Animal Study on Primary Dysmenorrhoea Treatment at Different Administration Times

The new methods of different administration times for the treatment of primary dysmenorrhea are more widely used clinically; however, no obvious mechanism has been reported. Therefore, an animal model which is closer to clinical evaluation is indispensable. A novel animal experiment with different administration times, based on the mice oestrous cycle, for primary dysmenorrhoea treatment was explored in this study. Mice were randomly divided into two parts (one-cycle and three-cycle part) and each part includes five groups (12 mice per group), namely, Jingqian Zhitong Fang (JQF) 6-day group, JQF last 3-day group, Yuanhu Zhitong tablet group, model control group, and normal control group. According to the one-way ANOVAs, results (writhing reaction, and PGF2α, PGE2, NO, and calcium ions analysis by ELISA) of the JQF cycle group were in accordance with those of JQF last 3-day group. Similarly, results of three-cycle continuous administration were consistent with those of one-cycle treatment. In conclusion, the consistency of the experimental results illustrated that the novel animal model based on mice oestrous cycle with different administration times is more reasonable and feasible and can be used to explore in-depth mechanism of drugs for the treatment of primary dysmenorrhoea in future.

Introduction
Dysmenorrhoea, which is caused by female endocrine disorders, is one of the unresolved problems in medical science. Approximately 80% of women globally had dysmenorrhoea in different degrees according to a report from the British medical authority [1]. This condition is classified into two categories: primary and secondary dysmenorrhoea. The incidence of primary dysmenorrhoea is higher. In an epidemiological study, 53.2% of women in China have dysmenorrhoea during their menstrual period, and the incidence rate is about 43% to 70% overseas [2]. Primary dysmenorrhoea can be mostly experienced by young women, which occurs without an identifiable pelvic pathology. Such condition is mainly characterised by cramping pain in the lower abdomen immediately before or during menstruation, which does affect the quality of their life and work [3].
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly used drugs for primary dysmenorrhoea treatment [4], particularly for cases in which subjective symptoms are moderate. However, NSAIDs have side effects, such as damage of liver and kidney after discontinuing medication [5].
Chinese medical treatment of primary dysmenorrhoea has a long history. Based on the rich clinical experience and syndrome differentiation theory, primary dysmenorrhoea was recently divided into five types, including the qi stagnation and blood stasis type, cold-damp stagnation type, qi and blood deficiency type, and liver and kidney deficiency type [6]. Traditional Chinese medicine (TCM) treats primary dysmenorrhoea by fundamentally regulating the female physique and body balance of Yin and Yang, and TCM mostly has no obvious side effects. Therefore, such treatment has elicited considerable attention worldwide.
Jingqian Zhitong Fang (JQF) prescription is a typical and relatively simple TCM formula. Such prescription originally came from Fo Shou San and has been proven effective for treating primary dysmenorrhoea. Radix Angelicae Sinensis (Angelica sinensis Diels) is the main drug in JQF and the main constituent that affects blood [7]. It could enrich the blood and alleviate menstrual pain [8]. Rhizoma Chuan Xiong (Ligusticum chuanxiong Hort.) can treat menstruation, amenorrhea, dysmenorrhoea, and headache [9]. Herba Leonuri Motherwort (Leonurus japonicus Houtt.) can also regulate menstruation [10]. Radix Paeoniae Rubra (Paeonia lactiflora Pall.) is used in clinical treatment of heat and cold blood, as well as for healing bruises and relieving pain [11]. We developed an ultra-performance liquid chromatographyquadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) method to construct the chromatographic fingerprint and to identify the constituents of JQF. Based on experimental pharmacological studies [12][13][14], JQF has a wide variety of actions, including antinociceptive, antiinflammatory spasmolysis and vasorelaxation, all of which may contribute to its therapeutic action.
Some restricted problems such as slow onset, long treatment cycle (the period of TCM treatment is three months per course), and poor taste of drugs hinder the modernization and internationalization of TCM [15,16]. Such problems need to be resolved. Recently, through continuous exploration, short-term medication and effective treatment method at different administration times for primary dysmenorrhoea are widely used clinically [17][18][19][20][21]. The administration time is 3 days (d) to 7 d before menstruation instead of the entire physiological cycle during medication. This treatment was only reported in clinical trials and never occurred in animal experiments. An animal experiment for primary dysmenorrhoea could facilitate not only observational changes of induced pain factor content but also an in-depth study of the pathway, receptor cells, and uterine tissues. After many years of discussion and research, animal experiments for primary dysmenorrhoea have already found a certain research foundation [22][23][24]. Animal experiments are important means for investigating the mechanisms of action underlying primary dysmenorrhoea.
Recently, animal studies have been conducted to explore the basis and mechanism of primary dysmenorrhoea treatment and developed a general animal model of this condition. Therefore, the current experiment selected the point of different administration times to treat primary dysmenorrhoea on the basis of the formal clinical studies [25][26][27]. The modelling time was selected in line with oestrous cycle by observing the mice vaginal smears and provided medicine in groups combined with clinical treatment. The scientific connotation of primary dysmenorrhoea treatment can then be explored at different administration times by animal experimental study. This treatment provides basic ideas for further research and also facilitates new information and methods for animal studies of primary dysmenorrhoea, which have explored the mechanism of action and dosage of drugs in recent years. Such treatment also provides further experimental data for TCM treatment of primary dysmenorrhoea and promotes modernisation and globalisation of TCM application.
In this study, we aimed to explore and establish an animal model by evaluating primary dysmenorrhoea treatment at different administration times, in which the primary dysmenorrhoea model was based on the proestrus stage of mice. Furthermore, the present study was a preliminary study on the mechanism of the treatment and optimized the animal primary dysmenorrhoea model. For outcome, prostaglandin E 2 assay kit, prostaglandin F 2 assay kit, nitric oxide assay kit (nitrate reductase method), and calcium colorimetric assay kit (Nanjing Jiancheng Bioengineering Institute); YXJ-type high speed centrifuge (Changzhou Jiancheng Teaching Instrument Factory); OLYMPUS U-CMAD3 optical microscope; Japanese OLYMPUS, OLYMPUS C5060-ADU optical camera; Japanese OLYMPUS; gas bath thermostats oscillator (Jintan Ronghua Instrument Manufacturing Co., Ltd.); and Infinite 200 PRO automatic enzyme mark instrument (Tecan Group, Ltd.) were applied.

Determination of Oestrous Cycle and Experimental
Groups. All 140 mice were fasted for 2 d and were randomly divided into groups before starting the experiments. At 8:00 AM, the mice vaginal mucus were collected on a glass slide fixed with methanol and stained with HE to determine the oestrous cycle time and to divide the same oestrous cycle mice in a group [28]. As a result of individual differences of the oestrous cycle of mice, 128 mice were confirmed with an oestrous cycle of 6 d, among which 120 mice were selected for experiment.
The entire experiments were departed into two parts to investigate the efficiency of treatment for one and three cycles. In each treatment cycle, mice were randomized into five groups, namely, JQF cycle group (6 d group), JQF last 3 d group (3 d group), Yuanhu Zhitong tablet group (Yuanhu group), model control group, and normal control group ( Figure 1).

