Effective reads from all samples were selected from high-quality reads and clustered into operational taxonomic units (OTUs) based on 97% sequence similarity according to MiSeq standard operating procedures. 28 In brief, high-quality reads were selected by removing sequences that lacked V3 –V4 primers or barcode sequences as well as sequences that contained undetermined bases ( 42 bases) or a short variable region ( o90 bp) in the V3–V4 region. For reducing noise signals, a 50-bp sliding window was used to reduce sequencing error in combination with an average quality score of 35 within that window for sequence trimming. High-quality sequences were aligned using the nearest alignment space termination multi-aligner in SILVA-compatible database alignment, 29 and reads that were not aligned to the anticipated reference region were deleted, followed by removal of chimera sequences identi ﬁed using the UCHIME algorithm. 30 All reads were classi ﬁed using a Bayesian classiﬁer with a homemade RDP database. Reads that could not be classi ﬁed at the kingdom level were deleted. Alpha diversity analysis including rarefaction analysis and Shannon index were calculated using QIIME as described before. 31 UniFrac-based principal coordinate analysis was performed and the phylogenetic tree was constructed by inserting the representative of each OTU generated using QIIME. 31 Based on published statistical methods, 22,25 the statistical signi ﬁcance of the separation among animal groups in the principal coordinate analysis score plots was assessed using multivariate analysis of variance test according to UniFrac matrix differences. OTUs with statistically signi ﬁcant differences were analyzed using Tukey ’s post hoc test (SPSS, Chicago, IL, USA). Log 10 -transformed relative abundance of each OTU was used to construct RDA models with Canoco-assessed OTUs that were different between animal groups (Microcomputer Power, Ithaca, NY, USA). Statistical signi ﬁcance was determined using the Monte Carlo Permutation Procedure with 499 random permutations.
feeding. Apart from a reduction in body weight, treatment with aqueous extract of Moringa oleifera leaf was observed to attenuate the levels of total cholesterol and LDL significantly and increased the level of HDL level in micefed on HFD. According to Farooq F et al., the hypolipidaemic effect of different medicinal plants has been related to their bioactive components . A mechanism by which these compounds may decrease plasma cholesterol in our obese mice is that flavonoids that are contained in Moringa leaf extract may augment the activity of lecithin acyltransferase (LCAT), which regulates blood lipids. LCAT plays a key role in the incorporation of free cholesterol into HDL (this may increase HDL) and transferring it back to VLDL and LDL which are taken back later in liver cells  .
Bacterial DNA was isolated from fecal samples from 54 mice from the different groups (30 Sv129 mice: 10 fed the LF diet, 10 fed the HF diet, and 10 fed the HFI diet ; 24 BL6 mice: 7 fed the LF diet, 8 fed the HF diet, and 9 fed the HFI diet) and subjected to whole genome sequencing (WGS) using the Illumina HiSeq2000 plat- form . In total, 200.91 Gb high-quality data were generated with an average of 3.72 Gb (46.10 million reads) per sample. We employed de novo assembly as previously described  to generate a non-redundant gene catalog containing 793,847 genes (Additional file 1: Table S1). A rarefaction analysis revealed a curve ap- proaching saturation with 45 samples, and ICE and Chao 1 indices indicated that we captured 99.53% of the total gut microbial genes in the cohort (Additional file 2: Figure S1). We carried out taxonomical assignment and functional annotation using the Integrated Microbial Ge- nomes (IMG) database (v3.4) , the NR database (v3), and KEGG  database (release 59.0), respectively. 6.53% of the genes in the catalog, which covered 14.85% in gene abundance, could be annotated using the IMG database, while 75.14% of the genes could be annotated using the NR database. At the functional level, we iden- tified 4846 KEGG orthologues (KOs) covering 46.43% of genes. For each sample, an average of 46.9 ± 0.2% (mean ± s.e.m.) of genes could be annotated in the KEGG data- base. The taxonomic and functional profiles were con- structed by summarizing the relative abundance of genes.
