extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Analysis of endocrine disruption effect of Roundup® in adrenal gland of male rats

The effect of Roundup® on adrenal gland steroidogenesis and signaling pathway associated with steroid production was investigated. Doses of 10, 50, 100 and 250 mg/kg bw/d Roundup® were administered for two weeks to adult male rats. The 10 mg/kg bw/d dose which reduced circulatory corticosterone levels, but did not change food consumption and body weight, was selected for further study. The expression of cholesterol receptor (low density lipoprotein receptor), de novo cholesterol synthesis enzyme (3-hydroxy-3-methylglutaryl-coenzyme A synthase), hormone-sensitive lipase, steroidogenic acute regulatory protein (StAR) mRNA and phosphorylated form was decreased. Adrenocorticotropic hormone receptor (ACTH), melanocortin-2 receptor, expression was not changed but circulatory ACTH levels and adrenal cortex protein kinase A (PKA) activity were reduced. Surprisingly, exogenous ACTH treatment rescued steroidogenesis in Roundup®-treated animals. Apoptosis was evident at 250 mg/kg bw/d, but not at 10 mg/kg bw/d dose. These results suggest that Roundup® may be inhibitory to hypothalamic–pituitary axis leading to reduction in cyclic adenosine monophosphate (cAMP)/PKA pathway, StAR phosphorylation and corticosterone synthesis in the adrenal tissue.

Introduction
Agricultural advancements have increased production and correspondingly increased the usage and release of herbicides into the environment. Among the herbicides, the glyphosate-based herbi-cide Roundup ® is most extensively used world over [14]. Roundup ® is non-selective and broad spectrum herbicide utilized in agricultural fields, gardens, play grounds, road sides etc. [10]. The half life of Roundup ® is ∼47 days in soil and up to 90 days in water with low microbial metabolism and disintegration [18,37,39]. In rats, Roundup ® whole body pharmacokinetics is biphasic for single 10 mg/kg bw dose with half-life of the alpha phase is 6 h and 79-106 h for beta phase [43]. Roundup ® and its metabolite, aminomethyl-phosphonic acid, have been detected in water and crops [11,25,26,35]. Therefore, there is increased probability of Roundup ® exposure to animals and human, and it becomes of interest to study its toxic effects, if any.
Glyphosate or its formulation Roundup ® acts via specific inhibition of plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase which is essential for synthesis of aromatic amino acids [19,34,36] and thus, considered non toxic to animals. However in recent past, several studies have suggested glyphosate toxic effects such as carcinogen [28,38,40], teratogen [12,13] and as an endocrine disruptor (ED). The endocrine disrupting chemicals (EDC) represent a broad class of exogenous substances that adversely affect the endocrine system by interfering with hormone biosynthesis, metabolism or action [23]. As an ED, glyphosate and its formulation, Roundup ® was reported to decrease testosterone hormone levels in adult rats [9]. The prenatal exposure of glyphosate disrupted the masculinization process and caused endocrine dysfunction in reproductive parameters of male offspring [32]. Moreover, various formulations of glyphosate including Roundup ® were reported to disrupt aromatase activity, enzyme required for estrogen synthesis, in human liver HepG2 cells [15]. The human placental JEG3 cells treated with Roundup ® altered aromatase mRNA levels and enzymatic activity by interacting with the active site of the purified enzyme [31]. A study involving MA-10 Leydig tumor cell line reported down regulation of StAR mRNA levels, a key regulatory steroidogenic gene, and dibutyryl cAMP-stimulated progesterone production upon treatment [42]. It has been observed that the commercial formulation had more adverse effects than the active ingredient i.e., glyphosate [3]. The EDC effects of Roundup ® in male reproductive system have been described; however, studies detailing EDC effect on the adrenal gland steroidogenesis have not been reported in animals.
In the present study, experiments have been conducted to examine effects of Roundup ® on adrenal steroidogenesis at systemic as well as at the tissue level. Different doses of Roundup ® were orally administered to adult male rats for 14 days daily although, only the lowest dose required for disrupting the major adrenal gland steroid hormone i.e., corticosterone level was selected for further detailed study. The EDC effect of Roundup ® was examined on important regulatory genes for steroidogenesis; StAR and p 450scc , the steroid precursor, cholesterol levels, and cholesterol homeostasis genes and the signaling involved. An ACTH challenge experiment was also performed to evaluate Roundup ® action to be via hypothalamic pituitary axis or directly upon the gland.

Animal experiments
All procedures in animals were approved by the Institutional Animal Ethical Committee, Indian Institute of Science, Bangalore, India. In this study, 2-2.5 months old male Harlan Wistar rats were used. One rat per cage was housed in the room with L:D cycle of 12 h each under a controlled temperature of 24-26 • C. Rats were allowed free access to standard chow diet and drinking water ad libitum. For this study, rats were randomly divided into five groups with 5 or more animals per group. Rats were administered with 24.4, 121.9, 244 and 609.8 l of Roundup ® (41% w/w) which corresponds to 10, 50, 100 and 250 mg/kg bw/d of glyphosate, respectively. The highest dose tested was well below LD 50 of 4900 mg/kg bw. Roundup ® was dissolved in deionized water to make the total volume of 300 l except for 250 mg/kg bw/d dose, in which Roundup ® was administered directly. The vehicle (0 mg/kg bw/d) group received 300 l of deionized water only. The treatments were oral, once daily (0800-0900 h) for 14 days. Body weight and food consumption were monitored twice weekly. Food consumption was calculated by subtracting food pellet weight remaining in the cage mesh from the total food pellet weight provided to the each rat cage mesh. A total 10 rats for control, n = 10 rats for 10 mg/kg, n = 5 rats for 50 mg/kg, n = 10 rats for 100 mg/kg and n = 10 rats for 250 mg/kg bw/d dose group were utilized in the study. On day 15 of treatment, animals were weighed and anesthetized with 50 mg/kg bw/d pentobarbitone sodium (Sigma Chemical Co., St. Louis, MO) and blood was collected by cardiac puncture. Plasma was separated by centrifugation at 2000 × g, 4 • C for 15 min and stored at −20 • C until analyzed. The anaesthetized animals were killed by cervical dislocation, adrenal glands were dissected out, weighed, transferred to neutral buffered formaldehyde (NBF) solution or snap frozen in liquid nitrogen and stored in −70 • C freezer until analysis. To examine the effect of exogenous adrenocorticotropic hormone (ACTH) on adrenal gland steroidogenesis, a standardized dose of 5 IU of Porcine ACTH was injected i.v., based on the protocol reported by others [21,30,41] to vehicle (n = 4 rats) as well as Roundup ® 10 mg/kg bw/d (n = 3 rats) treated animals. Blood sample and adrenal glands were collected after 60 min of ACTH treatment.

Hormone assays
Two different kits have been utilized for determining plasma corticosterone levels. The steroids extraction from plasma was carried out by using diethyl ether or methylene chloride (Merck, Billerica, MA) as per requirement of the kits. The corticosterone levels obtained from different sources gave similar concentration of corticosterone. The inter-and intra-coefficient of variations of assay were <15%. Plasma concentrations of testosterone were measured by testosterone RIA kit according to the manufacturer's protocol. ACTH in plasma was measured using luminescence based ELISA kit for rats.

Cholesterol assay
Adrenal gland tissue lysate was prepared by homogenizing 0.5 mg tissue in unit ml of 10% SDS containing phosphate buffered saline (PBS) (Sigma-Aldrich Co., Bangalore, India). Tissue debris was removed by centrifugation. The tissue lysate and plasma were analyzed for total cholesterol and esterified cholesterol by using the Amplex ® red cholesterol assay kit as per the manufacturer's instructions. Briefly, plasma or tissue sample were mixed with equal volume of Amplex ® red working reagent with and without cholesterol esterase. The reaction mixture was then incubated for 30 min at 37 • C in the dark. The fluorescence values were read at an excitation wavelength of 545 nm and an emission wavelength of 590 nm (Tecan Infinite F200 Microplate Reader, Männedorf, Switzerland). A series of cholesterol standards were prepared that were provided in the kit and ran alongside the plasma and tissue lysate samples. Plasma HDL (high-density lipoprotein) and LDL (low-density lipoprotein) were analyzed by commercially available HDL and LDL/VLDL quantitation kit according to the manufacturer's instruction.

qPCR analysis
mRNA expression of key regulatory receptors, enzymes and carrier proteins involved in the cholesterol homeostasis and steroidogenesis were determined by qPCR analysis as previously described from the laboratory Priyanka et al. [46] using ABI 7500 Real-Time PCR instrument. Briefly, total RNA was isolated from adrenal glands by TRIzol ® method, treated with DNaseI before performing reverse transcription for cDNA preparation with oligo(dT). Real time PCR was performed with each reaction carrying 10 ng of cDNA. Primers have been preferably designed from exon junction sequences, except for gene. The details of primers employed along with the sequence source are provided in Table 1. Expression level of individual gene was normalized to Rpl19 expression which was used as calibrator (internal control) for each cDNA sample. PCR for each sample was set up in duplicates and the average C t value was used in the C t equation.

Oil Red O staining
The 5 m thick cryosections of frozen adrenal glands were prepared and fixed in NBF, washed with PBS (Sigma-Aldrich Co., Bangalore, India) and stained with Oil Red O (ORO) (Sigma-Aldrich Co., Bangalore, India) in 60% isopropanol (Sigma-Aldrich Co., Bangalore, India) for lipid detection. For nuclear staining, sections were equilibrated in McIlvaine's Citric acid; Na 2 HPO 4 buffer and stained with DAPI and the sections were visualized under florescent microscope (Olympus IX81 inverted microscope, Tokyo, Japan).

