Anti-inflammatory effects of immature Citrus unshiu fruit extracts via suppression of NF-κB and MAPK signal pathways in LPS-induced RAW264.7 macrophage cells.

This study examined the anti-inflammatory effects of 70% ethanol crude extract of immature Citrus unshiu fruits (ICE) and its solvent fractions in LPS-stimulated RAW 264.7 cells. In addition, we analyzed the active compounds related to suppression of inflammation. It was found that the ethyl acetate (EtOAc) fraction showed the highest level of inhibition of NO production, and this inhibitory activity was concentration-dependent. Moreover, the EtOAc fraction not only inhibited TNF-α and IL-6 production but also inhibited iNOS and COX-2 protein expression. Furthermore, inhibition of NF-κB activity and MAPK phosphorylation was also observed. In addition, ß-sitosterol, campesterol and isoferulic acid were identified as major anti-inflammatory components in the EtOAc fraction. These results suggested that the EtOAc fraction of immature C. unshiu fruit extract exerts anti-inflammatory effects by inhibiting NF-κB and MAPK signaling pathways, and that this fruit could be used as a natural anti-inflammatory material.

Recent advancement in OMICS approaches to enhance abiotic stress tolerance in legumes.

The world is facing rapid climate change and a fast-growing global population. It is believed that the world population will be 9.7 billion in 2050. However, recent agriculture production is not enough to feed the current population of 7.9 billion people, which is causing a huge hunger problem. Therefore, feeding the 9.7 billion population in 2050 will be a huge target. Climate change is becoming a huge threat to global agricultural production, and it is expected to become the worst threat to it in the upcoming years. Keeping this in view, it is very important to breed climate-resilient plants. Legumes are considered an important pillar of the agriculture production system and a great source of high-quality protein, minerals, and vitamins. During the last two decades, advancements in OMICs technology revolutionized plant breeding and emerged as a crop-saving tool in wake of the climate change. Various OMICs approaches like Next-Generation sequencing (NGS), Transcriptomics, Proteomics, and Metabolomics have been used in legumes under abiotic stresses. The scientific community successfully utilized these platforms and investigated the Quantitative Trait Loci (QTL), linked markers through genome-wide association studies, and developed KASP markers that can be helpful for the marker-assisted breeding of legumes. Gene-editing techniques have been successfully proven for soybean, cowpea, chickpea, and model legumes such as Medicago truncatula and Lotus japonicus. A number of efforts have been made to perform gene editing in legumes. Moreover, the scientific community did a great job of identifying various genes involved in the metabolic pathways and utilizing the resulted information in the development of climate-resilient legume cultivars at a rapid pace. Keeping in view, this review highlights the contribution of OMICs approaches to abiotic stresses in legumes. We envisage that the presented information will be helpful for the scientific community to develop climate-resilient legume cultivars.

HYL1-CLEAVAGE SUBTILASE 1 (HCS1) suppresses miRNA biogenesis in response to light-to-dark transition.

The core plant microprocessor consists of DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1) and plays a pivotal role in microRNA (miRNA) biogenesis. However, the proteolytic regulation of each component remains elusive. Here, we show that HYL1-CLEAVAGE SUBTILASE 1 (HCS1) is a cytoplasmic protease for HYL1-destabilization. HCS1-excessiveness reduces HYL1 that disrupts miRNA biogenesis, while HCS1-deficiency accumulates HYL1. Consistently, we identified the HYL1K154A mutant that is insensitive to the proteolytic activity of HCS1, confirming the importance of HCS1 in HYL1 proteostasis. Moreover, HCS1-activity is regulated by light/dark transition. Under light, cytoplasmic CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase suppresses HCS1-activity. COP1 sterically inhibits HCS1 by obstructing HYL1 access into the catalytic sites of HCS1. In contrast, darkness unshackles HCS1-activity for HYL1-destabilization due to nuclear COP1 relocation. Overall, the COP1-HYL1-HCS1 network may integrate two essential cellular pathways: the miRNA-biogenetic pathway and light signaling pathway.

