Targeting lipid-protein interaction to treat Syk-mediated acute myeloid leukemia.

Membrane lipids control the cellular activity of kinases containing the Src homology 2 (SH2) domain through direct lipid-SH2 domain interactions. Here we report development of new nonlipidic small molecule inhibitors of the lipid-SH2 domain interaction that block the cellular activity of their host proteins. As a pilot study, we evaluated the efficacy of lipid-SH2 domain interaction inhibitors for spleen tyrosine kinase (Syk), which is implicated in hematopoietic malignancies, including acute myeloid leukemia (AML). An optimized inhibitor (WC36) specifically and potently suppressed oncogenic activities of Syk in AML cell lines and patient-derived AML cells. Unlike ATP-competitive Syk inhibitors, WC36 was refractory to de novo and acquired drug resistance due to its ability to block not only the Syk kinase activity, but also its noncatalytic scaffolding function that is linked to drug resistance. Collectively, our study shows that targeting lipid-protein interaction is a powerful approach to developing new small molecule drugs.

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways.

De novo genome assembly of the bioluminescent mushroom Omphalotus guepiniiformis reveals an Omphalotus-specific lineage of the luciferase gene block.

Omphalotus guepiniiformis, a bioluminescent mushroom species, is a source of the potentially valuable anticancer chemical. To provide genome information, we de novo assembled the high-quality O. guepiniiformis genome using two Next-Generation sequencing techniques, PacBio and Illumina sequencing. Our draft O. guepiniiformis genome comprises 42.5 Mbp of sequence with only 80 contigs and an N50 sequence length of over 1 Mbp. There were 15,554 predicted coding genes, and 7693 genes were functionally annotated with Gene Ontology terms. We performed a genomic study focusing on mushroom bioluminescent pathway cluster genes by comparing 17 luminescent and 23 non-luminescent Agaricales species belonging to 23 genera. Synteny analysis of genomic regions near the luminescent pathway cluster genes inferred that the Omphalotus lineage was genus-specific. In summary, our de novo assembled O. guepiniiformis genome provides significant biological insights into this organism, including the evolution of the luciferase gene block, and forms the basis for future analyses.

First report of fusarium wilt disease caused by Fusarium equiseti on grafted watermelon in Korea.

In Korea, most of the grafted watermelons are a fusion of bottle gourd (Lagenaria siceraria) as a rootstock and watermelon (Citrullus lanatus) as a scionstock (Lee et al., 2010). Currently, we have collected several samples from grafted watermelon displaying symptoms of yellowing, withered and wilting leaves. When the symptomatic stem was excised, browning vascular tissues were observed due to the colonization of fungal pathogen. From the samples obtained, 25 fungal isolates were identified as species of Fusarium. Among 25 isolates, 18 were identified as Fusarium oxysporum, four as Fusarium solani, and three as Fusarium equiseti (F. equiseti) . Initial assessment showed that one of the F. equiseti isolates (NIHHS 16-126) was highly virulent to rootstock. Interestingly, this is the first time F. equiseti has been identified pathogenic to grafted watermelon. NIHHS 16-126 isolate was collected from watermelon cultivation field around Buyeo-gun (36.25951°N, 126.92044°E) county. Disease incident was estimated to infect approximately 10% of the watermelon plants cultivated in this area. NIHHS 16-126 isolate was examined to confirm its identity. On potato dextrose agar, colonies appeared yellowish-brown while the aerial mycelium was whitish to peach in color. Macroconidia were relatively long (20.21 - 51.13 × 2.30 - 4.5 µm, n=50), comprise of 3-6 septa, curvature shape and its conidiophores were with monophialides. However, microconidia formation was not observed. These morphological characteristics resemble F. equiseti characters as described by Hyun (2019). For molecular identification, an internal transcribed spacer of ribosomal DNA (ITS-rDNA), elongation factor-1α (EF-1α), and beta-tubulin (ß-tub) genes were sequenced using primer pairs of ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Glass and Donaldson 1995), and Bt2a/Bt2b (Carbone and Kohn 1999). BLASTN analysis revealed that ITS-rDNA (LC648248), EF-1α (LC648250), and ß-tub (LC648249) sequences were 99-100% identical to F. equiseti reference sequences (KF515650, KF747331, and KF747330) infected Avicennia marina in China (Lu 2014). Phylogenetic analysis of concatenated ITS-rDNA, EF-1α and ß-tub sequences showed that this isolate clustered in the same clade as F. equiseti, confirming its identity as F. equiseti. For the inoculation, roots of 12-days-old seedlings (watermelon and bottle gourd, n=10 each) were dipped in the conidia suspension (1x106 conidia/µL) for 30 min. Inoculated seedlings were planted in the soil before being transferred to the greenhouse (temperature; 30°C, daylight; 14 hours). Control plants were inoculated with sterile water. Results showed that after 21 days post-inoculation, all inoculated bottle gourd seedlings (n=10) wilted and eventually died. In contrast, none of the inoculated watermelons or control seedlings were affected. Re-isolation of three fungal isolates (infected root) showed that their morphology and gene markers sequence were identical to the original isolates thus fulfilled Koch's postulates. Bottle gourd is the most preferred rootstock for grafted watermelons among Korean farmers due to its ability to resist Fusarium spp. infection. Therefore, the identification of F. equiseti as a fungal that is pathogenic to rootstock is crucial information to manage fusarium wilt disease among grafted watermelon. To our knowledge, this is the first report confirming F. equiseti infection in grafted watermelon plants in Korea.