Primary Dysmenorrhoea Mice Model and Administration.
The primary dysmenorrhoea mice model is improved from reference [29] of building the dysmenorrhoea model and assessment methods. Firstly, the model should determine the proestrus stage of the mice via vaginal smear test, and then dysmenorrhoea mice were modelled. In detail, oestradiol benzoate injection was subcutaneous injected to mice except those in the normal control group to establish the animal Evidence-Based Complementary and Alternative Medicine 3   model of primary dysmenorrhoea. For the JQF last 3 d group (in one cycle, mice were given saline for the first 3 days, and JQF was replaced for the remaining last 3 days) and 6 d group, mice were intragastrically given JQF (0.218 g/kg). As a positive control, Yuanhu Zhitong tablet group (0.1 g/kg) were intragastrically administered (0.02 mL/g). Simultaneously, saline at the same dose was given to mice in the normal control group (Figure 1 and Table 1).

Writhing Response and Preparation for Determination.
The drug effect on the behavioural pattern of mice was investigated. Writhing response complied with the Schmauss and Yaksh's [30] standard. The mice were fasted with water access for 12 h to 16 h prior to initiation of the study. On the sixth day, after the injection of oestradiol benzoate moulding for 40 min, the mice were administered oxytocin 2 U/mice (0.01 mL/g) by intraperitoneal injection. After 5 min, the writhing reaction times were recorded for 30 min. Blood was then extracted from the retroorbit sinus. Plasma specimens were separated by centrifugation at 3500 r⋅min −1 for 15 min at −4 ∘ C and then stored at −20 ∘ C until analysis. The specific method for sample detection strictly complied with the kit operation. The experiment complied with the Guide for the Care and Use of Laboratory Animals (National Research 2.6. Statistical Management. SPSS 13.0 software was used for statistical analysis. One-way ANOVA was used to analyse the difference between the two groups, and data were expressed as mean ± standard error of mean. A value less than 0.05 or 0.01 was considered as significance.

Stage of Oestrous Cycle Typical Vaginal Smear.
The characteristic of proestrus smear was almost nucleated epithelial and occasionally had few keratinocytes (Figure 2(a)). The smear was almost entirely covered with nucleus-free keratinocytes in the estrus (Figure 2(b)). White blood cells, keratinocytes, and nucleated epithelial cells simultaneously existed in the metestrus (Figure 2(c)). The diestrus mainly had white blood cells (Figure 2(d)).

Effects of Different Administration Times on Oxytocin-Induced Dysmenorrhoea Mice Model.
Compared with the normal control group, the writhing times of mice in the model control group were significantly increased ( < 0.01) (Figure 3). Compared with the model control group, the writhing times of the mice were significantly decreased in the one-cycle parts (6 d and the last 3 d group, < 0.01). Results of the three-cycle continuous moulding administration experiment were similar to those of the one-cycle treatment, but without significance between one-and three-cycle parts. Compared with the model control group, moreover, Yuanhu group decreased the writhing times of the mice ( < 0.05 or < 0.01), not only in the one-cycle part but also in the three-cycle part.

Effects of PGF 2 and PGE 2 at Different Administration
Times on Dysmenorrhoea Mice. Compared with the normal control group, PGF 2 of the model control group was significantly increased, and PGE 2 was significantly decreased, whereas the serum of PGF 2 and PGE 2 ratio was significantly increased ( < 0.01). Compared with the model control group, PGF 2 was significantly decreased, and PGE 2 was significantly increased; the serum of PGF 2 and PGE 2 ratio tended to be normal in the 6 d group and the last 3 d group ( < 0.01). The results of the three-cycle continuous moulding administration experiment were similar to those of the one-cycle treatment. Compared with the model control group, the one-cycle experiment of the Yuanhu group decreased the PGF 2 , whereas PGE 2 increased ( < 0.05). However, no obvious difference was found in the serum of PGF 2 and PGE 2 ratio. After three cycles of continuous administered treatment, the effects of all indexes were significantly modulated ( < 0.01) (Figures 4, 5, and 6).

Effects of NO and Calcium Ions at Different Administration
Times on Dysmenorrhoea Mice. Compared with the normal control group, NO contents in the serum of the model control group were significantly decreased, whereas calcium ions were significantly increased ( < 0.01). Compared with the model control group, NO contents in the serum were significantly increased, whereas calcium ions were significantly decreased in the 6 d and the last 3 d group ( < 0.01). The results of the three-cycle continuous moulding administration experiment were similar to those of the onecycle treatment. Compared with the model control group, the Yuanhu group significantly decreased the calcium ions in both one-cycle and three-cycle experiments ( < 0.01). However, no obvious difference was found in the NO contents of the serum in the one-cycle experiment. The NO contents  were significantly increased after three cycles of continuous administered treatment ( < 0.05) (Figures 7 and 8).

Screening of Experimental Animal and Basis of Another
Administration Time. In this study, experimental animal selection was defined based on our previous study on vaginal smear oestrous cycle of female rats and mice. The oestrous cycle of mice was generally from 5 d to 6 d, whereas that of rats was from 4 d to 5 d [31]. Owing to the fact that each stage of the oestrous cycle of rats was shorter and almost the same, the drug cannot completely play its role, and the results may be affected. On the basis of the time of primary dysmenorrhoea in animal model, the mice need only 6 d, and the rat usually takes about 10 d [32], which is not conducive to perform three cycles of continuous administration experiment. Finally, the female mice were selected as the experimental animals. The female physiology cycle is divided into four phases [33]: menstrual period (1 d to 4 d of the menstrual cycles); estrin phase (5 d to 14 d of the menstrual cycles, follicular maturation stage); progestational stage (15 d to 24 d of the menstrual cycles, corpus luteum maturation stage); and premenstrual period (25 d to 28 d of the menstrual cycles, degeneration of the corpus luteum period). In this study, on the basis of this theory and the result of JQF in previous clinical studies, the medication in luteal regression period of patients was effective, and the diestrus of mice was at the last 3 d of the cycle through vaginal smear. Therefore, the last 3 d in the cycle group (the last 3 d group) that equals administration in the corpus luteum period [34] was finally defined as another time of administration. To explore the different administration times of primary dysmenorrhoea, the results of the experiment cycle group (6 d group) were compared with those of the animal experiment.

Relationship between Related Pain Factors and Primary
Dysmenorrhoea. This research determined four kinds of wide pain factors from the existing primary dysmenorrhoea animal experiment references to explore the scientific connotation of different administration times for primary dysmenorrhoea treatment from the mechanism perspective. Prostaglandin was the most relevant pain factor to primary dysmenorrhoea [35]. PGF 2 combined with PGF 2 receptor on the spiral arterioles increased uterine contractility, resulting in ischemia pain [36]; PGE 2 could inhibit uterine contraction and induce uterine relaxation. The content of PGF 2 /PGE 2 in endometrium of dysmenorrhoea patients is higher than that of healthy women [37]. NO influenced the primary dysmenorrhoea from many aspects. NO could regulate uterine contraction and execute pain modulation of peripheral and central levels [38]. Through the NO-CGMP pathway, NO can cause pain and analgesia. The decrease in NO could promote nociceptive transmission and pain, whereas the increase in NO could inhibit analgesia [39]. Calcium ions also play an important role in primary dysmenorrhoea. Intracellular calcium overload induced cell membrane damage and cell energy metabolism; it also contributed to the ischemia-reperfusion injury, causing vasoconstriction of uterine blood vessels (uterine ischemia), increasing uterine smooth muscle contraction, and reducing the endometrial blood supply, which lead to the occurrence of dysmenorrhoea [40].