Effects of Cooking and Processing Methods on Phenolic Contents and Antioxidant and Anti-Proliferative Activities of Broccoli Florets.

We investigated the effects of cooking (steaming and microwaving) and processing (freeze-drying and hot-air-drying) methods on the antioxidant activity of broccoli florets. 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•), and alkyl• free radical scavenging assays were employed to assess anti-oxidant potentials. The cytoprotective effect against oxidative damage induced by H2O2 was studied using hepatocellular carcinoma (HepG2) cells. Anti-proliferative effects were assessed in MCF-7 and MDA-MB-231 breast cancer cells. L-sulforaphane in broccoli extracts was quantified using high-performance liquid chromatography (HPLC). Steam and microwave treatments caused increases in total polyphenol content (TPC), whereas the total flavonoid content (TFC) decreased following steam treatment. A slight increase in TFC was observed in the microwaved samples. Extracts of all broccoli samples showed almost identical radical scavenging and cytoprotective effects. HPLC demonstrated that steamed (3 min)-freeze-dried (F-S3) and microwaved (2 min)-freeze-dried (F-M2) samples exhibited elevated levels of L-sulforaphane. In addition, the F-S3 and F-M2 extracts displayed strong anti-proliferative effects in MCF-7 cells, which correlated with L-sulforaphane content. As we observed no significant decrease in the antioxidant activity of broccoli florets, the cooking and processing methods and conditions studied here are recommended for broccoli.

Toxicity of dietary selenomethionine in juvenile steelhead trout, Oncorhynchus mykiss: tissue burden, growth performance, body composition, hematological parameters, and liver histopathology.

The steelhead trout (Oncorhynchus mykiss) is the species most at risk from selenium (Se) exposure in the San Francisco Bay Delta (SFBD). However, although steelhead trout are usually exposed to environmental Se in the juvenile stage, data to test their sensitivity to excess Se, especially its organic form, in the juvenile stage are scarce. Therefore, the objective of the current study was to assess the sensitivity of juvenile steelhead trout to ecologically relevant forms of Se using integrated sensitive endpoints. Fish (mean weight: 22.3 g) were fed one of five diets containing 1.1 (control), 8.8, 15.4, 30.8, and 61.6 µg Se/g diet dw (Se1.1, Se8.8, Se15.4, Se30.8, and Se61.6, respectively) in the form of selenomethionine for 4 weeks. After 4 weeks, Se significantly accumulated in a dose-dependent manner in all tissues at different rates. The growth rate and plasma cholesterol were significantly depressed in fish fed diets containing Se30.8 and above. Hematological parameters and mortality were significantly elevated in fish fed the Se61.6 diet. Marked histopathological alterations were observed in fish fed the Se8.8 diet (the lowest observed effect concentration, LOEC) and above. The current results suggest that the steelhead trout is more sensitive to excess Se than nonanadromous rainbow trout used in previous studies because of its lower LOEC despite the use of selenomethionine and the shorter experimental duration. Additionally, it should be noted that the current Se levels found in the SFBD are already a threat to the threatened population of steelhead trout on the central California coast.

PDC1, a pyruvate/α-ketoacid decarboxylase, is involved in acetaldehyde, propanal and pentanal biosynthesis in melon (Cucumis melo L.) fruit.

Plant pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO2 and are well known to play a key role in energy supply via fermentative metabolism in oxygen-limiting conditions. In addition to their role in fermentation, plant PDCs have also been hypothesized to be involved in aroma formation although, to date, there is no direct biochemical evidence for this function. We investigated the role of PDCs in fruit volatile biosynthesis, and identified a melon pyruvate decarboxylase, PDC1, that is highly expressed in ripe fruits. In vitro biochemical characterization of the recombinant PDC1 enzyme showed that it could not only decarboxylate pyruvate, but that it also had significant activity toward other straight- and branched-chain α-ketoacids, greatly expanding the range of substrates previously known to be accepted by the plant enzyme. RNAi-mediated transient and stable silencing of PDC1 expression in melon showed that this gene is involved in acetaldehyde, propanal and pentanal production, while it does not contribute to branched-chain amino acid (BCAA)-derived aldehyde biosynthesis in melon fruit. Importantly, our results not only demonstrate additional functions for the PDC enzyme, but also challenge the long standing hypothesis that PDC is involved in BCAA-derived aldehyde formation in fruit.

