Extracellular vesicles of Pseudomonas aeruginosa downregulate pyruvate fermentation enzymes and inhibit the initial growth of Staphylococcus aureus.

Staphylococcus aureus and Pseudomonas aeruginosa are well-known opportunistic pathogens that frequently coexist in chronic wounds and cystic fibrosis. The exoproducts of P. aeruginosa have been shown to affect the growth and pathogenicity of S. aureus, but the detailed mechanisms are not well understood. In this study, we investigated the effect of extracellular vesicles from P. aeruginosa (PaEVs) on the growth of S. aureus. We found that PaEVs inhibited the S. aureus growth independently of iron chelation and showed no bactericidal activity. This growth inhibitory effect was also observed with methicillin-resistant S. aureus but not with Acinetobacter baumannii, Enterococcus faecalis, S. Typhimurium, E. coli, Listeria monocytogenes, or Candida albicans, suggesting that the growth inhibitory effect of PaEVs is highly specific for S. aureus. To better understand the detailed mechanism, the difference in protein production of S. aureus between PaEV-treated and non-treated groups was further analyzed. The results revealed that lactate dehydrogenase 2 and formate acetyltransferase enzymes in the pyruvate fermentation pathway were significantly reduced after PaEV treatment. Likewise, the expression of ldh2 gene for lactate dehydrogenase 2 and pflB gene for formate acetyltransferase in S. aureus was reduced by PaEV treatment. In addition, this inhibitory effect of PaEVs was abolished by supplementation with pyruvate or oxygen. These results suggest that PaEVs inhibit the growth of S. aureus by suppressing the pyruvate fermentation pathway. This study reported a mechanism of PaEVs in inhibiting S. aureus growth which may be important for better management of S. aureus and P. aeruginosa co-infections.

Differential proteomic analysis and pathogenic effects of outer membrane vesicles derived from Acinetobacter baumannii under normoxia and hypoxia.

Acinetobacter baumannii is a major causative agent of nosocomial infections and its outer membrane vesicles (AbOMVs) have been shown to be involved in pathogenicity by transporting virulence factors and transferring information for communication between pathogens and host cells. Despite the fact that the infected sites of A. baumannii such as lungs and skin soft tissues are hypoxic, most studies on AbOMV virulence have used AbOMVs prepared under aerobic conditions. The present study aims to elucidate the protein profile and pathogenic impact of AbOMVs released under hypoxic condition. AbOMVs were isolated from A. baumannii under normoxic and hypoxic conditions, and their protein profiles were compared. The different effects of both normoxic and hypoxic AbOMVs in cytokine response from mouse macrophages, cytotoxicity to the human lung epithelial cells, and bacterial invasion were then investigated. Our results showed that A. baumannii under hypoxia released larger amounts of OMVs with different protein profiles. Although the cytotoxic effect of AbOMVs from normoxia and hypoxia were comparable, AbOMVs from normoxia induced higher TNF-α production and invasion of Staphylococcus aureus and Pseudomonas aeruginosa than those from hypoxia. On the other hand, AbOMVs significantly enhanced A. baumannii invasion into lung epithelial cells in a dose-dependent manner. These results clearly demonstrate that AbOMVs released from normoxic and hypoxic have different impacts in pathogenesis. This finding provides new insight into the complex interactions between A. baumannii, coinfecting pathogens and host cells via OMVs, in particular the different pathogenic effects of AbOMVs under normoxic and hypoxic conditions.

Miniaturized Synthetic Palmitoylation Motifs for Small-Molecule Localization in Living Cells.