Oricola thermophila sp. nov., a marine bacterium isolated from tidal flat sediment and emended description of the genus Oricola Hameed et al. 2015.

A Gram-stain-negative, facultatively anaerobic, rod-shaped (1.8-4.4×0.5-0.7 µm) and motile marine bacterium, designated as MEBiC13590T, was isolated from tidal flat sediment sampled at Incheon City, on the west coast of the Republic of Korea. The 16S rRNA gene sequence analysis revealed that strain MEBiC13590T showed high similarity to Oricola cellulosilytica CC-AMH-0T (98.2 %), followed by Oceaniradius stylonematis StC1T (97.5 %); however, it clustered with Oricola cellulosilytica. The phylogenomic tree inferred by the up-to-date bacterial core gene set suggested that strain MEBiC13590T shared a phyletic line with Oricola cellulosilytica. Average nucleotide identity and digital DNA-DNA hybridization values (75.0 and 19.3 %, respectively) between strain MEBiC13590T and Oricola cellulosilytica CC-AMH-0T were below the respective species delineation cutoffs. Growth was observed at 22-50 °C (optimum, 45 °C), at pH 5-9 (optimum, pH 7) and with 1-6 % (optimum, 3 %) NaCl. The predominant cellular fatty acids were C16 : 0 (7.6 %), C18 : 0 (12.2 %), 11-methyl C18 : 1 ω7c (5.7 %), C19 : 0 cyclo ω6c and summed feature 8 (comprising C18 : 1 ω7c and/or C18 : 1 ω6c; 38 %). The DNA G+C content was 63.5 mol%. The major respiratory quinone was Q-10. Several phenotypic characteristics such as growth temperature, oxygen requirement, enzyme activities of urease, gelatinase, lipase (C14), α-chymotrypsin, acid phosphatase, ß-galactosidase, ß-glucosidase etc. differentiate strain MEBiC13590T from Oricola cellulosilytica CC-AMH-0T. Based on this polyphasic taxonomic data, strain MEBiC13590T should be classified as representing a novel species in the genus Oricola for which the name Oricola thermophila sp. nov. is proposed . The type strain is MEBiC13590T (=KCCM 43313T=JCM 33661T).

Effectiveness of Information and Communication Technology on Obesity in Childhood and Adolescence: Systematic Review and Meta-analysis.

