Network analysis, in vivo, and in vitro experiments identified the mechanisms by which Piper longum L. [Piperaceae] alleviates cartilage destruction, joint inflammation, and arthritic pain.

Osteoarthritis (OA) is characterized by irreversible joint destruction, pain, and dysfunction. Piper longum L. [Piperaceae] (PL) is an East Asian herbal medicine with reported anti-inflammatory, analgesic, antioxidant, anti-stress, and anti-osteoporotic effects. This study aimed to evaluate the efficacy of PL in inhibiting pain and progressive joint destruction in OA based on its anti-inflammatory activity, and to explore its potential mechanisms using in vivo and in vitro models of OA. We predicted the potential hub targets and signaling pathways of PL through network analysis and molecular docking. Network analysis results showed that the possible hub targets of PL against OA were F2R, F3, MMP1, MMP2, MMP9, and PTGS2. The molecular docking results predicted strong binding affinities for the core compounds in PL: piperlongumine, piperlonguminine, and piperine. In vitro experiments showed that PL inhibited the expression of LPS-induced pro-inflammatory factors, such as F2R, F3, IL-1ß, IL-6, IL-17A, MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, NOS2, PTGS2, PGE2, and TNF-ß. These mechanisms and effects were dose-dependent in vivo models. Furthermore, PL inhibited cartilage degradation in an OA-induced rat model. Thus, this study demonstrated that multiple components of PL may inhibit the multilayered pathology of OA by acting on multiple targets and pathways. These findings highlight the potential of PL as a disease-modifying OA drug candidate, which warrants further investigation.

Pectin Methylesterification Impacts the Relationship between Photosynthesis and Plant Growth.

Photosynthesis occurs in mesophyll cells of specialized organs such as leaves. The rigid cell wall encapsulating photosynthetic cells controls the expansion and distribution of cells within photosynthetic tissues. The relationship between photosynthesis and plant growth is affected by leaf area. However, the underlying genetic mechanisms affecting carbon partitioning to different aspects of leaf growth are not known. To fill this gap, we analyzed Arabidopsis plants with altered levels of pectin methylesterification, which is known to modulate cell wall plasticity and plant growth. Pectin methylesterification levels were varied through manipulation of cotton Golgi-related (CGR) 2 or 3 genes encoding two functionally redundant pectin methyltransferases. Increased levels of methylesterification in a line over-expressing CGR2 (CGR2OX) resulted in highly expanded leaves with enhanced intercellular air spaces; reduced methylesterification in a mutant lacking both CGR-genes 2 and 3 (cgr2/3) resulted in thin but dense leaf mesophyll that limited CO2 diffusion to chloroplasts. Leaf, root, and plant dry weight were enhanced in CGR2OX but decreased in cgr2/3. Differences in growth between wild type and the CGR-mutants can be explained by carbon partitioning but not by variations in area-based photosynthesis. Therefore, photosynthesis drives growth through alterations in carbon partitioning to new leaf area growth and leaf mass per unit leaf area; however, CGR-mediated pectin methylesterification acts as a primary factor in this relationship through modulation of the expansion and positioning of the cells in leaves, which in turn drive carbon partitioning by generating dynamic carbon demands in leaf area growth and leaf mass per unit leaf area.

CGR2 and CGR3 have critical overlapping roles in pectin methylesterification and plant growth in Arabidopsis thaliana.

Pectins are critical polysaccharides of the cell wall that are involved in key aspects of a plant's life, including cell-wall stiffness, cell-to-cell adhesion, and mechanical strength. Pectins undergo methylesterification, which affects their cellular roles. Pectin methyltransferases are believed to methylesterify pectins in the Golgi, but little is known about their identity. To date, there is only circumstantial evidence to support a role for QUASIMODO2 (QUA2)-like proteins and an unrelated plant-specific protein, cotton Golgi-related 3 (CGR3), in pectin methylesterification. To add to the knowledge of pectin biosynthesis, here we characterized a close homolog of CGR3, named CGR2, and evaluated the effect of loss-of-function mutants and over-expression lines of CGR2 and CGR3 in planta. Our results show that, similar to CGR3, CGR2 is a Golgi protein whose enzyme active site is located in the Golgi lumen where pectin methylesterification occurs. Through phenotypical analyses, we also established that simultaneous loss of CGR2 and CGR3 causes severe defects in plant growth and development, supporting critical but overlapping functional roles of these proteins. Qualitative and quantitative cell-wall analytical assays of the double knockout mutant demonstrated reduced levels of pectin methylesterification, coupled with decreased microsomal pectin methyltransferase activity. Conversely, CGR2 and CGR3 over-expression lines have markedly opposite phenotypes to the double knockout mutant, with increased cell-wall methylesterification levels and microsomal pectin methyltransferase activity. Based on these findings, we propose that CGR2 and CGR3 are critical proteins in plant growth and development that act redundantly in pectin methylesterification in the Golgi apparatus.

