The impact of sexual reproduction-induced vertical migration on the complexity of harmful algal blooms in Heterosigma akashiwo.

Vertical migration behaviour, which is integral to marine energy circulation, is a prevalent trait among marine organisms. However, the behaviour of phytoplankton, particularly beyond diel vertical migration (DVM), remain underexplored compared to groups like zooplankton. Through the lens of the harmful alga Heterosigma akashiwo, which exhibits unique vertical migrations and fluctuating red tide patterns, this study aimed to explore the ecological intricacies and diverse benefits of phytoplankton vertical migration behaviours. During the bloom period of H. akashiwo, we unexpectedly observed a dense concentration of cells at bottom layer during daytime. This phase coincided with the emergence of cells related to this species' sexual reproduction. Laboratory experiments further showed an elevated frequency of sexual reproduction in the cell populations that migrated to deeper depths compared to those at the surface. This finding implies a connection between dense bottom accumulation (BA) and the life cycle transitions of the species. This BA phase persisted for two days, after which the populations returned to their standard DVM behaviour, providing insight into the unique fluctuating red tide patterns of H. akashiwo. Our study suggests that phytoplankton vertical migrations are not strictly dictated by DVM, revealing diverse vertical migration behaviours that may contribute to the complexity of harmful algal bloom patterns.

Insights into phylogenetic relationships and genome evolution of subfamily Commelinoideae (Commelinaceae Mirb.) inferred from complete chloroplast genomes.

BACKGROUND: Commelinaceae (Commelinales) comprise 41 genera and are widely distributed in both the Old and New Worlds, except in Europe. The relationships among genera in this family have been suggested in several morphological and molecular studies. However, it is difficult to explain their relationships due to high morphological variations and low support values. Currently, many researchers have been using complete chloroplast genome data for inferring the evolution of land plants. In this study, we completed 15 new plastid genome sequences of subfamily Commelinoideae using the Mi-seq platform. We utilized genome data to reveal the structural variations and reconstruct the problematic positions of genera for the first time. RESULTS: All examined species of Commelinoideae have three pseudogenes (accD, rpoA, and ycf15), and the former two might be a synapomorphy within Commelinales. Only four species in tribe Commelineae presented IR expansion, which affected duplication of the rpl22 gene. We identified inversions that range from approximately 3 to 15 kb in four taxa (Amischotolype, Belosynapsis, Murdannia, and Streptolirion). The phylogenetic analysis using 77 chloroplast protein-coding genes with maximum parsimony, maximum likelihood, and Bayesian inference suggests that Palisota is most closely related to tribe Commelineae, supported by high support values. This result differs significantly from the current classification of Commelinaceae. Also, we resolved the unclear position of Streptoliriinae and the monophyly of Dichorisandrinae. Among the ten CDS (ndhH, rpoC2, ndhA, rps3, ndhG, ndhD, ccsA, ndhF, matK, and ycf1), which have high nucleotide diversity values (Pi > 0.045) and over 500 bp length, four CDS (ndhH, rpoC2, matK, and ycf1) show that they are congruent with the topology derived from 77 chloroplast protein-coding genes. CONCLUSIONS: In this study, we provide detailed information on the 15 complete plastid genomes of Commelinoideae taxa. We identified characteristic pseudogenes and nucleotide diversity, which can be used to infer the family evolutionary history. Also, further research is needed to revise the position of Palisota in the current classification of Commelinaceae.

Characteristics of the completed chloroplast genome sequence of Xanthium spinosum: comparative analyses, identification of mutational hotspots and phylogenetic implications.