Open Problems.
The following problems need further investigation: whether only the selected pain factors could reflect the results which were consistent with the clinical treatment; whether the association with drug resistance resulted in no difference between the one-cycle group and the last 3 d of cycle group of primary dysmenorrhoea model mice; the main reason and whether it is related to the mechanism of possible hormone regulation. Given the preliminary results of this experimental study, further clinical and animal experimental studies will be conducted.

Conclusion
Both in one-cycle and three-cycle experiments, comparing with the model control group, the writhing times and contents of PGF 2 and calcium ion in serum were significantly decreased by administration of JQF (last 3 d group and 6 d group) or Yuanhu Zhitong tablet. Similarly, levels of PGE 2 , NO as well as PGF 2 and PGE 2 ratio were normalized in Evidence-Based Complementary and Alternative Medicine 7 the JQF last 3 d group and 6 d group. Moreover, results in the three-cycle part were the same as that of one cycle, which proved that the cycles and the different times in the same cycle (the last 3 d group and 6 d group) of administration with JQF had the same treatment effect and all remained valid. What we found confirmed the clinical effective treatment of selecting 3 d to 7 d before menstruation instead of the entire physiological cycle therapy in primary dysmenorrhoea treatment. The animal model designed according to the mice oestrous cycle, in this study, may be more feasible and closer to the evaluation of clinical curative effect. It supplies a good foundation for further exploration of in-depth mechanisms of primary dysmenorrhoea treatment.


extfieldsofstudy: Biology Medicine

text:
Reproductive biology of the Suez Canal spider crab Schizophrys aspera (H. Milne Edwards, 1834: Crustacea: Brachyura: Majidae)

A reproductive biology study of the spider crab Schizophrys aspera (H. Milne Edwards, 1834) was conducted in the Suez Canal from July 2012 to June 2013. The annual sex ratio (Male:Female) of S. aspera was female biased with values of 1:1.25. Out of the four ovarian development stages of this crab, two stages were observed in the Suez Canal throughout the whole year. The ovigerous crab’s carapace width varied from 28 to 52 mm. This crab species can spawn during most of the year in the canal water, with a peak during late spring and early winter. The fecundity of ovigerous females ranged between 2349 and 13600 eggs with a mean of 5494 ± 1486 eggs. Female crabs that reached sexual maturity exhibited a minimum carapace width varying between 22 and 46 mm, and fifty percentage of all ovigerous females showed a carapace width of 36 mm.

Introduction
Crustaceans are the most dominant and successful aquatic group. They are usually represented by a large number of species, exhibiting a great array of life styles, and occupying quite dissimilar habitats. This diversity in the crustacean groups is a result of their life patterns and reproductive strategies (Sastry, 1983). Crustacean decapods form the major and important component of commercial fisheries in the region of the Suez Canal. Fishing for brachyuran crabs in the Suez Canal is mainly concentrated in the area of the eastern and western coasts of the Bitter Lakes in the southern sector of the canal, and is also dominated by a single family, Portunidae, and especially a single edible species, the blue swimming crab, Portunus pelagicus (Linnaeus, 1758). Catches in the canal, however, often contain specimens of a few of the twelve species of non-edible brachyuran crabs, principally the spider crab Schizophrys aspera (H. Milne Edwards, 1834) (El-Serehy et al., 2012). The spider crab is not edible but is common in the Suez Canal environment, and has a negative impact on fishing nets and the broader fishing industry.
The study of the reproductive biology of these crabs, with particular reference to the seasons of their spawning and fecundity, is important in order to gain complete understanding of their population fluctuations and dynamics. This is vital in order to enable managers and policy makers to develop and establish regulatory measures to conserve the population of the edible and economically important brachyuran crabs in canal water and to help reduce the negative impact of the nonedible brachyuran crabs on the fisheries of the Suez Canal.
Although studying different biological aspects of the spider crabs from the Suez Canal attracted the attention of many authors (Ibrahim, 2012(Ibrahim, , 2014Madkour et al., 2012;Ibrahim and Amin, 2013), there appears to be no published reproductive biology study for S. aspera in the Suez Canal, and this study is, therefore, an attempt to provide reproductive biology information. Particularly, aspects of the ratio of males and females (sex ratio), developmental stages of the gonads, season during which spawning occurs, size at first sexual maturation and size at 50 percentage maturation of ovigerous females were investigated, along with their fecundity.

Collection and processing of crab samples
The study was undertaken in the Suez Canal from July 2012 to June 2013 at six localities. These locations represent the best known fishing sites along the Suez Canal: El-Qntra and El-Ferdan (on the Suez Canal proper); Ismailia on Timsah Lake; and three localities on the Bitter Lake: Deversoir, Fayed and Fanara (Fig. 1). The crabs were collected as discard products from artisanal fishery (gillnets, trammel nets, crab nets and beach seine), moreover few specimens were provided by scuba divers.
Crab samples were randomly selected as they were being brought outside the lift nets. The selected samples were immediately kept in ice. In the laboratory crabs were transferred to a deep freezer and kept at À20°C until analysis. A total of 3047 crabs of S. aspera were collected and preserved this way. Each crab's sex was determined and its body weight and the width of its carapace were measured and recorded. The width of the carapace was measured to the nearest mm using a Vernier Calliper. Individual wet weights of the collected crabs were recorded to the nearest gram. The ovigerous females were counted, and their carapace width was recorded.
Four major parts of the reproductive biology of the Suez Canal spider crab S. aspera were studied as described below:

Sex ratio
The analysis of S. aspera sex ratio was done using monthly data sets of the total number of female and male individuals. Chi-square (x 2 ) test was applied to detect the significant differences between the ratios of male and female crabs.

Female maturity stages and determination the season of spawning
Female crabs sampled from the Suez Canal were measured for their biometric data. The carapace was opened to observe the stages of ovarian maturity, the latter were grouped into four main classes according their size and color, as follows: stage I-immature gonads: colorless and very thin ovary; stage II-early gonad maturation: ovary enlarged in size and its color changed to creamy; stage III-mature gonad: ovary with yellow color covering most parts of the hepatic region; stage IV-ovigerous females: all females carry eggs. The spawning season of this spider crab species was determined from the percentage of females in each month who were either at ovary stage III or stage IV.

Fecundity
For each ovigerous S. aspera female, carapace width was measured to the nearest mm and the eggs were weighed to the nearest 0. 001 g. In order to calculate the total number of eggs carried externally by each ovigerous female, a sub-sample (5%) of the total egg weight was examined and counted under a Wild M40 inverted microscope. These data were used to calculate the total number of eggs carryined by each ovigerous female Kumar et al. (2000).

Size at first sexual maturity and Lm 50 percentage
During the present study, the minimum size class data obtained from 603 females were used to determine the size (Carapace width) at first sexual maturity of S aspera in the Suez Canal. The Lm 50 percentage (i.e. the length at first reproduction) or the length at which fifty percentage of all females in the crab stock from the Suez Canal are ovigerous was estimated by using the formula given below (King, 1995): where, P is the probability of existing mature crabs, L is the mean carapace width (mm), r m is the slope of the curve, and L m is the mean carapace width (mm) at sexual maturity.

