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Department of Production and Characterization of Novel Foods, CIAL – Research Institute in Food Science (UAM+CSIC), C/Nicolas Cabrera 9, Campus de Cantoblanco, Universidad Autonoma de Madrid, 28049 Madrid, Spain. E-mail: firstname.lastname@example.com
First published on 8th November 2017
Cholesterol levels are strictly regulated to maintain its homeostasis; therefore, if it is not absorbed with the diet, the cholesterol biosynthetic pathway is enhanced and vice versa. Nowadays, the commonly prescribed therapeutic treatments for hypocholesterolemic patients are targeted toward the reduction of both cholesterol intestinal absorption and/or its endogenous biosynthesis. But, when hypercholesterolemia is still moderate the consumption of food products with cholesterol-lowering capacities is more desirable than using drugs. Marketed foods supplemented with hypocholesterolemic compounds are only inhibiting mechanisms for cholesterol absorption (i.e. phytosterols and cereal β-glucans). However, certain fungal extracts obtained from edible mushrooms might be able to modulate cholesterol levels by both strategies, pharmaceutical drugs and functional foods. In vitro and in vivo studies indicated that fungal sterols down-regulated genes involved in cholesterol homeostasis (such as Srebf2 and Nr1h4 (FXR)) and other specific mushroom extracts (β-glucans and other water-soluble compounds) also stimulated transcriptional profiles similar to simvastatin or ezetimibe (two hypocholesterolemic drugs). These and other observations suggested that the hypocholesterolemic effect of mushroom extracts could be due to transcriptional and post-transcriptional modulations besides other indirect effects.
CVD risks are influenced by genetic factors such as specific tendencies to obesity, hypertension, etc., and gender or age; however, many risk factors are also modulated by lifestyle habits such as smoking, sedentary/involved in sports or in-/adequate diets2 and this is the reason that CVDs are considered as multifactorial diseases.
Several biomarkers are used to evaluate the real individual CVD risk, i.e. elevated homocysteine, coronary artery calcium-phosphate calcification or elevated fibrinogen blood level but, the most useful marker for CVD risk is the LDL (low-density lipoprotein)-cholesterol concentration in serum, one of the major transport cholesterol complexes. LDL-cholesterol constitutes 60–70% of total serum cholesterol and a blood concentration higher than 200 mg dL−1 is considered, nowadays, as a risky level.3,4
Due to the increasing interest of modern society for natural products as agents able to decrease the consumption of pharmaceutical drugs, new food products with high levels of hypocholesterolemic compounds are being explored. Nowadays, the cholesterol-lowering effect of compounds such as plant sterols (phytosterols, phytostanols and derivatives);5,6 dietary fibers such as β-glucans or chitosans;7 peptides such as those derived from soy proteins8 or bovine milk β-lactoglobulins9 have been studied and therefore, several functional food products supplemented with these compounds are available at the supermarkets.10–14
Some edible mushroom species are able to lower cholesterol levels in serum since they are a good source of fungal sterols (with high structural similarity to phytosterols), dietary fibers such as β-glucans and chitins (fungal polysaccharides), β-lactones, specific proteo/glucan complexes or compounds such as eritadenine.10 Recent studies pointed out that ergosterol-enriched extracts and other fungal compounds were also able to modulate cholesterol-related gene expression in animal intestine and liver.15
Therefore, in this work, the molecular events occurring during cholesterol synthesis and absorption are reviewed analyzing the influence of some hypocholesterolemic compounds obtained from edible mushrooms.
Cholesterol molecules from diet, bile, intestinal secretions and desquamated cells together with the bile secreted compounds such as phospholipids (lecithin) and bile acids (salts of taurocholic and deoxycholic acids, etc.) form small emulsified droplets. Then, pancreatic lipase, phospholipase A2 and cholesterol esterase transform the emulsified particles into a series of colloidal structures including vesicles, micelles or dietary mixed micelles (DMM).10
NPC1L1 is involved in this transmembrane sterol efflux due to a sterol-sensing domain (SSD) and it is co-localized at the cellular and intracellular vesicular membranes. The distribution of non-esterified cholesterol determines the main location of NPC1L1 proteins. At low intracellular concentrations, NPC1L1 will be mostly exposed at the brush-border enterocyte membrane and it will be translocate inside the cell at high levels of non-esterified cholesterol.24 In humans, NPC1L1 genes are not only expressed in the enterocytes of the small intestine but also in the liver where they are expressed in large amounts. Human hepatocytic NPC1L1 protein is located at the canalicular membrane facilitating the uptake of newly secreted biliary cholesterol and therefore, showing a role similar to intestinal NPC1L1.20 The transcriptional regulation of NPC1L1 has not yet been elucidated but, it seems to be influenced by sterol regulatory element-binding protein or SREBP2 (encoded by SREBF2 gene in humans) that are sensors activating different answers depending on intracellular cholesterol concentrations. At low cholesterol levels, SCAP (integral membrane protein) goes along with SREBP2 from the endoplasmic reticulum (ER) to the Golgi body (GB) for subsequent processing and activation. In contrast, at high or enough cholesterol levels the SCAP-SREBP2 complex is retained by INSIG proteins (Insulin induced gene 1 protein located in the ER membrane) to avoid SREBP2 maturation impairing its transcription.25 Moreover, other reports indicated PPARδ (peroxisome-proliferator-activated receptor δ) as another NPC1L1 modulator since down-regulation of the cholesterol transporter has been induced by PPARδ activation in mice.11
Intracellular non-esterified cholesterol concentrations could also be modulated by ATP-binding cassette (ABC) transporters such as ABCG5/ABCG8 and ABCA1, located respectively at apical and basolateral enterocyte sides. ABCG5 and ABCG8 proteins, expressed in the liver and small intestine, are involved in the reverse cholesterol transport (RCT) of sterols, from the intracellular environment to the lumen. Independent expression of both genes is necessary for the proper function of this heterodimer.26 Over-expression of the ABCG5/8 heterodimer increases non-esterified cholesterol excretion to the lumen reducing its internal concentration and consequently inducing activation of the cholesterol synthesis rate.27 In the small intestine, ABCG5/8 gene expression seems to be regulated by an LXR-dependent member of the nuclear receptor family named RXR (retinoid X receptor)26–28 while in the liver, the heterodimer is directly modulated by LXR.30 Apparently, the latter receptor, along with PPARδ, is also a modulator of ABCA1 expression.10,26 ABCA1 is a transport protein directly involved in the excretion of exceeding non-esterified cholesterol into HDL. LXR agonist administration or high cholesterol concentrations in the cytosol stimulate a direct effect on the transcriptional modulation of these transport proteins although its specific regulatory mechanisms remain still unclear. A protein–protein interaction with another transcription factor affecting the transcription rate of the ABC proteins has been hypothesized.29
Due to confusions noticed in several publications, it is worth defining the specific role of two widely mentioned enzymes involved in the transferring of acyl groups within the cholesterol metabolism. Acetyl-Coenzyme A transferase (ACAT) and sterol O-acyltransferase (SOAT) are two enzymes belonging to the acyltransferase family; however, they do not catalyze the same reaction (Table 1). ACAT isoforms (ACAT1/ACAT2) are responsible for the reversible formation of acetoacetyl-CoA from two molecules of acetyl-CoA. ACAT1 and ACAT2 genes are respectively located in chromosome 11 (11q22.3) and chromosome 6 (6q25.3).30,31 However, SOAT isoforms (SOAT1/SOAT2) catalyze the formation of fatty acid-cholesterol esters from cholesterol and acyl-CoA molecules and are encoded by two genes located at loci 1q25.2 and 12q13.13.32,33 Then, since in some of the mentioned works the text wrongly referred to ACAT when it should indicate SOAT, personal advice (AvS mark) was given pointing attention where required.
Once non-esterified cholesterol reaches the cytoplasm it becomes a substrate of ACAT2(AvS), an integral membrane protein mainly expressed in the small intestine and liver. ACAT2(AvS) decreases cytoplasmic amounts of non-esterified cholesterol promoting its esterification and integration into the ER pre-chylomicrons modulating the cholesterol transmembrane absorption rate from the intestinal lumen.34 It also plays an important role in maintaining the dynamic equilibrium (homeostasis) between free-cholesterol and esterified-cholesterol.35 More than 50% of sterol esterification within the enterocytes is carried out by ACAT2(AvS) with a higher affinity for cholesterol esterification rather than for other non-cholesterol sterols.