ABSTRACT The intestinal microbiota and the expanded endocannabinoid (eCB) sys- tem, or endocannabinoidome (eCBome), have both been implicated in diet-induced obesity and dysmetabolism. These systems were recently suggested to interact dur- ing the development of obesity. We aimed at identifying the potential interactions between gutmicrobiota composition and the eCBome during the establishment of diet-induced obesity and metabolic complications. Male mice were fed a high-fat, high-sucrose (HFHS) diet for 56 days to assess jejunum, ileum, and cecum micro- biomes by 16S rRNA gene metataxonomics as well as ileum and plasma eCBome by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). The HFHS diet induced early (3 days) and persistent glucose intolerance followed by weight gain and hyperinsulinemia. Concomitantly, it induced the elevation of the two eCBs, anandamide, in both ileum and plasma, and 2-arachidonoyl-glycerol, in plasma, as well as alterations in several other N-acylethanolamines and 2-acylglycerols. It also promoted segment-speciﬁc changes in the relative abundance of several genera in intestinal microbiota, some of which were observed as early as 3 days following HFHS diet. Weight-independent correlations were found between the relative abun- dances of, among others, Barnesiella, Eubacterium, Adlercreutzia, Parasutterella, Propi- onibacterium, Enterococcus, and Methylobacterium and the concentrations of anandamide and the anti-inﬂammatory eCBome mediator N-docosahexaenoyl-ethanolamine. This study highlights for the ﬁrst time the existence of potential interactions between the eCBome, an endogenous system of multifunctional signaling lipids, and several intestinal genera during early and late HFHS-induced dysmetabolic events, with potential impact on the host capability of adapting to increased intake of fat and sucrose.
In the current research, dietary treatment with pure polyphenolic compounds, especially when jointly administered, decreased body weight gain. The lack of anti‐obesity effect of quercetin is in agreement with data in the literature, which suggest the need of treatment periods longer than 9 weeks for this polyphenol to achieve an effective body fat lowering effect . As far as resveratrol is concerned, the dose used in the present experiment was effective in reducing body fat after 6 weeks of treatment, but in a model of genetic obesity (Zucker fa/fa rats) [40, 54]. In a model of obesity induced by using the same high‐fathigh‐sucrose diet than that used in the present study, our group previously showed that a dose of 6 mg/kg bw/day resveratrol did not induce changes in adipose tissue weight . By contrast, a dose of 30 mg/kg bw/day was very effective . It seems that in this obesity model a dose higher than 15 mg/kg bw/day is needed to effectively reduce body fat. The dosage used in the current experiment for resveratrol and quercetin was effective to decrease insulin levels and also, to improve insulin sensitivity, these findings support results from previous studies describing resveratrol and quercetin as potential candidates for the treatment of metabolic diseases such as insulin resistance [30, 31]. To our knowledge, few studies have explored the effects of pure resveratrol and quercetin consumption alone or combined, on resident microbiota composition in order to find a possible association between their beneficial properties in the host and specific gut microbial modifications . In contrast, there is strong evidence supporting the role of gutmicrobiota in several metabolic functions . Furthermore, modifications carried out in microbial ecology have been demonstrated to derive in host metabolic phenotype modulation, influencing host biochemistry and increasing host susceptibility to diseases [57, 58]. The obese metabolic phenotype transmissibility in germ‐free mice that were transplanted the gutmicrobiota from obese (ob/ob) mice has been also proven . Nevertheless, gutmicrobiota modifications have been attributed both, to genetic obesity , and to diet‐induced alterations independent of obesity .
Animals. Male wild-type (WT) mice of the C57BL/6J genetic lineage were bred in the speciﬁc- pathogen-free animal (SPF) facility of the Third Military Medical University, Chongqing, People’s Republic of China, and maintained in a temperature-controlled room (22 to 24°C) with a strictly followed 12-h light/12-h dark diurnal cycle with food and water ad libitum. Germfree (GF) mice were created using caesarean rederivation from existing SPF mouse lines. A total of 10 1-day-old GF mice of the C57BL/6J background were bred in the Department of Laboratory Animal Science of the Third Military Medical University and were used as recipients for the fecal microbiota transplantation. Sterile plastic ﬁlm isolators, which help to avoid cross contamination with other bacteria or fungi, were used to house the mice in a completely germfree environment; this provides an environment that allowed us to conduct experiments without competing background levels of microbiota. They were given ad libitum access to sterilized water during the whole course of the experiment. Food and water and other sterile supplies were imported into the isolators by docking autoclaved supply cylinders to a double-door port built into the isolator wall (Fig. S6A). GF foster mice were used to breastfeed 1-day-old GF mice until weaning (which occurred at 3 weeks of age); they were then fed ad libitum with a sterilized normal chow diet for 5 weeks postweaning. Culture and PCR analysis of feces amplifying the 16S rRNA gene were used to routinely test the sterility of GF isolators. The animal experiments were approved by the Animal Care and Use Committee of the Third Military Medical University (Chongqing, China).