Sex ratio
During the present study a total of 3047 crabs of S. aspera were collected from the canal, with 1354 and 1693 individuals of males and females, respectively. Thus the overall ratio between male and female S. aspera in the Suez Canal during the whole year is not equal. Rather, females comprise 55.6% of the total. Moreover, the Chi square analysis (Table 1) confirmed that the overall sex ratio of the Suez Canal S. aspera differed significantly from unity (p < 0.05).

Female maturity stages and spawning season
Monthly variations in the different maturity stages of female ovaries of S. aspera species are shown in Fig. 2. Eggs of the female crab have various colors of yellow, orange and brown depending on the stage of maturity. Four different stages of maturation were recognized: stage I (immature), white to ivory; stage II (maturing or developing), creamy; stage III (mature), yellow to orange; and stage IV (ripe), brown. Immature females were recorded in all months, but the highest percentage was in January. The proportion of maturing females increased during the winter months to reach a maximum in April. Mature females were observed during summer with the highest percentages occurring in August. Specimens at the spent stage (ovigerous females) appeared in considerable numbers for most of the year except between January and April. This indicates that S. aspera has a prolonged breeding season that commences in May and terminates in December.

Size at first sexual maturity and Lm 50 %
The size of female S. aspera at first sexual maturity was determined using the minimum size class data of 603 ovigerous females collected from the Suez Canal. During the present study, female S. aspera crabs attained maturation when their carapace width (CW) varied between 22 and 62 mm. The minimum carapace width of female S. aspera attaining maturation in the Suez Canal varied between 22 and 46 mm. The logistic curve fitting the estimation of carapace width at which 50 percentage of female S. aspera are sexually mature is shown in Fig. 3. Fifty percent maturity was attained at a female carapace width of 36 mm. Table 2 shows the relationships between the width of the carapace, the egg size and the number of females' eggs for S. aspera recorded during the period of this investigation. Determination of fecundity of the female S. aspera from the Suez Canal showed that each female with a carapace width ranging between 28 and 52 mm can produce a total number of eggs varying between 2349 and 13,600. During the present study, the mean fecundity of S. aspera was 5494 ± 1486 eggs.

Fecundity
In the Suez Canal, the smallest ovigerous female had a carapace width of 28 mm, 14.3 g total weight, 0.36 g gonad weight, a fecundity of 2349 eggs and an egg size of 890 lm. The largest ovigerous female, meanwhile, achieved a carapace width of 52 mm, 81.34 g total weight, 4.95 g gonad weight, a fecundity of 13,600 eggs and an egg size of 1263 lm.

Discussion
In the majority of brachyuran crab species, the sex ratio is usually close to unity (Hartnoll, 1978;Lawal-Are, 2010), however some variations between different populations and from one year to the other in the same population have been detected (Sastry, 1983;Varisco and Vinuesa, 2011). During the present study the overall sex ratio in S. aspera (with the values of M:F = 1:1.25) differed significantly from the expected 1:1 ratio, with most months revealing a higher number of females, i.e. the sex ratio of the Suez Canal spider crab S. aspera is female biased. An overall female bias in the sex has been recorded for many brachyuran crab species (Safaie et al., 2013;Chatterjee and Chakraborty, 2015). There are two main possible reasons for this difference from the expected 1:1 ratio between the two sexes. On the one hand, it may result from differences in habitat preferences and feeding behavior between male and female crabs. Mature crabs are known to display different habitat preferences between male and females. Generally, females migrate from inshore areas toward offshore to spawn (Weng, 1992). Moreover, most female crabs are  known to require a sandy substrate for successful egg extrusion (Kangas, 2000). Females of many crab species are more abundant in shallow water (Potter et al., 1983). All the six fishing localities of the Suez Canal exhibited sandy substrates, shallow water depths (5-25 m), and most of the fishing process occurred offshore. Alternatively, the unequal sex ratio of S. aspera in the Suez Canal could be attributed to the effects of fishing gear. Fishermen in the Suez Canal use a variety of gears such as gillnets, trammel nets, crab nets and beach seine. Gillnets and trammel nets usually operate in the deeper area, while crab nets and beach seine perform in shallow water. This difference in operating fishing gears produces unequal brachyuran sex ratio (Kangas, 2000;Lawal-Are, 2010;Safaie et al., 2013). The immature stages of S. aspera were observed through 2012 and 2013, revealing that the mature stages occurred from May and extended to December (Fig. 2). This pattern of ovarian development in S. aspera from the Suez Canal indicates that the third and fourth stages of ovarian development is usually achieved in conjunction with rising sea water temperatures, which in their turn indicate that the development of ovaries and eggs in S. aspera is controlled by water temperatures. Brachyuran crab spawning takes place during the whole year in tropical and subtropical waters, while it appears to be more restricted to the warmer months in the temperate regions (Kangas, 2000;Lawal-Are, 2010;Safaie et al., 2013).
In the present study, the range of the minimum carapace width (CW) of ovigerous female S. aspera was 22-46 mm, and the CW at which 50% of all females in the stock of that spider crab are ovigerous was 36 mm. According to Campbell and Fielder (1986), Sukumaran andNeelakantan (1996), andSal-Moyano et al. (2014), the size at first sexual maturity differs with latitude and/or location and within individuals at any location.
The fecundity of brachyuran crabs is usually recognized to be an important parameter for measuring their reproductive output (Mantellatto and Fransozo, 1997). Determination of the fecundity of female S aspera from the Suez Canal showed that each female with a carapace width varying from 28 to 52 mm can produce 2349-13,600 eggs. In the Suez Canal water, mean fecundity of S. aspera attained 5149 ± 1367 eggs. The spider crab S. aspera, therefore, seems to be a crab species with relatively low fecundity. Fecundity can vary among crab species from less than 5000 eggs, described for Aratus pisonii (H. Milne Edwards, 1837) in Jamaica (Warner, 1967) to more than 4,000,000 eggs, as reported by Haynes et al. (1976) for Chionoecetes opilio (Fabricius, 1788) in the Gulf of Saint Lawrence, Canada. Fecundity of female crabs is size dependent (Kumar et al., 2003), moreover fecundity is strongly correlated with body size of the crab species and weight within the species (Hartnoll, 1985). The analysis of the relationship between carapace width and fecundity for S. aspera in the Suez Canal with Fecundity = 2.2808 (CW) 2.09 indicates that the fecundity increases with an increase in carapace width (R 2 = 0.62). Thus, it can be stated that higher fecundity was found in larger female crabs due to the longer inter-molt period between population and egg extrusion. In other words, larger females usually have more time than the smaller ones in order to accumulate the energy reserves. The latter are usually required by female crabs (small and/or large) to produce their eggs. This difference might account for the higher number of eggs often produced by large sized females (De Lestang et al., 2003;Sal-Moyano et al., 2014). As discussed above, fecundity of crabs not only varies from one species to the other, but also varies within the same species. Many factors can affect fecundity in brachyuran crabs including: size, age, climatic regime, nourishment, and even ecological properties of the water body (Arshad et al., 2006;Safaie et al., 2013;Gonzalez-Pisani and Lopez-Greco, 2014).
In conclusion, the annual sex ratio (Male:Female) of S. aspera is not 1:1 but rather, 55.6% are females, i.e. the sex ratio in this species is biased toward females and differed significantly from the expected unity, with most months sustaining a higher female occurrence. The range of the minimum carapace width (CW) of ovigerous female S. aspera was 22-46 mm, and the carapace width at which 50% of all females in the stock of that spider crab are ovigerous was 36 mm. Despite having a low fecundity, S. aspera has a prolonged breeding season in the Suez Canal that commences in May and terminates in December.