The internal ER triglyceride re-assemblage is carried out by several enzymes such as lysophosphatidate acyltransferase (AGPAT), phosphatidate phosphatase (LPAP), 2-acylglycerol O-acyltransferase 2 (MGAT2) and diacylglycerol O-acyltransferase 1 (DGAT1). Then, esterified-cholesterol together with the triglycerides is packed into pre-chylomicrons by the microsomal triglyceride transfer protein (MTTP) and apolipoprotein B48 (an isoform derived from the APOB gene characteristic of enterocytes). MTTP and APOB48 proteins constitute an active heterodimeric complex linked by ionic interactions with a particular feedback assembly and secretion system i.e. the larger the APOB48 subunit is, the lower binding capacity with MTTP is noticed and less APOB48 is secreted. Therefore, the APOB48-MTTP binding process plays an important role in lipoprotein biogenesis.36–38
The combined regulatory effect of NPC1L1, ABCA1, ABCG5/8 and ACAT2(AvS) activities play a critical role in modulating the amount of esterified cholesterol that will be integrated in the pre-chylomicrons with the assistance of apolipoporotein B48 (APOB48), microsomal triglyceride transfer protein (MTTP) and diacylglycerol-O-acyltransferase (DGAT1/2).39
Once prechylomicrom structure is assembled, it is further transformed into chylomicrons in the GB and excreted by exocytosis into the lymph system through the enterocyte basolateral membrane. On the other hand, the non-esterified cholesterol remaining in the cytoplasm could bind to the APOA1 protein for further transport to the lymphatic vessels leading to nascent HDL lipoproteins. Thus, HDL as well as chylomicrons are released free into the bloodstream and transported to the liver and peripheral organs such as adrenal glands.11
3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is considered the key enzyme of cholesterol synthesis although the activities of many enzymes involved in the biosynthetic pathway, such as ACAT2, hydroxymethylglutaryl-CoA synthase (HMGCS), delta24-sterol reductase (DHCR24), farnesyl-diphosphate farnesyltransferase (FDFT1/SQS) or 7-dehydrocholesterol reductase (DHCR7), are also susceptible to modulation. Recently, Gill et al. (2011) suggested that squalene monoxygenase (SQLE) might be the second critical modulatory point despite its lower specificity within cholesterol metabolism compared to HMGCR (after in vitro experiments).41
However, other enzymes such as SQS and SQLE are also gaining attention as a potential stop-point of cholesterol biosynthesis because SQS is involved in the transformation of farnesyl pyrophosphate into squalene, being the first specific reaction at the branching point between sterol and non-sterol biosynthesis. The SQS transcriptional product and protein are modulated by cholesterol since low levels of this sterol activate the SQS promoter by sterol regulatory element binding proteins (mostly SREBP2). In contrast, SQS mRNA concentration decreases in response to cholesterol excess. SQLE is a monoxygenase (also named squalene epoxidase) that catalyzes the next step after squalene transformation by SQS, a crucial oxygenation process yielding squalene 2,3-epoxide (Fig. 2). Modulation of SQLE activity seems also cholesterol-dependent and apparently regulation is mediated by proteosome activity.41
HMGCR includes seven domains inserted in the ER (endoplasmic reticulum) membrane with an active carboxyl chain located at the cytosol. HMGCR is ubiquitously expressed i.e. in immune (white blood cells), nervous, muscle, small intestine or reproductive (ovary cells) human tissues.42 HMGCR transcription and degradation depends on a sterol/non-sterol feedback regulation but is not directly controlled by the cholesterol molecule. Under cholesterol depleted conditions, the SREBP-SCAP complex is formed in the ER membrane (without any INSIG interaction); then, it is transported to GB where SREBP is activated by proteolytic events facilitating its translocation into the nucleus to activate HMGCR transcription (Fig. 3a). In contrast, under cholesterol exceeding conditions, high amount of oxysterols are synthesized by the mitochondrial sterol 27-hydroxylase (CYP27A) and several mechanisms reducing HMGCR transcriptional rates are activated.43 HMGCR regulatory mechanisms could be classified as INSIG-dependent (modulating at transcriptional and post-transcriptional levels), or INSIG-independent.
INSIG-dependent HMGCR regulation mechanisms (Fig. 3b):
- INSIG can disrupt SREBP activation by binding to SCAP when high sterol concentrations are noticed in the cytosol. Once the INSIG-SCAP complex is formed, SCAP is structurally altered impairing SREBP recognition and stopping the assembly of the SREBP-SCAP complex for its further transport from the ER (endoplasmic reticulum) to the GB (Golgi body). In consequence, SREBP is not translocated to the nucleus and HMGCR transcription is inactivated.17,44
- Under similar conditions, INSIG can also bind to the N-terminal region of HMGCR and conjugate it with ubiquitin. Ubiquitation is carried out by gp78 (membrane-bound ubiquitin E3 ligase) assisted by Ubc7 (an E2 ubiquitin conjugating enzyme) providing active ubiquitins and a few other enzymes. Then, ubiquitinated HMGCR is released into the cytosol and subsequently proteosome-degraded with the participation of p97/VCP (ATPase associated to the membrane).
However, the presence of high levels of sterols is not mandatory for the ubiquitination process but it stimulates HMGCR degradation by enhancing INSIG-HMCR bindings.44
INSIG-independent HMGCR regulation mechanisms (Fig. 3b):
- HMGCR activity could be also modulated in situations of cellular stress (low ATP levels) by AMP kinase (AMP-activated protein kinase). In this case, HMGCR is inactivated by a serine phosphorylation due to AMP kinase activity. It is a reversible reaction and HMGCR can also be activated by protein phosphatase 2A (PP2A).44,45
- Non-sterol isoprenoids might also modulate HMGCR translation by a mechanism still unclear, according to Burg et al. (2011).44
RCT is a derivative branch of the hepatobiliary pathway. Lipoproteins such as HDL or LDL make available cholesterol for hepatic absorption as esterified or non-esterified molecules. Esterified cholesterol is transformed by the hepatic cholesteryl ester hydrolase (NCEH1) into the non-esterified form after hydrolysis of the ester linkage. Thus, the generated forms are directly excreted through the ABCG8/5 heterodimer protein or transformed into bile salts.46 CYP7A1 (cholesterol 7-alpha-monooxygenase) is the enzyme responsible for cholesterol transformation into primary bile salts (cholic and chenodeoxycholic acid) within the neutral bile acid pathway in the liver. Synthesized bile salts are secreted to the bile canaliculus by the bile salt export pump (BSEP) or the multidrug resistance-associated protein 2 (MRP2) and become part of bile fluids. Recent in vivo studies have suggested the involvement in the RCT of other cholesterol transporters such as NPC1L120,47 although its role in biliary cholesterol excretion has not yet been elucidated. But, it might involve adjustments in cholesterol balance to avoid excessive loss of the metabolite through the intestinal track.
TICE has been suggested as an alternative cholesterol excretion mechanism where the sterol is directly eliminated from blood through the intestinal mucosa and excreted via the feces.17 The hypothesis was drawn after unexpected results obtained by several authors that showed an unwarranted balance between cholesterol inputs and outputs in mouse models. They showed a higher amount of fecal cholesterol than the sum of dietary intake and biliary secretion48 or an unaltered cholesterol excretion rate in knockout NPC1L1 mice with a decreasing of 90% biliary excretion.49
These results questioned the complete RCT classical concept50 as well as the contribution of biliary or non-biliary cholesterol to its RCT excretion.51 Although the involvement of several membrane transports in TICE have been studied (i.e. SR-BI,52 NPC1L1,53 APOA1,54 LDLR or ABCG5/855), it is still unknown whether TICE is carried out through basolateral or apical transporters or whether HDLs are involved;56 therefore, the mechanism is not yet elucidated. There is also scientific controversy about the importance of TICE; some authors suggest that it could be only a compensatory cholesterol excretion mechanism in case of biliary cholesterol depletion but, other authors indicated it as the main mechanism in cholesterol excretion. Van der Velde et al. (2007, 2010) estimated the contribution of TICE as 70 and 30% of total cholesterol were excreted in mice and humans, respectively.48,56
The non-esterified cholesterol eliminated by ABCA1 through the basolateral membrane of enterocytes is bound to APOA1 generating nascent-HDL and the esterified cholesterol is similarly assembled with APOB48 in ER synthetized pre-chylomicrons further transformed into chylomicrons in GA and excreted by exocytosis to the intracellular space. Therefore, both structures become cholesterol transporters and they distribute it via the bloodstream to the rest of the organism.
