of device size. METHODS: Four hundred and seventeen patients with ASD who underwent transcatheter closure were enrolled. Septal malalignment was defined as separation between the septum primum and the septum secundum on transesophageal echocardiography. RESULTS: One hundred and eighty-four patients had septal malalignment. The frequency of septal malalignment increased with age reaching around 50% in adult patients. Septal malalignment was related to aortic rim deficiency. The distance of separation between the septum primum and the septum secundum was 5 ± 2 mm (range, 1-11 mm). In patients with septal malalignment, the ASD diameter measured at the septum primum was 19 ± 6 mm, while the ASD diameter measured at the septum secundum was 16 ± 6 mm. There was a difference of 4 ± 2 mm (range, 0-8 mm) between the ASD diameter measured at the septum primum and that measured at the septum secundum. For transcatheter closure, the Amplatzer Septal Occluder device size 2-3 mm larger and the Occlutech Figulla Flex II device size 4-7 mm larger than the ASD diameter measured at the septum primum were frequently used. During the study period, erosion or device embolization did not occur in all of the patients. CONCLUSIONS: Septal malalignment is highly prevalent in adult patients with aortic rim deficiency. The measurement of ASD diameter at the septum primum can be valuable for the selection of device size in patients with septal malalignment.

Importance of Abdominal Compression Valsalva Maneuver and Microbubble Grading in Contrast Transthoracic Echocardiography for Detecting Patent Foramen Ovale.

BACKGROUND: Although transthoracic echocardiography (TTE) may be useful for patent foramen ovale (PFO) screening, the optimal methodologies remain unclear. The aims of this study were to evaluate the efficacy of the abdominal compression Valsalva maneuver and identify the optimal cutoff value of microbubbles in contrast TTE for detecting PFO, compared with transesophageal echocardiography and catheterization as the reference. METHODS: One hundred thirty-four patients with cryptogenic stroke or migraine who had suspected PFO and underwent TTE and transesophageal echocardiography plus catheterization were enrolled. The sensitivity, specificity, and accuracy of TTE for PFO detection were analyzed according to different provocations (spontaneous Valsalva maneuver, abdominal compression Valsalva maneuver) and different cutoff values of microbubbles for a positive result (at least one microbubble, at least five microbubbles). RESULTS: Eighty patients had PFO confirmed by transesophageal echocardiography and catheterization. When the cutoff was at least one microbubble, the sensitivity of TTE in detecting PFO was 93% with the spontaneous Valsalva maneuver and 99% with the abdominal compression Valsalva maneuver. When the cutoff was at least five microbubbles, sensitivity was 85% with the spontaneous Valsalva maneuver and 99% with the abdominal compression Valsalva maneuver. With the abdominal compression Valsalva maneuver, specificity was increased using the cutoff of at least five microbubbles compared with at least one microbubble (89% vs 57%). The abdominal compression Valsalva maneuver with the cutoff of at least 5 microbubbles provided the greatest accuracy of 95%. CONCLUSIONS: TTE with the abdominal compression Valsalva maneuver had excellent sensitivity. The cutoff of at least five microbubbles increased specificity. Our findings suggest that TTE with these criteria is valuable for PFO diagnosis.

Higher load operation by adoption of ethanol fermentation pretreatment on methane fermentation of food waste.

The study aims to examine whether ethanol fermentation pretreatment (EP) of food waste can contribute to high load operation on methane fermentation using an anaerobic membrane bioreactor (AnMBR). The hydraulic retention time was reduced stepwise from 20 to 5 days to increase the load by increasing the feeding volume. The corresponding organic loading rate (OLR) ranged from 6.6 to 26.5 g-COD/L/day. The control series (without pretreatment) was operable to an OLR of 8.8 g-COD/L/day versus 26.5 g-COD/L/day for the EP series. In the control series, the major volatile fatty acid (VFA) produced by acidogenesis in the reactor was propionic acid because based on Gibbs free energy variations for the methane conversion, acetic acid conversion was not as easy as from propionic acid as from ethanol in the EP series. EP proved effective in avoiding VFA accumulation and subsequent decreased pH. Therefore, EP significantly improves AnMBR performance.

Identification of High-Risk Patent Foramen Ovale Associated With Cryptogenic Stroke: Development of a Scoring System.

