T and N1T) are described. Strain CD3 : 22T was isolated from a biopsy of the small intestine of a child with coeliac disease, and strain N1T from the saliva of a healthy young man. The cells of both strains were observed to be filamentous, approximately 5 to >20 µm long, some of them curving and with swellings. The novel organisms produced H2S, NH3, butyric acid and acetic acid as major metabolic end products. Phylogenetic analyses, based on comparative 16S rRNA gene sequencing, revealed close relationships (98 % sequence similarity) between the two isolates, as well as the type strain of Eubacterium saburreum and four other Lachnospiraceae bacterium-/E. saburreum-like organisms. This group of bacteria were clearly different from any of the 19 known genera in the family Lachnospiraceae. While Eubacterium species are reported to be non-spore-forming, reanalysis of E. saburreum CCUG 28089T confirmed that the bacterium is indeed able to form spores. Based on 16S rRNA gene sequencing, phenotypic and biochemical properties, strains CD3 : 22T and N1T represent novel species of a new and distinct genus, named Lachnoanaerobaculum gen. nov., in the family Lachnospiraceae . Strain CD3 : 22T ( = CCUG 58757T  = DSM 23576T) is the type strain of the type species, Lachnoanaerobaculum umeaense gen. nov., sp. nov., of the proposed new genus. Strain N1T ( = CCUG 60305T = DSM 24553T) is the type strain of Lachnoanaerobaculum orale sp. nov. Moreover, Eubacterium saburreum is reclassified as Lachnoanaerobaculum saburreum comb. nov. (type strain CCUG 28089T  = ATCC 33271T  = CIP 105341T  = DSM 3986T  = JCM 11021T  = VPI 11763T).Two novel obligately anaerobic, Gram-stain-positive, saccharolytic and non-proteolytic spore-forming bacilli (strains CD3 : 22 Clostridiales, Prevotella and Actinomyces. Any or all of these bacteria may contribute to the aetiology or pathogenesis of CD in children. One of these rod-shaped bacteria was a spore-forming bacterium initially believed to belong to the clostridia  to the type strain of Eubacterium saburreum (CCUG 28089T  = JCM 11021T  = ATCC 33271T) and to several isolates and clones of the Lachnospiraceae than to the so-called ‘true’ clostridia belonging to cluster I of the subphylum Clostridium  and from the tongue dorsum (Coeliac disease (CD) in children has features analogous to those of an infectious disease. Between 1985 and 1996, the incidence of childhood CD in Sweden among children younger than 2 years was four times higher than it was before or after  and immediately plated onto selective and non-selective agar media. Strain N1T was isolated from the saliva of a healthy young man in 1998 at the Karolinska Institute, Karolinska University Hospital, Huddinge, Stockholm, Sweden. E. saburreum CCUG 28089T, initially isolated from human dental plaque, was obtained from the Culture Collection University of Gothenburg (CCUG), Sweden. Pure cultures of the three strains grew well on blood agar plates [Columbia or Brucella agar base (BBL) supplemented with 5 % horse blood], on chocolate agar plates and in Brucella broth, under an anaerobic atmosphere  at 37 °C. Vitamin K and haemin were not required for growth and were not added to the media.Strain CD3 : 22T and N1T and E. saburreum CCUG 28089T are all strictly anaerobic; they did not grow in the presence of oxygen. However, all three strains survived exposure to air for more than 12 h.Colony morphology and presumptive identification tests by diagnostic discs . Colonies of strain N1T had a similar size and speckled structure to those of CD3 : 22T but with edges more rhizoid and with sometimes pyramidal centres, making the colony morphology clostridium-like. The colony morphology of E. saburreum CCUG 28089T was also similar to that of CD3 : 22T, although the colonies lacked the compact appearance at the centre observed for CD3 : 22T .When the bacterial strains were cultured on a blood agar medium for 3 days, individual colonies of strain CD3 : 22T and N1T were filamentous, 5 to >20 µm long. Some of the cells were curved and with swellings; chain forms were occasionally observed . The cells of strain N1T were frequently observed in aggregates. Cells of E. saburreum CCUG 28089T were longer, more slender and did not display the swellings or spore-like structures observed in CD3 : 22T and N1T. All three strains were easily decolorized during Gram-staining.The morphology of bacterial cells was investigated by light microscopy after Gram-staining, dark-field microscopy, SEM and transmission electron microscopy (TEM). Cells of strains CD3 : 22T and N1T  but not in E. saburreum CCUG 28089T. However, spores were detected in all three strains by spore-staining . To confirm the presence of spores, TEM was performed on cultures of CD3 : 22T and E. saburreum CCUG 28089T, identifying spores inside the cells and extracellular spores in both cultures . Treatment of bacterial cells with heat (70 °C for 10 min) killed the spores of the novel strains as well as spores from spore-forming positive-control strains of Clostridium perfringens and C. innocuum. Spores of the novel strains were killed by 75 % ethanol treatment for 30 min, but not by 50 % ethanol, whereas the clostridial spores were resistant to 75 % ethanol treatment but not 85 %.The presence of spores was studied by Gram-staining, a specific spore-staining test, using malachite green , and TEM. Spore formation was enhanced by growing the bacteria in Brucella broth at 37 °C for more than 4 days. Gram-staining detected spore-like structures in strains CD3 : 22T and N1T and 7.0–7.5 for E. saburreum CCUG 28089T. Motility was not observed. None of the strains was haemolytic. All three strains produced H2S and NH3. Growth on glucose as the sole carbon source yielded the following volatile fatty acids: for strain CD3 : 22T, butyric acid and small amounts of acetic acid; for N1T and E. saburreum CCUG 28089T, butyric acid, acetic acid, lactic acid and small amounts of succinic acid.All three strains exhibited a temperature optimum for growth at 37 °C. The pH optima were 6.5–7.0 for CD3 : 22T and N1T and E. saburreum CCUG 28089T were resistant to 10 µg colistin and susceptible to 5 µg vancomycin, 1 mg kanamycin, 5 µg metronidazole and 20 % bile. Susceptibilities to penicillin G  were determined to be 0.06 mg l−1 for strain CD3 : 22T, 0.3 mg l−1 for N1T and 0.004 mg l−1 for E. saburreum CCUG 28089T.Strains CD3 : 22T and N1T were determined by primer walking, covering the gene, and by cloning and sequencing of PCR amplification fragments, also covering the gene  than most bacterial 16S rRNA genes. It contained an insert of 110 nt at position 1436 compared with the sequence of E. saburreum CCUG 28089T. The sequence outside the insert exhibited 98 % similarity to that of E. saburreum CCUG 28089T. The sequence of strain N1T did not contain this insert and was 98 % identical to the sequences of the other two strains (for CD3 : 22T outside the insert). T and N1T, E. saburreum JCM 11021T ( = CCUG 28089T), E. saburreum-like isolate C27KA . Strains CD3 : 22T and N1T and E. saburreum CCUG 28089T and related bacteria form a separate group distinct from the 19 known genera in the family Lachnospiraceae. This phylogenetic group is also distinct from members of Clostridium clusters XIVa and I. Based on cluster analyses and 16S rRNA gene sequence similarities between CD3 : 22T, N1T and E. saburreum CCUG 28089T, these bacteria can be proposed to represent a novel genus, for which the name Lachnoanaerobaculum gen. nov. is proposed.The nucleotide sequences of the 16S rRNA genes of strains CD3 : 22T, 38 mol% for strain N1T and 37 mol% for E. saburreum CCUG 28089T.The DNA G+C contents of the three strains were determined by HPLC as 35 mol% for strain CD3 : 22T and N1T exhibited β-glucuronidase activity, while E. saburreum CCUG 28089T did not. E. saburreum CCUG 28089T and N1T displayed indole production and pyroglutamic acid arylamidase activity, in contrast to CD3 : 22T. Acid production from carbohydrates was not detected in strain N1T by the API 20A test, whereas strains CD3 : 22T and E. saburreum CCUG 28089T were more active. However, when grown in peptone yeast glucose broth, strain N1T also formed volatile fatty acids. Strain N1T was the only urease-positive strain. Another difference was that only CD3 : 22T produced acid from mannose and raffinose. Finally, E. saburreum CCUG 28089T displayed α-fucosidase activity, in contrast to the other two strains. Differentiating characteristics between the three strains are summarized in Analyses of biochemical characteristics  showed that the three strains were saccharolytic and non-proteolytic (Table S1). However, they differed from each other in several respects. CD3 : 22http://www.ccug.se/pages/CFA_method_2008 and in File S1)  under the same conditions, using chocolate agar as a cultivation medium at 37 °C, and harvested after 40±2 h. Cellular fatty acids (CFAs) were extracted and saponified by mild alkaline methanolysis and the released fatty acids were methylated. CFAs were identified and quantified by GC (Hewlett Packard HP 5890). Retention times of CFA peaks were converted to equivalent chain-length (ECL) values and the relative amount (w/w) of each fatty acid was expressed as a percentage of the total fatty acids in the profile of the respective strain (Table S2). The major CFAs detected in strains CD3 : 22T, N1T and E. saburreum CCUG 28089T were C14 : 0, C16 : 0 and C18 : 1ω7c dimethylacetal (DMA). CFAs occurred in different relative amounts in the three strains. Thus, C18 : 1ω7c DMA constituted 16, 12 and 8 %, respectively, of the total CFAs for CD3 : 22T, N1T and E. saburreum CCUG 28089T. There were also distinct differences in minor fatty acids (Table S2). The CFA profiles of the three strains differed, primarily, only in the relative amounts of any given CFA. The overall profiles of the three strains were distinctly similar. The isolation and characterization of more strains will enable more evidence to be presented as to whether the quantitative differences noted between the strains are characteristic of the individual strains or whether they represent species-level differences. Moreover, the fatty acid profiles of strains CD3 : 22T and N1T and E. saburreum CCUG 28089T were distinct from the CFA compositions of type strains from the genera Eubacterium and Clostridium (Table S2). Overall, these data support the conclusion from the phylogenetic analyses that they indeed represent different bacterial species in a distinct and new genus.Cell fatty acid methyl ester analyses were performed using a standardized protocol, similar to that of the MIDI Sherlock MIS system (described at File S1) . StrainsT and N1T and E. saburreum CCUG 28089T at 60 °C for 16 h .14 : 0, C16 : 0 and C18 : 1ω7c DMA, accounting for 56–58 % of the total CFA profile. The G+C content of the DNA is 35–38 mol%. Strains have been isolated from the oral cavity, small intestine, blood and amniotic fluid , spore-forming rods. Spores may be difficult to detect by Gram-staining. The cells are filamentous (5 to >20 µm long), sometimes curving and with swellings. Colony morphology on blood agar appears variably speckled and spreading; the edges are erose or rhizoid. Some colonies appear flat and others pyramidal. Temperature and pH optima for growth are 37 °C and pH 6.5–7.5. Saccharolytic and non-proteolytic, with butyric acid and acetic acid as major end products from glucose metabolism. Predominant CFAs are CLachnoanaerobaculum umeaense .2S and NH3. Acid is produced from glucose, mannose, xylose, arabinose, lactose, sucrose, maltose, trehalose, raffinose and melezitose. Activities for α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase and β-glucuronidase are observed but indole production and urease activity are not observed.Has the following properties in addition to those described for the genus. On blood agar, colonies are 1–3 mm in diameter, flat, spreading and circular with erose edges after 72 h at 37 °C. Colonies are non-haemolytic. Optimum growth at pH 6.5–7.0. Produces butyric acid, acetic acid, HT ( = CCUG 58757T  = DSM 23576T), isolated from a biopsy of the small intestine of a child with CD. The DNA G+C content of the type strain is 35 mol%.The type strain is CD3 : 22Lachnoanaerobaculum orale .2S and NH3. Acid production is not detected from any of 17 common mono- or oligosaccharides or sugar alcohols. Activity for α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-glucuronidase and pyroglutamic acid arylamidase is observed. Urease activity is observed.Has the following properties in addition to those described for the genus. Cells frequently occur in aggregates. On blood agar, colonies are 1.5–3 mm in diameter, spreading and circular with rhizoid edges and pyramidal centres after 72 h at 37 °C. Colonies are non-haemolytic. Optimum growth at pH 6.5–7.0. Produces butyric acid, acetic acid, lactic acid, indole, HT ( = CCUG 60305T  = DSM 24553T), isolated from the saliva of a healthy young man. The DNA G+C content of the type strain is 38 mol%.The type strain is N1Laehnoanaerobaculum saburreum . Basonym: Eubacterium saburreum , originally isolated from dental plaque. The DNA G+C content of the type strain is 37 mol%.The type strain is CCUG 28089