Nascent HDL is transformed into mature HDL by the accumulation of non-esterified cholesterol molecules secreted by hepatocyte and enterocyte ABCA1 protein. Once mature HDLs are formed, some cholesterol molecules are esterified by the action of lecithin–cholesterol acyltransferase (LCAT) along with their blood transport. Esterified and non-esterified cholesterol are detected by SR-B1 located in the basolateral membrane of cells from the liver, small intestine or gland adrenals allowing the incorporation of esterified cholesterol inside the cell. Intracellular esterified cholesterol is transformed into non-esterified cholesterol (by SOAT action) and HDL is turned into LDL. The non-esterified cholesterol can be used for bile salt synthesis and their further intestinal secretion by BSEP or to be directly excreted via ABCA1 (basolateral membrane) and ABCG5/ABCG8 (apical membrane) activity. In turn, secreted non-esterified cholesterol again could be attached to nascent HDL to create mature HDL and continue with cholesterol transport.
LDL molecules are recognized by the liver, intestine and adrenal gland LDLRs as esterified cholesterol suppliers. LDL particles can be generated not only by VLDL transformation (by SR-B1 activity) but also by the addition of esterified cholesterol to APOB100 (apolipoprotein isoform characteristic of hepatocytes) leading to very low density lipoproteins (VLDL). These structures are secreted to the bloodstream by hepatocytes. VLDL lipids are used by the muscle and peripheral tissues as they pass through the bloodstream generating IDL and LDL by lipoprotein lipase (LPL) activity. Moreover, the esterified cholesterol of chylomicron structures is also recognized by LDLR providing to the hepatocyte those cholesterol molecules assembled in enterocytic ER after the digestion process.
The complex cholesterol biosynthesis and absorption engineering and the multifactorial regulation system make the control of cholesterol metabolism a difficult challenge for the scientific community, particularly because some of the involved compounds are also intermediates of other metabolic pathways i.e LXR modulates ABCG5/8 activity but also DIO1, a selenoprotein involved in thyroid hormone metabolism. However, these factors also make it a flexible system that could be modulated from different critical points.
The intake of dietary fibers (DF) from cereals etc., increases cholesterol and bile acid excretion rates lowering their bioavailability because they might act as bile acid scavengers. This mechanism impairs bile acid re-absorption stimulating the hepatic synthesis of new bile acids from cholesterol reducing its blood concentration10,60,61 although for particular β-glucans, modulation of cholesterol-related gene expressions have been also noticed.62
Plant sterols (phytosterols and phytostanols) are considered direct cholesterol competitors for their incorporation into DMMs because of their structural similarity and due to the limited capacity of DMMs to solubilize lipophilic and water-insoluble molecules.63–65 Some reports also suggested that phytosterols might modulate SOAT activities although it still remains partially unclear. Cholesterol enterocytic esterification by ACAT(AvS) was decreased by sterol competition although the enzyme showed lower esterification efficiency for plant sterol than cholesterol. Other authors suggested that plant sterols influenced MTTP and APOB48 lipoprotein in vitro models.65
Compounds such as novel amino β-lactam derivates66 or curcuminoid polyphenols were described as potential NPC1L1 inhibitiors by showing high binding affinity or acting by indirect influence on SREBP1.37 ABCG5/8 postranscriptional regulations were also described for spironolactones or polyphenols from Aronia melanocarpa.67,68
Other compounds from natural sources have been reported as ACAT(AvS) inhibitors reducing cholesterol esterification rates such as alkamides from Piper nigrum,69 shikonin derivatives from Lithospermum erythrorhizon,70 an isoprenyl flavonoid identified as grabol from licorice roots,71 ursolic acid (indirect inhibition via (PPAR)-α activation)72 or flucoxanthin from marine plants.73
Ergosterol (ergosta-5,7,22-trien-3β-ol) is considered the major fungal sterol (from 53% up to 80% of the fungal sterols, w/w) followed by ergosterol derivatives such as ergosta-5,8,22-trien-3-ol, ergosta-7,22-dien-3-ol, ergosta-5,7-dien-3-ol and ergosta-7-en-3-ol (fungisterol).74 However, their precise concentration in the different mushroom strains was dependent on environmental conditions (developmental stage, cultivation conditions, etc.) or species. For instance, Chantharellus cibarius and Craterellus cornucopioides showed almost exclusively ergosterol, while other species showed more ergosta-5,7-dienol than fungisterol (Lyophyllum shimeji or Pleurotus ostreatus). Flammulina velutipes contained high amounts of ergosta 5,8,22-trien-3β-ol.10
Apparently, the hypocholesterolaemic activities of fungal sterols were mainly due to their structural similarity with cholesterol as noticed for plant sterols. Therefore, these compounds such as phytosterols and phytostanols were able to hinder cholesterol incorporation into DMMs. Ergosterol-enriched fractions obtained using green technologies, such as supercritical fluid extraction (SFE), were more effective than β-sitosterol in displacing cholesterol from DMMs during an in vitro digestion model.59 Moreover, fungal sterol-enriched extracts also showed HMGCR inhibitory activities in vitro75 and ergosterol was also considered as a competitive inhibitor of C24-reductase due to its double bond at C-22 in the side chain of its structure (as also noticed for stigmasterol and brasicasterol). C24-Reductase is another enzyme involved in the cholesterol biosynthetic pathway down-stream from HMGCR.76
Fungal β-glucans showed different molecular structures from cereal polysaccharides. Their branching profiles are (1→3) and (1→3)(1→6) conferring on them many biological activities such as antitumoral, antioxidant, immunomodulatory, etc. apart from hypocholesterolaemic properties.10,77,78 Some reports suggested that those bioactive properties are due to their different tridimensional configuration depending on their monomer composition, branching degree or conformation while other authors considered their water solubility (that is also partially dependent on their glycosidic linkages) a more relevant factor, considering those water-insoluble as responsible for the cholesterol lowering activities by impairing cholesterol absorption. Moreover, other publications indicated β-glucan degradation products (generated i.e. after a digestion process) as hypocholesterolemic molecules more bioactive than their larger precursors.10,79 However, until now the precise structural requirements for the observed hypocholesterolemic action of fungal β-glucans remain unclear awaiting further studies.
β-Glucans from a large number of mushroom species have been studied such as lentinan from shiitake mushrooms (Lentinula edodes), schizophyllan from Schizophyllum commune, grifolan from Grifola frondosa, and many others from Agaricus blazei, Ganoderma lucidum (a medicinal mushroom consumed as a nutraceutical or dietary supplement), and P. ostreatus as well as (1→3) β-glucans and glucuronoxylomannans from Auricularia polytricha and Tremella fuciformis.80,81 All showed different conformations, and solubilities and therefore biological properties.
Chitin, a special type of water-insoluble β-(1→4)-glucan including N-acetylglucosamine monomers, is also considered a hypocholesterolaemic polysaccharide while its derivative, named chitosan, was more studied because of its antitumor and immunomodulation activities.10,77 Several polysaccharide fractions (containing high percentages of fungal β-, α-glucans and chitooligosacharides among others) from L. edodes, P. ostreatus and A. bisporus extracted by using pressurized technologies (PSE) showed bile acid binding capacities in vitro similar to those shown by cereal fibers82 indicating that this might impair cholesterol absorption as previously suggested for plant β-glucans.