Most of the experiment that evaluate the role of diet in the gutmicrobiota and type 2 diabetes, obesity use western type of diet, which is full of processed foods and devoid of fibers . Such foods are filled with calories from the saturation of sucrose and fat, and they illustrate that gutmicrobiota significantly regulates the occurrence of obesity through additional pathways [99, 100]. For instance, research shows that germ- free mice that have been fed with a diet that is low in sucrose and high in fiber show partial protection from obesity, primarily microbiota-dependent obesity . This type of protection is withdrawn upon the omission of sucrose from the diet. For purposes of this analysis, it is very important that the source of the dietary fats be understood. This is because the two types of dietary fats; saturated and unsaturated deliver diverse influence on the gutmicrobiota [102, 103]. Moreover, the modified microbiota caused by intake of unsaturated fats plays a role in protecting human beings from weight gain resulting from such feeding [76, 104]. Subsequently, weight gain becomes inevitable with consumption of unsaturated fats than when saturated fats are consumed. The review of previous
Forty-eight 12-week-old male C57BL6/W mice were transferred to a non-SPF breeding facility and given 2 weeks to acclimate, during which time all animals were fed the standard diet (normal diet [ND]; 10 % of calories from fat) containing 22 % protein and 4.4 % fat (Labo- feed H, Morawski, Poland). Animals were then randomly divided into three groups of 16 mice. Two experimental groups were fed a high-fatdiet (HFD) containing 22 % protein and 30 % fat (Morawski, Poland), while the con- trol group was fed ND. The diets of mice in one HFD group were supplemented with feces (HFDS) excreted by micefed ND. Mice are coprophagic and, therefore, five fecal droppings per recipient mouse were added to the cage every week. Each animal was weighed weekly to an accuracy of 0.1 g. Every 4 weeks, each animal was placed into a metabolic cage with full access to feed and water for 24 h. The feces were collected and stored at −80 °C until use. After 28 weeks, mice were weighed
Veillonella, Prevotella, Fusobacterium, Leptotrichia and promote tumorigenesis in various ways
, but this still needs further study.
He et al. found that, after 12 weeks of HFD, the gastric flora was abnormal and the diversity of the flora decreased . Besides, HFD-induced overgrowth of Enterobacteriaceae can increase endotoxin production, thereby further triggering chronic inflammation and accelerating obesity . The research of Xiao et al. showed that Desulfovibrionaceae may be an important group of endotoxin producers that can produce LPS and induce chronic inflammation and metabolic endotoxemia , which can be observed in the stomach of micefed with HFD. LPS stimulation induces the expression of CXCR7 in gastric cancer and promotes the proliferation and migration of gastric cancer cells through the TLR4/MD-2 signaling pathway . In addition, TLR4/CD74/MIF can also promote cell proliferation . TLR4 signal activation can also promote gastric cancer cell proliferation by generating mROS ; LPS-NFκB-PD-L1 axis can also affect immune escape . Meanwhile, Desulfovibrionaceae reduces sulfate to H 2 S and destroys the intestinal barrier
Although, there was an overall uniformity in the distri- bution of bacterial groups across all samples, one notable exception was the class of Tenericutes, and more specific- ally the genus Anaeroplasma, which were only detected in NC mice. Previously, the presence of Anaeroplasma , was associated with diet-induced obesity . Another notable exception was the Firmicutes genus Turicibacter , which was present in all HC mice but not detected in the NC group. An increase in Turicibacter has previously been correlated to the amount of cecal butyrate in rats fed a barley-malt based diet with a highfat content . How- ever, it has also been found to decrease in response to high-fat feeding in mice  suggesting that Turicibacter might be responsive to components in the diet other than fat. According to our results the increase in Turicibacter could be related to cholesterol abundance of the diet. This suggests a role of the bacterium for assimilation or sequestration of cholesterol, as it has been previously demonstrated for other colonic bacteria of the Firmicutes group . Clearly more research is needed to delineate the pathophysiological importance of these bacteria with low abundance and their potential products for their role in cholesterol metabolism and their potential relevance for the development of cardio-metabolic disease. However, such studies are technically difficult, since the Turicibacter genus consists of strict anaerobes with poor survival under laboratory conditions .