Biosynthesis and accumulation of 20-hydroxyecdysone in individual male and female spinach plants during the reproductive stage.

The steroid 20-hydroxyecdysone (20E) is a major component of phytoecdysteroid in plants and may play a defensive role against insect pests in higher plants. In spinach, the biosynthesis and accumulation of 20E have been investigated during the vegetative stage; however, these processes have not been clearly studied during the reproductive stage, particularly in male and female individuals. In this study, we analyzed the level and distribution of 20E in individual male and female spinach plants during the reproductive stage via high performance liquid chromatography (HPLC). We found that 20E biosynthesis and accumulation were markedly different between male and female spinach during the late flowering stage. Compared with the male plant, biosynthesis of 20E in the leaves was more active and its accumulation in the floral parts was higher in female plants during the late flowering stage. These results indicate that the female reproductive organs at least in PE-positive plants could be effectively protected against harmful insects via active biosynthesis and accumulation of PE during the late flowering stage to protect floral parts from harmful insects for seed formation and store the available 20E in seeds for the next generation.

HIGLE is a bifunctional homing endonuclease that directly interacts with HYL1 and SERRATE in Arabidopsis thaliana.

A highly coordinated complex known as the microprocessor precisely processes primary transcripts of MIRNA genes into mature miRNAs. In plants, the microprocessor minimally consists of three components: Dicer-like protein 1 (DCL1), HYPONASTIC LEAF 1 (HYL1), and SERRATE (SE). To precisely modulate miRNA maturation, the microprocessor cooperates with at least 12 proteins in plants. In addition, we here show the involvement of a novel gene, HYL1-interacting GIY-YIG-like endonuclease (HIGLE). The encoded protein has a GIY-YIG domain that is generally found within a class of homing endonucleases. HIGLE directly interacts with the microprocessor components HYL1 and SE. Unlike the functions of other GIY-YIG endonucleases, the catalytic core of HIGLE has both DNase and RNase activities that sufficiently processes miRNA precursors into short fragments in vitro.

In vitro plant regeneration of Aster scaber via somatic embryogenesis.

We established an in vitro plant regeneration system via somatic embryogenesis of Aster scaber, an important source of various biologically active phytochemicals. We examined the callus induction and embryogenic capacities of three explants, including leaves, petioles, and roots, on 25 different media containing different combinations of α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The optimum concentrations of NAA and BA for the production of embryogenic calli were 5.0 µM and 0.05 µM, respectively. Media containing higher concentrations of auxin and cytokinin (such as 25 µM NAA and 25 µM BA) were suitable for shoot regeneration, especially for leaf-derived calli, which are the most readily available calli and are highly competent. For root induction from regenerated shoots, supplemental auxin and/or cytokinin did not improve rooting, but instead caused unwanted callus induction or retarded growth of regenerated plants. Therefore, plant growth regulator-free medium was preferable for root induction. Normal plants were successfully obtained from calli under the optimized conditions described above. This is the first report of the complete process of in vitro plant regeneration of A. scaber via somatic embryogenesis.

Demineralized bone matrix, as a graft enhancer of auto-local bone in posterior lumbar interbody fusion.

