La polyveoline,nouvel indolosesquiterpene isole du polyalthiasuaveolens,annonacees

The structure and absolute configuration of polyveoline, new indolosesquiterpene from Polyalthiasuaveolena, Annonaceae, were determined by X-ray crystal lographic study.     

Mycomedicine: A Unique Class of Natural Products with Potent Anti-tumour Bioactivities

Mycomedicine is a unique class of natural medicine that has been widely used in Asian countries for thousands of years. Modern mycomedicine consists of fruiting bodies, spores, or other tissues of medicinal fungi, as well as bioactive components extracted from them, including polysaccharides and, triterpenoids, etc. Since the discovery of the famous fungal extract, penicillin, by Alexander Fleming in the late 19th century, researchers have realised the significant antibiotic and other medicinal values of fungal extracts. As medicinal fungi and fungal metabolites can induce apoptosis or autophagy, enhance the immune response, and reduce metastatic potential, several types of mushrooms, such as Ganoderma lucidum and Grifola frondosa, have been extensively investigated, and anti-cancer drugs have been developed from their extracts. Although some studies have highlighted the anti-cancer properties of a single, specific mushroom, only limited reviews have summarised diverse medicinal fungi as mycomedicine. In this review, we not only list the structures and functions of pharmaceutically active components isolated from mycomedicine, but also summarise the mechanisms underlying the potent bioactivities of several representative mushrooms in the Kingdom Fungi against various types of tumour.     

Anti-Cancer and Medicinal Potentials of Moringa Isothiocyanate

Moringa oleifera (M. oleifera), which belongs to the Moringaceae family, is a common herb, rich in plant compounds. It has a variety of bioactive compounds that can act as antioxidants, antibiotics, anti-inflammatory and anti-cancer agents, etc., which can be obtained in different body parts of M. oleifera. Isothiocyanates (ITCs) from M. oleifera are one class of these active substances that can inhibit cancer proliferation and promote cancer cell apoptosis through multiple signaling pathways, thus curbing cancer migration and metastasis, at the same time they have little adverse effect on normal cells. There are multiple variants of ITCs in M. oleifera, but the predominant phytochemical is 4-(α-L-rhamnosyloxy)benzyl isothiocyanate, also known as moringa isothiocyanate (MIC-1). Studies have shown that MIC-1 has the possibility to be used clinically for the treatment of diabetes, neurologic diseases, obesity, ulcerative colitis, and several cancer types. In this review, we focus on the molecular mechanisms underlying the anti-cancer and anti-chronic disease effects of MIC-1, current trends, and future direction of MIC-1 based treatment strategies. This review combines the relevant literature of the past 10 years, in order to provide more comprehensive information of MIC-1 and to fully exploit its potentiality in the clinical settings.     

An Insight into the Anti-Angiogenic and Anti-Metastatic Effects of Oridonin: Current Knowledge and Future Potential

Cancer is one of the leading causes of death worldwide, with a mortality rate of more than 9 million deaths reported in 2018. Conventional anti-cancer therapy can greatly improve survival however treatment resistance is still a major problem especially in metastatic disease. Targeted anti-cancer therapy is increasingly used with conventional therapy to improve patients’ outcomes in advanced and metastatic tumors. However, due to the complexity of cancer biology and metastasis, it is urgent to develop new agents and evaluate the anti-cancer efficacy of available treatments. Many phytochemicals from medicinal plants have been reported to possess anti-cancer properties. One such compound is known as oridonin, a bioactive component of Rabdosia rubescens. Several studies have demonstrated that oridonin inhibits angiogenesis in various types of cancer, including breast, pancreatic, lung, colon and skin cancer. Oridonin’s anti-cancer effects are mediated through the modulation of several signaling pathways which include upregulation of oncogenes and pro-angiogenic growth factors. Furthermore, oridonin also inhibits cell migration, invasion and metastasis via suppressing epithelial-to-mesenchymal transition and blocking downstream signaling targets in the cancer metastasis process. This review summarizes the recent applications of oridonin as an anti-angiogenic and anti-metastatic drug both in vitro and in vivo, and its potential mechanisms of action.     

