Systematics and evolutionary dynamics of insect-fern interactions in the specialized fern-spore feeding Cuprininae (Lepidoptera, Stathmopodidae).

Fern-spore-feeding (FSF) is rare and found in only four families of Lepidoptera. Stathmopodidae is the most speciose family that contains FSF species, and its subfamily Cuprininae exclusively specializes on FSF. However, three species of Stathmopodinae also specialize on FSF. To better understand the evolutionary history of FSF and, more generally, the significance of specialization on a peculiar host, a phylogenetic and taxonomic revision for this group is necessary. We reconstructed the most comprehensive molecular phylogeny, including one mitochondrial and four nuclear genes, of Stathmopodidae to date, including 137 samples representing 62 species, with a particular focus on the FSF subfamily, Cuprininae, including 33 species (41% of named species) from 6 of the 7 Cuprininae genera. Species from two other subfamilies, Stathmopodinae and Atkinsoniinae, were also included. We found that FSF evolved only once in Stathmopodidae and that the previous hypothesis of multiple origins of FSF was misled by inadequate taxonomy. Moreover, we showed that (1) speciation/extinction rates do not differ significantly between FSF and non-FSF groups and that (2) oligophage is the ancestral character state in Cuprininae. We further revealed that a faster rate of accumulating specialists over time, and thus a higher number of specialists, was achieved by a higher transition rate from oligophagages to specialists compared to the transition rate in the opposite direction. We finish by describing three new genera, Trigonodagen. nov., Petalagen. nov., and Pediformisgen. nov., and revalidating five genera: Cuprina, Calicotis, Thylacosceles, Actinoscelis, Thylacosceloides in Cuprininae, and we provide an updated taxonomic key to genera and a revised global checklist of Cuprininae.

Characterization of Lipid Nanoparticles Containing Ionizable Cationic Lipids Using Design-of-Experiments Approach.

Lipid nanoparticles (LNPs) containing short-interfering RNA (LNP-siRNA systems) are a promising approach for silencing disease-causing genes in hepatocytes following intravenous administration. LNP-siRNA systems are generated by rapid mixing of lipids in ethanol with siRNA in aqueous buffer (pH 4.0) where the ionizable lipid is positively charged, followed by dialysis to remove ethanol and to raise the pH to 7.4. Ionizable cationic lipids are the critical excipient in LNP systems as they drive entrapment and intracellular delivery. A recent study on the formation of LNP-siRNA systems suggested that ionizable cationic lipids segregate from other lipid components upon charge neutralization to form an amorphous oil droplet in the core of LNPs. This leads to a decrease in intervesicle electrostatic repulsion, thereby engendering fusion of small vesicles to form final LNPs of increased size. In this study, we prepared LNP-siRNA systems containing four lipid components (hydrogenated soy phosphatidylcholine, cholesterol, PEG-lipid, and 1,2-dioleoyl-3-dimethylammonium propane) by microfluidic mixing. The effects of preparation parameters [lipid concentration, flow rate ratio (FRR), and total flow rate], dialysis process, and complex formation between siRNA and ionizable cationic lipids on the physicochemical properties [siRNA entrapment on the particle size and polydispersity index (PDI)] were investigated using a design of experiments approach. The results for the preparation parameters showed no impact on siRNA encapsulation, but lipid concentration and FRR significantly affected the particle size and PDI. In addition, the effect of FRR on the particle size was suppressed in the presence of anionic polymers such as siRNA as compared to the case of LNPs alone. More intriguingly, unlike empty LNPs, a decrease in the PDI and an increase in the particle size occurred after dialysis in the LNP-siRNA systems. Such changes by dialysis were suppressed at FRR = 1. These findings provide useful information to guide the development and manufacturing conditions for LNP-siRNA systems.

Protective Effect of Edaravone against Cationic Lipid-Mediated Oxidative Stress and Apoptosis.