BACKGROUND: The invasive species Xanthium spinosum has been used as a traditional Chinese medicine for many years. Unfortunately, no extensive molecular studies of this plant have been conducted. RESULTS: Here, the complete chloroplast (cp) genome sequence of X. spinosum was assembled and analyzed. The cp genome of X. spinosum was 152,422 base pairs (bp) in length, with a quadripartite circular structure. The cp genome contained 115 unique genes, including 80 PCGs, 31 tRNA genes, and 4 rRNA genes. Comparative analyses revealed that X. spinosum contains a large number of repeats (999 repeats) and 701 SSRs in its cp genome. Fourteen divergences (Π > 0.03) were found in the intergenic spacer regions. Phylogenetic analyses revealed that Parthenium is a sister clade to both Xanthium and Ambrosia and an early-diverging lineage of subtribe Ambrosiinae, although this finding was supported with a very weak bootstrap value. CONCLUSION: The identified hotspot regions could be used as molecular markers for resolving phylogenetic relationships and species identification in the genus Xanthium.

Implications of plastome evolution in the true lilies (monocot order Liliales).

The families of the monocot order Liliales exhibit highly contrasting characteristic of photosynthetic and mycoheterotrophic life histories. Although previous phylogenetic and morphological studies of Liliales have been conducted, they have not examined molecular evolution associated with this contrasting phenomenon. Here, we conduct the first comparative plastome study of all ten families of Liliales using 29 newly sequenced plastid genomes analyzed together with previously published data. We also present a phylogenetic analysis for Liliales of 78 plastid genes combined with 22 genes from all three genomes (nuclear 18S rDNA and phyC; 17 plastid genes; and mitochondrial matR, atpA, and cob). Within the newly generated phylogenetic tree of Liliales, we evaluate the ancestral state changes of selected morphological traits in the order. There are no significant differences in plastid genome features among species that show divergent characteristics correlated with family circumscriptions. However, the results clearly differentiate between photosynthetic and mycoheterotrophic taxa of Liliales in terms of genome structure, and gene content and order. The newly sequenced plastid genomes and combined three-genome data revealed Smilacaceae as sister to Liliaceae instead of Philesiaceae and Ripogonaceae. Additionally, we propose a revised familial classification system of Liliales that consists of nine families, considering Ripogonaceae a synonym of Philesiaceae. The ancestral state reconstruction indicated synapomorphies for each family of Liliales, except Liliaceae, Melanthiaceae and Colchicaceae. A taxonomic key for all nine families of Liliales is also provided.

Difference in joint line convergence angle between the supine and standing positions is the most important predictive factor of coronal correction error after medial opening wedge high tibial osteotomy.

PURPOSE: Coronal correction errors after medial opening wedge high tibial osteotomy (MOWHTO) occasionally occur even with the assistance of navigation. The purpose of the present study was to determine the navigation accuracy in MOWHTO and to identify factors that affect the coronal correction error after navigation-assisted MOWHTO. METHODS: A total of 114 knees treated with navigation-assisted MOWHTO were reviewed retrospectively. Mechanical axis (MA) on standing radiograph and medial proximal tibial angle (MPTA) were measured preoperatively and at 6 months postoperatively, and the differences (ΔMA and ΔMPTA) were calculated. Joint line convergence angle (JLCA) on supine and standing radiographs was measured preoperatively, and their difference (ΔJLCA) was calculated. To assess the navigation accuracy, ΔMA and ΔMPTA were compared with the coronal correction by navigation (ΔNMA) using intraclass correlation coefficients (ICCs). Univariable and multivariable regression analyses were used to identify factors that affect coronal correction discrepancy (ΔMA - ΔNMA). RESULTS: The reliability of navigation was good in terms of bony correction (ICC between ΔNMA and ΔMPTA, 0.844) and fair in terms of MA correction (ICC between ΔNMA and ΔMA, 0.706). The mean coronal correction discrepancy was 2.0° ± 2.4°. In the multivariable analysis, ΔJLCA was shown to be a predictive factor of coronal correction discrepancy (unstandardized coefficient, 1.026; R2, 0.470). CONCLUSION: Navigation in MOWHTO provided reliable information about bony correction; however, MA tended to be overcorrected. The difference in JLCA between the supine and standing radiographs was the most important preoperative factor that predicted the coronal correction discrepancy after MOWHTO. In patients with larger ΔJLCA, each degree of ΔJLCA should be subtracted from the planned amount of correction angle when preoperative planning is performed using standing radiographs. LEVEL OF EVIDENCE: IV.

