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Direct oral factor Xa inhibitors are replacing vitamin K-dependent antagonists as anticoagulation treatment in many clinical scenarios. Trauma centers are noting an increase in patients presenting on these medications. The 2018 Food and Drug Administration approval of andexanet alfa provides an alternative anticoagulation reversal. Barriers may limit utilization of new medications including a lack of grade 1A evidence supporting the use of prothrombin complex concentrate (PCC) versus andexanet alfa and cost. To evaluate barriers of andexanet alfa utilization by trauma surgeons, a 15-question survey was conducted. There was a 9% completion rate (n = 89). The results revealed 23.5% would choose andexanet alfa as first-line treatment in children, and 25.8% as first-line treatment in adults. The majority of respondents, 64.7% and 67.4%, would use PCC preferentially in children and adults, respectively. Respondents indicated that cost burden was an overriding factor (76.3%); 42.4% cited lack of high-level efficacy data of andexanet alfa for reversal of factor Xa inhibitors. Additional double-blinded multi-institutional randomized controlled trials comparing 4F-PCC and andexanet alfa for factor Xa inhibitor reversal are needed to support efficacy especially with the increased cost associated.

Background:. Keloids are disfiguring, scar-like lesions that are challenging to treat, with low response rates to current interventions and frequent recurrence. It has been widely reported that keloids are characterized by myofibroblasts, specialized contractile fibroblasts that express alpha-smooth muscle actin (α-SMA). However, evidence supporting a role for myofibroblasts in keloid pathology is inconclusive, with conflicting reports in the literature. This complicates development of more effective therapies, as the benefit of interventions targeting myofibroblasts is unclear. This study was undertaken to determine whether myofibroblasts can be considered characteristic of keloids. Methods:. Myofibroblasts in tissue sections from keloids, hypertrophic scars (HTSs), and normal skin were localized by α-SMA immunostaining. Expression of α-SMA mRNA (ACTA2 gene) in normal skin and keloid tissue, and in fibroblasts from normal skin, keloid, and HTSs, was measured using quantitative polymerase chain reaction. Results:. Normal skin did not exhibit α-SMA-expressing myofibroblasts, but myofibroblasts were identified in 50% of keloids and 60% of HTSs. No significant differences in ACTA2 expression between keloid and normal skin tissue were observed. Mean ACTA2 expression was higher in HTS (2.54-fold, P = 0.005) and keloid fibroblasts (1.75-fold, P = 0.046) versus normal fibroblasts in vitro. However, α-SMA expression in keloids in vivo was not associated with elevated ACTA2 in keloid fibroblasts in vitro. Conclusions:. Despite elevated ACTA2 in cultured keloid fibroblasts, myofibroblast presence is not a consistent feature of keloids. Therefore, therapies that target myofibroblasts may not be effective for all keloids. Further research is required to define the mechanisms driving keloid formation for development of more effective therapies.

Cyst nematodes remain a major threat to global agricultural production, causing huge losses. To understand the parasitism of the cyst nematodes Heterodera trifolii (HT) and Heterodera schachtii (HS), we constructed whole-genome assemblies using short- and long-read sequencing technologies. The nematode genomes were 379 Mb and 183 Mb in size, with the integrated gene models predicting 40,186 and 18,227 genes in HT and HS, respectively. We found more than half of the genes predicted in HT (64.7%) and HS (53.2%) were collinear to their nearest neighbor H . glycines (HG). Large-scale duplication patterns in HT and segmental duplications of more than half of the orthologous genes indicate that the genome of HT is polyploid in nature. Functional analysis of the genes indicated that 65.6% of the HG genes existed within the HT genome. Most abundant genes in HT and HS were involved in gene regulation, DNA integration, and chemotaxis. Differentially expressed genes showed upregulation of cuticle structural constituent genes during egg and female stages and cytoskeletal motor activity-related genes in juvenile stage 2 (J2). Horizontal gene transfer analyses identified four new vitamin biosynthesis genes, pdxK , pdxH , pdxS , and fabG , of bacterial origin, to be first reported in HT and HS. Mitogenomes of HT, HS, and HG showed similar structure, composition, and codon usage. However, rates of substitution of bases in the gene nad4l were significantly different between HT and HS. The described genomes, transcriptomes, and mitogenomes of plant-parasitic nematodes HT and HS are potential bio-resources used to identify several strategies of control of the nematode.

Leaf mustard, characterized by its purple/red/green leaves with a green/white midrib, is known for its thick, tender, and spicy leaves with a unique taste and flavor. There were only a few studies reported on leaf mustard for its morphological and biochemical traits from Korea. A total of 355 leaf mustard accessions stored at the GenBank of the National Agrobiodiversity Center were evaluated for 25 agro-morphological traits and seven intact glucosinolates (GSLs). The accessions showed a wide variation in terms of most of the traits. The quantitative agro-morphological traits varied from 16.0 (leaf length) to 48.7% (petiole width) of the coefficient of variation (CV). The highest variation was observed in glucoiberin (299.5%, CV), while the total GSL showed a CV of 66.1%. Sinigrin, followed by gluconapin and gluconasturtiin, was the most abundant GSL, accounting for as high as 75% of the total GSLs, while glucobrassicanapin and glucoiberin were the least abundant, contributing 0.7% and 0.1% on average, respectively. Sinigrin had a positive significant correlation with all GSLs but gluconasturtiin, while glucobarbarin and gluconasturtiin were highly positively correlated to each other, but least correlated with other GSLs. The leaf length was negatively correlated with sinigrin and glucoiberin. The width of the petiole showed a positive correlation with gluconapin, glucobrassicanapin, and glucobrassicin, while the length of the petiole had a negative correlation with sinigrin, glucobrassicanapin, glucoiberin, glucobrassicin, and the total GSLs. A higher width of the midrib was associated with higher contents of gluconapin, glucobrassicanapin, and glucobrassicin. A PCA analysis based on the agro-morphological traits showed that the first and second principal components accounted for 65.2% of the overall variability. Accessions that form a head tend to exhibit a longer leaf length, a larger plant weight, a thicker midrib, and higher widths of the midrib, petiole, and leaf. The GSLs showed inconsistent inter-and intra-leaf variation. Accessions that identified for various traits in their performance, such as, for example, Yeosu66 and IT259487 (highest total glucosinolates) and IT228984 (highest plant weight), would be promising lines for developing new varieties.

The National Agrobiodiversity Center under the Rural Development Administration (RDA) in Jeonju, Republic of Korea stands as the foremost national genebank in the country. Over the years, the National Agrobiodiversity Center has remained committed to enriching its collection with foreign genetic resources, elevating its status to a world-class plant genetic resources (PGR)- holding genebank. Currently, several steps are being undertaken to improve the accessibility of the collection to national as well as international researchers, improve the data available on the resources, and amend the passport information for the accessions. With the implementation of the Nagoya Protocol, the origin of genetic resources is being highlighted as an important input in the passport information. The RDA-Genebank actively responds to the Nagoya Protocol by supplementing passport data for resources lacking information on their origin. In addition, a large number of conserved resources are continuously multiplied, and agronomic traits are investigated concurrently. With the traditional methods of characterization of the germplasm requiring a significant amount of time and effort, we have initiated high-throughput phenotyping using digital techniques to improve our germplasm data. Primarily, we have started adding seed phenotype information followed by measuring root phenotypes which are stored under agronomic traits. This may be the initial step toward using largescale high-throughput techniques for a germplasm. In this study, we aim to provide an introduction to the RDA-Genebank, to adopted international standards, and to the establishment of high-throughput phenotyping techniques for the improvement of passport information.