Boesenbergia Pandurata Roxb., An Indonesian Medicinal Plant: Phytochemistry,Biological Activity,Plant Biotechnology

Boesenbergia pandurata Roxb. (Zingiberaceae), known as “temu kunci”, is one of the Indonesian medicinal plants. Its rhizome has been traditionally used in folk medicine for treatment of several diseases. Rhizome of B. pandurata contains essential oils and many flavonoid compounds that showed many interesting pharmacological activities, such as antifungal, antibacterial, antioxidant, etc. Interestingly, this plant has several prenylated flavonoid compounds, panduratins, that showed very promising of biological activities, especially as strong antifungal and antibacterial, anti-inflammatory, and anti-cancer. This paper aims to review chemical constituents of this plant and their pharmacological activities and also to give a brief view through biotechnological perspective concerning the several possibilities to produce several valuable prenylated flavonoids from this plant.     

3D-QSAR study,docking molecular and simulation dynamic on series of benzimidazole derivatives as anti-cancer agents

Benzimidazole is an important heterocyclic organic compound which has a structural analogy to nucleotides found in human body and hence is an important pharmacophore in medicinal chemistry. The anti-cancer activities for a diverse set of benzimidazole as anti-cancer agents against breast cancer cell line (MCF7) assay have been subjected to 3D-QSAR (3-Dimensional Quantitative Structural-Activity Relationship) studies. Both CoMFA and CoMSIA models exhibit significant results in terms of statistical parameters as determination coefficients R² > 0.9 and Leave One Out cross-validation determination coefficients Q²> 6. The predictive quality of both 3D QSAR models have been assessed by external validation and Y-randomization test. Five new compounds have been designed and predicted by in silico ADMET method. In the second part, we have used the docking molecular and simulation dynamics (MD) to investigate the bonding interactions and stability of the designed compounds into the Pin1. Then, we have compared them to Trastuzumab and Tamoxifen as a standard inhibitors drug of breast cancer. The designed compounds form stable hydrogen and hydrophobic bonding interactions with the residues Lys63, Gln131, Ser154, Arg 68 and Arg69 of Pin1 receptor during 100 ns as a time of the simulation. The obtained results showed that the new benzimidazole are useful as a template for future design of more potent inhibitors against breast cancer cell lines (MCF7).     

Acanthopanax henryi: Review of Botany,Phytochemistry and Pharmacology

Acanthopanax henryi (Oliv.) Harms (Araliaceae), also known as Eleutherococcus henryi and Caoyewujia (Hengliwujia) in Chinese, is a widely used traditional Chinese herb with the effects of expelling wind and removing dampness, relaxing the muscles and stimulating the blood circulation, and regulating the flow of qi to alleviate pain in the theory of Traditional Chinese Medicine. Acanthopanax henryi (AH, thereafter) possesses ginseng-like activities and is known as ginseng-like herb. In the past decade, a great number of phytochemical and pharmacological studies on AH have been carried out. Several kinds of chemical compositions have been reported, including terpenoids (monoterpenoids, diterpenoids, and triterpenoid saponins), phenylpropanoids, caffeoyl quinic acid derivatives, flavonoids, lignans, sterols, fatty acids, etc., among which, triterpenoid saponins were considered to be the most active components. Considerable pharmacological experiments in vitro have demonstrated that AH possessed anti-neuroinflammatory, anti-adipogenic, anti-inflammatory, antibacterial, anti-cancer, anti-oxidation, anti-AChE, anti-BuChE, and antihyaluronidase activities. The present review is an up-to-date and comprehensive analysis of the botany, phytochemistry, and pharmacology of AH.     

Fluorescent probes for the stabilization and detection of G-quadruplexes and their prospective applications

This review article discusses the non-covalent interaction of various probe molecules of different structural diversity with G-quadruplex forming Guanine rich DNA sequences, revealing remarkable stimuli responsive fluorescence changes which are appropriate for sensing and other technological applications. Tailor-made probes having quadruplex inducing/stabilizing attachments of well-known dye molecules and its derivatives such as, coumarins, cyanines, thiazole orange, pyrazines, thioflavin T, triphenylmethane, tetraphenylethene, dimethylindole red etc have been employed. These probes express their modulations due to the binding to the topologically distinct G-quadruplexes through structural rigidization, aggregation propensity, binding strengths, tunability and other competitive interactions and are viewed as remarkable changes particularly in their fluorescence features. Based on this concept, several studies have reported the development of label free fluorescence sensor for the selective detection of topology specific G-quadruplexes, therapeutic, early diagnostic of cancer, cation-sensing, trace level detection of an anti-cancer drugs etc. have been accomplished. In vivo imaging is also achieved using a cholesterol attached G-quadruplex forming oligonucleotide probe labelled with specific dyes. Since the fine details of the topological information and control mechanisms of G-quadruplex forming sequences are very much essential for targeting and tuning several important biological processes relevant to cancer proliferation and developing stimuli responsive sensors, it is sure that many more contributions in this field will emerge in the coming years.     