s2fieldsofstudy: Biology Medicine

text:
Long-term primary culture of a clear cell ovarian carcinoma reveals an epithelial–mesenchymal cooperative interaction

Background We studied a primary culture developed from a biopsy of a clear cell carcinoma of the ovary (O-CCC) by (a) assessing its capacity to retain in vitro pathological features of the tumor of origin; (b) characterizing the main cells released from the complex mass without forced purification of any particular cellular entity; and (c) investigating its long-term proliferative capacity. Methods A primary cell culture was developed from a pelvic mass diagnosed as an O-CCC. The morphological analysis of the cell culture was carried out by phase contrast microscopy. Markers of epithelial, mesenchymal, and tumor initiating cells were evaluated by immunocytochemistry. Cell proliferation was studied by detection of bromodeoxyuridine (BrdU) incorporated into newly synthesized DNA. As a biomarker of O-CCC, we assessed the expression of hepatocyte nuclear factor (HNF) 1β. Results We show that cells with epithelial morphological features express E-cadherin and expand with time in culture, a fact that the incorporation of BrdU confirms. Cells with mesenchymal-like characteristics that express the mesenchymal marker vimentin, however, allocate to the edges of the epithelial compartment. Moreover, we found that some cells with epithelial features also expressed vimentin. At the beginning of incubation, over 60 % of primary cells expressed the O-CCC marker HNF1β; such percentage declined upon passaging. We show that epithelial not mesenchymal cells undergo DNA replication, and that few cells in both epithelial and mesenchymal compartments express the stem-like tumor antigen CD133. Conclusions We provide proof-of-principle that cells separated in bulk from a biopsy of an O-CCC can be maintained in culture for several months, and that two consistent cellular compartments—one epithelial that retains the O-CCC marker HNF1β, and another mesenchymal—persist, and seem to have a cooperative interaction leading to the multiplication of epithelial cells within a mesenchymal cellular environment.

Background
Cell lines that can be propagated indefinitely in culture are a helpful resource with which to study biological processes in cancer biology and experimental therapy. A large number of human cancer cell lines has been generated in the past few decades, and vast scientific information has been gathered from experimental exploitation of these cell lines; however, expanding literature suggests that predictions made based on the behavior of cancer cell lines in vitro, or xenografted into immunosuppressed mice, do not always materialize in clinical applications. In the case of ovarian cancer, this occurrence is highly remarkable. Over 100 human epithelial ovarian cancer (EOC) cell lines have been characterized [1][2][3] in the past few decades, facilitating the discovery of many pathobiological mechanisms and allowing preclinical testing of novel anti-cancer drugs. Nevertheless, the standard of care for this disease has not changed since Open Access *Correspondence: alicia.goyeneche@mail.mcgill.ca the introduction of the platinum/taxane drug combination in the mid-1990s [4]. This leads us to reflect that the experimental models we are using to study cancer in general and ovarian cancer in particular, while useful, are not entirely comprehensive.
A common practice in generating EOC cell lines from primary cultures has been to use a reductionist approach. Within this scope, efforts have always been made to separate the epithelial from the non-epithelial cellular component of the samples, more often than not considered cellular "contaminants" since the role of the tumor environment had yet to be uncovered. Thus, the first cellular components to be separated from the epithelial cells were blood cells, fibroblasts, mesenchymal, and mesothelial cells [5]. The purification of EOC cells has been typically achieved utilizing strategies that take advantage of the differential adhesion and proliferation capacities of the epithelial cells against the non-epithelial cells, leading to the generation of a highly homogenous population of EOC cells that adapt to grow in culture indefinitely [5].
We now know more about the relevance of the interaction between the epithelial component of the tumor and the surrounding stroma in terms of cancer survival, progression, and metastasis; hence, it is important to study the behavior of cancer cells when accompanied by tumor-associated, non-epithelial stromal cells. Herein we provide proof-of-principle for the successful longterm primary culture established from a tumor biopsy of a patient diagnosed with stage IIIc EOC, without purifying any cellular entity in particular. We observed that the complexity of the cellular components of the tumor is what allows the epithelial primary culture phenotype to succeed in growing for a substantial period of time in vitro. We also present evidence suggesting that cells within the epithelial compartment are in an epithelialmesenchymal hybrid stage, indicating that cells within the tumor biopsy retain phenotypic heterogeneity and plasticity in vitro.

Surgical specimen
A 50-year-old patient had a preoperative diagnosis of an ovarian mass. The patient underwent total abdominal hysterectomy, bilateral salpingo-oophorectomy, and omentectomy. A 20-cm left ovarian mass revealed a centrally solid, yellow-white, papillary structure. The postoperative diagnosis was a stage IIIc optimally debulked, clear cell ovarian carcinoma (O-CCC). The patient received standard IP/IV chemotherapy, but relapsed 13 months after initial surgery and succumbed to the disease 6 months thereafter. The use of the surgical specimen for research purposes was approved by the Institutional Review Board (2007-138) and informed consent from the patient.

Primary cell culture
A sample from the main ovarian tumor mass was placed in saline in the operating room, sat on ice to maintain viability, transported to the tissue culture room, and handled in a sterile environment. The tissue biopsy was divided into two. One part was fixed in 4 % paraformaldehyde (PFA) and embedded in paraffin; serial paraffin sections (thickness, 5 μm) were obtained and stained with hematoxylin. The second sample of tumoral tissue was enzymatically digested with collagenase Type II. Without further purification, the bulk of the cells was allowed to attach to regular tissue culture flasks and was maintained in RPMI-1640/Ham's F12 1:1 media supplemented with 10 % fetal bovine serum and antibiotics.

Cytospin preparations
A 5-day-old primary culture was trypsinized. Suspended cells were fixed at room temperature (RT) for 30 min with 4 % PFA. Finally, approximately 50,000 cells were immobilized in glass slides by cytospin, dried, and subjected to hematoxylin staining or immunostaining for hepatocyte nuclear factor (HNF) 1β.

Bromodeoxyuridine labeling
Cells isolated from the primary biopsy of O-CCC, after residing in culture for 2 months, were trypsinized and seeded in chamber slides for 11 days. Chamber slides containing cells were exposed to 10 μM 5-bromo-2′deoxyuridine (BrdU; Molecular Probes, #B23151, Grand Island, NY, USA) for 7 days. At the end of incubation, the cells were fixed with 4 % PFA for 30 min at RT. BrdU incorporated into the DNA was visualized by immunocytochemistry as previously described [6].

