Computed tomography-guided cutting needle biopsy for lung nodules: when the biopsy-based benign results are real benign.

BACKGROUND: Computed tomography (CT)-guided cutting needle biopsy (CNB) is an effective diagnostic method for lung nodules (LNs). The false-negative rate of CT-guided lung biopsy is reported to be up to 16%. This study aimed to determine the predictors of true-negative results in LNs with CNB-based benign results. METHODS: From January 2011 to December 2015, 96 patients with CNB-based nonspecific benign results were included in this study as the training group to detect predictors of true-negative results. From January 2016 to December 2018, an additional 57 patients were included as a validation group to test the reliability of the predictors. RESULTS: In the training group, a total of 96 patients underwent CT-guided CNB for 96 LNs. The CNB-based results were true negatives for 82 LNs and false negatives for 14 LNs. The negative predictive value of the CNB-based benign results was 85.4% (82/96). Univariate and multivariate logistic regression analyses revealed that CNB-based granulomatous inflammation (P = 0.013, hazard ratio = 0.110, 95% confidential interval = 0.019-0.625) was the independent predictor of true-negative results. The area under the receiver operator characteristic (ROC) curve was 0.697 (P = 0.019). In the validation group, biopsy results for 47 patients were true negative, and 10 were false negative. When the predictor was used on the validation group, the area under the ROC curve was 0.759 (P = 0.011). CONCLUSIONS: Most of the CNB-based benign results were true negatives, and CNB-based granulomatous inflammation could be considered a predictor of true-negative results.

Ceramide-1-phosphate transfer protein promotes sphingolipid reorientation needed for binding during membrane interaction.

Lipid transfer proteins acquire and release their lipid cargoes by interacting transiently with source and destination biomembranes. In the GlycoLipid Transfer Protein (GLTP) superfamily, the two-layer all-α-helical GLTP-fold defines proteins that specifically target sphingolipids (SLs) containing either sugar or phosphate headgroups via their conserved but evolutionarily-modified SL recognitions centers. Despite comprehensive structural insights provided by X-ray crystallography, the conformational dynamics associated with membrane interaction and SL uptake/release by GLTP superfamily members have remained unknown. Herein, we report insights gained from molecular dynamics (MD) simulations into the conformational dynamics that enable ceramide-1-phosphate transfer proteins (CPTPs) to acquire and deliver ceramide-1-phosphate (C1P) during interaction with 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. The focus on CPTP reflects this protein's involvement in regulating pro-inflammatory eicosanoid production and autophagy-dependent inflammasome assembly that drives interleukin (IL-1ß and IL-18) production and release by surveillance cells. We found that membrane penetration by CPTP involved α-6 helix and the α-2 helix N-terminal region, was confined to one bilayer leaflet, and was relatively shallow. Large-scale dynamic conformational changes were minimal for CPTP during membrane interaction or C1P uptake except for the α-3/α-4 helices connecting loop, which is located near the membrane interface and interacts with certain phosphoinositide headgroups. Apart from functioning as a shallow membrane-docking element, α-6 helix was found to adeptly reorient membrane lipids to help guide C1P hydrocarbon chain insertion into the interior hydrophobic pocket of the SL binding site.These findings support a proposed 'hydrocarbon chain-first' mechanism for C1P uptake, in contrast to the 'lipid polar headgroup-first' uptake used by most lipid-transfer proteins.

Preoperative computed tomography-guided coil localization of sub-centimeter lung nodules.

