Expression, purification and preliminary crystallographic analysis of bacterial transmembrane protein EfeU for iron import.

The bacterial Efe system functions as an importer of free Fe2+ into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding protein EfeO, peroxidase EfeB, and transmembrane permease EfeU. While we and other researchers reported crystal structures of EfeO and EfeB, that of EfeU remains undetermined. In this study, we constructed expression system of EfeU derived from Escherichia coli, selected E. coli Rosetta-gami 2 (DE3) as an expression host, and succeeded in purification of the proteins which were indicated to form an oligomer by blue native PAGE. We obtained preliminary data of the X-ray crystallography, suggesting that expression and purification methods we established in this study enable structural analysis of the bacterial Efe system.

Incidence of interstitial lung disease in patients with breast cancer: a nationwide database study in Japan.

Aim: This study estimated the incidence of moderate-to-severe drug-induced interstitial lung disease (ILD) among patients with breast cancer in Japan. Methods: We analyzed a large nationwide database of patients with breast cancer treated with anticancer therapies between 2009 and 2022. ILD was identified using diagnostic codes and treatment records. Results: Of the 81,601 patients, 1042 developed ILD requiring corticosteroids, corresponding to an incidence rate of 1.41 per 100 person-years. The incidence varied across years and treatment regimens. Most ILD incidents occurred within the initial 90-day period post-anticancer therapy initiation. Conclusion: Increase in ILD cases and potential risk variations among treatments underline the importance of continued monitoring, especially during treatment onset, and ILD management in patients with breast cancer undergoing therapy.

This article investigates how often a lung condition known as interstitial lung disease (ILD) occurs in patients treated for breast cancer in Japan. ILD can cause inflammation and damage to the lungs and can be a side effect of some cancer treatments. The study looked at over 81,000 patients with breast cancer from 2009 to 2022. A total of 1042 patients developed ILD that required treatment with steroids to reduce inflammation. This number suggests that ILD occurred in 1.41 out of every 100 patients treated each year. The study noted that the chances of developing ILD varied over the years and depended on the type of cancer treatment. The findings showed that ILD is a risk factor for patients undergoing breast cancer treatment, and the risk can change depending on the treatment they receive. This highlights the importance of doctors keeping a close eye on their patients, especially early in the treatment process, to identify and manage any signs of ILD. Careful monitoring can help improve the health and treatment outcomes of patients with breast cancer. The study also points to the need for more research to understand why ILD occurs and how to prevent or treat it.

Crystal structures of EfeB and EfeO in a bacterial siderophore-independent iron transport system.

EfeUOB is a siderophore-independent iron uptake mechanism in bacteria. EfeU, EfeO, and EfeB are a permease, an iron-binding or electron-transfer protein, and a peroxidase, respectively. A Gram-negative bacterium, Sphingomonas sp. strain A1, encodes EfeU, EfeO, EfeB together with alginate-binding protein Algp7, a truncated EfeO-like protein (EfeOII), in the genome. The typical EfeO (EfeOI) consists of N-terminal cupredoxin and C-terminal M75 peptidase domains. Here, we detail the structure and function of bacterial EfeB and EfeO. Crystal structures of strain A1 EfeB and Escherichia coli EfeOI were determined at 2.30 Å and 1.85 Å resolutions, respectively. A molecule of heme involved in oxidase activity was bound to the C-terminal Dyp peroxidase domain of EfeB. Two domains of EfeOI were connected by a short loop, and a zinc ion was bound to four residues, Glu156, Glu159, Asp173, and Glu255, in the C-terminal M75 peptidase domain. These residues formed tetrahedron geometry suitable for metal binding and are well conserved among various EfeO proteins including Algp7 (EfeOII), although the metal-binding site (HxxE) is proposed in the C-terminal M75 peptidase domain. This is the first report on structure of a typical EfeO with two domains, postulating a novel metal-binding motif "ExxE-//-D-//-E" in the EfeO C-terminal M75 peptidase domain.

Hsp104-dependent ability to assimilate mannitol and sorbitol conferred by a truncated Cyc8 with a C-terminal polyglutamine in Saccharomyces cerevisiae.

