A systems biology-based mathematical model demonstrates the potential anti-stress effectiveness of a multi-nutrient botanical formulation.

Stress is an adaptive response to the stressors that adversely affects physiological and psychological health. Stress elicits HPA axis activation, resulting in cortisol release, ultimately contributing to oxidative, inflammatory, physiological and mental stress. Nutritional supplementations with antioxidant, anti-inflammatory, and stress-relieving properties are among widely preferred complementary approaches for the stress management. However, there is limited research on the potential combined impact of vitamins, minerals and natural ingredients on stress. In the present study, we have investigated the effect of a multi-nutrient botanical formulation, Nutrilite® Daily Plus, on clinical stress parameters. The stress-modulatory effects were quantified at population level using a customized sub-clinical inflammation mathematical model. The model suggested that combined intervention of botanical and micronutrients lead to significant decline in physical stress (75% decline), mental stress (70% decline), oxidative stress (55% decline) and inflammatory stress (75% decline) as evident from reduction in key stress parameters such as ROS, TNF-α, blood pressure, cortisol levels and PSS scores at both individual and population levels. Further, at the population level, the intervention relieved stress in 85% of individuals who moved towards a healthy state. The in silico studies strongly predicts the use of Gotukola based Nutrilite® Daily Plus as promising anti-stress formulation.

Elevated H3K4me3 Through MLL2-WDR82 upon Hyperglycemia Causes Jagged Ligand Dependent Notch Activation to Interplay with Differentiation State of Endothelial Cells.

Endothelial-to-mesenchymal transition (EndMT) is a hallmark of diabetes-associated vascular complications. Epigenetic mechanisms emerged as one of the key pathways to regulate diabetes-associated complications. In the current study, we aimed to determine how abrupt changes in histone 3 lysine 4 tri-methylation (H3K4me3) upon hyperglycemia exposure reprograms endothelial cells to undergo EndMT. Through in vitro studies, we first establish that intermittent high-glucose exposure to EC most potently induced partial mesenchyme-like characteristics compared with transient or constant high-glucose-challenged endothelial cells. In addition, glomerular endothelial cells of BTBR Ob/Ob mice also exhibited mesenchymal-like characteristics. Intermittent hyperglycemia-dependent induction of partial mesenchyme-like phenotype of endothelial cells coincided with an increase in H3K4me3 level in both macro- and micro-vascular EC due to selective increase in MLL2 and WDR82 protein of SET1/COMPASS complex. Such an endothelial-specific heightened H3K4me3 level was also detected in intermittent high-glucose-exposed rat aorta and in kidney glomeruli of Ob/Ob mice. Elevated H3K4me3 enriched in the promoter regions of Notch ligands Jagged1 and Jagged2, thus causing abrupt expression of these ligands and concomitant activation of Notch signaling upon intermittent hyperglycemia challenge. Pharmacological inhibition and/or knockdown of MLL2 in cells in vitro or in tissues ex vivo normalized intermittent high-glucose-mediated increase in H3K4me3 level and further reversed Jagged1 and Jagged2 expression, Notch activation and further attenuated acquisition of partial mesenchyme-like phenotype of endothelial cells. In summary, the present study identifies a crucial role of histone methylation in hyperglycemia-dependent reprograming of endothelial cells to undergo mesenchymal transition and indicated that epigenetic pathways contribute to diabetes-associated vascular complications.

Intermittent High Glucose Elevates Nuclear Localization of EZH2 to Cause H3K27me3-Dependent Repression of KLF2 Leading to Endothelial Inflammation.

Epigenetic mechanisms have emerged as one of the key pathways promoting diabetes-associated complications. Herein, we explored the role of enhancer of zeste homolog 2 (EZH2) and its product histone 3 lysine 27 trimethylation (H3K27me3) in high glucose-mediated endothelial inflammation. To examine this, we treated cultured primary endothelial cells (EC) with different treatment conditions-namely, constant or intermittent or transient high glucose. Intermittent high glucose maximally induced endothelial inflammation by upregulating transcript and/or protein-level expression of ICAM1 and P-selectin and downregulating eNOS, KLF2, and KLF4 protein levels. We next investigated the underlining epigenetic mechanisms responsible for intermittent hyperglycemia-dependent endothelial inflammation. Compared with other high glucose treatment groups, intermittent high glucose-exposed EC exhibited an increased level of H3K27me3 caused by reduction in EZH2 threonine 367 phosphorylation and nuclear retention of EZH2. Intermittent high glucose also promoted polycomb repressive complex-2 (PRC2) assembly and EZH2's recruitment to histone H3. Abrupt enrichment of H3K27me3 on KLF2 and KLF4 gene promoters caused repression of these genes, further supporting endothelial inflammation. In contrast, reducing H3K27me3 through small molecule and/or siRNA-mediated inhibition of EZH2 rescued KLF2 level and inhibited endothelial inflammation in intermittent high glucose-challenged cultured EC and isolated rat aorta. These findings indicate that abrupt chromatin modifications cause high glucose-dependent inflammatory switch of EC.