Catechol-Type Flavonoids from the Branches of Elaeagnus glabra f. oxyphylla Exert Antioxidant Activity and an Inhibitory Effect on Amyloid-ß Aggregation.

Elaeagnus glabra f. oxyphylla (Elaeagnaceae) is a small evergreen tree with narrow lanceolate leaves that is native to Korea. In this work, we studied the chemical composition of E. glabra f. oxyphylla branches (EGFOB) for the first time. Additionally, we evaluated the effects of the ethanol extract of EGFOB and each of its chemical components on key mediators of Alzheimer's disease (AD), namely, amyloid-ß (Aß) aggregation and oxidative stress. The ethanol extract of EGFOB decreased Aß aggregation (IC50 = 32.01 µg/mL) and the levels of the oxidative free radicals 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (IC50 = 11.35 and 12.32 µg/mL, respectively). Sixteen compounds were isolated from EGFOB. Among them, procyanidin B3 (8), procyanidin B4 (9), and helichrysoside (13) significantly inhibited Aß aggregation (IC50 = 14.59, 32.64, and 44.45 µM, respectively), indicating their potential as bioactive compounds to control Aß aggregation. Furthermore, these compounds markedly enhanced in vitro scavenging activity against ABTS (IC50 = 3.21-4.61 µM). In the DPPH test, they showed lower scavenging activity than in the ABTS test (IC50 ≥ 54.88 µM). Thus, these results suggest that EGFOB and specifically compounds 8, 9, and 13 may be beneficial in AD prevention and treatment through their antioxidant and anti-Aß aggregation activities.

Osteomeles schwerinae Extract and Its Major Compounds Inhibit Methylglyoxal-Induced Apoptosis in Human Retinal Pigment Epithelial Cells.

The accumulation and formation of advanced glycation end products (AGEs) are related to diabetes and age-related disease. Osteomeles schwerinae C. K. Schneid. (Rosaceae, OSSC) is used traditionally for the treatment of various diseases in Asia. Previous studies have shown that OSSC elicits preventive effects in an in vivo model of diabetes. This study was to evaluate the antiapoptotic effects of dried leaves and twigs of OSSC extract and its major compounds in ARPE-19 cells-spontaneously arising human retinal pigment epithelial cells-under diabetic conditions. To examine the effects of an OSSC extract and its active compounds (acetylvitexin, hyperoside and quercitrin) on apoptosis in methylglyoxal (MG, the active precursor in the formation of AGEs)-treated ARPE-19 cells and the mechanism by which these effects occur, apoptosis was measured using flow cytometry analysis. Protein expression levels of phospho-p53 (p-p53), Bax and Bcl-2 were determined by western blot analyses. The OSSC extract inhibited apoptosis in MG-treated ARPE-19 cells in a dose-dependent manner. The major compounds also reduced the rate of apoptosis. Both the extract and major compounds also inhibited the expression of p-p53 and Bax and increased the levels of Bcl-2 that had been previously reduced by MG treatment. The OSSC extract (0.1 µg/mL) and its major compounds (0.01 µM) attenuated apoptosis in ARPE-19 cells under toxic diabetic conditions by downregulating of expression of p-p53 and Bax. OSSC may serve as an alternative therapy to retard the development of diabetic retinopathy.

Difficulties During Ponseti Casting for the Treatment of Idiopathic Clubfoot.

