The Radical S-Adenosyl-L-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds.

The bacterial enzyme designated QhpD belongs to the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes and participates in the post-translational processing of quinohemoprotein amine dehydrogenase. QhpD is essential for the formation of intra-protein thioether bonds within the small subunit (maturated QhpC) of quinohemoprotein amine dehydrogenase. We overproduced QhpD from Paracoccus denitrificans as a stable complex with its substrate QhpC, carrying the 28-residue leader peptide that is essential for the complex formation. Absorption and electron paramagnetic resonance spectra together with the analyses of iron and sulfur contents suggested the presence of multiple (likely three) [4Fe-4S] clusters in the purified and reconstituted QhpD. In the presence of a reducing agent (sodium dithionite), QhpD catalyzed the multiple-turnover reaction of reductive cleavage of SAM into methionine and 5'-deoxyadenosine and also the single-turnover reaction of intra-protein sulfur-to-methylene carbon thioether bond formation in QhpC bound to QhpD, producing a multiknotted structure of the polypeptide chain. Homology modeling and mutagenic analysis revealed several conserved residues indispensable for both in vivo and in vitro activities of QhpD. Our findings uncover another challenging reaction catalyzed by a radical SAM enzyme acting on a ribosomally translated protein substrate.

Initial O2 Insertion Step of the Tryptophan Dioxygenase Reaction Proposed by a Heme-Modification Study.

L-Tryptophan 2,3-dioxygenase (TDO) is a protoheme-containing enzyme that catalyzes the production of N-formylkynurenine by inserting O2 into the pyrrole ring of L-tryptophan. Although a ferrous-oxy form (Fe²âº-O2) has been established to be an obligate intermediate in the reaction, details of the ring opening reaction remain elusive. In this study, the O2 insertion reaction catalyzed by Pseudomonas TDO (PaTDO) was examined using a heme-modification approach, which allowed us to draw a quantitative correlation between the inductive electronic effects of the heme substituents and the substituent-induced changes in the functional behaviors of the ferrous-oxy form. We succeeded in preparing reconstituted PaTDO with synthetic hemes, which were different with respect to the inductive electron-withdrawing nature of the heme substituents at positions 2 and 4. An increase in the electron-withdrawing power of the heme substituents elevated the redox potential of reconstituted PaTDO, showing that the stronger the electron-withdrawing ability of the heme substituents, the lower the electron density on the heme iron. The decrease in the electron density of the heme iron resulted in a higher frequency shift of the C-O stretch of the heme-bound CO and enhanced the dissociation of O2 from the ferrous-oxy intermediate. This result was interpreted as being due to weaker π back-donation from the heme iron to the bound CO or O2. More importantly, the reaction rates of the ferrous-oxy intermediate to oxidize L-Trp were increased with the electron-withdrawing ability of the heme substituents, implying that the more electron-deficient ferrous-oxy heme is favored for the PaTDO-catalyzed oxygenation. On the basis of these results, we propose that the initial step of the dioxygen activation by PaTDO is a direct electrophilic addition of the heme-bound O2 to the indole ring of L-Trp.

(E)- and (Z)-stereodefined enol phosphonates derived from ß-ketoesters: stereocomplementary synthesis of fully-substituted α,ß-unsaturated esters.

A versatile, robust, and stereocomplementary synthesis of fully-substituted (E)- and (Z)-stereodefined α,ß-unsaturated esters 3 from accessible α-substituted ß-ketoesters 1via (E)- and (Z)-enol phosphonates was achieved. The present method involves two accessible reaction sequences: (i) (E)- and (Z)-stereocomplementary enol phosphorylations of a wide variety of ß-ketoesters 1 (24 examples; 71­99% yield, each >95: 5 ds), and (ii) (E)- and (Z)-stereoretentive Suzuki­Miyaura cross-coupling (16 examples; 71­91% yield, >81/19 ds) and Negishi cross-coupling (32 examples; 65­96% yield, >95 : 5 ds) using (E)- and (Z)-enol phosphonates 2. 1H-NMR monitoring for a key reactive N-phosphorylammonium (imidazolium) intermediate I and an application in the synthesis of both (E)- and (Z)-tamoxifen precursors 6 are described.

Effect of high-fat diet prior to pregnancy on hepatic gene expression and histology in mouse offspring.