Conjugating small-molecule ligands to synthetic motifs that can localize to specific organelles or membranes in living cells is a practical approach to develop compounds as chimeric tools or drugs that can manipulate biological processes in a subcellular site-specific manner. However, the number of available organelle-targeted synthetic motifs for small-molecule localization is limited. We have recently developed a synthetic myristoyl-DCys motif for small-molecule localization that undergoes S-palmitoylation via the cellular palmitoylation machinery and localizes to the Golgi surface. Herein, we show that the lipid acyl chain of the myristoyl (C14)-DCys motif can be as short as 10-carbons and still retain the palmitoylation-dependent Golgi localization property in cells. This discovery led to the identification of four new derivatives for small-molecule localization: tridecanoyl (C13)-, dodecanoyl (C12)-, undecanoyl (C11)-, and decanoyl (C10)-DCys motifs. We demonstrated that even the short decanoyl-DCys palmitoylation motif could be used to generate small-molecule ligand conjugates that functioned as chemical tools for controlling protein localization and cell signaling. The miniaturized synthetic palmitoylation motifs identified in this work may find applications in creating various Golgi-localizable chimeric molecules for use in chemical biology and drug development.

Feasibility and safety of platinum-doublet therapy in patients with small-cell lung cancer in the third-line setting: A multi-institutional retrospective study.

Small-cell lung cancer (SCLC) is a highly malignant tumor, and no standard third-line therapy has been established. The present study retrospectively analyzed the efficacy and safety of platinum-based regimens in patients with third-line SCLC who received third-line chemotherapy. The association of regimen type with overall survival (OS) or time to treatment failure (TTF) was evaluated using the Cox hazard proportional method, including well-known covariates affecting the prognosis of SCLC. TTF and OS analyses were conducted using the Kaplan-Meier method. The data cutoff date was June 30, 2020. As a result, from January 2015 to August 2019, 111 patients were diagnosed with SCLC, and 37 received third-line chemotherapy. Subsequently, 15 patients received a platinum-doublet regimen, and 22 patients received a single-agent regimen. Only the type of regimen was significantly associated with TTF in univariate analysis (odds ratio, 0.44; 95% confidence interval, 0.20-0.95; P=0.03). There were no significant factors associated with OS. The median TTF of patients receiving a platinum-doublet regimen and those receiving a single-agent regimen were 3.9 and 2.3 months, respectively (P=0.03). The overall response rates of the platinum-doublet and single-agent regimens were 20.0 and 4.5%, respectively. Similarly, the disease control rates were 73.3 and 36.4% for platinum-doublet and single-agent regimens, respectively. There was a tendency for adverse events (AEs) with any grade to occur more often in platinum-based regimens compared with in single-agent regimens. Severe AEs of grade 3 or higher were observed more often in the platinum-based regimen, especially in myelosuppression. In conclusion, the present study demonstrated the feasibility and safety of platinum-doublet regimens in patients with SCLC in a third-line setting (Registration no. 2020-048. Date of registration, June 5, 2020).

A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation.

Chemogenetic methods enabling the rapid translocation of specific proteins to the plasma membrane (PM) in a single protein-single ligand manner are useful tools in cell biology. We recently developed a technique, in which proteins fused to an Escherichia coli dihydrofolate reductase (eDHFR) variant carrying N-terminal hexalysine residues are recruited from the cytoplasm to the PM using the synthetic myristoyl-d-Cys-tethered trimethoprim (mDcTMP) ligand. However, this system achieved PM-specific translocation only when the eDHFR tag was fused to the N terminus of proteins, thereby limiting its application. In this report, we engineered a universal PM-targeting tag for mDcTMP-induced protein translocation by grafting the hexalysine motif into an intra-loop region of eDHFR. We demonstrate the broad applicability of the new loop-engineered eDHFR tag and mDcTMP pair for conditional PM recruitment and activation of various tag-fused signaling proteins with different fusion configurations and for reversibly and repeatedly controlling protein localization to generate synthetic signal oscillations.

Chemogenetic Control of Protein Localization and Mammalian Cell Signaling by SLIPT.

