LAG-3xPD-L1 bispecific antibody potentiates antitumor responses of T cells through dendritic cell activation.

Several preclinical studies demonstrate that antitumor efficacy of programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade can be improved by combination with other checkpoint inhibitors. Lymphocyte-activation gene 3 (LAG-3) is an inhibitory checkpoint receptor involved in T cell exhaustion and tumor immune escape. Here, we describe ABL501, a bispecific antibody targeting LAG-3 and PD-L1 in modulating immune cell responses against tumors. ABL501 that efficiently inhibits both LAG-3 and PD-L1 pathways enhances the activation of effector CD4+ and CD8+ T cells with a higher degree than a combination of single anti-LAG-3 and anti-PD-L1. The augmented effector T cell responses by ABL501 resulted in mitigating regulatory-T-cell-mediated immunosuppression. Mechanistically, the simultaneous binding of ABL501 to LAG-3 and PD-L1 promotes dendritic cell (DC) activation and tumor cell conjugation with T cells that subsequently mounts effective CD8+ T cell responses. ABL501 demonstrates its potent in vivo antitumor efficacy in a humanized xenograft model and with knockin mice expressing human orthologs. The immune profiling analysis of peripheral blood reveals an increased abundance of LAG-3hiPD-1hi memory CD4+ T cell subset in relapsed cholangiocarcinoma patients after gemcitabine plus cisplatin therapy, which are more responsive to ABL501. This study supports the clinical evaluation of ABL501 as a novel cancer immunotherapeutic, and a first-in-human trial has started (NCT05101109).

Halomonas getboli sp. nov., a halotolerant bacteria isolated from a salt flat.

A novel Gram-stain-negative, rod-shaped, cream-coloured, motile, halotolerant bacterium, designated as YJPS3-2T, was isolated from saltern sediment of the Yellow sea in Yongyu-do, Republic of Korea. Strain YJPS3-2T grew at pH 5.0-10.0 (optimum, pH 7.0), 4-40 °C (optimum, 30 °C) and with 1-15% (w/v) NaCl (optimum 3 %). The 16S rRNA gene sequence analysis indicated that strain YJPS3-2T was closely related to those of Halomonas halophila F5-7T (98.75 %), Halomonas salina F8-11T (98.74 %), Halomonas smyrnensis AAD6T (98.66 %), Halomonas organivorans G-16.1T (98.34 %), Halomonas koreensis SS20T (97.98 %) and Halomonas beimenensis NTU-107T (96.93 %). The average nucleotide identity and digital DNA-DNA hybridization values between YJPS3-2T and related type strains were 86.9-91.6 % and 32.0-44.8 %. Strain YJPS3-2T was characterized as having Q-9 as the predominant respiratory quinone and the principal fatty acids (>10 %) were C16 : 0 (31.4 %), C19 : 0 ω8c cyclo (16.3 %), C17 : 0 cyclo (11.9 %) and C12 : 0 3-OH (10.4 %). The polar lipids consisted of phosphatidylcholine, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of strain YJPS3-2T is 68.1mol %. Based on the polyphasic taxonomic evidence presented in this study, YJPS3-2T should be classified as representing a novel species within the genus Halmonas, for which name Halomonas getboli is proposed, with the type strain YJPS3-2T (= KCTC 92124T=KACC 22561T=JCM 35085T).

Novel anti-4-1BB×PD-L1 bispecific antibody augments anti-tumor immunity through tumor-directed T-cell activation and checkpoint blockade.

BACKGROUND: Stimulation of 4-1BB with agonistic antibodies is a promising strategy for improving the therapeutic efficacy of immune checkpoint inhibitors (ICIs) or for overcoming resistance to ICIs. However, dose-dependent hepatotoxicity was observed in clinical trials with monoclonal anti-4-1BB agonistic antibodies due to the activation of 4-1BB signaling in liver resident Kupffer cells. METHODS: To avoid this on-target liver toxicity, we developed a novel bispecific antibody (4-1BB×PD-L1 bispecific antibody, termed "ABL503") uniquely designed to activate 4-1BB signaling only in the context of PD-L1, while also blocking PD-1/PD-L1 signaling. RESULTS: Functional evaluation using effector cells expressing both 4-1BB and PD-1 revealed superior biological activity of ABL503 compared with the combination of each monoclonal antibody. ABL503 also augmented T-cell activation in in vitro assays and further enhanced the anti-PD-L1-mediated reinvigoration of tumor-infiltrating CD8+ T cells from patients with cancer. Furthermore, in humanized PD-L1/4-1BB transgenic mice challenged with huPD-L1-expressing tumor cells, ABL503 induced superior anti-tumor activity and maintained an anti-tumor response against tumor rechallenge. ABL503 was well tolerated, with normal liver function in monkeys. CONCLUSION: The novel anti-4-1BB×PD-L1 bispecific antibody may exert a strong anti-tumor therapeutic efficacy with a low risk of liver toxicity through the restriction of 4-1BB stimulation in tumors.