BACKGROUND: Internet or mobile device use as a form of information and communication technology (ICT) can be more effective in weight loss and weight maintenance than traditional obesity interventions. OBJECTIVE: The study aims to assess the effectiveness of child-centered ICT interventions on obesity-related outcomes. METHODS: Articles were retrieved from the Cochrane Central Register of Controlled Trials, Embase, and PubMed web-based databases. We selected randomized controlled trials in which the participants were aged <18 years. The primary outcomes were BMI, body weight, BMI z-score, waist circumference, and percentage body fat. RESULTS: In total, 10 of the initial 14,867 studies identified in the databases were selected according to the inclusion criteria. A total of 640 participants were included in the intervention group and 619 in the comparator group. Meta-analyses were conducted considering various subgroups (intervention type, comparator type, target participants, mean age, sex, BMI status, and follow-up period). Overall, ICT interventions demonstrated no significant effect on BMI, body weight, BMI z-score, waist circumference, and percentage body fat. Subgroup analyses revealed that the effect of the intervention was statistically significant for the following: web intervention (weighted mean difference [WMD]=-1.26 kg/m2, 95% CI -2.24 to -0.28), lifestyle modification comparator (WMD=-1.75, 95% CI -2.76 to -0.74), intervention involving both boys and girls (WMD=-1.30, 95% CI -2.14 to -0.46), and intervention involving obesity only (WMD=-1.92, 95% CI -3.75 to -0.09). CONCLUSIONS: The meta-analysis results for children with obesity who used the web intervention program confirmed significant effects on BMI reduction compared with lifestyle modification. Evidence from the meta-analysis identified internet technology as a useful tool for weight loss in children with obesity.

Description of Luteithermobacter gelatinilyticus gen. nov., sp. nov., and Paremcibacter congregatus gen. nov., comb. nov. via reclassification of the genus Emcibacter.

Strain MEBiC09520T, which was isolated from a tidal sediment in Incheon, Korea, is a pale yellow, rod-shaped bacterium, cells of which are 0.4-0.5 µm in width and 1.5-2 µm in length. Strain MEBiC09520T shared 95.17 and 92.57% 16S rRNA gene sequence similarity with Emcibacter nanhaiensis and E. congregatus, respectively. It grew optimally at pH 6.0, at 55 °C and with 2.5-3.5% (w/v) NaCl. Its polar lipid components included phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), an unidentified phospholipid (PL), three unidentified aminolipids (ALs) and two unidentified lipids (L). The fatty acids C16:0, C19:0 cyclo ω8c, C14:0 2-OH and summed feature 8 (C18:1ω7c and/or C18:1ω6c) were predominantly present in its cell wall. Strain MEBiC09520T was thermophilic, while E. nanhaiensis and E. congregatus were mesophilic. Although E. nanhaiensis showed no nitrate reduction activity, MEBiC09520T and E. congregatus showed a positive reaction. These strains differed in carbohydrate utilization. In particular, E. congregatus was able to thrive on various carbohydrate substrates as compared to the other strains. The average nucleotide identity value was 69.92% between strain MEBiC09520T and E. congregatus ZYLT, 70.38% between E. congregatus ZYLT and E. nanhaiensis HTCJW17T, and 72.83% between strain MEBiC09520 and E. nanhaiensis HTCJW17T. Considering these differences, strain MEBiC09520T (=KCCM 43320T=MCCC 1K03920T) is suggested to represent and novel species of a new genus, Luteithermobacter gelatinilyticus gen. nov., sp. nov., and E. congregatus should be reclassified as Paremcibacter congregatus gen. nov., comb. nov.

First report of bacterial shot-hole disease caused by Xanthomonas arboricola pv. pruni on plumcot in South Korea.