Chemical control of protein localization is a powerful approach for manipulating mammalian cellular processes. Self-localizing ligand-induced protein translocation (SLIPT) is an emerging platform that enables control of protein localization in living mammalian cells using synthetic self-localizing ligands (SLs). We recently established a chemogenetic SLIPT system, in which any protein of interest fused to an engineered variant of Escherichia coli dihydrofolate reductase, DHFRiK6, can be rapidly and specifically translocated from the cytoplasm to the inner leaflet of the plasma membrane (PM) using a trimethoprim (TMP)-based PM-targeting SL, mDcTMP. The mDcTMP-mediated PM recruitment of DHFRiK6-fusion proteins can be efficiently returned to the cytoplasm by subsequent addition of free TMP, enabling temporal and reversible control over the protein localization. Here we describe the use of this mDcTMP/DHFRiK6-based SLIPT system for inducing (1) reversible protein translocation and (2) synthetic activation of the Raf/ERK pathway. This system provides a simple and versatile tool in mammalian synthetic biology for temporally manipulating various signaling molecules and pathways at the PM.

Clinical Usefulness of Preoperative MCA Anatomical Scanning MRI in Thrombectomy Therapy for Acute Anterior Circulation Vessel Occlusion.

Objective: The aim of the present study was to evaluate the usefulness of the T2-weighted three-dimensional sequence method, known as "basi-parallel anatomical scanning (BPAS)-magnetic resonance imaging (MRI)," in demonstrating the running course of the obstructed middle cerebral artery (MCA) before acute mechanical thrombectomy. Methods: Patients whose M1 part and internal carotid artery (ICA) were occluded on preprocedural MRA, but well demonstrated on MCA anatomical scanning (MAS)-MRI were enrolled in this study. The MAS-MR images for patients in whom thrombectomy was performed were compared with the post-thrombectomy angiography. We compared the running course of the C1-M2 bifurcation on MAS-MRI and angiography after thrombectomy, and the results were classified into 3 groups (Excellent, Good, and Poor). Results: A total of 13 patients (range: 54-89) were enrolled, among whom 12 underwent thrombectomy. We compared MAS-MRI and post-thrombectomy angiography in 10. On comparison between MAS-MRI and post-procedural angiography, visualization was excellent in six (60%) patients. The mean age was 75.7 years, ranging from 54 to 89, and 6 were males. 3 patients had ICA occlusion and seven had MCA occlusion. Conclusion: MAS-MRI was considered useful to clarify the running course of the MCA before acute mechanical thrombectomy.

An Improved Intracellular Synthetic Lipidation-Induced Plasma Membrane Anchoring System for SNAP-Tag Fusion Proteins.

The ability to chemically introduce lipid modifications to specific intracellular protein targets would enable the conditional control of protein localization and activity in living cells. We recently developed a chemical-genetic approach in which an engineered SNAP-tag fusion protein can be rapidly relocated and anchored from the cytoplasm to the plasma membrane (PM) upon post-translational covalent lipopeptide conjugation in cells. However, the first-generation system achieved only low to moderate protein anchoring (recruiting) efficiencies and lacked wide applicability. Herein, we describe the rational design of an improved system for intracellular synthetic lipidation-induced PM anchoring of SNAP-tag fusion proteins. In the new system, the SNAPf protein engineered to contain an N-terminal hexalysine (K6) sequence and a C-terminal 10-amino acid deletion, termed K6-SNAPΔ, is fused to a protein of interest. In addition, a SNAP-tag substrate containing a metabolic-resistant myristoyl-DCys lipopeptidomimetic, called mDcBCP, is used as a cell-permeable chemical probe for intracellular SNAP-tag lipidation. The use of this combination allows significantly improved conditional PM anchoring of SNAP-tag fusion proteins. This second-generation system was applied to activate various signaling proteins, including Tiam1, cRaf, PI3K, and Sos, upon synthetic lipidation-induced PM anchoring/recruitment, offering a new and useful research tool in chemical biology and synthetic biology.

Golgi recruitment assay for visualizing small-molecule ligand-target engagement in cells.

The development of methods that allow detection of ligand-target engagement in cells is an important challenge in chemical biology and drug discovery. Here, we present a Golgi recruitment (G-REC) assay in which the ligand binding to the target protein can be visualized as Golgi-localized fluorescence signals. We show that the G-REC assay is applicable to the detection of various ligand-target interactions, ligand affinity comparison among distinct protein isoforms, and the monitoring of unmodified drug-target engagement in cells.