The past decade has seen an observable loss of plant biodiversity which can be attributed to changing climate conditions, destroying ecosystems to create farmlands and continuous selective breeding for limited traits. This loss of biodiversity poses a significant bottleneck to plant biologists across the globe working on sustainable solutions to address the current barriers of agricultural productivity. Plant genetic resources centers or genebanks that conserve plant germplasm can majorly contribute towards addressing this problem. Second only to soybean, Brassica remains the largest oil-seed crop and is cultivated across 124 countries, and FAO estimates for a combined gross production values of broccoli, cabbages, cauliflower, mustard and rape seeds stands at a staggering 67.5 billion US dollars during the year 2020. With such a global status, wide variety of uses and more recently, growing importance in the health food sector, the conservation of diverse genetic resources of Brassica appeals for higher priority. Here we review the current status of Brassica conservation across plant genebanks. At present, at least 81,752 accessions of Brassica are recorded to be conserved in 148 holding institutes spread across only 81 countries. Several aspects that need to be addressed to improve proper conservation of the Brassica diversity was well as dissemination of germplasm are discussed. Primarily, the number of accessions conserved across countries and the diversity of Brassica taxa most countries has been highly limited which may lead to biodiversity loss in the longer run. Moreover, several practical challenges in Brassica germplasm conservation especially with respect to taxonomic authorities have been discussed. The current review identifies and highlights areas for progress in Brassica conservation, which include but are not limited to, distribution of conserved Brassica biodiversity, challenges faced by conservation biologists, conservation methods, technical hurdles and future avenues for research in diverse Brassica species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Subramanian, Kim and Hahn.)

The aim of this study was to analyze glucosinolates (GSLs) in germplasm that are currently conserved at the RDA-Genebank. The analysis focused on the glucosinolate diversity among the analyzed germplasms, with the goal of identifying those that would be most useful for future breeding efforts to produce nutritionally rich Choy sum plants. In total, 23 accessions of Choy sums that possessed ample background passport information were selected. On analyzing the glucosinolate content for 17 different glucosinolates, we observed aliphatic GSLs to be the most common (89.45%) and aromatic GSLs to be the least common (6.94%) of the total glucosinolates detected. Among the highly represented aliphatic GSLs, gluconapin and glucobrassicanapin were found to contribute the most (>20%), and sinalbin, glucoraphanin, glucoraphasatin, and glucoiberin were detected the least (less than 0.05%). We identified one of the accessions, IT228140, to synthesize high quantities of glucobrassicanapin and progoitrin, which have been reported to contain several therapeutic applications. These conserved germplasms are potential bioresources for breeders, and the availability of information, including therapeutically important glucosinolate content, can help produce plant varieties that can naturally impact public health.

The genebank at the National Agrobiodiversity Center (RDA-Genebank, Jeonju, Republic of Korea), conserves approximately 8000 germplasms of Brassica spp., of which Chinese cabbage ( Brassica rapa L. ssp. pekinensis ) is one of the major crops actively used as food in Northeast Asia, including Korea, as the main ingredient for kimchi. Glucosinolates are a major class of compounds in Chinese cabbage that are responsible for their distinctive flavor, and RDA-Genebank is constantly building a related database (DB) to select suitable germplasms required by consumers and provide resources for breeding programs. In this study, ten glucosinolates were analyzed in sixty Chinese cabbage germplasms. Six aliphatic glucosinolates were the major components, accounting for 85.00% to 91.98% of total glucosinolates in each germplasm. Among them, gluconapin (333.26 to 23,501.58 μmol∙kg -1 DW) was highly represented, followed by glucobrassicanapin (545.60 to 10,344.70 μmol∙kg -1 DW) and progoitrin (155.28 to 8536.51 μmol∙kg -1 DW). In addition, we selected germplasms with a high content of each studied glucosinolate. To analyze the diversity and distribution of glucosinolates among the studied germplasms, Pearson's correlation was performed, and the related results were interpreted through their biosynthetic pathways. The k-means clustering indicated four optimal clusters, which were confirmed through principal component analysis. Orthogonal projection to latent structure discriminant analysis (OPLS-DA) was also performed on the status (landrace and cultivar) and origin (Korea, China, Taiwan, and Japan) passport data of the germplasms, followed by the calculation of variable importance in the projection (VIP) values. These results are part of a continuous series of studies to analyze the glucosinolates of Brassica germplasms that are being conserved at RDA-Genebank. We aim to provide related results through a public platform accessible to everyone and thereby improve the distribution of Brassica germplasms.

Pepper ( Capsicum spp.; Family: Solanaceae; 2n = 24) is an important crop cultivated worldwide for the consumption of its fresh and dried processed fruits. Pepper fruits are used as raw materials in a wide variety of industrial processes. As a multipurpose vegetable crop, there is a need to increase the yield. However, yield productivity of pepper is severely constrained by infectious plant pathogens, including viruses, bacteria, fungi, and oomycetes. The pepper mild mottle virus (PMMoV) is currently one of the most damaging pathogens associated with yield losses in pepper production worldwide. In addition to impacts on pepper productivity, PMMoV has been detected in domestic and aquatic water resources, as well as in the excreta of animals, including humans. Therefore, PMMoV has been suggested as a potential indicator of domestic water quality. These findings present additional concerns and trigger the need to control the infectious pathogen in crop production. This review provides an overview of the distribution, economic impacts, management, and genome sequence variation of some isolates of PMMoV. We also describe genetic resources available for crop breeding against PMMoV.

Soybean [ Glycine max (L.) Merr.], an important oilseed crop, is a low-cost source of protein and oil. In Southeast Asia and Africa, soybeans are widely cultivated for use as traditional food and feed and industrial purposes. Given the ongoing changes in global climate, developing crops that are resistant to climatic extremes and produce viable yields under predicted climatic conditions will be essential in the coming decades. To develop such crops, it will be necessary to gain a thorough understanding of the genetic basis of agronomic and plant root traits. As plant roots generally lie beneath the soil surface, detailed observations and phenotyping throughout plant development present several challenges, and thus the associated traits have tended to be ignored in genomics studies. In this study, we phenotyped 357 soybean landraces at the early vegetative (V2) growth stages and used a 180 K single-nucleotide polymorphism (SNP) soybean array in a genome-wide association study (GWAS) conducted to determine the phenotypic relationships among root traits, elucidate the genetic bases, and identify significant SNPs associated with root trait-controlling genomic regions/loci. A total of 112 significant SNP loci/regions were detected for seven root traits, and we identified 55 putative candidate genes considered to be the most promising. Our findings in this study indicate that a combined approach based on SNP array and GWAS analyses can be applied to unravel the genetic basis of complex root traits in soybean, and may provide an alternative high-resolution marker strategy to traditional bi-parental mapping. In addition, the identified SNPs, candidate genes, and diverse variations in the root traits of soybean landraces will serve as a valuable basis for further application in genetic studies and the breeding of climate-resilient soybeans characterized by improved root traits.

Soybean ( Glycine max L.) is a crop native to Northeast Asia, including China, Korea, and Japan, but currently cultivated all over the world. The National Agrobiodiversity Center in Korea at the Rural Development Administration (RDA) conserves approximately 26,000 accessions and conducts characterizations of its accessions, to accumulate new information. Roots are essential organs of a plant, providing mechanical support, as well as aiding water and nutrient acquisition. Currently, not much information is available in international gene banks regarding root characterization. We studied the root phenotype of 374 soybean accessions, using a high-throughput method. Eight root morphological traits (RMT) were studied and we observed that the surface area (SA), number of forks (NF), and number of tips (NT) had a positive correlation with total length (LENGTH), and that link average length (LAL) and other traits all had a negative correlation. Additionally, the correlation between seed traits (height, width, and 100-seed weight) and root traits was confirmed for the first time in this experiment. The germplasms were divided into three clusters by k-means clustering, and orthogonal projections to latent structures discriminant analysis (OPLS-DA) was used to compare clusters. The most distinctive characteristics between clusters were total lateral average length (LAD) and total lateral average length (DIAM). Cluster 3 had the highest LENGTH, SA, NF, and NF, whereas cluster 1 had the smallest LENGTH, SA, and NF. We selected the top 10 accessions for each RMT, and IT208321, IT216313, and IT216137 were nominated as the best germplasms. These accessions can be recommended to breeders as materials for breeding programs. This is a preliminary report on the characterization of the root phenotype at an international gene bank and will open up the possibility of improving the available information on accessions in gene banks worldwide.