BACKGROUND: Transcatheter closure of patent foramen ovale (PFO) has become an effective therapeutic strategy for cryptogenic stroke (CS). The identification of high-risk PFO is essential, but the data are limited. This study aimed to clarify the factors related to CS and to develop a score for high-risk PFO. METHODS: We retrospectively analyzed 57 patients with prior CS and 50 without CS who were scheduled for transcatheter closure. PFO characteristics were evaluated by transesophageal echocardiography. Based on factors related to CS, we estimated the risk score. RESULTS: Patients with CS had a greater frequency of large-size PFO (≥2 mm in height), long-tunnel PFO (≥10 mm in length), atrial septal aneurysm, hypermobile interatrial septum, prominent Eustachian valve or Chiari's network, the large right-to-left shunt at rest and during Valsalva maneuver, and low-angle PFO (≤10° of PFO angle from inferior vena cava), compared with patients without CS. Multivariate analysis showed that long-tunnel PFO, the presence of hypermobile interatrial septum, the presence of prominent Eustachian valve or Chiari's network, the large right-to-left shunt during Valsalva maneuver, and low-angle PFO were independently related to CS. When the score was estimated based on 1 point for each factor, the proportion of CS was markedly elevated with a score of ≥2 points. The probability of CS was markedly different between scores of ≤1 or ≥2 points. CONCLUSIONS: PFO risk can be assessed with a score based on high-risk features. The presence of two or more high-risk PFO features is associated with CS.

High-pressure protein crystal structure analysis of Escherichia coli dihydrofolate reductase complexed with folate and NADP.

A high-pressure crystallographic study was conducted on Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP+ in crystal forms containing both the open and closed conformations of the M20 loop under high-pressure conditions of up to 800 MPa. At pressures between 270 and 500 MPa the crystal form containing the open conformation exhibited a phase transition from P21 to C2. Several structural changes in ecDHFR were observed at high pressure that were also accompanied by structural changes in the NADP+ cofactor and the hydration structure. In the crystal form with the closed conformation the M20 loop moved as the pressure changed, with accompanying conformational changes around the active site, including NADP+ and folate. These movements were consistent with the suggested hypothesis that movement of the M20 loop was necessary for ecDHFR to catalyze the reaction. In the crystal form with the open conformation the nicotinamide ring of the NADP+ cofactor undergoes a large flip as an intermediate step in the reaction, despite being in a crystalline state. Furthermore, observation of the water molecules between Arg57 and folate elucidated an early step in the substrate-binding pathway. These results demonstrate the possibility of using high-pressure protein crystallography as a method to capture high-energy substates or transient structures related to the protein reaction cycle.

Structural basis of chimpanzee APOBEC3H dimerization stabilized by double-stranded RNA.

APOBEC3H (A3H) is a mammal-specific cytidine deaminase that potently restricts the replication of retroviruses. Primate A3Hs are known to exert key selective pressures against the cross-species transmission of primate immunodeficiency viruses from chimpanzees to humans. Despite recent advances, the molecular structures underlying the functional mechanisms of primate A3Hs have not been fully understood. Here, we reveal the 2.20-Å crystal structure of the chimpanzee A3H (cpzA3H) dimer bound to a short double-stranded RNA (dsRNA), which appears to be similar to two recently reported structures of pig-tailed macaque A3H and human A3H. In the structure, the dsRNA-binding interface forms a specialized architecture with unique features. The analysis of the dsRNA nucleotides in the cpzA3H complex revealed the GC-rich palindrome-like sequence preference for dsRNA interaction, which is largely determined by arginine residues in loop 1. In cells, alterations of the cpzA3H residues critical for the dsRNA interaction severely reduce intracellular protein stability due to proteasomal degradation. This suggests that cpzA3H stability is regulated by the dsRNA-mediated dimerization as well as by unknown cellular machinery through proteasomal degradation in cells. Taken together, these findings highlight unique structural features of primate A3Hs that are important to further understand their cellular functions and regulation.

Potential benefit of a simultaneous, side-by-side display of contrast MDCT and echocardiography over routine sequential imaging for assessment of adult congenital heart disease: A preliminary study.