Immunoblot analysis
The adrenal gland tissue was homogenized using RIPA buffer with protease inhibitors as per the procedure reported earlier [44] and the lysates were stored at −70 • C until further use. Total protein estimation was performed by Bradford method (Bio Rad lab. Inc, Berkeley, CA). Tissue lysates (30 g protein) were resolved on 12% SDS PAGE and transferred onto PVDF membrane using a wet transfer unit (Bio Rad Laboratories, Berkeley, CA). Non specific sites on the membrane were blocked using 10% milk in TBST (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20) by incubating it for 1 h at room temperature. The membrane was incubated overnight at 4 • C with primary antibody at 1:1000 dilution specific for pCREB (Ser133), CREB, Cleaved Caspase-3, pStAR, StAR and ␤-actin. The membrane was washed with TBST and incubated with secondary antibody (horseradish peroxidase labeled anti rabbit IgG) at 1:3000 dilutions. The bands were visualized using Western blot imaging system (Flourchem FC2, Cell Biosciences Inc., Santa Clara, CA) and the band intensity was quantitated by Gene tool software (Syngene, Cambridge, UK).

PKA assay
PKA assays were performed as per the previously published procedure [29] using SignaTECT ® cAMP dependent protein kinase (PKA) assay kit (Promega, Madison, Wisconsin). The activity of PKA was determined by measuring the incorporation of 32 P from [␥ 32 P] ATP via adrenal gland PKA to biotinylated kemptide, a highly specific peptide substrate. Briefly, the medullary region of adrenal gland was removed carefully from the decapitated gland under dissecting microscope in ice cold PBS solution. The adrenal medulla region specific gene, PNMT expression was found to be undetectable in the dissected cortical fraction (data not shown). The cortical fraction was homogenized in cold extraction buffer containing 25 mM Tris-HCl, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM ␤ ME, 1 g/ml Aprotinin and 1 g/ml Leupeptin and centrifuged at 4 • C, 14,000 × g for 5 min. After protein estimation, 10 g of protein was used to perform the assay by incubating with [␥ 32 P] ATP at 30 • C for 5 min. The reaction was terminated by adding stopping buffer provided with the kit and 10 l of reaction mixture was spotted onto SAM biotin capture membrane. The individual membrane square was dried and then transferred to liquid scintillation vials for counting in ␤ counter (Tri Carb B2910TR liquid scintillation analyzer, Waltham, MA). A control reaction without the substrate was also performed for determining the background activity which was subtracted from the total activity of samples.

TUNEL assay
Assay was performed using TACS ® TdT DAB in situ apoptosis detection kit (Trevigen Inc., Gaithersburg, MD) according to manufacturer's protocol. Briefly, NBF fixed adrenal gland from vehicle, 10 and 250 mg/kg bw/d treated animals was sectioned into 5 m thickness. Sections were cleared in xylene and rehydrated using ascending grades of alcohol solutions and PBS. Sections were treated with Proteinase K followed by TdT labeling reaction mix and buffer incubation. The reaction was stopped by TdT Stop Buffer provided with the kit. The sections were washed thoroughly in PBS and treated with HRP conjugated streptavidin in a humid chamber. After PBS washing, the sections were exposed to DAB provided in the kit, till development of brown color, followed by haematoxylin staining for nuclei and the sections were observed under light microscope. The positive and negative controls for the technique were included by addition of nuclease during incubation and removal TdT enzyme from the labeling mix, respectively.

Statistical analysis
Data were expressed as mean ± SEM. A t-test was used to calculate p value between two groups. Multiple comparisons were made between vehicle and Roundup ® treatment groups using Bonferroni's test after one way ANOVA. Prism version 5 (GraphPad, California) was utilized for statistical analysis. A p value <0.05 was considered statistically significant throughout.

Effect of Roundup ® on body weight and food consumption
No overt signs of toxicity were observed during oral administration of Roundup ® up to dose of 250 mg/kg bw/d for 14 days. However, food consumption and body weight were significantly lower beginning with 50 mg/kg bw/d dose (Table 2A and B). This might be due to toxic effects of Roundup ® other than EDC effect, hence doses higher than 50 mg/kg bw/d were not included for further analysis.

Roundup ® effect on circulatory levels of steroid
Plasma testosterone and corticosterone, the representative steroid hormone from major endocrine sources i.e., testis and adrenal gland of male rat, were assayed ( Fig. 1A and B). Both hormone concentrations were lowered after Roundup ® treatment in a dose dependent manner. The lowest dose 10 mg/kg bw/d itself could decrease corticosterone levels significantly (p < 0.05), therefore this dose was selected for detailed EDC studies.

Roundup ® effect on expression of genes associated with steroidogenesis
The key regulatory steps in steroidogenesis are transport of cholesterol from outer to inner mitochondrial membrane by StAR protein and cholesterol side chain cleavage step by P 450scc enzyme. The mRNA expression of P 450scc was unchanged, while StAR mRNA and total protein were down regulated in Roundup ® (10 mg/kg bw/d) treated animals compared to vehicle group ( Fig. 2A and  B). Phosphorylated form of CREB, the transcriptional regulator of StAR expression, was down regulated in 10 mg/kg bw/d Roundup ®treated rats and phosphorylated StAR protein was also found to be significantly lower (p < 0.05) in the 10 mg/kg bw/d treatment group (Fig. 2C and D).

Roundup ® effect upon circulatory and adrenal gland lipid & cholesterol content
Together with steroidogenesis process, the homeostasis of steroid precursor cholesterol was also examined. The level of cholesterol in the circulation as well as in the adrenal gland was measured in vehicle and Roundup ® -treated animals. Circulatory levels of total, free and esterified cholesterol were unchanged except for 100 mg/kg bw/d dose (Fig. 3A). A dose-dependent increase in HDL and LDL levels were observed ( Fig. 3B and C). Cholesterol was found to be moderately higher in the adrenal gland (Fig. 4A). The weight of adrenal glands (paired weight) was observed to be significantly higher (p < 0.05) (Fig. 4B) in Roundup ® -treated animals. H&E and ORO staining indicated moderately higher number of lipid droplets present in the adrenal gland of 10 mg/kg bw/d Roundup ® -treated rats ( Fig. 4C and D).

Effect of Roundup ® treatment on genes involved in cholesterol intake and de novo synthesis
The RNA expression of receptors for cholesterol uptake i.e., lowdensity lipoprotein receptor (Ldlr) found to be significantly lower (p < 0.05) however, expression of high-density lipoproteins receptor (Srb1) was unaltered in the adrenal gland of 10 mg/kg bw/d Roundup ® -treated group compared to vehicle group ( Fig. 4A and B). Gene expression for enzymes involved in cholesterol de novo synthesis were found to be unchanged (Hmgcr) or significantly   lower (p < 0.05) (Hmgcs) in the adrenal gland of 10 mg/kg bw/d Roundup ® -treated animals ( Fig. 4C and D). Although expression of genes associated with cholesterol mobilization in the gland was lower, there was a moderately higher accumulation of cholesterol in adrenal cells which could be due to decreased utilization of cholesterol in Roundup ® -treated animals.

Effect of Roundup ® treatment on esterified cholesterol (CE) and ester hydrolase
Stored form of cholesterol i.e., CE was estimated by calculating free from total cholesterol. CE tended to be moderately higher in the adrenal gland of Roundup ® -treated animals (Fig. 5A). Cholesterol ester hydrolase or hormone sensitive lipase (Hsl), catalyzes the hydrolysis of CE into their free form, was found to be lower (Fig. 5B) even though changes in both, CE and Hsl, were not statistically significant (p > 0.05).

Roundup ® effect on adrenal gland ACTH receptor expression and circulatory ACTH levels
The ACTH receptor, Mc2r, expression in the adrenal gland was found to be unchanged in 10 and 50 mg/kg bw/d Roundup ® -treated rats (Fig. 6A), however the circulatory levels of ACTH were lower in Roundup ® -treated rats (Fig. 6B). The results suggest that Roundup ® treatment might have decreased the synthesis or release of ACTH from the pituitary gland. Lower ACTH levels might explain observed down regulation in StAR and Hsl expression, expression of both genes regulated by ACTH in the adrenal gland post Roundup ® treatment.

Effect of exogenous ACTH upon circulatory corticosterone levels in Roundup ® -treated animals
To examine ACTH responsiveness in the adrenal glands of Roundup ® -treated animals, a 5 IU dose [based on previous literature as well as pilot study carried out for the present work (data not presented)] of porcine ACTH administered i.v. to vehicle and Roundup ® -treated animals. Administration of exogenous ACTH increased corticosterone levels significantly (p < 0.05) at 60 min in Roundup ® -treated rats compared to vehicle treated rats (Fig. 7). Exogenous ACTH treatment also increased StAR, Hsl expression ( Fig. 8A and B) and the expression of cholesterol homeostasis genes (Srb1, Ldlr, Hmgcr, Hmgcs, Hsl) in the adrenal gland of Roundup ®treated rats (Fig. 8D-G). The results indicate that responsiveness of the adrenal gland to ACTH was intact in the Roundup ® -treated rats.