Purpose: This study was undertaken to identify factors that affect segmental lordosis (SL) after minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) by comparing patients whose postoperative SL increased with those whose decreased., Materials and Methods: Fifty-five patients underwent MIS-TLIF at our institute from January 2018 to September 2019. Demographic, pre- and postoperative radiologic, and cage-related factors were included. Statistical analyses were designed to compare patients whose SL increased with decreased after surgery., Results: After surgery, SL increased in 34 patients (group I) and decreased in 21 patients (group D). The index level, disc lordosis, SL, lumbar lordosis, proximal lordosis (PL), and Y-axis position of the cage (Yc) differed significantly between groups I and D. The cage in group I was more anterior than that in group D (Yc: 55.84% vs. 51.24%). Multivariate analysis showed that SL decreased more significantly after MIS-TLIF when the index level was L3/4 rather than L4/5 [odds ratio (OR): 0.46, p =0.019], as preoperative SL (OR: 0.82, p =0.037) or PL (OR: 0.68, p =0.028) increased, and as the cage became more posterior (OR: 1.10, p =0.032)., Conclusion: Changes in SL after MIS-TLIF appear to be associated with preoperative SL and PL, index level, and Yc. An index level at L4/5 instead of L3/4, smaller preoperative SL or PL, and an anterior position of the cage are likely to result in increased SL after MIS-TLIF., Competing Interests: The authors have no potential conflicts of interest to disclose., (© Copyright: Yonsei University College of Medicine 2022.)

One of the most serious pepper diseases is Phytophthora blight, which is caused by Phytophthora capsici. It is crucial to assess the resistance of pepper genetic resources to Phytophthora blight, understand the genetic resistances, and develop markers for selecting resistant pepper materials in breeding programs. In this study, the resistance of 342 pepper accessions to P. capsici was evaluated. The disease severity score method was used to evaluate the phenotypic responses of pepper accessions inoculated with the KCP7 isolate. A genome-wide association study (GWAS) was performed to identify single nucleotide polymorphisms (SNPs) linked to P. capsici (isolate KCP7) resistance. The pepper population was genotyped using the genotype-by-sequencing (GBS) method, and 45,481 SNPs were obtained. A GWAS analysis was performed using resistance evaluation data and SNP markers. Significantly associated SNPs for P. capsici resistance at 4 weeks after inoculation of the GWAS pepper population were selected. These SNPs for Phytophthora blight resistance were found on all chromosomes except Chr.05, Chr.09, and Chr.11. One of the SNPs found on Chr.02 was converted into a high-resolution melting (HRM) marker, and another marker (QTL5-1) from the previous study was applied to pepper accessions and breeding lines for validation and comparison. This SNP marker was selected because the resistance phenotype and the HRM marker genotype matched well. The selected SNP was named Chr02-1126 and was located at 112 Mb on Chr.02. The Chr02-1126 marker predicted P. capsici resistance with 78.5% accuracy, while the QTL5-1 marker predicted resistance with 80.2% accuracy. Along with the marker for major quantitative traits loci (QTLs) on Chr.05, this Chr02-1126 marker could be used to accurately predict Phytophthora blight resistance in pepper genetic resources. Therefore, this study will assist in the selection of resistant pepper plants in order to breed new phytophthora blight-resistant varieties., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ro, Haile, Hur, Geum, Rhee, Hwang, Kim, Lee, Hahn, Lee and Kang.)

Lettuce is an important dietary source of bioactive phytochemicals. Screening and identification of the health beneficial metabolites and evaluating the relationships with phenotypic characters can help consumers adjust their preferences for lettuce plant types. Thus, we explored the major health-beneficial individual metabolites and antioxidant potential of 113 red pigmented lettuce leaf samples. A UV-Vis spectrophotometer and UPLC-DAD-QTOF/MS (TQ/MS) instruments were used for the identification and quantification of metabolites and antioxidant activity accordingly. The metabolites were quantified against their corresponding external standards. The contents of metabolites varied significantly among lettuce samples. Cyanidin 3- O -(6″- O -malonyl)glucoside (4.7~5013.6 μg/g DW), 2,3-di- O -caffeoyltartaric acid (337.1~19,957.2 μg/g DW), and quercetin 3- O -(6″- O -malonyl)glucoside (45.4~31,121.0 μg/g DW) were the most dominant in red pigmented lettuce samples among anthocyanins, hydroxycinnamoyl derivatives, and flavonols, respectively. Lettuces with dark and very dark red pigmented leaves, circular leaf shape, a strong degree of leaf undulation, and highly dense leaf incisions were found to have high levels of flavonoids and hydroxycinnamoyl derivatives. Principal component analysis was used to investigate similarities and/or differences between samples, and the partial least square discriminant analysis classified them into known groups. The key variables that contributed highly were determined. Our report provides critical data on the bioactive constituents of red pigmented lettuce to breeders developing varieties with enhanced bioactive compounds and to nutraceutical companies developing nutrient dense foods and pharmaceutical formulations.

The cyst nematodes Heterodera schachtii and Heterodera trifolii , whose major hosts are sugar beet and clover, respectively, damage a broad range of plants, resulting in significant economic losses. Nematodes synthesize metabolites for organismal development and social communication. We performed metabolic profiling of H. schachtii and H. trifolii in the egg, juvenile 2 (J2), and female stages. In all, 392 peaks were analyzed by capillary electrophoresis time-of-flight mass spectrometry, which revealed a lot of similarities among metabolomes. Aromatic amino acid metabolism, carbohydrate metabolism, choline metabolism, methionine salvage pathway, glutamate metabolism, urea cycle, glycolysis, gluconeogenesis, coenzyme metabolism, purine metabolism, pyrimidine metabolism, and tricarboxylic acid (TCA) cycle for energy conversion (β-oxidation and branched-chain amino acid metabolism) energy storage were involved in all stages studied. The egg and female stages synthesized higher levels of metabolites compared to the J2 stage. The key metabolites detected were glycerol, guanosine, hydroxyproline, citric acid, phosphorylcholine, and the essential amino acids Phe, Leu, Ser, and Val. Metabolites, such as hydroxyproline, acetylcholine, serotonin, glutathione, and glutathione disulfide, which are associated with growth and reproduction, mobility, and neurotransmission, predominated in the J2 stage. Other metabolites, such as SAM, 3PSer, 3-ureidopropionic acid, CTP, UDP, UTP, 3-hydroxy-3-methylglutaric acid, 2-amino-2-(hydroxymethyl-1,3-propanediol, 2-hydroxy-4-methylvaleric acid, Gly Asp, glucuronic acid-3 + galacturonic acid-3 Ser-Glu, citrulline, and γ-Glu-Asn, were highly detected in the egg stage. Meanwhile, nicotinamide, 3-PG, F6P, Cys, ADP-Ribose, Ru5P, S7P, IMP, DAP, diethanolamine, p -Hydroxybenzoic acid, and γ-Glu-Arg_divalent were unique to the J2 stage. Formiminoglutamic acid, nicotinaminde riboside + XC0089, putrescine, thiamine 2,3-dihydroxybenzoic acid, 3-methyladenine, caffeic acid, ferulic acid, m -hydrobenzoic acid, o - and p -coumaric acid, and shikimic acid were specific to the female stage. Overall, highly similar identities and quantities of metabolites between the corresponding stages of the two species of nematode were observed. Our results will be a valuable resource for further studies of physiological changes related to the development of nematodes and nematode-plant interactions.