Plumcot is an interspecific hybrid product between Japanese plums (Prunus salicina) and apricots (Prunus armeniaca) obtained by the NIHHS, Korea in 1999 [1]. At the early of 2017, black spots-like symptoms were observed on plumcot fruits and leaves at cultivation areas in Naju (34.965595, 126.665853) province. Further investigation shows that approximately 60% of the plumcot leaves in the affected orchard were infected, which caused 40% total production loss. At the early stage of infection, disease symptoms appear as small, angular and water-soaked spots and develop into circular brown spots at the later stages of infection. As the disease progresses, the leaf tissues around the spots became yellow and the lesions enlarged. When the adjacent lesions merged and the necrotic tissues fall off, shot-hole symptoms appear on the leaves. To identify the causal agent of this disease, infected leaf tissues were excised and surface-sterilized with 1% NaOCl for 30 secs prior to rinsing with sterile water, thrice . Tissue samples were then placed in sterile water (0.5 mL) for 5 min before its aliquots were streaked onto Luria-Bertani (LB) agar. Plates then were incubated at 28°C. To obtain pure colonies, bacteria were re-streak into a new LB agar and colonies showing typical Xanthomonas spp. morphology (i.e. convex, smooth, yellow, and mucoid) were subjected to Gram staining assay. For molecular identification, 16S ribosomal DNA (16S-rDNA) and gyrase B (gyrB) genes were amplified using a 9F/1512r and UP-1/UP-2Sr primers [2,3] respectively from 5 gram-negative isolates. PCR products were sequenced and analysed using BLASTN. Result shows that 16S-rDNA and gyrB genes are 99-100% identical to a similar genomic region of Xanthomonas arboricola pv. pruni (Xap) isolated in almond (MK156163), peach (MG049922) and apricot (KX950802) respectively [4,5,6]. 16S-rDNA and gyrB gene sequences were deposited in the GenBank (LC485472 and LC576824), whereas pathogen isolate was deposited into Korean Agricultural Culture Collection (KACC19949). Pathogenicity test was performed using Xap bacterial suspension (108 cfu/mL) inoculated on the abaxial and adaxial surface of plumcot detached leaves. For inoculation, 10 healthy young leaves were used whereas, 5 young leaves mock-inoculated with sterile LB broth were used as a control. Both leaf samples were kept in a closed container to maintain 100% humidity before being incubated at 25°C. The water-soaked symptoms were observed visually on the inoculated leaves 2 to 3 days post-inoculation. No water-soaked symptoms were observed on the control leaves. Morphology and sequences of molecular markers used showed that the 3 bacterial colonies re-isolated from the inoculated leaves were identical to the original isolate, fulfilling Koch's postulate. Pathogenicity tests were repeated twice and the results obtained were consistent with the first experiment. As a new variety of stone fruit cultivated in Korea, information about pathogens and registered agrochemicals to control disease outbreak in plumcot are still limited. Therefore, the identification of Xap as a causal agent to the black spot disease is critical for the development of disease management strategies and to identify appropriate agrochemicals to control the occurrence of this disease in the field. To our knowledge, this is the first report of Xap as a causal agent to the shot-hole disease on the plumcot in Korea.

First Report of Verticillium Wilt Caused by Verticillium dahliae Infection on Chinese Cabbage in Korea.

Chinese cabbage (Brassica rapa L.) is one of the most important vegetables in Korea due to its role as the main ingredient for the making of Kimchi. In June 2014, disease symptoms of leaves wilt, dry, and drop off on Chinese cabbage were observed in a Chinese cabbage farm located at Taebeak (37°26'50.7"N 128°95'50.0"E), Gangwon province, Korea. This disease was observed on approximately 35% of the plants in the field, causing an almost 10% decrease in total production. At the early stage of infection, the color at the edge of the plant foliage changed from green to yellow. As the disease progressed, infected leaves wilted, dried off, and detached from the plant. Soft rot that occurred at the base of the leaf stem and root tissues caused the infected leaves to dry and fell off the plant. To identify the causal agent, a small piece of infected leaf tissues was sterilized with 1% sodium hypochlorite solution for 1 min and rinsed with sterile water before it was transferred onto potato dextrose agar (PDA) media. The plates were then incubated at 25°C for 10 days in the dark. Fungal colonies grown on PDA media were of white-creamy in color with an abundance of mycelia and later develop into black color due to the formation of microsclerotia embedded in the media. Microscopic examination showed conidiophores and phialides were both appeared in a verticillate arrangement, whereas conidia were hyaline, smooth-walled, and ellipsoidal to oval with average size 5.4×2.5 µm (n=100). Microsclerotia appeared in elongate to an irregularly spherical shape and greatly variable in size. The morphological attributes of the fungal isolate described above were comparable to the characteristics of Verticillium dahliae Kleb. (V. dahliae) described by Hawksworth and Talboys (1970), and V. dahliae isolated from Chinese cabbage in Japan reported in Kishi (1998). Pathogenicity test was performed by soaking twelve individual Chinese cabbage seedlings for 15 min into fungal pathogen conidial suspension (1x106 conidium/ml) before transferred into soil tray. The same number of non-inoculated seedlings on the soil tray was used as a control. Inoculated and control plants were then covered with a plastic bag for 24 hours to maintain high humidity before transferred into the greenhouse (25°C). Seven days post-inoculation (dpi), treated plant leaves turned yellow, and soft rot was observed. At 10-dpi, plant leaf tissues dried off and severe soft rot occurred. Pathogenicity test was repeated three times and consistent results were obtained. The re-isolated fungal pathogen from the inoculated plants showed identical morphological characteristics to the original isolates, thus fulfilling Koch's postulates. For further identification, PCR amplification targeting Internal Transcribed Spacer (ITS) and RNA polymerase II gene (RPB2) regions were performed (Liu et al., 1999; White et al., 1990). Each PCR product was sequenced and deposited in the GenBank under the accession LC549667 and LC061275, respectively. Sequence analysis using BLAST showed that the nucleotide sequences of ITS and RPB2 DNA fragments are 99-100% identical to the reference strain of V. dahliae available in the NCBI database (MG585719, HE972023, XM_009652520 and DQ522468, respectively). Therefore, based on the results of morphological and molecular analyses, the fungal pathogen isolated from Chinese cabbage in this study was identified as V. dahliae and deposited in the National Institute of Horticultural and Herbal Science germplasm collection (NIHHS 13-252). Recently, due to high demand and a more competitive price, more Chrysanthemum farmers in Korea switch their crops to Chinese cabbage. Interestingly, the occurrence of V. dahliae infection was also reported to occur in Chrysanthemum plants in Korea (Han et al. 2007), which indicates a serious problem ahead to these farmers. Therefore, in this current study, the identification of V. dahliae pathogenic to Chinese cabbage will provide vital knowledge for the development of disease management strategies to minimize the loss of crop production. To our knowledge, this is the first report that V. dahliae causes Verticillium wilt disease on Chinese cabbage in Korea.