Immunocytochemistry
Cells isolated from the primary biopsy, after spending 2 months in culture, were trypsinized, counted, and allowed to grow for 11 days into chamber slides. Slides containing either cultured cells or cytocentrifuged cells were washed in PBS, fixed with 4 % PFA, and subjected to immunostaining for E-cadherin, vimentin, CD133, BrdU, or HNF1β. Permeabilization and reduction of the non-specific binding was achieved by incubating the cells with 2.5 % normal horse serum, 0.5 % Triton-100 for 20 min at RT. Cells were incubated with the specific primary antibodies in a moist incubation chamber for either 1 h at RT or overnight at 4 °C. Endogenous peroxidase activity was blocked with 3 % H 2 O 2 for 20 min at RT following incubation with the primary antibody. Antibodies In negative controls the primary antibody was replaced with 2.5 % normal horse serum (Vector Laboratories).

Culture of SKOV-3 cells and development of SKOV-3 xenografts
The origin and culture conditions of the SKOV-3 cell line, as well as the establishment of SKOV-3 xenografts in immunosuppressed mice, were previously published in detail [6].

Tumor histology
The histopathological assessment of the sections, performed on various samples representing the ovarian mass, reported an ovarian clear cell carcinoma (O-CCC). Hematoxylin-and eosin-stained (H&E) sections of the tumor mass, as processed in the laboratory of pathology, show an overall clear cell phenotype, with dominant cells having clear cytoplasm and growing in a solid, tubular or papillary pattern, with hobnail cells lining tubules and cysts in a hyalinized stroma. Most epithelial cells are cuboidal and have eccentric and hyperchromatic pleomorphic nuclei, some containing pseudo-inclusions ( Fig. 1a-c). A sample from an omental metastasis confirmed the papillary and tubulocystic pattern of the tumor with abundant cells of clear cytoplasm (Fig. 1d). Representative hematoxylin-stained sections of the tumor biopsy utilized for primary culture confirmed the diagnosis of O-CCC, characterized by tubulocystic architecture of the tissue with epithelial cells having little stratification, hobnail cells, and cells with abundant clear cytoplasm (Fig. 2a, b). Positive immuno-detection of a biomarker of O-CCC, HNF1β [7,8], added to the histopathological diagnosis of the specimen (Fig. 2c, d).
HNF1β positivity was also observed in a mouse xenograft developed from the human cell line SKOV-3 (Fig. 2g, h), which depicts the solid morphological features of O-CCC with eccentric nuclei (Fig. 2e-h).

Primary cell culture findings
Upon enzymatic cellular dispersion of the solid tumor, and after 5 days in culture, the cells that adhered to the plate formed colonies of epithelial appearance, but showed in their surroundings cells with mesenchymal phenotype (Fig. 3a). We considered the mesenchymal cells neighboring the cells with epithelial phenotype a representation of a cellular network preexisting within the in vivo environment; hence, we did not intend to separate them-a method that has been traditionally used to generate epithelial cancer cell lines. Cytospin preparations from a suspension of trypsinized cells, after being cultured for 5 days, revealed the presence of cells of different sizes, most of which displayed a clear cytoplasm and eccentric hyperchromatic nuclei (Fig. 3b). We then assessed the expression of HNF1β in cells from cytospin preparations of a 5-day old culture. About 60 % of the cultured cells displayed positive nuclear HNF1β staining (Fig. 3c, and left column in Fig. 3f ). As an in vitro control for HNF1β staining, we utilized a culture of SKOV-3 cells, which shows positive nuclear labelling in the majority of the cells (Fig. 3g). After 30 days of incubation, a primary culture showed an apparent increase in the proportion of cells having a mesenchymal morphology when compared to a 5-day primary culture (Fig. 3d). These changes were associated with a significant decline in the percentage of cells that labeled positive for the epithelial O-CCC maker HNF1β (Fig. 3e, and right column in Fig. 3f ).
After 60 days of incubation, and with periodic media changes, the culture still displayed a combination of epithelial foci surrounded by mesenchymal-like cells (Fig. 4a, b). When we compared the phase contrast micrographs taken 5 days after initial plating versus those taken 2 months later (Figs. 3a vs. 4a, b), the net growth of the epithelial component of the culture was evident; yet, mesenchymal cells seemed to increase in relative proportion, as well, accompanying and surrounding the epithelial compartment.
After 2 months in culture, when we sub-cultured the cells shown in Fig. 4a, b in chamber-slides, the cells with epithelial morphology-but not those with mesenchymal morphology-stained positive for the epithelial marker E-cadherin (Fig. 4c, d). The mesenchymal marker vimentin stained positive mostly in the mesenchymal compartment; however, positive staining also appeared in the cytoplasm of some cells of the epithelial compartment (Fig. 4e, f ), suggesting the existence of a transitional epithelial-mesenchymal phenotype.
The synthesis of DNA within the epithelial compartment, but not the mesenchymal compartment, was confirmed by BrdU incorporation (Fig. 5a-c). Finally, after 2 months of incubation, the heterogeneous culture retained cells that stained positive for the stem cell marker CD133, described by many as being expressed in ovarian tumor-initiating cells [9][10][11][12]. CD133 expression was observed in the plasma membrane of few cells, which were associated with both the epithelial and the mesenchymal compartments of the cell culture (Fig. 5d-f ).

Discussion
Most EOC cell lines available for research purposes have been developed from cancer cells found within ascites and pleural effusions of patients diagnosed with EOC. A limited number of EOC cell lines were developed from solid primary and metastatic biopsies. In both cases, the main efforts placed into the development of the EOC cell lines were geared at isolating clones with epithelial-only phenotype, with the purpose of selecting the ovarian cancer cells.
During the past 20 years, the critical role of the tumor microenvironment, mainly composed of tumor-associated cells, has been unveiled (reviewed in [11]). It is now clear that the relative low heterogeneity of EOC lines seems insufficient to reflect the inter-cellular complexity found within a tumor. Thus, in the present work, we provide a snapshot of the behavior of a primary cell culture derived from a biopsy, aiming to preserve the main cellular components of the original tumor mass. The bulk  of cells released form the biopsy had clear cytoplasm, which is characteristic of a clear cell carcinoma. Furthermore, 60 % of the cells showed nuclear staining for the biomarker HNF1β, a protein considered to be pivotal in the pathogenesis of O-CCC as evolving from endometrioid lesions [8,13], and found to have high sensitivity and specificity to differentiate O-CCC from high-grade serous ovarian cancers [7].
We provide new evidence that, in addition to the epithelial phenotype denoted by a pavement-like arrangement of the cells, there was a second phenotype with mesenchymal features composed of vimentin-positive, E-cadherin negative cells always located in close proximity to the epithelial edge. This suggests that a cooperative interaction might exist between these two cellular compartments with the likelihood for the mesenchymal cells to molecularly prompt the epithelial cells to undergo proliferation followed by transition to, again, a mesenchymal phenotype (i.e. EMT). These observations are in agreement with a recent study in which Fig. 4 a, b Phase contrast images depicting the coexistence of epithelial (asterisks) and mesenchymal (arrowheads) cellular compartments after two well-known EOC lines, when co-cultured with primary mesenchymal stem cells, reprogrammed their transcriptome leading to the acquisition of traits that promote migration, invasion, proliferation, and resistance to platinum [14]. Significantly enough in this last study, the mesenchymal cells were able to sustain proliferation of EOC in vitro in the absence of serum and cytokines, unveiling the involvement of cells from the microenvironment that are likely hijacked by the tumor cells to promote their own persistence via paracrine mediators.
Another study suggests that resident fibroblasts can be reprogrammed into cancer-associated fibroblasts upon epigenetic changes triggered by factors released from the EOC cells. Subsequently, the reprogrammed fibroblasts upregulate the expression of cytokines and chemokines that promote growth and metastasis of the EOC cells [15]. We observed DNA-synthesizing EOC cells in the epithelial compartment but not in the mesenchymal compartment, suggesting that paracrine pro-proliferative factors may be provided by the mesenchymal cells to the EOC cells.
The present study also suggests that there is a mesenchymal-epithelial plasticity in culture based on two key observations. On the one hand, cells with epithelial-like morphology stain for the mesenchymal marker vimentin, denoting that they are in a hybrid, epithelial-mesenchymal stage. Such a hybrid stage was described in human ovarian cancer cells freshly isolated from mouse xenografts, or, in situ, in human biopsies in which cancer cells simultaneously express the epithelial marker EpCAM and the mesenchymal marker vimentin [16]. On the other hand, we observed that the overall proportion of cells expressing the O-CCC epithelial biomarker HNF1β declines with time in culture in association with an apparent expansion of the mesenchymal compartment. This observation suggests the likely existence of a transdifferentiating phenomenon, taking into account that the epithelial but not he mesenchymal cells are the ones that divide in culture. This plasticity may provide an explanation for the retention, within both epithelial and mesenchymal compartments, of cells expressing the O-CCC stem-like marker CD133 [12], which is usually lost when cells undergo lineage-associated differentiation [16].