Root-knot nematodes (RKNs) are a group of plant-parasitic nematodes that cause damage to various plant species and extensive economical losses. In this study, we performed integrated analysis of miRNA and mRNA expression data to explore the regulation of miRNA and mRNA in RKNs. In particular, we aimed to elucidate the mRNA targets of Meloidogyne incognita miRNAs and variations of the RKN transcriptome during five stages of its life cycle. Stage-wise RNA sequencing of M. incognita resulted in clean read numbers of 56,902,902, 50,762,456, 40,968,532, 47,309,223, and 51,730,234 for the egg, J2, J3, J4, and female stages, respectively. Overall, stage-dependent mRNA sequencing revealed that 17,423 genes were expressed in the transcriptome of M. incognita . The egg stage showed the maximum number of transcripts, and 12,803 gene transcripts were expressed in all stages. Functional Gene Ontology (GO) analysis resulted in three main GO classes: biological process, cellular components, and molecular function; the detected sequences were longer than sequences in the reference genome. Stage-wise selected fragments per kilobase of transcript per million mapped reads (FPKM) values of the top 10 stage-specific and common mRNAs were used to construct expression profiles, and 20 mRNAs were validated through quantitative real-time PCR (qRT-PCR). Next, we used three target prediction programs (miRanda, RNAhybrid, and PITA) to obtain 2431 potential target miRNA genes in RKNs, which regulate 8331 mRNAs. The predicted potential targets of miRNA were generally involved in cellular and metabolic processes, binding of molecules in the cell, membranes, and organelles. Stage-wise miRNA target analysis revealed that the egg stage contains heat shock proteins, transcriptional factors, and DNA repair proteins, whereas J2 includes DNA replication, heat shock, and ubiquitin-conjugating pathway-related proteins; the J3 and J4 stages are represented by the major sperm protein domain and translation-related proteins, respectively. In the female stage, we found proteins related to the maintenance of molybdopterin-binding domain-containing proteins and ubiquitin-mediated protein degradation. In total, 29 highly expressed stage-specific mRNA-targeting miRNAs were analyzed using qRT-PCR to validate the sequence analysis data. Overall, our findings provide new insights into the molecular mechanisms occurring at various developmental stages of the RKN life cycle, thus aiding in the identification of potential control strategies.

Objective: To investigate the radiographic characteristics of the uppermost instrumented vertebrae (UIV) and UIV+1 compression fractures that are predictive of revision surgery following long-segment spinal fixation., Methods: A total 27 patients who presented newly developed compression fracture at UIV, UIV+1 after long segment spinal fixation (minimum 5 vertebral bodies, lowest instrumented vertebra of L5 or distal) were reviewed retrospectively. Patients were divided into two groups according to following management : revisional surgery (group A, n=13) and conservative care (group B, n=14). Pre- and postoperative images, and images taken shortly before and after the occurrence of fracture were evaluated for radiologic characteristics., Results: Despite similar degrees of surgical correction of deformity, the fate of the two groups with proximal junctional compression fractures differed. Immediately after the fracture, the decrement of adjacent disc height in group A (32.3±7.6 mm to 23.7±8.4 mm, Δ=8.5±6.9 mm) was greater than group B (31.0±13.9 mm to 30.1±15.5 mm, Δ=0.9±2.9 mm, p=0.003). Pre-operative magnetic resonance imaging indicated that group A patients have a higher grade of disc degeneration adjacent to fractured vertebrae compared to group B (modified Pfirrmann grade, group A : 6.10±0.99, group B : 4.08±0.90, p=0.004). Binary logistic regression analysis indicated that decrement of disc height was the only associated risk factor for future revision surgery (odds ratio, 1.891; 95% confidence interval, 1.121-3.190; p=0.017)., Conclusion: Proximal junctional vertebral compression fractures with greater early-stage decrement of adjacent disc height were associated with increased risk of future neurological deterioration and necessity of revision. The condition of adjacent disc degeneration should be considered regarding severity and revision rate of proximal junctional kyphosis/proximal junction failures.

Recently, spine surgery has gradually evolved from conventional open surgery to minimally invasive surgery, and endoscopic spine surgery (ESS) has become an important procedure in minimally invasive spine surgery. With improvements in the optics, spine endoscope, endoscopic burr, and irrigation pump, the indications of ESS are gradually widening from lumbar to cervical and thoracic spine. ESS was not only used previously for disc herniations that were contained without migration but is also used currently for highly migrated disc herniations and spinal stenosis; thus, the indications of ESS will be further expanded. Although ESS has certain advantages such as less soft tissue dissection and muscle trauma, reduced blood loss, less damage to the epidural blood supply and consequent less epidural fibrosis and scarring, reduced hospital stay, early functional recovery, and improvement of quality of life as well as better cosmesis, several obstacles remain for ESS to be widespread because it has a steep learning curve and surgical outcome is strongly dependent on the surgeon's skillfulness. A solid surgical technique requires reproducibility and ensured safety in addition to surgical outcomes. In this review article, how to improve ESS was investigated by grafting novel technologies such as navigation, robotics, and 3-dimensional and ultraresolution visualization., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cabbage (Brassica oleracea var. capitate L.) is an important vegetable crop that is widely cultivated throughout the world. In August 2019, wilting symptoms on cabbage (stunted growth, withered leaves, and wilted plants) were observed in a cabbage field of Pyeongchang, Gangwon Province, with an incidence of 5 to 10%. To identify the cause, symptomatic root tissue was excised, surface-sterilized with 70% ethanol, and rinsed thrice with sterile distilled water. The samples were dried on blotter paper, placed onto potato dextrose agar (PDA), and incubated at 25°C for 1 week. Five morphologically similar fungal isolates were sub-cultured and purified using the single spore isolation method (Choi et al. 1999). The fungus produced colonies with abundant, loosely floccose, whitish-brown aerial mycelia and pale-orange pigmentation on PDA. Macroconidia had four 4 to six 6 septa, a foot-shaped basal cell, an elongated apical cell, and a size of 20.2 to 31.8 × 2.2 to 4.1 μm (n = 30). No microconidia were observed. Chlamydospores were produced from hyphae and were most often intercalary, in pairs or solitary, globose, and frequently formed chains (6.2? to 11.7 μm, n = 10). Based on these morphological characteristics, the fungus was identified as Fusarium equiseti (Leslie and Summerell 2006). A representative isolate was deposited in the Korean Agricultural Culture Collection (KACC48935). For molecular characterization, portions of the translation elongation factor 1-alpha (TEF-1α) and second largest subunit of RNA polymerase II (RPB2) genes were amplified from the representative isolate using the primers pair of TEF-1α (O'Donnell et al. 2000) and GQ505815 (Fusarium MLST database), and sequenced. Searched BLASTn of the RPB2 sequence (MT576587) to the Fusarium MLST database showed 99.94% similarity to the F. incarnatum-equiseti species complex (GQ505850) and 98.85 % identity to both F. equiseti (GQ505599) and F. equiseti (GQ505772). Further, the TEF-1α sequence (MT084815) showed 100% identity to F. equiseti (KT224215) and 99.85% identity to F. equiseti (GQ505599), respectively. Therefore, the fungus was identified as F. equiseti based on morphological and molecular identification. For pathogenicity testing, a conidial suspension (1 × 106 conidia/ml) was prepared by harvesting macroconidia from 2-week-old cultures on PDA. Fifteen 4-week-old cabbage seedlings (cv. 12-Aadrika) were inoculated by dipping roots into the conidial suspension for 30 min. The inoculated plants were transplanted into a 50-hole plastic tray containing sterilized soil and maintained in a growth chamber at 25°C, with a relative humidity of >80%, and a 12-h/12-h light/dark cycle. After 4 days, the first wilt symptoms were observed on inoculated seedlings, and the infected plants eventually died within 1 to 2 weeks after inoculation. No symptoms were observed in plants inoculated with sterilized distilled water. The fungus was re-isolated from symptomatic tissues of inoculated plants and its colony and spore morphology were identical to those of the original isolate, thus confirming Koch's postulates. Fusarium wilt caused by F. equiseti has been reported in various crops, such as cauliflower in China, cumin in India, and Vitis vinifera in Spain (Farr and Rossman 2020). To our knowledge, this is the first report of F. equiseti causing Fusarium wilt on cabbage in Korea. It This disease poses a threat to cabbage production in Korea, and effective disease management strategies need to be developed.

