A minor product, rebaudioside M2 (2), from the bioconversion reaction of rebaudioside A (4) to rebaudioside D (3), was isolated and the complete structure of the novel steviol glycoside was determined. Rebaudioside M2 (2) is considered an isomer of rebaudioside M (1) and contains a relatively rare 1→6 sugar linkage. It was isolated and characterized with NMR (1H, 13C, COSY, HSQC-DEPT, HMBC, 1D-TOCSY, and NOESY) and mass spectral data. Additionally, we emphasize the importance of 1D and 2D NMR techniques when identifying complex steviol glycosides. Numerous NMR spectroscopy studies of rebaudioside M (1), rebaudioside D (3), and mixture of 1 and 3 led to the discovery that SG17 which was previously reported in literature, is a mixture of rebaudioside D (3), rebaudioside M (1), and possibly other related steviol glycosides.
Immobilized lipases were applied to the enzymatic conversion of oils from spent coffee ground into biodiesel. Two lipases were selected for the study because of their conformational behavior analysed by Molecular Dynamics (MD) simulations taking into account that immobilization conditions affect conformational behavior of the lipases and ultimately, their efficiency upon immobilization. The enzymatic synthesis of biodiesel was initially carried out on a model substrate (triolein) in order to select the most promising immobilized biocatalysts. The results indicate that oils can be converted quantitatively within hours. The role of the nature of the immobilization support emerged as a key factor affecting reaction rate, most probably because of partition and mass transfer barriers occurring with hydrophilic solid supports. Finally, oil from spent coffee ground was transformed into biodiesel with yields ranging from 55% to 72%. The synthesis is of particular interest in the perspective of developing sustainable processes for the production of bio-fuels from food wastes and renewable materials. The enzymatic synthesis of biodiesel is carried out under mild conditions, with stoichiometric amounts of substrates (oil and methanol) and the removal of free fatty acids is not required.
Lead (Pb) is a toxic, environmental heavy metal that induces serious clinical defects in all organs, with the nervous system being its primary target. Curcumin is the main active constituent of turmeric rhizome (Curcuma longa) with strong antioxidant and anti-inflammatory properties. This study is aimed at evaluating the therapeutic potentials of curcumin on Pb-induced neurotoxicity. Thirty-six male Sprague Dawley rats were randomly assigned into five groups with 12 rats in the control (normal saline) and 6 rats in each of groups, i.e., the lead-treated group (LTG) (50 mg/kg lead acetate for four weeks), recovery group (RC) (50 mg/kg lead acetate for four weeks), treatment group 1 (Cur100) (50 mg/kg lead acetate for four weeks, followed by 100 mg/kg curcumin for four weeks) and treatment group 2 (Cur200) (50 mg/kg lead acetate for four weeks, followed by 200 mg/kg curcumin for four weeks). All experimental groups received oral treatment via orogastric tube on alternate days. Motor function was assessed using a horizontal bar method. The cerebellar concentration of Pb was evaluated using ICP-MS technique. Pb-administered rats showed a significant decrease in motor scores and Superoxide Dismutase (SOD) activity with increased Malondialdehyde (MDA) levels. In addition, a marked increase in cerebellar Pb concentration and alterations in the histological architecture of the cerebellar cortex layers were recorded. However, treatment with curcumin improved the motor score, reduced Pb concentration in the cerebellum, and ameliorated the markers of oxidative stress, as well as restored the histological architecture of the cerebellum. The results of this study suggest that curcumin attenuates Pb-induced neurotoxicity via inhibition of oxidative stress and chelating activity.
Epithelial cell adhesion molecule (EpCAM) is a cell surface protein that was discovered as a tumour marker of epithelial origins nearly four decades ago. EpCAM is expressed at basal levels in the basolateral membrane of normal epithelial cells. However, EpCAM expression is upregulated in solid epithelial cancers and stem cells. EpCAM can also be found in disseminated tumour cells and circulating tumour cells. Various OMICs studies have demonstrated that EpCAM plays roles in several key biological processes such as cell adhesion, migration, proliferation and differentiation. Additionally, EpCAM can be detected in the bodily fluid of cancer patients suggesting that EpCAM is a pathophysiologically relevant anti-tumour target as well as being utilized as a diagnostic/prognostic agent for a variety of cancers. This review will focus on the structure-features of EpCAM protein and discuss recent evidence on the pathological and physiological roles of EpCAM in modulating cell adhesion and signalling pathways in cancers as well as deliberating the clinical implication of EpCAM as a therapeutic target.