Liposomes containing ionizable cationic lipids have been widely used for the delivery of nucleic acids such as small-interfering RNA and mRNA. The utility of cationic lipids with a permanent positive charge, however, is limited to in vitro transfection of cultured cells due to its dose-limiting toxic side effects observed in animals. Several reports have suggested that the permanently charged cationic lipids induce reactive oxygen species (ROS) and ROS-mediated toxicity in cells. We therefore hypothesized that the concomitant use of ROS inhibitor could reduce toxicity and improve drug efficacy. In this study, suppression of the cationic toxicity was evaluated using an ROS scavenger, edaravone, which is a low-molecular-weight antioxidant drug clinically approved for acute-phase cerebral infarction and amyotrophic lateral sclerosis. Cell viability assay in the mouse macrophage-like cell line RAW264 indicated that the concomitant use of edaravone were not able to suppress the cytotoxicity induced by cationic liposomes comprised of monovalent cationic lipid N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTMA) over a short period of time. Cationic lipids-induced necrosis was assumed to be involved in the cytotoxicity upon short-term exposure to cationic liposomes. On the other hand, the significant improvement of cell viability was observed when the short treatment with cationic liposomes was followed by exposure to edaravone for 24 h. It was also confirmed that apoptosis inhibition by ROS elimination might have contributed to this effect. These results suggest the utility of continuous administration with edaravone as concomitant drug for suppression of adverse reactions in therapeutic treatment using cationic liposomes.

Identification of novel long chain N-acylhomoserine lactones of chain length C20 from the marine phototrophic bacterium Rhodovulum sulfidophilum.

Gram-negative bacterial quorum sensing is mainly regulated by an extracellularly produced N-acylhomoserine lactone (AHL). AHL consists of a lactone ring and an acyl chain, which generally varies from C4 to C18 in length and affords species-specific variety. In this study, we developed an ultra-high performance liquid chromatography tandem mass spectrometry system and detected two kinds of long chain AHLs with chain length C20 from the reverse-phase thin layer chromatography-fractionated cultured supernatant of the marine photosynthetic bacterium Rhodovulum sulfidophilum. By fragmentation search analysis to detect compounds with a homoserine lactone ring moiety for data dependent acquisition, a minor AHL, presumed to be 3-OH-C18-homoserine lactone (HSL), was also found. Among the detected C20-HSLs, 3-OH-C20-HSL was structurally identified and 3-OH-C20:1-HSL was strongly suggested. To our knowledge, this is the first report to show a novel AHL with the longest C20 acyl side chain found to date. ABBREVIATIONS: AGC: automatic gain control; AHL: N-acylhomoserine lactone; CD: cyclodextrin; CID: collision induced dissociation; DDA: data dependent acquisition; EPI: enhanced product ion; FISh: fragment ion search; HCD: high energy collisional dissociation; HSL: homoserine lactone; IT: injection time; LC: liquid chromatography; MS: mass spectrometry; PRM: parallel reaction monitoring; RP: reverse phase; SRM: selected reaction monitoring; TLC: thin layer chromatography; UHPLC: ultra high performance liquid chromatography.

Hot-alkaline DNA extraction method for deep-subseafloor archaeal communities.

A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods.

Carbon and nitrogen assimilation in deep subseafloor microbial cells.

Remarkable numbers of microbial cells have been observed in global shallow to deep subseafloor sediments. Accumulating evidence indicates that deep and ancient sediments harbor living microbial life, where the flux of nutrients and energy are extremely low. However, their physiology and energy requirements remain largely unknown. We used stable isotope tracer incubation and nanometer-scale secondary ion MS to investigate the dynamics of carbon and nitrogen assimilation activities in individual microbial cells from 219-m-deep lower Pleistocene (460,000 y old) sediments from the northwestern Pacific off the Shimokita Peninsula of Japan. Sediment samples were incubated in vitro with (13)C- and/or (15)N-labeled glucose, pyruvate, acetate, bicarbonate, methane, ammonium, and amino acids. Significant incorporation of (13)C and/or (15)N and growth occurred in response to glucose, pyruvate, and amino acids (∼76% of total cells), whereas acetate and bicarbonate were incorporated without fostering growth. Among those substrates, a maximum substrate assimilation rate was observed at 67 × 10(-18) mol/cell per d with bicarbonate. Neither carbon assimilation nor growth was evident in response to methane. The atomic ratios between nitrogen incorporated from ammonium and the total cellular nitrogen consistently exceeded the ratios of carbon, suggesting that subseafloor microbes preferentially require nitrogen assimilation for the recovery in vitro. Our results showed that the most deeply buried subseafloor sedimentary microbes maintain potentials for metabolic activities and that growth is generally limited by energy but not by the availability of C and N compounds.