The Ponseti method has resulted in excellent outcomes in the treatment of idiopathic clubfoot. To evaluate the process of serial casting, most studies previously used the number of casts required to achieve correction. In contrast, difficulties that occurred during serial casting were rarely studied. We reviewed 47 patients with severe idiopathic clubfoot (Dimeglio score ≥10) and a follow-up period of ≥4 years by focusing on difficulties during the Ponseti casting and their related factors. All events requiring temporary discontinuation and/or delay of scheduled casting were considered difficulties. Among the 47 patients, difficulties occurred in 18 patients (38.3%). Initial Dimeglio score (14.9 ± 2.5 [vs 13.5 ± 2.3, p = .042]), its subcomponent of equinus (3.8 ± 0.4 [vs 3.3 ± 0.6, p = .005]), and the number of casts (6.3 ± 2.0 [vs 4.9 ± 1.2, p = .009]) in the 18 patients with difficulties were greater than those of the remaining 29 patients. In addition, the foot length was shorter (7.2 ± 0.6 cm [vs 7.7 ± 0.8 cm, p = .030]) in patients with difficulties. In the regression analysis, both subcomponents of equinus and foot length showed the significance for the occurrence of difficulties. During 8.7 ± 3.8 years, further surgery was needed in 35 patients (74.5%) because of residual or recurrent deformity. The ratio was higher in patients with difficulties (94.4% [vs 62.1%, p = .017]). In our experience, difficulties frequently occurred in feet with rigid equinus and small length, and further surgery was required after the initial correction in almost all of the feet with difficulties.

The newly developed single nucleotide polymorphism (SNP) markers for a potentially medicinal plant, Crepidiastrum denticulatum (Asteraceae), inferred from complete chloroplast genome data.

Medicinal effects of Crepidiastrum denticulatum have been previously reported. However, the genomic resources of this species and its applications have not been studied. In this study, based on the next generation sequencing method (Miseq sequencing system), we characterize the chloroplast genome of C. denticulatum which contains a large single copy (84,112 bp) and a small single copy (18,519 bp), separated by two inverted repeat regions (25,074 bp). This genome consists of 80 protein-coding gene, 30 tRNAs, and four rRNAs. Notably, the trnT_GGU is pseudogenized because of a small insertion within the coding region. Comparative genomic analysis reveals a high similarity among Asteraceae taxa. However, the junctions between LSC, SSC, and IRs locate in different positions within rps19 and ycf1 among examined species. Also, we describe a newly developed single nucleotide polymorphism (SNP) marker for C. denticulatum based on amplification-refractory mutation system (ARMS) technique. The markers, inferred from SNP in rbcL and matK genes, show effectiveness to recognize C. denticulatum from other related taxa through simple PCR protocol. The chloroplast genome-based molecular markers are effective to distinguish a potentially medicinal species, C. denticulatum, from other related taxa. Additionally, the complete chloroplast genome of C. denticulatum provides initial genomic data for further studies on phylogenomics, population genetics, and evolutionary history of Crepidiastrum as well as other taxa in Asteraceae.

Evaluation of skin sensitization potential of chemicals by local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry.

The local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA used to identify skin sensitizers. This assay measures the proliferation of auricular lymph node cells (LNCs) during the induction phase of skin sensitization and the number of BrdU-positive LNCs using flow cytometry. We determined if LLNA: BrdU-FCM can evaluate the skin sensitization potential of 20 substances, including 16 sensitizers and 4 non-sensitizers, that were tested using LLNA: DA and LLNA: BrdU-ELISA but not listed in OECD TG 429. After selecting appropriate vehicles and conducting pre-screen tests in 2 phases, solvents and test concentrations for the main test were determined. In the main study, we measured changes in LN weight, the number of LNCs, and the proportion of BrdU incorporated into LNCs to calculate stimulation indexes (SI). SI was calculated based on the total number of LNCs and BrdU incorporation in LNCs. We found that all substances were correctly classified as sensitizers or non-sensitizers. Overall, we confirmed that the LLNA: BrdU-FCM can evaluate skin sensitization potential of the 20 substances. Additionally, our results of combining 22 reference substances listed in OECD TG 429 and 20 additional substances showed that concordance of LLNA: BrdU-FCM with the LLNA was higher than before.