The genus Carthamus is a diverse group of plants belonging to the family Compositae. Florets of Carthamus species exhibit various colors, including white, yellow, orange, and red, which are related to their metabolite compositions. We aimed to investigate the metabolites accumulated in florets of three wild ( C. lanatus , C. palaestinus, and C. turkestanicus ) and one cultivated ( C. tinctorius ) species of safflower at three developmental stages. Metabolites were extracted from freeze-dried florets using 70% methanol; qualification and quantification were carried out using liquid chromatography quadrupole time-of-flight mass spectrometry in positive and negative ion modes followed by extraction of the peaks. Fifty-six metabolites, including phenylpropanoids, chalcones, isoflavonoids, flavanones, flavonols, flavones, and other primary metabolites, were identified for the first time in safflower wild species. The orange florets contained high abundances of safflomin A, anhydrosafflor yellow B, and baimaside, whereas white/cream and light-yellow pigmented florets had high abundances of 1,5-dicaffeoylquinic acid, luteolin 7- O -glucuronide, and apigenin 7- O -β- D -glucuronide. The principal component analysis clearly distinguished the samples based on their pigment types, indicating that color is a dominant factor dictating the identity and amount of the metabolites. Pearson correlation data based on levels of metabolites showed that orange and yellow florets were significantly correlated to each other. White and cream pigmented species were also highly correlated. Comparison between three developmental stages of safflower wild species based on their metabolite profile showed inconsistent. The findings of this study broaden the current knowledge of safflower metabolism. The wide diversity of metabolites in safflower materials also helps in efforts to improve crop quality and agronomic traits.

Meloidogyne incognita is a devastating plant parasitic nematode that causes root knot disease in a wide range of plants. In the present study, we investigated host-induced RNA interference (RNAi) gene silencing of chitin biosynthesis pathway genes (chitin synthase, glucose-6-phosphate isomerase, and trehalase) in transgenic tobacco plants. To develop an RNAi vector, ubiquitin (UBQ1) promoter was directly cloned, and to generate an RNAi construct, expression of three genes was suppressed using the GATEWAY system. Further, transgenic Nicotiana benthamiana lines expressing dsRNA for chitin synthase (CS), glucose-6-phosphate isomerase (GPI), and trehalase 1 (TH1) were generated. Quantitative PCR analysis confirmed endogenous mRNA expression of root knot nematode (RKN) and revealed that all three genes were more highly expressed in the female stage than in eggs and in the parasitic stage. In vivo, transformed roots were challenged with M . incognita . The number of eggs and root knots were significantly decreased by 60-90% in RNAi transgenic lines. As evident, root galls obtained from transgenic RNAi lines exhibited 0.01- to 0.70-fold downregulation of transcript levels of targeted genes compared with galls isolated from control plants. Furthermore, phenotypic characteristics such as female size and width were also marginally altered, while effect of egg mass per egg number in RNAi transgenic lines was reduced. These results indicate the relevance and significance of targeting chitin biosynthesis genes during the nematode lifespan. Overall, our results suggest that further developments in RNAi efficiency in commercially valued crops can be applied to employ RNAi against other plant parasitic nematodes.

Safflower ( Carthamus tinctorius L.) has long been grown as a crop due to its commercial utility as oil, animal feed, and pharmacologically significant secondary metabolites. The integration of omics approaches, including genomics, transcriptomics, metabolomics, and proteomics datasets, has provided more comprehensive knowledge of the chemical composition of crop plants for multiple applications. Knowledge of a metabolome of plant is crucial to optimize the evolution of crop traits, improve crop yields and quality, and ensure nutritional and health factors that provide the opportunity to produce functional food or feedstuffs. Safflower contains numerous chemical components that possess many pharmacological activities including central nervous, cardiac, vascular, anticoagulant, reproductive, gastrointestinal, antioxidant, hypolipidemic, and metabolic activities, providing many other human health benefits. In addition to classical metabolite studies, this review focuses on several metabolite-based working techniques and updates to provide a summary of the current medical applications of safflower.

In an effort to isolate novel natural antibiotics, we searched for antibacterial long-chain N-acyl amino acid synthase (NAS) genes from 70,000 soil metagenome clones by Bacillus subtilis-overlaying screening. In an antibacterial cosmid clone, YS92B, a single gene nasYPL was responsible for the production of the Nas. nasYPL was 903 bp long, and the deduced amino acid sequence showed the highest 71% identity with a hypothetical protein from Massilia niastensis. Phylogenetic analysis demonstrated that NasYPL belongs to Group 1 Nas. Heterologous expression of the same nasYPL gene in Escherichia coli and two Pseudomonas strains (P. putida and P. koreensis) conferred antibacterial activities against Listeria monocytogenes, Staphylococcus epidermidis, and Bacillus subtilis. Mass spectral analysis of the antibacterial fractions identified 7 peaks corresponding to long-chain N-acyl tyrosine, 5 peaks to N-acyl phenylalanine, and 3 peaks to N-acyl leucine (or isoleucine) derivatives linked with 7 fatty acids, indicating enzymatic products derived by NasYPL. Therefore, NasYPL expression by host-specific manner may provide applicable antibacterial characteristics to biotechnologically important Pseudomonas strains., (Copyright © 2019. Published by Elsevier B.V.)

The original version of this article contained a mistake. The bands for HA Tag and t-ERK in Figs. 2d, 2h, 3d are incorrect. The author informs that these errors had no influence in the scientific content of the paper. The corrected figures (Figs. 2 and 3) are given below.

Objective: Hermetia illucens is a voracious insect scavenger that efficiently decomposes food waste. To exploit novel hydrolytic enzymes from this insect, we constructed a fosmid metagenome library using unculturable H. illucens intestinal microorganisms., Results: Functional screening of the library on carboxymethyl cellulose plates identified a fosmid clone with a product displaying hydrolytic activity. Fosmid sequence analysis revealed a novel mannan-degrading gene (ManEM17) composed of 1371 base pairs, encoding 456 amino acids with a deduced 54 amino acid N-terminal signal peptide sequence. Conceptual translation and domain analysis revealed that sequence homology was highest (46%) with endo-1,4-β-mannosidase of Anaerophaga thermohalophila. Phylogenetic and domain analysis indicated that ManEM17 belongs to a novel β-mannanase containing a glycoside hydrolase family 26 domain. The recombinant protein (rManEM17) was expressed in Escherichia coli, exhibiting the highest activity at 55 °C and pH 6.5. The protein hydrolyzed substrates with β-1,4-glycosidic mannoses; maximum specific activity (5467 U mg -1 ) occurred toward locust bean gum galactomannan. However, rManEM17 did not hydrolyze p-Nitrophenyl-β-pyranosides, demonstrating endo-form mannanase activity. Furthermore, rManEM17 was highly stable under stringent conditions, including polar organic solvents as well as chemical reducing and denaturing reagents., Conclusions: ManEM17 is an attractive candidate for mannan degradation under the high-organic-solvent and protein-denaturing processes in food and feed industries.