Effect of Roundup ® on adrenal gland PKA activity
The cAMP/PKA pathway activated by ACTH in the adrenal gland was examined by quantifying PKA activity. Equal amount of gland cortical region total protein from vehicle and 10 mg/kg bw/d Roundup ® and/or ACTH treated rats, was utilized for the assay. The PKA activity was significantly lower (p < 0.01) in Roundup ® -treated rats, while exogenous ACTH treatment increased the activity (p < 0.001) (Fig. 8C). The results suggested that Roundup ® decreased endogenous ACTH levels and in turn cAMP/PKA pathway in the adrenal gland tissue Figs. 9 and 10.   5. Expression of genes associated with cholesterol import and de novo synthesis in the adrenal gland of vehicle and Roundup ® -treated animals. qPCR analysis was performed to quantitate the expression of genes involved in cholesterol transport (Srb1, Ldlr) (A and B) and Cholesterol de novo synthesis (Hmgcr, Hmgcs) (C and D). Rpl19 was used as internal control. mRNA expression in the vehicle treated group was set as 1 fold and the expression in treatment group was calculated in relation to the vehicle group. Data presented as mean ± SEM (n = 5 rats per group). 't' test was performed to compare treatment group from vehicle group. *, significantly different from the vehicle group (p < 0.05).

Fig. 6.
Esterified cholesterol levels (A) of adrenal gland from vehicle and 10 mg/kg bw/d Roundup ® -treated animals. Adrenal glands were isolated post treatment and lysate was prepared. Esterified cholesterol levels were obtained by subtracting free cholesterol levels from total cholesterol values. qPCR was performed to detect Hsl gene expression in the adrenal gland of vehicle and 10 mg/kg bw/d Roundup ® -treated animals (B). Rpl19 was used as internal control. qPCR value for vehicle was set as 1 fold and Roundup ® -treated group values were plotted in relation to the vehicle treated group. Values are presented in mean ± SEM (n = 6 rats per group). 't' test was performed to compare the two groups.

Analysis of Roundup ® effect on cell toxicity
Immunoblot analysis of cleaved caspase 3 (as marker of apoptosis) was performed on the adrenal gland tissue lysate of vehicle and Roundup ® -treated animals (Supplementary Fig.  1A and B). It was observed that cleaved caspase-3 level was up regulated in 250 mg/kg bw/d dose treated rats while the levels were unchanged in the 10 mg/kg bw/d treated group. TUNEL assay for in vivo apoptosis was performed to determine DNA fragmentation (Supplementary Fig. 1C). The incidence of apoptotic cells in the adrenal gland cortical region was higher in 250 mg/kg bw/d treated rats compared to vehicle and 10 mg/kg bw/d dose of Roundup ® -treated rats.
Supplementry material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.toxrep.2015.07.021 Fig. 7. Dose dependent adrenal ACTH receptor expression (A) and circulatory ACTH levels (B) present in vehicle and Roundup ® -treated animals. Blood plasma was collected and ACTH levels were measure utilizing luminescence based assay kit. ACTH receptor, Mc2r, expression was examined by qPCR analysis. Rpl19 was used as internal control.
Vehicle group values were set as one fold and values in the treated groups were expressed in relation to the vehicle group. One way ANOVA was performed to compare the vehicle group with the treatment groups.

Discussion
4.1. The effect of Roundup ® is demonstrable at the low dose of 10 mg/kg bw/d, while toxic effects are evident at the high dose The present study was conducted to examine EDC effect of Roundup ® on adrenal gland steroidogenesis and to determine its mechanism of action. For this purpose, after determining the effect of ranges of doses on different parameters such as food consumption, body weight etc., the lowest dose was selected which was devoid of obvious toxic effects other than the EDC effect. A dose of 50 mg/kg bw/d Roundup ® manifested significant loss in body weight but not in food consumption, while doses higher than 50 mg/kg bw/d caused decrease in food consumption as well as body weight of rats in the second week of the treatment. In previous studies, it was observed that Roundup ® exposure did not change body weight of male Spraque-Dawley rats at a dose of 560 mg/ kg bw Roundup ® for 91 days [4], while female Spraque-Dawley pregnant rats exposed orally to Roundup ® with 500 mg/kg bw/d and higher doses for 10 days of pregnancy, reduced food consumption as well as body weight [12]. The discrepancy of the effect of Roundup ® observed in the present as well as in other studies may be related to the differences in strains of rats, age of rats and duration of treatment employed. In order to circumvent possible effects of Roundup ® on causing stress and other toxicity related effects, the doses of Roundup ® higher than 50 mg/kg were not used for studying the endocrine disrupting effect.
Further, circulatory corticosterone levels in the doses 10 and 50 mg/kg bw/d were observed to be lower compared to the vehicle treated group. To verify that the decrease in corticosterone is due to possible EDC effect of Roundup ® , another steroid hormone, testosterone level was also determined. The Roundup ® has previously been reported to inhibit testosterone levels and the results in present study are in agreement with others [9,5,32]. The result observed suggest suitability of the dose 10 mg/kb bw to assess the EDC effect of the Roundup ® .
In cancer cell line studies, Roundup ® has been reported to have apoptotic effect [7,27]. We examined one of the makers of apoptosis and performed TUNEL assay in adrenal glands and observed evidence for increased apoptosis at higher dose of 250 mg/kg bw/d compared to vehicle but not in 10 mg/kg bw/d treated rats. Therefore, decreased corticosterone level that was seen at low dose may not be attributed to toxicity or cell death induced by Roundup ® treatment. Fig. 8. Circulatory corticosterone levels in ACTH administered and/or vehicle and Roundup ® -treated groups. Animals were treated with vehicle and Roundup ® (10 mg/kg bw/d). 5 IU of porcine ACTH was administered intravenously. Post 1 h of ACTH treatment, corticosterone levels were measured in the plasma. Data presented as mean ± SEM (n = 3-4 rats per group). 't' test was performed to compare vehicle and treatment groups. *, ** & ***, significantly different from the vehicle group by p < 0.05. p < 0.01, p < 0.001 viz.   The analysis of expression of genes associated with steroidogenesis such as StAR and P 450scc , revealed no change in P 450scc expression, but decrease in StAR expression, both at mRNA and protein levels. Moreover, phosphorylated StAR expression showed greater degree of down regulation compared to total protein and RNA levels. The difference between total StAR and phosphorylated StAR expression levels post Roundup ® treatment, suggests gene regulation at two levels; one at transcription and another at phosphorylation process. Phosphorylation of StAR at serine 194/195 enhances the cholesterol transport capacity to at least 40-50% [2]. Significant down regulation in phosphorylated StAR levels observed in the present study suggest decreased pStAR could be responsible for down regulation of steroidogenesis in the adrenal gland of Roundup ® -treated rats. Interestingly, in the present study higher lipid droplet accumulation was observed in Roundup ®treated rats and this is one of the characteristic of the StAR gene knockout mice [17]. The finding that phosphorylated CREB was lower, may be correlated to StAR expression down regulation, since CREB phosphorylation is involved in StAR transcription [24]. However to what extent that CREB down regulation contributed to decrease in StAR mRNA levels, remains to be explored. It should be pointed out that in the adrenal gland, ACTH upon binding to its cognate receptor Mc2r, activates cAMP/PKA pathway leading phosphorylation of CREB and StAR proteins [2]. In the adrenal gland of Roundup ® -treated rats, the lowered PKA activity was reversed post ACTH treatment confirming cAMP/PKA pathway to be disrupted in the adrenal gland of Roundup ® -treated animals.

Roundup ® alters cholesterol homeostasis moderately
Roundup ® -treated animals did not show altered total cholesterol levels in circulation at the lowest dose i.e., 10 mg/kg bw/d, but it was observed to be moderately higher in the adrenal gland. With this observation, it can be hypothesized that the cholesterol homeostasis within gland may be altered by increased cholesterol intake and/or de novo synthesis. Interestingly, there was down regulation of genes associated with cholesterol intake (Ldlr, Srb1) and de novo synthesis (Hmgcs, Hmgcr). Also, analysis revealed higher levels of esterified or stored form of cholesterol in the adrenal gland of Roundup ® -treated rats. The data taken together suggest that increased levels of stored cholesterol might be due to lowered utilization and/or lowered hydrolysis of esterified form. Hsl expression was not significantly altered in the present study. Hsl or lipe gene is involved in cholesterol ester hydrolysis [22] and reported to be regulated by cAMP/PKA pathway [20]. Therefore, Roundup ® appears to act via cAMP/PKA pathway and regulate StAR phosphorylation negatively leading to decrease in cholesterol utilization and increase in cholesterol stored in adrenal glands.
Interestingly, increase in the weight of adrenal gland was observed in Roundup ® -treated animals, but its significance is not clear. The study examining diethylstilbestrol effects on the adrenal gland steroidogenesis has suggested steroid metabolic changes to be the contributing factor for increased weight [16].