Novosphingobium aureum sp. nov., a marine bacterium isolated from salt flat sediment.

A Gram-stain-negative, aerobic, pale yellow-coloured, rod-shaped marine bacterium designated strain YJ-S2-02T was isolated from salt flat sediment sampled in Yongyu-do, Republic of Korea. Strain YJ-S2-02T grew at pH 6.0-9.0 (optimum, pH 7.0), 10-40 °C (optimum, 30 °C) and with optimum 1 % (w/v) NaCl. The 16S rRNA gene sequence analysis indicated that strain YJ-S2-02T was closely related to Novosphingobium naphthalenivorans NBRC 102051T (97.8 %) followed by Novosphingobium mathurense SM117T (97.5 %), Novosphingobium indicum H25T (97.3 %), Novosphingobium pentaromativorans US6-1T (96.8 %), Novosphingobium fontis STM-14T (96.6 %), Novosphingobium endophyticum EGI60015T (96.5 %), Novosphingobium naphthae D39T (96.5 %) and Novosphingobium malaysiense MUSC 273T (95.9 %). The average nucleotide identity and estimated DNA-DNA hybridization values between YJ-S2-02T and related type strains were 77.0-77.9 % and 19.1-24.0 %. Strain YJ-S2-02T was characterized as having Q-10 as the predominant respiratory quinone and the principal fatty acids (>10 %) were summed feature 8 (C18 : 1 ω6c/ω7c, 20.7 %), C18 : 3 ω6c (16.3 %) and C17 : 1 ω6c (11.8 %). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, sphingolipids and two unidentified lipids. The DNA G+C content of strain YJ-S2-02T was 65.6 mol%. On the basis of the polyphasic taxonomic evidence presented in this study, YJ-S2-02T should be classified as representing a novel species within the genus Novosphingobium, for which name Novosphingobium aureum is proposed, with the type strain YJ-S2-02T (=KACC 21677T =KCTC 72891T=JCM 33996T).

Marinobacter halodurans sp. nov., a halophilic bacterium isolated from sediment of a salt flat.

A Gram-staining-negative, aerobic, cream-coloured, marine bacterium, with rod-shaped cells, designated strain YJ-S3-2T, was isolated from salt flat sediment of Yongyu-do, Republic of Korea. YJ-S3-2T grew at pH 5.0-9.0 (optimum pH 7.0), 4-45 °C (optimum 30 °C) and with 1-18 % (w/v) NaCl (optimum 6 %). The results of 16S rRNA gene sequence analysis indicated that YJ-S3-2T was closely related to Marinobacter segnicrescens SS011B1-4T (97.0 %) followed by, 'Marinobacter nanhaiticus' D15-8W (96.7 %), Marinobacter bryozoorum 50-11T (96.7 %), Marinobacter koreensis DSMZ 179240T T (96.5 %) and Marinobacter bohaiensis T17T (96.5 %). The average nucleotide identity (ANI) and the genome to genome distance calculator (GGDC) estimate values between YJ-S3-2T and related type strains were 73.7-79.8 and 19.9-22.5 %, and also 73.5 and 20.7 % with Marinobacter hydrocarbonoclasticus. YJ-S3-2T was characterized as having Q-9 as the predominant respiratory quinone and the principal fatty acids (>10 %) were C16 : 0 (22.3 %), summed feature 9 (C17 : 1iso ω9c/C16 : 0 10-methyl, 13.8 %) and 3 (C16 : 1ω7c/C16 : 1ω6c, 11.9 %). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids and two unidentified phospholipids. The DNA G+C content of YJ-S3-2T is 60.9 mol%. On the basis of the polyphasic taxonomic evidence presented in this study, YJ-S3-2T should be classified as representing a novel species within the genus Marinobacter, for which name Marinobacter halodurans sp. nov. is proposed, with the type strain YJ-S3-2T (=KACC 19883T=KCTC 62937T=JCM 33109T).