INTRODUCTION: Lung nodules (LNs) are often identified in at-risk patients via low-dose computed tomography (CT) approaches. Sub-centimeter (≤ 1 cm) LNs (SCLNs) are particularly difficult for surgeons and pathologists to accurately treat and diagnose. AIM: To evaluate the clinical efficacy of preoperative CT-guided coil localization for SCLNs. MATERIAL AND METHODS: Between January 2015 and December 2019, consecutive patients at our hospital with SCLNs underwent CT-guided coil localization followed by video-assisted thoracoscopic surgery (VATS). We then assessed rates of technical success corresponding to the localization and VATS-guided wedge resection procedures and measured rates of localization-related complications. RESULTS: In total, 52 patients were analyzed in this study, with 66 total SCLNs being localized with one coil each. CT-guided coil localization achieved a 93.9% (62/66) technical success rate, and a mean duration of 15.2 ±4.5 minutes. Following coil localization, 6 (11.5%) patients experienced pneumothorax and 4 (7.7%) patients suffered hemoptysis, with 1 patient requiring the insertion of a chest tube to alleviate pneumothorax. VATS-guided wedge resection was associated with a 100% technical success rate, and no patients needed to undergo conversion to thoracotomy. One-stage VATS-guided wedge resection was conducted in the 12 patients with multiple SCLNs. The mean VATS duration was 128.9 ±66.7 minutes, and mean blood loss associated with this procedure was 83.0 ±67.7 ml. CONCLUSIONS: Preoperative CT-guided coil localization can safely and effectively achieve high rates of success when conducting the diagnostic VATS wedge resection of SCLNs.

Purification of Cytosolic Phospholipase A2α C2-domain after Expression in Soluble Form in Escherichia coli.

Previous expression/purification strategies for cytosolic phospholipase A2α C2-domain in Escherichia coli have relied on refolded protein recovered from inclusion bodies and sometimes containing C-terminal Cys139Ala and Cys141Ser substitutions to eliminate potential refolding complications induced by Cys residues. The protocol presented herein describes an effective method for the expression of cytosolic phospholipase A2α C2-domain in soluble form in E. coli and subsequent purification to homogeneity. This protocol, which utilizes a cleavable 6xHis-SUMO tag, has recently been used to gain insights into the structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase A2α ( Hirano et al., 2019 ).

Ceramide-1-phosphate transfer protein (CPTP) regulation by phosphoinositides.

Ceramide-1-phosphate transfer proteins (CPTPs) are members of the glycolipid transfer protein (GLTP) superfamily that shuttle ceramide-1-phosphate (C1P) between membranes. CPTPs regulate cellular sphingolipid homeostasis in ways that impact programmed cell death and inflammation. CPTP downregulation specifically alters C1P levels in the plasma and trans-Golgi membranes, stimulating proinflammatory eicosanoid production and autophagy-dependent inflammasome-mediated cytokine release. However, the mechanisms used by CPTP to target the trans-Golgi and plasma membrane are not well understood. Here, we monitored C1P intervesicular transfer using fluorescence energy transfer (FRET) and showed that certain phosphoinositides (phosphatidylinositol 4,5 bisphosphate (PI-(4,5)P2) and phosphatidylinositol 4-phosphate (PI-4P)) increased CPTP transfer activity, whereas others (phosphatidylinositol 3-phosphate (PI-3P) and PI) did not. PIPs that stimulated CPTP did not stimulate GLTP, another superfamily member. Short-chain PI-(4,5)P2, which is soluble and does not remain membrane-embedded, failed to activate CPTP. CPTP stimulation by physiologically relevant PI-(4,5)P2 levels surpassed that of phosphatidylserine (PS), the only known non-PIP stimulator of CPTP, despite PI-(4,5)P2 increasing membrane equilibrium binding affinity less effectively than PS. Functional mapping of mutations that led to altered FRET lipid transfer and assessment of CPTP membrane interaction by surface plasmon resonance indicated that di-arginine motifs located in the α-6 helix and the α3-α4 helix regulatory loop of the membrane-interaction region serve as PI-(4,5)P2 headgroup-specific interaction sites. Haddock modeling revealed specific interactions involving the PI-(4,5)P2 headgroup that left the acyl chains oriented favorably for membrane embedding. We propose that PI-(4,5)P2 interaction sites enhance CPTP activity by serving as preferred membrane targeting/docking sites that favorably orient the protein for function.

A Simple and Straightforward Approach for Generating Small, Stable, Homogeneous, Unilamellar 1-Palmitoyl 2-Oleoyl Phosphatidylcholine (POPC) Bilayer Vesicles.