An improved cell separation technique for marine subsurface sediments: applications for high-throughput analysis using flow cytometry and cell sorting.

Development of an improved technique for separating microbial cells from marine sediments and standardization of a high-throughput and discriminative cell enumeration method were conducted. We separated microbial cells from various types of marine sediment and then recovered the cells using multilayer density gradients of sodium polytungstate and/or Nycodenz, resulting in a notably higher percent recovery of cells than previous methods. The efficiency of cell extraction generally depends on the sediment depth; using the new technique we developed, more than 80% of the total cells were recovered from shallow sediment samples (down to 100 meters in depth), whereas ~50% of cells were recovered from deep samples (100-365 m in depth). The separated cells could be rapidly enumerated using flow cytometry (FCM). The data were in good agreement with those obtained from manual microscopic direct counts over the range 10(4)-10(8) cells cm(-3). We also demonstrated that sedimentary microbial cells can be efficiently collected using a cell sorter. The combined use of our new cell separation and FCM/cell sorting techniques facilitates high-throughput and precise enumeration of microbial cells in sediments and is amenable to various types of single-cell analyses, thereby enhancing our understanding of microbial life in the largely uncharacterized deep subseafloor biosphere.

Label-Free Identification of Spore-Forming Bacteria Using Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy.

Spore-forming bacteria accumulate dipicolinic acid (DPA) to form spores to survive in extreme environments. Vibrational spectroscopy is widely used to detect DPA and elucidate the existence of the bacteria, while vegetative cells, another form of spore-forming bacteria, have not been studied extensively. Herein, we applied coherent anti-Stokes Raman scattering (CARS) microscopy to spectroscopically identify both spores and vegetative cells without staining or molecular tagging. The spores were identified by the strong CARS signals due to DPA. Furthermore, we observed bright spots in the vegetative cells in the CARS image at 1735 cm-1. The vegetative cells contained molecular species with C=O bonds because this vibrational mode was associated with the carbonyl group. One of the candidate molecular species is diketopimelic acid (DKP), a DPA precursor. This hypothesis was verified by comparing the spectrum obtained by the vegetative cells with that of the DKP analogue (ketopimelic acid) and with the result obtained by DFT calculation. The results indicate that the observed vegetative cell is in the sporulation process. CARS spectra can be used to monitor the maturation and preformation of spores.

Connecting the Dots between Barriers to W.I.C. Access and Adult and Child Food Insecurity: A Survey of Missouri Residents.

Background. Previous research has explored the impact of W.I.C. on recipients' health, but less is known about the connection between barriers to W.I.C. access and health outcomes. We fill in a gap in the literature by studying the relationship between barriers to Special Supplemental Nutrition Program for Women, Infants, and Children (W.I.C.) access and adult and child food insecurity. Methods. After survey administration, we analyzed a cross-sectional sample of 2244 residents in Missouri who have used W.I.C. or lived in a household with a W.I.C. recipient in the past three years. We ran logistic regression models to understand the relationships among barriers to W.I.C. utilization, adult food insecurity, and child food insecurity. Results. Having special dietary needs (for adults), lacking access to technology, encountering inconvenient clinic hours of operation, and experiencing difficulties taking off work were associated with increased adult food insecurity. Difficulties finding WIC-approved items in the store, technological barriers, inconvenient clinic hours, difficulties taking off work, and finding childcare were associated with increased child food insecurity. Conclusion. Barriers to accessing and utilizing W.I.C. are associated with adult and child food insecurity. However, current policies suggest promising approaches to curbing these barriers.