Dicranopteris linearis leaf has been reported to exert antinociceptive activity. The present study elucidates the possible mechanisms of antinociception modulated by the methanol extract of D. linearis leaves (MEDL) using various mouse models. The extract (25, 150, and 300 mg/kg) was administered orally to mice for 30 min priot to subjection to the acetic acid-induced writhing-, hot plate- or formalin-test to establish the antinociceptive profile of MEDL. The most effective dose was then used in the elucidation of possible mechanisms of action stage. The extract was also subjected to the phytochemical analyses. The results confirmed that MEDL exerted significant (p < 0.05) antinociceptive activity in those pain models as well as the capsaicin-, glutamate-, bradykinin- and phorbol 12-myristate 13-acetate (PMA)-induced paw licking model. Pretreatment with naloxone (a non-selective opioid antagonist) significantly (p < 0.05) reversed MEDL effect on thermal nociception. Only l-arginine (a nitric oxide (NO) donor) but not N(ω)-nitro-l-arginine methyl ester (l-NAME; a NO inhibitor) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a specific soluble guanylyl cyclase inhibitor) significantly (p < 0.05) modified MEDL effect on the writhing test. Several polyphenolics and volatile antinociceptive compounds were detected in MEDL. In conclusion, MEDL exerted the opioid/NO-mediated antinociceptive activity, thus, justify D. linearis as a potential source for new analgesic agents development.
Stone leaf (Tetracera scandens) is a Southeast Asian medicinal plant that has been traditionally used for the management of diabetes mellitus. The underlying mechanisms of the antidiabetic activity have not been fully explored yet. Hence, this study aimed to evaluate the α-glucosidase inhibitory potential of the hydromethanolic extracts of T. scandens leaves and to characterize the metabolites responsible for such activity through gas chromatography-mass spectrometry (GC-MS) metabolomics. Crude hydromethanolic extracts of different strengths were prepared and in vitro assayed for α-glucosidase inhibition. GC-MS analysis was further carried out and the mass spectral data were correlated to the corresponding α-glucosidase inhibitory IC50 values via an orthogonal partial least squares (OPLS) model. The 100%, 80%, 60% and 40% methanol extracts displayed potent α-glucosidase inhibitory potentials. Moreover, the established model identified 16 metabolites to be responsible for the α-glucosidase inhibitory activity of T. scandens. The putative α-glucosidase inhibitory metabolites showed moderate to high affinities (binding energies of -5.9 to -9.8 kcal/mol) upon docking into the active site of Saccharomyces cerevisiae isomaltase. To sum up, an OPLS model was developed as a rapid method to characterize the α-glucosidase inhibitory metabolites existing in the hydromethanolic extracts of T. scandens leaves based on GC-MS metabolite profiling.
: Oxidative stress plays an important role in retinal neurodegeneration and angiogenesis associated with diabetes. In this study, we investigated the effect of the tocotrienol-rich fraction (TRF), a potent antioxidant, against diabetes-induced changes in retinal layer thickness (RLT), retinal cell count (RCC), retinal cell apoptosis, and retinal expression of vascular endothelial growth factor (VEGF) in rats. Additionally, the efficacy of TRF after administration by two different routes was compared. The diabetes was induced in Sprague-Dawley rats by intraperitoneal injection of streptozotocin. Subsequently, diabetic rats received either oral or topical treatment with vehicle or TRF. Additionally, a group of non-diabetic rats was included with either oral or topical treatment with a vehicle. After 12 weeks of the treatment period, rats were euthanized, and retinas were collected for measurement of RLT, RCC, retinal cell apoptosis, and VEGF expression. RLT and RCC in the ganglion cell layer were reduced in all diabetic groups compared to control groups (p < 0.01). However, at the end of the experimental period, oral TRF-treated rats showed a significantly greater RLT compared to topical TRF-treated rats. A similar observation was made for retinal cell apoptosis and VEGF expression. In conclusion, oral TRF supplementation protects against retinal degenerative changes and an increase in VEGF expression in rats with streptozotocin-induced diabetic retinopathy. Similar effects were not observed after topical administration of TRF.