Hermetia illucens is a voracious insect scavenger, decomposing food waste efficiently. To survey novel hydrolytic enzymes, we constructed a fosmid metagenome library using unculturable intestinal microorganisms from H. illucens in our previous study (Lee et al., 2014). Functional screening of the library on carboxymethyl cellulose plates identified a fosmid clone the product of which displayed hydrolytic activity. Sequence analysis of the fosmid revealed a novel α-galactosidase gene, Agas2. The Agas2 gene is composed of 2,007 base pairs encoding 668 amino acids with a deduced 25 amino acid N-terminal signal peptide sequence. The conceptual translation and domain analysis of Agas2 showed the highest sequence identity (84%) with the putative α-galactosidase of Dysgonomonas sp. HGC4, exhibiting well-conserved domain homology with glycosyl hydrolase family 97. Phylogenetic analysis indicated that Agas2 may be a currently uncharacterized α-galactosidase. The recombinant protein, rAgas2, was successfully expressed in E. coli. rAgas2 showed the highest activity at 40 °C and pH 7.0. It displayed great pH stability within a pH range of 5-11 for 15 h at 4 °C. rAgas2 was highly stable under stringent conditions, including polar organic solvents, non-ionic detergents, salt, and proteases. rAgas2 hydrolyzed α-d-galactose substrates, showing the maximum enzymatic activity toward p-nitrophenyl α-d-galactopyranoside (specific activity 128.37 U/mg). However, rAgas2 did not hydrolyze substrates linked with β-glucose moieties. Overall, Agas2 may be an attractive candidate for the degradation of α-galactose family oligosaccharides in high-salt, protease-rich and high-organic-solvent processes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

N -linked glycosylation is one of the key post-translational modifications. α1,3-Fucosyltransferase (OsFucT) is responsible for transferring α1,3-linked fucose residues to the glycoprotein N -glycan in plants. We characterized an Osfuct mutant that displayed pleiotropic developmental defects, such as impaired anther and pollen development, diminished growth, shorter plant height, fewer tillers, and shorter panicle length and internodes under field conditions. In addition, the anthers were curved, the pollen grains were shriveled, and pollen viability and pollen number per anther decreased dramatically in the mutant. Matrix-assisted laser desorption/ionization time-of-flight analyses of the N -glycans revealed that α1,3-fucose was lacking in the N -glycan structure of the mutant. Mutant complementation revealed that the phenotype was caused by loss of Osfuct function. Transcriptome profiling also showed that several genes essential for plant developmental processes were significantly altered in the mutant, including protein kinases, transcription factors, genes involved in metabolism, genes related to protein synthesis, and hypothetical proteins. Moreover, the mutant exhibited sensitivity to an increased concentration of salt. This study facilitates a further understanding of the function of genes mediating N -glycan modification and anther and pollen development in rice.

Meloidogyne incognita is a common root-knot nematode with a wide range of plant hosts. We aimed to study the metabolites produced at each stage of the nematode life cycle to understand its development. Metabolites of Meloidogyne incognita were extracted at egg, J2, J3, J4, and female stages and 110 metabolites with available standards were quantified using CE-TOF/MS. Analyses indicated abundance of stage-specific metabolites with the exception of J3 and J4 stages which shared similar metabolic profiles. The egg stage showed increased abundance in glycolysis and energy metabolism related metabolites while the J2 metabolites are associated with tissue formation, motility, and neurotransmission. The J3 and J4 stages indicated amino acid metabolism and urea cycle- related metabolites. The female stage was characterized with polyamine synthesis, antioxidant activity, and synthesis of reproduction related metabolites. Such metabolic profiling helps us understand the dynamic physiological changes related to each developmental stage of the root-knot nematode life cycle., Competing Interests: The authors declare no conflict of interest.

Main Conclusion: Genome-wide screening of Saccharomyces cerevisiae revealed that signaling pathways related to the alkaline pH stress contribute to resistance to plant antimicrobial peptide, Pn-AMP1. Plant antimicrobial peptides (AMPs) are considered to be promising candidates for controlling phytopathogens. Pn-AMP1 is a hevein-type plant AMP that shows potent and broad-spectrum antifungal activity. Genome-wide chemogenomic screening was performed using heterozygous and homozygous diploid deletion pools of Saccharomyces cerevisiae as a chemogenetic model system to identify genes whose deletion conferred enhanced sensitivity to Pn-AMP1. This assay identified 44 deletion strains with fitness defects in the presence of Pn-AMP1. Strong fitness defects were observed in strains with deletions of genes encoding components of several pathways and complex known to participate in the adaptive response to alkaline pH stress, including the cell wall integrity (CWI), calcineurin/Crz1, Rim101, SNF1 pathways and endosomal sorting complex required for transport (ESCRT complex). Gene ontology (GO) enrichment analysis of these genes revealed that the most highly overrepresented GO term was "cellular response to alkaline pH". We found that 32 of the 44 deletion strains tested (72 %) showed significant growth defects compared with their wild type at alkaline pH. Furthermore, 9 deletion strains (20 %) exhibited enhanced sensitivity to Pn-AMP1 at ambient pH compared to acidic pH. Although several hundred plant AMPs have been reported, their modes of action remain largely uncharacterized. This study demonstrates that the signaling pathways that coordinate the adaptive response to alkaline pH also confer resistance to a hevein-type plant AMP in S. cerevisiae. Our findings have broad implications for the design of novel and potent antifungal agents.

In this study, we investigated global changes in miRNAs of Meloidogyne incognita throughout its life cycle. Small RNA sequencing resulted in approximately 62, 38, 38, 35, and 39 Mb reads in the egg, J2, J3, J4, and female stages, respectively. Overall, we identified 2724 known and 383 novel miRNAs (read count > 10) from all stages, of which 169 known and 13 novel miRNA were common to all the five stages. Among the stage-specific miRNAs, miR-286 was highly expressed in eggs, miR-2401 in J2, miR-8 and miR-187 in J3, miR-6736 in J4, and miR-17 in the female stages. These miRNAs are reported to be involved in embryo and neural development, muscular function, and control of apoptosis. Cluster analysis indicated the presence of 91 miRNA clusters, of which 36 clusters were novel and identified in this study. Comparison of miRNA families with other nematodes showed 17 families to be commonly absent in animal parasitic nematodes and M. incognita . Validation of 43 predicted common and stage-specific miRNA by quantitative PCR (qPCR) indicated their expression in the nematode. Stage-wise exploration of M. incognita miRNAs has not been carried out before and this work presents information on common and stage-specific miRNAs of the root-knot nematode., Competing Interests: The authors declare no conflict of interest.

A novel pullulanase gene, PulSS4, was identified from the gut microflora of Hermetia illucens by a function-based metagenome screening. The PulSS4 gene had an open reading frame of 4455 base pairs, and encoded a mature protein of 1484 amino acids, with a signal peptide sequence of 44 amino acids. The deduced amino acid sequence of PulSS4 gene showed 51% identity with that of the amylopullulanase of Amphibacillus xylanus, exhibiting no significant sequence homology to already known pullulanases. A conserved domain analysis revealed it to be a pullulanase type II with respective active sites at the N-terminal pullulanase and C-terminal amylase domain. PulSS4 was active in the temperature range of 10-50°C, with an optimum activity at 40°C. It was active in the pH range of 6.5-10.5, with optimum pH at 9.0, and retained more than 80% of its original activity in a broad pH range of 5-11 for 24h at 30°C. Also, PulSS4 was highly stable against many different chemical reagents, including 10% polar organic solvents and 1% non-ionic detergents. Overall, PulSS4 is expected to have the strong potential for application in biotechnological industries that require high activity at moderate temperature and alkaline conditions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We expressed the heat-labile enterotoxin B (LTB) subunit from enterotoxigenic Escherichia coli and the cholera toxin B (CTB) subunit from Vibrio cholerae under the control of the rice (Oryza sativa) globulin (Glb) promoter. Binding of recombinant LTB and CTB proteins was confirmed based on GM1-ganglioside binding enzyme-linked immunosorbent assays (GM1-ELISA). Real-time PCR of three generations (T3, T4, and T5) in homozygous lines (LCI-11) showed single copies of LTB, CTB, bar and Tnos. LTB and CTB proteins in rice transgenic lines were detected by Western blot analysis. Immunogenicity trials of rice-derived CTB and LTB antigens were evaluated through oral and intraperitoneal administration in mice, respectively. The results revealed that LTB- and CTB-specific IgG levels were enhanced in the sera of intraperitoneally immunized mice. Similarly, the toxin-neutralizing activity of CTB and LTB in serum of orally immunized mice was associated with elevated levels of both IgG and IgA. The results of the present study suggest that the combined expression of CTB and LTB proteins can be utilized to produce vaccines against enterotoxigenic strains of Escherichia coli and Vibrio cholera, for the prevention of diarrhea.