The Newly Recorded Fern-spore Feeding Moths in the Genus Calicotis, Meyrick 1889 (Lepidoptera: Stathmopodidae) from Taiwan, with Notes on Life History of Three Species.

Three newly recorded species of the genus Calicotis, Meyrick 1889 are reported from Taiwan: C. attiei (Guillermet, 2011), C. rotundinidus Terada, 2016, and C. exclamationis Terada, 2016. C. biserraticola Terada, 2016 is treated as a junior subjective synonym for C. attiei based on both morphological and molecular data. The life history of these three species is presented as well as the first observation of fern-feeding stathmopodid eggs in the world.

Microfluidic Preparation of Nanoparticles Using Poly(ethylene Glycol)-distearoylphosphatidylethanolamine for Solubilizing Poorly Soluble Drugs.

Microfluidic systems have shown promise for the production of nanoparticles from mixtures of aqueous and organic solutions, including liposomes, oil-in-water nanoemulsions, and lipid nanoparticles. They offer important practical advantages, including low reagent consumption, parallelization, and automation, and are ideally suited to high-throughput optimization and scale-up. In this study, we developed a new method for the formulation of nanoparticles of poorly soluble drug compounds. The nanoparticles, prepared by microfluidic mixing using only poly(ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE), were highly stable and uniform in size. By mixing an organic solution of poorly soluble cyclosporine A and PEG-DSPE with water in the microfluidic device, amorphous cyclosporine A nanoparticles (CsA-NPs), with an encapsulation efficiency of approximately 90% and a particle size of 100-200 nm, were obtained. Analysis of the microfluidic process parameters revealed that particle size distribution was significantly controlled by the flow rate ratio. The obtained CsA-NPs were stable for up to 150 days at room temperature, and the pharmacokinetic profile was similar to that of the commercial formulation containing Cremophor EL, which has been reported to induce serious adverse effects after intravenous administration. These findings provide a useful technical platform for the safe solubilization of poorly soluble compounds and their subsequent pharmaceutical development.

Simple In-liquid Staining of Microbial Cells for Flow Cytometry Quantification of the Microbial Population in Marine Subseafloor Sediments.

Microbial cell counting provides essential information for the study of cell abundance profiles and biogeochemical interactions with the surrounding environments. However, it often requires labor-intensive and time-consuming processes, particularly for subseafloor sediment samples, in which non-cell particles are abundant. We developed a rapid and straightforward method for staining microbial intracellular DNA by SYBR Green I (SYBR-I) to enumerate cells by flow cytometry (FCM). We initially examined the efficiency of microbial cell staining at various dye/sediment ratios (volume ratio of SYBR-I/sediment [vSYBR/vSed]). Non-cell particles in sediment strongly and preferentially adsorbed SYBR-I dye, resulting in the unsuccessful staining of microbial cells when an insufficient ratio (<1.63 vSYBR/vSed) of SYBR-I dye was present per volume of sediment. SYBR-I dye at an abundance of 10 vSYBR/vSed successfully and stably stained microbial cells in green fluorescence, while the fluorescent color of non-cell particles red-shifted to yellow-orange with the overaccumulation of SYBR-I dye. A low vSYBR/vSed ratio was quickly recognized by a colorless supernatant after centrifugation. At the appropriate vSYBR/vSed ratio, FCM-measured cell concentrations in subseafloor sediments were consistently similar to microscopy counts (>106 cells cm-3). Samples with low cell abundance (<105 cells cm-3) still require cell separation. This modified staining allows us to efficiently process and perform the microbial cell counting of sediment samples to a depth of a few hundred meters below the seafloor with a higher throughput and capability to scale up than procedures employing microscopy-based observations.

Construction of Aerobic/Anaerobic-Substrate-Induced Gene Expression Procedure for Exploration of Metagenomes From Subseafloor Sediments.

Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

EDTA-FISH: A Simple and Effective Approach to Reduce Non-specific Adsorption of Probes in Fluorescence in situ Hybridization (FISH) for Environmental Samples.