Various methods have been developed to generate phosphoglyceride liposomes. Approaches resulting in homogeneous populations of unilamellar bilayer vesicles are generally preferred to mimic various cell membrane situations, as well as to optimize aqueous solute trapping efficiency using the least amount of lipid for biotechnological purposes. Most are time-consuming, often tedious, or require specialized equipment, and produce vesicles with limited shelf-life at room temperature or in cold storage. Herein, we describe a straightforward approach that avoids the preceding complications and streamlines the construction of unilamellar bilayer vesicles from 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/dihexanoyl phosphatidylcholine (DHPC) bicelle mixtures at room temperature. The resulting vesicles are small (32-36 nm diameter), unilamellar, bilayer vesicles that are homogeneous, stable, and resistant to freeze-thaw alterations. Graphic abstract: Cryo-EM of POPC vesicles formed by dilution of 0.5 q-value POPC/DHPC bicelle mix.

[12]aneN3-Based Gemini-Type Amphiphiles with Two-Photon Absorption Properties for Enhanced Nonviral Gene Delivery and Bioimaging.

Although a plethora of nonviral gene vectors have been developed for potential gene therapy, imageable gemini surfactants with stimuli-responsiveness and high transfection efficiency are still scarce for gene delivery. Herein, three gemini amphiphiles (DEDPP-4/8/12) consisting of an aggregation-induced emission (AIE) central fluorophore: 5,6-diphenylpyrazine-2,3-diester (DEDPP), decorated with triazole-[12]aneN3 as the hydrophilic moiety and alkyl chains of various lengths as the hydrophobic moiety, were designed and synthesized for trackable gene delivery via optical imaging. All three amphiphiles exhibited ultralow critical micelle concentrations (CMCs) (up to 3.40 × 10-6 M), prominent two-photon absorption properties, and solvatochromic fluorescence. Gel electrophoresis assays demonstrated that the migration of plasmid DNA was completely retarded after condensation with these gemini amphiphiles at low concentrations (up to 10 µM). In addition, the ester bond in these amphiphiles may facilitate vector degradation and DNA release, in response to esterase and the acidic environment inside cells. Upon self-assembly with DOPE to form liposomes, DEDPP-8/DOPE achieved the best transfection efficiency in four cell lines, and the transfection efficiency of DEDPP-8/DOPE in HeLa cell lines was 23.5-fold higher than that of Lipo2000, which is unusually high for small organic molecule-based nonviral vectors. Furthermore, excellent transfection efficiency of DEDPP-8/DOPE was obtained in the presence of serum, and the red fluorescence protein (RFP) gene was successfully transfected in zebrafish embryos. Both one- and two-photon fluorescence imaging clearly demonstrated the delivery process of plasmid DNA. This study demonstrated that gemini-type amphiphiles composed of a two-photon fluorophore core conjugated with triazole-[12]aneN3 via an ester bond afforded an unprecedentedly high transfection efficiency with excellent biocompatibility, which may provide new insights for the design and development of multifunctional nonviral gene vectors for imageable gene delivery.

The Bone Extracellular Matrix in Bone Formation and Regeneration.

Bone regeneration repairs bone tissue lost due to trauma, fractures, and tumors, or absent due to congenital disorders. The extracellular matrix (ECM) is an intricate dynamic bio-environment with precisely regulated mechanical and biochemical properties. In bone, ECMs are involved in regulating cell adhesion, proliferation, and responses to growth factors, differentiation, and ultimately, the functional characteristics of the mature bone. Bone ECM can induce the production of new bone by osteoblast-lineage cells, such as MSCs, osteoblasts, and osteocytes and the absorption of bone by osteoclasts. With the rapid development of bone regenerative medicine, the osteoinductive, osteoconductive, and osteogenic potential of ECM-based scaffolds has attracted increasing attention. ECM-based scaffolds for bone tissue engineering can be divided into two types, that is, ECM-modified biomaterial scaffold and decellularized ECM scaffold. Tissue engineering strategies that utilize the functional ECM are superior at guiding the formation of specific tissues at the implantation site. In this review, we provide an overview of the function of various types of bone ECMs in bone tissue and their regulation roles in the behaviors of osteoblast-lineage cells and osteoclasts. We also summarize the application of bone ECM in bone repair and regeneration. A better understanding of the role of bone ECM in guiding cellular behavior and tissue function is essential for its future applications in bone repair and regenerative medicine.