Induction of G2/M Cell Cycle Arrest via p38/p21Waf1/Cip1-Dependent Signaling Pathway Activation by Bavachinin in Non-Small-Cell Lung Cancer Cells

Lung cancer is the most commonly diagnosed malignant cancer in the world. Non-small-cell lung cancer (NSCLC) is the major category of lung cancer. Although effective therapies have been administered, for improving the NSCLC patient’s survival, the incident rate is still high. Therefore, searching for a good strategy for preventing NSCLC is urgent. Traditional Chinese medicine (TCM) are brilliant materials for cancer chemoprevention, because of their high biological safety and low cost. Bavachinin, which is an active flavanone of Proralea corylifolia L., possesses anti-inflammation, anti-angiogenesis, and anti-cancer activities. The present study’s aim was to evaluate the anti-cancer activity of bavachinin on NSCLC, and its regulating molecular mechanisms. The results exhibited that a dose-dependent decrease in the cell viability and colony formation capacity of three NSCLC cell lines, by bavachinin, were through G2/M cell cycle arrest induction. Meanwhile, the expression of the G2/M cell cycle regulators, such as cyclin B, p-cdc2^Y15, p-cdc2^T161, and p-wee1, was suppressed. With the dramatic up-regulation of the cyclin-dependent kinase inhibitor, p21^Waf1/Cip1, the expression and association of p21^Waf1/Cip1 with the cyclin B/cdc2 complex was observed. Silencing the p21^Waf1/Cip1 expression significantly rescued bavachinin-induced G2/M cell accumulation. Furthermore, the expression of p21^Waf1/Cip1 mRNA was up-regulated in bavachinin-treated NSCLC cells. In addition, MAPK and AKT signaling were activated in bavachinin-added NSCLC cells. Interestingly, bavachinin-induced p21^Waf1/Cip1 expression was repressed after restraint p38 MAPK activation. The inhibition of p38 MAPK activation reversed bavachinin-induced p21^Waf1/Cip1 mRNA expression and G2/M cell cycle arrest. Collectively, bavachinin-induced G2/M cell cycle arrest was through the p38 MAPK-mediated p21^Waf1/Cip1-dependent signaling pathway in the NSCLC cells.     

Cancer chemoprevention by carotenoids

Carotenoids are natural fat-soluble pigments that provide bright coloration to plants and animals. Dietary intake of carotenoids is inversely associated with the risk of a variety of cancers in different tissues. Preclinical studies have shown that some carotenoids have potent antitumor effects both in vitro and in vivo, suggesting potential preventive and/or therapeutic roles for the compounds. Since chemoprevention is one of the most important strategies in the control of cancer development, molecular mechanism-based cancer chemoprevention using carotenoids seems to be an attractive approach. Various carotenoids, such as β-carotene, a-carotene, lycopene, lutein, zeaxanthin, β-cryptoxanthin, fucoxanthin, canthaxanthin and astaxanthin, have been proven to have anti-carcinogenic activity in several tissues, although high doses of β-carotene failed to exhibit chemopreventive activity in clinical trials. In this review, cancer prevention using carotenoids are reviewed and the possible mechanisms of action are described.     