Change in adduction moment following medial open wedge high tibial osteotomy: a meta-analysis.

BACKGROUND: This meta-analysis was designed to quantify adduction moment loss, to evaluate the relationship between changes in mechanical axis alignment and adduction moment, and to assess whether sagittal plane moment is altered after medial open wedge high tibial osteotomy (HTO). METHODS: Following preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines, all studies reporting preoperative and postoperative peak knee adduction moment or change in peak knee adduction moment from before to after surgery in patients who underwent medial open wedge HTO were included. RESULTS: Nine studies were included in the meta-analysis. The pooled mean difference in adduction moment from before to after medial open wedge HTO was 1.44% Nm/body weight (BW)xheight (HT) (95% confidence interval [CI]: 1.33 to 1.55% Nm/BWxHT; P < 0.001; I2 = 4%). However, flexion (0.18% Nm/BWxHT, 95% CI: -0.50 to 0.86% Nm/BWxHT; P = 0.61; I2 = 79%) and extension (0.15% Nm/BWxHT, 95% CI, - 0.37 to 0.68% Nm/BWxHT; P = 0.56; I2 = 46%) moments did not differ significantly from before to after surgery. Alignment correction amount and postoperative final valgus alignment were not significantly associated with difference in adduction moment from before to after surgery. CONCLUSION: Knee adduction moment after medial open wedge HTO decreased to 60% of the preoperative level. However, this adduction moment decrement was not affected by the magnitude of alignment correction. In addition, there was no change in sagittal plane knee moment, including flexion and extension moments, from before to after medial open wedge HTO. LEVEL OF EVIDENCE: Meta-analysis (Level II).

A Minimum of 15-Year Results of Cementless Total Hip Arthroplasty Using a 28-mm Metal-On-Metal Articulation.

BACKGROUND: We previously reported unexpected early failures of total hip arthroplasty (THA) utilizing a 28-mm Ultima metal-on-metal (MoM) articulation. However, long-term results of small-diameter MoM THAs still remain unclear. The purpose of this study was to evaluate the clinical and radiographic outcomes of the previously reported cohort at a minimum follow-up of 15 years. METHODS: The original cohort consisted of 171 primary THAs (167 patients) using a 28-mm MoM articulation performed between April 2000 and March 2002. Of these, 130 hips (126 patients) were reviewed at an average follow-up of 17.1 (range, 15-18) years. Clinical score, complications, presence of osteolytic lesion, serum metal ion concentrations, and implant survivorships were evaluated. RESULTS: The mean Harris Hip Score improved from 44.9 points preoperatively to 90.5 points at the latest follow-up. During the entire period since the original surgery, a total of 5 revisions (3.8%) were associated with adverse reaction to metal debris (ARMD). The last revision surgery for symptomatic ARMD was performed at 6 years postoperatively. The implant survivorships with an end point of revision for aseptic failure and for any reason were 95.4% and 93.8%, respectively. Radiographic osteolytic lesions were detected in 27 hips (20.8%). The average serum metal concentration was 2.50 (range, 0.12-9.86) µg/L for cobalt and 2.81 (range, 0.82-12.3) µg/L for chromium. CONCLUSIONS: THA using a 28-mm MoM articulation showed favorable long-term outcomes with a relatively high revision-free survival rate. There was no significant symptomatic ARMD after the last acetabular revision performed at 6 years postoperatively.

Clinical and Radiographic Characteristics of Atypical Slipped Capital Femoral Epiphysis.