Shortwave infrared polymethine fluorophores matched to excitation lasers enable non-invasive, multicolour in vivo imaging in real time.

High-resolution, multiplexed experiments are a staple in cellular imaging. Analogous experiments in animals are challenging, however, due to substantial scattering and autofluorescence in tissue at visible (350-700 nm) and near-infrared (700-1,000 nm) wavelengths. Here, we enable real-time, non-invasive multicolour imaging experiments in animals through the design of optical contrast agents for the shortwave infrared (SWIR, 1,000-2,000 nm) region and complementary advances in imaging technologies. We developed tunable, SWIR-emissive flavylium polymethine dyes and established relationships between structure and photophysical properties for this class of bright SWIR contrast agents. In parallel, we designed an imaging system with variable near-infrared/SWIR excitation and single-channel detection, facilitating video-rate multicolour SWIR imaging for optically guided surgery and imaging of awake and moving mice with multiplexed detection. Optimized dyes matched to 980 nm and 1,064 nm lasers, combined with the clinically approved indocyanine green, enabled real-time, three-colour imaging with high temporal and spatial resolutions.

Shortwave Infrared Imaging with J-Aggregates Stabilized in Hollow Mesoporous Silica Nanoparticles.

Tissue is translucent to shortwave infrared (SWIR) light, rendering optical imaging superior in this region. However, the widespread use of optical SWIR imaging has been limited, in part, by the lack of bright, biocompatible contrast agents that absorb and emit light above 1000 nm. J-Aggregation offers a means to transform stable, near-infrared (NIR) fluorophores into red-shifted SWIR contrast agents. Here we demonstrate that J-aggregates of NIR fluorophore IR-140 can be prepared inside hollow mesoporous silica nanoparticles (HMSNs) to result in nanomaterials that absorb and emit SWIR light. The J-aggregates inside PEGylated HMSNs are stable for multiple weeks in buffer and enable high resolution imaging in vivo with 980 nm excitation.

The Chrysanthemum nankingense Genome Provides Insights into the Evolution and Diversification of Chrysanthemum Flowers and Medicinal Traits.

The Asteraceae (Compositae), a large plant family of approximately 24 000-35 000 species, accounts for ∼10% of all angiosperm species and contributes a lot to plant diversity. The most representative members of the Asteraceae are the economically important chrysanthemums (Chrysanthemum L.) that diversified through reticulate evolution. Biodiversity is typically created by multiple evolutionary mechanisms such as whole-genome duplication (WGD) or polyploidization and locally repetitive genome expansion. However, the lack of genomic data from chrysanthemum species has prevented an in-depth analysis of the evolutionary mechanisms involved in their diversification. Here, we used Oxford Nanopore long-read technology to sequence the diploid Chrysanthemum nankingense genome, which represents one of the progenitor genomes of domesticated chrysanthemums. Our analysis revealed that the evolution of the C. nankingense genome was driven by bursts of repetitive element expansion and WGD events including a recent WGD that distinguishes chrysanthemum from sunflower, which diverged from chrysanthemum approximately 38.8 million years ago. Variations of ornamental and medicinal traits in chrysanthemums are linked to the expansion of candidate gene families by duplication events including paralogous gene duplication. Collectively, our study of the assembled reference genome offers new knowledge and resources to dissect the history and pattern of evolution and diversification of chrysanthemum plants, and also to accelerate their breeding and improvement.

IM-133N - A Useful Herbal Combination for Eradicating Disease-triggering Pathogens in Mice via Immunotherapeutic Mechanisms.