Roundup ® acts via HPA axis
Since Roundup ® treatment at a dose of 10 mg/kg bw/d decreased corticosterone levels, it became of interest to examine the responsiveness of adrenal gland to exogenous ACTH treatment. The findings that the adrenal gland was responsive to ACTH treatment in Roundup ® treated animals suggest that Roundup ® acts at a site higher than the adrenal gland and this indirectly sug-gests that ACTH synthesis and/or release may be affected. Since the adrenal gland responsiveness to external ACTH was found to be similar or higher compared to vehicle treated animals suggest that the process of steroidogenesis in the adrenal gland appears to be intact post herbicide treatment. Therefore, it can be inferred that the stimulation of adrenal gland i.e., ACTH synthesis and release appears to be impaired rather than defects in the steroidogenesis machinery of the adrenal gland. A significantly higher increase in corticosterone levels in response to supraphysiological dose of ACTH was observed in Roundup ® -treated rats compared to vehicle treated rats is perhaps due to higher stored cholesterol content in the adrenal gland of Roundup ® -treated animals. The mechanism of action may vary with different experimental system e.g., [33] observed higher testosterone, LH and FSH concentrations in second generation or pups of Roundup ® -treated Wistar rat dams in contrast to lowered testosterone observed in the present study as well as other studies where adult rats or different cell lines have been studied. Nonetheless, the results are in agreement with a pilot study of glyphosate exposure to Jundiá fish [6]. In sum, the data suggest Roundup ® appears to act at the hypothalamo-pituitary level under in vivo conditions.

Implications of the study
The recent information regarding Roundup ® and its metabolites detection in food, water and in human urine [1] signifies the relevance of toxicological studies as one presented. The findings that Roundup ® treatment down regulates endogenous ACTH, is similar to the condition known as adrenal insufficiency in humans. This condition manifests as fatigue, anorexia, sweating, anxiety, shaking, nausea, heart palpitations and weight loss. Chronic adrenal insufficiency could be fatal, if untreated. A progressive increase in its prevalence has been observed in humans [8], while a very few studies relating to xenobiotic exposure and adrenal insufficiency development have been reported. The present study describes one of the possible mechanisms of adrenal insufficiency due to Roundup ® and suggests more systematic studies, to investigate the area further.


Single-particle cryo-EM of the ryanodine receptor channel in an aqueous environment

Ryanodine receptors (RyRs) are tetrameric ligand-gated Ca 2+ release channels that are responsible for the increase of cytosolic Ca 2+ concentration leading to muscle contraction. Our current understanding of RyR channel gating and regulation is greatly limited due to the lack of a high-resolution structure of the channel protein. The enormous size and unwieldy shape of Ca 2+ release channels make X-ray or NMR methods difficult to apply for high-resolution structural analysis of the full-length functional channel. Single-particle electron cryomicroscopy (cryo-EM) is one of the only effective techniques for the study of such a large integral membrane protein and its molecular interactions. Despite recent developments in cryoEM technologies and break-through single-particle cryo-EM studies of ion channels, cryospecimen preparation, particularly the presence of detergent in the buffer, remains the main impediment to obtaining atomic-resolution structures of ion channels and a multitude of other integral membrane protein complexes. In this review we will discuss properties of several detergents that have been successfully utilized in cryo-EM studies of ion channels and the emergence of the detergent alternative amphipol to stabilize ion channels for structurefunction characterization. Future structural studies of challenging specimen like ion channels are likely to be facilitated by cryo-EM amenable detergents or alternative surfactants.

Membrane proteins have always been attractive, yet challenging subjects in structural biology. Membrane proteins contain specialized domains that are specifically adapted to reside within the hydrophobic environment of the lipid bilayer allowing for the protein to communicate cell signals across the biological membrane. Ion channels are integral membrane proteins that are responsible for movements of ions across the cell membrane and for maintaining the ion concentration gradients within cells, driving numerous physiological processes such as muscle contraction, fertilization, learning and memory. Given the significant role of ion channels in myriad cell functions, knowledge of their high-resolution 3D structures is a critical prerequisite to our understanding of channel functions and molecular mechanisms underlying membrane transport in both normal and disease states. While membrane proteins are abundant, representing 20-30% of the proteome, high-resolution structures solved for them are disproportionally less, ~2% of unique proteins in the PDB to date, proving them to be more difficult targets than soluble proteins. 1 Hindering efforts to uncover the structures of ion channels is their association with the lipid bilayer, the need to extract them from the membrane, their unwieldy size and often dynamic nature. Large protein size, flexibility and membrane-associated domains make X-ray crystallography or NMR methods difficult to apply for structure determination of full-length membrane proteins. To date, single-particle electron cryomicroscopy (cryo-EM) has emerged as an effective and straightforward approach for the study of membrane protein complexes, their interactions and dynamics in different functional states in a vitrified aqueous solution or lipid environment. [2][3][4][5] Recent advances in cryo-EM technology in combination with continuing developments in image processing software have made it possible to obtain structures of protein complexes beyond 5 Å resolution, with some achieving near-atomic resolutions of 3-4 Å, allowing for high-resolution structural models to be derived de novo from cryo-EM density maps. [6][7][8][9][10][11][12][13][14] Applications of these enhanced technologies by singleparticle cryo-EM include protein assemblies within a wide range of molecular weights (~170 kDa -~4 MDa) and complex symmetry. [14][15][16] Among recently reported near-atomic resolution structures are a 3.4 Åresolution structure of the tetrameric TRPV1 ion channel and a 4.5 Å-structure of the -secretase, a ~170 kDa` membrane-embedded protease, determined by single-particle cryo-EM. 13,14 The ryanodine receptor (RyR), a homo-tetrameric Ca 2+ release channel, was one of the first non-icosahedral proteins to be solved by single-particle cryo-EM, in part owing to its massive size of 2.3 MDa. However, despite rigorous efforts spent to investigate structurefunctional characteristics of RyR channels, there are major gaps in our knowledge about the structure of these ion channels, their ion-conducting pore and modulator-binding sites, largely due to the lack of atomic-level structural details for the entire channel assembly. Several low-to moderate-resolution structures of the full-length channel have been solved and some functional regions mapped to the 3D structure. In addition, atomic models of small soluble portions of the channel have also been determined by X-ray crystallography representing ~10% of the entire protein.
Among the obstacles for achieving a high-resolution structure of RyR channels are its inherent flexibility and location within the biological membrane. RyR ion channels can be conceptualized as integral membrane scaffolding protein assemblies that function in tight association with a large array of multiple intracellular modulatory proteins/ligands, interacting with the channel complex in a dynamic manner to provide specific functional feedback. Thus, obtaining biochemically homogeneous and functionally stable channel protein from its native source (i.e. muscle cell) suitable for structure determination by single-particle cryo-EM, remains one of the major challenges in pursuit of a high-resolution structure of the entire RyR channel. Detergents are traditionally used to make membrane proteins water soluble and suitable for Xray crystallography, NMR or cryo-EM. However, detergents tend to destabilize and inactivate membrane proteins. 17 While single-particle cryo-EM remains the most viable methodology for structural analysis of large membrane protein complexes such as RyRs, the presence of detergent in the buffer is an impediment to producing high-resolution cryo-EM structures of membrane proteins. This review will focus on the structure determination of the Ca 2+ release channel by single-particle cryo-EM with an emphasis on cryospecimen preparation. We will discuss how the choice of surfactant may affect cryospecimen preparation and the success of cryo-EM imaging of membrane proteins.