Main Conclusion: The aim of this study is to demonstrate the feasibility of down-regulating endogeneous prefoldin-2 root-knot nematode transcripts by expressing dsRNA with sequence identity to the nematode gene in tobacco roots under the influence of strong Arabidopsis ubiquitin (UBQ1) promoter. Root-knot nematodes (RKNs) are sedentary endoparasites infecting a wide range of plant species. They parasitise the root system, thereby disrupting water and nutrient uptake and causing major reductions in crop yields. The most reliable means of controlling RKNs is via the use of soil fumigants such as methyl bromide. With the emergence of RNA interference (RNAi) technology, which permits host-mediated nematode gene silencing, a new strategy to control plant pathogens has become available. In the present study, we investigated host-induced RNAi gene silencing of prefoldin-2 in transgenic Nicotiana benthamiana. Reductions in prefoldin-2 mRNA transcript levels were observed when nematodes were soaked in a dsRNA solution in vitro. Furthermore, nematode reproduction was suppressed in RNAi transgenic lines, as evident by reductions in the numbers of root knots (by 34-60 % in independent RNAi lines) and egg masses (by 33-58 %). Endogenous expression of prefoldin-2, analysed via real-time polymerase chain reaction and Western blotting, revealed that the gene was strongly expressed in the pre-parasitic J2 stage. Our observations demonstrate the relevance and potential importance of targeting the prefoldin gene during the nematode life cycle. The work also suggests that further improvements in silencing efficiency in economically important crops can be accomplished using RNAi directed against plant-parasitic nematodes.

Although lumbar discectomy is an effective treatment for lumbar disc herniation, complications exist, including postoperative disc height loss, facet joint degeneration, and recurrent disc herniation. To solve these problems, annular closure devices have been utilized in other countries, producing satisfactory results, but there has been no report of annular closure device use in our country. Here, we demonstrate the preliminary reports of Barricaid® insertion in 3 patients who underwent surgery for lumbar disc herniation.

Objective: To study practical guidelines and strategies in the treatment of cervical osteomyelitis., Methods: We retrospectively reviewed 14 patients who underwent surgical treatment for cervical osteomyelitis from May 2000 to July 2008. We investigated their clinical course, antibiotic regimen, surgical methods, and laboratory and radiologic findings including X-ray, CT and MRI., Results: 5 patients had primary spondylodiscitis, 5 patients had post operative spondylodiscitis and 4 patients had tuberculosis in cervical spine. The causative microorganisms were MRSA (5), P. aeruginosa (1), Methicillin resistant coagulase negative streptococcus (1), P. aeruginosa changed to MRSA (1), and 2 patients showed no growth on culture studies. Patients were treated 13.8 weeks (range, 5.4-25.8) with IV antibiotics and then treated for 58.2 days (range, 13-106) with oral antibiotics. Antituberculotic medications were used for a mean of 383.8 days. Patients were treated with anterior debridement and fusion (5), irrigation and debridement (5), simultaneous cervical anterior interbody and transthoracic thoracic interbody fusion (1). 3 patients underwent the planned 2-staged operation, which included an anterior debridement with or without fusion for the 1(st) operation and posterior instrumentation for 2(nd) operation. 10 patients (71.4%) had neurologic deficits at the time of diagnosis and 7 patients (70%) among them improved post-operatively., Conclusion: Anterior cervical spine surgery is the preferable treatment option in patients with neurological deterioration, extensive bony destruction with expected kyphotic deformity, and uncontrolled infection being managed only with antibiotics. Antibiotics are also important for thorough treatment.

A metagenomic fosmid library was constructed using genomic DNA isolated from the gut microflora of Hermetia illucens, a black soldier fly. A cellulase-positive clone, with the CS10 gene, was identified by extensive Congo-red overlay screenings for cellulase activity from the fosmid library of 92,000 clones. The CS10 gene was composed of a 996 bp DNA sequence encoding the mature protein of 331 amino acids. The deduced amino acids of CS10 showed 72% sequence identity with the glycosyl hydrolase family 5 gene of Dysgonomonas mossii, displaying no significant sequence homology to already known cellulases. The purified CS10 protein presented a single band of cellulase activity with a molecular mass of approximately 40 kDa on the SDS-PAGE gel and zymogram. The purified CS10 protein exhibited optimal activity at 50°C and pH 7.0, and the thermostability and pH stability of CS10 were preserved at the ranges of 20~50°C and pH 4.0~10.0. CS10 exhibited little loss of cellulase activity against various chemical reagents such as 10% polar organic solvents, 1% non-ionic detergents, and 0.5 M denaturing agents. Moreover, the substrate specificity and the product patterns by thinlayer chromatography suggested that CS10 is an endo-β-1,4-glucanase. From these biochemical properties of CS10, it is expected that the enzyme has the potential for application in industrial processes.

A functional screen of a metagenomic library from "Upo" swamp sediment in Korea identified a gene EstL28, the product of which displayed lipolytic properties on a tributyrin-supplemented medium. The EstL28 sequence encodes a 290 amino acid protein (designated as EstL28), with a predicted molecular weight of 31.3 kDa. The encoded EstL28 protein exhibited the highest sequence similarity (45 %) to a hydrolase found in Streptococcus sanguinis. Phylogenetic analysis indicated that EstL28 belongs to a currently uncharacterized family of esterases. Within the conserved α/β-hydrolase 6 domain, the EstL28 retains the catalytic triad Ser₁₀₃-Asp₂₄₈-His₂₆₈ that is typical of esterases. The Ser103 residue in the catalytic triad is located in the consensus pentapeptide motif GXSXG. The purified EstL28 enzyme worked optimally at 35 °C and pH 8.5 and remained stable at temperatures lower than 20 °C. The catalytic activity of EstL28 was maximal with p-nitrophenyl butyrate, indicating that it was an esterase. This enzyme also exhibited stable activity in the presence of methanol, ethanol, isopropanol, and dimethyl sulfoxide. Therefore, the level of stability in organic solvents and cold temperature suggests that EstL28 has potential for many biotechnological applications.

Cyclophilin, a cytosolic receptor for the immunosuppressive drug cyclosporin A, plays a role in diverse pathophysiologies along with its receptor, CD147. Although the interaction between cyclophilin A and CD147 is well established in inflammatory disease, that of cyclophilin B (CypB) with CD147 has not been fully explored, especially in cancer cell biology, and the exact molecular mechanism underlying such an association is poorly understood. In this study, we first identified high expression levels of CypB in 54 % of hepatocellular carcinoma patient tissues but in only 12.5 % of normal liver tissues. Then, we demonstrated that CypB overexpression protects human hepatoma cells against oxidative stress through its binding to CD147; this protective effect depends on the peptidyl prolyl isomerase activity of CypB. siRNA-mediated knockdown of CypB expression rendered hepatoma cells more vulnerable to ROS-mediated apoptosis. Furthermore, we also determined that a direct interaction between secreted CypB and CD147 regulates the extracellular signal-regulated kinase intracellular signaling pathway and is indispensible for the protective functions of CypB. For the first time, we demonstrated that CypB has an essential function in protecting hepatoma cells against oxidative stress through binding to CD147 and regulating the ERK pathway.

Endometrial cancer is a common type of malignant tumor of the human female genital tract, which typically occurs after menopause. Asian nations, including Korea, Japan and China, have a 4-5 times lower incidence than Western industrialized nations. However, in recent years, there has been a marked increase in the incidence of the disease. Peroxiredoxin (Prx) is an antioxidant enzyme that consists of six isoforms in mammals. These enzymes share a common reactive Cys residue in the N-terminal region, and are capable of breaking down H(2)O(2) as a peroxidase and involving thioredoxin or glutathione as the electron donor. In the present study, we evaluated the expression of Prx isoforms in normal endometrium, endometrial hyperplasia and endometrial cancer. A total of 240 patients, diagnosed with endometrial cancer by immunohistochemistry, were enrolled in this study. Results showed that Prx I, III, IV and V were negative or weakly expressed in normal endometrium, whereas levels of Prx II and VI were strongly expressed. Notably, the expression levels of Prx III and V were upregulated in endometrial cancer, compared with normal endometrium and endometrial hyperplasia. However, no differences in the staining intensities according to the grade of lesion were observed in the other Prx isoforms. The Kaplan-Meier survival analysis demonstrated that Prx V expression in endometrial cancer is significantly associated with survival rate. Therefore, we suggest that Prx V is a clinically significant prognostic marker for the development of endometrial cancer.