Palm oil is natural oil packed with important compounds and fatty acids ready to be exploited in lipid-based formulations and drug delivery. Palm oil and palm kernel oil contain long-chain and medium-chain triglycerides, respectively, including phytonutrients such as tocotrienol, tocopherol and carotenes. The exploitation of these compounds in a lipid-based formulation would be able to address hydrophobicity, lipophilicity, poor bioavailability and low water-solubility of many current drugs. The utilisation of palm oil as part of the drug delivery system seemed to improve the bioavailability and solubility of the drug, stabilising emulsification of formulation between emulsifier and surfactant, promoting enhanced drug permeability and performance, as well as extending the shelf-life of the drug. Despite the complexity in designing lipid-based formulations, palm oil has proven to offer dynamic behaviour in providing versatility in drug design, form and delivery. However, the knowledge and application of palm oil and its fractions in lipid-based formulation are scarce and interspersed. Therefore, this study aims to focus on the research and outcomes of using palm oil in lipid-based formulations and drug delivery systems, due to the importance of establishing its capabilities and benefits.
Continuously increasing energy demand and growing concern about energy resources has attracted much research in the field of clean and sustainable energy sources. In this context, zero-emission fuels are required for energy production to reduce the usage of fossil fuel resources. Here, we present the synthesis of Pd-Ag-decorated reduced graphene oxide (rGO) nanostructures using a green chemical approach with stevia extract for hydrogen production and antibacterial studies under light irradiation. Moreover, bimetallic nanostructures are potentially lime lighted due to their synergetic effect in both scientific and technical aspects. Structural characteristics such as crystal structure and morphological features of the synthesized nanostructures were analyzed using X-ray diffraction and transmission electron microscopy. Analysis of elemental composition and oxidation states was carried out by X-ray photoelectron spectroscopy. Optical characteristics of the biosynthesized nanostructures were obtained by UV-Vis absorption spectroscopy, and Fourier transform infrared spectroscopy was used to investigate possible functional groups that act as reducing and capping agents. The antimicrobial activity of the biosynthesized Pd-Ag-decorated rGO nanostructures was excellent, inactivating 96% of Escherichia coli cells during experiments over 150 min under visible light irradiation. Hence, these biosynthesized Pd-Ag-decorated rGO nanostructures can be utilized for alternative nanomaterial-based drug development in the future.
As the most recognizable natural secondary carotenoid astaxanthin producer, the green microalga Haematococcus pluvialis cultivation is performed via a two-stage process. The first is dedicated to biomass accumulation under growth-favoring conditions (green stage), and the second stage is for astaxanthin evolution under various stress conditions (red stage). This mini-review discusses the further improvement made on astaxanthin production by providing an overview of recent works on H. pluvialis, including the valuable ideas for bioprocess optimization on cell growth, and the current stress-exerting strategies for astaxanthin pigment production. The effects of nutrient constituents, especially nitrogen and carbon sources, and illumination intensity are emphasized during the green stage. On the other hand, the significance of the nitrogen depletion strategy and other exogenous factors comprising salinity, illumination, and temperature are considered for the astaxanthin inducement during the red stage. In short, any factor that interferes with the cellular processes that limit the growth or photosynthesis in the green stage could trigger the encystment process and astaxanthin formation during the red stage. This review provides an insight regarding the parameters involved in bioprocess optimization for high-value astaxanthin biosynthesis from H. pluvialis.