BACKGROUND: Slipped capital femoral epiphysis (SCFEs) can be idiopathic or atypical (associated with renal failure, radiation therapy, and endocrine disorders). It is important to estimate the likelihood that atypical SCFE shows different clinical and radiographic characteristics and complications with typical SCFEs. METHODS: We retrospectively reviewed 53 patients (66 hips) with SCFEs managed between 1995 and 2016. Sex, age, weight, height, the duration of symptoms the laterality of the slip, and complications were recorded. Bilateral hip and lower extremity long bone radiographs were utilized to record slip angle, genu valgus deformity. Statistical analyses were performed. RESULTS: Among 53 children (32 boys and 21 girls) with 66 SCFEs, 22 children had 29 atypical SCFEs, and 31 children had 38 typical SCFEs. The average age at the time of presentation for the initial SCFEs was 10.9±0.3 years. The average duration of symptoms was 14.1±21.8 weeks. The average Southwick slip angle difference was 23.3±14.8 degrees. There are 43 mild, 13 moderate, and 10 severe SCFEs. 58 were classified as stable, and 8 were unstable. There were significant differences in terms of average age (10.4±2.6 y for atypical SCFEs, 11.2±1.4 for typical SCFEs; P=0.036), average weight (36.2±22.3 kg for patients with atypical slips, compared with 56.9±9.9 kg for those with typical slips; P<0.001), height (132.1±17.6 cm for those with atypical slips, compared with 148.9±12.1 for those with typical slips; P<0.001), and the body mass index (19.4±5.6 kg/cm for those with atypical slips, compared with 25.8±4.6 for those with typical slips; P<0.001). In the group of 22 children with atypical slipped capital epiphysis, there were 19 children with neoplasm (8 neuroblastomas, 3 medulloblastomas, 3 retinoblastomas, 2 ependymomas, 1 craniopharyngioma, 1 Wilms tumor, 1 immature teratoma), 4 children with primary endocrine disorder. We categorized atypical SCFEs in 3 groups by etiology; endocrine disorder-associated (ENDA) SCFEs, chemotherapy-associated (CTA) SCFEs, and radiation therapy-associated (RTA) SCFEs. Subjects of RTA were included if exposed to pelvic irradiation or total body irradiation. There were 4 ENDA, 9 RTA, and 16 CTA. Atypical SCFEs had significantly more valgus slips (P<0.001) and genu valgus deformity (P<0.001) compared with in the group of typical slipped capital epiphysis. Among the group of atypical SCFEs, children with CTA had significantly low body weight, height, and body mass index. CONCLUSIONS: The markedly greater incidence of valgus displacement of proximal femoral epiphysis and genu valgum in childhood cancer survivors with pelvic irradiation and chemotherapy may be associated with atypical SCFE. The authors conclude that in pediatric cancer survivors with hip pain, consideration should be given to atypical SCFE with valgus slip and long-term follow-up of lower extremity alignment. LEVEL OF EVIDENCE: Level III-retrospective comparative study. See instructions for authors for a complete description of levels of evidence.

Autophagy Activation by Crepidiastrum Denticulatum Extract Attenuates Environmental Pollutant-Induced Damage in Dermal Fibroblasts.

Pollution-induced skin damage results in oxidative stress; cellular toxicity; inflammation; and, ultimately, premature skin aging. Previous studies suggest that the activation of autophagy can protect oxidation-induced cellular damage and aging-like changes in skin. In order to develop new anti-pollution ingredients, this study screened various kinds of natural extracts to measure their autophagy activation efficacy in cultured dermal fibroblast. The stimulation of autophagy flux by the selected extracts was further confirmed both by the expression of proteins associated with the autophagy signals and by electron microscope. Crepidiastrum denticulatum (CD) extract treated cells showed the highest autophagic vacuole formation in the non-cytotoxic range. The phosphorylation of adenosine monophosphate kinase (AMPK), but not the inhibition of mammalian target of rapamycin (mTOR), was observed by CD-extract treatment. Its anti-pollution effects were further evaluated with model compounds, benzo[a]pyrene (BaP) and cadmium chloride (CdCl2), and a CD extract treatment resulted in both the protection of cytotoxicity and a reduction of proinflammatory cytokines. These results suggest that the autophagy activators can be a new protection regimen for anti-pollution. Therefore, CD extract can be used for anti-inflammatory and anti-pollution cosmetic ingredients.