Tup1-Cyc8 (also known as Tup1-Ssn6) is a general transcriptional corepressor. D-Mannitol (mannitol) and D-sorbitol (sorbitol) are the major polyols in nature. Budding yeast Saccharomyces cerevisiae is unable to assimilate mannitol or sorbitol, but acquires the ability to assimilate mannitol due to a spontaneous mutation in TUP1 or CYC8. In this study, we found that spontaneous mutation of TUP1 or CYC8 also permitted assimilation of sorbitol. Some spontaneous nonsense mutations of CYC8 produced a truncated Cyc8 with a C-terminal polyglutamine. The effects were guanidine hydrochloride-sensitive and were dependent on Hsp104, but were complemented by introduction of CYC8, ruling out involvement of a prion. Assimilation of mannitol and sorbitol conferred by other mutations of TUP1 or CYC8 was guanidine hydrochloride-tolerant. It is physiologically reasonable that S. cerevisiae carries this mechanism to acquire the ability to assimilate major polyols in nature.

Binding mode of metal ions to the bacterial iron import protein EfeO.

The tripartite EfeUOB system functions as a low pH iron importer in Gram-negative bacteria. In the alginate-assimilating bacterium Sphingomonas sp. strain A1, an additional EfeO-like protein (Algp7) is encoded downstream of the efeUOB operon. Here we show the metal binding mode of Algp7, which carries a M_75 metallopeptidase motif. The Algp7 protein was purified from recombinant E. coli cells and was subsequently characterized using differential scanning fluorimetry, fluorescence spectrometry, atomic absorption spectroscopy, and X-ray crystallography. The fluorescence of a dye, SYPRO Orange, bound to denatured Algp7 in the absence and presence of metal ions was measured during heat treatment. The fluorescence profile of Algp7 in the presence of metals such as ferric, ferrous, and zinc ions, shifted to a higher temperature, suggesting that Algp7 binds these metal ions and that metal ion-bound Algp7 is more thermally stable than the ligand-free form. Algp7 was directly demonstrated to show an ability to bind copper ion by atomic absorption spectroscopy. Crystal structure of metal ion-bound Algp7 revealed that the metal ion is bound to the cleft surrounded by several acidic residues. Four residues, Glu79, Glu82, Asp96, and Glu178, distinct from the M_75 motif (His115xxGlu118), are coordinated to the metal ion. This is the first report to provide structural insights into metal binding by the bacterial EfeO element.

A solute-binding protein in the closed conformation induces ATP hydrolysis in a bacterial ATP-binding cassette transporter involved in the import of alginate.

The Gram-negative bacterium Sphingomonas sp. A1 incorporates alginate into cells via the cell-surface pit without prior depolymerization by extracellular enzymes. Alginate import across cytoplasmic membranes thereby depends on the ATP-binding cassette transporter AlgM1M2SS (a heterotetramer of AlgM1, AlgM2, and AlgS), which cooperates with the periplasmic solute-binding protein AlgQ1 or AlgQ2; however, several details of AlgM1M2SS-mediated alginate import are not well-understood. Herein, we analyzed ATPase and transport activities of AlgM1M2SS after reconstitution into liposomes with AlgQ2 and alginate oligosaccharide substrates having different polymerization degrees (PDs). Longer alginate oligosaccharides (PD ≥ 5) stimulated the ATPase activity of AlgM1M2SS but were inert as substrates of AlgM1M2SS-mediated transport, indicating that AlgM1M2SS-mediated ATP hydrolysis can be stimulated independently of substrate transport. Using X-ray crystallography in the presence of AlgQ2 and long alginate oligosaccharides (PD 6-8) and with the humid air and glue-coating method, we determined the crystal structure of AlgM1M2SS in complex with oligosaccharide-bound AlgQ2 at 3.6 Å resolution. The structure of the ATP-binding cassette transporter in complex with non-transport ligand-bound periplasmic solute-binding protein revealed that AlgM1M2SS and AlgQ2 adopt inward-facing and closed conformations, respectively. These in vitro assays and structural analyses indicated that interactions between AlgM1M2SS in the inward-facing conformation and periplasmic ligand-bound AlgQ2 in the closed conformation induce ATP hydrolysis by the ATP-binding protein AlgS. We conclude that substrate-bound AlgQ2 in the closed conformation initially interacts with AlgM1M2SS, the AlgM1M2SS-AlgQ2 complex then forms, and this formation is followed by ATP hydrolysis.