According to suggestions by Gunde-Cimerman et al. (1993, 1995), lovastatin an inhibitor of HMGCR (3-hydroxy-3-methylglutaryl-Coenzime A reductase) was naturally present in some Pleurotus spp. fruiting bodies such as P. ostreatus or P. eryngii83,84 and in other species such as A. bisporus or B. edulis. However, recent studies did not detect any statin in mushroom species showing interesting HMGCR inhibitory activity pointing to some water soluble polysaccharides and proteoglucans as responsible for the noticed in vitro inhibition.10,75
HMGCR inhibition was also mediated by an AMP kinase via phosphorylation and green and black tea polyphenols induced a direct increase of HMGCR phosphorylation possibly via AMP kinase phosphorylation. Their precise mechanism of action is still unclear but, it seems to involve the activation of regulatory factors such as PPAR.85,86
Only a few compounds are still nowadays indicated as potential inhibitors of SQS such as resveratrol from wine87 as well as zaragozic acids isolated from the liquid broth of certain ascomycetes.88
24(S),25-Epoxycholesterol was also pointed out as an inhibitor of DHCR24 activity. This enzyme catalyzes the transformation of desmosterol into cholesterol. The inhibitor did not modify DHCR24 protein levels, but increased desmosterol accumulation decreasing cholesterol levels in in vitro studies due to its structural similarity with desmosterol.89
Mushrooms such as Pleurotus ostreatus, Grifola frondosa and Hypsizigus marmoreus were able to differently modulate the gene expression patterns of livers of mice fed with each mushroom (10–14%) for 4 weeks. Triglyceride levels in the liver and plasma decreased in the mice fed with P. ostreatus compared with those in the control group. Moreover, liver cholesterol decreased while plasma total cholesterol increased probably due to HDL values that were also increased. Cholesterol in the liver was lower in the group fed with G. grondosa than in the control group but no changes were found in the H. marmoreus-fed group. DNA microarray analysis of the livers revealed that CTP1A and FABP families were upregulated in the P. ostreatus-fed group, which were considered to promote lipid transport and β-oxidation. In the G. frondosa-fed group, not only the gene involved in signal transduction of innate immunity via TLR3 and interferon but also virus resistance genes (such as MX1, RSAD2 and OAS1) were upregulated.90
Administration of Agaricus brasiliensis (also known as Agaricus blazei) to hypercholesterolemic albino Fischer rats during 6 weeks lowered cholesterol levels in serum and induced significant changes in the expression of cholesterol-related genes. HMGCR mRNA expression was not influenced but LDLR upregulation was noticed together with the upregulation of CYP7A1, the rate-limiting enzyme for bile acid synthesis, and mRNA levels of the ABCG5/8 carriers. These increases were accompanied by a significant increase in the content of cholesterol excreted in the faeces and by a concomitant increase in NR1H3 (LXR) mRNA levels. However, in this case PPAR-α was not significantly upregulated compared with levels noticed in the hypercholesterolemic control group.91
In a few reports specific extracts, and not the whole fruiting body, were studied. For instance, ethanol (95%) extracts obtained from P. ostreatus were administered to hypercholesterolemic male Wistar rats for 14 days and a reduction of triglycerides and HDL cholesterol was noticed in the plasma. Moreover, the extract was able to upregulate the genes that were downregulated after hypercholesterolemia induction such as those related to fatty acid oxidation such as acyl CoA oxidase (ACO) and synthetase (ACS) together with carnityl palmityl transferase-1 (CPT-1) and PPAR-α. They also downregulated genes related to fatty acid biosynthesis and cholesterol metabolism (FAS fatty acid synthase, SREBF1, APOC3) to similar expression than during normocholesterolemia.92
Hericium erinaceus hot water and ethanol extracts were administered, together with a high-fat diet, to C57BL76J mice for 4 weeks. Incorporation of the extracts in the diet resulted in a significant decrease in body weight, fat and serum and hepatic trigliceride levels compared to control. The ethanol extract acted as an agonist of PPAR-α since it was able to up-regulate mRNAs usually modulated by PPAR-α (ACAT, ApoA1, LPL or SREBP1) in spite of the fact that the PPAR-α expression itself did not change.93
An extract obtained from the cauliflower mushroom (Sparassis crispa) significantly enhanced hepatic cholesterol catabolism when administered to male Sprague-Dawley rats fed a cholesterol-rich diet for 4 weeks because they were able to up-regulate CYP7A1 mRNA expression concomitant with HMGCR downregulation after. Additionally, the extract supplementation resulted in cholesterol and bile acid fecal excretion.94
Ergosterol was able to regulate sterol regulatory element binding protein (SREBP) cleavage in yeast as response to cellular oxygen levels95 suggesting that it might also modulate these elements in mammalians. Studies carried out using cell cultures revealed that DMMs isolated from the in vitro digestion of ergosterol and extracts containing ergosterol mixed with cholesterol did not influence the transcriptional levels of SREBF1 differently than when only cholesterol was administered. Moreover, only a slight inhibition was noticed for SREBF2 mRNA of Caco2 cells when treated with an ergosterol-containing extract but not with ergosterol itself. However, other cholesterol related genes were also overexpressed such as LDLR (when an ergosterol-enriched extract from Agaricus bisporus was applied).15 When the lower compartment of the Caco2 cells supplemented with the sterol-containing digested extracts were added to HepG2 cells, higher modulation of genes related to lipid metabolism was noticed than those more directly related to cholesterol homeostasis such as DGAT2 upregulation.
When similar extracts were given for 4 weeks to C57BL/6JRj mice previously fed for 4 weeks with hypercholesterolemic diets, inhibition of SREBF2 and NR1H4 (gene enconding FXR) were noticed in the jejunum up to similar levels than hypocholesterolemic drugs such as ezetimibe and simvastatin.15 This downregulation was due to their ergosterol content because supplementation with purified ergosterol induced similar modulation. FXR is the farnesoid X receptor, a nuclear receptor also involved in the regulation of cholesterol homeostasis.96 FXR is involved in the activation or inhibition of bile acid synthesis and transport acting as a sensor of their concentration.97 The extract induced the downregulation of DGAT2 in the liver as observed in cell cultures but upregulation of FDFT1 might be to compensate for the reduction of the ratio TC/HDL recorded. Reduction of the triglyceride levels was also noticed in the liver, perhaps because of the observed modulation of DGAT2 gene transcription. Moreover, this extract induced the overexpression of DIO1 mRNA in the jejunum, the tissue where cholesterol absorption is higher.98 Type I iodothyronine deiodinase is a selenoprotein encoded by the DIO1 gene and plays a major role in normal thyroid function, but it also indirectly influences cholesterol homeostasis by regulating LDLR gene expression. Increased DIO1 activity in the liver correlated with up-regulation of LDLR mRNA and lowering of TC, TG and LDL levels in serum.15 However, no significant effect was noticed by Gil-Ramirez et al., 201598 on the latter gene; perhaps the supplemented concentration was sufficient to stimulate DIO1 but it did not revert on LDLR readjustments. In contrast, ergosterol administered as a single compound induced the downregulation of DIO1 mRNA; therefore the effect of the ergosterol enriched extract could be due to some other sterol or compound present in the extract.98
However, when ergosterol containing extracts were incorporated into lard and the mixture was supplemented together with the diet to induce hypercholesterolemia, insignificant effects were noticed on any SREBP, other cholesterol regulatory nuclear receptor or genes involved in cholesterol metabolism except for ABCG5 and 8 mRNAs expression in cecum that were up-regulated. Perhaps the addition of the extract as an ingredient inside a lipid matrix prevented their proper bioavailability (because it was also unable to lower cholesterol levels in serum, HDLc or LDLc) or perhaps the beneficial properties of ergosterol were not sufficient to overcome the detrimental effect of lard and the hypercholesterolemic diet when administered simultaneously.99
The transcription of some ABC transporters (ABCG5/8 etc.) was induced by the LXR factor in enterocytes (but not in hepatocytes)65 although, there is a controversy about its influence on other transporters (ABCA1). Oxysterols such as 22(R)-hydroxycholesterol, 24(S),25-epoxycholesterol or 27-hydroxycholesterol, etc., are considered as endogenous natural LXR agonists; however, plant sterol derivatives showed higher LXR agonist activity. Brasicasterols from unicellular algae and Brassica sp. (i.e. rapeseed) induced large variations in the gene expression of ABC transporters due to their ability to act as the LXR factor in mice. Moreover, sitostanol (in mice), sitosterol (in Caco2 cells) and a few 4-desmethylsterol derivatives were also able of inducing ABCA1 up-regulation using their LXR agonist activity.5,11,13,65 However, no direct effects of phytosterols or fungal sterols were noticed on the transcriptional levels of the transporters because, on the one hand, in ABCA1 and ABCG5/8-deficient mice decreasing of cholesterol intestinal absorption was also noticed after phytosterol administration indicating that ABC transporters were not their direct targets. On the other hand, no significant overexpression or repression was noticed in hypercholesterolemic C57BL/6JRj mice treated with ergosterol-containing extracts.15
