Changes in the Harpagide, Harpagoside, and Verbascoside Content of Field Grown Scrophularia lanceolata and Scrophularia marilandica in Response to Season and Shade.

Scrophularia lanceolata Pursh and Scrophularia marilandica L. are two common species within the Scrophulariaceae family that are endemic to North America. Historically, these species were used by indigenous peoples and colonialists to treat sunburn, sunstroke, frostbite, edema, as well as for blood purification, and in women's health. Several iridoid and phenylethanoid/phenylpropanoid glycosides detected in these species, such as harpagoside and verbascoside, possess anti-inflammatory and anti-nociceptive properties. Due to the presence of anti-inflammatory metabolites and the historical uses of these species, we performed a two-year field study to determine the optimal production of these important compounds. We subjected the plants to shade treatment and analyzed differences in the metabolite composition between the two species and each of their leaves, stems, and roots at various times throughout the growing seasons. We determined that S. lanceolata plants grown in full sun produced 0.63% harpagoside per dried weight in their leaves compared to shade-grown plants (0.43%). Furthermore, S. lanceolata accumulated more harpagoside than S. marilandica (0.24%). We also found that verbascoside accumulated in the leaves of S. lanceolata and S. marilandica as the growing season progressed, while the production of this metabolite remained mostly seasonally unchanged in the roots of both species.

Bacopa monnieri (L.) Wettst. Extract Improves Memory Performance via Promotion of Neurogenesis in the Hippocampal Dentate Gyrus of Adolescent Mice.

Bacopa monnieri L. Wettst. (BM) is a botanical component of Ayurvedic medicines and of dietary supplements used worldwide for cognitive health and function. We previously reported that administration of BM alcoholic extract (BME) prevents trimethyltin (TMT)-induced cognitive deficits and hippocampal cell damage and promotes TMT-induced hippocampal neurogenesis. In this study, we demonstrate that administration of BME improves spatial working memory in adolescent (5-week- old) healthy mice but not adult (8-week-old) mice. Moreover, improved spatial working memory was retained even at 4 weeks after terminating 1-week treatment of adolescent mice. One-week BME treatment of adolescent mice significantly enhanced hippocampal BrdU incorporation and expression of genes involved in neurogenesis determined by RNAseq analysis. Cell death, as detected by histochemistry, appeared not to be significant. A significant increase in neurogenesis was observed in the dentate gyrus region 4 weeks after terminating 1-week treatment of adolescent mice with BME. Bacopaside I, an active component of BME, promoted the proliferation of neural progenitor cells in vitro in a concentration-dependent manner via the facilitation of the Akt and ERK1/2 signaling. These results suggest that BME enhances spatial working memory in healthy adolescent mice by promoting hippocampal neurogenesis and that the effects of BME are due, in significant amounts, to bacopaside I.

Toxicology studies of aqueous-alcohol extracts of Harpagophytum procumbens subsp. procumbens (Burch.) DC.Ex Meisn. (Pedaliaceae) in female and male rats.

BACKGROUND: A variety of medicinal products prepared from secondary tubers of Harpagophytum procumbens subsp. procumbens (Burch.) DC.ex Meisn. (Devil's Claw) and H. zeyheri are marketed in Africa, Europe, the United States, South America and elsewhere, where they are used for inflammatory and musculoskeletal conditions such as arthritis, lower back pain, rheumatism and neuralgia, etc. While clinical studies conducted over the last twenty years support the general safety of such products, infrequent gastrointestinal disturbances (diarrhea, nausea, vomiting, abdominal pain), headache, vertigo and hypersensitivity (allergic) reactions (rash, hives and face swelling) have been documented. Sex-related differences occur in the health conditions for which Devil's Claw products are used, so it is likely that usage is similarly sex-related and so might be side effects and potential toxicities. However toxicologic studies of Devil's Claw products have been conducted primarily with male animals. To address this deficit, we report toxicological studies in female and male rats of several H. procumbens (HP) aqueous-alcohol extracts chemically analyzed by UPLC-MS. METHODS: Female and male Sprague Dawley rats were studied for one and three months in groups differing by consumption of diets without and with HP extracts at a 7-10-fold human equivalent dose (HED). Sera were analyzed for blood chemistry, and heart, liver, lung, kidney, stomach, and small and large intestine tissues were examined for histopathology. Treatment group differences for blood chemistry were analyzed by ANOVA with Dunnett's test and significant group differences for endpoints with marginal distributional properties were verified using the Kruskal-Wallis test. Group differences for histopathology were tested using Chi Square analysis. RESULTS: Significant group by sex-related differences in blood chemistry were detected in both studies. Additionally, several sex-related differences occurred between the studies. However, significant histopathology effects associated with the consumption of the extracts were not detected. CONCLUSION: Toxicologic analysis of Devil's Claw extracts cause significant sex-related effects in blood chemistry. However, in our judgement, none of the observed effects suggest serious toxicity at these doses and durations. Subsequent toxicologic and clinical studies of H. procumbens and other medicines with similar properties should explore in greater detail the basis and consequences of potential sex-related effects.