The medicinal Umbelliferae plant Fennel (Foeniculum vulgare Mill.): Cultivation,traditional uses,phytopharmacological properties,and application in animal husbandry

Foeniculum vulgare Mill., commonly called fennel is one of the most popular perennial herbaceous plants used as herbal medicine and spices worldwide. It belongs to the family Umbelliferae or Apiaceae and is characterized by a distinct floral arrangement with an umbrella-like shape. In addition to other well-known Umbelliferae plants, such as carrot, celery, and angelica, fennel has been utilized ethnobotanically to cure various ailments, including gastrointestinal issues, hormonal disorders, reproductive, and respiratory diseases. This review aims to update the information on conventional usage, cultivation, phytopharmacological properties, and other applications of F. vulgare Mill. in animal husbandry. Ethnobotanical studies have shown its medicinal uses worldwide; some tribes have reported its medicinal uses and how they use different parts of the plant. Alkaloids, essential oils, phenols, fatty acids, and amino acids are examples of natural phytochemicals that have been characterized and biologically tested for their medicinal potential. Fennel has been used to treat ten categories of diseases over the last two decades. Pharmacological studies revealed that the Umbelliferae plant has antiviral, antimicrobial, antioxidant, anti-inflammatory, anti-anxiety, gastro-protective, estrogenic-like, cardiovascular, lipid, anti-mutagenic, anti-diabetic, anti-cancer activity, hepatoprotective, and memory-protective properties. Fennel extract is used as a potential phytogenic agent to enhance the productivity and quality of animal husbandry. Finally, the present study may provide up-to-date information on the medicinal application of F. vulgare from ethnobotanical and phytopharmacological perspectives and could be further explored to provide a broader benefit to humankind.     

New diterpene pyrone-type compounds, metarhizins A and B, isolated from entomopathogenic fungus, Metarhizium flavoviride and their inhibitory effects on cellular proliferation

To date, entomopathogenic fungi have not been extensively examined by natural product chemists. In this study, we isolated novel pyrone diterpene-type compounds, metarhizins A (1) and B (2), from methanol extracts of entomopathogenic fungus, Metarhizium flavoviride. They showed potent and selective antiproliferative activity against both insect and human cancer cell lines. These results indicate that metarhizins A (1) and B (2) can be used as novel lead compounds for anti-cancer agents and probes for cell cycle regulation. To further investigate the structural requirements for this inhibitory activity, we synthesized many metarhizin derivatives and evaluated their antiproliferative activities.     

Anticancer Activity of Continentalic Acid in B-Cell Lymphoma

Aralia continentalis has been used in Korea as a folk remedy for arthralgia, rheumatism, and inflammation. However, its anti-lymphoma effect remains uncharacterized. Here, we demonstrate that A. continentalis extract and its three diterpenes efficiently kill B-lymphoma cells. Our in vitro and in vivo results suggest that the cytotoxic activities of continentalic acid, a major diterpene from A. continentalis extract, are specific towards cancer cells while leaving normal murine cells and tissues unharmed. Mechanistically, continentalic acid represses the expression of pro-survival Bcl-2 family members, such as Mcl-1 and Bcl-xL. It dissociates the mitochondrial membrane potential, leading to the stimulation of effector caspase 3/7 activities and, ultimately, cell death. Intriguingly, this agent therapeutically synergizes with roflumilast, a pan-PDE4 inhibitor that has been successfully repurposed for the treatment of aggressive B-cell malignancies in recent clinical tests. Our findings unveiled that A. continentalis extract and three of the plant’s diterpenes exhibit anti-cancer activities. We also demonstrate the synergistic inhibitory effect of continentalic acid on the survival of B-lymphoma cells when combined with roflumilast. Taken in conjunction, continentalic acid may hold significant potential for the treatment of B-cell lymphoma.     

Extraction,Isolation and Characterization of Bioactive Compounds from Artemisia and Their Biological Significance: A Review

Diverse medicinal plants such as those from the genus Artemisia have been employed globally for centuries by individuals belonging to different cultures. Universally, Artemisia species have been used to remedy various maladies that range from simple fevers to malaria. A survey conducted by the World Health Organization (WHO) demonstrated that 80% of the global population is highly reliant on herbal medicine for their primary healthcare. WHO recommends artemisinin-based combination therapies (ACT) for the treatment of global diseases such as malaria. Artemisinin is a bioactive compound derived from Artemisia annua leaves. It is a sesquiterpene endoperoxide with potent antimalarial properties. This review strives to instill natural products to chemists and others in diverse fields with a heterogeneous set of knowledge compiled from multifaceted researchers and organizations in literature. In particular, the various Artemisia species and effective extraction, isolation, and characterization methodologies are discussed in detail. An in-depth investigation into the literature reveals that divergent species of Artemisia exhibit a vast array of biological activities such as antimalarial, antitumor, and anti-inflammatory activities. There is substantial potential for bioactive compounds from Artemisia to provide significant relief from differing human ailments, but more meticulous research in this field is needed.     