Effects of air cleaners and school characteristics on classroom concentrations of particulate matter in 34 elementary schools in Korea.

Exposure to particulate matter (PM) in school environments has been associated with respiratory illnesses among children. Although using air cleaners was reported to reduce PM exposure and improve residents' health in homes, their effects in classrooms are not well understood. We examined how the use of air cleaners in classrooms and school/classroom characteristics affect the levels of indoor PM. Our environmental study included 102 classrooms from 34 elementary schools located on the mainland peninsula and an island in Korea. Indoor and outdoor PM were monitored simultaneously with portable aerosol spectrometers, and indoor gravimetric PM levels were measured with low volume, size-selective samplers during the class hours. Correlations among PM measurements were computed and final multiple regression models for indoor PM were constructed with a model building procedure. Correlation between indoor and outdoor PM2.5 (PM < 2.5 µm in aerodynamic diameter) was higher (r = 0.78, p < 0.01) than that of PM10 (PM < 10 µm) (r = 0.49, p < 0.01). School location, classroom occupant density, and ambient PM levels significantly (p-values<0.05) affected classroom PM concentrations. The adjusted PM levels in classrooms using air cleaners were significantly (p-values<0.01) lower by approximately 35% than in classrooms not using them. However, air cleaners appeared to remove PM2.5 more effectively than PM10, perhaps because coarse particles settle more rapidly than fine particles on surfaces, or their resuspension and generation rate by occupants exceeds the removal rate by air cleaners. Our study suggests that routine cleaning to remove surface dust along with the use of air cleaners might be required to effectively reduce occupants' exposure in classrooms.

Roseithermus sacchariphilus gen. nov., sp. nov. and proposal of Salisaetaceae fam. nov., representing new family in the order Rhodothermales.

A novel bacterium with cells that were pinkish-cream-coloured, aerobic, rod-shaped, 0.62-1.00 µm wide and 2.3-3.3 µm long, designated as strain MEBiC09517T, was isolated from Buksung-Po, a small port in Incheon, Republic of Korea. Strain MEBiC09517T had low 16S rRNA gene sequence similarity to validly reported strains; among them, Rubrivirgaprofundi SAORIC-476T displayed highest sequence similarity (89.9 %). Nevertheless, the novel strain shared a phylogenetic line with members of the genus Rhodothermus, not the genus Rubrivirga. Optimum growth conditions of strain MEBiC09517T were at 50-55 °C, pH 7 and in 2.0-4.0 % salt concentration. Strain MEBiC09517T was found to be an obligate marine bacterium that requires KCl, MgCl2 and CaCl2 as well as NaCl for growth. A phosphatidylethanolamine, a diphosphatidylglycerol, three glycolipids and four unidentified lipids were the strain's predominant polar lipid components. The fatty acid of the cell wall mainly consisted of carbons with 16 or 18 chain lengths such as C16 : 0, C18 : 0, C18 : 1 and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). The predominant menaquinone was MK-7. The DNA G+C content is 68.65 mol%. Strain MEBiC09517T differs from genera of the order Rhodothermales in terms of fatty acid composition, growth conditions, and range of carbon source utilization. Based on phylogenetic analysis using the strain's 16S rRNA gene sequence and results of physiological tests, strain MEBiC09517T (KCCM=43267T, JCM=32374T) is proposed as Roseithermus sacchariphilus gen. nov., sp. nov. Additionally, the novel family Salisaetaceae fam. nov. based on phylogenetic analysis and physiological characteristics is suggested.