Ryanodine Receptor Biology -From Discovery to Structure
The ryanodine receptor is an intracellular Ca 2+ release channel that resides in the sarcoplasmic reticulum (SR) membrane and is integral to the Ca 2+ dependent signaling process of muscle contraction. In skeletal muscle, type 1 RyR (RyR1) forms a macromolecular complex with voltage-gated Ca 2+ channels, Ca V 1.1, located in the adjacent T-tubule membrane, whereby Ca V 1.1 senses membrane depolarization and transmits a mechanical signal to RyR1 resulting in the release of Ca 2+ ions from SR stores through RyR1. The voltagemediated rapid release of Ca 2+ into the cytosol from SR stores allows for the contractile apparatus to operate, a process called excitation-contraction coupling. Excitation-contraction coupling in cardiac muscle differs in that the voltage sensor is not physically coupled to the Ca 2+ release channel, instead, coupling relies upon Ca 2+ entry through the Ca V 1.2 channel to initiate type 2 RyR (RyR2) openings. Disruption of excitation-contraction coupling can result in several pathological consequences. Mice lacking RyR1 or RyR2 protein expression die either prenatally or perinatal. 18,19 Mutations in the RyR channel protein can cause abnormal Ca 2+ handling and lead to several conditions in humans including Malignant Hyperthermia, Central Core Disease, Multi-minicore Disease, catecholaminergic polymorphic ventricular tachycardia and arrhythmogenic right ventricular cardiomyopathy. 20 Structure-function studies of RyR to date have been conducted in the absence of highresolution information about the molecular arrangement of the channel, yet this level of information is necessary to understand the molecular basis for excitation-contraction coupling and channel dysfunction. The RyR channel functions as a homotetrameric channel with a total molecular mass of 2.3 MDa, each monomer containing ~5000 amino acids. The channel consists of a large cytoplasmic N-terminal region (~80% of the protein mass), several membranespanning segments and a small cytoplasmic Cterminus. The N-terminal domain serves as a scaffold for many regulatory proteins and molecules to bind, including Ca 2+ , ATP, caffeine, Mg 2+ , PKA, ryanodine, calmodulin, FK506 binding protein and Ca V 1.1. The transmembrane region of RyR contains the ionconducting pore across the SR membrane, and based on sequence analysis the channel is predicted to contain an even number of membrane spanning helices per subunit, likely between 4 and 12, encompassed within the amino acid sequence 3985-4940. [21][22][23][24] However, the exact number of transmembrane helices has yet to be confirmed by direct structural analysis of the channel. Three mammalian isoforms of RyR exist (RyR1-3) and have a relatively high amino acid sequence homology (~65%). Divergent sequences between the three isoforms occur in domains known as D1 (residues 4254-4631 in RyR1), D2 (residues 1342-1403) and D3 (residues 1872-1923) and are likely responsible for the differences in regulation by the channels. RyR1 is predominantly expressed in skeletal muscle; RyR2 is predominantly expressed in cardiac muscle, and RyR3 is found in brain and diaphragm.
None of the isoforms are completely tissue specific, although, RyR1 is the most thoroughly studied isoform due to its abundance in fast-twitch skeletal muscle and relative ease of purification. The first visual documentation of RyR1 occurred over 40 years ago where RyR1 was identified using thinsection electron microscopy as a large "foot" structure RyR1 was first identified as large electron-dense "foot" observed between the junctions of T-tubule and SR membranes. 25 Over the next two decades many efforts were made to molecularly identify the structure observed in the triad junctions and its role in muscle physiology.
Eventually, through the use of 3 H-ryanodine, differential centrifugation and biophysical characterizations, RyR1 was solidified as the intracellular Ca 2+ release channel responsible for the release of Ca 2+ preceding muscle contraction. [27][28][29] Due to its relative ease of purification and large size, the structure of RyR1 was investigated by single-particle electronmicroscopy. The first 3D structure was obtained by negative stain microscopy, reveling basic morphological features, albeit in a stained and dehydrated state. 33 The first depictions of RyR1 in the more native, hydrated conditions came ~4 years later by cryo-EM and were solved to ~30 Å resolution. 34,36 Low-resolution structural dynamics of RyR1 gating were described by adding ligands that affect channel open probability to the cryospecimen prior to virtification. 54,55 Several additional low-resolution structures of RyR1 were solved that localized small molecule binding sites (CaM, FKBP12 and imperatoxin) and functional domains on the 3D structure of RyR1. 46,47,[50][51][52][53] Structures of RyR2 and RyR3 isoforms were also determined by cryo-EM and appear similar in nature to RyR1. 43,45 In two decades since the first structure of RyR1 by cryo-EM was determined, ~1 nm resolution structures were obtained and density based models of channel gating were proposed. 37 spanning the gap between skeletal muscle T-tubule and SR membranes (Fig. 1). 25 Initially, the composition and role of the foot structure was unclear until biochemical isolation and characterization led to its identification as the Ca 2+ release channel, which was greatly facilitated by the protein's ability to bind the plant alkaloid ryanodine with high affinity and specificity. [26][27][28][29][30][31] Early studies on purified single channels by electron microscopy provided a snapshot into the channel ultrastructure and basic quaternary features, followed by the first 3D reconstruction of negatively stained channel particles determined to ~40 Å resolution. 32,33 These studies paved the way for determining the structure of the detergent-solubilized RyR1 under frozen, hydrated conditions by cryo-EM. Extensive efforts have been made to study the 3D structure of the detergent-solubilized RyR1 by using electron microscopy of both single particles [34][35][36][37][38][39][40][41] and 2D crystals. 42 The first structures of RyR1 by single-particle cryo-EM were generated for the 'closed' Ca 2+depleted state by two different groups to ~40 Å resolution elucidating its now characteristic mushroom-shape appearance 34,36 The general structure of the RyR1 channel exhibits 4fold symmetry with an overall square-mushroom shape top (280 Å × 280 Å × 120 Å) connected to a stalk structure (120 Å × 120 Å × 60 Å) (Fig 2). Much of RyR1's mass is contained in the cytoplasmic domain and is connected to the transmembrane domain through the stalk structure that is composed of four column domains and rotated ~40° with respect to the cytoplasmic domain. The cytoplasmic region contains several cavities and globular sub-regions with highly flexible clamp-shaped structures present at the corners. Structures of the RyR2 and RyR3 isoforms determined by cryo-EM have an overall architecture similar to that of the RyR1 channel. [43][44][45][46][47][48][49] The cytoplasmic portion of RyR1 serves as a scaffold for many of the channel regulators. Several domains 46,47,50 and modulator binding sites (e.g. FKBP12, calmodulin, imperatoxin A have been mapped to the 3D structure of the channel. [51][52][53] While these structures were determined at low resolutions, they have provided an abundance of information concerning the regulation of the channel. Moreover, in cases where the binding/regulatory sequences are known, localization allows for the assignment of sequence to the 3D structure. Conformational changes associated with RyR1 channel opening were also addressed by cryo-EM. Based on biochemical and electrophysiological studies, RyR channel gating can be controlled by the presence of Ca 2+ , adenine nucleotides and ryanodine. The addition of channel activators to the RyR1 cryospecimen permitted samples to be driven into a channel 'open' state, allowing for the investigation of the molecular motions associated with several states of channel gating. 54,55 These studies detailed the first direct evidence of the channel's long-range structural changes under activating conditions revealing general allosteric mass rearrangements that occur with channel gating. At ~30 Å resolution the RyR1 structure exhibited a global conformational change, opening of the cytoplasmic clamp domains, mass depletion and a 4 twist of the transmembrane domain, twisting like the iris of a camera. 54,55 Our current knowledge of RyR1's structure has progressed from a rather hollow appearing cytoplasmic domain nested atop a dense stalk containing the transmembrane domain to moderately high resolution structures where some secondary structure features are detectable, domain and subunit boundaries are more clearly distinguishable and a putative pore region is described. From the initial data set by Serysheva et al. 36 algorithmic improvements in image processing correcting for microscope aberrations using CTF correction resulted in a 14 Å resolution map of RyR1 38 (Fig. 2, left). The improved map exhibited more structural detail and allowed for generation and fitting of a homologous structural model for the RyR1 Nterminal domain (residues 216-572) within the clamp region. The arrangement of -helices in the transmembrane domain were still ambiguous in the 14 Å resolution density map of RyR1, however, two groups achieving 10.2 Å and 9.6 Å resolution structures (Fig. 2 37 Subtle differences in handling the protein, such as freeze-thawing the sample prior to vitrification, cryospecimen preparation or imaging conditions may be at the root of such ambiguity, and higher resolution structures are ultimately necessary to address the transmembrane domain helical arrangement and gating mechanism. Furthermore, modeling of ion permeation pathway of the RyR channels has substantially advanced our understanding of RyR1 gating and ion conduction. 99,100 While these studies are highly important and combine vast research data on RyR function and based primary on available high-resolution crystal structures of the KcsA/KvAP/Shaker channels, they are out of scope of this review and will not be discussed here. Although all of the previous studies used conventionally accepted methods for resolution determination, currently a more stringent estimation of resolution via the 'gold standard' method has now been adopted by the cryo-EM field making the highest resolution maps currently available for RyR1 more appropriately estimated at ~12-15 Å resolution. 56,57 It is also critical that as cryo-EM produces higher resolution structures that these maps be evaluated for the resolvability of features at the claimed resolution whereby -helices should appear as rod-like densities at ~9 Å and a helical pitch detectable at ~6-7 Å resolution; β-sheets should appear as planar density at 8-9 Å resolution and strand separation should occur at ~5 Å resolution or better; connectivity between structural features should be identifiable at ~6 Å resolution or better and at ~4 Å resolution bulky side chain densities will begin to appear and the path through the density should be clearly resolved. 57,58 Prior to any soluble domains of the RyR1 channel solved by X-ray crystallography, bioinformatics and density-constrained comparative modeling were utilized to generate a homology model of the RyR1 Nterminal domain (residues 12-572) based on its high structural homology to the N-terminus of the IP3 receptor, which contains the IP 3 binding and suppressor domains. 39 This model was later confirmed by X-ray crystallography of the RyR1 N-terminus. The N-terminal structure contains three domains composed of two β-trefoil structures (residues 1-205 and 206-394) followed by a bundle of five α-helices (residues 395-532). 59 While structurally similar to the IP 3 binding domain, RyR1 channel cannot bind or be regulated by IP 3 . However, over 30 disease-associated mutations are found in the RyR1 N-terminal domain that are proposed to affect channel function by protein misfolding or destabilization of inter-and intradomain interactions. Fitting of the X-ray structure into the RyR1 cryo-EM density maps yielded two feasible locations, one in the clamp domain and one in the central cytoplasmic vestibule. 39,59 A structural model of an additional functional domain of the channel was determined by X-ray crystallography for RyR1 residues 2734-2940, which contains 11 disease-associated mutations and a PKA phosphorylation site. 60 The domain is a two-fold symmetrical structure with each half containing two αhelices, one or more short 310 helices and a C-terminal β-strand with the halves separated by a long, flexible loop containing the phosphorylation site. This structure was computationally fitted into the clamp domain of the cryo-EM 3D structure of the tetrameric RyR1 channel. 60 However, caution should be used when docking small domains into very large density maps. 61 At intermediate resolutions (coarser than 1 nm), secondary structure elements are marginally detectable and remain unresolved in a majority of protein densities, resulting in too little structural information to anchor and unambiguously validate fits. In this situation, the validity of the X-ray model fit-to-density is limited by the resolution of the cryo-EM density map and the low resolvability of features in the map can leads to an imperfect match. It is clear that a sufficiently high-resolution cryo-EM map of the fulllength channel (<1 nm resolution based on the gold standard criteria), such that secondary structures become better resolved, is required to reconcile the placement of domains and to provide a structural context for RyR1 function. While the current state of the art technology is available for producing nearatomic resolution structures using cryo-EM, cryospecimen preparation is now, more than ever, key to achieving improved resolution density maps for RyR1.