Emerging roles for human glycolipid transfer protein superfamily members in the regulation of autophagy, inflammation, and cell death.

Glycolipid transfer proteins (GLTPs) were first identified over three decades ago as ~24kDa, soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. Upon discovery that GLTPs use a unique, all-α-helical, two-layer 'sandwich' architecture (GLTP-fold) to bind glycosphingolipids (GSLs), a new protein superfamily was born. Structure/function studies have provided exquisite insights defining features responsible for lipid headgroup selectivity and hydrophobic 'pocket' adaptability for accommodating hydrocarbon chains of differing length and unsaturation. In humans, evolutionarily-modified GLTP-folds have been identified with altered sphingolipid specificity, e. g. ceramide-1-phosphate transfer protein (CPTP), phosphatidylinositol 4-phosphate adaptor protein-2 (FAPP2) which harbors a GLTP-domain and GLTPD2. Despite the wealth of structural data (>40 Protein Data Bank deposits), insights into the in vivo functional roles of GLTP superfamily members have emerged slowly. In this review, recent advances are presented and discussed implicating human GLTP superfamily members as important regulators of: i) pro-inflammatory eicosanoid production associated with Group-IV cytoplasmic phospholipase A2; ii) autophagy and inflammasome assembly that drive surveillance cell release of interleukin-1ß and interleukin-18 inflammatory cytokines; iii) cell cycle arrest and necroptosis induction in certain colon cancer cell lines. The effects exerted by GLTP superfamily members appear linked to their ability to regulate sphingolipid homeostasis by acting in either transporter and/or sensor capacities. These timely findings are opening new avenues for future cross-disciplinary, translational medical research involving GLTP-fold proteins in human health and disease. Such avenues include targeted regulation of specific GLTP superfamily members to alter sphingolipid levels as a therapeutic means for combating viral infection, neurodegenerative conditions and circumventing chemo-resistance during cancer treatment.

Macrocyclic polyamine [12]aneN3 modified triphenylamine-pyrazine derivatives as efficient non-viral gene vectors with AIE and two-photon imaging properties.

With the aim to develop a novel multifunctional gene delivery system that may overcome the common barriers of gene transfection, near-infrared fluorescent triphenylamine-pyrazine was modified with a DNA condensing triazole-[12]aneN3 moiety through different length alkyl ester linkages to afford three new non-viral gene vectors, TDM-A/B/C. All compounds showed prominent solvatochromic fluorescence (Stokes shift of up to 383 nm) and two-photon absorption properties (σ2P to 101 GM), and exhibited strong aggregation-induced emission (AIE). Gel electrophoresis demonstrated that plasmid DNA was completely condensed at a concentration of 10 µM (TDM-A), 14 µM (TDM-B) and 16 µM (TDM-C), and released in esterase and acidic environment. SEM demonstrated that the three compounds were able to self-assemble and co-aggregate with DNA to form regular nanoparticles. Experiments demonstrated that TDM-A/B/C was able to integrate with DNA through electrostatic interactions and supramolecular stacking, and the short alkyl linkage favored the strong interaction with DNA. Among the three compounds, TDM-B showed the best luciferase and GFP transfection activities in the presence of DOPE, which were 156% and 310% higher than those of Lipo2000, respectively. The transfection process of DNA was clearly traced through one- and two-photon fluorescence microscopy imaging. Cellular uptake inhibition assay indicated that the DNA complex entered the cell mainly via clathrin-independent endocytosis. Furthermore, the in vivo transfection experiments of TDM-B/DOPE were successfully implemented in zebra fish embryos, and the GFP gene expression level was superior to that of Lipo2000 (200%). Finally, this study clearly unraveled that the length of the alkyl linkage affected the DNA condensation and transfection activity, which can serve as a base for the future rational design of non-viral gene vectors.

Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes.