Fluorescence in situ hybridization (FISH) is a widely used molecular technique in microbial ecology. However, the non-specific adsorption of fluorescent probes and resulting high intensity of background signals from mineral particles hampers the specific detection of microbial cells in grain-rich environmental samples, such as subseafloor sediments. We herein demonstrated that a new buffer composition containing EDTA efficiently reduced the adsorption of probes without compromising the properties of the FISH-based probing of microbes. The inclusion of a high concentration of EDTA in the buffer in our protocol provides a simple and effective approach for reducing the background in FISH for environmental samples.

Aerobic microbial life persists in oxic marine sediment as old as 101.5 million years.

Sparse microbial populations persist from seafloor to basement in the slowly accumulating oxic sediment of the oligotrophic South Pacific Gyre (SPG). The physiological status of these communities, including their substrate metabolism, is previously unconstrained. Here we show that diverse aerobic members of communities in SPG sediments (4.3‒101.5 Ma) are capable of readily incorporating carbon and nitrogen substrates and dividing. Most of the 6986 individual cells analyzed with nanometer-scale secondary ion mass spectrometry (NanoSIMS) actively incorporated isotope-labeled substrates. Many cells responded rapidly to incubation conditions, increasing total numbers by 4 orders of magnitude and taking up labeled carbon and nitrogen within 68 days after incubation. The response was generally faster (on average, 3.09 times) for nitrogen incorporation than for carbon incorporation. In contrast, anaerobic microbes were only minimally revived from this oxic sediment. Our results suggest that microbial communities widely distributed in organic-poor abyssal sediment consist mainly of aerobes that retain their metabolic potential under extremely low-energy conditions for up to 101.5 Ma.

Dehalogenation activities and distribution of reductive dehalogenase homologous genes in marine subsurface sediments.

Halogenated organic compounds serve as terminal electron acceptors for anaerobic respiration in a diverse range of microorganisms. Here, we report on the widespread distribution and diversity of reductive dehalogenase homologous (rdhA) genes in marine subsurface sediments. A total of 32 putative rdhA phylotypes were detected in sediments from the southeast Pacific off Peru, the eastern equatorial Pacific, the Juan de Fuca Ridge flank off Oregon, and the northwest Pacific off Japan, collected at a maximum depth of 358 m below the seafloor. In addition, significant dehalogenation activity involving 2,4,6-tribromophenol and trichloroethene was observed in sediment slurry from the Nankai Trough Forearc Basin. These results suggest that dehalorespiration is an important energy-yielding pathway in the subseafloor microbial ecosystem.

Microbial Metabolism and Community Dynamics in Hydraulic Fracturing Fluids Recovered From Deep Hydrocarbon-Rich Shale.

Hydraulic fracturing is a prominent method of natural gas production that uses injected, high-pressure fluids to fracture low permeability, hydrocarbon rich strata such as shale. Upon completion of a well, the fluid returns to the surface (produced water) and contains natural gas, subsurface constituents, and microorganisms (Barbot et al., 2013; Daly et al., 2016). While the microbial community of the produced fluids has been studied in multiple gas wells, the activity of these microorganisms and their relation to biogeochemical activity is not well understood. In this experiment, we supplemented produced fluid with 13C-labeled carbon sources (glucose, acetate, bicarbonate, methanol, or methane), and 15N-labeled ammonium chloride in order to isotopically trace microbial activity over multiple day in anoxic incubations. Nanoscale secondary ion mass spectrometry (NanoSIMS) was used to generate isotopic images of 13C and 15N incorporation in individual cells, while isotope ratio monitoring-gas chromatography-mass spectrometry (IRM-GC-MS) was used to measure 13CO2, and 13CH4 as metabolic byproducts. Glucose, acetate, and methanol were all assimilated by microorganisms under anoxic conditions. 13CO2 production was only observed with glucose as a substrate indicating that catabolic activity was limited to this condition. The microbial communities observed at 0, 19, and 32 days of incubation did not vary between different carbon sources, were low in diversity, and composed primarily of the class Clostridia. The primary genera detected in the incubations, Halanaerobium and Fusibacter, are known to be adapted to harsh physical and chemical conditions consistent with those that occur in the hydrofracturing environment. This study provides evidence that microorganisms in produced fluid are revivable in laboratory incubations and retained the ability to metabolize added carbon and nitrogen substrates.