Clinical evaluations of autologous fibrin glue and polyglycolic acid sheets as oral surgical wound coverings after partial glossectomy.

Polyglycolic acid (PGA) sheets and commercial fibrin glue are commonly used to cover open wound surfaces in oral surgery. Compared to commercial fibrin glue composed of pooled allogeneic blood, autologous fibrin glue is less expensive and poses lower risks of viral infection and allergic reaction. Here, we evaluated postoperative pain, scar contracture, ingestion, tongue dyskinesia, and postoperative bleeding in 24 patients who underwent partial glossectomy plus the application of a PGA sheet and an autologous fibrin glue covering (autologous group) versus 11 patients in whom a PGA sheet and commercial fibrin glue were used (allogeneic group). The evaluated clinical measures were nearly identical in both groups. Remarkable wound surface granulation was recognized in two cases in the autologous group. No complications were observed in either group, including viral infection or allergic reaction. Abnormal postoperative bleeding in the wound region was observed in one case in the allogeneic group. Coagulation and adhesion of the autologous fibrin glue were equivalent to those of conventional therapy with a PGA sheet and commercial fibrin glue. Thus, our results show that covering wounds with autologous fibrin glue and PGA sheets may help avoid the risks of viral infection and allergic reaction in partial glossectomy cases.

Structure of a Bacterial ABC Transporter Involved in the Import of an Acidic Polysaccharide Alginate.

The acidic polysaccharide alginate represents a promising marine biomass for the microbial production of biofuels, although the molecular and structural characteristics of alginate transporters remain to be clarified. In Sphingomonas sp. A1, the ATP-binding cassette transporter AlgM1M2SS is responsible for the import of alginate across the cytoplasmic membrane. Here, we present the substrate-transport characteristics and quaternary structure of AlgM1M2SS. The addition of poly- or oligoalginate enhanced the ATPase activity of reconstituted AlgM1M2SS coupled with one of the periplasmic solute-binding proteins, AlgQ1 or AlgQ2. External fluorescence-labeled oligoalginates were specifically imported into AlgM1M2SS-containing proteoliposomes in the presence of AlgQ2, ATP, and Mg(2+). The crystal structure of AlgQ2-bound AlgM1M2SS adopts an inward-facing conformation. The interaction between AlgQ2 and AlgM1M2SS induces the formation of an alginate-binding tunnel-like structure accessible to the solvent. The translocation route inside the transmembrane domains contains charged residues suitable for the import of acidic saccharides.

Structural studies on Laz, a promiscuous anticancer Neisserial protein.

Azurin and Laz (lipidated azurin) are 2 bacterial proteins with anticancer, anti-viral and anti-parasitic activities. Azurin, isolated from the bacterium Pseudomonas aeruginosa, termed Paz, demonstrates anticancer activity against a range of cancers but not against brain tumors. In contrast, Laz is produced by members of Gonococci/Meningococci, including Neisseria meningitides which can cross the blood-brain barrier to infect brain meninges. It has been previously reported that Laz has an additional 39 amino acid moiety, called an H.8 epitope, in the N-terminal part of the azurin moiety that allows Laz to cross the entry barrier to brain tumors such as glioblastomas. Exactly, how the H.8 epitope helps the azurin moiety of Laz to cross the entry barriers to attack glioblastoma cells is unknown. In this paper, we describe the structural features of the H.8 moiety in Laz using X-ray crystallography and demonstrate that while the azurin moiety of Laz adopts a ß-sandwich fold with 2 ß-sheets arranged in the Greek key motif, the H.8 epitope was present as a disordered structure outside the Greek key motif. Structures of Paz and H.8 epitope-deficient Laz are well superimposed. The structural flexibility of the H.8 motif in Laz explains the extracellular location of Laz in Neisseria where it can bind the key components of brain tumor cells to disrupt their tight junctions and allow entry of Laz inside the tumors to exert cytotoxicity.

Are octogenarians in good condition after cardiac valvular surgery?