In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short "shelf life". Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different glycolipid transfer protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5 q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol injection. Among the notable advantages of using bicelle-generated model membranes are (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation and (ii) long "shelf life" after production (≥6 days) and resilience to freeze-thaw storage. The bicelle-dilution-based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs but also for LTP activity detection in crude cytosolic fractions of cell homogenates.

A 1,8-naphthalimide-[12]aneN3 derivative for efficient Cu2+ recognition, lysosome staining and siRNA delivery.

Development of multifunctional compounds as both fluorescence probes and non-viral vectors is still difficult till date. It is necessary to overcome many hurdles such as the balance of hydrophilic and hydrophobic moieties, binding affinity between multifunctional compound and targeting substrate, the cytotoxicity of multifunctional compound, and so on. In this work, the performances of compound 1 on Cu2+ recognition, lysosome staining and siRNA (small interfering RNA) delivery were investigated. It was found that compound 1 exhibited high selectivity and sensitivity toward Cu2+ in aqueous solutions. The fluorescence emission of 1 was quenched by a factor of 42-fold in the presence of Cu2+ ions. Even in the common pure organic solutions, still more than 8-fold fluorescence quenching was achieved. Due to its high sensitivity to the pH, the complex of 1-Cu was also successfully applied in selective staining of lysosome in HeLa cells. Furthermore, cellular uptake experiment revealed that compound 1 showed good RNA delivery ability in HeLa, HepG2, U2Os and MC3T3-E1 cells, and its performance was better than commercial agents lipofectamine 2000 and 25 kDa PEI (Polyethylenimine). The RNA interference effect mediated by compound 1 was further evaluated by real-time fluorescent quantitative PCR experiment. Compound 1 showed much higher transfection efficacy than lipofectamine 2000 in MC3T3-E1 cells. Our study demonstrated that 1,8-naphthalimide- [12]aneN3 compound 1 with low cytotoxicity, high specificity towards Cu2+ and lysosome, high transfection efficacy, and low cost is an efficient multifunctional material both in molecular recognition and gene delivery.

[12]aneN3-based multifunctional compounds as fluorescent probes and nucleic acids delivering agents.

A series of multifunctional compounds (MFCs) 1a-1e based on 1,8-naphthalimide and [12]aneN3 building blocks were designed and synthesized. They were used as not only fluorescent probes for recognition of Cu2+ ions but also as non-viral gene vectors for DNA and RNA delivery. Furthermore, their complexes with Cu2+ (1-Cu) could also selectively stain lysosome in HeLa cells. In order to achieve high performance multifunctional materials, structure-performance relationship of MFCs 1a-1e was studied. It was found that MFCs 1a-1e exhibited highly selective fluorescence turn-off for Cu2+, without interference by other metal ions in aqueous solution. The fluorescence emission of 1a-1e was quenched by a factor of 10-fold, 47-fold, 6-fold, 64-fold, and 15-fold respectively in the presence of Cu2+ ions. Due to high sensitivity, good water solubility, and low cytotoxicity, MFCs 1a-1d were successfully applied in the recognition of Cu2+ and selectively staining lysosome in HeLa cells. Most importantly, MFCs 1a and 1b had excellent HeLa cell selectivity in RNA delivery, and their performances were far better than lipofectamine 2000 and 25 kDa PEI.

The Development of Functional Non-Viral Vectors for Gene Delivery.

Gene therapy is manipulation in/of gene expression in specific cells/tissue to treat diseases. This manipulation is carried out by introducing exogenous nucleic acids, such as DNA or RNA, into the cell. Because of their negative charge and considerable larger size, the delivery of these molecules, in general, should be mediated by gene vectors. Non-viral vectors, as promising delivery systems, have received considerable attention due to their low cytotoxicity and non-immunogenicity. As research continued, more and more functional non-viral vectors have emerged. They not only have the ability to deliver a gene into the cells but also have other functions, such as the performance of fluorescence imaging, which aids in monitoring their progress, targeted delivery, and biodegradation. Recently, many reviews related to non-viral vectors, such as polymers and cationic lipids, have been reported. However, there are few reviews regarding functional non-viral vectors. This review summarizes the common functional non-viral vectors developed in the last ten years and their potential applications in the future. The transfection efficiency and the transport mechanism of these materials were also discussed in detail. We hope that this review can help researchers design more new high-efficiency and low-toxicity multifunctional non-viral vectors, and further accelerate the progress of gene therapy.