Other reports indicated that phytosterol/stanols modulated HMGCR expression via ACAT(AvS) inhibition. Apparently, lower ACAT(AvS) activity led to higher free cholesterol amounts, inhibiting the cholesterol biosynthetic pathway and HMGCR expression beside others. It also reduced chylomicron assembling, and promoted the back efflux of non-esterified sterols to the lumen.11,65,100 However, their influence on SOAT seemed to be by chemical inhibition more than by molecular modulation. Direct down-regulation of HMGCR mRNA (together with inhibition of the enzyme) was only noticed in organic extracts obtained from the Reishi mushroom (Ganoderma lucidum) containing oxygenated lanosterol derivatives. They also inhibited cholesterol synthesis in T9A4 hepatocytes and reduced total cholesterol in hamsters.101
Several in vitro studies pointed out that SREBP2, NPC1L1 and SR-B1 gene expression was modulated by plant sterols such as stigmasterol and β-sitosterol toward the reduction of cholesterol absorption. Surprisingly, HepG2 cells treated with these sterols showed simultaneous down-regulation of NPC1L1 and SR-B1 when an opposite effect on these two molecules could be expected. This modulation was not noticed when fungal sterols were applied to hepatic cells15 or when administered to mice together with a hypercholesterolemic diet.99 Studies using homozygous and heterozygous knockout mice (NPC1L1−/− and NPC1L1+/−) showed a lower cholesterol absorption in homozygous animals; however, heterozygous mice showed higher HMGCR mRNA levels in gut and liver tissues than wild-type animals without changes in ABC transporter expression rates. Authors explained their results by compensatory effects: difficulties for cholesterol absorption were compensated by a stimulation of endogenous cholesterol synthesis to maintain physiological plasma levels.102,103 Moreover, when the knockout mice were treated with plant sterols or ezetimibe, the wild-type and heterozygous animals showed a reduction in cholesterol and TG levels. Absorption of campesterol and β-sitosterol was reduced in NPC1L1+/− and almost absent in NPC1L1−/− mice indicating that certain modulation of NPC1L1 took place.102,103 However, no modulation of NPC1L1 expression was noticed in C57BL/6 mice fed a fungal sterol extract together with a hypercholesterolemic diet.99
Moreover, β-sitosterol addition to Caco2 cultures induced the down-regulation of HMGCR expression although this effect was not noticed in mice. Only sitosterolemic individuals showed reduction in HMGCR activity in the ileum; so apparently only large amounts of plant sterols or long-term 2% (w/w) plant sterol administration can induce such an effect.11,104
Other reports indicated that phytosterols might modulate the expression of other closely related genes such as those encoding the hepatic cholesterelogenic farnesyl phosphate synthase (FFPS), liver CYP7A1 (cytochrome P450 family 7 subfamily A polypeptide 1) or the annexin 2-caveolin 1 (ANXA2-CAV1) protein complex.11 Therefore, fungal sterols might be involved in the modulation of CYP7A1 noticed during the administration of the complete mushroom fruiting bodies to hypercholesterolemic mice.91
Fukushima et al. (2000)105 examined the effects of a fiber extract obtained from Agaricus bisporus on LDLR mRNA expression after feeding rats with cholesterol-free diets during 4 weeks. Despite a higher relative liver weight in the control group (cellulose supplementation) compared with those animals fed with the mushroom fiber extract as supplement, serum total cholesterol, VLDL and LDL concentrations in the control group were significantly greater than in animals fed the A. bisporus fiber. Added to this, once LDLR mRNA hepatic levels were analyzed, results showed that mushroom LDLR up-regulation was significantly higher than that induced by cellulose fiber supplement.105 One year later, the same group reported that maitake (Grifola frondosa) and enokitake (Flammulina velutipes) fiber extracts lowered total serum cholesterol levels by two mechanisms, by scavenging cholesterol molecules inducing their fecal excretion and by enhancement of LDLR mRNA expression in rat liver.106 However, no up-regulation was significantly noticed when rats were fed shiitake (Lentinula edodes) fiber extracts.
Dietary fibre fractions from P. ostreatus, L. edodes and A. bisporus fruiting bodies obtained as described by Jeurink et al. (2008)107 including a low α-glucan amount and high concentrations of chitin derivatives and other β-glucans82 were also added to Caco2 cells to study their effect on the most interesting cholesterol-related genes. The transcriptomic profile was studied after 1 and 24 h application and results indicated that the fiber extracts obtained from P. ostreatus were able to modulate the transcription of more genes related to cholesterol metabolism than the other two mushrooms studied at longer incubation times. Up-regulation of FDFT1 and NPC1L1 was noticed together with slight modulation of a few other mRNAs; therefore, in vivo experiments were carried out using P. ostreatus fiber extract using two different experimental settings. In the first one, C57BL/6J mice were firstly administered hypercholesterolemic diets and then, supplemented with the fiber extract (as palliative treatment) and in the second experiment, mice were simultaneously fed with hypercholesterolemic diet plus fiber extract (preventive treatment). Results indicated that their molecular responses were completely dependent on the supplementation setting. In the palliative setting, administration of the fiber extract reduced hepatic triglyceride levels and it might be because of the DGAT1 downregulation also recorded. In the preventive setting, the fiber extract modulated cholesterol-related gene expression similar to simvastatin and ezetimibe in the liver (i.e. by inhibiting FDFT1, NR1H3 (LXR) and NR1H4 (FXR) mRNA expression) although no changes in plasma and liver biochemical data were recorded.108 Later on, when a similar extract was applied in a higher concentration and mixed with lard, a hypocholesterolemic effect was noticed (as reduction of TC and LDLc levels) but no relevant modulation of cholesterol-related gene transcription was noticed despite the overexpression of NPC1L1 in mice liver and jejunum.99
The molecular modulations noticed for fungal β-glucans seemed to be different from other dietary fibers obtained from plants i.e. high viscosity oat or barley β-glucan extracts demonstrated their ability to down-regulate SREBF2 gene expression in intestinal cells (NCl-H716)62 but no with in vivo testing. However, according to Hu, Wang & Xu (2008) corn bran dietary fiber up-regulated the expression of other genes such as FXR in ileal cells or PPAR in liver.109
Hepatic HMGCR up-regulation was observed after the administration of hydroxylpropylmethycellulose or inulin-oligofructose (1:1) to animal models;110,111 however, barley β-glucan administration induced no changes in HMGCR expression.112 Jones (2008)112 noticed hepatic SREBF2 up-regulation when soluble dietary fiber (guar gum, a galactomannan) was administered to pigs. In consequence, LDLR expression was enhanced and reduced the LDLs from the bloodstream. Fungal fibers instead seemed to modulate LDLR mRNA expression directly.105,106 Hepatic ABCG5/8 gene expression was also enhanced by guar gum consumption leading a higher cholesterol efflux from the liver to the intestinal lumen.112 A similar enhancement together with ABCA1 mRNA was noticed in jejunum from mice administered P. ostreatus fibers (palliative treatment) but without significant cholesterol reduction.108
Other studies indicated that the scavenging of bile acids by plant fiber (β-glucans) activated CYP7A1 to convert cholesterol into new bile acids109,113 provoking a hepatic cholesterol decrease that up-regulated LDLR expression and reduced cholesterol blood levels. Due to these changes, the biosynthetic pathway was also activated via HMGCR up-regulation to compensate for the lack of hepatic cholesterol.113,114 However, the effect of fungal fibers on CYP7A1 is still, at the present, unknown.
Other studies were carried out using extracts containing water soluble polysaccharides from L. edodes. This fraction contained α- and β-glucans and fucomannogalactans that when digested (in vitro) were applied to Caco2 cell cultures and no significant effect was noticed on the modulation of cholesterol-related gene expression. But, when the lower compartment of the cell monolayer was applied to HepG2, modulation of some mRNAs was noticed after 24 h incubation. However, the modulatory pattern fitted more with a low cholesterol level response since enzymes such as HMGCR, FDFT1 and ACAT1 were overexpressed; perhaps the HMGCR inhibitory activity showed by this extract induced a posttranscriptional cholesterol reduction that activated the biosynthetic pathway after 24 h.115 Later on, when this extract was administered to normocholesterolemic mice for 4 weeks similar overexpression of HMGCR (in liver) and FDFT1 (in jejunum) was noticed. However, when the extract was administered to hypercholesterolemic mice the modulation profile showed similarities with those generated after the administration of ezetimibe, simvastatin or both such as for instance the down-regulation of FDFT1 (in the ileum), SREBF2 or NR1H4 (in the jejunum) or the up-regulation of ABCG8 or ACAT1 in the jejunum.