Use of 3D Human Liver Organoids to Predict Drug-Induced Phospholipidosis.

Drug-induced phospholipidosis (PL) is a storage disorder caused by the formation of phospholipid-drug complexes in lysosomes. Because of the diversity of PL between species, human cell-based assays have been used to predict drug-induced PL in humans. We established three-dimensional (3D) human liver organoids as described previously and investigated their liver characteristics through multiple analyses. Drug-induced PL was initiated in these organoids and in monolayer HepG2 cultures, and cellular changes were systemically examined. Organoids that underwent differentiation showed characteristics of hepatocytes rather than HepG2 cells. The organoids also survived under PL-inducing drug conditions for 48 h and maintained a more stable albumin secretion level than the HepG2 cells. More cytoplasmic vacuoles were observed in organoids and HepG2 cells treated with more potent PL-induced drugs, but to a greater extent in organoids than in HepG2 cells. Lysosome-associated membrane protein 2, a marker of lysosome membranes, showed a stronger immunohistochemical signal in the organoids. PL-distinctive lamellar bodies were observed only in amiodarone-treated organoids by transmission electron microscopy. Human liver organoids are thus more sensitive to drug-induced PL and less affected by cytotoxicity than HepG2 cells. Since PL is a chronic condition, these results indicate that organoids better reflect metabolite-mediated hepatotoxicity in vivo and could be a valuable system for evaluating the phospholipidogenic effects of different compounds during drug development.

A proposed stepwise screening framework for the selection of polycyclic aromatic hydrocarbon (PAH)-degrading white rot fungi.

This study suggests a simple three-step screening protocol for the selection of white rot fungi (WRF) capable of degrading polycyclic aromatic hydrocarbons (PAHs), which combines easily applicable bioassay techniques, and verifies that protocol by evaluating the PAH degradation activity, ligninolytic enzyme secretion, and relevant gene expressions of the selected PAH-degraders. Using 120 fungal strains, a sequence of bioassay techniques was applied: Bavendamm's reaction (Step 1), remazol brilliant blue R (RBBR) decolorization (Step 2); assays for tolerance to four mixed PAHs-phenanthrene, anthracene, fluoranthene, and pyrene (Step 3). This stepwise protocol selected 14 PAH-degrading WRF, including Microporus vernicipes, Peniophora incarnata, Perenniporia subacida, Phanerochaete sordida, Phlebia acerina, and Phlebia radiata. Of these, P. incarnata exhibited the highest PAH degradative activity, ranging from 40 to > 90%, which was related to the time-variable secretions of three extracellular ligninolytic enzymes: laccase, manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP). Laccase and MnP production by P. incarnata tended to be greater in the early stages of PAH degradation, whereas its LiP production became intensified with decreasing laccase and MnP production. Pilc1 and pimp1 genes encoding laccase and MnP were expressed, indicating the occurrence of extracellular enzyme-driven biodegradation of PAH by the fungal strains.

ABL001, a Bispecific Antibody Targeting VEGF and DLL4, with Chemotherapy, Synergistically Inhibits Tumor Progression in Xenograft Models.

Delta-like-ligand 4 (DLL4) is a promising target to augment the effects of VEGF inhibitors. A simultaneous blockade of VEGF/VEGFR and DLL4/Notch signaling pathways leads to more potent anti-cancer effects by synergistic anti-angiogenic mechanisms in xenograft models. A bispecific antibody targeting VEGF and DLL4 (ABL001/NOV1501/TR009) demonstrates more potent in vitro and in vivo biological activity compared to VEGF or DLL4 targeting monoclonal antibodies alone and is currently being evaluated in a phase 1 clinical study of heavy chemotherapy or targeted therapy pre-treated cancer patients (ClinicalTrials.gov Identifier: NCT03292783). However, the effects of a combination of ABL001 and chemotherapy on tumor vessels and tumors are not known. Hence, the effects of ABL001, with or without paclitaxel and irinotecan were evaluated in human gastric or colon cancer xenograft models. The combination treatment synergistically inhibited tumor progression compared to each monotherapy. More tumor vessel regression and apoptotic tumor cell induction were observed in tumors treated with the combination therapy, which might be due to tumor vessel normalization. Overall, these findings suggest that the combination therapy of ABL001 with paclitaxel or irinotecan would be a better clinical strategy for the treatment of cancer patients.