However, the gender differences in AT2R and BW changes have not been studied. To understand the gender based role of AT2R involving BW changes, we fed male and female wild type (WT) and AT2R knock out (AT2KO) mice with C57BL6 background with high fat diet (HFD) for 16 weeks. The male AT2KO had higher HFD calorie intake  but gained less BW compared with the WT . Contrary to the male animals, the female AT2KO mice with equivalent caloric intake  gained significantly more BW than the WT mice . The male AT2KO on HFD displayed lower plasma insulin level, less impaired glucose tolerance (GT), and higher plasma T3 compared with WT males on HFD; whereas the female AT2KO mice on HFD showed elevated levels of plasma insulin, more impaired GT, lower plasma T3 and higher free fatty acid and hepatic triglycerides compared with WT females on HFD. Interestingly, compared with WT, AT2KO female mice had significantly lower estrogen, which was further reduced by HFD. These results suggest that AT2R in female mice via potentially regulating estrogen may have protective role against BW gain and impaired glucose tolerance and lipid metabolism.Obesity is a major disease condition, in turn leading to pathological changes collectively recognized as metabolic syndrome. Recently angiotensin receptor AT While obesity is described as an overall disorder of energy balance resulting from increase in food intake and/or reduced energy expenditure, it is also symptomatically characterized by impaired glucose tolerance, hyperinsulinemia and enhanced plasma free fatty acids (FFA) that in turn contributes further towards insulin resistance.2R in body weight changes and metabolic parameters between male and female mice. We found that at the end of 16 weeks of high fat diet (HFD), absence of AT2R had opposite effects on the body weight gain, glucose tolerance, and plasma insulin of males and females; and these changes were inversely related to several factors including changes in the estrogen levels, and physiological role of AT2R in males and females.Gender based differences in both humans and animal models are known to exist with regards to BP regulation, progression of renal damage, inflammation, weight gain and obesity.2 knockout females and males with C57BL6 background were used to breed AT2R knockout mice. 2KO mice  were placed either on normal diet  or high-fat diet  for 16 weeks. The mice were housed in the University of Houston animal care facility and maintained under a 12-hr light/dark cycle. The ND/HFD and water were provided ad libitum throughout the experiment period. The Institutional Animal Care and Use Committee approved the animal experimental protocols at the University of Houston, Houston, TX.C57BL6 breeder pairs (11–12 weeks of age) were obtained from Harlan  to create F1 WT mice population. The ATTo determine the direct impact of food intake differing in caloric content on body weight, we measured the food intake throughout the length of the study. Since mice thrive in a social group for a long-term study like the present, with the exception of a few aggressive male mice, the mice was housed in social groups of 2–4 animals in each cage. Weekly 100 g of food in the form of pallet was placed in the food slot of the mice cages. At the end of each week the remaining food, including any fallen bits, was weighed on an electronic weighing scale (Denver instrument P-602) and the amount subtracted from the initial 100 g to account for the food consumed. The total intake of each cage was then divided by the number of mice in the cage. Three days prior to sacrifice, the mice were placed individually in metabolic cages to record the food consumption, water intake, urine output and other parameters for single mouse. The food intake in the different groups was calculated in terms of calorie consumed. We measured the change in body weight in the different groups of mice to assess the effect of the diet and treatment on the body weight. Body weight in the different groups also was measured every week for each animal on a triple beam veterinary scale  and measured as change in body weight over basal, which was weight measured at the start of the dietary regimen.While obesity is described as an overall disorder of energy balance resulting from increase in food intake and/or reduced energy expenditure, it is also symptomatically characterized by impaired glucose metabolism which further contributes towards glucose intolerance.Prior to the termination of experiment all animals were individually housed in mouse metabolic cages for 3 consecutive days for urine collection. At the end of 16-weeks treatment, the mice were euthanized by cervical dislocation under anesthesia following 6 hours of fasting. Plasma was collected and stored at −80°C until further use. Gonadal fat depot is the largest dissectable fat pad in rodents and accounts for 30% of all removable fat whose mass has been used as an index of obesity. Therefore, to correlate changes in body weight with the fat content, we measured the mass of gonadal fat depot in the different groups. Paired-gonadal adipose depot  were removed, pat dry on tissue paper and weighed.2R deficiency has been shown to prevent diet-induced insulin resistance in male mice. 2R on insulin resistance in HFD-induced obesity. Plasma insulin and T3 levels in different groups were determined using ELISA kit as per the manufacturer’s instructions. For insulin, 5 µl of blank, standard, and sample with 95 µl of sample diluent was added in duplicates to appropriate wells. The plate was incubated for 2 hours at room temperature (RT). The wells were washed 5 times and 100 µl of anti-insulin enzyme conjugate was added to each well and incubated for 30 min at room temperature. The wells were washed 7 times, 100 µl of enzyme substrate was added and incubated in dark for 40 min. 100 µl of stop solution was added to stop the reaction and absorbance was measured on a micro-plate reader  at A450 and subtracted from A630 values. Since obesity is associated with metabolic/physiological changes either contributed by or as a result of alterations in the endocrine system, we measured a critical metabolic hormone T3, whose plasma level is known to positively correlate with metabolic rate. 450 within 15 min on the micro-plate reader.Hyperinsulinemia is an index of insulin resistance which is known to correlate with obesity. Furthermore, AT2O for quantification.Obesity is known to be associated with enhanced plasma FFA, which in turn contributes towards insulin resistance.2R. 410 on the micro-plate reader. The data was calculated for 17-β-estradiol excreted in 24 hr urine.Low levels of estrogen could lead to accumulation of fat mass and development of obesity and hyperlipidemia in females. The HFD feed Teklad custom research diet  with adjusted calorie diet (60/Fat) containing 18.4% protein, 21.3% carbohydrate and 60.3% fat, and normal diet 7022 with isocaloric 29% protein, 56% carbohydrate and 15% fat were purchased from Harlan, Indianapolis, IN. Both diets contained similar mineral mix AIN-93G-MX (94046) and vitamin mix AIN93-VX (94047). The Ultra-Sensitive mouse ELISA kit catalog # 90080 from Crystal Chem. Inc. Downers Grove, IL was used for plasma insulin. The Mouse/Rat T3 total, ELISA kit catalog # T3043T-100 from CALBIOTECH, Spring Valley, CA was used for T3. Plasma free fatty acid levels was measured by free fatty acid quantification kit . Hepatic TG was analyzed using the triglyceride quantification kit . The Estradiol EIA kit 96 solid wells, EIA kit catalog # 582251 from Cayman Chemical, Ann Arbor, MI was used for estradiol.t test. A p value of less than 0.05 was considered statistically significant. Power analysis was performed on groups with higher internal variation to determine statistical significance.Data are presented as mean±SEM. The data were analyzed using GraphPad Prism 5  and subjected to one-way ANOVA with Newman-Keuls post hoc test and Student’ unpaired 2KO . Furthermore, the AT2KO male mice on HFD had significantly higher kcal consumption compared with WT males on HFD (P = 0.01)  consumed over 16 weeks. We found no significant difference in the kcal consumption of ND among the males and females of either WT or AT2KO . However = 0.01) .2KO mice on HFD clearly was opposite. In males HFD had a similar effect on body weight gain in both WT and AT2KO. In WT starting from comparable body weights, HFD caused significant increase (28±1 g) compared with ND (15±0.8 g) (P = 0.002) with net difference in 13 g. On the other hand, the difference between AT2KO males on HFD (27±0.3 g) and AT2KO on ND (19±3 g) was attenuated to 8 g. This difference in AT2KO weight on ND and HFD, in part could be attributed to increased weight in AT2KO on ND. Contrary to the males, AT2KO female mice on ND (15±0.3 g) as well as on HFD (30±1 g) gained body weight significantly greater than WT on ND (11±1.2g) and WT on HFD (20±1 g), respectively  (P = 0.0001); females  (P = 0.0001);  (P = 0.0001). The data suggest that HFD caused changes in the slope of weight gain in both WT and AT2KO males and females. However, the slope of weight gains in WT males on HFD and AT2KO males on HFD are similar, but there was a significant increase in AT2KO females on HFD compared WT females on HFD. To determine the overall pattern of weight changes in various groups, we determined area under the curve (AUC) of weight gain over 16 weeks. The AUC data in males are: WT-ND 151±1.6 vs. WT-HFD 236±2.7 (P = 0.001) and AT2KO-ND 177±1.8 vs. AT2KO-HFD 226±2.4 and in females are: WT-ND 102±1.3 vs. WT-HFD 151±1.6 and AT2KO-ND 127±1.3 vs. AT2KO-HFD 193±2) (P = 0.001). The data clearly suggests that changes in AUC pattern in males and females are opposite and follow the end point weight gain data in males and females after 16-weeks of dietary regimen. In males, the AUC increase (49 units) in AT2KO HFD over ND was lesser than the AUC increase (85 units) in WT HFD over ND. Contrarily, in females the AUC increase (66 units) in AT2KO HFD over ND was greater than the AUC increase (49 units) in WT HFD over ND.Weight gain pattern in male and female ATectively . We also2KO males on HFD gained (0.9±0.06 g) significantly higher fat mass compared with AT2KO mice on ND (P = 0.001); but there was a significant decrease in epididymal fat weight between male AT2KO and WT on HFD (P = 0.01). On the other hand, AT2KO female mice on HFD (1.2±0.13 g) gained fat weight significantly greater than WT on HFD (0.6±0.09 g) (P = 0.02).As shown in 2KO males (307±15 mg/dL) was significantly higher compared with ND fed WT (217±22 mg/dL) (P = 0.01) and AT2KO (224±17 mg/dL), respectively  were modestly lower than WT ; however, the decrease was not statistically significant.ectively . Both th2KO (438±25 mg/dL) was significantly higher compared with their respective ND-fed control groups, WT (235±5 mg/dL) (p<0.04) and AT2KO (304±32 mg/dL) (P = 0.02) (2KO group with the AUC of HFD fed (49550±3236) being significantly greater than the ND fed (33080±1269) (P = 0.004)  in response to HFD was profoundly greater than the increase detected in WT females on HFD  (P = 0.003 and P = 0.03) respectively)  and AT = 0.02) . The AUC= 0.004) . Howeverctively) .2KO males and females, exhibited greater plasma insulin levels compared with their respective ND control groups (P = 0.01- P = 0.001). However, plasma insulin in AT2KO males on HFD (11±1.3 ng/ml) was modestly lower than WT on HFD (13±1.4 ng/ml)  was significantly higher than WT on HFD (2.4±0.3 ng/ml) (P = 0.04)  exhibited modestly but significantly higher plasma insulin compared with WT on ND (1.3±0.2 ng/ml) (P = 0.02)  . Contrar = 0.04) . Also, A = 0.02) .2KO males  (P = 0.008)  exhibited lower plasma T3 levels compared with WT females on ND (1.26±0.01 ng/ml) (P = 0.001)  but caused a modest increase in T3 in the AT= 0.008) . On the es on ND . However= 0.001) .2KO males. AT2KO males on HFD had significantly (P = 0.05) lower TG (21.2±1 mg/g tissue) compared with HFD fed WT (TG: 25±0.1 mg/g tissue). The FFA in AT2KO males on HFD (20.1±1.3 mg/dL) were modestly, but non-significantly, reduced compared with HFD WT (22±0.6 mg/dL). In females, both WT and AT2KO on HFD had elevated plasma FFA ; TG: 49±4 mg/g (P = 0.002) compared with WT females on HFD (FFA: 18±0.4 mg/dL (P = 0.0001); TG: 37±2 mg/g (P = 0.003).The HFD caused a significant increase in plasma FFA  and hepaasma FFA  and hepaasma FFA  compared2KO female mice on ND (27±1.3 ng/24 hr) was significantly lower than WT females (P = 0.0001) and was further reduced by HFD (9±0.6 ng/24 hr) (P = 0.0001)  was significantly lower compared with AT2KO males fed on ND (0.33±0.01 ng/24 hr) (P = 0.0001), but was significantly greater than WT on HFD (P = 0.0001) (The urinary estradiol (E2) level in the WT female mice on HFD (19±1.5 ng/24 hr) was lower compared with the WT females on ND (46±4 ng/24 hr). Interestingly, the urinary E2 level in the AT 0.0001) . The uri 0.0001) . The E2  0.0001) .2KO males on HFD had lesser body weight gain, lower gonadal adipose depot weight, lower levels of plasma insulin less impaired glucose tolerance (GT), higher plasma T3 and higher urinary estrogen levels, compared with HFD fed WT males. In contrast to the males, AT2KO females on HFD had significantly higher body weight, higher gonadal adipose depot weight, elevated levels of plasma insulin, more impaired GT, lower plasma T3 and estrogen levels, compared to HFD fed WT female mice. Additionally, the urinary E2 in AT2KO females on ND was lower compared with WT on ND.In the present study, we found that AT2KO males following 16 wk of HFD treatment , ACE2 and renin, which are inter-regulatory. The present study paves way for future studies that could expand the scope of therapeutic potential of AT2R in metabolic disorders particularly in postmenopausal women.The present study provides a unique and novel perspective of the gender biased role of AT