Other polysaccharides extracted from 3 different strains of Pleurotus tuber-regium, when administered to obese-diabetic rats for 8 weeks also showed a hypolipidemic effect that was associated with up-regulated liver PPAR-alpha mRNA expression and protein levels. Moreover, hyperglycemia was also attenuated by polysaccharides; the elevated serum total cholesterol, triglycerides and low-density lipoprotein (LDL) concentrations were controlled, and parallel restoration of decreased high-density lipoprotein (HDL) levels was noticed after their supplementation.116
Water extracts obtained by PWE (pressurized water extractions) from normal and selenium-enriched A. bisporus were tested in vitro as HMGCR inhibitors. Selenium supplementation enhanced the inhibitory activity of statins and therefore, the latter extracts might improve their HMGCR inhibitory capacity. However, no significant differences were found; both similarly inhibited the enzyme only if the extracts were not thermally treated. When the extracts were applied to HepG2 cells to study their effect at the molecular level they also induced similar responses in most of the cholesterol-related genes. However, after 1 h application overexpression of LDLR mRNA was noticed in non Se-fortified extracts. After 24 h LDLR mRNA was downregulated together with FDFT1. Similar downregulation of the SQS mRNA was also noticed in Se-fortified extracts indicating that non-thermal water extracts from A. bisporus could inhibit transcription of this enzyme that is also a key enzyme downstream the cholesterol metabolic pathway.121 Reduced expression of GPX3 (glutathione peroxidase, a selenoprotein largely influenced by the presence of selenium in the media) was shown to increase the cell-mediated oxidation of LDL and selenium supplementation (1 ppm) also induced downregulation of APOB and HMGCR expression during hypercholesterolemia in rat models. However, no significant modulation of GPX398 and APOB121 expression was noticed either in non- or Se-fortified extracts; only a slight up-regulation of HMGCR after 1 h for non-fortified extracts and after 24 h for Se-fortified ones perhaps to compensate the inhibition of the FDFT1 transcription was noticed.121
This journal is © The Royal Society of Chemistry 2018 |


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I'm pleased and excited to be able to display the badge above - which was awarded to my debut novel Never Coming Home for achieving the final of the Colorado Romance Writers annual Awards of Excellence, in the category romantic suspense. The winners of the awards will be announced later in the year. Do I have hopes? Of course - but I know that all those other books in my category will have as much heart- stopping romance, and be as thrilling, as their authors could make them. The competition will be strong, and the ultimate winners are the readers, as everyone strives to produce the very best book they can.
Just to be in the final, with my first published book, is an honour. I wish every success to all my fellow nominees, in all categories, and my thanks to the organizers and judges who give their time to make the contest possible.

subset: commoncrawl


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This week's top hits from Eater Seattle, Curbed's sibling bar, restaurant, and nightlife blog.
Image: Suzi Pratt
VITAL UPDATES -- Cat cafe mania is officially here. Well, almost: Seattle Meowtropolitan, the city's first cat cafe, is set to open in Wallingford around the last week of October. Owner Matt Lai hasn't announced an address yet; check back in with Eater for updates as the historic day draws closer.
EATER INSIDE -- The newest addition to the McMenamins empire opens today in Bothell, and it sure is a beauty. Once an actual school, McMenamins Anderson School is now a 5-acre-wide, 78,000-square-foot constellation of bars, restaurants, event spaces, and lodging (converted from classrooms). There's even a movie theater and heated saltwater pool that's free to overnight guests and available for a fee to others. Eater has the inside look.
COMING ATTRACTIONS -- JarrBar, the latest by Bryan Jarr (owner of the former Madison Park Conservatory), is set to open within weeks. The menu is based on favorites from a trip to Spain and Portugal that Jarr took last year, focused especially on preserved seafood and Spanish meats and cheeses. The bar will have wines from Spain, Portugal, and elsewhere; Spanish Estrella Damm beer on draft, another rotating tap, and more brews in bottles; cocktails with infusions; sangria; and beyond.


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The employees of the Greenpoint-based tech company Kickstarter (58 Kent St.) voted to unionize on Tuesday, becoming one of the first U.S. tech companies to join a union.
A vote on Tuesday by employees of the crowdfunding website resulted in 46 in favor of and 37 against joining the Office and Professional Employees International Union, which represents over 100,000 white collar workers according to VICE.
#KickstarterUnited has now been certified by the NLRB:
We Are A Union!!! pic.twitter.com/l6W91lILKU — Kickstarter United (@ksr_united) February 18, 2020
A year-and-a-half process leading up to Tuesday's unionization vote began in 2018 when Kickstarter employees and management disagreed on the decision to allow a campaign for a book entitled "Always Punch Nazis," as NBC New reports:
The seeds for the Kickstarter union were sown in August 2018 during an internal debate about a comic book called "Always Punch Nazis." Conservative news website Breitbart accused Kickstarter of violating its terms of service for approving a fundraiser for the comic book. Breitbart argued that the book, described as an "anthology about our country's battle against racism," violated Kickstarter's rule barring projects that encourage violence.
Although the Kickstarter employees who reviewed the comic book project decided it did not violate company guidelines, the management disagreed and decided to pull the fundraiser from the platform. Workers grew concerned that their bosses were giving in to demands from far-right trolls.
Ultimately, the management reversed its decision and let the comic book fundraiser remain. But the company's approximately 150 staff started discussing the possibility of starting a union.
Two union organizers at Kickstarter were fired in September of 2019 and filed complaints with the National Labor Relations Board, "alleging that their rights as employees to organize had been violated," according to the Washington Post.
"You discover it's not just the neo-Nazis and unjust termination," Taylor Moore, a former employee told the Washington Post. "It's also some women saying there's a gender pay disparity, someone saying they've had their sexual harassment claims ignored, another saying that working remotely is not fairly applied on their team. It really became clear that a union was not only a good idea but necessary."
A statement from CEO Aziz Hasan posted to Kickstarter's website on Tuesday says that the company respects the decision:
Today we learned that in a 46 to 37 vote, our staff has decided to unionize. We support and respect this decision, and we are proud of the fair and democratic process that got us here. We've worked hard over the last decade to build a different kind of company, one that measures its success by how well it achieves its mission: helping to bring creative projects to life. Our mission has been common ground for everyone here during this process, and it will continue to guide us as we enter this new phase together


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Show Dem Camp
Show Dem Camp is a rap duo based in Nigeria. Tec (Wale Davies) and Ghost (Olumide Ayeni), who make up the group were were born in Nigeria and lived in cities in Europe and the US before returning to Lagos to pursue a career in music.
After the underground success of 'street releases' 'Clone Wars volume 1 and 2', the duo released their debut studio album The Dreamer Project in 2011. Its first single 'Farabale' was well-received and the album featured artists such as 2face, MI and Temi Dollface.
In 2013 they released the song 'Feel Alright' featuring Poe and the alternative artist Boj. It met a warm response, and a humorous video followed.
In 2015, Show Dem Camp joined a half-dozen other artists to form The Collective (stylised as The Collectiv3.) The duo also contributed a song to the debut album of the group.


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The Supreme Court as Sitting Constitutional Convention
Debating the proper role of the judiciary.
I was traveling most of yesterday, so missed Steven Taylor's thoughtful post "On Constitutional Language," which argues against my contention that the Supreme Court's finding a right to same-sex marriage is judicial activism. As he notes in the post, it's an issue we've debated in person as well as in the blogosphere numerous times over the past seventeen years.
We're in agreement that democracy is aspirational and that our perception of even our most fundamental civil liberties has evolved substantially over the years, mostly in the direction of broader freedom for groups that were once marginalized. We also agree that, while it's preferable for a variety of reasons for these changes to be enacted by the legislature, the judiciary has a role. The disagreement is simply over how these should be balanced.
My general sense is that the people have the right, through their representatives, to make public policy and that the Constitution serves merely to constrain the limits of public policy. First, because we're a federal system, the Constitution guides which areas are under the purview of the central government, the governments of the several states, or prohibited to government altogether. Second, because of separation of powers, the Constitution guides how those policy areas under the purview of the central government are managed. Third, various provisions of the Constitution set forth parameters under which the central government and, in a handful of cases, the several states must operate. Because much of the language of the Constitution is subject to interpretation, the judicial branch serves, in the words of Chief Justice Roberts, as an umpire, issuing rulings when there are disputes over these areas.