BACKGROUND: Management of adult congenital heart disease (ACHD) patients requires understanding of its complex morphology and functional features. An innovative imaging technique has been developed to display a virtual multi-planar reconstruction obtained from contrast-enhanced multidetector-computed tomography (MDCT) corresponding to the same cross-sectional image from transthoracic echocardiography (TTE). The aim of this study is to assess the usefulness of this imaging technology in ACHD patients. METHODS: This study consisted of 46 consecutive patients (30 women; mean age, 52±18 years old) with ACHD who had undergone contrast MDCT. All patients underwent TTE within a week of MDCT. An experienced sonographer who did not know the results of MDCT conducted a diagnosis using TTE and, then, using the new imaging technology. We studied whether this imaging technology provided additional or unexpected findings or makes more accurate diagnosis. RESULTS: In this imaging technology, MDCT cross-section provides higher-resolution image to the deep compared to corresponding TTE image. Depending on the MDCT section which can be arbitrarily set under the echo guide, we can diagnose unexpected or incremental lesions or more accurately assess the severity of the lesion in 27 patients (59%) compared to TTE study alone. This imaging technology was useful in the following situations: CONCLUSIONS: This integrated imaging technology provides incremental role over TTE in complex anatomy, and allows functional information in ACHD patients.

Similar structural stabilities of 3-isopropylmalate dehydrogenases from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 and its atmospheric congener S. oneidensis strain MR-1.

We previously found that the enzymatic activity of 3-isopropylmalate dehydrogenase from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 (SbIPMDH) was pressure-tolerant up to 100 MPa, but that from its atmospheric congener S. oneidensis strain MR-1 (SoIPMDH) was pressure-sensitive. Such characteristics were determined by only one amino acid residue at position 266, serine (SoIPMDH) or alanine (SbIPMDH) [Y. Hamajima et al. Extremophiles 20: 177, 2016]. In this study, we investigated the structural stability of these enzymes. At pH 7.6, SoIPMDH was slightly more stable against hydrostatic pressure than SbIPMDH, contrary to the physiological pressures of their normal environments. Pressure unfolding of these IPMDHs followed a two-state unfolding model between a native dimer and two unfolded monomers, and the dimer structure was pressure-tolerant up to 200 MPa, employing a midpoint pressure of 245.3 ±â€¯0.1 MPa and a volume change of -225 ±â€¯24 mL mol-1 for the most unstable mutant, SbIPMDH A266S. Thus, their pressure-dependent activity did not originate from structural perturbations such as unfolding or dimer dissociation. Conversely, urea-induced unfolding of these IPMDHs followed a three-state unfolding model, including a dimer intermediate. Interestingly, the first transition was strongly pH-dependent but pressure-independent; however, the second transition showed the opposite pattern. Obtained volume changes due to urea-induced unfolding were almost equal for both IPMDHs, approximately +10 and -30 mL mol-1 for intermediate formation and dimer dissociation, respectively. These results indicated that both IPMDHs have similar structural stability, and a pressure-adaptation mechanism was provided for only the enzymatic activity of SbIPMDH.

Thermal mineralization behavior of PFOA, PFHxA, and PFOS during reactivation of granular activated carbon (GAC) in nitrogen atmosphere.

Waste disposal site is one of the important sinks of chemicals. A significant amount of perfluoroalkyl and polyfluoroalkyl substances (PFASs) such as perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorohexanoic acid (PFHxA) have been brought into it. Because of their aqueous solubility, PFASs are released to landfill effluent waters, from which PFASs are efficiently collected by adsorption technique using granular activated carbon (GAC). The exhausted GAC is reactivated by heating processes. The mineralization of PFASs during the reactivation process was studied. Being thermally treated in N2 atmosphere, the recovery rate of mineralized fluorine and PFC homologues including short-chained perfluorocarboxylic acids was determined. If the reagent form of PFOA, PFHxA, and PFOS were treated at 700 °C, the recovery of mineralized fluorine was less than 30, 46, and 72 %, respectively. The rate increased to 51, 74, and 70 %, if PFASs were adsorbed onto GAC in advance; moreover, addition of excess sodium hydroxide (NaOH) improved the recovery to 74, 91, and 90 %. Residual PFAS homologue was less than 1 % of the original amount. Steamed condition did not affect destruction. The significant role of GAC was to suppress volatile release of PFASs from thermal ambient, whereas NaOH enhanced destruction and retained mineralized fluorine on the GAC surface. Comparing the recovery of mineralized fluorine, the degradability of PFOS was considered to be higher than PFOA and PFHxA. Whole mass balance missing 9~26 % of initial amount suggested formation of some volatile organofluoro compounds beyond analytical coverage.