Multiple evaluation of the potential toxic effects of sediments and biota collected from an oil-polluted area around Abu Ali Island, Saudi Arabia, Arabian Gulf.

After the Gulf War Oil Spill, there have been many investigations about distributions of oil-derived pollutants nearby areas, but lacking in ecotoxicological assessment. We evaluated the potential toxicity of asphalt mats, sediments, and biota (polychaetes, chitons, snapping shrimps, and crabs) by combining two bioassays (H4IIE-luc and Vibrio fischeri) and in situ microbial community (eDNA). Samples were collected from Abu Ali Island, and organic extracts were bioassayed and further fractionated according to the chemical polarity using silica gel column. Great aryl hydrocarbon receptor (AhR)-mediated potencies and inhibition of bioluminescence were mainly found in aromatics (F2) and saturates (F1) fractions of asphalt mat and sediments, respectively, while great toxicological responses in biota samples were found in resins and polar (F3) fraction. We also confirmed that potential toxicities of biota were species-specific; great AhR-mediated potencies were found in polychaetes and great bioluminescence inhibitions were found in crabs. In microbial communities, most genera (up to 90%) were associated with polycyclic aromatic hydrocarbons (PAHs)-degrading bacteria, supporting that PAHs are the primary stressors of the benthic community around Abu Ali Island. The present study provides useful information on the contamination status, risk assessment of environmental matrices and benthic organisms in Abu Ali Island.

Serinicoccus sediminis sp. nov., isolated from tidal flat sediment.

A novel Gram-strain-positive, non-spore-forming bacterial strain, designated GP-T3-3T, was isolated from sediment sampled at a tidal flat in Gopado, Republic of Korea. Cells were aerobic, catalase-negative, oxidase-positive, non-motile cocci that occurred singly, in pairs or in clusters. Strain GP-T3-3T grew at 4-45 °C (optimum, 28-37 °C), at pH 4.0-12.0 (pH 8.0-9.0) and in the presence of 0-15 % (w/v) NaCl (3-5 %). Colonies of strain GP-T3-3T were deep-yellow, circular, smooth and pulvinate. The results of the phylogenetic analyses based on 16S rRNA gene sequences indicated that strain GP-T3-3T was closely related to Serinicoccus profundi MCCC 1A05965T (99.1 %), Serinicoccus chungangensis CAU 9536T (99.0 %) and Serinicoccus marinus JC1078T (98.0 %). The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The predominant respiratory quinone was MK-8(H4) and the major fatty acids were anteiso-C17 : 0, iso-C16 : 0, iso-C15 : 0 and anteiso-C15 : 0. The polar lipid profile consisted of diphosphadidylglycerol, phosphadidylglycerol, phosphatidylcholine, phosphatidylinositol and two unidentified phospholipids. The DNA G+C content was 72.9 mol%. DNA-DNA relatedness values between strain GP-T3-3T and type strains of the genus Serinicoccus ranged from 28.9 to 50.5 %. On the basis of the phenotypic differences and DNA-DNA relatedness data, the isolate represents a new species of the genus Serinicoccus, for which the name Serinicoccussediminis sp. nov. is proposed. The type strain is GP-T3-3T (=KCTC 49173T=JCM 32825T=KCCM 43309T=KACC 19850T).

Comparative evaluation of bioremediation techniques on oil contaminated sediments in long-term recovery of benthic community health.

While various bioremediation techniques have been widely used at oil spill sites, the in situ efficiency of such techniques on recovering the benthic communities in intertidal areas has not been quantified. Here, the performance of several bioremediation tools such as emulsifiers, multi-enzyme liquid (MEL), microbes, and rice-straw was evaluated by a 90-days semi-field experiment, particularly targeting recovery of benthic community. Temporal efficiency in the removal of sedimentary total petroleum hydrocarbons (TPH), reduction of residual toxicity, and recovery of bacterial diversity, microalgal growth, and benthic production was comprehensively determined. Concentrations of TPH and amphipod mortality for all treatments rapidly decreased within the first 10 days. In addition, the density of bacteria and microphytobenthos generally increased over time for all treatments, indicating recovery in the benthic community health. However, the recovery of some nitrifying bacteria, such as the class Nitrospinia (which are sensitive to oil components) remained incomplete (13-56%) during 90 days. Combination of microbe treatments showed rapid and effective for recovering the benthic community, but after 90 days, all treatments showed high recovery efficiency. Of consideration, the "no action" treatment showed a similar level of recovery to those of microbe and MEL treatments, indicating that the natural recovery process could prevail in certain situations.

Subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelium (MA09-hRPE): A safety and tolerability evaluation in minipigs.

This study aimed to determine the safety and tolerability of the subretinal injection of hESC-derived RPE cells at higher doses than the established clinical dose (5 × 104 cells/150 µL) by using minipigs. The hESC-derived RPE cells (60 or 120 × 104 cells/150 µL) were injected in subretinal region, and minipigs were sacrificed at Weeks 4, 8, and 12 post-surgery. Time-course examination was performed by using fundus photography, optical coherence tomography (OCT), histopathology, and fluorescence in situ hybridization (FISH). After surgery, retinal bleb and pigmentation were seen and retinal bleb became smaller gradually. In histopathology, cell clusters consisting of a uniform population of the round to oval cells were seen at the subretinal injection site. In immunohistochemistry, most of the cells were positive for anti-CD3 and CD45 antibodies but negative for anti-human nuclei antibody; transplanted cells were not detectable by DNA probe in FISH assay. Cell clusters were thought to be a host immune response to trauma or transplanted cells. There were no other changes related to subretinal RPE cell injection. These results suggested that subretinal injection of hESC-derived RPE cells (60 and 120 × 104 cells/150 µL) in minipigs is well-tolerated and safe.

Gemmobacter lutimaris sp. nov., a marine bacterium isolated from a tidal flat.

A novel cream-pigmented marine bacterium, designated strain YJ-T1-11T, was isolated from a tidal flat at Yeongjong-do, Republic of Korea. Cells were rod-shaped, non-motile, aerobic, Gram-reaction-negative, oxidase-positive and catalase-positive. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain YJ-T1-11T clustered with Gemmobacter fontiphilus JS43T (98.3 %) within the genus Gemmobacter and its closest neighbours were G.emmobacter aquatilis DSM 3857T (98.5 %), Gemmobacter aquaticus A1-9T (98.4 %), Gemmobacterlanyuensis Orc-4T (98.4 %), Gemmobacterfontiphilus JS43T (98.3 %), Gemmobactercaeni DCA-1T (98.2 %), Gemmobacternanjingensis Y12T (97.5 %) and Gemmobactertilapiae Ruye-53T (97.2 %). Average nucleotide identity values between the genome sequences of strain YJ-T1-11T and the related type strains ranged from 77.08 to 90.48 %. The predominant fatty acid of strain YJ-T1-11T was summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c). The major isoprenoid quinone was Q-10 and the DNA G+C content was 65.6 mol%. The polar lipid profile consisted of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, diphosphatidylglycerol and three unidentified lipids. The DNA-DNA relatedness values between strain YJ-T1-11T and the type strains of the 12 phylogenetically related species of the genus Gemmobacter were 23.6-53.7 %. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain YJ-T1-11T is considered to represent a novel species of the genus Gemmobacter, for which the name Gemmobacter lutimaris sp. nov. is proposed. The type strain is YJ-T1-11T (=KCTC 62715T=JCM 32828T).

Importance of functional diversity in assessing the recovery of the microbial community after the Hebei Spirit oil spill in Korea.

Over 10 years after the Hebei Spirit oil spill (HSOS), the concentrations of pollutants, such as TPH and PAHs, in spilled crude oil have recovered to background levels, but in some areas, the environment has not fully recovered. In particular, PAHs were more resistant to degradation, and their persistence could have deleterious impacts on the sediment ecosystem. This study aimed to evaluate the microbial recovery of coastal sediments from the HSOS by analyzing the structure and diversity of the microbial community and its functional contribution to PAHs degradation. High-throughput sequencing on the MiSeq platform was conducted using tidal flat sediments collected in 2014 and 2016 from the area contaminated by the HSOS. The microbial recovery was evaluated by various diversity factors, including microbial composition and structure and functional diversity based on PICRUSt analysis. The abundance of microbial taxa associated with TPH degradation was higher in 2014 than that in 2016, but the taxa associated with PAHs degradation were similar between years. These results are consistent with the dynamics of microbes associated with the fate of pollutants, and they also showed similar tendency in functional profiles. That is, even if the pollutants are completely degraded, the microbial community has not yet completely recovered from the HSOS. The evaluation of microbial ecosystems in contaminated environments should consider both the fate of pollutants and the dynamics of microbial species that make functional contributions to the degradation of pollutants.