There are millions of microbes in the host intestine, such as bacteria, eukaryotes, and archaea, with bacteria being the most predominant. The colon is the last part of the digestive system and is full of gut mi- crobes; the bacteria concentration is approximately 109–1012 CFU/mL, containing at least 1000 different species . In the adult gut, approximately 90% of bacterial species are from the Firmicutes and Bacter- oidetes phyla . The Firmicutes phylum contains gram-positive bacteria from > 200 various genera, in- cluding Clostridium, Catenibacterium, Eubacterium, Lactobacillus, Faecalibacterium, Ruminococcus, Rose- buria, Dorea, and Veillonella . The Bacteroidetes phylum, which contains approximately 20 genera of gram-negative bacteria, such as Bacteroides, Prevo- tella, Tannerella and Odoribacter, is the second most widespread bacterial phylum. Other less abundant but common phyla of the intestinal flora include Actino- bacteria, Verrucomicrobia and Proteobacteria . The gutmicrobiota plays important roles in physi- ology and metabolism  by extracting energy from indigestible dietary compounds, mediating immunity and synthesizing vitamins. However, more and more research indicates that a change in gutmicrobiota is closely related to a variety of diseases. Abnormal changes in the gut flora usually affect host health by inducing an immune response [14, 17]. Interestingly, numerous studies have demonstrated the role of gut bacteria in obesity and metabolic dysfunction. In fact, some researchers have demonstrated a relationship between gutmicrobiota and the abundance of two bacterial phyla, Bacteroidetes and Firmicutes . The first evidence of a change in gutmicrobiota compos- ition in response to an obese phenotype was shown in genetically obese ob/ob mice; these mice displayed fewer Bacteroidetes and more Firmicutes bacteria . The idea of an obesogenic gut microbial popula- tion emerged when the same authors discovered that the obese phenotype could be transmitted by gutmicrobiota transplantation in mice . The increase in Firmicutes is related to an increase in enzymes that can disintegrate polysaccharides from food and produce short chain fatty acids (SCFA) .
Abstract: Objective: Our aim was to explore the effects of Lactobacillus delbrueckii on blood lipids and gut micro- biota in high-fatdiet-fedmice. Methods: A total of 60 male C57BL/6 mice were divided into four groups (n=15 each): normal control group (CON group), Lactobacillus group (LAC group), high-fat group (MOD group), and high-fat group with Lactobacillus (MOD+LAC group). Intestinal microbiota were analyzed using traditional culturing methods. After 12 weeks, serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were measured using enzymatic methods. Results: Mice in the MOD group were significantly obese, compared with mice in the CON, MOD+LAC, and LAC groups (all P<0.05). TG, TC, and LDL-C levels were significantly higher in the MOD group than those in CON group (P<0.01), while HDL-C was decreased (P<0.01). TG, TC, and LDL-C levels were significantly lower and HDL-C was higher in MOD+LAC group than in the MOD group (P<0.05). Mice in the MOD group had significantly less Lactobacillus, Bifidobacterium, and Enterococcus but an increase of Enterobacter, compared with the other three groups (P<0.05). Conclusion: Our results suggest that Lactobacillus could reduce blood lipid levels in mice, effectively improve their gutmicrobiota, and inhibit occurrence of obesity.