Maternal overnutrition and obesity are associated with fetal development and cause long-term effects in offspring. However, the effects of a high-fat diet specific to the pre-pregnancy period are not determined. The present study aimed to examine the effect of high-fat diet prior to pregnancy on the liver of mouse offspring. Female C57BL/6J mice were fed a normal chow (15.2% fat by energy) [control diet (CTR) and CTR pre-pregnancy (PP) groups] or a high-fat chow (31.2% fat by energy) [high-fat diet (HFD) and HFD-pre-pregnancy (PP) groups] for 3-4 weeks and then mated with male C57BL/6J mice fed normal chow. Some mothers continued on the same diet until pups reached 21 days of age (CTR and HFD), and others were fed the different chows from gestational day 0 (CTR-PP and HFD-PP) to determine the effects of a high-fat diet during the pre-pregnancy period in HFD-PP/CTR and HFD/CTR-PP comparisons. Liver tissues from pups were subjected to gene expression analysis by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and microarray, and histological analysis using Oil Red O staining (Sigma Chemical Co., Ltd., Balcatta, WA, USA). Lipid droplets were increased in hepatocytes of mice in HFD-PP compared to CTR and those in HFD compared to CTR-PP. Expression of stearoyl-coenzyme A desaturase 1 (Scd1), acetyl-coenzyme A carboxylase beta (Acacb), and fatty acid binding protein 5 (Fabp5) was increased by maternal high-fat diet during pre-pregnancy. The results showed that maternal high-fat diet intake prior to pregnancy uniquely affects metabolic phenotype related to health and disease in the liver of the next generation.

Effectiveness of initiating advance care planning among older outpatients through intervention by physicians trained in a model discussion video: A randomized controlled trial.

AIM: This study aimed to assess the impact of a physician-led intervention on advance care planning (ACP) introduction among older outpatients using a model discussion video. METHODS: This prospective interventional study included Japanese general practice outpatients aged ≥65 years. All participants received an ACP leaflet. Co-researchers were trained using a model discussion video based on the "Serious Illness Conversation Guide," and provided ACP introduction explanations to the intervention group to ensure intervention content consistency. The control group received the ACP leaflet only, with no standardized explanations. The primary outcome was ACP discussion occurrence. Secondary outcomes included ACP engagement (assessed through the 4-item ACP Engagement Survey), engagement score for advance directives acquisition and score for surrogate decision-maker identification, and anxiety incidence (assessed through Generalized Anxiety Disorder-7). RESULTS: This study included 48 participants: 25 (52.1%) in the intervention group and 23 (47.9%) in the control group. Regarding primary outcome, the intervention group had significantly more ACP discussions compared with the control group (92.0% vs 26.1%, adjusted odds ratio 50.2, P = 0.0012). Regarding secondary outcomes, the intervention group showed greater readiness (mean scores for the 4-item Engagement Survey [P = 0.0001], engagement score for advance directives acquisition [P = 0.01] and score for surrogate decision-maker determination [P = 0.03]) than the control group, without increasing anxiety. CONCLUSIONS: This physician-led ACP introduction for older patients in a Japanese general practice setting enhanced ACP readiness and stimulated discussions, without increasing anxiety. The model discussion video for ACP introduction might effectively train physicians in general practice settings. Geriatr Gerontol Int 2024; ••: ••-••.

Novel evaluation of nephrolithiasis as a complication of gout: a cross-sectional study using helical computerized tomography.

PURPOSE: We clarified whether the clinical profiles of patients with a history of urolithiasis (stone formers) truly reflect those of patients who currently have renal stones (stone carriers). MATERIALS AND METHODS: We evaluated 463 patients with gout using helical computerized tomography, urolithiasis history and relevant clinical parameters. RESULTS: Nephrolithiasis was observed in 157 of the 463 patients (34%) on helical computerized tomography but only 75 (16%) had a urolithiasis history. Of the 157 stone carriers 107 (68%) did not have a urolithiasis history. In those 157 patients serum urate and serum creatinine were higher than in the 306 nonstone carriers (p = 0.017), and the estimated glomerular filtration rate and urinary pH were lower (p = 0.0096 and 0.0249, respectively). However, there was no significant difference in laboratory findings between the 75 stone formers and 388 nonstone formers. Serum urate and creatinine were higher, and the estimated glomerular filtration rate and urine pH in bilateral stone carriers were lower than in unilateral stone carriers. According to HU density attenuation values on computerized tomography, an estimated third of the calculi that complicated 31 recent gout cases was uric acid. CONCLUSIONS: The concept of stone formers may lead to underestimating the prevalence of urolithiasis. Our analysis of stone carriers showed that a higher stone burden is associated with greater renal derangement, as determined by serum urate and creatinine, the estimated glomerular filtration rate and urine pH. To accurately clarify the correlation of gout and urolithiasis, it is advantageous to select stone carriers as subjects of study.