Novel lipolytic enzymes identified from metagenomic library of deep-sea sediment.

Metagenomic library was constructed from a deep-sea sediment sample and screened for lipolytic activity. Open-reading frames of six positive clones showed only 33-58% amino acid identities to the known proteins. One of them was assigned to a new group while others were grouped into Families I and V or EstD Family. By employing a combination of approaches such as removing the signal sequence, coexpression of chaperone genes, and low temperature induction, we obtained five soluble recombinant proteins in Escherichia coli. The purified enzymes had optimum temperatures of 30-35°C and the cold-activity property. Among them, one enzyme showed lipase activity by preferentially hydrolyzing p-nitrophenyl palmitate and p-nitrophenyl stearate and high salt resistance with up to 4 M NaCl. Our research demonstrates the feasibility of developing novel lipolytic enzymes from marine environments by the combination of functional metagenomic approach and protein expression technology.

Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome.

To search for new cold-active lipases, a metagenomic library was constructed using cold-sea sediment samples at Edison Seamount and was screened for lipolytic activities by plating on a tricaprylin medium. Subsequently, a fosmid clone was selected, and the whole sequence of 36 kb insert of the fosmid clone was determined by shotgun sequencing. The sequence analysis revealed the presence of 25 open reading frames (ORF), and ORF20 (EML1) showed similarities to lipases. Phylogenetic analysis of EML1 suggested that the protein belonged to a new family of esterase/lipase together with LipG. The EML1 gene was expressed in Escherichia coli, and purified by metal-chelating chromatography. The optimum activity of the purified EML1 (rEML1) occurred at pH 8.0 and 25 degrees C, respectively, and rEML1 displayed more than 50% activity at 5 degrees C. The activation energy for the hydrolysis of olive oil was determined to be 3.28 kcal/mol, indicating that EML1 is a cold-active lipase. rEML1 preferentially hydrolyzed triacylglycerols acyl-group chains with long chain lengths of > or = 8 carbon atoms and displayed hydrolyzing activities toward various natural oil substrates. rEML1 was resistant to various detergents such as Triton X-100 and Tween 80. This study represents an example which developed a new cold-active lipase from a deep-sea sediment metagenome.

Yeosuana aromativorans gen. nov., sp. nov., a mesophilic marine bacterium belonging to the family Flavobacteriaceae, isolated from estuarine sediment of the South Sea, Korea.

A marine bacterium, GW1-1T, capable of degrading benzo[a]pyrene (BaP), was isolated from estuarine sediments of the South Sea (the Korea Strait), Korea, after an enrichment culture maintained for 2 years in a medium supplemented with a mixture of BaP and pyrene. The strain formed yellowish-brown colonies on marine agar 2216. Cells were strictly aerobic, non-motile, Gram-negative rods and produced non-diffusible carotenoid pigments. Optimal growth occurred in the presence of 1 % (w/v) NaCl and at pH 7 and 33-36 degrees C. No growth occurred without supplementation with either CaCl2 or MgCl2, even in the presence of NaCl. Phylogenetic analysis based on the nearly complete sequence of the 16S rRNA gene revealed that the isolate formed a phyletic lineage with the genera Gelidibacter (93.9-94.7 % gene sequence similarity), Subsaximicrobium (93.3 %) and Subsaxibacter (93.9 %). The isolate also showed high sequence similarities to Gaetbulibacter saemankumensis (94.5 %), Algibacter lectus (94.2 %), members of the genus Bizionia (93.6-94.3 %) and Formosa algae (93.2 %), even though it belonged to a different phyletic line. The major respiratory quinones of the isolate were menaquinones MK-5 and MK-6. The DNA G+C content was 51.4 mol%. Dominant fatty acids were i-15 : 0, a-15 : 0, i-15 : 1omega10c and 16 : 1. On the basis of this polyphasic taxonomic evidence, strain GW1-1T is classified as a member of a novel genus and species in the family Flavobacteriaceae, for which the name Yeosuana aromativorans gen. nov., sp. nov. is proposed. The type strain of the type species is GW1-1T (=KCCM 42019T = JCM 12862T).