Selecting an appropriate surgical treatment instead of carotid artery stenting alone according to the patient's risk factors contributes to reduced perioperative complications in patients with internal carotid stenosis: a single institutional retrospective analysis.

This retrospective study was aimed to compare the perioperative complications for internal carotid artery stenosis (ICS) in a Japanese single institute between the use of carotid artery stenting (CAS) alone or the use of an appropriate individualized treatment method allowing either carotid endarterectomy (CEA) or CAS based on patient risk factors. Based on the policy at our hospital, only CAS was performed on patients (n = 33) between January 2005 and November 2009. From December 2009 to December 2012, either CEA or CAS (tailored treatment) was selected for patients (n = 61) based on individual patient risk factors. CEA was considered the first-line treatment in all cases. In high-risk CEA cases, CAS was performed instead (n = 11), whereas in low-risk CEA cases, CEA was performed (n = 19). Further, in moderate-risk CEA cases based on own criteria, CAS was considered first, whereas for high-risk CAS cases, CEA was performed (n = 17). For low-risk CAS cases, CAS was performed (n = 9). Perioperative clinical complications (any stroke, myocardial infarction, or death within 30 days) were compared between both periods. Significantly reduced perioperative complications were observed during the tailored period (4/61 sites, 6.6%) as compared with the CAS period (8/33 sites, 24.2%) [Fisher's exact test p = 0.022; odds ratio, 4.56 (CAS/tailored); 95% confidence interval, 1.26-16.5]. Selecting an appropriate individualized treatment method according to patient risk factors, as opposed to adhering to a single treatment approach such as CAS, may contribute to improved overall outcomes in patients with ICS.

Impact of platelet transfusion on survival of patients with intracerebral hemorrhage after administration of anti-platelet agents at a tertiary emergency center.

This study examined the impact of platelet transfusion (PLT) on the survival of intracerebral hemorrhage (ICH) patients who had been administered anti-platelet agents (APA). This retrospective cohort analysis investigated 432 patients (259 men, 60%) who were newly diagnosed with ICH between January 2006 and June 2011 at the tertiary emergency center of Kitasato University Hospital. Median age on arrival was 67.0 years (range, 40-95 years). ICH was subcortical in 72 patients (16.7%), supratentorial in 233 (53.9%), and infratentorial in 133 (30.8%). PLT was performed in 16 patients (3.7%). Within 90 days after admission to the center, 178 patients (41.2%) had died due to ICH. Before the onset of ICH, 66 patients had been prescribed APA because of atherosclerotic diseases. Multivariate regression analysis indicated APA administration was an independent risk factor for death within 7 days (odds ratio, 5.12; P = 0.006) and within 90 days (hazard ratio, 1.87; P = 0.006) after arrival. Regarding the effect of a PLT in ICH patients with APA, no patient with PLT died. PLT had a survival benefit on patients with ICH, according to our analysis. Further prospective analysis is necessary to confirm the effects of PLT on survival in ICH with APA.

Pathophysiological evaluation of cerecyte coil embolization for experimental broad neck aneurysms.

Cerecyte second-generation coils feature inner surfaces coated with an absorbable polyglycolic acid (PGA) polymer. Their use is expected to accelerate aneurysm organization, but time course data are limited. The present experimental study was therefore conducted to clarify the processes by pathological examination. Methods. Two types of experimental aneurysms were initially generated in adult mongrel dogs, one bifurcation and another of lateral wall type. Long-term persistence of each was defined by follow-up angiography for more than 1 year. Embolization of the aneurysms was then performed using only cerecyte coils, and follow-up angiography was conducted after 2 and 4 weeks followed by pathological examination. Results. Organization of both types of broad neck aneurysm was apparent 4 weeks after embolization, which is earlier as compared with already reported data for bare coils.

Altered hemodynamics associated with pathogenesis of the vertebral artery dissecting aneurysms.