STUDY DESIGN: A case controlled study with prospective data collection. PURPOSE: To evaluate the early influence and the final consequence of demineralized bone matrix (DBM) on auto-local bone as a graft enhancer in posterior lumbar interbody fusion (PLIF). OVERVIEW OF LITERATURE: DBM is known as an osteoinductive material; however, it has not been clearly recognized to enhance auto-local bone with a small amount. METHODS: Patients who had a PLIF were allocated into two groups. Group I (70 cases) used auto-local bone chips and group II (44 cases) used DBM as an additive to auto-local bone, 1 mL per a segment. Group selection was alternated. Early assessment was performed by computed tomography at 6 months and final assessment was done by simple radiography after 24 months at least. The degree of bone formation was assessed by 4 grade scale. RESULTS: The subjects of both groups were homogenous and had similar Oswestry Disability Index at final assessment. The ratio of auto-local bone chips and DBM was 6:1. The degree of bone formation at 6 months after surgery was superior in group II. However, there was no significant difference between the two groups at the final assessment. CONCLUSIONS: DBM was not recognized to enhance auto-local bone with small amount.

Ossification of the ligamentum flavum.

Ossification of the ligamentum flavum is a rare cause of thoracic myelopathy. It develops in East Asians more frequently than in people from other areas. The exact pathophysiology has not been elucidated yet; however, it largely depends on biomechanical alterations, especially changes in the tensile force. Because the spinal cord is compressed from the posterior side, the first and most common clinical manifestation is usually loss of functional gait and spastic paralysis, which develop as the spinal cord compression progresses. The choice of diagnostic imaging is T2 sagittal magnetic resonance imaging scanning. Whole spine scanning is mandatory to identify multiple areas of compression and any associated distal lumbar diseases. Fine computed tomography imaging is necessary to make a differential diagnosis and set up a precise surgical plan. Conservative treatment does not work in this disorder. Decompression surgery is the only option and prognosis after surgical treatment is better with this disorder than with other causes of thoracic myelopathy. The severity of preoperative symptoms and the time interval before surgical treatment are the most important prognostic factors.

The degree of bacterial contamination while performing spine surgery.

STUDY DESIGN: Prospective experimental study. PURPOSE: To evaluate bacterial contamination during surgery. OVERVIEW OF LITERATURE: The participants of surgery and ventilation system have been known as the most significant sources of contamination. METHODS: Two pairs of air culture blood agar plate for G(+) bacteria and MacConkey agar plate for G(-) bacteria were placed at 3 different locations in a conventional operation room: in the surgical field, under the airflow of local air conditioner, and pathway to door while performing spine surgeries. One pair of culture plates was retrieved after one hour and the other pair was retrieved after 3 hours. The cultured bacteria were identified and number of colonies was counted. RESULTS: There was no G(-) bacteria identified. G(+) bacteria grew on all 90 air culture blood agar plates. The colony count of one hour group was 14.5±5.4 in the surgical field, 11.3±6.6 under the local air conditioner, and 13.1±8.7 at the pathway to the door. There was no difference among the 3 locations. The colony count of 3 hours group was 46.4±19.5, 30.3±12.9, and 39.7±15.2, respectively. It was more at the surgical field than under the air conditioner (p=0.03). The number of colonies of one hour group was 13.0±7.0 and 3 hours group was 38.8±17.1. There was positive correlation between the time and the number of colonies (r=0.76, p=0.000). CONCLUSIONS: Conventional operation room was contaminated by G(+) bacteria. The degree of contamination was most high at the surgical field. The number of bacteria increased right proportionally to the time.

The influence of postoperative tibiofemoral alignment on the clinical results of unicompartmental knee arthroplasty.

PURPOSE: To evaluate the influence of postoperative tibiofemoral alignment on the clinical results and failure in patients who underwent unicompartmental knee athroplasty (UKA). MATERIALS AND METHODS: We reviewed 246 cases of medial UKA which were followed up for at least 5 years after the operation. The clinical results were compared between 5 groups classified according to the tibiofemoral angle that was measured at 3 months after surgery. We analyzed the relationship between the tibiofemoral alignment and the failure after UKA. RESULTS: The preoperative tibiofemoral angle was changed from 0.4° of varus to 5.4° of valgus after surgery and the average correction angle was 5.8°. During the follow-up, which averaged 7 years and 5 months, the knee score and function score were improved significantly in all groups regardless of the tibiofemoral angle (p<0.01). There were no significant difference between the groups in the clinical results (p>0.05). However, there were significant differences in the cumulative survival rate of implants between the groups and the highest rate was found in the group with a tibiofemoral angle of 4° to 6° of valgus (p<0.01). CONCLUSIONS: The tibiofemoral angle after UKA had no significant influence on the midterm clinical scores, but there was a significant relationship between the postoperative tibiofemoral angle and failure rate of implant.