Interactions of soluble guanylate cyclase with a P-site inhibitor: effects of gaseous heme ligands, azide, and allosteric activators on the binding of 2'-deoxy-3'-GMP.

Nitric oxide (NO) elicits a wide variety of physiological responses by binding to the heme in soluble guanylate cyclase (sGC) to stimulate cGMP production. Although nucleotides, such as ATP or GTP analogues, have been reported to regulate the signaling of NO binding from the heme site to the catalytic site, the other regulatory functions of nucleotides remain unexamined. Among the nucleotides tested, we found that 2'-d-3'-GMP acted as a potent noncompetitive inhibitor with respect to Mn-GTP, when the ferrous enzyme combined with NO, CO, or allosteric activator BAY 41-2272. 2'-d-3'-GMP also displayed nearly identical patterns of inhibition for the ferric enzyme, in which the binding of N(3)(-) or BAY 41-2272 significantly increased the inhibitory effects of the nucleotide. Equilibrium dialysis measurements using the CO-ligated enzyme in the presence of allosteric activators demonstrated that 2'-d-3'-GMP exclusively binds to the catalytic site of sGC. Furthermore, the affinity of 2'-d-3'-GMP for the enzyme was found to increase upon addition of foscarnet, an analogue of PP(i). These findings together with other kinetic results imply that 2'-d-3'-GMP acts as a P-site inhibitor probably by forming a dead-end complex, sGC-2'-d-3'-GMP-PP(i), in the catalytic reaction. The formation of the complex of the enzyme with 2'-d-3'-GMP does not seem to be associated with changes in the Fe-proximal His bond strength, because the CO coordination state or the redox potentials of the enzyme-heme complex are virtually unaffected.

Oxygen binding and redox properties of the heme in soluble guanylate cyclase: implications for the mechanism of ligand discrimination.

Soluble guanylate cyclase is an NO-sensing hemoprotein that serves as a NO receptor in NO-mediated signaling pathways. It has been believed that this enzyme displays no measurable affinity for O(2), thereby enabling the selective NO sensing in aerobic environments. Despite the physiological significance, the reactivity of the enzyme-heme for O(2) has not been examined in detail. In this paper we demonstrated that the high spin heme of the ferrous enzyme converted to a low spin oxyheme (Fe(2+)-O(2)) when frozen at 77 K in the presence of O(2). The ligation of O(2) was confirmed by EPR analyses using cobalt-substituted enzyme. The oxy form was produced also under solution conditions at -7 °C, with the extremely low affinity for O(2). The low O(2) affinity was not caused by a distal steric protein effect and by rupture of the Fe(2+)-proximal His bond as revealed by extended x-ray absorption fine structure. The midpoint potential of the enzyme-heme was +187 mV, which is the most positive among high spin protoheme-hemoproteins. This observation implies that the electron density of the ferrous heme iron is relatively low by comparison to those of other hemoproteins, presumably due to the weak Fe(2+)-proximal His bond. Based on our results, we propose that the weak Fe(2+)-proximal His bond is a key determinant for the low O(2) affinity of the heme moiety of soluble guanylate cyclase.

FAK-mediated extracellular signals are essential for interkinetic nuclear migration and planar divisions in the neuroepithelium.

During the development of the vertebrate nervous system, mitosis of neural progenitor cells takes place near the lumen, the apical side of the neural tube, through a characteristic movement of nuclei known as interkinetic nuclear migration (INM). Furthermore, during the proliferative period, neural progenitor cells exhibit planar cell divisions to produce equivalent daughter cells. Here, we examine the potential role of extracellular signals in INM and planar divisions using the medaka mutant tacobo (tab). This tab mutant shows pleiotropic phenotypes, including neurogenesis, and positional cloning identified tab as laminin gamma1 (lamc1), providing a unique framework to study the role of extracellular signals in neurogenesis. In tab mutant neural tubes, a number of nuclei exhibit abnormal patterns of migration leading to basally mislocalized mitosis. Furthermore, the orientation of cell division near the apical surface is randomized. Probably because of these defects, neurogenesis is accelerated in the tab neural tube. Detailed analyses demonstrate that extracellular signals mediated by the FAK pathway regulate INM and planar divisions in the neuroepithelium, possibly through interaction with the intracellular dynein-motor system.

Structural analyses of mannose pentasaccharide of high mannose type oligosaccharides by 1D and 2D NMR spectroscopy.