PURPOSE: With the aging of society in developed countries and advances in surgical technology in recent years, surgery is increasing in elderly patients. When performing surgery in older patients, both surgical outcomes and the maintenance of postoperative quality of life (QOL) are important issues. This study investigated surgical outcomes and postoperative QOL in octogenarians who underwent cardiac valvular surgery. METHODS AND RESULTS: Fifty-nine (16 males) octogenarians (80-89 years old, mean age, 82.4 ± 2.4 years) underwent cardiac valvular surgery between August 1999 and June 2011. A QOL questionnaire, which included the Barthel Index (BI), Fillenbaum Instrumental Activities of Daily Living (FIADL), and the Vitality Index (VI), was sent to all survivors. Kaplan-Meier analysis was used to assess survival. Hospital mortality was 1.6% (1 patient). The 3-, 5-, and 7-year survival rates were 81.2%, 75.4%, and 67.8%, respectively. The BI showed that 87.5% of patients did not require caregiving, the FIADL showed that 32.5% were highly independent, and the VI showed that 87.5% were motivated to live. CONCLUSIONS: Short-term outcomes were satisfactory, with low complication and mortality rates. Mid-term outcomes showed maintenance of the minimal required ADL and good motivation for living. However, independence in social activities was decreased, suggesting the need for comprehensive social support.

A novel bleb-dependent polysaccharide export system in nitrogen-fixing Azotobacter vinelandii subjected to low nitrogen gas levels.

The alginate biofilm-producing bacterium Azotobacter vinelandii aerobically fixes nitrogen by oxygen-sensitive nitrogenases. Here we investigated the bacterial response to nitrogen/oxygen gas mixtures. A. vinelandii cells were cultured in nitrogen-free minimal media containing gas mixtures differing in their ratios of nitrogen and oxygen. The bacteria did not grow at oxygen concentrations >75% but grew well in the presence of 5% nitrogen/25% oxygen. Growth of wild-type and alginate-deficient strains when cultured with 50% oxygen did not differ substantially, indicating that alginate is not required for the protection of nitrogenases from oxygen damage. In response to decreasing nitrogen levels, A. vinelandii produced greater amounts of alginate, accompanied by the formation of blebs on the cell surface. The encystment of vegetative cells occurred in tandem with the release of blebs and the development of a multilayered exine. Immunoelectron microscopy using anti alginate-antibody revealed that the blebs contained alginate molecules. By contrast, alginate-deficient mutants could not form blebs. Taken together, our data provide evidence for a novel bleb-dependent polysaccharide export system in A. vinelandii that is activated in response to low nitrogen gas levels.

Pharmacokinetics of linezolid in the mediastinum and pleural space.

The purpose of this study was to investigate the penetration of linezolid into the mediastinum and pleural space by comparing its concentration in the serum, mediastinum, and pleural space. The linezolid area under the concentration-versus-time curve from zero to 12h (AUC)(mediastinum fluid/serum) and AUC(pleural fluid/serum) ratio were 1.32 and 1.64, respectively. The results suggest that the linezolid concentration in the mediastinum varies as in the serum, and that the concentration in the mediastinum is the same as or greater than that in the serum.

Recognition of heteropolysaccharide alginate by periplasmic solute-binding proteins of a bacterial ABC transporter.

Alginate is a heteropolysaccharide that consists of ß-D-mannuronate (M) and α-L-guluronate (G). The Gram-negative bacterium Sphingomonas sp. A1 directly incorporates alginate into the cytoplasm through the periplasmic solute-binding protein (AlgQ1 and AlgQ2)-dependent ABC transporter (AlgM1-AlgM2/AlgS-AlgS). Two binding proteins with at least four subsites strongly recognize the nonreducing terminal residue of alginate at subsite 1. Here, we show the broad substrate preference of strain A1 solute-binding proteins for M and G present in alginate and demonstrate the structural determinants in binding proteins for heteropolysaccharide recognition through X-ray crystallography of four AlgQ1 structures in complex with saturated and unsaturated alginate oligosaccharides. Alginates with different M/G ratios were assimilated by strain A1 cells and bound to AlgQ1 and AlgQ2. Crystal structures of oligosaccharide-bound forms revealed that in addition to interaction between AlgQ1 and unsaturated oligosaccharides, the binding protein binds through hydrogen bonds to the C4 hydroxyl group of the saturated nonreducing terminal residue at subsite 1. The M residue of saturated oligosaccharides is predominantly accommodated at subsite 1 because of the strict binding of Ser-273 to the carboxyl group of the residue. In unsaturated trisaccharide (ΔGGG or ΔMMM)-bound AlgQ1, the protein interacts appropriately with substrate hydroxyl groups at subsites 2 and 3 to accommodate M or G, while substrate carboxyl groups are strictly recognized by the specific residues Tyr-129 at subsite 2 and Lys-22 at subsite 3. Because of this substrate recognition mechanism, strain A1 solute-binding proteins can bind heteropolysaccharide alginate with different M/G ratios.