Structural insights into the binding of lauric acid to CYP107L2 from Streptomyces avermitilis.

Streptomyces avermitilis is an actinobacterium known to produce clinically useful macrolides including avermectins. CYP107L2 from S. avermitilis shares a high sequence similarity with the PikC (CYP107L1) from S. venezuelae. To elucidate the structural features of CYP107L2, we conducted biochemical and structural characterization of CYP107L2 from S. avermitilis. The CYP107L2 gene was cloned, and its recombinant protein was expressed and purified. The CYP107L2 showed a low-spin state of heme, and the reduced form yielded the CO difference spectra with a maximal absorption at 449 nm. Binding of pikromycin and lauric acid yielded the typical type I spectra with Kd values of 4.8 ± 0.3 and 111 ± 9 µM, respectively. However, no metabolic product was observed in the enzyme reaction. X-ray crystal structures of the ligand-free CYP107L2 and its complex with lauric acid were determined at the resolution of 2.6 and 2.5 Å, respectively. CYP107L2 showed a well-conserved CYP structure with a wide-open substrate-binding cavity. The lauric acid is bound mainly via hydrophobic interactions with the carboxylate group of lauric acid coordinated to the heme of P450. Glu-40 and Leu-382 residues in the CYP107L2 complex with lauric acid showed significant conformational changes to provide plentiful room for the lauric acid in the substrate-binding site.

Molecular systematics and historical biogeography of Maianthemum s.s.

PREMISE OF THE STUDY: Understanding the underlying mechanisms for the evolution of intercontinental disjunct patterns has long fascinated botanists. We present a molecular phylogenetic reconstruction of Maianthemum s.l. (including Smilacina) with a focus on Maianthemum s.s. species, which have a disjunct distribution between Eurasia and North America. Within this evolutionary framework, we clarify the systematic classification and biogeographical history of Maianthemum s.s. METHOD: Data from two nuclear and five chloroplast DNA regions were analyzed using the programs PAUP*, RAxML, MrBayes, BEAST, and RASP. KEY RESULTS: Our molecular phylogeny supports Maianthemum s.s. as monophyletic. Maianthemum bifolium and M. canadense are grouped according to their taxonomic species, but the accessions of M. dilatatum are divided into two well-defined groups, i.e., one comprising western North American accessions, and the other composed of northeast Asian accessions. Molecular dating and biogeographic reconstructions suggest a northeast Asian origin for Maianthemum s.s. and that a complicated pattern of divergent evolution began approximately in the late Miocene. Intercontinental disjunctions of Maianthemum s.s. in the Northern Hemisphere appear to have occurred two times during the Pliocene. CONCLUSIONS: Based on our results, two possible ways to treat Maianthemum s.s. species can be envisioned: (1) elevate Asian populations of M. dilatatum to specific rank; or (2) combine the three Maianthemum s.s. species into a broadly defined single species. We recommend treatment of Maianthemum s.s. as a single species. The biogeographic patterns of Maianthemum s.s. coupled with molecular dating suggest both vicariance and long-distance dispersal events as key mechanisms for diversification of the clade.

Flavonoids from Litsea japonica Inhibit AGEs Formation and Rat Lense Aldose Reductase In Vitro and Vessel Dilation in Zebrafish.