INK4A overexpression in BEAS-2B cells as demonstrated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB). In A549 cells we observed downregulation of p53 but not a significant increase of hTERT transcripts. In addition, the HPV16 E6/E7 transfected cell lines showed an increased proliferation rate and anchorage-independent growth in a HPV16 E6 and E7 expression-dependent manner. Moreover, both HPV16 E6/E7 and mock transfected cells showed an increased proliferation rate and anchorage-independent growth in the presence of 0.1 and 10 µg/mL CSC. However, this increase was significantly greater in HPV16 E6/E7 transfected cells (p<0.001). Data were confirmed by FCSE proliferation assay. The results obtained in this study are suggestive of a functional interaction between tobacco smoke and HPV16 E6/E7 oncoproteins for malignant transformation and tumorigenesis of lung epithelial cells. More studies are warranted in order to dissect the molecular mechanisms involved in this cooperation.The smoking habit is the most important, but not a sufficient cause for lung cancer development. Several studies have reported the human papillomavirus type 16 (HPV16) presence and E6 and E7 transcripts expression in lung carcinoma cases from different geographical regions. The possible interaction between HPV infection and smoke carcinogens, however, remains unclear. In this study we address a potential cooperation between tobacco smoke and HPV16 E6 and E7 oncoproteins for alterations in proliferative and tumorigenic properties of lung epithelial cells. A549  and BEAS-2B  cell lines were stably transfected with recombinant pLXSN vectors expressing HPV16 E6 and E7 oncoproteins and exposed to cigarette smoke condensate (CSC) at different concentrations. HPV16 E6 and E7 expression was associated with loss of p53 stability, telomerase (hTERT) and p16 Human Papilloma Virus (HPV) infection and persistence are the necessary conditions for cervix-uterine cancer development INK4a up-regulation by a negative feedback mechanism So far, HPV16 has been the most frequent high-risk HPV genotype found in lung carcinomas around the world The oncogenic role of tobacco smoking and cigarette smoke components (CSC) in lung cancer has been demonstrated in previous epidemiological and functional studies Transfection of A549 and BEAS-2B lung cell lines with a pLXSN recombinant vector containing the HPV16 E6 and E7 open reading frames (ORF) allowed an efficient and stable expression of the corresponding transcripts Fig.2A.INK4a. As shown in INK4a showed a significant increase after transfection of BEAS-2B cells with E6 and E7 oncogenes  identified tobacco smoke a as carcinogen to humans Even though in this study a significant increase in proliferative rate and anchorage-independent growth was found, the mere expression of E6 and E7 oncoproteins was sufficient for alterations in the analyzed properties of lung cells. In consequence, is not possible to discard the possibility that E6 and E7 oncoproteins act independently of tobacco smoke. In fact, Ciotti et al using proteomic analysis reported that E6 and E7 oncogenes when overexpressed in A549 lung cells are able to upregulate the levels of Annexin III, gp96, transaldolase 1, between others A very important issue is if the CSC concentrations that were used in this study represent a real physiological exposure to tobacco smoke. The concentration of CSC is 40 mg/mL which corresponds to 6.4% of nicotine. In this report, lung cells were exposed to 0.1 and 10 µg/mL CSC which corresponds to 0.064 and 0.64 µg/mL nicotine. It has been reported that nicotine concentration ranged between 0.6 to 1.0 µg/mL into the tissue of smokers and 0.04 to 0.072 µg/mL in serum In conclusion, in this study a functional cooperation between HPV16 E6/E7 oncoproteins and CSC, leading to an increase of proliferation rate and anchorage-independent growth of lung cells was shown. According to our knowledge, this is the first report addressing a potential cooperation between HPV and tobacco smoke in lung epithelial cells. Considering that HPV16 E6 and E7 oncoproteins are detected in a subset of lung carcinomas from subjects around the world who smoke or are exposed to tobacco smoke, they appear to be able to modulate the proliferative and tumoral behavior of cells exposed to tobacco smoke in bronchial epithelia. So it is plausible that in smoking subjects exposed to HPV infection, a functional interaction increases the possibility of carcinogenic progression. Additional functional and epidemiological studies are warranted to resolve this question.2 atmosphere incubator. The cells were checked for mycoplasma infection using standardized protocols. A549 and BEAS-2B cells were transfected with the retroviral vectors pLXSN and pLXSN containing the HPV-16 E6/E7 ORFs  . Transfection assays were made using lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, one day before transfection cells were seeded in six-well plates with 3 mL of basal RPMI-1640 medium without antibiotics. For each plate, 4 μg of pLXSN recombinant plasmids were mixed with 250 μL of RPMI-1640 basal medium without antibiotics. The mixture was then combined with 10 μL of lipofectamine 2000 (Invitrogen) in 250 μL of RPMI-1640 and incubated for 20 min at room temperature. The plasmid mixture was directly added to the cells containing RPMI-1640 supplemented with 5% FBS without antibiotics. The cells were incubated for 18 h at 37°C and then the cell culture medium was discarded; the cells were washed with phosphate buffered saline pH 7.4 (PBS) and incubated with RPMI-1640 supplemented with 5% FBS and 0.5 mg/mL G418 for selection. After 2 to 3 weeks, the resulting colonies were pooled and used for subsequent analysis.Human cell lines A549 , BEAS-2B  and SiHa  were directly obtained from the American Type Culture Collection (ATCC) . These cell lines were maintained in RPMI-1640 (Invitrogen) supplemented with 10% inactivated fetal bovine serum (FBS) (Hyclone), 0.1 μg/mL gentamicin (Invitrogen), 1 U/mL penicillin and 1 μg/mL streptomycin (Invitrogen) and incubated at 37°C in a 5% COCellular RNA was isolated using 1 mL of Trizol reagent (Invitrogen) according to the manufacturer's instructions. The RNA concentration and purity was determined using a Nanodrop1000 spectrophotometer (Thermo Scientific) and 2 μg of RNA was treated with RQ1 DNAse (Promega) at 37°C for 1 h and denatured at 65°C. cDNA was synthesized in a 20 μL reaction volume containing DNAse-treated RNA (2 μg), 1 U RNAse inhibitor (Promega), 0.04 μg/μL random primers (Promega), 2 mM dNTP (Promega) and 10 U Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega). The reaction mixture was incubated for 1 h at 37°C followed by incubation at 65°C for 10 min. The cDNA samples were subjected to PCR amplification with specific primers for HPV16 E6/E7, p53 and GAPDH Table1.INK4a amplification were described in INK4a, the PCR amplification was performed as follows: 10 min incubation at 95°C; 45 cycles of 10 s at 95°C, 10 s at 55°C and 10 s at 72°C. For E6 and E7 the amplification conditions were the same that were used for hTERT. GAPDH amplification was used for data normalization. The PCR amplification was performed as follows: 10 min incubation at 95°C; 45 cycles of 15 s at 95°C, 20 s at 55°C and 20 s at 72°C. The PCR reactions were carried out using Rotor Gene 6000 (Corbett Research) real-time PCR equipment. The specificity of amplification was confirmed using dissociation analysis starting at 60°C.The primers used for E6, E7, hTERT and p16Total protein was extracted from cells with a lysis buffer  containing a protease inhibitor cocktail (Sigma-Aldrich). The cells were incubated at 4°C for 1 h, sonicated on ice for 20 s and centrifuged at 12,000 G for 10 min. The protein concentration was determined using Pierce BCA protein Assay Reagent kit (Thermo). For p53 blots; 25 µg of the extract was loaded per well on 10% polyacrylamide gels. Following SDS-PAGE, proteins were transferred onto a nitrocellulose membrane in a Trans-Blot semi-dry electrophoretic transfer cell (BioRad) . The membranes were blocked in 5% dry milk–TBS–Tween 20 and incubated for 16 h at 4°C with a primary monoclonal anti-p53 antibody (DO7 clone) diluted 1∶1000 (BD Biosciences). After extensive washing with TBS-Tween 20, the membrane was developed using an ECL kit according to the manufacturer's instructions (Amersham Biosciences). β-actin (1/10.000) (Abcam) was probed in all samples to exclude false negative results and cell lysates from CasKi cells (containing HPV16) were used as positive controls.−3 to 50 µg/mL using a 40 mg/mL stock solution. Dimethyl-sulfoxide (DMSO) was used as negative control. The CSC stock solution was aliquoted and stored light-protected at −80°C.The transfected A549 and BEAS-2B cell lines were maintained at 80–90% confluence with RPMI-1640 basal medium, serum-starved for 24 h previous to CSC  exposition. The CSC dilutions in basal RPMI-1640 medium were freshly prepared for each experiment in a range from 103 cells were prepared and incubated on 96-well plates with RPMI-1640  supplemented with 10% FBS , penicillin/streptomycin mixture and 0.1 µg/mL gentamicin (Invitrogen). The CSC was added to the cells and incubated at 37°C in a 5% C02 atmosphere incubator. After 72 h of incubation, thirty µL of MTS reagent (Promega) were added to each well and the plate was incubated for three h at 37°C; the absorbance was measured at 492 nm using a microplate reader (model Anthos 2001).A549 and BEAS-2B transfected cells were trypsinized with an EDTA-trypsin mixture (Invitrogen) and counted using Trypan blue exclusion staining with a Neubauer chamber. Briefly, 5×103 cells were cultured in a 96-well flat-bottomed microtiter plate with RPMI-1640 basal medium and alternately supplemented with 0.1 and 10 µg/ml of CSC for periods ranging from 24 to 120 h, using DMSO as control. Finally, thirty µL of  (Promega) were added to each well, the plate was incubated for 3 h at 37°C and the absorbance was measured at 492 nm using a microplate reader (Anthos 2001).Transfected cell lines were grown to 80–90% confluence and incubated with serum-starved RPMI-1640 medium in 60 mm plates for 24 h. The cells were trypsinized with EDTA-trypsin mixture (Invitrogen) and counted using Trypan blue exclusion staining with a Neubauer chamber. 5×102 atmosphere incubator. Then the cells were trypsinized with EDTA-trypsin 1X (Invitrogen) and counted using trypan blue staining. 1×106 cells were treated with 0.5 µM carboxifluorescein succinimidyl ester (CFSE) for 10 min at 37°C. Then 5×104 cells were deposited in 6-well plates in RPMI-1640 basal medium with 10% FCS and exposed to 0.01 and 10 µg/mL CSC during 48 h. The cells were washed with 1X PBS, suspended with 1X trypsin-EDTA and analyzed by flow cytometry in the FACS Cantoll equipment. , considering a universe of 10,000 cells.A549 transfected and non-transfected cells were passaged at 80–90% confluence in 60 mm plates without serum for 24 h at 37°C in a 5% CO4 cells were suspended in 0.33% Bacto-agar dissolved in a mixture containing 2X RPMI-1640 basal medium, 12.5% FBS, 1X RPMI-1640 supplemented with 0.01 or 10 µg/mL CSC. The cells were cultured in six-well plates containing 0.5% agar for 1 month. The cells were fed twice each week with 0.5 mL of RPMI-1640-10% FBS supplemented with 0.01 or 10 µg/mL CSC. Finally, the cells were stained using 0.005% crystal violet for 1 h and photographed.The transfected cells exposed to 0.01 and 10 µg/mL of CSC for 72 h were trypsinized using 1X Trypsin-EDTA mixture and counted using trypan blue staining. Thus 1×10Overall numeric data were analyzed considering averages and standard deviations. The comparison between CSC treated groups and controls were made considering a non-paired statistical analysis with 95% confidence interval using Prism 4.0 software . A p value less than 0.05 was considered as statistically significant.


