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.

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.

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.

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.

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.

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.

[Effect of Chai-ling-tang (Sairei-to) for methotrexate concentration degradation delay by methotrexate high-dose therapy].

When methotrexate (MTX) salvage chemotherapy is performed for primary brain malignant lymphoma, use of leucovonrin rescue must often be extended due to delays in the degradation of blood concentration. We examined whether delay in MTX blood concentration degradation could be prevented by chai-ling-tang (Sairei-to) which has diuretic action. In the five cases examined were MTX blood concentration 72 hours after MTX administration was more than 1 x 10(-7) M. A single dose of 3 g of chai-ling-tang was administered three times on the day the MTX salvage chemotherapy was subsequently performed. MTX blood concentration at 72 hours post MTX administration and subsequent chai-ling-tang administration was less than 1 x 10(-7) M in all five cases. In addition, urea nitrogen and creatinine levels in serum increased and creatinine clearance decreased following MTX administration, however these changes induced by MTX administration were reduced by chai-ling-tang administration. Chai-ling-tang was effective in preventing an MTX deferent delay in MTX high-dose therapy by improving renal blood flow.

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.

Shortening of ventricular shunt catheter associated with cranial growth: effect of the frontal and parieto-occipital access route on long-term shunt patency.

OBJECT: The authors present the difference of shortening the ventricular shunt catheter associated with growth of the cranium between the frontal and parieto-occipital access, a key for long patency of the shunt implanted in children. MATERIALS AND METHOD: Our retrospective study included 28 children. In group A (n=9), the catheter was inserted through a frontal burr hole and in group B (n=19), through a parieto-occipital burr hole. To compare changes that occurred in the interval between the time of insertion and follow-up in the length of the ventricular catheter in the cranium and to assess displacement of the burr used for catheter entry. RESULTS: The results show that ventricular catheter shortening and burr-hole displacement were more pronounced in group A. CONCLUSIONS: This study documents that insertion of the ventricular catheter via the frontal route in children resulted in a higher incidence of shortening due to greater displacement of the burr hole adjacent to the coronal suture. Therefore, we recommend that the parieto-occipital route be used to maintain long-term shunt function.

Auditory alert system for fluorescence-guided resection of gliomas.

Protoporphyrin IX (PPIX) fluorescence-guided brain tumor resection using 5-aminolevulinic acid labeling is one of the most valuable tools available to determine the extent of glioma infiltration, but requires repeated spectroscopic evaluation of the tissue. The present method informs the surgeon of residual tumor in real time using spectrum analysis of PPIX fluorescence and an audible alert system. The target region was illuminated with a laser with a peak wavelength of 405 +/- 1 nm in addition to the usual microscope halogen lamp during tumor resection. Analysis of the spectrum detected the PPIX peak using a difference in relative intensity exceeding 500 at 636 nm and 632 nm, when an audible alert was transmitted to the surgeon. Using this method, infiltration of glioma was detected and confirmed histologically in three of six glioblastomas. The surgeon can detect tumor infiltration far more objectively and with less effort using this system during tumor resection.

Malignant transformation of intracranial mature teratoma to yolk sac tumor after late relapse. Case report.

The recurrence of intracranial mature teratomas as germ cell tumors of different histological types is rarely reported. The authors describe the first case of the malignant transformation of an intracranial mature teratoma into a yolk sac tumor in a 16-year-old boy who presented with a 1-month history of anorexia and somnolence. Seven years prior to this presentation, the boy had undergone surgery for extirpation of a mature pineal teratoma. Computed tomography images obtained at his second presentation revealed a homogeneously enhanced mass within the third ventricle. The tumor was resected and the results of a histological examination were consistent with a yolk sac tumor. After resection, the patient underwent radiation therapy followed by chemotherapy with cisplatin and etoposide but died of tumor progression 15 months after his second hospitalization.

Fluorescence-guided resection of metastatic brain tumors using a 5-aminolevulinic acid-induced protoporphyrin IX: pathological study.

We performed a pathological study to identify the locus of production of protoporphyrin IX (PPIX) in human metastatic brain tumors. Patients with metastatic brain tumors (n = 11) received 1 g of 5-aminolevulinic acid (5-ALA) perorally 2 h before undergoing surgery. The target region was exposed to laser light with a peak wavelength of 405 +/- 1 nm and an output of 40 mW. Tissue samples from the tumor bulk and surrounding areas were examined by histological and fluorescence methods. Of the 11 tumors, 9 manifested PPIX fluorescence in the tumor bulk and peritumoral brain tissue. Our findings indicate that PPIX fluorescence can be observed in peritumoral edematous areas that are free of neoplastic cells, because PPIX produced by neoplastic cells leaks into the surrounding edematous area.

Efficacy of a direct puncture approach for anterior circulation aneurysms using a newly developed guiding catheter - especially for geriatric patients.