1,8-Naphthalimide-Based Multifunctional Compounds as Cu2+ Probes, Lysosome Staining Agents, and Non-viral Vectors.

A series of multifunctional compounds (MFCs) 1a-1d based on 1,8-naphthalimide moiety were designed and synthesized. Due to the good fluorescence property and nucleic acid binding ability of 1,8-naphthalimide, these MFCs were applied in Cu2+ ion recognition, lysosome staining as well as RNA delivery. It was found that these MFCs exhibited highly selective fluorescence turn-off for Cu2+ in aqueous solution. The fluorescence emission of 1a-1d was quenched by a factor of 116-, 20-, 12-, and 14-fold in the presence of Cu2+ ions, respectively. Most importantly, 1a-Cu and 1b-Cu could be used as imaging reagents for detection of lysosome in live human cervical cancer cells (HeLa) using fluorescence microscopy. Furthermore, in order to evaluate the RNA delivery ability of 1a-1d, cellular uptake experiments were performed in HeLa, HepG2, U2Os, and MC3T3-E1 cell lines. The results showed that all the materials could deliver Cy5-labled RNA into the targeted cells. Among them, compound 1d modified with long hydrophobic chain exhibited the best RNA delivery efficiency in the four tested cell lines, and the performance was far better than lipofectamine 2000 and 25 kDa PEI, indicating the potential application in non-viral vectors.

Structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase A2α.

Ca2+-stimulated translocation of cytosolic phospholipase A2α (cPLA2α) to the Golgi induces arachidonic acid production, the rate-limiting step in pro-inflammatory eicosanoid synthesis. Structural insights into the cPLA2α preference for phosphatidylcholine (PC)-enriched membranes have remained elusive. Here, we report the structure of the cPLA2α C2-domain (at 2.2 Å resolution), which contains bound 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and Ca2+ ions. Two Ca2+ are complexed at previously reported locations in the lipid-free C2-domain. One of these Ca2+ions, along with a third Ca2+, bridges the C2-domain to the DHPC phosphate group, which also interacts with Asn65. Tyr96 plays a key role in lipid headgroup recognition via cation-π interaction with the PC trimethylammonium group. Mutagenesis analyses confirm that Tyr96 and Asn65 function in PC binding selectivity by the C2-domain and in the regulation of cPLA2α activity. The DHPC-binding mode of the cPLA2α C2-domain, which differs from phosphatidylserine or phosphatidylinositol 4,5-bisphosphate binding by other C2-domains, expands and deepens knowledge of the lipid-binding mechanisms mediated by C2-domains.

Computed tomography-guided simultaneous coil localization as a bridge to one-stage surgery for multiple lung nodules: a retrospective study.

BACKGROUND: Video-assisted thoracoscopic surgery (VATS) has been widely used for diagnostic wedge resection of lung nodules. When VATS is performed for multiple lung nodules, preoperative localization for each target nodule is required. In this study, we evaluated the clinical effectiveness of computed tomography (CT)-guided simultaneous coil localization in one-stage VATS wedge resection for multiple lung nodules. METHODS: Between November 2015 to March 2018, 19 patients with multiple target nodules underwent CT-guided simultaneous coil localization and one-stage VATS resection at our center. Data on the technical success of simultaneous localization and wedge resection, complications, and pathological results were collected. RESULTS: A total of 43 nodules were localized. The localization was successfully achieved in 42 of 43 nodules (97.7%). The technique of simultaneous localization was successfully achieved in 18 of 19 patients (94.7%). Fifteen patients underwent unilateral lung localization and four patients underwent bilateral lung localization. Three patients (15.8%) experienced asymptomatic pneumothorax after localization. All patients successfully underwent one-stage wedge resection for all target nodules. The mean duration of one-stage VATS procedure was 171.8 ± 84.0 min. The mean volume of blood loss was 94.2 ± 58.0 mL. Three patients experienced pleural effusion after VATS. During a follow-up of 6-31 months (median 18 months), no patient developed new lung nodules or distant metastasis. CONCLUSIONS: Preoperative simultaneous coil implantation is a safe and simple method for localization of multiple lung nodules. Simultaneous coil localization could effectively guide a one-stage VATS diagnostic wedge resection procedure.