Early institution of therapy has been advocated worldwide. However, until recently, NACO guidelines recommended therapy in HIV positive patients with CD4 count <250/µL. In this study we observed the clinical and immunological disease progression for a year in treatment naïve patients with CD4 count between 250-500/µL.3 in WHO clinical stage 1 or 2 were included in the study and enrolled into 2 groups at 2:1 ratio and followed for 1 year. Group A with CD4 count between 250-350/mm3 & Group B with CD4 count between 350-500/mm3. At the end of study each group was evaluated for number of opportunistic infection, ART conversion, conversion to stage III and IV, lost to follow up (LFU) and death. Statistical analysis was done by SPSS 16.0 version. Data were expressed as mean (±SD) for continuous variable and percentage for categorical variable. An independent sample t-test was used to detect differences in clinical and laboratory results.150 treatment naïve adults PLHIV with CD4 count between 250 -500/mmAs compared to Group B, Group A had significantly higher number of opportunistic infection , ART conversion , death  and LFU (20% vs 6% p=0.037).This study reinforces the need of early initiation of treatment in patients with CD4 <350/ µL.


The role of dynorphin/kappa opioid receptors in epilepsy and addiction are well accepted, but their function in emotional control is not yet fully understood. Data obtained from different strains of prodynorphin (Pdyn)- and kappa opioid receptor (KOP)-deficient mice do not provide a consistent picture of the functions of Dyn/KOP in anxiety, suggesting the influence of testing conditions and/or genetic background. Therefore, we investigated the behaviour and neurochemistry of male and female Pdyn KO mice on the balb/c and C57Bl/6N background. Consistent with our results obtained from male mice on the C57bl/6N background, we observed a less anxious phenotype in the elevated plus maze, open-field and light-dark test in male mice on the balb/c background. Female mice on the balb/c background also displayed less anxiety like behaviour; however these data reflect high trait anxiety and inter-individual differences. In contrast, female mice on the C57Bl/6N background displayed low trait anxiety and a paradigm-dependent reduction of anxiety. No differences were observed in the forced swim test, while balb/c Pdyn KO mice displayed prolonged immobility in the tail suspension test. In line with our previous results, we observed reduced CRH mRNA in the central amygdala in all groups of mice. In contrast, the recently observed CRH mRNA reduction in the hypothalamic paraventricular nucleus appears restricted to male, but not female mice. Our data support previous data suggesting a pronounced impact of endogenous prodynorphin-derived peptides on anxiety. Moreover, our data support the idea that the less anxious phenotype manifests only at elevated stress levels. The role of Dyn/KOP in anxiety control is not well understood. Data obtained from Pdyn- and KOP-deficient mice are relatively rare and do not provide a consistent picture of the functions of Dyn/KOP in anxiety. In Pdyn KO mice on a C57bl/6J background In addition, anxiety testing is strongly influenced by epigenetic and environmental conditions The neurochemical background of the involvement of Dyn in anxiety control appears fairly complex and KOP activation in different brain areas affects behaviour differently. Therefore, the behaviour of Pdyn KO mice hardly can be pinned down to single brain areas. However, involvement of the amygdala is very likely. Thus, reduced expression of corticotropin-releasing hormone (CRH) was observed in the hypothalamic paraventricular nucleus independently in two different strains of Pdyn KO mice Similar unequivocal as the influence on anxiety-like behaviour are influences on stress-induced behaviour in Pdyn KO mice. While we observed no differences in stress-induced hyperthermia Tests for stress-induced immobility (depression-like behaviour) revealed further subtle phenotypes in Pdyn KO mice. Again, these data appear unequivocal in the forced swim test. While McLaughlin et al. propose anti-depressant phenotype in Pdyn KO mice, this could not be confirmed in our study. In our hands, Pdyn KO mice displayed increased immobility at a single measurement There is vast literature on the influence of testing conditions, sex and genetic background on the outcome of behavioural testing (for review see The generation of Pdyn KO mice has been described elsewhere http://empress.har.mrc.ac.uk) where available. Due to the high trait anxiety of balb/c mice, the illumination in all anxiety tests was reduced to 50 lux . All tests were video monitored and evaluated by an experimenter blinded to the genotype of the animals. A fixed order of tests was kept to guarantee comparable conditions: open field, elevated-plus maze, light dark tests with one week intervals, tail suspension test and forced swim test with two weeks interval. Number of animals in different tests may vary because data of mice showing injuries (resulting from fighting), reduced whiskers or problems at the testing day (for example irregular noise outside) were rejected.Unless stated otherwise, mice were group-housed before testing and transferred to the ante-room of the testing facility 24–72 h before the commencement of experiments, where they were given free access to food and water. The climate and light-dark cycle remained constant. Tests were performed between 9 am and 1 pm. Test settings were in accordance with the recommendations of EMPRESS . The pretested animals were not included in this study. Explorative behaviour was analysed using Video-Mot 2 equipment and software . Arenas were subdivided into the border , centre , and intermediate area.Behaviour was tested over 5 min in an elevated plus maze 0.7 m above the ground, consisting of two closed and two open arms, each 50×5 cm in size. The test instrument was built from grey PVC, the height of the closed arm walls was 20 cm, while the open arms were enclosed by a rim of 3 mm. Illumination was set to 50 lux for balb/c and 180 lux for C57Bl/6N mice. Animals were placed in the centre, facing an open arm. Analysis of open and closed arm entries and time spent on the open arm was conducted automatically using Video-Mot 2 equipment and software. Entry into the open arm was recorded only when all four legs of the mouse left the neutral central area.Explorative behaviour in a (brightly) lit area  was investigated by insertion of a black box into the open-field arena, covering one third of the space. One small field directly at the entrance to the black box was assigned as the transition zone. To reach the larger compartment assigned as the open area, the mouse had to completely leave the dark area. The number of entries, time spent and distance travelled in the light area were evaluated for C57Bl/6N mice. For balb/c mice entry or lack thereof into this field was recorded, as the number of entries was too low to statistically evaluate the time spent and distance travelled in this compartment in wild-type mice.The tip (c.a. 1.0–1.5 cm) of the tail of the mice was securely fastened with medical adhesive tape to a metallic surface. Mice were suspended for 6 min approximately 30 cm above the surface. The illumination on the floor of the table was about 100 lux. Immobility (lasting over 2 sec) and latency to the first immobile phase of the mice was evaluated.This test was performed in a single 15-min trial. To increase stress we performed the test in water held at 25°C (30°C in previous experiments). Immobility, defined as no activity for at least 2 sec, was independently evaluated from video clips for the final 4 min of the test by two investigators blinded to the genotype of the animals.In situ hybridization was carried out as described elsewhere 5′-GAGGGTCAGTCCACACAGCCCCATTCGCTTGTTACCTAGCAT-3′; CRH: 5′-CCGATAATCTCCATCAGTTTCCTGTTGCTGTGAGCTTGCTGAGCT-3′.in situ hybridization, digitized images of the areas of interest were acquired from photo-emulsion dipped and superficially Nissl counter-stained brain slices at 200× magnification using a digital camera  mounted onto a Zeiss Axiophot 2 microscope. The density of silver grains was evaluated by an experimentally blinded observer by outlining single neurons and measuring the percentage of area covered by silver grains .The values of at least 30 single neurons were used to calculate the mean value for each animals. Thus, n represents the number of animals. Expression levels are given as mean percent of control. If not stated differently, brain samples were collected from testing naïve, unstressed animals.To evaluate 2 anaesthesia. Trunk blood was captured and serum was stored at −20°C until analysis. Corticosterone serum levels were measured using a commercial radioimmunoassay  according to the manufacturer's instructions. Each serum was analysed in duplicate.Testing naïve, unstressed animals were killed by decapitation between 12.00 and 14.00 hours while under deep CO2 test to the distribution frequency of classification data. Comparison of more than two groups was carried out by One-Way-ANOVA, followed by Bonferroni's multiple comparison test, using GraphPad Prism 5.0 software. P-values of <0.05 were accepted as statistically significant. All data are given as mean ± SEM (n).The Student's t test was used for all comparisons of the two genotypes, with the exception of the analysis of the behaviour of male balb/c mice in the light-dark test. Here we applied a ChiPdyn KO mice spent significantly more time (intermediate 11±3.9 sec (15) vs. 47.7±13.8 sec (12); p = 0.0001; centre 9.7±3.0 sec (15) vs. 28.4±7.3 sec (12); p = 0.0084; WT vs. Pdyn KO, respectively) and travelled more distances (intermediate 3.2±0.8% (15) vs. 10.0±2.5% (12); p = 0.0014; centre 2.7±0.5% (15) vs. 6.2±1.4% (12); p = 0.0049; WT vs. Pdyn KO, respectively) in the intermediate and central areas of the open field than WT mice. In addition, the number of entries into these areas was higher in Pdyn KO mice (intermediate 10.8±3.6 (15) vs. 34.2±9.7 (12); p = 0.0029; centre 3.3±0.9 (15) vs. 11.2±3.8 (12); P<0.0001; WT vs. Pdyn KO, respectively). Due to high inter-individual differences statistical significance was only reached for the border and intermediate areas, and not for the centre .Pdyn KO mice displayed more exploratory behaviour on the open arm than WT mice . This is2 test).Balb/c mice are known to be very shy in the light-dark test. In fact, balb/c WT mice very rarely exit the dark compartment. Therefore, the usual evaluation of entries, time and distance in the light area could not be applied. However, we still observed a significantly higher number of animals entering the light area in the Pdyn KO group (9 out of 15) as compared to WT (1 out of 20) animals  vs. 71.7±23.6 sec. (n = 7); p = 0.8964 WT vs. Pdyn KO, respectively).Statistically significant differences were observed for both the time spent immobile and the latency to the first immobile phase between WT and Pdyn KO mice. While WT mice spent 15.8±6.0 sec (n = 9) immobile, Pdyn KO mice were immobile for an average of 56.4±10.8 sec (n = 7) (p = 0.0038). The onset of the first immobile period was observed in WT mice after 239±23.6 sec (n = 9), while Pdyn KO mice displayed the first immobile phase after 149±117 sec . One of the WT, but none of the Pdyn KO mice climbed along their tail to the bar and had to be removed from evaluation.In situ hybridization revealed a reduction of CRH mRNA in the central amygdala (100±11.5 (n = 4) vs. 78±8.7 (n = 5); p = 0.0154; WT vs. Pdyn KO, respectively) and in the paraventricular nucleus (100±8.2 (n = 5) vs. 82±8.8 (n = 5); p = 0.0088; WT vs. Pdyn KO, respectively) of balb/c Pdyn KO mice. NPY mRNA was increased in neurons of the basolateral amygdala of Pdyn KO mice (100±6.5 (n = 5) vs. 117±3.3 (n = 5); p = 0.0008; WT