Enhancing the intrinsic bioremediation of PAH-contaminated anoxic estuarine sediments with biostimulating agents.

Estuarine sediments are frequently polluted with hydrocarbons from fuel spills and industrial wastes. Polycyclic aromatic hydrocarbons (PAHs) are components of these contaminants that tend to accumulate in the sediment due to their low aqueous solubility, low volatility, and high affinity for particulate matter. The toxic, recalcitrant, mutagenic, and carcinogenic nature of these compounds may require aggressive treatment to remediate polluted sites effectively. In petroleum-contaminated sediments near a petrochemical industry in Gwangyang Bay, Korea, in situ PAH concentrations ranged from 10 to 2,900 microg/kg dry sediment. To enhance the biodegradation rate of PAHs under anaerobic conditions, sediment samples were amended with biostimulating agents alone or in combination: nitrogen and phosphorus in the form of slow-release fertilizer (SRF), lactate, yeast extract (YE), and Tween 80. When added to the sediment individually, all tested agents enhanced the degradation of PAHs, including naphthalene, acenaphthene, anthracene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, and benzo[a]pyrene. Moreover, the combination of SRF, Tween 80, and lactate increased the PAH degradation rate 1.2-8.2 times above that of untreated sediment (0.01-10 microg PAH/kg dry sediment/day). Our results indicated that in situ contaminant PAHs in anoxic sediment, including high molecular weight PAHs, were degraded biologically and that the addition of stimulators increased the biodegradation potential of the intrinsic microbial populations. Our results will contribute to the development of new strategies for in situ treatment of PAH-contaminated anoxic sediments.

Evaluation of bioremediation effectiveness on crude oil-contaminated sand.

A treatability study was conducted using sea sand spiked with 3% or 6% (w/w) of Arabian light crude oil to determine the most effective bioremediation strategies for different levels of contamination. The sea sand used in the study was composed of gravel (0.1%), sand (89.0%), and silt and clay (10.9%). The water content of the sea sand was adjusted to 12.6% (w/w) for the study. Different combinations of the following treatments were applied to the sand in biometer flasks: the concentration of oil (3% or 6%), the concentration of a mixture of three oil-degrading microorganisms (Corynebacterium sp. IC-10, Sphingomonas sp. KH3-2 and Yarrowia sp. 180, 1x10(6) or 1x10(8) cells g-1 sand), the concentration of the surfactant Tween 80 (1 or 10 times the critical micelle concentration), and the addition of SRIF in a C:N:P ratio of 100:10:3. Three biometer flasks per combination of experimental conditions were incubated, and the performance of each treatment was examined by monitoring CO2 evolution, microbial activity, and oil degradation rate. The results suggest that the addition of inorganic nutrients accelerated the rate of CO2 evolution by a factor of 10. The application of oil-degrading microorganisms in a concentration greater than that of the indigenous population clearly increased biodegradation efficiency. The application of surfactant slightly enhanced the oil degradation rate in the contaminated sand treated with the higher concentration of oil-degrading microorganisms. The initial CO2 evolution rate was shown to efficiently evaluate the treatability test by providing significant data within a short period, which is critical for the rapid determination of the appropriate bioremediation approach. The measurements of microbial activity and crude oil degradation also confirmed the validity of the CO2 evolution rate as an appropriate criterion.

Novel method for determining pyrene biodegradation using synchronous fluorimetry.

To study the biodegradation rate of pyrene dissolved in liquid medium supplemented with mineral salts, a synchronous fluorimetry (SF) method was established. The limit of detection for pyrene dissolved in mineral salts medium (MSM) was determined as 0.19 ng/ml with a relative standard deviation of less than 1.3% (n = 9). The pyrene degrading rates of four bacterial strains were investigated using this method under the same experimental conditions. The degradation rates of the three active strains ranged from 76% to 87% after a 14-h incubation. The results were confirmed by the gas chromatography with a flame ionized detector (GC/FID) method. This implies that pyrene degradation can be directly monitored by the SF method without the solvent extraction of samples. The advantages of SF are that it is less laborious, faster, and less expensive than the GC/FID determination method with solvent extraction. The SF method provides a new tool for studying the degradation of polynuclear aromatic hydrocarbons (PAHs) in the natural environment and under experimental conditions.