Nanovaccines for cancer immunotherapy: Current knowledge and future perspectives

Cancer immunotherapy harnesses the immune system to attack tumors and has received extensive attention in recent years. Cancer vaccines as an important branch of immunotherapy are designed for delivering tumor antigens to antigen-presenting cells (APCs) to stimulate a strong immune response to against tumors, representing a potentially therapeutic and prophylactic effect with the long-term anti-cancer benefits. Nevertheless, the disappointing outcomes of their clinical use might be attributed to dilemma in antigen selection, immunogenicity, lymph nodes (LNs) targeting ability, lysosomal escape ability, immune evasion, etc. Nanotechnology, aiming to overcome these barriers, has been utilized in cancer vaccine development for decades. Numerous preclinical and clinical studies demonstrate positive results in nanomaterials-based cancer vaccines with considerable improvement in the vaccine efficacy. In this review, we systematically introduced the characteristics of nanovaccines and highlighted the different types of nanomaterials used for cancer vaccine design. In addition, the opportunities and challenges of the emerging nanotechnology-based cancer vaccines were discussed.     

Natural product extracts of the Canadian prairie plant,Thermopsis rhombifolia,have anti-cancer activity in phenotypic cell-based assays

Many plant species within the terrestrial ecological zones of Canada have not yet been investigated for anti-cancer activity. We examined the scientific literature describing the endemic flora from the prairie ecological zone and selected the species, Thermopsis rhombifolia, locally known as the buffalo bean, for investigation of its anti-cancer potential. We tested it in cell-based assays using phenotypic screens that feature some of the hallmarks of cancer. An ethanolic extract prepared from T. rhombifolia was cytotoxic to HT-29 (colon) and SH-SY5Y (brain) cancer cell lines, and showed little cytotoxicity to a normal human cell line (WI-38). In phenotypic assays, we identified activities in the extracts that target cell death, cell cycle and cell adhesion. These data highlight the anti-cancer potential of previously untested plants found in northern ecological zones and the feasibility of using pertinent phenotypic assays to examine the anti-cancer potential of natural product extracts.     

Applications of stable,nonradioactive isotope tracers in in vivo human metabolic research

The human body is in a constant state of turnover, that is, being synthesized, broken down and/or converted to different compounds. The dynamic nature of in vivo kinetics of human metabolism at rest and in stressed conditions such as exercise and pathophysiological conditions such as diabetes and cancer can be quantitatively assessed with stable, nonradioactive isotope tracers in conjunction with gas or liquid chromatography mass spectrometry and modeling. Although measurements of metabolite concentrations have been useful as general indicators of one''s health status, critical information on in vivo kinetics of metabolites such as rates of production, appearance or disappearance of metabolites are not provided. Over the past decades, stable, nonradioactive isotope tracers have been used to provide information on dynamics of specific metabolites. Stable isotope tracers can be used in conjunction with molecular and cellular biology tools, thereby providing an in-depth dynamic assessment of metabolic changes, as well as simultaneous investigation of the molecular basis for the observed kinetic responses. In this review, we will introduce basic principles of stable isotope methodology for tracing in vivo kinetics of human or animal metabolism with examples of quantifying certain aspects of in vivo kinetics of carbohydrate, lipid and protein metabolism.     

Genus Chloranthus: A comprehensive review of its phytochemistry,pharmacology, and uses

This paper is intended to review advances in the botanical, traditional uses, phytochemical, pharmacological and development and utilization studies of the genus Chloranthus. Chloranthus, a genus of the family Chloranthaceae, which is mainly distributed in the temperate and tropical regions of Asia, has been used as a folk remedy for swollen boils, snake bites and bruises. Up to now, 418 compounds have been reported from the genus Chloranthus, including 383 terpenoids, 4 coumarins, 6 lignans, 2 simple phenylpropanoids, 4 flavonoids, 6 amides, 5 organic acids and some other types of compounds. Among them, the main chemical constituents are sesquiterpenes and their diterpenoids. Modern pharmacological studies have shown that most of the Chloranthus plants possessed anti-cancer, anti-inflammatory, antibacterial, antiviral, and antimalarial activities. As one of the most important genera in China, Chloranthus should be paid further attention to gathering information about the pharmacological mechanism and value active compounds. This paper summarized the phytochemistry, pharmacology, and uses of genus Chloranthus in order to lay a foundation and provide reference for the follow-up research and wide application of the genus.     