Catalpol isolated from Rehmannia glutinosa is a potent antioxidant and investigated against many disorders. This review appraises the key molecular pathways of catalpol against diabetes mellitus and its complications. Multiple search engines including Google Scholar, PubMed, and Science Direct were used to retrieve publications containing the keywords "Catalpol", "Type 1 diabetes mellitus", "Type 2 diabetes mellitus", and "diabetic complications". Catalpol promotes IRS-1/PI3K/AKT/GLUT2 activity and suppresses Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose 6-phosphatase (G6Pase) expression in the liver. Catalpol induces myogenesis by increasing MyoD/MyoG/MHC expression and improves mitochondria function through the AMPK/PGC-1α/PPAR-γ and TFAM signaling in skeletal muscles. Catalpol downregulates the pro-inflammatory markers and upregulates the anti-inflammatory markers in adipose tissues. Catalpol exerts antioxidant properties through increasing superoxide dismutase (sod), catalase (cat), and glutathione peroxidase (gsh-px) activity in the pancreas and liver. Catalpol has been shown to have anti-oxidative, anti-inflammatory, anti-apoptosis, and anti-fibrosis properties that in turn bring beneficial effects in diabetic complications. Its nephroprotective effect is related to the modulation of the AGE/RAGE/NF-κB and TGF-β/smad2/3 pathways. Catalpol produces a neuroprotective effect by increasing the expression of protein Kinase-C (PKC) and Cav-1. Furthermore, catalpol exhibits a cardioprotective effect through the apelin/APJ and ROS/NF-κB/Neat1 pathway. Catalpol stimulates proliferation and differentiation of osteoblast cells in high glucose condition. Lastly, catalpol shows its potential in preventing neurodegeneration in the retina with NF-κB downregulation. Overall, catalpol exhibits numerous beneficial effects on diabetes mellitus and diabetic complications.
Antifreeze proteins (AFPs) are specific proteins, glycopeptides, and peptides made by different organisms to allow cells to survive in sub-zero conditions. AFPs function by reducing the water's freezing point and avoiding ice crystals' growth in the frozen stage. Their capability in modifying ice growth leads to the stabilization of ice crystals within a given temperature range and the inhibition of ice recrystallization that decreases the drip loss during thawing. This review presents the potential applications of AFPs from different sources and types. AFPs can be found in diverse sources such as fish, yeast, plants, bacteria, and insects. Various sources reveal different α-helices and β-sheets structures. Recently, analysis of AFPs has been conducted through bioinformatics tools to analyze their functions within proper time. AFPs can be used widely in various aspects of application and have significant industrial functions, encompassing the enhancement of foods' freezing and liquefying properties, protection of frost plants, enhancement of ice cream's texture, cryosurgery, and cryopreservation of cells and tissues. In conclusion, these applications and physical properties of AFPs can be further explored to meet other industrial players. Designing the peptide-based AFP can also be done to subsequently improve its function.
Catalpol was tested for various disorders including diabetes mellitus. Numerous molecular mechanisms have emerged supporting its biological effects but with little information towards its insulin sensitizing effect. In this study, we have investigated its effect on skeletal muscle mitochondrial respiration and insulin signaling pathway. Type-2 diabetes (T2DM) was induced in male C57BL/6 by a high fat diet (60% Kcal) and streptozotocin (50 mg/kg, i.p.). Diabetic mice were orally administered with catalpol (100 and 200 mg/kg), metformin (200 mg/kg), and saline for four weeks. Fasting blood glucose (FBG), HbA1c, plasma insulin, oral glucose tolerance test (OGTT), insulin tolerance test (ITT), oxygen consumption rate, gene (IRS-1, Akt, PI3k, AMPK, GLUT4, and PGC-1α) and protein (AMPK, GLUT4, and PPAR-γ) expression in muscle were measured. Catalpol (200 mg/kg) significantly (p < 0.05) reduced the FBG, HbA1C, HOMA_IR index, and AUC of OGTT whereas, improved the ITT slope. Gene (IRS-1, Akt, PI3k, GLUT4, AMPK, and PGC-1α) and protein (AMPK, p-AMPK, PPAR-γ and GLUT4) expressions, as well as augmented state-3 respiration, oxygen consumption rate, and citrate synthase activity in muscle was observed in catalpol treated mice. The antidiabetic activity of catalpol is credited with a marked improvement in insulin sensitivity and mitochondrial respiration through the insulin signaling pathway and AMPK/SIRT1/PGC-1α/PPAR-γ activation in the skeletal muscle of T2DM mice.