The etiology of the vertebral dissecting aneurysms is largely unknown, and they frequently occurs in relatively healthy young men. Objectives and Methods. A series of 57 consecutive cases defined by angiography were evaluated with regard to deviation in the course of the affected and contralateral vertebral arteries. Division was into 3 types: Type I without any deviation, Type II with mild-to-moderate deviation but not over the midline; and Type III with marked deviation over to the contralateral side beyond the midline. Results. The most frequent type of VA running was Type III for the affected and Type I nonaffected side, with this being found in all 17 patients except one. All of the Type III dissections occurred just proximal to a tortuous portion, while in cases with Type-I- and Type-II-affected sides, the majority (33 of 39) occurred near the union of the vertebral artery. In 10 of 57, a non-dominant side was affected, all except one being of Type I or II. With 12 recent patients assessed angiographically in detail for hemodynamics, eleven patients showed contrast material retrograde inflowing into the pseudolumen from the distal portion of the dissection site. Turbulent blood flow was recognized in all of these patients with retrograde inflow. Conclusions. Turbulent blood flow is one etiology of vertebral artery dissection aneurysms, with the sites in the majority of the cases being just proximal to a tortuous portion or union of vessels. In cases with dissection proximal to the tortuous course of the vertebral artery, retrograde inflow will occur more frequently than antegrade, which should be taken into account in designing therapeutic strategies.

Efficacy of endovascular surgery for ruptured aneurysms with vasospasm of the parent artery.

INTRODUCTION: In the presence of vasospasm it is recommended that surgical clipping for a ruptured aneurysm should be delayed until it disappears, but this may be associated with re-rupture of the aneurysm resulting in a poor outcome. The indications for endovascular coil embolization in such cases are discussed. METHODS: Since November 2002, endovascular coil embolization has been used in 18 consecutive patients with ruptured aneurysm with vasospasm of the parent artery ranging from 2 to 28 days (mean 9 days) after the initial subarachnoid hemorrhage. After successful obliteration of the aneurysm, a microcatheter preceded by a guidewire was introduced into the peripheral vessels with vasospasm of the A2 or M2 portions in order to release the vasospasm mechanically. RESULTS: Endovascular procedures were performed successfully in all but one of the cases (94%), resulting in complete occlusion in 14 of 17 patients and mild dilation of the vasospasm in all 17 patients without technical complications or re-rupture of the aneurysm. In the one case of failure because of a tortuous artery, surgical clipping was performed after disappearance of the vasospasm. Cerebral infarction occurred in four patients, but only one correlated with the distribution of catheterization and the neurological deficits had completely disappeared 3 months after the onset. CONCLUSION: Catheterization of parent vessels in cases of vasospasm is safe for coiling and also mechanically releases vasospasm. Vasospasm of M2 and A2 segments can be treated with microcatheterization only.

A new transvenous approach to the carotid-cavernous sinus via the inferior petrooccipital vein.

OBJECT: The transvenous approach via the inferior petrosal sinus (IPS) is commonly used as the most appropriate for carotid-cavernous fistula (CCF) or cavernous sinus sampling. However, sometimes the IPS is not accessible because of anatomical problems and/or complications, therefore an alternative route is needed. In this paper, the authors present and discuss the utility of a transvenous approach to the cavernous sinus via the inferior petrooccipital vein. METHODS: Four patients, 3 with dural CCFs and the other with Cushing disease, in whom endovascular surgical attempts failed using a conventional venous approach via the IPS, underwent a transvenous approach to the cavernous sinus via the inferior petrooccipital vein (IPOV). One dural CCF case had only cortical venous drainage, the second CCF also mainly drained into the cortical vein with slight inflow into the superior ophthalmic vein and inferior ophthalmic vein, and the third demonstrated drainage into the superior and inferior ophthalmic veins and IPOV. RESULTS: In all cases, the cavernous sinus could be accessed successfully via this route and without complications. CONCLUSIONS: The transvenous approach to the cavernous sinus via the IPOV should be considered as an alternative in cases when use of the IPS is precluded by an anatomical problem and there are no other suitable venous approach routes.

Endosaccular embolization for a ruptured distal anterior inferior cerebellar artery aneurysm.