Parashewanella tropica sp. nov., a mesophilic bacterium isolated from a marine sponge from Chuuk lagoon, Federated States of Micronesia, and emended description of the genus Parashewanella.

A mesophilic, straight-rod-shaped, non-flagellated bacterium, designated MEBiC05444T, was isolated from a marine sponge collected from Chuuk lagoon, Federated States of Micronesia. The strain was Gram-negative, catalase- and oxidase-positive, and facultative anaerobic. The isolate aerobically grew at 8-38 °C (optimum, 24-32 °C), pH 4.0-10.0 (pH 7.0-7.5) with an absolute requirement for Na+ up to 6 % (w/v) NaCl (2 %). Phylogenetic analyses based on 16S rRNA gene sequences revealed that MEBiC05444T belonged to the family Shewanellaceae, within the class Gammaproteobacteria. Strain MEBiC05444T showed highest 16S rRNA gene sequence similarity to Parashewanella curva C51T, followed by [Shewanella] irciniae UST040317-058T and Parashewanella spongiae HJ039T (98.9 %, 97.2 and 95.7 %, respectively). In the phylogenetic tree based on the 16S rRNA gene sequences, MEBiC05444T formed a cluster with P. curva C51T, but the average nucleotide identity value between the two strains was 82 %, thus confirming their separation at species level. The major fatty acids were iso-C15 : 0 (19.7 %), summed feature 3 (composed of C16 : 1 ω7c and/or C16 : 1ω6c; 16.1 %) and C17 : 1ω8c (10.2 %). The only detected respiratory quinone was ubiquinone Q-8. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, three unidentified aminoglycolipids, two unidentified glycolipids, an unidentified aminoglycophospholipid and an unidentified lipid. The genomic DNA G+C content of strain MEBiC05444T was 40.8 mol%. Based on the results of polyphasic analysis, the strain represents a novel species of the genus Parashewanella, distinct from P. curva C51T, [Shewanella]irciniae UST040317-058T and P. spongiae HJ039T for which the name Parashewanellatropica sp. nov. is proposed with type strain MEBiC05444T (=KCCM 43304T=JCM 16653T).

Anaerosacchariphilus polymeriproducens gen. nov., sp. nov., an anaerobic bacterium isolated from a salt field.

A Gram-stain-negative oval-rod-shaped, spore-forming anaerobic bacterium, designated as strain MCWD5T, was isolated from sediment of a salt pond in the Republic of Korea (35° 7' 18″ N 126° 19' 4″ E). The 16S rRNA gene sequence analysis revealed that strain MCWD5T had low similarity values to members in the family Lachnospiraceae, such as Robinsoniella peoriensis PPC31T (94.8 %), Ruminococcusgauvreauii CCRI-16110T (94.2 %) and Lachnotalea glycerini DLD10T (94.0 %), and its phylogenetic position is unstable. The strain could grow at 20-42 °C (optimum, 38-42 °C), pH 5.5-10.0 (pH 7.0) and with 0-6 % (2.0 %) NaCl. Strain MCWD5T could not use nitrate, nitrite, sulfate or sulfite as electron acceptors. The strain could utilize various carbohydrates, such as arabinose, cellobiose, glucose, etc., and polymers such as pectin and starch. The major fatty acids of strain MCWD5T were C14 : 0, C16 : 0, C16 : 1ω7c, C18 : 1ω7c DMA and summed feature 8 (C17 : 1ω8c and/or C17 : 2), which was clearly different from those of related genera. The major polar lipids were diphosphatidyglycerol, phosphatidyglycerol and an unknown phospholipid. Based on the results of phylogenetic, physiologic and chemotaxonomic studies, Anaerosacchariphilus polymeriproducens gen. nov., sp. nov. with the type strain MCWD5T (=KCTC 15595T=DSM 105757T) is proposed in the family Lachnospiraceae.