Inhibition of Interleukin-6-Induced Matrix Metalloproteinase-2 Expression and Invasive Ability of Lemon Peel Polyphenol Extract in Human Primary Colon Cancer Cells

Among matrix metalloproteinases (MMPs), MMP-9/2 are key enzymes involved in the proteolysis of extracellular matrices in the inflammatory process and in cancer. Since MMP-9/2 expression levels, activity, and secretion is up-regulated during inflammation in response to pro-inflammatory cytokines, such as interleukin-6 (IL-6), many efforts have been devoted to identifying factors that could inhibit the IL-6-induced MMP-9/2 expression. Up to now, several reports indicated that polyphenols from fruits and vegetables are among the major components of health promotion for their antioxidant properties and also for their anti-inflammatory and anti-cancer agents. Among plant derived polyphenols, lemon (Citrus limon) peel extract (LPE) shows anti-cancer properties in various cancer types. In our previous work, we demonstrated that LPE can reduce IL-6-induced migration/invasiveness and MMP-9/2 up-regulation in some gastric cancer cell lines. This study aims to exploit the anti-cancer properties of LPE using an in vitro system model of inflammation, consisting of IL-6-exposed human primary colon cancer cells. We first analyzed the effect of LPE on IL-6-induced cell migration and invasiveness by wound healing and Boyden chamber assay, respectively. The MMP-2 mRNA expression levels and gelatinolytic activity in the cell culture media were determined by q-PCR analysis and gelatin zymography, respectively, and finally, the effects of LPE on IL-6-induced JAK2/STAT3 signaling pathways have been investigated by Western blotting analysis. Our results show that LPE is able to inhibit the IL-6-dependent cell migration and invasiveness associated with the up-regulation of MMP-2 expression levels and that these effects are correlated to the STAT3 phosphorylation in human primary T88 and T93 colon cancer cells.     

Genus Morinda: An insight to its ethnopharmacology,phytochemistry, pharmacology and Industrial Applications

Background of the studyThe genus Morinda of the Madder family, (Rubiaceae) has been widely documented in traditional medicine due to its therapeutic properties and also, contributed a great deal in chemical industry. Different parts of Morinda species have traditionally been used to treat malaria, diabetes, memory loss, cancer, inflammation, skin infections, and typhoid fever.Aim and ObjectivesThe review provide a critical and innovative information on the traditional uses, phytochemical constituents, and industrial applications of the genus Morinda. This will help researchers understand future research trends by bridging the gap between documented literature and contemporary uses.MethodologyAll the systematic literature data or information on the genus Morinda was collected via selected electronic databases, including Scopus, PubMed, Web of Science, Springer, Medline, ChemSpider, Taylor and Francis, Google Scholar, SciFinder, ScienceDirect and Wiley. Relevant book chapters, Wikipedia and books were also explored.ResultsThe study reveals that different parts of Morinda plants have been extensively used for folkloric therapeutic purposes and are a plethora of mineral or nutritional benefits and secondary metabolites. Several classes of bioactive compounds have been elucidated from Morinda plants via spectroscopic and chromatographic phytochemical analyses. Compounds such as terpenoids, glycosides, anthraquinones, polyphenols, steroids, saponins and reducing sugars are among the bioactive substances reported in the genus. Plant extracts, fractions and isolates of Morinda plants have exhibited pronounced antidiabetic, antioxidant, antiplasmodial, antidepressant, wound healing, anticancer, and anti-inflammatory effects (in vitro and in vivo). These pharmacological activities exhibited could be linked to the presence of secondary metabolites reported. The applications of this genus is well documented in textile, metallurgical, agrochemicals and food industries.ConclusionA number of reports on Morinda showed significant therapeutic effect against several diseases such as cancer, dermal infection, diabetes, inflammation, malaria, typhoid, cholera and memory loss, however, there were several drawback in previous reports including mechanism of action, drug dose, controls and toxicological data of extracts or isolates. In view of this, further studies should emphasis on targeting active secondary metabolites which responsible for the therapeutic activities, structural elucidation and toxicological assessment. Furthermore, industrial uses require authentication.