vs. Pdyn KO, respectively), but not in the central amygdaloid nucleus (100±3.0 (n = 4) vs. 99±5.1 (n = 5); p = 0.7406; WT vs. Pdyn KO, respectively). Data represent the mean percent of control ± SD (n).No marked differences in serum corticosterone levels were observed between Pdyn KO and WT mice (73.3±34.4 ng cort./ml (n = 11) vs. 52.0±23.3 ng cort./ml (n = 11); p = 0.1046; WT vs. Pdyn KO, respectively).Female mice on C57Bl/6N and balb/c backgrounds were tested. However, valid data for all tests were only obtained from the C57Bl/6N mice. Balb/c mice were very shy, even at reduced light intensities, and therefore allowed only limited evaluation.Female mice on the balb/c background rarely left the border zone, and therefore statistical analysis of ambulation in the intermediate and central zones did not reveal significant differences. On the other hand, Pdyn KO mice spent less time (588±2.21 sec (n = 12) vs. 546±17.4 sec (n = 8); p = 0.0102; WT vs. Pdyn KO, respectively) and travelled a shorter distance (95±2.79% (n = 12) vs. 87±3.07% (n = 8); p = 0.0019; WT vs. Pdyn KO, respectively) in the border zone, reflecting increased exploration of non-border areas  regarding time (9.0±1.92 sec (n = 12) vs. 49.4±16.7 sec (n = 8); p = 0.0065 and distance travelled (4.9±0.81% (n = 12) vs. 13.1±3.07% (n = 8); p = 0.0065; WT vs. Pdyn KO, respectively; Female Pdyn KO mice on the C57Bl/6N background displayed significantly increased ambulation on the open arms compared to WT mice . This waStatistically significant differences between WT and PdynKO mice on the C57Bl/6N background were observed for the number of entries into the light area (3.4±0.52 (n = 16) vs. 6.1±1.14 (n = 18); p = 0.0473; WT vs. Pdyn KO, respectively), but not for the distance travelled in this area (298±42.2 cm (n = 16) vs. 344±67.2 cm (n = 18); p = 0.5773; WT vs. Pdyn KO, respectively). Only 4 PdynKO mice and none of the WT out of 22 mice on the balb/c background entered the light area, and therefore, this test could not be evaluated.The forced swim test was performed in a single 15-min session. No marked differences in immobility were observed over the last 4 min of this test between WT and Pdyn KO mice on the C57Bl/6N (89±49.8 sec (n = 17) vs. 78±44.2 sec (n = 19); p = 0.4952; WT vs. Pdyn KO, respectively) or on the balb/c (91±31.8 sec (n = 11) vs. 94±43.0 sec (n = 15); p = 0.8319; WT vs. Pdyn KO, respectively) background.Pdyn KO mice on the balb/c background spent a prolonged time immobile (43.2±28.9 sec (n = 10) vs 74.5±40.8 sec (n = 15); p = 0.0474; WT and Pdyn KO respecitively), while the delay to to onset of the first immobile phase did not reach statistical significance  vs 88±60.7 sec (n = 15); p = 0.1266; WT and Pdyn KO respecitively). No statistically significant differences were observed for either the time spent immobile or the latency to the first immobile phase between WT and Pdyn KO mice on the C57Bl/6N background. While WT mice spent 73±49.9 sec (n = 22) immobile, Pdyn KO mice were immobile for an average of 74±43.1 sec (n = 32) (p = 0.8954). The onset of the first immobile period was observed in WT mice after 81±39.6 sec (n = 22), while in Pdyn KO mice the first immobile phase occurred after 78±33.7 sec .In situ hybridization revealed a reduction in the levels of CRH mRNA in the central amygdala (100±5.8% (n = 10) vs. 74±3.8% of control (n = 10); p = 0.0014; WT vs. Pdyn KO, respectively) but not in the paraventricular nucleus (100±3.2% (n = 10) vs. 92±4.2% of control (n = 10); p = 0.1472; WT vs. Pdyn KO, respectively) of C57Bl/6N Pdyn KO mice. Similar results were obtained from Balb/c mice (CeA: 100±2.6% (n = 6) vs. 85±1.35% of control (n = 4); p = 0.0029; WT vs. Pdyn KO, respectively; PVN: 100±4.7% (n = 6) vs. 96±5.0% of control (n = 4); p = 0.5874; WT vs. Pdyn KO, respectively). Levels of NPY mRNA were unchanged in neurons of the basolateral amygdala of female mice on both backgrounds (97±5.0% (n = 10) and 93±3.1% of control (n = 6); C57Bl/6N and Balb/c, respectively).Investigation of the involvement of dynorphin in anxiety control has produced conflicting results to date. The phenotypes observed in different Pdyn KO mouse lines range from markedly anxiolytic Our present data confirm an anxiogenic role for prodynorphin-derived peptides. Thus balb/c Pdyn KO mice displayed increased ambulation in all three anxiety-related tests, backing up the data obtained for mice on the c57bl/6N background In contrast to balb/c mice of both sexes and male C57Bl/6N mice, female Pdyn KO C57Bl/6N mice displayed reduced anxiety in dependence of the testing paradigm. Increased ambulation in aversive zones in the elevated plus maze was opposed by unchanged behaviour in the open-field or light-dark choice tests. This may be due to the lower overall anxiety observed in female rodents 2 receptor antagonists 1 receptor deficient mice In a recent study In line with our recent results obtained from C57Bl/6N mice, male Pdyn KO balb/c mice also displayed longer lasting and earlier immobility than wild-type mice in the tail suspension test. This phenotype appeared less pronounced in female balb/c mice and was not observed in female Pdyn KO C57Bl/6N. In contrast, no marked differences in behaviour were observed in the forced swim test, regardless of genetic background or sex. However there are contrary findings regarding activation of KOP and the behavioural response to stress. Thus, McLaughlin et al. reported disrupted stress response from Pdyn deficient mice Based on the behavioural and neurochemical data of our recent and present studies and the data obtained by other groups, we suggest that the influence of dynorphin on anxiety control predominantly depends on the relationship between the trait anxiety and stress resistance of mice and the level of aversion in the test setup. Thus, Pdyn KO on the balb/c background with high trait anxiety displayed anxiolytic-like phenotypes in all tests and both sexes. In contrast, Pdyn KO on the less anxious C57Bl/6N background do not. Female mice  only appeared less anxious in the elevated plus maze test.It is noteworthy that male Pdyn KO on this background only displayed increased entry into the light area of the light-dark choice test at 400, and not at 150 lux


Intrinsic disorder is abundant in viral genomes and provides conformational plasticity to its protein products. In order to gain insight into its structure-function relationships, we carried out a comprehensive analysis of structural propensities within the intrinsically disordered N-terminal domain from the human papillomavirus type-16 E7 oncoprotein (E7N). Two E7N segments located within the conserved CR1 and CR2 regions present transient α-helix structure. The helix in the CR1 region spans residues L8 to L13 and overlaps with the E2F mimic linear motif. The second helix, located within the highly acidic CR2 region, presents a pH-dependent structural transition. At neutral pH the helix spans residues P17 to N29, which include the retinoblastoma tumor suppressor LxCxE binding motif (residues 21–29), while the acidic CKII-PEST region spanning residues E33 to I38 populates polyproline type II (PII) structure. At pH 5.0, the CR2 helix propagates up to residue I38 at the expense of loss of PII due to charge neutralization of acidic residues. Using truncated forms of HPV-16 E7, we confirmed that pH-induced changes in α-helix content are governed by the intrinsically disordered E7N domain. Interestingly, while at both pH the region encompassing the LxCxE motif adopts α-helical structure, the isolated 21–29 fragment including this stretch is unable to populate an α-helix even at high TFE concentrations. Thus, the E7N domain can populate dynamic but discrete structural ensembles by sampling α-helix-coil-PII-ß-sheet structures. This high plasticity may modulate the exposure of linear binding motifs responsible for its multi-target binding properties, leading to interference with key cell signaling pathways and eventually to cellular transformation by the virus. In 1984, Emil Fisher proposed that proteins must acquire a unique three-dimensional globular structure in order to achieve functionality. This hypothesis was first challenged by the discovery of gene sequences that encoded for unfolded proteins The most widely used experimental techniques for IDP studies include protease mapping, Far-UV circular dichroism spectroscopy (CD), nuclear magnetic resonance (NMR), In-cell NMR, analytical ultracentrifugation (AUC), fluorescence correlation and vibrational spectroscopy, among others The proportion of IDDs and IDPs was noticed to be particularly high in viral genomes, where a small number of gene products or their combination is sufficient for completion of the viral life cycle. This increased proportion of disordered regions has been associated with high adaptability and mutation rates, and also with the high structural flexibility that allows interaction with multiple cellular and viral targets conferring a multifunctional nature 2+ binding protein composed of two domains, the N-terminal domain  and the C-terminal domain  bona fide domain despite the lack of canonical secondary or tertiary structure, or cooperative unfolding The human papillomavirus type-16 (HPV16) E7 oncoprotein, is a small, 98-amino acid acidic ZnWe have shown that HPV16 E7 can self-assemble into defined spherical oligomers (E7SOs) upon removal of its coordinated zinc atoms Given the medical relevance of HPV and the prototypic nature of the HPV E7 oncoprotein as a model viral IDP In order to dissect the structural elements within the intrinsically disordered E7N domain, we recombinantly expressed or synthesized a series of proteins and peptide fragments spanning different regions of the HPV-16 E7N domain and the E7 protein. The sequences of E7N and its sub-fragments are listed and shown in 2·dmol−1 resembling those of the previously described E7 (1–40) domain 2·dmol−1) characteristic of PII conformation Comparison of the far-UV CD spectra of the E7N sub-fragments in 10 mM Tris.Cl buffer at pH 7.5 showed a lack of canonical secondary structure with a general appearance of disorder . Most suH20G and m-values for helix formation were obtained. The thermodynamic parameters of the E7N sub-fragments were similar to those of the E7N domain . One of the most widely used co-solvents, 2,2,2-trifuoroethanol (TFE), is known to stabilize α-helical populations in sequences with intrinsic α-helical propensity N domain . The perN domain , and incN domain . It mustN domain , suggestN domain . These r2·dmol−1, In order to assess the influence of the globular E7C domain on the structural determinants of E7N, we analyzed helix stabilization in full-length E7 and in the truncated version E7 (27–98). Far-UV CD spectra of both species are shown in comparison to the E7 (1–40) domain  A–C. WhipKa of 4.4±0.2, while E7 (16–31) showed a single transition with an average pKa of 5.5±0.1,  or to the decrease in the dielectric constant produced by TFE pKa value of 5.2±0.1 , which is known to stabilize α-helix in E7N at supra-micellar concentrations (CMC  = 5 mM), and may stabilize ß-sheet conformations at sub-micellar concentrations appM)  of all peptides, indicating that all fragments were monomeric in solution  sedimentation velocity experiments Mapp) . The estsolution . Peptidesolution , which cminf/f, which is a useful parameter for describing the shape of macromolecules in solution f/fmin values of globular proteins are nearly constant, increasing from 1.15 to 1.3 for the 5- to 1000 kDa molecular mass range, while f/fmin values for IDPs are significantly larger and increase from 1.5 to 3 for molecular mass ranging from 5 to 200 kDa minf/f values for all E7N fragments had values ranging between 1.40 and 1.53 (H) for the different fragments by using AUC at pH 7.5 and for the E7N domain by pulsed field gradient NMR experiments (PFG-NMR) at pH 7.5 and pH 