Although industrialized trans fats are being phased out in some countries, many will continue to produce trans-fat-containing foods and little is known about the impact of their consumption on metabolic stress and dysbiosis. The aim of this study was to evaluate the effects of a whey protein hydrolysate (WPH) on HSP expression, inflammation and gutmicrobiota of mice consuming a hyperlipidic diet containing partially hydrogenated oil. Mice were divided into five groups: control (AIN 93-G) diet, two receiving hyperlipidic- hydrogenated-oil diets, but containing either casein or WPH (HOCAS, HOWPH) as sole protein source, and two other groups receiving hyperlipidic-unhydrogenated-oil diets with either casein or WPH (OCAS, OWPH). Results indicated that WPH increased HSP90, HSP60 and HSP25 expressions in animals consuming the hyperlipidic-unhydrogenated oil, while no influence was noticed when the oil was hydrogenated. Contrasting with the unhydrogenated, the hydrogenated oil inhibited JNK phosphorylation in the experimental casein diets though no alteration was observed in IKK. Neither lipid nor protein was found to influence the pro- inflammatory TLR4, CD14, MyD88, NF-B and TNF-α biomarkers. Both lipid sources did impair glucose tolerance, but only when WPH was the protein source, although without altering insulin levels, basal glucose and GLUT-4 translocation. Changes in gutmicrobiota suggest that this hyperlipidic diet does not invert the Bacteroidetes:Firmicutes ratio. We conclude that high intake of trans fatty acids did not trigger inflammation, but antagonized the WPH-induced HSPs expression.
lipids, proteins and DNA, which initiates the processes of atherogenesis through cell dysfunction . In fact, oxidative stress is the unifying mechanism for many CVD risk factors . For example, free radicals med- iate many signaling pathways which underlie vascular inflammation in atherogenesis . However, the dele- terious effects of oxidative stress can be prevented by enzymatic and non-enzymatic antioxidant defense mechanism. In mammals, the most important antioxi- dant enzymes include SOD which converts superoxide to hydrogen peroxide, GPx and CAT which are respon- sible for converting hydrogen peroxide to water . As a very important non-enzymatic antioxidant, GSH can react directly with free radical or act an electron donor in the reduction of peroxides catalyzed by GPx . In addition to scavenge free radicals directly, supplement of these micronutrients in the present study also signifi- cantly elevated plasma activities of SOD, CAT and GPx as well as level of GSH, which led to the remarkable increase of T-AOC and were thus favorable to attenuate oxidative stress. As a result, plasma lipid peroxidation levels were significantly decreased with the supplement of these micronutrients. In accord with these findings, these micronutrients naturally abundant in rapeseed oil have been reported to increase antioxidant status and lipid peroxidation in plasma  as well as in brain . Hyperlipemia is also well known to be closely linked to arteriosclerosis and other cardiovascular disease. It has been reported that rapeseed oil possessed hypolipi- demic properties [7,31] and which could be due to its optimum fatty acid composition. In line with expecta- tions, the further reduction in plasma TG, TC and LDL- C were observed with the fortification of rapeseed oil with micronutrients in this study. The micronutrients phytosterols and polyphenols were both responsible for these beneficial changes. Many studies have shown that intake of phytosterols is effective at lowering plasma TC and LDL-C. Phytosterols have a similar chemical struc- ture with cholesterol but themselves are absorbed only in trace amounts , thus they inhibit cholesterol absorption including recirculating endogenous biliary cholesterol which is a key step in cholesterol elimination . These meant that although rodent diet contained little cholesterol in this study, inhibition of intestinal cho- lesterol absorption was still the main mechanism respon- sible for the cholesterol-lowering effect of phytosterols. Besides, the hypolipidaemic effect of the phytosterols were also associated with the down-regulation of hepatic acyl CoA:cholesterol acytransferase activity  and the increasing LDL receptor expression . Polyphenols have been shown to reduce plasma TG, TC and LDL-C by altering hepatic triglyceride assembly and secretion, cholesterol absorption and the processing of lipoproteins in plasma . Although these micronutrients did not
Obesity is primarily a consequence of unhealthy diet and lack of physical activity and the modern best-practice treatment of obesity includes a low-fatdiet, increasing physical activity and behavior modification. A recent study found the use of functional foods which have been reported to reduce the overall cardiovascular risk in- duced by dyslipidemia by acting in synergy with statins . Exercise is an effective approach for prevention and treatment of obesity which could improve multiple in- dexes of human body. PGC-1α, which was first de- scribed in 1998, is reported to strongly correlate with adaptations induced by exercise training . Subse- quently, it was found that exercise induced PGC-1α ex- pression in skeletal muscle, which promoted FNDC5 expression, following which FNDC5 was spliced in vivo to a new form, irisin [2, 6]. It has been reported that iri- sin induces browning of subcutaneous white adipocytes, and enhances energy expenditure and fat oxidation. Iri- sin is therefore identified as a novel target for prevention and treatment of obesity .