We report a rare case of a ruptured distal anterior inferior cerebellar artery aneurysm that was successfully treated by endosaccular embolization, and review the literature on the results of endovascular surgery for this condition.

Contrast stasis in large and giant internal carotid artery aneurysms as a good prognostic factor for endovascular coil embolization: retrospective study.

OBJECTIVE: Before treatment for large and giant aneurysms, we need some of the predictors to prognose a good result. In this retrospective study, we attempted to determine criteria such as angiographic signs to identify good candidates for effective endovascular surgery. METHODS: This study involved 45 patients with large or giant aneurysms treated by endovascular embolization. For angiographic study, we delivered a bolus injection of contrast medium. All aneurysms were confirmed angiographically and the morphology was defined in detail before endovascular embolization. We divided the patients into two groups based on angiographic findings. Group A (n=16) manifested stasis of the contrast medium in the aneurysm on venous phase. Group B (n=29) exhibited other findings. We retrospectively evaluated the relationship between stasis of the contrast medium in the aneurysm and results of endovascular embolization. RESULTS AND DISCUSSION: There was no significant difference between the two groups with respect to the size of the aneurysm. However, the neck/dome ratio (P=0·04) and size of the neck (P=0·003) were significantly different between groups A and B. The morphological outcome was better in group A than group B (P=0·03). We demonstrate that contrast stasis is a good predictor of outcome in patients with large or giant aneurysms to consider the endovascular embolization. Hemodynamic studies on large patient populations may reveal other factors predictive of a good treatment outcome.

Effectiveness of interferon-beta therapy for recurrent glioblastoma: a case report.

AIMS AND BACKGROUND: Glioblastoma has a poor prognosis, with few therapeutic options if it recurs. We report a case in which we were able to inhibit the growth of a recurrent glioblastoma by weekly single-dose administration of interferon-beta. CASE REPORT: A patient with recurrent glioblastoma after radiation and chemotherapy was treated with nimustine and interferon-beta. After 2 cycles of nimustine, the patient's leukocyte, neutrophil, and platelet counts showed grade 4 toxicity according to the National Cancer Institute's Common Toxicity Criteria. The patient was treated with a weekly single dose of interferon-beta at 6 x 10(6) IU. The tumor showed no remarkable changes after 18 months, and the patient's Karnofsky performance status remained at 50%. CONCLUSIONS: The administration of interferon-beta produced long-term control in one case of glioblastoma and may be an effective therapy.

Anterior-wall aneurysm of the internal carotid artery successfully treated solely by stenting: With special reference to etiology.

In this case, a ruptured anterior wall aneurysm of the internal carotid artery disappeared on angiography immediately after stent placement. We focus on the underlying nature of the lesion and this possible alternative treatment.

Dural arteriovenous fistulas in the cavernous sinus: clinical research and treatment.

Introduction. The purpose of this paper is to clarify the clinical course, with the dural carotid cavernous fistula (CCF), featuring a pallet of symptoms, paying special attention to radiological findings. Methods. Seventy-six consecutive patients with dural CCFs were investigated in detail, all of whom were defined by angiography. Results. The most common initial symptom was diplopia in 47 patients (62%) and the most frequently observed on arrival were type II, featuring cranial nerve palsies followed by the classical triad in 27, and then type I only with cranial nerve palsies. The time until admission with type I (mean: 6.7 W ± 6.0) was significantly shorter than that with type II (mean: 25.1 W ± 23.5). Branches from bilateral carotid arteries widely inflowing into bilateral carotid cavernous sinus were present in 30 (39%), 20 (26%) of which also demonstrated direct inflow into the intercavernous sinus. type I and II had more multiple venous drainage routes as compared with type III (classical triad only on arrival) and IV (initial development of the classical triad followed by cranial nerve palsy). Conclusion. In our series of dural CCF patients, the most common initial symptom was cranial nerve palsy, mostly featuring multiple venous drainage including cortical drainage. Such palsies should be added to the classical triad as indicative symptoms. Bilateral carotid arteries often inflow into cavernous and intercavernous sinuses, which should be taken into account in choice of therapeutic strategy.