Harpagophytum procumbens Extract Ameliorates Allodynia and Modulates Oxidative and Antioxidant Stress Pathways in a Rat Model of Spinal Cord Injury.

Spinal cord injury (SCI) is a deliberating disorder with impairments in locomotor deficits and incapacitating sensory abnormalities. Harpagophytum procumbens (Hp) is a botanical widely used for treating inflammation and pain related to various inflammatory and musculoskeletal conditions. Using a modified rodent contusion model of SCI, we explored the effects of this botanical on locomotor function and responses to mechanical stimuli, and examined possible neurochemical changes associated with SCI-induced allodynia. Following spinal cord contusion at T10 level, Hp (300 mg/kg, p.o.) or vehicle (water) was administered daily starting 24 h post-surgery, and behavioral measurements made every-other day until sacrifice (Day 21). Hp treatment markedly ameliorated the contusion-induced decrease in locomotor function and increased sensitivity to mechanical stimuli. Determination of Iba1 expression in spinal cord tissues indicated microglial infiltration starting 3 days post-injury. SCI results in increased levels of 4-hydroxynonenal, an oxidative stress product and proalgesic, which was diminished at 7 days by treatment with Hp. SCI also enhanced antioxidant heme oxygenase-1 (HO-1) expression. Concurrent studies of cultured murine BV-2 microglial cells revealed that Hp suppressed oxidative/nitrosative stress and inflammatory responses, including production of nitric oxide and reactive oxygen species, phosphorylation of cytosolic phospholipases A2, and upregulation of the antioxidative stress pathway involving the nuclear factor erythroid 2-related factor 2 and HO-1. These results support the use of Hp for management of allodynia by providing resilience against the neuroinflammation and pain associated with SCI and other neuropathological conditions.

Yin-Yang Mechanisms Regulating Lipid Peroxidation of Docosahexaenoic Acid and Arachidonic Acid in the Central Nervous System.

Phospholipids in the central nervous system (CNS) are rich in polyunsaturated fatty acids (PUFAs), particularly arachidonic acid (ARA) and docosahexaenoic acid (DHA). Besides providing physical properties to cell membranes, these PUFAs are metabolically active and undergo turnover through the "deacylation-reacylation (Land's) cycle". Recent studies suggest a Yin-Yang mechanism for metabolism of ARA and DHA, largely due to different phospholipases A2 (PLA2s) mediating their release. ARA and DHA are substrates of cyclooxygenases and lipoxygenases resulting in an array of lipid mediators, which are pro-inflammatory and pro-resolving. The PUFAs are susceptible to peroxidation by oxygen free radicals, resulting in the production of 4-hydroxynonenal (4-HNE) from ARA and 4-hydroxyhexenal (4-HHE) from DHA. These alkenal electrophiles are reactive and capable of forming adducts with proteins, phospholipids and nucleic acids. The perceived cytotoxic and hormetic effects of these hydroxyl-alkenals have impacted cell signaling pathways, glucose metabolism and mitochondrial functions in chronic and inflammatory diseases. Due to the high levels of DHA and ARA in brain phospholipids, this review is aimed at providing information on the Yin-Yang mechanisms for regulating these PUFAs and their lipid peroxidation products in the CNS, and implications of their roles in neurological disorders.