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Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins

Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

Introduction
The interactions between RNA and protein molecules are essential for molecular processes both in cellular organisms, where they govern the assembly of the protein synthesizing macromolecular machineries, such as the ribosome and spliceosome [1], and in viruses, where they envelope the (RNA) genetic material by capsid formation [2], or enhance the efficiency of viral transcription [3]. Due to their central role, protein-RNA complexes have been extensively studied and it soon became apparent that RNA-binding proteins are enriched in intrinsic structural disorder [4]. As a consequence of conformational flexibility, these proteins often go through binding-induced folding [5]. Such disorder-to-order transitions appear ubiquitously, and in RNA-protein interactions conformational changes can occur either in the structure of the protein, the RNA partner, or both [6]. However, disorder-to-order transitions entail special energetic consequences on the interaction, because a fraction of the available binding enthalpy needs to compensate for the entropic cost of the conformational changes [5]. Therefore, even though structural disorder is prevalent in RNA-binding proteins, the advantages that would emanate from their prevalent bonding mode, remain elusive. Further, conformational flexibility could be the by-product of the optimization of electrostatic interactions, since RNA-binding proteins have to be enriched in positively charged amino acids in order to establish favourable electrostatic interactions with the highly negatively charged RNA surfaces [7,8]. Since charged residues are disorder promoting, they may destabilize the protein chain at the same time.
Understanding the role of structural disorder in protein-RNA interactions would be instructive in explaining the prevalence of conformational flexibility in general, and could provide valuable insights regarding complex assembly and the regulation of the molecular processes these complexes are involved in. Is disorder a by-product of binding optimization in terms of electrostatic interactions, or is there more to the prevalence of conformational flexibility, than meets the eye?
Here, we present a comprehensive computational analysis based on both sequence and structural data in an attempt to elucidate if protein function correlates with intrinsic disorder in RNA binding proteins. We demonstrate the extent of structural disorder in the known RNA-binding proteins in an evolutionary context, investigate the consequences of conformational flexibility and test a number of plausible explanations for the enrichment of intrinsic disorder in this family of proteins.

Materials and Methods Data Retrieval and Processing
The analyses were based on three data sets from the following sources: The Pfam27.0 database [9], the UniProt/SwissProt [10] database and the Protein Data Bank [11].
The Pfam dataset contained the full length protein sequences of 344 DNA-binding and 140 RNA-binding domain families. The sequences were filtered against redundancy using CD-HIT [12], and the domains were extracted using Pfam scan [9] yielding 406,736 unique DNA-binding and 210,962 unique RNA-binding protein domain sequences. The complete Pfam-A dataset (10,626,097 domain sequences), excluding the RNA-and DNA-binding domains, was used as a reference.
From UniProt/SwissProt we retrieved 43,111 unique DNA-binding protein sequences (GO:0003677) and 66,386 unique RNA-binding protein sequences (GO:0003723) based on GO-terms. The full SwissProt dataset excluding RNA-and DNA-binding proteins was composed of 542,782 protein sequences and served as a reference.
Finally, we retrieved 2877 unique DNA-protein complexes and 1605 unique RNA-protein complexes, along with 4278 protein-protein complexes. 1420 unique DNA-binding, and 1293 unique RNA-binding protein sequences were extracted from the complexes, and compared to the entire PDB dataset consisting of 57,041 unique protein sequences, excluding RNA-and DNA-binding proteins. The sequences in FASTA format for each dataset can be downloaded from http://pedb.vib.be/discons/data.tar.gz or from Dryad (doi:10.5061/dryad.33vn1).

Disorder Predictions and Conservation Analysis
Previously, we have developed DisCons [13](available at http://pedb.vib.be/discons), a novel and freely accessible tool that serves two purposes: first, it provides a position-specific conservation score of protein disorder in the context of a multiple sequence alignment (MSA). Second, it classifies each position by combining the conservation scores of the sequence and of the structural disorder, following the protocol of Bellay et al. [14].
Briefly, the calculation procedure is the following: during the initial step, DisCons performs a PSI-BLAST search against a specified collection of protein sequences; we used the UniProt/ SwissProt database for this purpose in our analysis. Hits that satisfy the pre-set thresholds are then aligned using MAFFT [15] and the MSA serves as an input for the next steps of the Dis-Cons pipeline. Position-specific sequence conservation scores (SCS) are calculated based on the MSA by the scoring method of Capra et al. [16]with Jensen-Shannon divergence and a window of size 3. Scores range from 0 (diverse) to 9 (strongly conserved). Next, disorder scores are calculated using IUPred [17] and are mapped onto the MSA. Position-specific disorder conservation scores (DCS) are determined by calculating the fraction of positions for which the disorder score is 0.5 or greater, with scores also ranging from 0 (non-conserved) to 9 (highly conserved).
Finally, for those MSA positions where the fraction of gaps across the aligned sequences is less than 30%, the sequence-and disorder conservation scores are combined, and positions are classified as having i.) 'Constrained' disorder, if both scores are 0.5 or greater; ii.) 'Flexible' disorder, if the sequence conservation score is lower than 0.5, but the disorder conservation score is 0.5 or greater; iii.) 'Non-conserved' disorder, if the disorder conservation score is lower than 0.5, but higher than 0; and finally, iv.) 'Structured', if the disorder conservation score is 0, indicating the complete lack of disorder at the given position.