Plasma concentrations of HDL-C have strong inverse correlations with risk of atherosclerotic cardiovascular disease, independently of LDL-C levels . ApoA-I is the major protein component of HDL-C in plasma. HDL-C and apoA-I exert anti-inflammatory, anti- oxidant, anti-thrombotic, promoting RCT and vasodilat- ing properties that could all potentially be involved in their anti-atherogenic effects . Here, we showed that simvastatin-treatment increased plasma levels of HDL-C and apoA-I. Our finding was in agreement with another report, which has shown that 8 weeks of simvastatin- treatment has a tendency to increase plasma HDL-C levels in apoE-/- micefed a high cholesterol diet . Furthermore, apoA-I is mainly synthesized by liver and hepatic ABCA1 exert anti-atherogenic effect via its con- tribution to HDL-C formation by promoting lipid efflux
The acute toxicity study in mice was performed as per the OECD guidelines (No. 423) to evaluate the undesirable effects or toxicity Ipomoea sepiaria aqueous extract. Swiss male albino mice were divided in to the groups of 3 animals per group and were administered once orally with dose of 2000 mg/kg of Ipomoea sepiaria aqueous extract. The mice were then critically observed for clinical signs, gross behavioral changes and mortality after 30min, 1hr, 2hr, 3hr and then after 24hr. These observations were continued for a period of 7 days.
What is Fecal Microbiota Transplantation? Historic perspective
Currently, the only existing non-antibiotic treatment for recurrent CDI that is recommended internationally is fecal microbiota transplantation. The accepted protocol is described in the annex section. The earliest reference of the use of human feces as a medical treatment dates back to fourth century in China where it was used to treat patients with food poisoning or severe diarrhea. (11) The first use of fecal enemas in humans for the treatment of pseudomembranous colitis was reported in 1958 in a four- patient case series. (12) Clostridium difficile as the causative agent of most pseudomembranous colitis was not identified until 1976. (13) A case report in which fecal enemas were used to successfully treat a patient with rCDI was published in 1984. (14,15) In the early 2000s the appearance of the BI/NAP1/027 strain of C. difficile was associated with widespread epidemics of CDI. This strain is characterized by high-level fluoroquinolone resistance, efficient sporulation, markedly high toxin production, and a mortality rate three times as high as that associated with less virulent strains. (5) Following this series of epidemics, the number of rCDI cases increased which led to the use of FMT in cases in which no other therapy was effective, prompting an increase of studies on FMT.
temperature . Food intake (on a per cage basis) and body weights were registered once a week. After 5, 11 and 22 weeks, body fat mass was analyzed using dual- energy X-ray absorptiometry technique with a Lunar PIXImus densitometer (GE Medical Systems). Oral glu- cose tolerance tests were performed after 8 and 21 weeks and an intravenous insulin tolerance test was performed after 15 weeks. Two mice in the Lp group did not wake up from the anaesthesia after the first oral glucose toler- ance test. Faeces were collected at three different time points during the study. Mice were placed in clean cages with a minimum of bedding for 24 hours. Faeces from each cage was collected, lyophilized and grounded in a mortar and stored at −20°C until analysis. After 11 weeks, 10 mice in each group were sacrificed in order to study time dependency of the treatment, and after 22 weeks, the remaining mice (9–11 per group) were sacrificed. At the time of sacrifice, mice were fasted for 4 hours and thereafter anesthetized with an intraperito- neal injection of midazolam (Dormicum, Hoffman- La Roche, Basel, Switzerland) and a mixture of fluanison/ fentanyl (Hypnorm, Janssen, Beerse, Belgium). Blood was drawn by intraorbital puncture followed by cervical dislocation. Periovarian white adipose tissue, liver (rinsed with PBS) and spleen were weighed and snap-frozen in liquid nitrogen. A part of the duodenum (3–10 mm distally of pylorus) was collected in buffer containing 10 mM Tris-HCl and 1 mM EDTA, pH 8.0, and snap- frozen in liquid nitrogen. The caecum including its content was weighed and thereafter the wall was removed. Parts of the content were frozen in freezing medium for culture of bacteria and parts were snap frozen for extraction of bacterial DNA. All samples were stored at −80°C until analysis.