Isolation of sutherlandins A, B, C and D from Sutherlandia frutescens (L.) R. Br. by counter-current chromatography using spiral tubing support rotors.

Spiral countercurrent-chromatography has great potential for improving the capacity and efficiency of purification of secondary metabolites, and here we describe applications useful for the isolation of flavonoids from the widely used South African medicinal plant, Sutherlandia frutescens (L.) R. Br. In the spiral tubing support rotor, STS-4 for high-speed counter-current chromatography, several polar butanol aqueous solvent systems were selected using a logK plot, and the novel flavonol glycosides (sutherlandins A-D) were well separated by the optimized solvent system (ethyl acetate:n-butanol:acetic acid:water; 5:1:0.3:6 by vol.). The yield of purified flavonoids from 0.9g extract varied from 8.6mg to 54mg of the sutherlandins for a total of 85.3mg. The same extract was fractionated in the new STS-12 rotor of the same outside dimensions but with more radial channels forming 12 loops of the tubing instead of 4. The rotor holds more layers and increased length of tubing. From 0.9g extract the STS-12 rotor yielded more recovery of 110.4mg total with amounts varying from 11.2mg to 64mg of the sutherlandins and apparent increased separation efficiency as noted by less volume of each fraction peak. Thus from 1-g amounts of extract, good recovery of the flavonoids was achieved in the butanol aqueous solvent system.

Chilling susceptibility in mungbean varieties is associated with their differentially expressed genes.

BACKGROUND: Mungbean (Vigna radiata L. Wilczek) is an economically important legume of high nutritional value, however, its cultivation is limited by susceptibility to chilling. Varieties NM94 and VC1973A, with differential susceptibility to stress, serve as good materials for uncovering how they differ in chilling tolerance. This study aimed to identify the ultrastructural, physiological and molecular changes to provide new insights on the differential susceptibility to chilling between varieties VC1973A and NM94. RESULTS: Chilling stress caused a greater reduction in relative growth rate, a more significant decrease in maximum photochemical efficiency of PSII and DPPH scavenging activity and more-pronounced ultrastructural changes in VC1973A than in NM94 seedlings. Comparative analyses of transcriptional profiles in NM94 and VC1973A revealed that the higher expression of chilling regulated genes (CORs) in NM94. The transcript levels of lipid transfer protein (LTP), dehydrin (DHN) and plant defensin (PDF) in NM94 seedlings after 72 h at 4 °C was higher than that in its parental lines VC1973A, 6601 and VC2768A. CONCLUSIONS: Our results suggested that LTP, DHN and PDF may mediate chilling tolerance in NM94 seedlings.

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of Scrophularia and Verbascum species used medicinally in North America.

INTRODUCTION: Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability (YLDs), such as arthritis and lower back pain. OBJECTIVES: We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America. METHODS: Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI. RESULTS: Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus. CONCLUSIONS: Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

An Investigation into the Immunomodulatory Activities of Sutherlandia frutescens in Healthy Mice.

Sutherlandia frutescens is a medicinal plant that has been traditionally used in southern Africa for cancers, infections, and inflammatory conditions. We recently published experiments demonstrating that an aqueous extract of S. frutescens possessed potent immune-stimulatory activity. This work was carried out with murine macrophages, an immune cell type that plays a pivotal role in host defense from infection and in shaping host inflammatory and immune responses. Here, we conducted a series of follow-up experiments to explore the impact of consuming S. frutescens on host response to bacterial challenge using healthy mice. We found that feeding mice a diet containing S. frutescens failed to significantly alter host response to systemic infection by either a gram-positive or gram-negative bacterium (i.e., L. monocytogenes and E. coli, respectively). In contrast to the in vitro observations, we found no evidence that S. frutescens consumption stimulated in vivo inflammatory responses; instead, consumption of S. frutescens tended to diminish in vivo inflammatory responses. Several possible reasons for this are discussed.