Cryo-EM Imaging of Membrane Proteins in Detergents
Due to recent technical advances in the cryo-EM field, the achievable resolution for membrane proteins is now 3-7 Å, 13,14,62 however vitrification of membrane protein samples is a critical rate-limiting step. In single-particle cryo-EM, the sample is preserved in a layer of vitreous ice and visualized in the form of isolated unordered particles. In general, to achieve a 3D structure using single-particle cryo-EM, the object that is vitrified must be monodisperse in solution and exhibit random orientations. A 3D reconstruction can be obtained through image processing by combining of thousands of 2D projection images of the 3D object being imaged. Integral membrane proteins naturally reside in a lipid bilayer satisfying the hydrophobicity of their transmembrane domain. Thus, to achieve vitrification conditions for cryo-EM visualization, membrane proteins are extracted from their native membrane environment and imaged in detergentbound forms in aqueous solution. 17 However, the loss of the structural support provided by the native membrane environment can lead to destabilization and inactivation of membrane proteins, which contributes a significant barrier to membrane protein research in general. 63 Moreover, cryo-EM studies confront the additional hurdle of the presence of detergent in the sample, that can prevent the particles from being distributed evenly in the embedding ice and dramatically reduce the image contrast critical to producing reliable and high-resolution cryo-EM reconstruction. 64 Single-particle cryo-EM studies of membrane proteins are substantially reliant on the successful solubilization and purification of monodisperse, isotropic solutions of the target protein. Integral membrane proteins exhibit a complex distribution of surface charges where the hydrophobic transmembrane domains are separated by intervening hydrophilic regions that are typically exposed into aqueous intra-or extra-cellular environment. Thus, membrane proteins extracted from their membranous environment will not be watersoluble unless they are placed in a surrounding environment compatible with their hydrophobicity. Due to their amphipathic nature, detergents allow for the removal of membrane proteins from a lipid environment while maintaining their solubility in aqueous solution. Detergents bind to hydrophobic transmembrane domains of membrane proteins and form an amphipathic belt, solubilizing the protein and substituting for the lipid bilayer. 17 These detergent belts are in rapid equilibrium with free detergent monomers and detergent micelles. Thus, the molecular properties and physical behavior of the detergent in the form of micelle and protein-detergent complexes are critical parameters to be considered and controlled in determining the success of single-particle cryo-EM experiments. The primary difficulty encountered in the singleparticle cryo-EM study of detergent-bound membrane proteins is preparation of cryospecimen with particles being evenly distributed within a thin layer of vitreous ice. In general, vitrification of proteins depends on environmental conditions such as temperature, humidity, buffer composition, pH, ionic strength, and surface properties of a supporting film on EM grid. While recent development of semi-automated devices have significantly improved the search for optimal conditions for ice-embedding, the presence of detergent in the membrane protein sample adds to the complexity of the system by changing the surface properties of its components. The presence of detergent in solution reduces the surface tension of the buffer making it difficult to obtain uniform ice film with evenly distributed vitrified particles of membrane protein. Particles that do not adhere to the grid or form a narrow peripheral band close to the edge of the hole in the cryospecimen is a common obstacle in singleparticle cryo-EM studies of membrane proteins. This is disadvantageous to cryo-EM data collection since imaging fields often yields only few membrane protein particles per frame. To overcome this difficulty, a continuous carbon film is often used to improve adhering particles to the EM grid (reviewed in 65 ). However, in the presence of carbon supporting film, the RyR1 channel particles exhibit a strong preferred orientation with a majority of particles appearing along the four-fold channel axis. While oblique views are certainly present within the cryospecimen and can be retrieved via iterative image processing procedure, 36 their presence is highly variable and specimen dependent. It requires a collection of substantially larger data sets to bring these oblique views to statistical significance in order to achieve reliable isotropic reconstruction of RyR channels. Another difficulty in cryo-EM study of membrane proteins is that the presence of detergent in the cryospecimen substantially reduces signal to noise ratio in EM images, which is already considered very low for single-particle studies. This effect is particularly disastrous for the high-resolution signal where contrast is marginal even without detergent. For optimal stability of protein-detergent complexes, detergent concentrations are kept near or above the critical micelle-forming concentration (CMC), meaning the buffer is nearly saturated with detergent molecules, which produces a tremendous level of 'noise' in cryo-EM images and can reduce image contrast. This is by far a limiting aspect of cryo-EM visualization of membrane proteins in detergent. This point has not been widely understood in the cryo-EM community since the detergent also produces very strong Thon rings, giving such images an appearance of very good quality when judged by its power spectrum. While there is no accepted standard for assessing a minimal acceptable contrast level in the raw cryo-EM images, it is evident that as contrast is reduced, image processing becomes more susceptible to a model bias and producing a reliable 3D reconstruction becomes problematic. 64 Clearly, the physical behavior of detergents and properties of protein-detergent complexes must be considered and controlled during cryo-EM experiments. However, despite the increasing number of structures of membrane proteins solved by singleparticle cryo-EM, ice-embedding procedure for membrane proteins remains most difficult due to the lack straightforward, reproducible methodologies and strategies. The choice of the detergent chosen for the disruption of lipid bilayer and extraction of the membrane protein of interest is rather more empirical than knowledge-driven procedure. In light of the complexities of protein-detergent interactions, it is important to preserve structure and function of membrane proteins while maintaining their solubility.
However, no 'magic bullet' detergent exists for handling all membrane proteins in a manner suitable for single-particle cryo-EM experiments, and often trial-and-error is the only way to tell. Examples of successful single-particle EM studies of ion channels and some other integral membrane proteins are given in Table 1. Despite the fact that critical information on vitrification of membrane proteins is unpublished, bits of lore can be extracted from these studies to rationalize cryo-EM experiments with membrane proteins. The general conclusion is that detergents with low CMC and high aggregation number are often not compatible with single-particle cryo-EM (Table 2). Through our experiences with membrane proteins and cryo-EM we found that single-particle cryo-EM experiment should be performed at or just below CMC to improve adhesion of membrane protein particles within a layer of vitreous ice on the EM grid yet maintain a soluble membrane protein. Having too little detergent in solution can shift the equilibrium to favor detergent monomers over micelles, leading to unprotected transmembrane domains and protein aggregation while, too much detergent may dissociate some stabilizing lipids required to maintain the protein's structure and affect ice-embedding procedure. Overall, selection of the appropriate detergent for the study of membrane protein by single-particle cryo-EM requires the consideration of how it will impact the protein activity, quaternary structure and its propensity for vitrification. The limited current understanding of protein/detergent systems and forces that maintain the sample in a vitreous ice have frustrated efforts of many researches to exploit the structure of membrane proteins using single-particle cryo-EM and stimulated the developments of approaches to transfer membrane proteins to a more tractable environment that would satisfy the hydrophobic nature of the transmembrane domains and allow for reproducible successful vitrification. 5,[66][67][68] Alternatives to maintaining membrane proteins in non-detergent aqueous solutions, like amphipols, have begun to prove useful in cryo-EM.

Imaging of Membrane Proteins in Detergent-free Aqueous Solution
To circumvent detergent-imposed difficulties in singleparticle cryo-EM studies of membrane proteins, amphipols (Apols) have recently been adopted in cryospecimen preparation with much success. Cryo-EM structures of Apol bound TRPV1 channel and secretase complex were solved at 3.4 Å and 4.5 Å, respectively, resolving their transmembrane domains at resolutions where the cryo-EM density maps served as the basis for generating de novo molecular structures. 13,14 The use of Apols allows for a detergent-free approach to maintain membrane protein complexes in solution and provides several benefits for attaining high-resolution structures by single-particle cryo-EM. Apols are a new class of amphipathic polymers that are designed to substitute for detergents, keeping membrane protein complexes soluble and functional in aqueous solution. [69][70][71] Apols are considered more mild surfactants than detergents and exhibit a very high affinity for transmembrane surfaces due to a large number of hydrophobic chains. Apols interact noncovalently forming a protective belt around the transmembrane domain, and yet they bind almost irreversibly in the absence of detergent (≥ CMC) and have an exceedingly low rate of spontaneous desorption. 72 However, Apols are weak membrane solubilizing surfactants and detergents are still necessary to extract membrane proteins from the lipid membrane. Detergents can easily be exchanged for Apols and removed from solution allowing for the membrane protein to be in a milder detergent-free aqueous environment while preventing protein aggregation and preserving protein structure-function stability.69-71 Membrane proteins are structurally flexible by the very nature of their function, which can often be a detriment to producing a structurally homogenous sample. Apols have been shown to stabilize transmembrane domains and require virtually no excess solution concentration once they have been substituted for detergent. 70,[73][74][75] Based on these favorable properties, Apols are a promising option in the pursuit of membrane protein structures by cryo-EM. Amphipol 8-35 (A8-35), one of the most wellcharacterized Apol, appears to be particularly amenable for single-particle cryo-EM studies of ion channels. 70 Our group initiated studies of RyR1 in complex with A8-35, a highly water-soluble derivative of polyacrylic acid. 70,76,77 In cryo-EM studies, trapping detergent-solubilized RyR1 with A8-35 allowed reproducibility to vitrify RyR1 channel within the holes of a holey-carbon grids with particles well dispersed throughout the ice (Fig. 3A,B). Our initial studies of the vitrified RyR1/A8-35 complex showed a strongly preferred particle orientation, similar to RyR1 protein frozen on supporting continuous carbon-film. The mechanism behind why RyR1 adopts a preferred orientation in comparison to other membrane proteins, like the IP3R channel, is unknown, however, RyR1's complexity of surface charges combined with its awkward organization in 3D space due to its enormous cytoplasmic domain (>80% of protein mass) likely play a role in driving the preferred orientation. Furthermore, in the presence of A8-35, we believe that the polymer forms a negatively charged film at the water-air interface during sample application to the EM grid resulting in channel particles in a preferred orientation. The addition of octyl-glucoside (OG) in a ATPase 0.6 5335 9.7 DDM 0.02% 2.3 (96) 0.9 5476 11 DDM 0.03% 3.4 (97) loss of signal was observed. 72 Similar approaches for adding small amount of detergent have been used in other imaging projects to reduce preferred orientation. 7 Another approach is the use of short-chain phospholipids, such as DHPC, that are believed to not strongly interact with the membrane protein but can form a monolayer at the air-water interface to reduce surface interactions with the Apol. 78,79 Such monolayers represent a very low volumetric concentration and should not have any significant impact on image contrast. Increased image contrast in the presence of Apol is particularly critical in the pursuit of high-resolution structures of membrane proteins in general. In cryo-EM images of Apol bound RyR1, the background observed is dramatically lower than in images with detergent present. The Apol bound RyR1 data has ~2-6% contrast at 5-10 Å resolution as compared to 0-2% in the presence of CHAPS using a traditional CCD detector. Advancements in cryo-EM imaging detectors have led to an increased contrast in at high to intermediate resolutions and are one of the reasons the field is seeing an increase of near-atomic resolution structures solved by cryo-EM. With new technology in place, a more stabilized protein with higher image contrast in detergent-free solution will greatly benefit the structural studies of the RyR1 channel complex. In order to address the structure-function relationship using cryo-EM, it is important that proteins are assessed for their functionality in solution, when possible. We have tested the functionality of RyR1/A8-35 complexes by [ 3 H]-ryanodine binding assay, which yielded a Kd of 1.99 nM and Bmax of 60.4 pmol/mg, similar to that of purified RyR1 in the presence of 0.4% CHAPS (Fig. 4). 80,81 Since, ryanodine binds specifically and with high affinity preferentially to the open state of the intact RyR1 channel, this radioligand binding assay can be considered as an indirect measure of channel activation and indication of its tetrameric structure. 30 It is evident by recent structural studies by cryo-EM that Apols have a major advantage in stabilizing membrane proteins in aqueous solution, reducing detergent-based background in cryo-imaging and maintaining functionality. Determining the structure of many challenging membrane proteins, like the Ca 2+ release channel, by cryo-EM will no doubt be well served by detergent alternatives like Apols.