While Steven and I are in agreement that the interpretation of the Constitution simply has to keep up with current conceptions of words, my preference is for the executive and legislative branches of the central government and the several states to take the lead on that front rather than the judiciary. So, for example, I note in my post that:
Congress was certainly permitted to create and fund an Air Force, despite the Constitution only mentioning an Army and a Navy and the airplane not having been invented in 1787.
Steven reasonably rejoins:
If we are going to say that the words should be bound to how the Framers themselves would have understood the terms, then I think that, in fact, we do need new language to allow for the Air Force (or, for that matter, for the owning of semi-automatic weapons). There is simply no way to assume that the Framers could have conceived of these things. Further, if we stick to the military for a moment, the only permanent arm of the military that the Framers envisioned was the Navy, given the need to protect trading lanes from pirates. Armies were raised for specific purposes and wars only fought when declared. The modern military bears no resemblance to what the Framers would have envisioned or understood (and while that is not an issue of democracy's evolution, it is an example of how changing language influence what government does).
That's all true. Still, I find it quite reasonable for the judiciary to interpret the Constitution's language in this case broadly, not because I happen to be in support of fielding a modern military, but because it's the express will of the people and there's no Constitutional prohibition against it. The Framers clearly intended to give the Congress the power to legislate on matters related to the organizing and equipping of a military. Further, that power is constrained both by the fact that Congress is subject to election every two years and by the fact that any legislation needs the signature of the president, who stands for re-election every four years. There's simply no reason the courts ought to get in the way.
Indeed, as a general principle, the judiciary ought to defer to the elected representatives of the people unless they're taking action directly prohibited by the Constitution. But, of course, what precisely is prohibited by the Constitution is itself a debatable point. Steven observes,
I find the general notion that we have to get into the heads of men from the 18th and 19th centuries to interpret political questions of right in the 21st century to be unnecessarily constraining and problematic to the point that semantic arguments override the very concepts under discussion, especially when we are talking about abstract principles (e.g., freedom of speech, equal protection, due process, etc.). (Abstract in definition, but with obvious concrete applications).
But back to the main topic: did the authors of the 14th Amendment know what homosexuality was? Of course they did. Did they intend for the 14th Amendment to lead to same sex marriage? I can't imagine it entered their minds one little bit. However, I don't think that's the appropriate test or issue here. The question is, did they lay down a basic principle with the following words?
All persons born or naturalized in the United States, and subject to the jurisdiction thereof, are citizens of the United States and of the state wherein they reside. No state shall make or enforce any law which shall abridge the privileges or immunities of citizens of the United States; nor shall any state deprive any person of life, liberty, or property, without due process of law; nor deny to any person within its jurisdiction the equal protection of the laws.
The notions of "equal protection of the laws," for example, is a pretty clear principle that ultimately transcends the immediate thoughts in the mind of the person who penned them. Indeed, even if the person/persons who wrote the document did not envision the expansion of rights for Hispanics, women, homosexuals, or whomever else one might think of does not, in my opinion, obviate a deep principle that was laid down in constitutional law: that every citizens of the United States (every citizen) deserves to be treated by the law equally.
But, again, this is mostly solved by letting the people's representatives update the principles. There's simply no question that the 14th Amendment applies to Hispanics, women, and homosexuals. The question is what does "equal protection of the laws" mean with respect to those groups.
The question of women is an interesting one. Certainly, the people who wrote and ratified the 14th Amendment (in 1865 and 1868, respectively) were aware of the existence of woman. Most, presumably, were married to one and all of them had mothers. Yet, rather clearly, they did not intend to confer on women the right to vote. Doing so required the passage of the 19th Amendment in 1920. Was the 19th Amendment redundant?
As recently as 1972, Congress passed an Amendment to the effect that "Equality of rights under the law shall not be denied or abridged by the United States or by any State on account of sex." It was ratified by 35 states, 5 of which later rescinded their ratification, failing to get the required 37 states by the 1979 and then 1982 deadlines.
While the above history makes is crystal clear that the 14th Amendment wasn't intended to nor did it even in the near term grant full equality to women, there's little doubt that the Supreme Court could have preempted all of this debate at any time if it wanted to. Doing so was unnecessary, in that Congress has essentially done it for us. Various legislation over the years has made illegal discrimination on account of sex in almost every aspect of public policy. The handful of exceptions are relatively minor and, as with the roles available to women in our armed forces, changing with time. Congress clearly has this power under Section 5 of the 14th Amendment.
Should the courts have sped the process up for us? I don't think so. The Constitution manifestly didn't require them to do so and it's far preferable for fundamental social changes to happen organically, through the political process. It's highly problematic that Anthony Kennedy—an unelected individual appointed to the bench as Ronald Reagan's third choice for the job in 1986—can simply override the longstanding will of the people by claiming a Constitutional clause written 150-odd years ago supports his public policy preferences, notwithstanding that the people who wrote said clause meant no such thing.
The Framers set forth processes for amending the Constitution in Article V:
The Congress, whenever two thirds of both houses shall deem it necessary, shall propose amendments to this Constitution, or, on the application of the legislatures of two thirds of the several states, shall call a convention for proposing amendments, which, in either case, shall be valid to all intents and purposes, as part of this Constitution, when ratified by the legislatures of three fourths of the several states, or by conventions in three fourths thereof, as the one or the other mode of ratification may be proposed by the Congress; provided that no amendment which may be made prior to the year one thousand eight hundred and eight shall in any manner affect the first and fourth clauses in the ninth section of the first article; and that no state, without its consent, shall be deprived of its equal suffrage in the Senate.
This is, by design, exceedingly difficult. The Constitution, written in 1787 and ratified in 1789, has only been amended in this fashion 27 times. Ten of those Amendments, the Bill of Rights, were almost immediate and essentially part of the original Constitution (the 27th, interestingly, was part of the same package but not ratified until 1992). Another three (13-15) were in response to the Civil War.
Yet, the Supreme Court, often on the strength of a single Justice's deciding vote, effectively acts as a sitting Constitutional Convention. Simply be redefining "equal protection" to encompass same-sex marriage, last week's ruling in Obergefell v. Hodges effectively passed an equivalent of the Equal Rights Amendment, notwithstanding vehement objections in a sufficient number of states to preclude amending the Constitution.
It's worth noting, too, that this power works both ways. While the recent trend has been for the courts to use their power to expand the rights of the previously disenfranchised groups more rapidly than would happen through the political process, it has frequently worked the other way. Most notably, the Supreme Court of the post-Civil War era effectively ignored both the plain meaning and obvious intent of the 14th Amendment in striking down the Civil Rights Act of 1875 and in its ruling in Plessy vs. Ferguson.
As a practical matter, there's no limit to the judicial power aside from their own prudence. When the Supreme Court says the Constitution says something it clearly does not say, that only remedies are amending the Constitution or a refusal by the other branches or lower courts to enforce the ruling. The former, as noted, is nearly impossible and has only happened in direct response to a Supreme Court ruling twice (the 11th and 16th Amendments). The latter is dangerous, indeed.


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LONDON, June 23 (Reuters) – Britain's six-year drive to increase the number of women in senior management at financial firms is "stagnating" for the first time, a review for the finance ministry said on Thursday.
The ministry launched its voluntary Women in Finance Charter in 2016 and more than 400 firms have now signed up.
A review by New Financial think tank found that 78% of signed-up firms are meeting or are on track to meet their targets, up 5% on last year.
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The average level of female representation in senior management remained flat at 33% in 2021 compared with 2020, the review said.
Almost half of firms have committed to have 40% of their boardroom made up of women.
"I am concerned to see progress stagnating," Women in Finance Champion Amanda Blanc said.
"Frankly, up to now there has been too much tinkering at the edges and not enough fundamental change," said Blanc, who is also chief executive of insurer Aviva.
"There are some glimmers of hope with more ambitious targets being set and met. But for the sake of women, companies and society, we've got to work quicker and harder."
Signatories agree to support the progression of women into senior roles by setting targets to improve diversity and publicly report on their progress.
"I welcome this year's progress, but settings targets is just one part of the process – I am today calling on firms to double-down on their to commitments and continue to deliver greater gender-equality in the workplace," Britain's financial services minister John Glen said in a statement.