BACKGROUND: Endovascular surgery is being increasingly used as an alternative to craniotomy clipping surgery, especially for aged patients and complicated cases. However, tortuous atherosclerotic arteries sometimes interfere with advancement of catheters so that direct puncture may be necessary. Short guiding catheters for use with this approach have been newly developed, as discussed in this article. METHODS: One hundred twenty three anterior circulation aneurysms in 121 patients were consecutively treated by endovascular coil embolization, of which 42 (34%) were older than 70 years. RESULTS: With 21 aneurysms, coil embolization via the transfemoral approach failed, but all could be successfully treated with the direct puncture approach with minor complications such as 1 transient ischemic attack and 1 nonsymptomatic minor leakage. In the aged patients, the direct puncture approach with short guiding catheter resulted in complete obliteration of aneurysms in 20 (71%) of 28 with follow-up angiography. CONCLUSION: Direct puncture using newly developed short guiding catheters is an alternative to femoral approaches for patients with anterior circulation aneurysm with tortuous arteries and obvious atherosclerotic change at bifurcations of the common carotid artery.

Confirmation of the initial pressure setting in Codman-Hakim programmable valves under magnification--technical note.

Codman-Hakim programmable valves allow neurosurgeons to adjust the opening pressure to the patient's particular clinical needs. However, the pressure control cam is extremely small, so identification of the pressure indicator and its operation is somewhat difficult when initially setting the opening pressure before implantation. We employed a commercial loupe on the program transmitter unit to sufficiently enlarge the view of the cam for clear identification. We recommend that the manufacturer to incorporate a loupe to provide a more sophisticated product.

Histological examination of false positive tissue resection using 5-aminolevulinic acid-induced fluorescence guidance.

Intraoperative 5-aminolevulinic acid (5-ALA)-induced fluorescence guidance for resection of malignant brain tumors was correlated with histological examination to investigate false positive findings in 42 patients with malignant glioma and six patients with metastatic brain tumor. Patients received a single 1 g oral dose of 5-ALA 2 hours before surgery. The tumor site was illuminated with a laser with a peak wavelength of 405 +/- 1 nm and output of 40 mW. Samples with strong fluorescence were obtained from the tumor bulk and samples with weak fluorescence from the tumor cavity. Fluorescence was observed in 36 of the 42 malignant gliomas and four of the six metastatic brain tumors. No tumor cells were found in fluorescent samples from six of the 36 malignant gliomas and all four metastatic brain tumors. Five of the six malignant gliomas were recurrent cases. Fluorescence was found in areas of peritumoral edema or inflammatory cell and reactive astrocyte infiltration. Intraoperative 5-ALA-induced fluorescence guidance is useful for the resection of initial malignant glioma since false positive results are rare, but only non-eloquent weak positive areas should be resected. In contrast, all weak positive areas of recurrent malignant gliomas must be resected. Weak positive areas of the peritumoral edema surrounding metastatic brain tumors should be removed carefully as false positive results are common.

[Superior sagittal sinus thrombosis caused by Crohn's disease and macrocytic anemia : a case report].

A 32-years-old man with a past history of hemorrhoids presenting with hemiparesis was diagnosed as having sagittal sinus thrombosis with hemorrtagic infarction. Laboratory data revealed macrocytic anemia (Hb 11.2 g/d/) with hypoproteinernia (5.5 g/d). After discharge the patient developed abdominal pain, diarrhea, edema in the leg and sustained anemia. Final diagnosis through colon fiberscope findings was Crohn's disease Macrocytic anemia seemed to be induced by Vit. B12 deficiency due to malabsorption. The mechanism and causal relationship between Crohn's disease and sinus thrombosis is discussed.

Disparity between adjusted and actual opening cerebrospinal fluid pressure in a patient with the Codman Hakim programmable valve: occult form of shunt failure due to head banging. Case report.

Although the Codman Hakim programmable valve (CHPV) system is popular, several problems arising from its design have been described. The authors report an additional cause of shunt failure associated with the use of the CHPV system. A 7-year-old girl who had previously undergone placement of a ventriculoperitoneal shunt with the CHPV system was admitted in a comatose state. Radiography did not demonstrate any breakage or change in the opening pressure of the valve. Manual pumping of the valve resulted in neurological improvement and shrinkage of the girl's ventricles. However, ventriculomegaly was again observed several hours later, and an emergency valve revision was performed. The manufacturer's examination of the extracted valve revealed a crack on the surface of the hard plastic housing covering the valve chamber, which appeared to be suggestive of some type of blunt trauma. In addition, the flat spring that transmitted resistance from the pressure control cam to the valve ball was deformed in such a manner that it caused excessive pressure against the valve ball. Because of this deformity, the actual opening pressure in this patient had reached a maximum level of 226 mm H2O instead of the adjusted pressure of 60 mm H2O. In patients with the CHPV system whose medical history is difficult to ascertain, the possibility of damage to the valve spring and the need for a prompt revision surgery should be considered. The authors conclude that placement of the CHPV system may be contraindicated in patients with a known habit of head banging.