Label-free shotgun proteomics and metabolite analysis reveal a significant metabolic shift during citrus fruit development.

Label-free LC-MS/MS-based shot-gun proteomics was used to quantify the differential protein synthesis and metabolite profiling in order to assess metabolic changes during the development of citrus fruits. Our results suggested the occurrence of a metabolic change during citrus fruit maturation, where the organic acid and amino acid accumulation seen during the early stages of development shifted into sugar synthesis during the later stage of citrus fruit development. The expression of invertases remained unchanged, while an invertase inhibitor was up-regulated towards maturation. The increased expression of sucrose-phosphate synthase and sucrose-6-phosphate phosphatase and the rapid sugar accumulation suggest that sucrose is also being synthesized in citrus juice sac cells during the later stage of fruit development.

Ginsenoside Production and Morphological Characterization of Wild Ginseng (Panax ginseng Meyer) Mutant Lines Induced by γ-irradiation ((60)Co) of Adventitious Roots.

With the purpose of improving ginsenoside content in adventitious root cultures of Korean wild ginseng (Panax ginseng Meyer), the roots were treated with different dosages of γ-ray (5, 10, 25, 50, 75, 100, and 200 Gy). The growth of adventitious roots was inhibited at over 100 Gy. The irradiated adventitious roots showed significant variation in the morphological parameters and crude saponin content at 50 to100 Gy. Therefore, four mutant cell lines out of the propagation of 35 cell lines treated with 50 Gy and 100 Gy were selected on the basis of phenotypic morphology and crude saponin contents relative to the wild type control. The contents of 7 major ginsenosides (Rg1, Re, Rb1, Rb2, Rc, Rf, and Rd) were determined for cell lines 1 and 3 from 100 Gy and lines 2 and 4 from 50 Gy treatments. Cell line 2 showed more secondary roots, longer length and superior growth rate than the root controls in flasks and bioreactors. Cell line 1 showed larger average diameter and the growth rate in the bioreactor was comparable with that of the control but greater in the flask cultured roots. Cell lines 1 and 2, especially the former, showed much more ginsenoside contents than the control in flasks and bioreactors. Therefore, we chose cell line 1 for further study of ginsenoside contents. The crude saponin content of line 1 in flask and bioreactor cultures increased by 1.4 and 1.8-fold, respectively, compared to the control. Total contents of 7 ginsenoside types (Rg1, Re, Rb1, Rb2, Rc, Rf, and Rd) increased by 1.8 and 2.3-fold, respectively compared to the control. Crude saponin and ginsenoside contents in the bioreactor culture increased by about 1.4-fold compared to that the flask culture.

Distribution and biosynthesis of 20-hydroxyecdysone in plants of Achyranthes japonica Nakai.

There is increasing interest in phytoecdysteroids (PEs) because of their potential role in plant defense against insects. To understand the mechanism regulating their levels in plants, the fluctuation, distribution, and biosynthesis of PE 20-hydroxyecdysone (20E) examined in Achyranthes japonica. The total amount of 20E per individual plant initially remained at a constant level, and increased markedly after the first leaf pair (LP) stage, while the concentration of 20E in a given plant decreased rapidly during vegetative growth. In addition, the incorporation of [2-(14)C]-mevalonic acid into 20E did not differ significantly depending on plant organs and developmental stages, suggesting that biosynthesis of 20E is not restricted to particular organs or growth stages.