5.0. PFG-NMR measures molecular diffusion rates and accurately complements the overall analysis of size and shape of proteins in solution by AUC. The RH values for the E7N domain from AUC and NMR measurements at pH 7.5 and similar concentrations (∼ 1mM) were 17±1 Å and 15.3±0.2 Å, respectively. The small discrepancy of the RH values obtained may be due to differences intrinsic to both techniques. Both RH values obtained for E7N were significantly larger than the RH value expected for a 40 residue globular protein (13.8 Å) H value predicted for a fully unfolded polymer (18.1 Å) H value at pH 5.0 , with most peaks within the 7.8–8.5 ppm range, making assignment a rather challenging task. The methyl proton region also showed poor dispersion of peaks, confirming the expected disordered nature of E7N. Using two-dimensional 1H-1H and 1H-13C spectroscopy we were able to fully assign the proton and carbon resonances of E7N in aqueous solution at pH 5.0  NOEs for the trans-proline isomer was observed for P6 and P17 residues, while no Hα-Hα  cross peak, characteristic for the cis-proline conformation, was detected for either proline residue (data not shown). This is in contrast with the linker region joining the disordered N-terminal and the globular C-terminal domains of E7, which also harbors two proline residues (P41 and P47), that present a cis-trans isomeric equilibrium Interestingly, in all the experimental conditions under which we studied E7N (see below), both prolines, P6 and P17, were fully in 1H spectrum, suggesting that the peptide underwent a major structural change in this condition, in agreement with the results from Far-UV CD experiments. At pH 7.5 and 3 mM E7N , we were able to measure chemical shifts using 50% TFE, where maximal helical content is reached  and E7 (16–40) which contain these regions  where PII conformation is stabilized. Despite the large peak overlapping, we were able to measure four coupling constants in the PEST region  region are deprotonated at pH 7.5, the electrostatic repulsion stabilizes extended conformations such as PII and precludes the formation of α-helix. Conversely, at low pH where acidic side chains are neutralized, α-helix conformations are favored.In order to have further confirmation of α-helical propensities and to investigate the presence of PII structure in E7N, we measured spectrum . Figure analysis . In wateα values  yet no sα values , makes iIn the present work we performed a structural dissection of the HPV-16 E7N IDD minf/f values characteristic of IDPs. These results show that although E7N and its conserved regions present global hydrodynamic properties typical of IDPs, not “all disorder” within the E7N domain is similar.Most sub-fragments used for the structural dissection display similar molar ellipticity values, disordered-like Far-UV CD spectra with a minimum at ∼200 nm, and present transient α-helical populations that can be stabilized by TFE to different degrees  and 3. TWe found a pH-dependent increase in α-helical content within E7 (1–40) at high TFE that was not observed in either full-length E7 or in the truncated version E7 27–98) , which i , which Interestingly, fragment E7 (21–29) comprised within Helix II is not able to stabilize an α-helix in isolation even at high TFE , which sMost of the linear motifs identified in E7N are located within the regions that populate transient secondary structures . The ubiTwo of the main interaction motifs within E7N show different conformational properties related to their target-bound forms. The E2F-mimic motif forms a α-helix in the bound form in vivoCKII and PEST sites are contiguous in most HPV E7 proteins, evolved together, and are functionally coupled in Rb binding Interestingly, a very recent publication reported secondary α-helical structures from homology and ab initio modeling in the same regions that we describe here by solvent stabilization and NMR In summary E7 (1–40) is a paradigmatic example of an IDD defined through bioinformatic and experimental analyses, which is able to populate dynamic but discrete structural ensembles that are tuned by pH. As the acidic residues are deprotonated, their side chains are electrostatically repelled, and the peptide adopts a more extended conformation involving an increase in PII at the expense of a loss in α-helix. The PV virus life cycle depends on the differentiation from basal epithelial cells to keratinocytes, which may involve substantial changes in the physicochemical environment inside the cell including parameters such as crowding, redox state or pH. Our results suggest that these changes could impact on the conformational equilibria and function of E7 through sampling of α-helix-coil-PII-ß-sheet structures that may modulate the exposure of post-translational modification, degradation and protein interaction sites. This conformational plasticity may potentiate E7 interference with key cell signaling pathways required for completion of the viral life cycle and contribute to cellular transformation by the virus.E. coli BL21 strain. Cell cultures were grown in 2L of 2TY medium at 37°C containing 0.1 mg/ml ampicillin. The induction of cultures was performed four hours after inoculation by adding 0.4 mM IPTG. Cells were harvested by centrifugation after 12 h of induction. For the purification procedure the protocol described for the full length E7 (1–98) The recombinant E7 HPV-16 protein (E7 (1–98)) was expressed and purified as previously described The E7 peptides used in this work, E7 (1–40), E7 (1–20), E7 (16–40), E7 (16–31), E7 (21–29) and E7 (25–40), were synthesized by F-moc chemistry  and purified by reverse phase HPLC. The purity of all preparations was judged by MALDI-TOF MS. The peptides were dissolved in Buffer Tris.Cl 50 mM pH 8.0 to a concentration of approximately 1 mM and were stored at −80°C. Quantification was carried out by absorbance at 276 nm in buffer solution and 220 nm in HCl. Peptide sequences are shown in Unless stated otherwise, measurements at pH 7.5 were performed in 10 mM Tris.Cl buffer and at pH 5.0 in 10 mM Sodium formate buffer, both with 1mM DTT at 20+/−0.1°C. All chemical reagents were of analytical grade (purchased from Sigma Aldrich) and all solutions were prepared with distilled and deionized water (Milli-Q plus) and filtered through 0.22 µm membranes prior to use.CD measurements were carried out on a Jasco J-810 spectropolarimeter  with cell paths of 0.1 and 0.2 cm. CD spectra were recorded between 195 and 260 nm at standard sensitivity and at a 50 nm/min of scanning speed with a response time of 8 s, data pitch of 0.1 nm and a bandwidth of 2 nm. All spectra were an average of at least 8 scans. Baseline measurements using buffer alone were subtracted from the measured spectra. Protein concentrations used for the E7 (1–98) and E7 (27–98) were 10 µM. Peptide concentration ranged between 20 to 80 µM depending on the peptide size. Raw data were converted to molar ellipticity, using the following equation:Where deg is the raw signal in millidegs, [c] is protein concentration in molar units, # bonds is the number of peptide bonds (number of amino acids –1), and L is the path length in cm.2,2,2-trifluoroethanol (TFE) measurements. TFE stabilization of helical conformations is stronger at lower temperatures, with little variation between 5°C and 25°C Data analysis. The mean residue ellipticity at 222 nm, which is assumed to be proportional to helical content, was plotted as a function of [TFE]/[H2O] ratio and was fit to a two state “coil to helix” equilibrium model 0, H20G and m, molar ellipticity data was fitted to the following equation:H2O and [θ]TFE are the mean residue ellipticities in water and at high TFE concentration, R is the gas constant and T is the temperature in Kelvin. The average percentage of residues in alpha helix conformation in water and TFE for each peptide was calculated from the values [θ]H2O and [θ]TFE obtained from the fitting, using the following empirical relationship to define the molar ellipticity value expected for 100% of helix pH titration curves at 30% TFE. The samples were prepared in 10 mM Citrate phosphate buffer ranging from pH 3.0 to 8.0 mixed with 30% TFE and incubated for one hour at room temperature.Data analysis. The mean residue ellipticity at 222 nm as a function of pH was fit to a variant of the Henderson-Hasselbalch equation pKa value for each fragment, assuming that all ionizable groups are deprotonated in the initial state and protonated in the final state:Din and Pin are the spectroscopic signals of the deprotonated and protonated states, respectively and Dm and Pm account for the linear variation of the signals with pH. The fractions of deprotonated and protonated species (Df and Pf ) are defined as:Sodium dodecyl sulfate (SDS). Experiments were carried out in 10 mM sodium formate buffer at pH 3.0. Samples were equilibrated for one hour at room temperature with different concentrations of SDS (100 µM to 25 mM). The critical micelle concentration (CMC) for sodium formate buffer used in the present work was previously reported Low temperatures were used in order to stabilize PII structures 56]. CD spectra for the different E7 peptides were measured at temperatures ranging from 5 to 75°C. The propensity to adopt PII structure for each peptide was assessed by analyzing the difference spectrum (5°C–75°C) at 218nm http://www.jphilo.mailway.com/). Partial specific volume of peptides was calculated from the amino acid sequences using SEDNTERP. Analyses of SV experiments were made using the continuous distribution c(s) and the non-interacting species model analysis of SEDFIT software from P. Schuck . In order to study non-ideality effects in solution, various protein concentrations were assessed . The sedimentation and diffusion coefficients 0s and 0appD were derived from linear approximations to infinite dilution (appM) and hydrodynamic radius (HR) for each peptide according to the Stokes-Einstein and Svedberg equations:Sedimentation velocity (SV) analytical ulracentrifugation experiments (AUC) were performed for peptides E7 (1–40), E7 (1–20) and E7 (16–40) in 50 mM Tris.Cl buffer at pH 7.5 . All AUCdilution  and usedR is the gas constant, T the absolute temperature, NA Avogadro's number and η solvent viscosity , which depends on the size of the molecule in solution, to the minimum theoretical hydrodynamic radius of anhydrous volume (minR):minR depends on the cubic root of the molar mass (M) and non-hydrated volumes of the particle (V):13C-1H correlation experiments were carried out at natural abundance. Pulsed field gradients were appropriately employed to achieve suppression of the solvent signal and spectral artifacts. The proton carrier was centered on the H2O frequency. Quadrature detection in the indirectly detected dimensions was obtained using the States-TPPI or the echo-antiecho method and the spectra were processed with the NMRPipe software NMR experiments were performed on a Bruker 600 MHz Avance III spectrometer equipped with a 5 mm triple resonance cryoprobe incorporating shielded z-axis gradient coils. All the heteronuclear 2O pHs 5.00 and 7.50, containing 5% D2O; TFE-d2 (50%), final pH 7.5; TFE-d2 (12%), final pH 5.0. All NMR samples contained 10 mM TCEP for avoiding oxidation in the course of the spectra acquisition. The experimental conditions were carefully controlled such that no oxidation or precipitation of the peptide occurred during measurements and that the pH of the sample remained stable.In order to explore the conformational properties of E7 (1–40), all proton and carbon resonances of an unlabelled 3 mM sample of E7 (1–40) were assigned at 20°C, for which the best quality spectra were obtained, in the following solutions: H1H-1H NOESY (100 and 250 ms mixing times), 1H-1H TOCSY (30 and 70 ms mixing times), 1H-13C HMQC, optimized to observe aliphatic or aromatic regions, and HMQC-TOCSY (70 ms mixing time) spectra were acquired. The NOESY experiments were acquired with 4096 (t2), and 1024 (t1) complex points, with spectral widths of 9615 and 6000 Hz in the direct and indirect dimensions, respectively. The TOCSY data sets consisted of 2048 (t2), and 800 (t1) complex points with the same spectral widths used in the NOESY experiments. The 1H-13C HMQC and 1H-13C HMQC-TOCSY data were collected with 2048 (t2), and 800 (t1) complex data points, and spectral widths of 9615 (1H), and 8906 (13C) Hz, respectively. The 13C carrier was centered at 40.0 ppm. For the aromatic 1H -13C HMQC, 2048 (t2), and 256 (t1) complex data points were collected, the spectral widths were of 9615 and 3774 Hz, respectively, and the carbon carrier was 130.0 ppm. Chemical shifts in water pH 5.0 were deposited in the Biological Magnetic Resonance Bank (BMRB code: 19269). For the other experimental conditions see Proton and carbon chemical shift assignments were achieved using a combination of homonuclear and heteronuclear standard 2D experiments. 