Heliotropium is one of the most important plant genera to have conventional folklore importance, and hence is a potential source of bioactive compounds. Thus, the present study was designed to explore the therapeutic potential of Heliotropium crispum Desf., a relatively under-explored medicinal plant species. Methanolic extracts prepared from a whole plant of H. crispum were studied for phytochemical composition and possible in vitro and in silico biological properties. Antioxidant potential was assessed via six different assays, and enzyme inhibition potential against key clinical enzymes involved in neurodegenerative diseases (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)), diabetes (α-amylase and α-glucosidase), and skin problems (tyrosinase) was assayed. Phytochemical composition was established via determination of the total bioactive contents and reverse phase ultra-high performance liquid chromatography mass spectrometry (RP-UHPLC-MS) analysis. Chemical profiling revealed the tentative presence of 50 secondary metabolites. The plant extract exhibited significant inhibition against AChE and BChE enzymes, with values of 3.80 and 3.44 mg GALAE/g extract, respectively. Further, the extract displayed considerable free radical scavenging activity against DPPH and ABTS radicals, with potential values of 43.19 and 41.80 mg TE/g extract, respectively. In addition, the selected compounds were then docked against the tested enzymes, which have shown high inhibition affinity. To conclude, H. crispum was found to harbor bioactive compounds and showed potent biological activities which could be further explored for potential uses in nutraceutical and pharmaceutical industries, particularly as a neuroprotective agent.
Modulation of major histocompatibility complex (MHC) expression using drugs has been proposed to control immunity. Phytochemical investigations on Garcinia species have allowed the isolation of bioactive compounds such as polycyclic polyprenylated acylphloroglucinols (PPAPs). PPAPs such as guttiferone J (1), display anti-inflammatory and immunoregulatory activities while garcinol (4) is a histone acetyltransferases (HAT) p300 inhibitor. This study reports on the isolation, identification and biological characterization of two other PPAPs, i.e., xanthochymol (2) and guttiferone F (3) from Garcinia bancana, sharing structural analogy with guttiferone J (1) and garcinol (4). We show that PPAPs 1-4 efficiently downregulated the expression of several MHC molecules (HLA-class I, -class II, MICA/B and HLA-E) at the surface of human primary endothelial cells upon inflammation. Mechanistically, PPAPs 1-4 reduce MHC proteins by decreasing the expression and phosphorylation of the transcription factor STAT1 involved in MHC upregulation mediated by IFN-γ. Loss of STAT1 activity results from inhibition of HAT CBP/p300 activity reflected by a hypoacetylation state. The binding interactions to p300 were confirmed through molecular docking. Loss of STAT1 impairs the expression of CIITA and GATA2 but also TAP1 and Tapasin required for peptide loading and transport of MHC. Overall, we identified new PPAPs issued from Garcinia bancana with potential immunoregulatory properties.
Plants are rich in phytoconstituent biomolecules that served as a good source of medicine. More recently, they have been employed in synthesizing metal/metal oxide nanoparticles (NPs) due to their capping and reducing properties. This green synthesis approach is environmentally friendly and allows the production of the desired NPs in different sizes and shapes by manipulating parameters during the synthesis process. The most commonly used metals and oxides are gold (Au), silver (Ag), and copper (Cu). Among these, Cu is a relatively low-cost metal that is more cost-effective than Au and Ag. In this review, we present an overview and current update of plant-mediated Cu/copper oxide (CuO) NPs, including their synthesis, medicinal applications, and mechanisms. Furthermore, the toxic effects of these NPs and their efficacy compared to commercial NPs are reviewed. This review provides an insight into the potential of developing plant-based Cu/CuO NPs as a therapeutic agent for various diseases in the future.
The Krüppel-like factors (KLFs) family of proteins control several key biological processes that include proliferation, differentiation, metabolism, apoptosis and inflammation. Dysregulation of KLF functions have been shown to disrupt cellular homeostasis and contribute to disease development. KLF6 is a relevant example; a range of functional and expression assays suggested that the dysregulation of KLF6 contributes to the onset of cancer, inflammation-associated diseases as well as cardiovascular diseases. KLF6 expression is either suppressed or elevated depending on the disease, and this is largely due to alternative splicing events producing KLF6 isoforms with specialised functions. Hence, the aim of this review is to discuss the known aspects of KLF6 biology that covers the gene and protein architecture, gene regulation, post-translational modifications and functions of KLF6 in health and diseases. We put special emphasis on the equivocal roles of its full-length and spliced variants. We also deliberate on the therapeutic strategies of KLF6 and its associated signalling pathways. Finally, we provide compelling basic and clinical questions to enhance the knowledge and research on elucidating the roles of KLF6 in physiological and pathophysiological processes.