OBJECTIVES: The present study was undertaken to evaluate the immunomodulatory (IM) activity of IM-133N, a herbal combination in various immunotherapeutic experimental models. METHODS: The IM activity of IM-133N was evaluated against three experimental models namely, effect of IM- 133N against Escherichia coli (E. coli)-induced abdominal sepsis in mice, and carbon clearance test was performed in Wistar albino rats to evaluated the phagocytic potential of IM-133N, in addition IM-133N was evaluated for its immunoglobulin enhancing potential in rats, where the immunoglobulin levels were measured by zinc sulphate turbity (ZST) test. Further, IM-133N was subjected for detailed liquid chromatography-mass spectrometry (LC-MS)/MS analysis to identify the probable active constituents present in it. RESULTS: The findings of the present study has demonstrated very promising IM property of IM-133N in all the experimental models. Briefly, pretreatment with IM-133N at 125, 250, 500 and 1,000 mg/kg, p.o. doses had protected the mice against E. coli-induced abdominal sepsis and mortality, further the effect of IM- 133N was found to be significant and dose-dependent. In support of this, in another study administration of IM-133N showed a significant and dose-dependent increase in serum immunoglobulin levels, estimated by ZST test. In line with the above findings, in the carbon clearance test the low doses (125 and 250 mg/ kg, p.o.) of IM-133N increased the rate of carbon clearance, whereas the higher doses (500 and 1,000 mg/kg, p.o.) did not sustain the response, and saturation effect was considered as one of the possible reason for futility of higher doses for IM-133N. In addition, A detailed LC-MS/MS analysis of IM-133N showed 17 bioactive phytochemical constituents: namely, apigenin, chaulmoogric acid, mesquitol, quercetin, symphoxanthone, salireposide, ß-sitosterol, nonaeicosanol, ß-amyrin, betulic acid, oleanolic acid, symplososide, symponoside, symploveroside, symplocomoside, symconoside A and locoracemoside B. CONCLUSION: These findings suggest that IM-133N possesses significant IM activity and, hence, could be useful for eradicating opportunistic disease-triggering pathogens via immunotherapeutic mechanisms. The findings also suggest IM-133N may also useful in other immunity disorders.

Bone health nutraceuticals alter microarray mRNA gene expression: A randomized, parallel, open-label clinical study.

BACKGROUND AND OBJECTIVE: Dietary intake of fruits and vegetables has been suggested to have a role in promoting bone health. More specifically, the polyphenols they contain have been linked to physiological effects related to bone mineral density and bone metabolism. In this research, we use standard microarray analyses of peripheral whole blood from post-menopausal women treated with two fixed combinations of plant extracts standardized to polyphenol content to identify differentially expressed genes relevant to bone health. METHODS: In this 28-day open-label study, healthy post-menopausal women were randomized into three groups, each receiving one of three investigational fixed combinations of plant extracts: an anti-resorptive (AR) combination of pomegranate fruit (Punica granatum L.) and grape seed (Vitis vinifera L.) extracts; a bone formation (BF) combination of quercetin (Dimorphandra mollis Benth) and licorice (Glycyrrhiza glabra L.) extracts; and a fixed combination of all four plant extracts (AR plus BF). Standard microarray analysis was performed on peripheral whole blood samples taken before and after each treatment. Annotated genes were analyzed for their association to bone health by comparison to a gene library. RESULTS: The AR combination down-regulated a number of genes involved in reduction of bone resorption including cathepsin G (CTSG) and tachykinin receptor 1 (TACR1). The AR combination also up-regulated genes associated with formation of extracellular matrix including heparan sulfate proteoglycan 2 (HSPG2) and hyaluronoglucosaminidase 1 (HYAL1). In contrast, treatment with the BF combination resulted in up-regulation of bone morphogenetic protein 2 (BMP-2) and COL1A1 (collagen type I α1) genes which are linked to bone and collagen formation while down-regulating genes linked to osteoclastogenesis. Treatment with a combination of all four plant extracts had a distinctly different effect on gene expression than the results of the AR and BF combinations individually. These results could be due to multiple feedback systems balancing activities of osteoblasts and osteoclasts. CONCLUSION: In summary, this ex-vivo microarray study indicated that the pomegranate, grape seed, quercetin and licorice combinations of plant extracts modulated gene expression for both osteoclastic and osteogenic processes.

Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes.

Human skin is body's vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 µg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 µg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations.

LC-MS/MS profiling and neuroprotective effects of Mentat® against transient global ischemia and reperfusion-induced brain injury in rats.

OBJECTIVE: The aim of this study was to evaluate the possible beneficial effects of Mentat against transient global ischemia and reperfusion-induced brain injury in rats. METHODS: The neuroprotective effects of Mentat were evaluated against transient global ischemia and reperfusion (I/R)-induced brain injury in rats. Various neurobehavioral and biochemical parameters were assessed, followed by morphologic and histopathologic evaluation of brain tissue to conclude the protective effect of Mentat. Additionally, in vitro antioxidant assays were performed to explore the antioxidant capacity of Mentat and detailed liquid chromatography-mass spectrometry (LC-MS/MS) profiling was carried out to identify the active phytoconstituents responsible for the protective effects of Mentat. RESULTS: Sixty minutes of transient global ischemia followed by 24 h reperfusion (I/R) caused significant alterations in the cognitive and neurologic functions in the ischemia control group (P < 0.01) compared with the sham control. Furthermore, 2,3,5-triphenyltetrazolium chloride staining of the ischemia control group showed 20.85% ± 0.39% of cerebral infarct area (P < 0.01), increased brain volume (% edema 17.81% ± 1.576%; P < 0.01), and increased lipid peroxidation (P < 0.01) in the brain homogenate. Additionally, the histopathology of the ischemia control group showed severe brain injury compared with the sham control group. Interestingly, pretreatment with Mentat (250 and 500 mg/kg, p.o.) and quercetin (20 mg/kg, p.o.) for 7 d has alleviated all pathological changes observed due to I/R injury. Mentat also showed very good antioxidant activity in in vitro assays (2,2-diphenyl-l-picrylhydrazyl, ferric-reducing antioxidant power, and oxygen radical absorbance capacity assays). Furthermore, the detailed LC-MS/MS analysis of Mentat was performed and enclosed for identifying the actives responsible for its protective effects. CONCLUSIONS: These findings suggest that Mentat is a neuroprotective agent that may be a useful adjunct in the management of ischemic stroke and its rehabilitation especially with respect to associated memory impairment and other related neurologic conditions.