NMR spectroscopy is a very important and useful method for the structural analysis of oligosaccharides, despite its low sensitivity. We first applied conventional measuring methods, 2D DQF COSY, (1)H-(13)C HSQC, and (1)H-(13)C HMBC, and also the Double Pulsed Field Gradient Spin Echo (DPFGSE)-TOCSY and DPFGSE-NOESY/ROESY techniques to analyze a branched mannose pentasaccharide as a model of high mannose type N-glycans in natural abundance. The NMR spectra of the model compound are very complex and difficult to analyze owing to overlapping signals. The superior selective irradiation capability of the DPFGSE technique is useful for fine structural and conformational analyses of such complex oligosaccharides. We here introduce a novel technique called DPFGSE-Double-Selective Population Transfer (SPT)-Difference and DPFGSE-NOE/ROE-SPT-Difference spectroscopy. The DPFGSE-Double-SPT-Difference method involves irradiation of two peaks from one proton and the subtraction of higher and lower peaks from each spectrum. The DPFGSE-NOE/ROE-SPT-Difference method involves the transfer of the magnetization polarized by NOE/ROE from the nuclei to the spin-coupled nuclei through scalar spin-spin interaction using the SPT method. Even if the signals in the NMR spectra overlap, each signal can be accurately assigned. In particular, DPFGSE-NOE/ROE-SPT-Difference is very useful for identifying sugar connectivity.

Application of Point-of-Care Ultrasound Scanning for the Diagnosis of Perianal Abscess.

A sixty-year-old man with ulcerative colitis and polymyalgia rheumatica, for which he was receiving prednisolone, visited the emergency department complaining of a gradual worsening of pain in the right buttock. Physical examination revealed mild redness and tenderness at the right side of the anus. Ultrasound revealed an approximately 38-mm long, avascular subcutaneous collection with heterogeneous echogenicity in the tender region. A perianal abscess was diagnosed, the lesion was punctured, and pus was drained, after which the pain improved. Point-of-care ultrasonography was effective in the prompt diagnosis and subsequent treatment of this patient's perianal abscess.

Abdominal Muscle Fasciculations and Amyotrophic Lateral Sclerosis Diagnosis in a Patient Unable to Perform Sit-Ups.

While performing sit-ups, a 70-year-old man was unable to lift his upper body. The abdominal skin reflex was absent, and abdominal ultrasonography showed intermittent, irregular, and localized muscle twitches of the abdominal muscles. Further, electromyography (EMG) detected widespread fasciculations. Amyotrophic lateral sclerosis (ALS) was diagnosed. Muscle ultrasonography (MU) is useful in detecting fasciculations. This technique allows for repeated non-invasive imaging and the assessment of an expansive range of muscles in real-time. It also detects deep abdominal muscles, which are difficult to assess using EMG. MU is particularly beneficial to patients with atypical ALS who experience truncal muscle weakness.

Subcutaneous Chest Abscess Caused by Candida albicans Infection Following Laparoscopic Cholecystectomy in an Immunocompetent Patient: A Case Report.

Cases of subcutaneous abscess due to Candida albicans (C. albicans) infection are rare, even among immunocompromised patients. To our knowledge, there have only been eleven reports of such cases in adults, all of which presented with comorbidities of immunodeficiency, prior antibiotic administration, or skin breakdown following traumatic episodes or iatrogenic procedures. We report a rare case of a 42-year-old Japanese woman with a subcutaneous abscess due to C. albicans infection. The patient was referred to our hospital with a chief complaint of gradually worsening lower left-sided chest pain. Nine months before admission, she underwent laparoscopic cholecystectomy (Lap-C) for acute cholecystitis at another hospital. She developed fever and was treated with cefotiam for three days followed by cefoperazone/sulbactam for four days. One week after Lap-C, she began to feel pain in the lower left side of her chest. The chest pain worsened gradually and the fever persisted until two months before admission. On admission, enhanced chest computed tomography revealed a left chest subcutaneous abscess located between the seventh and ninth rib. She underwent surgical percutaneous drainage, and the abscess cavity was cleaned. The pus culture revealed C. albicans, but the blood cultures were negative. We administered intravenous micafungin (150 mg daily) for 10 days, followed by oral fluconazole (600 mg daily). She experienced telogen effluvium during the period of fluconazole treatment but recovered after the cessation of fluconazole. We also present a short review of the literature relating to subcutaneous candidal abscesses in patients over 15 years old.

Unusual heme binding in the bacterial iron response regulator protein: spectral characterization of heme binding to the heme regulatory motif.