In our ongoing efforts to identify effective naturally sourced agents for the treating of diabetic complications, two new (1 and 2) and 11 known phenolic compounds (3-13) were isolated from an 80 % ethanol extract of Litsea japonica leaves. The structures of the new compounds were established by spectroscopic and chemical studies. These isolates (1-13) were subjected to an in vitro bioassay evaluating their inhibitory activity on advanced glycation end products formation and rat lens aldose reductase activity. Of the compounds evaluated, the flavonoids (3, 4, 6-8, 11, and 12) markedly inhibited advanced glycation end products formation, with IC50 values of 7.4-72.0 µM, compared with the positive control, aminoguanidine (IC50 = 975.9 µM). In the rat lens aldose reductase assay, consistent with the inhibition of advanced glycation end products formation, the flavonoids (3, 4, 6-8, 11, and 12) exhibited considerable inhibition of rat lens aldose reductase activity, with IC50 values of 1.1-12.5 µM. In addition, the effects of kaempferol (4) and tiliroside (7) on the dilation of hyaloid-retinal vessels induced by high glucose in larval zebrafish were investigated. Only kaempferol significantly reduced the diameters of high glucose-induced hyaloid-retinal vessels, by 52.2 % at 10 µM, compared with those in the high glucose-treated control group.

Comparison of BALB/c and CBA/J mice for the local lymph node assay using bromodeoxyuridine with flow cytometry (LLNA: BrdU-FCM).

The local lymph node assay using 5-bromo-2-deoxyuridine (BrdU) with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA that is used to identify skin sensitizers by counting BrdU-incorporated lymph node cells (LNCs) with flow cytometry. Unlike other LLNA methods (OECD TG 429, 442A and 442B) in which the CBA/J mouse strain is used, LLNA: BrdU-FCM was originally designed to be compatible with BALB/c, a mouse strain that is more widely used in many countries. To justify the substitution of CBA/J for BALB/c, the equivalence of the test results between two strains shall be established prior to the official implementation of LLNA: BrdU-FCM. This study aims to compare the test results of LLNA: BrdU-FCM produced in BALB/c mice with those in CBA/J mice for 18 reference substances, including 13 sensitizers and 5 non-sensitizers, listed in OECD Test Guideline 429. Based on the LLNA: BrdU-FCM test procedure, we selected an appropriate solvent and then performed preliminary tests to determine the non-irritating dose ranges for the main study, which revealed the difference in the irritation responses to 8 of the 18 chemicals between the two strains. In the main study, we measured the changes in the number of total LNCs, which indicated differences in the responses to test chemicals between the two strains. However, the stimulation index obtained with the counts of BrdU-incorporated LNCs with 7-AAD using flow cytometry yielded comparable results and 100% concordance between the BALB/c and CBA/J mouse strains was achieved, suggesting that the performance of LLNA: BrdU-FCM using BALB/c mice was equivalent to that with CBA/J mice.

Oxidation of pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene by human cytochrome P450 2A13.

1. The polycyclic hydrocarbons (PAHs), pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene, were found to induce Type I binding spectra with human cytochrome P450 (P450) 2A13 and were converted to various mono- and di-oxygenated products by this enzyme. 2. Pyrene was first oxidized by P450 2A13 to 1-hydroxypyrene which was further oxidized to di-oxygenated products, i.e. 1,8- and 1,6-dihydroxypyrene. Of five other human P450s examined, P450 1B1 catalyzed pyrene oxidation to 1-hydroxypyrene at a similar rate to P450 2A13 but was less efficient in forming dihydroxypyrenes. P450 2A6, a related human P450 enzyme, which did not show any spectral changes with these four PAHs, showed lower activities in oxidation of these compounds than P450 2A13. 3. 1-Nitropyrene and 1-acetylpyrene were also found to be efficiently oxidized by P450 2A13 to several oxygenated products, based on mass spectrometry analysis. 4. Molecular docking analysis supported preferred orientations of pyrene and its derivatives in the active site of P450 2A13, with lower interaction energies (U values) than observed for P450 2A6 and that several amino acid residues (including Ala-301, Asn-297 and Ala-117) play important roles in directing the orientation of these PAHs in the P450 2A13 active site. In addition, Phe-231 and Gly-329 were found to interact with pyrene to orient this compound in the active site of P450 1B1. 5. These results suggest that P450 2A13 is one of the important enzymes that oxidizes these PAH compounds and may determine how these chemicals are detoxicated and bioactivated in humans.