1JCαHα coupling constants were also measured. In this case, proton coupled 1H -13C HMQC experiments were performed in H2O pH 7.5 (5% D2O) at 4°C; in H2O pH 5.0 (5% D2O) at 20°C; and in H2O TFE-d2 (50%) final pH 7.50 at 20°C. To increase resolution, the spectral width of the indirectly detected dimension was lowered to a fraction of the 13C spectral window. The alpha region was selected and the rest of the signals were folded into the reduced spectral window. Thus, the 1H-13C HMQCs were acquired with the 13C carbon carrier centered also at 40.0ppm, but with 2048 (t2), and 2048 (t1) complex data points, and spectral widths of 9615 (1H), and 2868 (13C) Hz, respectively.Typically, secondary chemical shifts are a convenient and accurate way to determine a polypeptide secondary structure 2O) was added to the sample (300 µL) as internal standard H) values for E7N in different experimental conditions were calculated as follows:The pulsed field gradient NMR self-diffusion measurements were performed using the PFG-SLED sequence diox and DE7 (1–40) are the measured diffusion coefficients of dioxane and E7 (1–40), respectively, and RHdiox is the effective hydrodynamic radius of dioxane, taken to be 2.12 Å And for unfolded proteins:HFigure S1TFE titration curves followed by Far UV-CD at pH 7.5 and pH 5.0. Rows represent the TFE titration curves for E7N and E7N sub-fragments. The name of the fragments is indicated at the right of each row. A) Far-UV CD spectra at different TFE percentage mixtures measured at pH 5.0. The arrows show the sense of change upon increasing TFE percentage. B) TFE Titration curves followed by ellipticity at 222 nm at different [TFE]/[buffer] molar ratios at pH 7.5 (black circles) and pH 5.0 (open circles). The data were fitted to a two-state coil-helix equilibrium model Click here for additional data file.Figure S2pH induction of α-helix structure within the E7N domain. Rows represent the pH transition curves at 30% TFE for E7N and the different sub-fragments. The name of the fragments is indicated at the right of each row. A) Far-UV CD spectra at different pH values after one hour of incubation. The arrows show the sense of change upon decreasing pH. B) pH equilibrium transition followed by molar ellipticity at 222 nm. The vertical dashed lines show the pH values selected for the CD and NMR studies. The buffers used for all spectra were 10 mM citrate phosphate with 30% TFE at different pH values ranging from 8.0 to 3.0. Fits for each signal obtained from global fitting of the data to (TIF)Click here for additional data file.Figure S3Analytical ultracentrifugation of the E7N domain and CR1 and CR2 regions. Sedimentation velocity profiles of E7 (1–40), E7 (1–20), and E7 (16–40) (left to right panels) at 50000 rpm and 20°C, in 50 mM Tris.Cl buffer at pH 7.5. A) Superposition of experimental (dots) and fitted (continuous line) profiles corrected for all systematic noise for E7 (1–40), E7 (1–20), and E7 (16–40) at 1.0, 3.4, and 2.2 mg ml−1 respectively. The last profile corresponds to 24 hours of sedimentation. The fit was obtained from the c(s) analysis of the SEDFIT program. For all peptides, the Lamm equation was simulated for 300 particles in the ranges  and , with a partial specific volume −1 for E7 (1–40), E7 (1–20), and E7 (16–40) respectively. Frictional ratio f/fmin was fitted for each sample. B) Corresponding c(s) distribution in the range 0–10 S. The signal was normalized to 1 cm optical path length. C) Superposition of the c(s) distributions for different concentrations of peptides in the range 0–2 S. The signal was normalized to 1 cm optical path length.(TIF)Click here for additional data file.Figure S4Analytical ultracentrifugation concentration dependency analysis. Concentration dependency of s−1 A) and Dapp B). The linear regressions (lines) provide values, which are given in s0, and D0app. The dependence of the inverse of the sedimentation coefficient s−1 with the concentration c (g ml−1) is analyzed by the equation s−1 =  s0−1+kss0−1c, where s0 is the sedimentation coefficient extrapolated to zero concentration, and ks is the concentration dependence coefficient for s (ml g−1). For the apparent diffusion coefficient Dapp, the dependence with the concentration c (g ml−1) is analyzed by the equation Dapp  =  D0app+kDD0appc, where D0app is the diffusion coefficient extrapolated to zero concentration, and kD is the concentration dependence coefficient for Dapp (ml g−1).(TIF)Click here for additional data file.Figure S5Analysis of polyproline type II (pII) propensity within the E7N domain. Difference spectrum between 5°C and 75°C for E7N and E7N sub-fragments performed in 10 mM Tris.Cl buffer at pH 7.5 (full line) and 10 mM sodium formate buffer at pH 5.0 (dotted line). The fragment names are indicated inside each panel.(TIF)Click here for additional data file.Table S11H and 13C chemical shifts assignments of the E7N in aqueous solution containing 102O at 20°C and pH 7.5.mM TCEP and 5% Da 1H Chemical shifts are reported in ppm with an accuracy of ±0.02 ppm. 13C Chemical shifts are reported in ppm with an accuracy of ±0.1 ppm. bCarbon chemical shifts first, and proton chemical shift in brackets. cThese signals may be interchangeable.(DOC)Click here for additional data file.Table S21H and 13C chemical shifts assignments of the E7N in aqueous solution containing 10mM TCEP, 12% TFE-d2 at 20°C and pH 5.0.a 1H Chemical shifts are reported in ppm with an accuracy of ±0.02 ppm. 13C Chemical shifts are reported in ppm with an accuracy of ±0.1 ppm. bCarbon chemical shifts first, and proton chemical shift in brackets.(DOC)Click here for additional data file.Table S31H and 13C chemical shifts assignments of the E7N in a 1:1 H2O: TFE-d2 solution containing 5mM TCEP at 20°C and pH 7.5. a 1H Chemical shifts are reported in ppm with an accuracy of ±0.02 ppm. 13C Chemical shifts are reported in ppm with an accuracy of ±0.1 ppm. bCarbon chemical shifts first, and proton chemical shift in brackets.(DOC)Click here for additional data file.Table S41JCαHα scalar coupling constant values (in Hz) measured for E7N. a The accuracy of the measurements is estimated to be ±0.5 Hz. bTFE-2d. c n.d.: not determined.(DOC)Click here for additional data file.


Verticillium longisporum represents an allodiploid hybrid with long spores and almost double the amount of nuclear DNA compared to other Verticillium species. V. longisporum evolved at least three times by hybridization. In Europe, virulent A1xD1 and avirulent A1xD3 hybrids were isolated from the oilseed crop Brassica napus. Parental A1 or D1 species are yet unknown whereas the D3 lineage represents Verticillium dahliae. Eleven V. longisporum isolates from Europe or California corresponding to hybrids A1xD1 or A1xD3 were compared. A single characteristic type of nuclear ribosomal DNA could be assigned to each hybrid lineage. The two avirulent A1xD3 isolates carried exclusively D3 ribosomal DNA (rDNA) which corresponds to V. dahliae. The rDNA of all nine A1xD1 isolates is identical but distinct from D3 and presumably originates from A1. Both hybrid lineages carry two distinct isogene pairs of four conserved regulatory genes corresponding to either A1 or D1/D3. D1 and D3 paralogues differ in several single nucleotide polymorphisms. Southern hybridization patterns confirmed differences between the A1 and D1/D3 isogenes and resulted in similar patterns for D1 and D3. Distinct signatures of the Verticillium transcription activator (VTA)2 regulatory isogene pair allow identification of V. longisporum hybrids by a single polymerase chain reaction and the separation from haploid species as V. dahliae or Verticillium albo-atrum. The combination between VTA2 signature and rDNA type identification represents an attractive diagnostic tool to discriminate allodiploid from haploid Verticillia and to distinguish between A1xD1 and A1xD3 hybrids which differ in their virulence towards B. napus.The cruciferous fungal pathogen The online version of this article (doi:10.1007/s00253-012-4530-1) contains supplementary material, which is available to authorized users. Speciation is a dynamic process, which requires the isolation of different populations to prevent the exchange of genetic material. Reproductive barriers are formed by changes in ploidy, chromosomal rearrangements or incompatibilities between genes. Reproductive barriers are prerequisites for the evolution of diverging genetic elements, which convey an ecological separation Mallet .Verticillium has a world-wide distribution and includes species which cause vascular wilting disease and early senescence in a broad range of economically important crops including alfalfa, lettuce, hops, olive trees, oilseed rape, potato, tomato or strawberries  forming branches at each node of the conidiophores  as long asexual spores (conidia) in comparison to V. dahliae  than those of V. dahliae or V. albo-atrum isolates  for genomic DNA or liquid SXM (Neumann and Dobinson V. longisporum genomic DNA by the high-fidelity Phusion polymerase with modifications optimized for interspecies hybrid microorganisms including increases in concentration of each primer (100 pmol) and each dNTP (20 mM) for each reaction of 25 μl, an increase in time for elongation step (4.5 min) together with a decrease in number of PCR cycles  was used to screen positive colonies from each cloning procedure. At least, 10–15 positive clones were selected and grown in LB (Luria-Bertani) liquid medium containing 100 μg/ml of ampicillin. Recombinant plasmids were purified with the QIAprep Spin Miniprep Kit  for sequencing. The sequencing was performed with the primers (forward and reverse) from the cloning kit by the in-house Göttingen Genomics Laboratory. DNA alignments with the Geneious Pro v5.4 software  amplifications were performed in 25 μl volumes with the high-fidelity Phusion polymerase  using the following general conditions: an initial denaturation at 98 °C for 3 min; a cycle including denaturation at 98 °C for 30 s, annealing at 55–60 °C for 40 s, and extension at 72 °C for 1–4.5 min was repeated for 25–30 times; a final extension at 72 °C for 7 min and stored at 4 °C until used. PCR products were analyzed on a 1 % agarose gel and the DNA fragments were excised and purified with QIAquick gel extraction kit . The purified DNA fragments were used for direct sequencing. For cloning, each DNA fragment of interest was amplified from The target genes contain no restriction sites for the enzymes used for genomic DNA digestion. A fragment (about 600 bp) of the target gene or the whole gene was amplified and labeled as probe for Southern hybridization. In brief, 20 μg of genomic DNA were digested with 3 μl of an appropriate restriction enzyme for overnight. The digested mixture was analyzed on a 1 % agarose gel, and DNA was transferred to an Amersham Hybond-N membrane  