Future outlook
While much progress has been made in the elucidation of the structure of RyR1 by cryo-EM and X-ray crystallography, there is still a need to pursue atomic level resolution structures of the channel in order to fully understand channel function. Cryo-EM and image processing technology, with their continuing advancements, are in their prime to be able to produced atomic-level details in density maps that can be directly utilized for de novo model building. Cryospecimen preparation, especially for membrane proteins, now appears to be a major bottleneck in the cryo-EM experiment. It is a formidable challenge to produce biochemically homogeneous and structurally stable large membrane protein complexes like the Ca 2+ release channel and is compounded by the structural flexibility inherent to RyRs complex allosteric regulation. The successful use of detergent alternatives, like Apols, will undoubtedly energize the field of the membrane protein structural biology by cryo-EM. The momentum in single-particle cryo-EM is currently remarkable and near-atomic resolution structures of the entire Ca 2+ release channel should be expected in near future.


extfieldsofstudy: Medicine Biology

text:
g-FLUA2H: a web-based application to study the dynamics of animal-to-human mutation transmission for influenza viruses

g-FLUA2H is a web-based application focused on the analysis of the dynamics of influenza virus animal-to-human (A2H) mutation transmissions. The application only requires the viral protein sequences from both the animal and human host populations as input datasets. The comparative analyses between the co-aligned sequences of the two viral populations is based on a sliding window approach of size nine for statistical significance and data application to the major histocompatibility complex (MHC) and T-cell receptor (TCR) immune response mechanisms. The sequences at each of the aligned overlapping nonamer positions for the respective virus hosts are classified as four patterns of characteristic diversity motifs, as a basis for quantitative analyses: (i) "index", the most prevalent sequence; (ii) "major" variant, the second most common sequence and the single most prevalent variant of the index, with at least one amino acid mutation; (iii) "minor" variants, multiple different sequences, each with an incidence (percent occurrence) less than that of the major variant; and (iv) "unique" variants, each with only one occurrence in the alignment. The diversity motifs and their incidences at each of the nonamer positions allow evaluation of the mutation transmission dynamics and selectivity of the viral sequences in relation to the animal and the human hosts. g-FLUA2H is facilitated by a grid back-end for parallel processing of large sequence datasets. The web-application is publicly available at http://bioinfo.perdanauniversity.edu.my/g-FLUA2H. It can be used for a detailed characterization of the composition and incidence of mutations present in the proteomes of influenza viruses from animal and human host populations, for a better understanding of host tropism.

Introduction
Influenza has a history as one of the world's most serious pathogens, with yearly regional infections and episodic global pandemics. Influenza viruses belong to the Orthomyxoviridae family of RNA viruses and occur as three major types: influenza A, infectious in birds and some mammals, including humans [1]; influenza B, a genus that infects only humans and seals [2]; and influenza C, a rare type that is known to have infected human and pigs [3].
Proliferation of influenza A is predominantly in avian hosts with very rapid mutation, resulting in a "quasispecies" [4], a vast number of viruses that are genetically related but differ in the amino acid sequences of the viral proteins. These mutations are a classic example of Darwinian evolution [5]; they occur in a random fashion and the variant viruses that have the best genetically endowed combination of efficient infection, rapid replication, and greatest survival become the dominant populations. The survival properties include virus escape from the immune responses of humans previously infected or immunized with an earlier virus strain.
Human infections by influenza A viruses commonly occur yearly, with a seasonal peak incidence [6], usually as a mild disease, but for some, as a more severe illness that may be fatal. A major complication is the occurrence of global pandemics resulting from the emergence of highly infectious subtypes of the virus, particularly those capable of human-to-human transmission. The history of influenza pandemics began with the H1N1 "Spanish Flu" strain of 1918-1919 that killed an estimated fifty million people [7]. This was followed by other less severe strains, the H2N2 "Asian influenza" of 1957-1958, H3N2 "Hong Kong flu" of 1968-1969, and H5N1 "bird flu" in 2006-2007, and recently the H1N1 "swine flu" of 2009-2010. It thus appears inevitable that, without means to prevent influenza infection, another pandemic will occur within the foreseeable future. It is simply a matter of chance that the mutations responsible for the infectivity and pathogenicity of a particular influenza virus in animals does not include the ability to efficiently infect humans with human-to-human transmission. If a strain comparable to that of the 1918 pandemic was to occur, the global consequences are inconceivable.
Sequence change (mutation) events can transform an animal-origin virus into a human virus, with varying levels of fitness to survive in the new host [7]. A number of mutations that facilitate the transmission from animal to human hosts have been described for the influenza A viral proteome. For example, specific amino acid substitutions can alter the host receptor binding site and specificity of hemagglutinin (HA) protein, a major determinant of host tropism, for preference from avian to human sialic acid linkages [1]. Additionally, a single amino acid change at position 627 of the viral polymerase complex subunit PB2 was found to enhance replication in human host [8]. There remains, however, the need for a comprehensive identification of sequence changes that allow or enhance human infection, with potential applications to influenza virus surveillance and possibly to prevention or treatment of human infection.
A number of computational models have been reported that discriminate between animal and human influenza A viruses based on molecular patterns in protein sequences [9][10][11][12] and genomic signatures [13]. Although these models are potentially useful for predicting interspecies transmission of influenza viruses, there is still a great difficulty in deciphering adaptation of viruses that show mixed signatures of both animal and human hosts. There remains the need for a greater understanding of the viral sequence diversity and the dynamics of sequence change, including the composition and incidence of mutations between the viral host populations.
Herein, we present g-FLUA2H, a web-based application, with a grid-backend, to analyze the dynamics of animalto-human (A2H) mutation transmission for influenza viruses. The application only requires the viral protein sequences from both the animal and human host populations as input datasets. g-FLUA2H is facilitated by a grid back-end to manage large datasets, as there are currently more than 500,000 sequences of influenza A virus reported in the public Influenza Research Database (IRD; http://www.fludb.org/). g-FLUA2H is publicly available at http://bioinfo.perdanauniversity.edu.my/g-FLUA2H. Figure 1 provides a schematic workflow summarizing the methodology employed by g-FLUA2H. Input protein sequences, in FASTA format, can be downloaded from the publicly available Influenza Research Database (IRD; http://www.fludb.org) for all worldwide, recorded animal and human influenza viruses. There is a specific format for the FASTA file header that needs to be followed: "> Strain name information | Accession number | Protein name" where the strain name is essential and follows the influenza nomenclature by WHO in the format "Antigenic type/Host/Location of sampling/Isolate ID code/Year of sampling". There is no specific format for accession number and protein name, both of which are optional. An example of a FASTA header format is "> A/blue_winged_teal/Ohio/566/2006 | A7IRT3 | HA". Instructions on how to download sequences from the FluDB in this header format are provided on the help page of g-FLUA2H.