Multilevel Assessment of Middle School Students' Interest in the Health Sciences: Development and Validation of a New Measurement Tool.

Using the context of a 2-wk instructional unit focused on eye and vision health, we developed and validated a multilevel measure of middle school students' interest in science and health careers. This survey contained three subscales positioned differently with respect to curricular content. Interest in Vision Care was most related, but less transferrable to other contexts. Interest in Science was most general, and Interest in Healthcare was positioned between the two. We found that, with two exceptions, items fitted well with validity expectations and were stable across a 2-wk intervention. Further, measures of interest in science, health, and vision-care careers were shown to be reliable and valid. We found that ease of facilitating change across the intervention was generally greater in subscales closely related to the curricular context but that the average magnitude of change in Interest in Healthcare and Interest in Science was not significantly different. We discuss use of these measures in informing instructional efforts and advise that changes in students' perceptions of how science and healthcare relate should be considered in longitudinal analyses.

Rapid and efficient Agrobacterium-mediated transformation of sorghum (Sorghum bicolor) employing standard binary vectors and bar gene as a selectable marker.

KEY MESSAGE: A rapid and efficient Agrobacterium -mediated transformation system in sorghum has been developed employing standard binary vectors and bar gene as a selectable marker. Sorghum (Sorghum bicolor) is an important food and biofuel crop worldwide, for which improvements in genetic transformation are needed to study its biology and facilitate agronomic and commercial improvement. Here, we report optimization of regeneration and transformation of public sorghum genotype P898012 using standard binary vectors and bar gene as a selectable marker. The tissue culture regeneration time frame has been reduced to 7-12 weeks with a yield of over 18 plants per callus, and the optimized transformation system employing Agrobacterium tumefaciens strain AGL1 and the bar with a MAS promoter achieved an average frequency over 14 %. Of randomly analyzed independent transgenic events, 40-50 % carry single copy of integrated T-DNA. Some independent transgenic events were derived from the same embryogenic callus lines, but a 3:1 Mendelian segregation ratio was found in all transgenic events with single copy as estimated by Southern blots. The system described here should facilitate studies of sorghum biology and agronomic improvement.

Phytochemicals and botanical extracts regulate NF-κB and Nrf2/ARE reporter activities in DI TNC1 astrocytes.

The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemicals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promoter activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells.

Does Concurrent Use of Some Botanicals Interfere with Treatment of Tuberculosis?

Millions of individuals with active TB do not receive recommended treatments, and instead may use botanicals, or use botanicals concurrently with established treatments. Many botanicals protect against oxidative stress, but this can interfere with redox-dependent activation of isoniazid and other prodrugs used for prophylaxis and treatment of TB, as suggested by results of a recent clinical trial of the South African botanical Sutherlandia frutescens (L.) R. Br. (Sutherlandia). Here we provide a brief summary of Sutherlandia's effects upon rodent microglia and neurons relevant to tuberculosis of the central nervous system (CNS-TB). We have observed that ethanolic extracts of Sutherlandia suppress production of reactive oxygen species (ROS) in rat primary cortical neurons stimulated by NMDA and also suppress LPS- and interferon γ (IFNγ)-induced ROS and nitric oxide (NO) production by microglial cells. Sutherlandia consumption mitigates microglial activation in the hippocampus and striatum of ischemic brains of mice. RNAseq analysis indicates that Sutherlandia suppresses gene expression of oxidative stress, inflammatory signaling and toll-like receptor pathways that can reduce the host's immune response to infection and reactivation of latent Mycobacterium tuberculosis. As a precautionary measure, we recommend that individuals receiving isoniazid for pulmonary or cerebral TB, be advised not to concurrently use botanicals or dietary supplements having antioxidant activity.

Inhibition of Gli/hedgehog signaling in prostate cancer cells by "cancer bush" Sutherlandia frutescens extract.