New Report of Three Unrecorded Species in Trichoderma harzianum Species Complex in Korea.

The genus Trichoderma (Hypocreaceae, Ascomycota) consists of globally distributed fungi. Among them, T. harzianum, one of the most commonly collected Trichoderma species, had been known as a polyphyletic or aggregate species. However, a total of 19 species were determined from the polyphyletic groups of T. harzianum. Thus, we explored Korean "T. harzianum" specimens that were collected in 2013-2014. These specimens were re-examined based on a recent study with translate elongation factor 1-alpha (EF1α) sequences to reveal cryptic Trichoderma species in Korea. As a result, four different species, T. afroharzianum, T. atrobruneum, T. pyramidale, and T. harzianum, were identified. Except T. harzianum, the other three species have not been reported in Korea. In this work, we describe these species and provide figures.

Bioaccumulation of Polycyclic Aromatic Hydrocarbons (PAHs) by the Marine Clam, Mactra veneriformis, Chronically Exposed to Oil-Suspended Particulate Matter Aggregates.

Dispersion and biodegradation of petroleum hydrocarbons are significantly enhanced by formation of oil-suspended particulate matter aggregates (OSAs), but little is known about their adverse effects on benthic invertebrates or microbes. In this study, we investigated: (1) bioaccumulation of polycyclic aromatic hydrocarbons (PAHs) by the marine bivalve, Mactra veneriformis and (2) changes in composition and relative abundances of microbes, during 50-d of an OSAs feeding experiment. Total concentrations of PAHs increased more rapidly during the first week of exposure, peaked at Day 30, then gradually declined to the end of experiment. While bioaccumulation of PAHs by clams varied among the 20 target compounds, two major groups of PAHs were identified by cluster analysis. One group including 3-methylphenanthrene, 1,6-dimethylphenanthrene, 1,2,6,9-tetramethylphenanthrene, and benzo[ a]anthracene showed a fairly constant rate of accumulation, while the second group including 2-methyldibenzothiophene, 2,4-dimethyldibenzothiophene, 2,4,7-trimethyldibenzothiophene, 3-methylchrysene, 6-ethylchrysene, and 1,3,6-trimethylchrysene exhibited a bell-shaped pattern. Bioaccumulation of PAHs by clams was dependent on changes in abundance of Gammaproteobacteria, indicating active degradations of hydrocarbons by selected species. Six key species included: Porticoccus litoralis, Porticoccus hydrocarbonoclasticus, Cycloclasticus spirillensus, Alcanivorax borkumensis, Alcanivorax dieselolei, and Alkalimarinus sediminis. These results are the first to demonstrate interactions of OSAs and macrofauna/microbe in oil cleanup operations.

Development of a Mouse Model of Prostate Cancer Using the Sleeping Beauty Transposon and Electroporation.

The Sleeping Beauty (SB) transposon system is non-viral and uses insertional mutagenesis, resulting in the permanent expression of transferred genes. Although the SB transposon is a useful method for establishing a mouse tumor model, there has been difficulty in using this method to generate tumors in the prostate. In the present study, electroporation was used to enhance the transfection efficiency of the SB transposon. To generate tumors, three constructs (a c-Myc expression cassette, a HRAS (HRas proto-oncogene, GTPase) expression cassette and a shRNA against p53) contained within the SB transposon plasmids were directly injected into the prostate. Electroporation was conducted on the injection site after the injection of the DNA plasmid. Following the tumorigenesis, the tumors were monitored by animal PET imaging and identified by gross observation. After this, the tumors were characterized by using histological and immunohistochemical techniques. The expression of the targeted genes was analyzed by Real-Time qRT-PCR. All mice subjected to the injection were found to have prostate tumors, which was supported by PSA immunohistochemistry. To our knowledge, this is the first demonstration of tumor induction in the mouse prostate using the electroporation-enhanced SB transposon system in combination with c-Myc, HRAS and p53. This model serves as a valuable resource for the future development of SB-induced mouse models of cancer.