Human tissue-plasminogen activator (t-PA) is a thrombolytic protein that plays an active role in dissolving fibrin clots by fibrinolysis and in activating plasminogen to plasmin in blood vessels. t-PA and synthetic t-PA (st-PA) genes were expressed as enzymatically active form in hairy roots of Oriental melon (Cucumis melo L. cv. Geumssaragi-euncheon) infected by Agrobacterium rhizogenes. The insertion of the t-PA genes in genomic DNA of transgenic hairy roots was verified by PCR. The presence and expression of t-PA-specific transcripts in the total RNAs of transgenic hairy roots were confirmed by RT-PCR. Western blot analysis of the transgenic hairy roots showed a single major band of 59-kDa recombinant t-PAs. ELISA demonstrated that the highest level of recombinant t-PA (798 ng mg⁻¹) was detected in hairy roots expressing t-PA. Similarly, the maximum fibrinolysis of recombinant t-PAs was observed in hairy roots transformed with t-PA. WPM medium was found to be more suitable for rapid growth of hairy roots among all the seven media types tested. The hairy root production was 5.8 times higher than that of White medium. The total yield of hairy roots grown on WPM medium was 621.8±8.7 g L⁻¹ at pH 7.0. These studies demonstrate that the hairy roots could be employed for the mass production of enzymatically active t-PA., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

We investigated the inhibitory activity of glycosaminoglycans (GAGs) in terms of growth, adhesion, and VacA vacuolation of Helicobacter pylori. Intact acharan sulfate (AS, MW:114 kDa) potently inhibited H. pylori adhesion to Kato III cells with IC(50) value of 1.4 mg/mL, while other GAGs did not show any inhibitory activity except for heparin which is a well-known inhibitor of H. pylori adhesion. To investigate whether low molecular weight acharan sulfate (LMWAS) can inhibit H. pylori adhesion, we performed chemical depolymerization of AS by radical reactions to obtain LMWAS. Its physicochemical properties were characterized by high-performance size exclusion chromatography (HPSEC), agarose gel electrophoresis, disaccharide compositional analysis after digestion with heparinase II, and (1)H-NMR spectroscopy. The most potent molecular size of LMWAS was 3 kDa with IC(50) value of 32 μg/mL, which is 44-fold more potent than intact AS. These results suggest that AS as well as other GAGs can be chemically depolymerized by free radicals and LMWAS compared to intact AS can be applied as a pharmaceutical candidate in order to inhibit H. pylori adhesion to Kato III cells.

Many aspects of plant metabolism that are involved in plant growth and development are influenced by light-regulated diurnal rhythms as well as endogenous clock-regulated circadian rhythms. To identify the rhythmic proteins in rice, periodically grown (12h light/12h dark cycle) seedlings were harvested for three days at six-hour intervals. Continuous dark-adapted plants were also harvested for two days. Among approximately 3000 reproducible protein spots on each gel, proteomic analysis ascertained 354 spots (~12%) as light-regulated rhythmic proteins, in which 53 spots showed prolonged rhythm under continuous dark conditions. Of these 354 ascertained rhythmic protein spots, 74 diurnal spots and 10 prolonged rhythmic spots under continuous dark were identified by MALDI-TOF MS analysis. The rhythmic proteins were functionally classified into photosynthesis, central metabolism, protein synthesis, nitrogen metabolism, stress resistance, signal transduction and unknown. Comparative analysis of our proteomic data with the public microarray database (the Plant DIURNAL Project) and RT-PCR analysis of rhythmic proteins showed differences in rhythmic expression phases between mRNA and protein, suggesting that the clock-regulated proteins in rice are modulated by not only transcriptional but also post-transcriptional, translational, and/or post-translational processes., (2011 Elsevier B.V. All rights reserved.)

Background: Foot-and-mouth disease virus (FMDV) is a small single-stranded RNA virus which belongs to the family Picornaviridae, genus Apthovirus. It is a principal cause of FMD which is highly contagious in livestock. In a wild type virus infection, infected animals usually elicit antibodies against structural and non-structural protein of FMDV. A structural protein, VP1, is involved in neutralization of virus particle, and has both B and T cell epitopes. A RNA-dependent RNA polymerase, 3D, is highly conserved among other serotypes and strongly immunogenic, therefore, we selected VP1 and 3D as vaccine targets., Methods: VP1 and 3D genes were codon-optimized to enhance protein expression level and cloned into mammalian expression vector. To produce recombinant protein, VP1 and 3D genes were also cloned into pET vector. The VP1 and 3D DNA or proteins were co-immunized into 5 weeks old BALB/C mice., Results: Antigen-specific serum antibody (Ab) responses were detected by Ab ELISA. Cellular immune response against VP1 and 3D was confirmed by ELISpot assay., Conclusion: The results showed that all DNA- and protein-immunized groups induced cellular immune responses, suggesting that both DNA and recombinant protein vaccine administration efficiently induced Ag-specific humoral and cellular immune responses.

Human ceruloplasmin, a copper binding alpha(2)-glycoprotein, was purified by a single-step procedure using acharan sulfate affinity chromatography. Acharan sulfate was immobilized to amine-functionalized agarose matrix through carboxylic acids. Ceruloplasmin in human plasma was obtained from 0.4 M NaCl salt elution and characterized by SDS-PAGE (132 and 125 kDa), isoelectric focusing (pI 4.6), Western blotting, and MALDI-TOF-MS peptide mass fingerprinting. Ceruloplasmin was purified 106 fold with a specific oxidase activity of 0.53 U/mg protein.

Chondroitin sulfate ABC lyase (ChonABC) is an enzyme with broad specificity that depolymerizes via beta-elimination chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans (GAGs). ChonABC eliminates the glycosidic bond of its GAG substrates on the nonreducing end of their uronic acid component. This lyase possesses the unusual ability to act on both epimers of uronic acid, either glucuronic acid present in CS or iduronic acid in DS. Recently, we cloned, purified, and determined the three-dimensional structure of a broad specificity chondroitin sulfate ABC lyase from Bacteroides thetaiotaomicron (BactnABC) and identified two sets of catalytic residues. Here, we report the detailed biochemical characterization of BactnABC together with extensive site-directed mutagenesis resulting in characterization of the previously identified active site residues. BactnABC's catalysis is stimulated by Ca(2+) and Mg(2+) cations, particularly against DS. It displays extremely low activity toward hyaluronic acid and no activity toward heparin/heparan sulfate. Degradation of CS and DS by BactnABC yields only disaccharide products, pointing to an exolytic mode of action. The kinetic evaluations of the active-site mutants indicate that CS and DS substrates bind in the same active site, which is accompanied by a conformational change bringing the two sets of active site residues together. Conservative replacements of key residues suggest that His345 plays the role of a general base, initiating the degradation by abstracting the C5 bound proton from DS substrates, whereas either Tyr461 or His454 perform the equivalent role for CS substrates. Tyr461 is proposed, as well, to serve as general acid, completing the degradation of both CS and DS by protonating the leaving group.

Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a beta-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: (alpha/alpha)(5) for CS (chondroitin lyases AC) and beta-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located approximately 12 A from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.

The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the -2,015 bp 5'-upstream promoter region of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and beta-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds. Progressive 5'-deletions up to the -963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression. Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both EGFP intensity and fluorometric GUS activity, respectively.