Structural analyses of 4-phosphate adaptor protein 2 yield mechanistic insights into sphingolipid recognition by the glycolipid transfer protein family.

The glycolipid transfer protein (GLTP) fold defines a superfamily of eukaryotic proteins that selectively transport sphingolipids (SLs) between membranes. However, the mechanisms determining the protein selectivity for specific glycosphingolipids (GSLs) are unclear. Here, we report the crystal structure of the GLTP homology (GLTPH) domain of human 4-phosphate adaptor protein 2 (FAPP2) bound with N-oleoyl-galactosylceramide. Using this domain, FAPP2 transports glucosylceramide from its cis-Golgi synthesis site to the trans-Golgi for conversion into complex GSLs. The FAPP2-GLTPH structure revealed an element, termed the ID loop, that controls specificity in the GLTP family. We found that, in accordance with FAPP2 preference for simple GSLs, the ID loop protrudes from behind the SL headgroup-recognition center to mitigate binding by complex GSLs. Mutational analyses including GLTP and FAPP2 chimeras with swapped ID loops supported the proposed restrictive role of the FAPP2 ID loop in GSL selectivity. Comparative analysis revealed distinctly designed ID loops in each GLTP family member. This analysis also disclosed a conserved H-bond triplet that "clasps" both ID-loop ends together to promote structural autonomy and rigidity. The findings indicated that various ID loops work in concert with conserved recognition centers to create different specificities among family members. We also observed four bulky, conserved hydrophobic residues involved in "sensor-like" interactions with lipid chains in protein hydrophobic pockets and FF motifs in GLTP and FAPP2, well-positioned to provide acyl chain-dependent SL selectivity for the hydrophobic pockets. In summary, our study provides mechanistic insights into sphingolipid recognition by the GLTP fold and uncovers the elements involved in this recognition.

Structure-activity relationship of novel low-generation dendrimers for gene delivery.

Structure-activity relationship (SAR) studies are very critical to design ideal gene vectors for gene delivery. However, It is difficult to obtain SAR information of low-generation dendrimers due to the lack of easy structural modification ways. Here, we synthesized a novel family of rigid aromatic backbone-based low-generation polyamidoamine (PAMAM) dendrimers. According to the number of primary amines, they were divided into two types: four-amine-containing PAMAM (DL1-DL5) and eight-amine-containing PAMAM (DL6-DL10). Due to the introduction of a rigid aromatic backbone, the low-generation PAMAM could be modified easier by different hydrophobic aliphatic chains. Several assays were used to study the interactions of the PAMAM dendrimers with plasmid DNA, and the results revealed that they not only had good DNA binding ability but also could efficiently condense DNA into spherical-shaped nanoparticles with suitable sizes and zeta potentials. The SAR studies indicated that the gene-transfection efficiency of the synthesized materials depended on not only the structure of their hydrophobic chains but also the number of primary amines. It was found that four-amine-containing PAMAM prepared from oleylamine (DL5) gave the best transfection efficiency, which was 3 times higher than that of lipofectamine 2000 in HEK293 cells. The cellular uptake mechanism mediated by DL5 was further investigated, and the results indicated that DL5/DNA complexes entered the cells mainly via caveolae and clathrin-mediated endocytosis. In addition, these low-generation PAMAMs modified with a single hydrophobic tail showed lower toxicity than lipofectamine 2000 in MC3T3-E1, MG63, HeLa, and HEK293 cells. These results reveal that such a type of low-generation polyamidoamines might be promising non-viral gene vectors, and also give us clues for the design of safe and high-efficiency gene vectors.