Biosurfactant-assisted bioremediation of crude oil by indigenous bacteria isolated from Taean beach sediment.

Crude oil and its derivatives are considered as one group of the most pervasive environmental pollutants in marine environments. Bioremediation using oil-degrading bacteria has emerged as a promising green cleanup alternative in more recent years. The employment of biosurfactant-producing and hydrocarbon-utilizing indigenous bacteria enhances the effectiveness of bioremediation by making hydrocarbons bioavailable for degradation. In this study, the best candidates of biosurfactant-producing indigenous bacteria were selected by screening of biochemical tests. The selected bacteria include Bacillus algicola (003-Phe1), Rhodococcus soli (102-Na5), Isoptericola chiayiensis (103-Na4), and Pseudoalteromonas agarivorans (SDRB-Py1). In general, these isolated species caused low surface tension values (33.9-41.3 mN m-1), high oil spreading (1.2-2.4 cm), and hydrocarbon emulsification (up to 65%) warranting active degradation of hydrocarbons. FT-IR and LC-MS analyses indicated that the monorhamnolipid (Rha-C16:1) and dirhamnolipid (Rha-Rha-C6-C6:1) were commonly produced by the bacteria as potent biosurfactants. The residual crude oil after the biodegradation test was quantitated using GC-MS analysis. The bacteria utilized crude oil as their sole carbon source while the amount of residual crude oil significantly decreased. In addition the cell-free broth containing biosurfactants produced by bacterial strains significantly desorbed crude oil in oil-polluted marine sediment. The selected bacteria might hold additional capacity in crude oil degradation. Biosurfactant-producing indigenous bacteria therefore degrade crude oil hydrocarbon compounds, produce biosurfactants that can increase the emulsification of crude oil and are thus more conducive to the degradation of crude oil.

EZH2 Expression in Naturally Occurring Canine Tumors.

Enhancer of zeste homolog 2 (EZH2) shows upregulated expression in tumors and is an important driver of tumor development and progression. However, the mechanism underlying the mediation of tumor aggressiveness by EZH2 remains unclear. We here investigated the levels of EZH2 in various normal and tumorous dog tissues and compared these patterns with those of the corresponding human tissues. Immunohistochemical analysis showed positive staining for EZH2 in 76 of 82 cases of canine tumors, whereas low or negligible staining occurred in normal tissues and other canine tumors, including hepatocellular adenoma and lipoma. In particular, canine lymphoma, melanoma, basal cell tumors, squamous cell carcinoma, and prostate cancer all show EZH2 overexpression, as do their human counterparts. Given the similarities of spontaneous canine tumors to human cancers, we believe that these canine tumors can be used as animal models in future research and clinical trials in the development of EZH2 inhibitors.

Efficacy of HL036 versus Cyclosporine A in the Treatment of Naturally Occurring Canine Keratoconjunctivitis Sicca.

PURPOSES: To (i) evaluate the efficacy and safety of HL036, a tumor-necrosis factor (TNF)-α-blocking protein, in the treatment of naturally occurring canine keratoconjunctivitis sicca (KCS) and (ii) compare these features with those of 1% cyclosporine A (CsA). MATERIALS AND METHODS: Dogs (n = 29) diagnosed with KCS were randomly assigned to receive one drop topical aqueous HL036 (0.2, 1, or 5 mg/mL) or 1% CsA in the affected eye(s) at 12-h intervals for 42 days. Schirmer's tear test (STT), fluorescein corneal staining (FCS), and clinical-sign scores were evaluated prior to application (day-0) and on days 14, 28, and 42 post-treatment. Of the 29 dogs enrolled, 19 (65.5%) received HL036 (HL036 group) and 10 (34.5%) received 1% CsA (CsA group). A linear mixed-effects model analysis was performed to determine score differences between groups and over time. RESULTS: After treatment, clinical-sign scores and STT values had significantly improved compared with baseline levels in dogs of both treatment groups. Decreases in total clinical-sign scores for the HL036-group were greater than those of 1% CsA group. No severe adverse reactions were noted in either group. CONCLUSIONS: Our findings suggest that topical aqueous HL036 is well-tolerated and more effective than 1% CsA for treating naturally occurring canine KCS.