Alternative splicing provides a proactive mechanism for the diurnal CONSTANS dynamics in Arabidopsis photoperiodic flowering.

The circadian clock control of CONSTANS (CO) transcription and the light-mediated stabilization of its encoded protein coordinately adjust photoperiodic flowering by triggering rhythmic expression of the floral integrator flowering locus T (FT). Diurnal accumulation of CO is modulated sequentially by distinct E3 ubiquitin ligases, allowing peak CO to occur in the late afternoon under long days. Here we show that CO abundance is not simply targeted by E3 enzymes but is also actively self-adjusted through dynamic interactions between two CO isoforms. Alternative splicing of CO produces two protein variants, the full-size COα and the truncated COß lacking DNA-binding affinity. Notably, COß, which is resistant to E3 enzymes, induces the interaction of COα with CO-destabilizing E3 enzymes but inhibits the association of COα with CO-stabilizing E3 ligase. These observations demonstrate that CO plays an active role in sustaining its diurnal accumulation dynamics during Arabidopsis photoperiodic flowering.

Lipids Regulate Lck Protein Activity through Their Interactions with the Lck Src Homology 2 Domain.

Lymphocyte-specific protein-tyrosine kinase (Lck) plays an essential role in T cell receptor (TCR) signaling and T cell development, but its activation mechanism is not fully understood. To explore the possibility that plasma membrane (PM) lipids control TCR signaling activities of Lck, we measured the membrane binding properties of its regulatory Src homology 2 (SH2) and Src homology 3 domains. The Lck SH2 domain binds anionic PM lipids with high affinity but with low specificity. Electrostatic potential calculation, NMR analysis, and mutational studies identified the lipid-binding site of the Lck SH2 domain that includes surface-exposed basic, aromatic, and hydrophobic residues but not the phospho-Tyr binding pocket. Mutation of lipid binding residues greatly reduced the interaction of Lck with the ζ chain in the activated TCR signaling complex and its overall TCR signaling activities. These results suggest that PM lipids, including phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, modulate interaction of Lck with its binding partners in the TCR signaling complex and its TCR signaling activities in a spatiotemporally specific manner via its SH2 domain.

LATE ELONGATED HYPOCOTYL regulates photoperiodic flowering via the circadian clock in Arabidopsis.

BACKGROUND: Plants constantly monitor changes in photoperiod or day length to trigger the flowering cycle at the most appropriate time of the year. It is well established that photoperiodic flowering is intimately associated with the circadian clock in Arabidopsis. In support of this notion, many clock-defective mutants exhibit altered photoperiodic sensitivity in inducing flowering. LATE ELONGATED HYPOCOTYL (LHY) and its functional paralogue CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) constitute the core of the circadian clock together with TIMING OF CAB EXPRSSION 1 (TOC1). While it is known that TOC1 contributes to the timing of flowering entirely by modulating the clock function, molecular mechanisms by which LHY and CCA1 regulate flowering time have not been explored. RESULTS: We investigated how LHY and CCA1 regulate photoperiodic flowering through molecular genetic and biochemical studies. It was found that LHY-defective mutants (lhy-7 and lhy-20) exhibit accelerated flowering under both long days (LDs) and short days (SDs). Consistent with the accelerated flowering phenotypes, gene expression analysis revealed that expression of the floral integrator FLOWERING LOCUS T (FT) is up-regulated in the lhy mutants. In addition, the expression peaks of GIGANTEA (GI) and FLAVIN-BINDING, KELCH REPEAT, F-BOX PROTEIN 1 (FKF1) genes, which constitute the clock output pathway that is linked with photoperiodic flowering, were advanced by approximately 4 h in the mutants. Furthermore, the up-regulation of FT disappeared when the endogenous circadian period is matched to the external light/dark cycles in the lhy-7 mutant. Notably, whereas CCA1 binds strongly to FT gene promoter, LHY does not show such DNA-binding activity. CONCLUSIONS: Our data indicate that the advanced expression phases of photoperiodic flowering genes are associated with the clock defects in the lhy mutants and responsible for the reduced photoperiodic sensitivity of the mutant flowering, demonstrating that LHY regulates photoperiodic flowering via the circadian clock, similar to what has been shown with TOC1. It is notable that while LHY regulates photoperiodic flowering in a similar manner as with TOC1, the underlying molecular mechanism would be somewhat distinct from that exerted by CCA1 in Arabidopsis.