Survival and quality of life of octogenarians who underwent mechanical valve replacement at a younger age.

PURPOSE: Mechanical valve replacement is associated with complications, however, there is little information on the quality of life (QOL) of octogenarians who had undergone mechanical valve replacement at a relatively younger age. We examined survival, valve-related events, and the QOL of octogenarians who had undergone mechanical valve replacement. METHODS: A total of 56 octogenarians who underwent mechanical valve replacement between 1969 and 1997 (age at the time of surgery, 65.6 ± 6.7 years), completed a questionnaire on survival, valve-related events, and QOL (basic activities of daily living, instrumental activities of daily living, mental health). RESULTS: The mean follow-up was 12.4 ± 6.6 years, and the cumulative follow-up period was 642.4 patient-years. Six valve-related deaths (0.9%/patient-year) were registered during the follow-up. Furthermore, 11 valve-related events (1.8%/patient-year) were recorded. The mean age of the 21 survivors was 82.9 ± 1.8 years, and 19 of the survivors lived at home. Their QOL was excellent. CONCLUSION: The valve-related deaths and events in octogenarians who had previously undergone mechanical valve replacement at a younger age were within acceptable limits. The QOL was similar to that of octogenarians described in previous studies.

Systolic anterior motion after mitral valve repair: predicting factors and management.

PURPOSE: The aim of this study was to determine the mechanism of systolic anterior motion (SAM) after mitral valve (MV) repair by analyzing the clinical data of patients with MV repair. METHODS: A total of 104 MV repairs were performed for patients with isolated degenerative posterior leaflet prolapse. Eight patients (7.7%) developed SAM with severe mitral regurgitation. We compared the preoperative and intraoperative findings of the two groups (8 patients in the SAM group, 96 in the non-SAM group) and reported the clinical courses of the SAM patients. RESULTS: Preoperative left ventricular end-diastolic and end-systolic diameters were significantly smaller and the preoperative left ventricular ejection fraction was significantly greater in the SAM group than in the non-SAM group. The number of patients with a sigmoid septum and the number with anterior leaflet-septal contact (LSC) during diastole were significantly larger in the SAM group. Incidence of billowing posterior leaflet, prolapsed segments, and operative techniques were comparable for the two groups. SAM improved with correction of hemodynamic status in four patients. In four other patients secondary cardiopulmonary bypass was required to resolve SAM. SAM resolved with additional repairs in two patients, whereas the other two required MV replacement. Of the six patients in whom conservative treatment or re-repair was successful, one had recurrent SAM 3 months after surgery. CONCLUSION: The sigmoid septum and LSC may predict SAM after MV repair. A strict follow-up is imperative for patients with persistent or recurrent SAM.

Primary cardiac osteosarcoma with imaging that revealed no calcification.

Osteosarcoma is a rare primary cardiac malignancy. Calcification on imaging is crucial to differentiating osteosarcoma, but we encountered a case that was difficult to diagnose because imaging revealed no calcification. A 67-year-old man was admitted for heart failure. Echocardiography demonstrated mitral regurgitation and a mass in the left atrium. A cardiac malignancy was suspected. Computed tomography revealed no calcification. Operation was performed, and histopathological examination identified the tumor as an osteosarcoma.

Ten years experience of aortic root replacement using a modified bentall procedure with a carrel patch and inclusion technique.