System description
Both full-length and incomplete partial sequences are considered for the analyses. Partial sequences are included because they provide additional data, however, they can be a source of spurious multiple sequence alignment, which require manual editing to correct the misalignment. Duplicate sequences for each protein are removed if present. The non-redundant sequences of each protein from both the animal and human hosts of the virus are then merged for a co-alignment by use of a local copy of MAFFT (obtained from http://mafft. cbrc.jp/alignment/software/); MAFFT is one of the most accurate multiple sequence alignment methods currently available [14]. The co-alignment provides for a unified alignment position, allowing comparison between the two host populations. Users are provided with parameters to disable the removal of redundant sequences and to remove alignment positions with a high fraction of gaps (insertions or deletions), considered to be of low statistical support. Additionally, users are provided with the option to upload manually edited co-alignments as input, in TALN format, if necessary. The sequences of each host population are eventually separated from the co-alignment for a position-by-position comparative analyses.
The comparative analyses is based on a sliding window approach of size nine (1-9, 2-10, 3-11, etc) on the separated alignments [15][16][17]. Each of the aligned overlapping nonamer (9-mer) positions represent a possible antigenic core binding domain for human leukocyte antigen (HLA; human MHC) molecules and T-cell receptors [16,17]; this assumption is based on the fact that there are large array of HLAs with different binding specificities in the human population [17]. Further, the repeated associations of each amino acid in a moving, overlapping 9-mer window can facilitate the detection of possible sequencing errors. The sequences at each of the 9-mer positions are classified as four patterns of characteristic diversity motifs that we previously defined in Hu, Y. et al. [18] and discussed the biological implications. In brief, the motifs are: (i) "index", the most prevalent sequence; (ii) "major" variant, the second most common sequence and the single most prevalent variant of the index, with at least one amino acid mutation; (iii) "minor" variants, multiple different sequences, each with an incidence (percent occurrence) less than that of the major variant; and (iv) "unique" variants, each observed only once. The quantification of the diversity motifs is detailed in [18] and also described on the g-FLUA2H help page (see Section D). The diversity motifs and their incidences at each of the nonamer positions allow evaluation of the mutation transmission dynamics and selectivity of the sequences in relation to the animal or human hosts. Distinct sequences of any motif that contain gaps (-) or any of the unresolved characters, including B (asparagine or aspartic acid), J (leucine or Isoleucine), X (unspecified or unknown amino acid), and Z (glutamine or glutamic acid) are excluded from all analyses.
The output page of g-FLUA2H provides users with a dropdown menu listing all the nonamer positions to view motif data compared between the animal and human hosts of the virus. The selection of a given nonamer position presents users with a detailed information on the distinct nonamer sequences at the position for both hosts, such as their motif classifications and incidences, amino acid substitutions relative to the index, strain name of origin virus in animal and human hosts, host species of the animal host, and the geographical distribution of the human host. Advanced functionalities include multi-motif analysis and transmission selection where users can analyse for any combination of the diversity motifs and incidences between the hosts, and filter for positive (the incidence of the corresponding motif is greater in human host) or negative selections (incidence of motif is greater in animal host). Snapshots of the key features of g-FLUA2H are provided in Figure 2.
g-FLUA2H was built on top of the gUSE/WS-PGRADE framework [19][20][21][22][23], a grid-backend with multiple CPU cores (several hundreds). This was implemented not as a key distinguishing feature, but for practical purpose to allow ease of handling large datasets, and thus reducing computation time. The g-FLUA2H pipeline employs a combination of parallel compute intensive and distributed compute less-intensive executions ( Figure 3).

g-FLUA2H Application: animal-to-human mutation patterns of amino acid substitution E627K in influenza A H5N1 and H7N9 subtypes
The amino acid substitution E627K in PB2 of the viral polymerase complex has been reported to be crucial for host tropism. Glutamic acid (E) is found at position 627 in most of the avian strains, whereas replacing the amino acid with lysine (K) enhances viral replication in humans [8,[24][25][26][27][28]. The dynamics of this substitution between the animal (avian) and human populations was analyzed and compared by use of g-FLUA2H for H5N1 and H7N9 subtypes, over the time period and the geographical range of the reported data in IRD (Figure 4). There were 2402 (avian: 2130, human: 272) and 127 (avian: 84, human: 43) PB2 sequences of H5N1 and H7N9 reported in IRD as of May 2015 and April 2014, respectively.
g-FLUA2H sample output for a representative nonamer position of H5N1 PB2 that contained this mutation is shown in Figure 4A. At this position (623-631; alignment numbering), there were 21 distinct nonamer sequences representing the reported populations of both avian and human viruses. Both populations contained the same index sequence AAPPEQSRM, present iñ 75% of the 1380 avian and~62% of the 208 human virus sequences reported in the public database for this position. Chicken (~24%) was the dominant avian source species for this shared index sequence, followed by duck (~20%, dropdown menu; number not shown in Figure 4A) and many other host species (~< 4% each). The human strains containing this sequence were isolated primarily in Indonesia (~22%) and from many other geographical regions in Asia.
The other dominant sequence AAPPKQSRM contained a mutation relative to the index that corresponded to the E627K substitution. This sequence was classified as the major variant of the avian index, present in~21% of the avian sequences and corresponded to the human major variant, present in~30% of the human sequences. Chicken (~14%) was also the dominant avian source for this sequence. The human strains containing this sequence were isolated primarily from Vietnam (~9%). Although the E627K substitution has been reported to be important for host tropism, it is clear here that a large majority of H5N1 viruses that infected human did not possess this mutation. Two of the six distinct nonamer sequences of human viruses at this position were not present in the avian viruses, suggesting human specific mutations. Similarly, as many as 15 distinct nonamer sequences appeared to be avian specific, non-existent in the reported human viruses.
In comparison, the corresponding aligned H7N9 PB2 position (623-631) contained only six distinct nonamer sequences representing both the avian and human viruses ( Figure 4B), and all but two (A624D; P626R & E627K) matched to H5N1. However, unlike H5N1, the avian and human viruses did not share the index sequence. The avian index sequence AAPPEQSRM, present in~94% of the 80 sequences reported in the public database for this position, was the same as the human major variant, present in~33% of the 40 human sequences reported for this position.
Chicken (~31%) was the dominant avian source species for this sequence, followed by duck (~16%), wild duck (~14%), and many other host species (< 8% each) (dropdown menu; numbers not shown in Figure 4B). The human virus strains containing this sequence were primarily isolated from various cities in mainland China. Notably, the index sequence of the human viruses (~68%), which contained the E627K substitution (AAPPKQSRM), was found in the avian host population solely as a unique sequence (~1%) in the Tree sparrow species. The other four distinct nonamers were unique sequences (~1% each) and appeared to be avian specific, not present in the human viruses. There was no report of human specific mutations for H7N9 at this position, possibly due to the short history of the virus in the human host. Figure 2 Snapshots of selected g-FLUA2H features. A. Homepage interface of g-FLUA2H, which includes a brief description of the application and links to start an analysis, check job status and get more information from the help page. B. Web-interface to upload input sequence files, parameters to disable the removal of redundant sequences and to remove alignment positions with a high fraction of gaps (insertions or deletions), and an option to receive email notification when the job submission is complete. C. A sample output page, providing users with a dropdown menu listing all the nonamer positions to view motif data compared between the animal and human hosts of the virus. The selection of a given nonamer position presents users with a detailed information on the distinct nonamer sequences at the position for both hosts, such as their motif classifications and incidences (% occurrence), amino acid substitutions relative to the index, strain name of origin virus in animal and human hosts, host species of the animal host, and the geographical distribution of the human host. D. Options for advanced analyses on the results, such as multi-motif analysis and transmission selection where users can analyse for any combination of the diversity motifs and incidences between the hosts, and filter for positive (the incidence of the corresponding motif is greater in human host) or negative selections (incidence of motif is greater in animal host).  In summary, the nonamer sequence (AAPPEQSRM) without the E627K substitution was conserved across both avian H5N1 and H7N9 viruses as the index sequences, with chicken as the dominant avian source species. However, the motif transmission pattern to human differed; it remained as index in human H5N1 viruses, but was a major variant in human H7N9 viruses. The sequence containing the E627K substitution (AAPPKQSRM) was a major variant in both the avian and human H5N1 viruses, and did not appear to be required by all human viruses. In contrast, this substitution was only seen once (a unique variant) in the reported avian H7N9 viruses, but was predominantly observed in the human viruses as the index, possibly indicating a role in host tropism. Furthermore, the analysis identified human specific mutations for H5N1, suggesting further adaptation in the human host. These results merit further investigation (Tan, S. et al., manuscript in preparation), and the small number of sequences for the reported human viruses, particularly for H7N9, should be treated as a caveat, with the possibility of sampling bias.

Conclusion
There is a rich metadata of information that can be obtained from a comparative analysis of animal and human host populations of influenza viruses. The data above reveal a complex dynamic of animal to human mutation transmission patterns. g-FLUA2H provides for a detailed proteome-wide characterization of the composition and incidence of mutations present in the animal and human host populations of influenza viruses, for a better understanding of host tropism and possibly identifying human adaptation mutations that are pan-subtype or subtype-specific. This may contribute to the development of tools for surveillance of influenza viruses, and possibly to efforts for the prevention or treatment of viral infection. Given that zoonosis (human disease caused by animal pathogens) is not unique to influenza viruses, g-FLUA2H may be applicable to other zoonotic diseases, which are mostly viral origin and are emerging and reemerging, such as rabies, Ebola, Rift valley fever, and Crimean-Congo hemorrhagic fever.