by blotting. The DNA on the membrane was overnight hybridized to a specific probe and the Amersham CDP-Star Detection reagent  was used to detect chemiluminescence signals according to the manufacturer’s instructions.p distance model was used and gaps/missing data were treated with complete deletion or pairwise deletion.Characterization of 18S rRNA gene, ITS1, 5.8S rRNA, ITS2, 28S rRNA, and intergenic region (IGS) of the rDNA was based on Genbank accession number AF104926  covering 5.8S rRNA gene and a large IGS were analyzed , ITS1 sequence (129 bp), 5.8S rRNA gene (158 bp), ITS2 sequence (165 bp), and a part of 28S rRNA (178 bp). Sequence analyses revealed that the 5.8S rRNA gene located between ITS1 and ITS2 is completely conserved within all three species. All nine sms Fig. .V. longisporum isolates. The ITS type 1 of the two European isolates Vl19 and Vl32 is identical to the ITS of V. dahliae. Vl19 and Vl32 have been isolated from oilseed rape in Europe and are both avirulent  within the 11 TS1 Fig. . The ITSTS1 Fig.  and threTS1 Fig. . A1 and sis Fig. . TherefoA closer inspection of the IGS separating rDNA repeats revealed PCR products ranging from 1.7 to 1.9 kb using the primer pair IGS-F/IGS-R. The IGS region is highly polymorphic within all three species and can be divided into two parts based on the sequence conservation Fig. . The varV. longisporum isolates. The IGS type 1 (two European isolates Vl19 and Vl32) groups within the V. dahliae clade and corresponds to D3  within the 11  D3 Fig. . It conttes Fig. .V. albo-atrum and corresponds to A1  in the IGS region from European oilseed rape and four repeat copies of the same sequence in the IGS of the American A1xD1 isolates from cauliflower. V. albo-atrum IGS differs from A1 IGS and has no specific repeats in the IGS region of rDNA  and A1xD3 (avirulent) by a single PCR reaction. The 18S rRNA gene-specific primer pair (18S-rRNA1/18S-rRNA2) resulted in a 0.44–1.28 kb fragment corresponding to two types of 18S rRNA genes for different V. longisporum isolates . The intron is absent in the 18S rRNA genes of the two A1xD3 V. longisporum isolates  carrying the V. dahliae D3 rDNA type and is also absent in all analyzed V. dahliae and V. albo-atrum isolates. The 839-bp intron is therefore typical for the A1 lineage but only conserved within the virulent V. longisporum A1xD1 hybrids which have lost the D1 rDNA  can be separated by their rDNA types. Only the two rDNA types A1 and D3 were maintained, the D1 rDNA type has been lost in all isolates. The virulent A1xD1 hybrids carried only the A1 rDNA including a specific intron of unknown origin which can be used to discriminate these strains from the avirulent A1xD3 hybrids carrying exclusively the D3 rDNA type which resembles the rDNA of the V. dahliae isolates.Our results demonstrate that the analyzed V. longisporum and there is hardly any comparison of regulatory genes between the different Verticillium isolates corresponding to A1xD1, A1xD3 hybrids and related haploid species. Two orthologues of the velvet genes VeA and VelB were examined, which are conserved in ascomycetes. For several fungi, these regulators are known to control secondary metabolism and fungal development including hyphal morphogenesis or the formation of the conidiophore producing asexual spores  from genomic DNA of all nine V. dahliae isolates as well as from all 11 V. longisporum isolates (A1xD1 and A1xD3), but not from the four V. albo-atrum isolates. DNA sequencing resulted in the internal primer set VEL1-F/VEL1-612-R which amplified a 612-bp fragment of all isolates. The 612-bp products were cloned and sequenced and Fig. V. longisporum A1xD1 and A1xD3 carry pairs of isogenes for VEL1 . The VlVEL1-1 sequence of A1xD3 lineage  corresponds to parental D3 and is identical to V. dahliae VdVEL1. The D1 derivative VlVEL1-1 of A1xD1 hybrids  has accumulated three nucleotide polymorphisms (SNPs). Both lineages of V. longisporum share the same VlVEL1-2 sequence which corresponds to the A1 parent and displays a 95 % identity to the single genes of V. dahliae VdVEL1 or V. albo-atrum VaVEL1  for VEL2 in V. longisporum isolates but only one in V. dahliae (VdVEL2) and V. albo-atrum (VaVEL2). The data are summarized in the phylogenetic tree of the Fig. VlVEL2-1 of A1xD3 corresponds to the D3 parent and is identical to V. dahliae VEL1 (VdVEL1). The corresponding D1 VlVEL2-1 allele of A1xD1 carries differences in some single nucleotides. Interestingly, the VEL2 sequences of V. dahliae Vd73 (from linseed) can be separated from other V. dahliae strains including the D3 parent of V. longisporum for VEL2 but not for VEL1. Similarly, V. dahliae isolate Vd14.01 (from pistachio) has accumulated even more SNPs which separates it even more from the other V. dahliae strains than the D1 parent of the V. longisporum A1xD1 hybrid for VEL2 but not for VEL1  with high identities (94–100 %) to each other and to the corresponding single genes of V. dahliae or V. albo-atrum as relatives to D1, D3, or A1. The reason why V. dahliae strains from specific hosts have accumulated more mutations in the VEL2 gene remains to be elucidated.Conclusively, our data demonstrate that in contrast to the rDNA repetitive genes, which are uniform, there are two isogenes for the putative regulators V. longisporum. Five thousand expressed sequence tags were sequenced from a cDNA library of V. longisporum 1 and VTA2. The corresponding genes encoding zinc finger family proteins which are conserved within filamentous ascomycetes 2Cys6 conserved domain. This domain is similar to the conserved domain of the AflR transcription factor in Aspergilli required for mycotoxin biosynthesis of the cancerogenic aflatoxin  is only present in V. dahliae. These VlVTA1-1 sequences of the two V. longisporum hybrids A1xD3  and A1xD1  are almost identical to V. dahliae VdVTA1 with differences in one (D3) or eight (D1) single nucleotide substitutions of the 1.278-bp sequences, respectively. The V. albo-atrum as well as all shorter A1 VTA1 sequences of V. longisporum (VlVTA1-2) are identical in both lineages A1xD1 and A1xD3 and include the small three-nucleotide deletion in comparison to the isoallele  when compared with V. albo-atrum VTA2. In addition, the third intron (I3) of the V. dahliae gene (217 bp) is 63–68 nucleotides shorter than V. albo-atrum I3 (280–285 bp). In contrast to the single parental genes, both types of V. longisporum rDNA carry two paralogues  varied in lengths slightly between 1.701 and 1.755 kb. The VlVTA2-1 sequences from the two V. longisporum lineages A1xD1 and A1xD3 carry the same characteristic ACTCTCACC insertion in the first exon and the shortened third intron of 217 bp as V. dahliae VTA2 gene. The V. dahliae VTA2 gene is absolutely conserved in this species. VlVTA2-1 of two V. longisporum A1xD3 isolates  carrying V. dahliae rDNA corresponds to D3 because it is identical to VdVTA2. This allele differs from the D1 allele of VlVTA2-1 of the A1xD1 isolates carrying V. albo-atrum-related rDNA by six single nucleotides. In contrast, the A1 allele VlVTA2-2 of the A1xD1 lineage including six European isolates  and three American isolates  is identical and differs from the other A1 allele of VlVTA2-2 of the A1xD3 lineage  by only a single nucleotide. This VlVTA2-2 sequence exhibits an identity of about 97 % to V. albo-atrum VaVTA2 gene. It carries the characteristic first exon of V. albo-atrum lacking the nine nucleotide insertion as well as the longer third intron of 282 bp  been changed during geographical separation. Sequencing of the corresponding VTA2 genes from nine V. dahliae isolates from different geographical regions also reveals an absolute conservation (100 % identity) of this gene. In contrast, V. albo-atrum isolates display minor changes in VTA2 genes depending on hosts. The VTA2 genes from the non-Alfalfa isolates  deriving from potato, hop, and tomato are completely identical to each other, but exhibit a slight reduction of 97.72 % identity compared to VTA2 sequence of the Alfalfa isolate Va2. However, the Alfalfa and the non-Alfalfa group still have conserved the signatures of the first exon and the third intron  and VlVTA2-2 resembles V. albo-atrum VaVTA2 . The difference in lengths between the two VTA2 isogenes is mainly due to differences in the third intron  that amplifies only the third intron region (I3) of VTA2 gene. These primers bind to the conserved sequences in the third (E3) and fourth exon (E4) of VTA2 gene for all the three species –PCR as groups lsp (A1xD1) or lsp* (A1xD3) which both produce longer conidia in comparison to the short spores of V. dahliae and V. albo-atrum. The lsp/A1xD1 group displays a high virulence on the oilseed rape Brassica napus by killing about 50 % plants after 42 days of infection, whereas the lsp*/A1xD3 group is significantly less virulent  and avirulent A1xD3 (lsp*) after analyses in morphology of conidia and the resting structures. There are two PCR bands of 315 and 380 bp for both the virulent A1xD1 and the avirulent A1xD3, but only one PCR band of 315 or 380 bp for V. dahliae and V. albo-atrum, respectively  or avirulent A1xD3 (D3 rDNA) V. longisporum strains.The A1xD1 and A1xD3 iae Fig. . These miae Fig. . Two priely Fig. . In a serum Fig. . This coV. longisporum hybrids A1xD1 and A1xD3 have both been isolated from oilseed rape but differ significantly in their pathogenicity towards this crop and can additionally be separated by their rDNA type. Both hybrid lineages have homogenized their rDNA to only one parental version. Two distinct rDNA types representing either the D3 V. dahliae parent or the A1 parent could be even isolated from the same site in Northern Germany. All A1xD1 V. longisporum isolates from Europe or California only represent the A1 rDNA type which is closer to V. albo-atrum than to V. dahliae. The D1 rDNA has not yet been found in any V. longisporum isolate. The loss of parental rDNA in V. longisporum hybrids might account for the described reduction from the diploid to the 1.8-fold ploidy of analyzed isolates. The recent classification of V. longisporum into the three different lineages A1xD1, A1xD2 and A1xD3  could be more separated from other V. dahliae strains as the V. longisporum parent D1  unavailable to the parents. It is currently unclear whether one parent had already accumulated some suitable specific mutations prior to hybridization, which supported the interaction to the new host or whether ecological speciation has only started after hybridization. In this study, we demonstrated that the V. dahliae VEL2 gene, an orthologue of A. nidulans VelB, has an increased accumulation of SNPs in isolates from linseed (Vd73) and pistachio (Vd14.01) in comparison to the other tested V. dahliae isolates. This increased SNP accumulation might play a role in host adaptation. The VelB protein interacts with VeA and acts as a light-dependent regulator of fungal development and secondary metabolism of A. nidulans  in F. fujikuroi results in reduction in virulence on the host plant  for virulent A1xD1 and a small band (0.44 kb) for avirulent A1xD3. The analysis of several regulatory gene pairs in V. longisporum resulted in a second tool for classification which allows the discrimination between the hybrids and haploid V. dahliae or V. albo-atrum strains. The PCR examination of the barcode marker VTA2 separates all hybrids by the resulting two bands from the single bands of V. dahliae and V. albo-atrum. Monitoring of the changes in the genomes of V. longisporum isolates will be an important part of a better understanding of host-specific growth of this fungus. Such diagnostic tools will support to oversee the dynamics of hybrid-mediated species formation of Verticillia as an emerging threat for agriculturally important crop plants.The interspecies hybrid ESM 1PDF 15,000 kbBelow is the link to the electronic supplementary material.