We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single "heme-regulatory motif", HRM, and plays a key role in the iron homeostasis of a nitrogen-fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where (29)Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside of the HRM. The Raman line for the Fe-S stretching mode observed at 333 cm(-1) unambiguously confirmed heme binding to Cys. The lower frequency of the Fe-S stretching mode corresponds to the weaker Fe-S bond, and the broad Raman line of the Fe-S bond suggests multiple configurations of heme binding. These structural characteristics are definitely different from those of typical hemoproteins. The unusual heme binding in Irr was also evident in the EPR spectra. The characteristic g-values of the 5-coordinate Cys-ligated heme and 6-coordinate His/His-ligated heme were observed, while the multiple configurations of heme binding were also confirmed. Such multiple heme configurations are not encountered for typical hemoproteins where the heme functions as the active center. Therefore, we conclude that heme binding to HRM in the heme-regulated protein, Irr, is quite different from that in conventional hemoproteins but characteristic of heme-regulated proteins using heme as the signaling molecule.

Anticoagulant Therapy for Frail Patients with Atrial Fibrillation.

The prevalence of atrial fibrillation (AF) increases with age, as does the proportion of patients with frailty. AF patients with frailty have a higher risk of stroke than those without frailty, and progressive frailty caused by stroke is also associated with a worse prognosis. Despite this, anticoagulant therapy tends to not be used in frail patients because of the risk of falls and bleeding complications. However, some studies have shown that anticoagulant therapy improves the prognosis in patients with frailty. An accurate assessment of the "net-clinical-benefits" is needed in patients with frailty, with the aim of improving the prognoses of patients with frailty by selecting those who will benefit from anticoagulant therapy and actively reducing the risk of bleeding. A comprehensive intervention that includes a team of doctors and social resources is required. We herein review the effectiveness and bleeding risk associated with anticoagulant therapy in frail patients investigated in clinical studies.

The effectiveness of transcutaneous cervical ultrasonography for diagnosing peritonsillar abscess in a patient complaining of sore throat.

Peritonsillar abscess should be considered as a differential in patients presenting with fever, sore throat, and other cold-like symptoms. Point-of-care transcutaneous cervical ultrasound is effective for diagnosing peritonsillar abscess in place of computed tomography (CT) imaging. Tongue movement during ultrasound examination will help confirm the presence of anatomical lesions on tonsils, and comparing the affected and unaffected sides will improve proper diagnosis.

Point-of-care ultrasound for prompt diagnosis and treatment monitoring of acute epiglottitis.

A 72-year-old woman presented with a 1-day history of acute throat pain, hoarseness. On cervical ultrasonography (transverse view), the epiglottis was swollen with anterior-posterior diameter of 3.5 and 3.8 mm at right and left edges, respectively. Laryngeal endoscopy showed marked swelling of the epiglottis, and acute epiglottitis was diagnosed.

A woman with adult-onset Still's disease and acute intestinal pseudo-obstruction.

Adult-onset Still's disease may cause intestinal pseudo-obstruction via a cytokine storm. Early diagnosis and treatment are the key for patient survival before the development of serious complications such as macrophage activation syndrome.

Comprehensive infection control measures prevent hospital-acquired severe acute respiratory syndrome coronavirus 2 infection: A single-center prospective cohort study and seroprevalence survey.

BACKGROUND: Coronavirus disease (COVID-19) is associated with a high mortality rate in older adults; therefore, it is important for medical institutions to take measures to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. This study aimed to assess the risk of SARS-CoV-2 infection among healthcare workers (HCWs) and the effectiveness of infection control measures. METHODS: This study had a cross-sectional component and a prospective cohort component. The cross-sectional component comprised an anti-SARS-CoV-2 antibody survey among HCWs at a medical center in Saitama City, Japan. In the prospective cohort component, HCWs at the same medical center were tested for anti-SARS-CoV-2 antibodies monthly over a 3-month period (May to July 2020) to assess the effectiveness of infection prevention measures, including personal protective equipment use. All participants in the cohort study also participated in the antibody survey. The primary outcome was anti-SARS-CoV-2 antibody (measured using Elecsys® Anti-SARS-CoV-2) positivity based on whether participants were engaged in COVID-19-related medical care. Other risk factors considered included occupational category, age, and sex. RESULTS: In total, 607 HCWs participated in the antibody survey and 116 doctors and nurses participated in the cohort study. Only one of the 607 participants in the survey tested positive for anti-SARS-CoV-2 antibodies. All participants in the cohort study were anti-SARS-CoV-2 antibody negative at baseline and remained antibody negative. Engaging in the care of COVID-19 patients did not increase the risk of antibody positivity. During the study period, a total of 30 COVID-19 in-patients were treated in the hospital. CONCLUSIONS: The infection control measures in the hospital protected HCWs from nosocomially acquired SARS-CoV-2 infection; thus, HCWs should engage in COVID-19-related medical care with confidence provided that they adhere to infectious disease precautions.