A self-regulatory circuit of CIRCADIAN CLOCK-ASSOCIATED1 underlies the circadian clock regulation of temperature responses in Arabidopsis.

The circadian clock synchronizes biological processes to daily cycles of light and temperature. Clock components, including CIRCADIAN CLOCK-ASSOCIATED1 (CCA1), are also associated with cold acclimation. However, it is unknown how CCA1 activity is modulated in coordinating circadian rhythms and cold acclimation. Here, we report that self-regulation of Arabidopsis thaliana CCA1 activity by a splice variant, CCA1ß, links the clock to cold acclimation. CCA1ß interferes with the formation of CCA1α-CCA1α and LATE ELONGATED HYPOCOTYL (LHY)-LHY homodimers, as well as CCA1α-LHY heterodimers, by forming nonfunctional heterodimers with reduced DNA binding affinity. Accordingly, the periods of circadian rhythms were shortened in CCA1ß-overexpressing transgenic plants (35S:CCA1ß), as observed in the cca1 lhy double mutant. In addition, the elongated hypocotyl and leaf petiole phenotypes of CCA1α-overexpressing transgenic plants (35S:CCA1α) were repressed by CCA1ß coexpression. Notably, low temperatures suppressed CCA1 alternative splicing and thus reduced CCA1ß production. Consequently, whereas the 35S:CCA1α transgenic plants exhibited enhanced freezing tolerance, the 35S:CCA1ß transgenic plants were sensitive to freezing, indicating that cold regulation of CCA1 alternative splicing contributes to freezing tolerance. On the basis of these findings, we propose that dynamic self-regulation of CCA1 underlies the clock regulation of temperature responses in Arabidopsis.

Tissue inhibitor of metalloproteinase-1 is responsible for residual pleural thickening in pleural tuberculosis.

Residual pleural thickening (RPT) is the most frequent complication associated with pleural tuberculosis, and may occur even after successful anti-tuberculosis medications. Matrix metalloproteinases (MMPs) are zinc-dependent proteinases capable of degrading all components of the extracellular matrix. The proteolytic action of MMPs may be involved in the pathogenesis of tuberculosis. MMP-9, secreted by monocytes and lymphocyte, may lead to long-term fibrosis. The aim of the present study was to determine whether MMP-2 and/or MMP-9 and their specific inhibitors, tissue inhibitors of metalloproteinase 1 (TIMP-1) and TIMP-2, could be used to predict RPT. This retrospective study enrolled 52 patients diagnosed with pleural tuberculosis. Levels of MMP-2, MMP-9, TIMP-1, and TIM-2 were determined in the pleural fluid by ELISA. The RPT was measured on chest X-ray at the completion of treatment and the final follow-up. The average periods of anti-tuberculosis medication and the follow-up after completion of treatment were 6.7 and 7.6 months, respectively. MMP-2 or MMP-9 levels had no significant correlation to RPT. The patients with RPT > 2 mm at the completion of anti-tuberculosis medication and the final follow-up had higher TIMP-1 levels (p = 0.00 and p = 0.001, respectively). However, patients with RPT > 2 mm at the completion of anti-tuberculosis medication had lower TIMP-2 levels (p = 0.005). In a logistic regression model, elevated TIMP-1 levels at the completion of anti-tuberculosis medications were associated with RPT. In conclusion, higher TIMP-1 levels are responsible for the development of RPT and may be helpful for predicting RPT in pleural tuberculosis.

Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis.

BACKGROUND: The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level. RESULTS: We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5' splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD. CONCLUSION: Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress adaptation in plants. It is also envisioned that alternative splicing of the clock genes plays more complex roles than previously expected.