This study investigated the renin-angiotensin-aldosterone system (RAAS) gene polymorphisms as possible genetic risk factors for the restenosis development in patients with drug-eluting stents. 113 participants had coronary artery disease and underwent stenting. The control group consisted of 62 individuals with intact coronary arteries. Patients were divided into two groups: with in-stent restenosis (ISR) and without it. The patients with ISR were classified into subgroups by the terms of the restenosis development and age. Real-time PCR and Restriction Fragment Length Polymorphism-PCR were used to genotype the study participants for RAAS gene polymorphisms. We found that the development of restenosis is generally associated with the minor A allele for renin (REN) rs2368564 and the major TT genotype for angiotensinogen (AGT) rs699. The heterozygous genotype for AGT rs4762 acts as a protective marker. A minor A allele for angiotensin II type 2 receptor (AGTR2) rs1403543 is associated with a risk of restenosis in people under 65 years old. Among patients with the early ISR, heterozygotes for angiotensin II type 1 receptor (AGTR1) rs5186 are more frequent, as well as A allele carriers for AGTR2 rs1403543. A minor homozygous genotype for REN rs41317140 and heterozygous genotype for aldosterone synthase (CYP11B2) rs1799998 are predisposed to the late restenosis. Thus, to choose the effective treatment tactics for patients with coronary artery disease, it is necessary to genotype patients for the RAAS polymorphisms, which, along with age and clinical characteristics, will allow a comprehensive assessment of the risk of the restenosis development after stenting.
The cerebral endothelium is an active interface between blood and the central nervous system. In addition to being a physical barrier between the blood and the brain, the endothelium also actively regulates metabolic homeostasis, vascular tone and permeability, coagulation, and movement of immune cells. Being part of the blood-brain barrier, endothelial cells of the brain have specialized morphology, physiology, and phenotypes due to their unique microenvironment. Known cardiovascular risk factors facilitate cerebral endothelial dysfunction, leading to impaired vasodilation, an aggravated inflammatory response, as well as increased oxidative stress and vascular proliferation. This culminates in the thrombo-inflammatory response, an underlying cause of ischemic stroke and cerebral small vessel disease (CSVD). These events are further exacerbated when blood flow is returned to the brain after a period of ischemia, a phenomenon termed ischemia-reperfusion injury. Purinergic signaling is an endogenous molecular pathway in which the enzymes CD39 and CD73 catabolize extracellular adenosine triphosphate (eATP) to adenosine. After ischemia and CSVD, eATP is released from dying neurons as a damage molecule, triggering thrombosis and inflammation. In contrast, adenosine is anti-thrombotic, protects against oxidative stress, and suppresses the immune response. Evidently, therapies that promote adenosine generation or boost CD39 activity at the site of endothelial injury have promising benefits in the context of atherothrombotic stroke and can be extended to current CSVD known pathomechanisms. Here, we have reviewed the rationale and benefits of CD39 and CD39 therapies to treat endothelial dysfunction in the brain.
Cancer development and progression are extremely complex due to the alteration of various genes and pathways. In most cases, multiple agents are required to control cancer progression. The purpose of this study is to investigate, using a mouse model, the synergistic interactions of anti-cancer agents, 1'-S-1'-acetoxychavicol acetate (ACA), Mycobacterium indicus pranii (MIP), and cisplatin (CDDP) in double and triple combinations to treat chemo-sensitize and immune-sensitize breast cancer. Changes in tumor volume and body weight were monitored. Organs were harvested and stained using hematoxylin-eosin for histopathological assessment. Milliplex enzyme-linked immunosorbent assay (ELISA) was performed to determine cytokine levels, while immunohistochemistry (IHC) was conducted on tumor biopsies to verify systemic drug effects. In vivo mouse models showed tumor regression with maintenance of regular body weight for all the different treatment regimens. IHC results provided conclusive evidence indicating that combination regimens were able to down-regulate nuclear factor kappa-B activation and reduce the expression of its regulated pro-inflammatory proteins. Reduction of pro-inflammatory cytokines (e.g., IL-6, TNF-α, and IFN-ɣ) levels were observed when using the triple combination, which indicated that the synergistic drug combination was able to significantly control cancer progression. In conclusion, ACA, MIP, and CDDP together serve as promising candidates for further development and for subsequent clinical trials against estrogen-sensitive breast cancer.