Assessment of Capacity to Capture DNA Aerosols by Clean Filters for Molecular Biology Experiments.

Experimental contamination by exogenous DNA is a major issue in molecular biological studies for data quality and its management. We herein assessed DNA aerosols for the risk of contamination and tested the capacity of clean air filters to trap and remove DNA aerosols. DNA aerosols were generated by atomizing a DNA solution and introduced into a laminar flow clean air unit. Capture and detection performed upstream and downstream of the clean air unit showed that a significant fraction (>99.96%) of introduced molecules was trapped and removed by the filter. Although DNA aerosols appear to be an avoidable source of exogenous contamination, a clearer understanding and careful experimental procedures are needed in order to perform contamination-free, high-quality molecular biology experiments.

Deep-biosphere methane production stimulated by geofluids in the Nankai accretionary complex.

Microbial life inhabiting subseafloor sediments plays an important role in Earth's carbon cycle. However, the impact of geodynamic processes on the distributions and carbon-cycling activities of subseafloor life remains poorly constrained. We explore a submarine mud volcano of the Nankai accretionary complex by drilling down to 200 m below the summit. Stable isotopic compositions of water and carbon compounds, including clumped methane isotopologues, suggest that ~90% of methane is microbially produced at 16° to 30°C and 300 to 900 m below seafloor, corresponding to the basin bottom, where fluids in the accretionary prism are supplied via megasplay faults. Radiotracer experiments showed that relatively small microbial populations in deep mud volcano sediments (102 to 103 cells cm-3) include highly active hydrogenotrophic methanogens and acetogens. Our findings indicate that subduction-associated fluid migration has stimulated microbial activity in the mud reservoir and that mud volcanoes may contribute more substantially to the methane budget than previously estimated.

Novel PEG-matrix metalloproteinase-2 cleavable peptide-lipid containing galactosylated liposomes for hepatocellular carcinoma-selective targeting.

In order to obtain an HCC-selective drug delivery system, a novel functional lipid, which is cleaved by the protease activity of matrix metalloproteinase-2 (MMP-2), was developed. The amino group of dioleoylphosphatidylethanolamine (DOPE) was conjugated with PEGylated MMP-2 substrate peptide (Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln), and MMP-2-cleavable PEG-Peptide-DOPE (PEG-PD) was synthesized. When PEG-PD was incorporated in galactosylated liposomes (Gal-PEG-PD-liposomes), we expected that Gal-PEG-PD-liposomes would not be taken up by normal hepatocytes due to the steric hindrance effect, but would be activated around HCC cells by secreted MMPs. In the pretreatment by hMMP2 (1, 5, and 10mug/ml), an hMMP2 concentration-dependent higher uptake of Gal-PEG-PD-liposomes was observed in HepG2 cells, suggesting PEG-PD cleavage. In the presence of an excess of galactose, the uptake of Gal-PEG-PD-liposomes with hMMP2 was significantly inhibited, suggesting asialoglycoprotein receptor-mediated uptake of Gal-PEG-PD-liposomes following the PEG-PD cleavage. Pretreatment of Gal-PEG-PD-liposomes with the conditioned medium of B16BL6, which contained secreted MMPs, enhanced the binding to HepG2 cells, as in the case of hMMP-2 treatment. Moreover, the cytotoxicity of N(4)-octadecyl-1-beta-d-arabinofuranosylcytosine (NOAC) incorporated Gal-PEG-PD-liposomes was enhanced by hMMPs (5mug/ml) and its cytotoxicity was significantly reduced by the presence of an excess of galactose in HepG2 cells. In conclusion, Gal-PEG-PD-liposomes were successfully developed for novel HCC-selective targeting.