OBJECTIVE: A modified Bentall procedure with a Carrel patch and inclusion technique (Modified Bentall Procedure) has been used to treat combined disease of the aortic valve and aortic root. The current study examined the outcomes of this surgical technique. MATERIALS AND METHODS: Between April 1999 and March 2009, 16 patients (10 males, 6 females; 63.3 ± 9.4 years) underwent elective surgery involving the Modified Bentall Procedure and no additional surgery, so they were included in the study. RESULTS: The mean cardiopulmonary bypass time was 140.2 ± 34.4 min (range: 97-232 min), and aortic cross-clamp time was 97.3 ± 16.6 min (range: 76-132 min). There were no hospital deaths. No patients required additional surgery to correct excessive bleeding. The follow-up rate was 100% (16/16). The mean follow-up period was 5.6 ± 2.8 years (range: 0.7-9.9 years). One of the 16 patients died (6.3%) due to lung cancer, and 1 of the 15 surviving patients required additional surgery (6.7%) for a thoracic aortic aneurysm. Kaplan-Meier analysis found that 1-year and 5-year survival and event-free survival rates were all 100%. CONCLUSIONS: The Modified Bentall Procedure provided satisfactory results over both the short term and long term.

Double patch technique for closing acute ventricular septal perforation.

PURPOSE: Ventricular septal perforation (VSP) is a rare but life-threatening complication of acute myocardial infarction (AMI). Even with assisted circulation heart failure often progresses quickly, and urgent surgical intervention is required to close the VSP. For several years, we have been performing a double patch closure technique using an equine pericardial patch. In this report, we present details of our patch closure technique and the VSP surgical results; we also examine the problems we encountered. METHODS: The present study was a review of nine patients who underwent our patch closure of VSP secondary to AMI. We used a large double-layered equine pericardial patch to close the VSP and did not exclude the infarction area from the left ventricular cavity. No necrotic myocardium was excised to avoid simultaneous excision of stunned myocardium. RESULTS: No patients died within 30 days of the surgery, and there were no in-hospital deaths. Over a mean clinical follow-up period of 4.5 years, no residual shunts were detected, and all patients were alive and had New York Heart Association functional class II. CONCLUSION: Our patch closure technique was effective for closing VSPs securely. The operating times and cardiopulmonary bypass times were short, and blood loss was minimal. Our patch closure technique may improve the prognosis of VSP.

Seven-year results of endovascular aneurysm repairs of abdominal aortic aneurysms with custom-made stent grafts.

BACKGROUND: The purpose of this study was to determine the long-term results of a 7-year follow-up of endovascular aneurysm repairs (EVARs) of abdominal aortic aneurysms (AAAs) using custom-made stent grafts (SGs). METHODS AND RESULTS: We performed a retrospective review of 17 patients (14 males, 3 females; mean age: 74.3 +/- 7.9 years; range: 53-85) undergoing EVAR of infrarenal aortic aneurysms at our institution from April 2000 to August 2006. The primary and secondary clinical success rates were 82.4% (14/17) and 100% (17/17). The initial and short-term clinical success rates were 100%. During follow-up (mean: 38.8 +/- 35.9 months; range: 0.8-90 months), 4 patients died, but there was no aneurysm-related death. In 2 patients, additional surgery was performed. The long-term clinical success rate was 83.3% (5/6). In the Kaplan-Meier curve, the 1- and 5-year survival rates were 55.0% and 45.8%, respectively. CONCLUSION: The initial and short-term clinical success rates were 100%; regarding the short-term, aneurysm-related death could be avoided. However, during long-term follow-up, aneurysm-related events did occur. Follow-up should be performed over a long period.

A patient in whom survival was achieved by acute-stage surgery for infective endocarditis complicated by a cerebral hemorrhage.

We report a patient who underwent mitral valve replacement despite acute-stage cerebral hemorrhage related to a complication of infective endocarditis (IE) and rupture of intracerebral mycotic aneurysm, achieving survival. The patient was a 24-year-old female. She consulted our hospital because of a fever and was diagnosed with IE and a cerebral hemorrhage. A blood culture test on admission revealed methicillin-sensitive Staphylococcus aureus. Transthoracic echocardiography revealed giant vegetation in the left atrium. Besides the management of an acute-stage cerebral hemorrhage, medical therapy for IE was performed. However, a severe infection was refractory, and mitral-regurgitation-related heart failure deteriorated. Despite acute-stage cerebral hemorrhage, mitral valve replacement was performed 10 days after admission because advanced disseminated intravascular coagulation syndrome made conservative management difficult. After surgery, neither exacerbation of cerebral symptoms nor recurrent IE was noted, and the patient was discharged.