A targeted approach for evaluating preclinical activity of botanical extracts for support of bone health.

Using a sequential in vitro/in vivo approach, we tested the ability of botanical extracts to influence biomarkers associated with bone resorption and bone formation. Pomegranate fruit and grape seed extracts were found to exhibit anti-resorptive activity by inhibiting receptor activator of nuclear factor-κB ligand (RANKL) expression in MG-63 cells and to reduce IL-1ß-stimulated calvarial (45)Ca loss. A combination of pomegranate fruit and grape seed extracts were shown to be effective at inhibiting bone loss in ovariectomised rats as demonstrated by standard histomorphometry, biomechanical and bone mineral density measurements. Quercetin and licorice extract exhibited bone formation activity as measured by bone morphogenetic protein-2 (BMP-2) promoter activation, increased expression of BMP-2 mRNA and protein levels, and promotion of bone growth in cultured mouse calvariae. A combination of quercetin and licorice extract demonstrated a potential for increasing bone mineral density in an intact female rat model as compared with controls. The results from this sequential in vitro/in vivo research model yielded botanical extract formulas that demonstrate significant potential benefits for bone health.

Novel experimental model of non-infectious pharyngitis in rats.

INTRODUCTION: Currently, there is a paucity of scientific literature and reports related to screening models for non-infectious type of pharyngitis. In this context, we made a sincere attempt to establish a novel animal model for screening drugs against non-infectious pharyngitis in rats. We have considered the use of pyridine, croton oil and their combination for inducing non-infectious pharyngitis in rats. METHODS: Various concentrations of pyridine were applied topically to the pharyngeal region of rats and the extent of inflammation was assessed by Evans Blue (EB) dye exudation test, evaluating the serum levels of proinflammatory cytokines and histopathology. Dexamethasone and diclofenac were used as reference standards. RESULTS: Upon pyridine application (2.5%, 5%, 10%, 20%, 40% and 80% in saline), dose-dependent increase in EB dye extravasation was observed (increased vascular permeability). In addition, the levels of TNF-α (P<0.01) and IL-6 (P<0.01) were significantly increased compared to control. Furthermore, the histopathology of pharyngeal tissue showed hypertrophy of submucosal glands, severe inflammation of the pharynx characterised by presence of mononuclear cells, neutrophils along with haemorrhages and congestion; however, normal control animals showed normal cytoarchitecture of pharynx. Indeed, dexamethasone (0.25, 0.5 and 1 mg/kg, i.v.) and diclofenac (1, 2.5 and 5 mg/kg, i.v.) showed dose-dependent protection against pyridine-induced pharyngitis. Further, the possible mechanism of pyridine-induced pharyngitis is thought to be primarily mediated through phospholipase A2 and cyclooxygenase (COX) pathway. CONCLUSION: These findings suggest that pyridine-induced pharyngitis is a simple and versatile novel animal model for screening the drugs against non-infectious pharyngitis in rats.

Overexpression of PH-4, a novel putative proline 4-hydroxylase, modulates activity of hypoxia-inducible transcription factors.

Hypoxia-inducible transcription factors (HIFs) are important for transcriptional adaptation to hypoxia. Availability of HIFs is regulated via posttranslational modification of their alpha subunits (HIF-1alpha and HIF-2alpha). Under normoxia, two highly conserved proline residues within the oxygen-dependent degradation domain (ODDD) are hydroxylated by oxoglutarate-dependent proline 4-hydroxylases EGLN1-3. Hydroxylated HIF-alpha interacts with the pVHL-E3 ubiquitin ligase complex and, subsequently, is degraded via the proteasomal pathway. We identified a novel putative proline 4-hydroxylase, PH-4, with an aminoterminal EF-hand motif and a carboxyterminal catalytic domain, which was highly expressed in most organs, and-unlike EGLNs which localize to the cytoplasm and nucleus-was associated with the endoplasmic reticulum. Like EGLNs, PH-4 overexpressed in cellular reporter assays suppressed the HIF transactivation activity, dependent on the consensus ODDD proline residues. Suppression of transactivation was correlated with decrease of cellular contents of HIF. Thus, PH-4 might be related to cellular oxygen sensing.