Pension Bee, Yorkshire Building Society and American Express headed the list of 33 signatories that have met their own internal targets ahead of deadline.
There were 31 firms who missed their own targets for 2021, though 19 of them were close, citing reasons such as restructuring, low turnover in senior management, and COVID-19.
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Reporting by Huw Jones;Editing by Elaine Hardcastle
Our Standards: The Thomson Reuters Trust Principles.


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What happened
After reporting fiscal third-quarter financial results on Aug. 9 and updating investors on its progress on developing marijuana-derived medicines, shares in GW Pharmaceuticals PLC (GWPH) fell 13.4% in August, according to S&P Global Market Intelligence.
So what
GW Pharmaceuticals is conducting a flurry of research into medicine derived from the chemical cannabinoids found in marijuana plants. Sales of the company's only approved marijuana medicine, Sativex, which is used to treat multiple sclerosis spasticity, are tepid, and as a result, GW Pharmaceuticals continues to lose money.
Its fiscal third-quarter revenue was about $3.1 million, down from $11.55 million last year, at current exchange rates. The drop-off in revenue is due to lower collaboration revenue from Otsuka following the shuttering of a program evaluating Sativex's use as a cancer pain therapy last year. Slightly offsetting that decline in collaboration revenue was a $500,000 increase in Sativex sales to $2 million.
The company spent $34.4 million on research and development last quarter, up from $28 million least year. Most of the increase in R&D spending was due to phase 3 studies of Epidiolex, a purified formulation of the cannabinoid CBD that GW Pharmaceuticals is evaluating as a treatment for rare forms of epilepsy.
Overall, GW Pharmaceuticals lost $16.4 million last quarter.
Now what
Sativex's sales are clocking in at an annualized pace of only about $8 million, so GW Pharmaceuticals $1.9 billion market cap is tied less to Sativex's potential than it is to Epidiolex's potential.
The company reported results earlier this year from two trials studying Epidiolex, and in both instances, epilepsy patients saw a substantial drop in monthly seizure rates. GW Pharmaceuticals is still waiting for data from two more epilepsy trials (one in Dravet syndrome and the other in Lennox-Gastaut syndrome), and if those trials are also positive, then a Food and Drug Administration filing for Epidiolex's approval could occur 2017.
There's a big need for new alternatives because many patients with rare forms of epilepsy don't respond to current treatment options. However, investors should remember that the patient population Epidiolex is targeting is small. Investors should also remember that there's no guarantee that the remaining two trials will back up the data that's already been released. Data from the Lennox-Gastaut trial is expected before the end of this quarter and the company hasn't said precisely when the Dravet syndrome results will be available.
Overall, GW Pharmaceuticals has a healthy $254 million cash stockpile, but it's spending heavily and that means it's going to keep losing money for a while. Undeniably, Epidiolex is the company's best shot at a commercial success so far, but with question marks remaining, GW Pharmaceuticals is still a risky bet.


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What a gorgeous, swirly tune!
Youtube info:
Published on Jan 6, 2014
Young the Giant's lyric video for 'Mind Over Matter' from the upcoming album, Mind Over Matter – available January 21st on Fueled By Ramen. Visit http://youngthegiant.com for more!
Pre-order the album at http://smarturl.it/mindovermatter2 and get instant downloads of 3 songs: Mind Over Matter, Crystallized and It's About Time.
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Site: http://youngthegiant.com
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Video by Andre Comtois and Christina Chi Craig (http://www.christinachi.com)


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As of late, I've started working on several Team Foundation Server API-based examples (some of which are starting to show up as blog postings). Although the Team Foundation Server SDK does include some examples, it's not always easy to locate the example you need (assuming it's even there). There are several blogs containing various TFS API examples, but again, the full details may be spread across several postings or even separate sites.
Several months ago, Grant Holliday created the TFSBuild.com site dedicated to MSBuild and Team Foundation Build recipes. I decided to take a similar approach and registered the site TFSExamples.com. I utilized the same ScrewTurn wiki software and customized it to the needs of the new site. The intent is to compile a library of Team Foundation Server API examples making it simple to get started with a project based on the TFS object model.
There are only a couple of examples on the site at the moment but I plan on adding several more over the next few days. I also hope that others will come across the site and add their own examples.
Some of the current functionality on TFSExamples.com includes:
- Add New Example – displays instructions on adding a new example to the wiki
- Browse All – displays a list of all pages in the wiki
- Browse Categories – displays a list of all categories in the wiki
- Request Example – allows you to request a specific example be added to the site
If you have any examples based on the TFS object model that you would like to publish, please check out TFSExamples.com and add them accordingly.


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Create a Website Account - Manage notification subscriptions, save form progress and more.
The City of Woodinville works cooperatively with Woodinville Fire & Rescue District to provide fire protection services and issue fire permits to residents and businesses inside the city.
Fire permits are required for many types of new construction as well as temporary tent installations. Annual fire inspections for businesses are required to verify fire-code compliance and protect the safety of the public.
The following fire permit types may be applied for through the city's online permit portal.
To ensure and maintain compliance with the Washington State Fire Prevention Code for the safety and welfare of all employees, residents, visitors, and emergency responders in the City of Woodinville, all businesses, churches and schools are required to receive an annual inspection. To schedule or pay for an annual fire & life safety inspection visit Woodinville Fire & Rescue's website.
Temporary tents that are 400 square feet or larger require a permit and inspection by Woodinville Fire & Rescue. Review by the City is sometimes also required. For more information on obtaining a temporary tent permit and scheduling an inspection visit Woodinville Fire & Rescue's website.


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Pipis and prejudice
The town of Venus Bay stands on a sand spit about two hours south east of Australia's fastest growing city Melbourne. In town, there are holiday lets and a caravan park, all serviced by 500 permanent inhabitants and supplemented by about 1500 part-time residents of holiday homes. But it is one of the smallest tenants that has been at the centre of tensions in the town for the last eight years: the pipi.
It's sometimes called a cockle, and digging them up from under the sand is one of the main drivers behind an annual total of 6-8000 day-visitors from Melbourne. These visitors mostly call on the long beach in front of the town on summer public holidays like Christmas and New Years, when the diversion of the asphalt bound city has nothing compared to the open sky and sand of the Bay.
But if you ask some locals, the visitors are not welcome.
In this story, local pride, racial prejudice and the joint pursuit of pipi hunting all intersect against the backdrop of an impossibly-long sandy beach on a summers day.
This program aired on Radio National's Off Track and was awarded Best Feature Story in the 2017 Rural Press Club of Victoria awards.
Read the online story


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Mauricio Pochettino has admitted that Tottenham could be forced to play the Manchester City game at Wembley the day after the NFL play on it.
Spurs are looking at their options after the new stadium was delayed by at least a further month, due to critical safety issues this week.
The grand opening game against Liverpool and the match with Cardiff City have both been moved to Wembley Stadium, but the Manchester City game, due to be played on Sunday, October 28, is set as TBC by Spurs, with no indication yet as to where it could be played.
That is because, with no indication as to whether or not the new stadium will be ready by then, Wembley is being used by the NFL on that day and the Premier League are against switching the game to the Etihad Stadium.
And Spurs cannot play at a third different 'home' stadium in the same season.
So the club have been forced to explore their options, with the boss admitting that playing at Wembley just a day after the Philadelphia Eagles take on the Jacksonville Jaguars in the national stadium might be the best option.
Pochettino said: "At the moment I cannot say that we're not going to play this game in our new stadium.
"If you ask me today if it's not possible to play in the new stadium, of course it's about finding a way to play at Wembley because I think to play at another place, maybe three stadiums in one season, will be too much.
"Maybe we need to adapt. If the day after the NFL game maybe the pitch is not in the best condition but we need to make the effort and try to play in the end because maybe to find another date will be difficult."
Spurs cannot move the game to earlier than October 28, as the NFL have rented the stadium for three weeks and if it was to be moved to Monday, October 29, it could have an impact on both clubs' League Cup games that midweek, should both teams make it through to the fourth round.
Pochettino is remaining calm, and positive about the situation.
He added: "Remember one year ago we were talking about Wembley, which was not a nice place for us to perform and get good results, but I think in the end we were the third team for [home] results in the league I think after Manchester City and Arsenal. It's so important to be positive. In football more than in other business, football is about faith, belief, willingness. Our responsibility is to be positive whether we play in one or another place."
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