Sutherlandia frutescens is a medicinal plant, traditionally used to treat various types of human diseases, including cancer. Previous studies of several botanicals link suppression of prostate cancer growth with inhibition of the Gli/hedgehog (Gli/Hh) signaling pathway. Here we hypothesized the anti-cancer effect of S. frutescens was linked to its inhibition of the Gli/Hh signaling in prostate cancer. We found a dose- and time-dependent growth inhibition in human prostate cancer cells, PC3 and LNCaP, and mouse prostate cancer cell, TRAMP-C2, treated with S. frutescens methanol extract (SLE). We also observed a dose-dependent inhibition of the Gli-reporter activity in Shh Light II and TRAMP-C2QGli cells treated with SLE. In addition, SLE can inhibit Gli/Hh signaling by blocking Gli1 and Ptched1 gene expression in the presence of a Gli/Hh signaling agonist (SAG). A diet supplemented with S. frutescens suppressed the formation of poorly differentiated carcinoma in prostates of TRAMP mice. Finally, we found Sutherlandioside D was the most potent compound in the crude extract that could suppress Gli-reporter in Shh Light II cells. Together, this suggests that the S. frutescens extract may exert anti-cancer effect by targeting Gli/Hh signaling, and Sutherlandioside D is one of the active compounds.

Inhibition of Hedgehog-Signaling Driven Genes in Prostate Cancer Cells by Sutherlandia frutescens Extract.

Sutherlandia frutescens (L) R. Br. (Sutherlandia) is a South African botanical that is traditionally used to treat a variety of health conditions, infections and diseases, including cancer. We hypothesized Sutherlandia might act through Gli/ Hedgehog (Hh)-signaling in prostate cancer cells and used RNA-Seq transcription profiling to profile gene expression in TRAMPC2 murine prostate cancer cells with or without Sutherlandia extracts. We found 50% of Hh-responsive genes can be repressed by Sutherlandia ethanol extract, including the canonical Hh-responsive genes Gli1 and Ptch1 as well as newly distinguished Hh-responsive genes Hsd11b1 and Penk.

Unveiling the anti-inflammatory activity of Sutherlandia frutescens using murine macrophages.

Sutherlandia frutescens is a botanical widely used in southern Africa for treatment of inflammatory and other conditions. Previously, an ethanolic extract of S. frutescens (SFE) has been shown to inhibit the production of reactive oxygen species (ROS) and nitric oxide (NO) by murine neurons and a microglia cell line (BV-2 cells). In this study we sought to confirm the anti-inflammatory activities of SFE on a widely used murine macrophage cell line (i.e., RAW 264.7 cells) and primary mouse macrophages. Furthermore, experiments were conducted to investigate the anti-inflammatory activity of the flavonol and cycloartanol glycosides found in high quantities in S. frutescens. While the SFE exhibited anti-inflammatory activities upon murine macrophages similar to that reported with the microglia cell line, this effect does not appear to be mediated by sutherlandiosides or sutherlandins. In contrast, chlorophyll in our extracts appeared to be partly responsible for some of the activity observed in our macrophage-dependent screening assay.

Consumption of Sutherlandia frutescens by HIV-Seropositive South African Adults: An Adaptive Double-Blind Randomized Placebo Controlled Trial.

BACKGROUND: Sutherlandia frutescens (L.) R. Br. is widely used as an over the counter complementary medicine and in traditional medications by HIV seropositive adults living in South Africa; however the plant's safety has not been objectively studied. An adaptive two-stage randomized double-blind placebo controlled study was used to evaluate the safety of consuming dried S. frutescens by HIV seropositive adults with CD4 T-lymphocyte count of >350 cells/µL. METHODS: In Stage 1 56 participants were randomized to S. frutescens 400, 800 or 1,200 mg twice daily or matching placebo for 24 weeks. In Stage 2 77 additional participants were randomized to either 1,200 mg S. frutescens or placebo. In the final analysis data from Stage 1 and Stage 2 were combined such that 107 participants were analysed (54 in the S. frutescens 1,200 mg arm and 53 in the placebo arm). RESULTS: S. frutescens did not change HIV viral load, and CD4 T-lymphocyte count was similar in the two arms at 24 weeks; however, mean and total burden of infection (BOI; defined as days of infection-related events in each participant) was greater in the S. frutescens arm: mean (SD) 5.0 (5.5) vs. 9.0 (12.7) days (p = 0.045), attributed to two tuberculosis cases in subjects taking isoniazid preventive therapy (IPT). CONCLUSION: A possible interaction between S. frutescens and IPT needs further evaluation, and may presage antagonistic interactions with other herbs having similar biochemical (antioxidant) properties. No other safety issues relating to consumption of S. frutescens in this cohort were identified. TRIAL REGISTRATION: ClinicalTrials.gov NCT00549523.

Immuno-stimulatory activity of a polysaccharide-enriched fraction of Sutherlandia frutescens occurs by the toll-like receptor-4 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Sutherlandia frutescens (L.) R. Br. is an indigenous plant of southern Africa that has been traditionally used for various cancers, infections, and inflammatory conditions. AIM OF THE STUDY: Our aim was to investigate the potential immuno-stimulatory activity of a polysaccharide-enriched fraction (SFPS) from a decoction of S. frutescens. MATERIALS AND METHODS: RAW 264.7 cells (a murine macrophage cell line) were used to determine the activities of SFPS on macrophage function. The production of reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines were evaluated in the cells treated with or without SFPS. CLI-095, a toll-like receptor (TLR) 4-specific inhibitor, was used to identify whether or not SFPS exerts its effects through TLR4. An antagonist of endotoxin, polymyxin B, was used to evaluate whether endotoxin present in SFPS contributed to its immune-stimulatory activity. RESULTS: SFPS exhibited potent immune-stimulatory activity by macrophages. The production of ROS, NO, and tumor necrosis factor (TNF-α) were increased upon exposure to SFPS in a dose-dependent manner. All of these activities were completely blocked by co-treatment with CLI-095, but only partially diminished by polymyxin B. CONCLUSION: We demonstrate for the first time potent immune-stimulatory activity in a decoction prepared from S. frutescens. We believe that this immune stimulatory activity is due, in part, to the action of polysaccharides present in the decoction that acts by way of TLR4 receptors and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. These findings provide a plausible mechanism through which we can understand some of the medicinal properties of S. frutescens.

From Gigabyte to Kilobyte: A Bioinformatics Protocol for Mining Large RNA-Seq Transcriptomics Data.

RNA-Seq techniques generate hundreds of millions of short RNA reads using next-generation sequencing (NGS). These RNA reads can be mapped to reference genomes to investigate changes of gene expression but improved procedures for mining large RNA-Seq datasets to extract valuable biological knowledge are needed. RNAMiner--a multi-level bioinformatics protocol and pipeline--has been developed for such datasets. It includes five steps: Mapping RNA-Seq reads to a reference genome, calculating gene expression values, identifying differentially expressed genes, predicting gene functions, and constructing gene regulatory networks. To demonstrate its utility, we applied RNAMiner to datasets generated from Human, Mouse, Arabidopsis thaliana, and Drosophila melanogaster cells, and successfully identified differentially expressed genes, clustered them into cohesive functional groups, and constructed novel gene regulatory networks. The RNAMiner web service is available at http://calla.rnet.missouri.edu/rnaminer/index.html.

Arabidopsis thaliana MSI4/FVE associates with members of a novel family of plant specific PWWP/RRM domain proteins.

AtMSI4/FVE/ACG1, one of five Arabidopsis thaliana genes encoding MSI1-like proteins, helps determine plant growth and development (including control of flowering), as well as responses to certain biotic and abiotic stresses. We reasoned that the product of this gene, AtMSI4, acts through protein partners, which we have co-immunopurified with AtMSI4 from A. thaliana suspension culture cells and identified by liquid chromatography-mass spectrometry (LC-MS). Many of the proteins associated with AtMSI4 have distinct RNA recognition motif (RRM) domains, which we determined to be responsible for association with AtMSI4; and most of the associated RRM domain proteins also contain PWWP domains that are specific to plants. We propose these novel ATMSI4-associated proteins help form nucleoprotein complexes that determine pleiotropic functional properties of AtMSI4/FVE/ACG1 involving plant development and responses to stress.