Identifying Secondary Structural Elements and Interface Residues
Secondary structure assignments for protein chains were obtained by the DSSP algorithm [18]. The secondary structure types considered were: alpha-and 3 10 -helix, beta-strand, turn (with or without hydrogen bonding), and unclassified. The two helical types and strands were considered as 'regular' secondary structures, whereas turns and unclassified types were labelled 'non-regular' secondary structures, according to the previously described protocol of Guharoy et al. [19].
Protein residues in direct contact with RNA were identified by pair-wise distance calculations between protein and RNA chains: contacts that consisted of 5 or more atom pairs within 5Å were recorded interface contacts, effectively identifying 19128 protein-RNA interfaces. The residues of these interfaces were investigated in terms of sequence-and disorder conservation using the DisCons pipeline described in the previous section.

Conformational Stability and Surface Accessibility Plots
Conformational stability of the protein chains in 1605 RNA-protein, 2877 DNA-protein and 4278 protein-protein complexes was calculated using FoldX [20]. The bound conformations of the proteins were separated from the complexes, and the calculations were performed on every single chain.
We plotted the accessible surface area as a function of the accessible interface area, normalized by the number of residues, as suggested by Nussinov [4,21], where structured proteins are located on the lower left side of the plot, below the threshold line of 80, while (disordered) proteins flexible in their free form are found in the upper right side [4,21]. In order to create the plots, the whole surface area, the chain surface area and the complement surface area have been calculated for each complex using in-house Python scripts, and the interface areas were defined as the whole complex area subtracted from the sum of the chain surface area and the complement surface area. This value was divided by two to take into account only one side of the interface. The calculations involved the usage of the PDBParser module of the Bio.PDB package [22] and the PyMol package to calculate the areas via The PyMol Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC. We used the parameter "dot solvent" set to "on" so that the solvent accessible surface area was taken into account. The accessible surface area and the interface area were both normalized to the number of residues.

Investigating the Number of Interaction Partners in RNA-Protein Complexes
The number of unique interaction partners for RNA-binding protein chains was calculated using in-house Python scripts that used the PDBParser module of Bio.PDB [22]. For PDB entries with multiple models, only the first model was taken into consideration. Interacting residues were defined as residues having at least one atom (each) with a maximum distance of 5 Å from each other. We considered two chains as interaction partners if they had at least 5 interacting residues.

Statistical Analyses
Data processing, exploratory data analyses and statistical tests were performed in the R statistical programming environment using RStudio. Welch t-tests were performed where the distributions were not Gaussian and we could not assume equal variances. Kolmogorov-Smirnov tests were performed in cases where the only valid assumption regarding the variables was their continuity. A p-value of 2.2e-16 is the lowest precision point allowed in R, and implies extremely high significance.

Large Scale Investigation of Structural Disorder Reveals Ubiquitous Enrichment of Conserved Flexibility in RNA-Binding Proteins
The prevalence of intrinsically disordered regions in RNA-binding proteins is well documented, yet the reasons behind this phenomenon and its consequences are not fully explored [5,23]. In order to comparatively investigate the functional implications of conformational flexibility in these proteins, we have assembled a comprehensive dataset of RNA-and DNAbinding protein sequences and structures.
The initial step of the large-scale computational investigation was to calculate residue-wise disorder scores for each protein sequence in our complete dataset. One of the meaningful descriptors of disorder that can be derived from such scores is the fraction of disordered residues, which provides information on the overall disorder content of a protein chain. The distribution of the disorder ratios for the sequences across the three datasets is displayed on Fig 1. In all three datasets, RNA-and DNA-binding proteins have significantly higher disorder contents than the reference datasets, as tested by two-sample Welch t-tests, yielding p-values lower than 2.2e-16. The domain sequences in the Pfam dataset have generally lower disorder ratios, compared to the PDB and SwissProt datasets. This is to be expected, since Pfam is hosting sequences of domains that most often have well-defined structures. However, RNA-binding regions are often found outside the boundaries of Pfam domains, and in fact most of the recently discovered RNA-binding sites are within such intrinsically disordered regions [24,25]. The PDB dataset contains relatively more disorder, as flexible segments can undergo induced folding upon binding or certain conformations might be selected from the dynamic ensemble (i.e. conformational selection). The highest proportion of disordered residues is observed in the dataset of the full length RBP protein sequences of the SwissProt dataset. Upon comparing RNA binding domains (RBDs)across taxonomic groups (viruses, bacteria, archaea and eukaryota), the Pfam and SwissProt datasets show distinct differences (Fig 2).
Viral RBDs of the Pfam dataset (Fig 2A) have surprisingly high fractions of disordered residues: almost 30% of all the amino acids are predicted to be disordered, compared to bacteria (5%) and eukaryota (8%). The SwissProt dataset (Fig 2B) is more balanced, with viral RNAbinding proteins having 20% disorder, while eukaryotic RNA-binding proteins 21% on average. When compared to DNA-binding domains/proteins, RNA-binders generally have higher disorder content, except for the known eukaryotic protein sequences, where the average ratio of disordered residues in DNA-binding proteins is significantly higher (33%).
Structural disorder is generally rather abundant in eukaryotic organisms; in fact, up to 30% of the eukaryotic proteins are predicted to have intrinsically disordered regions [26]. It has  been speculated, that disorder as a feature might be required for the coordination of signalling and regulatory pathways in the complex eukaryotic cells [27]. Viruses on the other hand might be enriched in disorder, because disordered regions are thought to face reduced selective pressure [28], enabling the rapid evolution of viral sequences, leading to enhanced adaptability. Additionally, viral genomes are selected to be compact, and in this regard disordered segments with a high density of functional motifs have an obvious advantage [29]. However, the unexpectedly high ratio of disorder in viral domains of Pfam could also indicate that viral proteins have less well-defined domains, and the boundary between short domains and (disordered) binding motifs is blurry. Thus we have seen that RNA-binding proteins are enriched in disorder, but is this conformational flexibility functional?
The conservation of an important feature, such as the amino acid sequence of a protein, or the presence of intrinsic disorder may help identify functionally important protein segments. We have used our recently developed disorder conservation analysis pipeline, DisCons [13], to quantify the conservation of sequence and of disorder in an evolutionary context. Following the nomenclature of Bellay et al. [14], DisCons classifies disordered positions into three relevant categories: i.) 'Constrained', if both disorder propensity and amino acid sequence are conserved; ii.) 'Flexible', if the sequence shows high degree of variability, yet disorder as a feature is conserved; and finally iii.) 'non-conserved', if the disorder of a position is not consistent. First, we analysed the PDB dataset to provide a background against which to quantify the conservation of disorder in RNA-, DNA-and protein-binding proteins. Disorder and sequence conservation score pairs of each position in the PDB dataset are displayed on Fig 3. As shown by two-sample Kolmogorov-Smirnov tests, disorder in RNA-binding proteins is significantly more conserved (p-value less than 2.2e-16), than in DNA-binding proteins, or proteins of PDB in general. Additionally, disorder in RBPs is often conserved even when the underlying amino acid sequence is not, indicating the functional importance of the lack of structure, rather than of specific residues within such disordered regions. The conservation of disorder is even more pronounced for residues that are in direct contact with RNA, i.e. RNAbinding interface residues (Fig 3D). On the binding interfaces 'constrained' disorder dominates, as both sequence and structural disorder are strongly conserved. Fig 4 provides two specific examples of 'constrained' disordered interfaces in protein-RNA complexes. The protein chain segments that border the members of the RNA Recognition Motif (RRM) domain family often also play important roles in RNA-binding. In many known examples these flanking segments undergo disorder-to-order transitions that juxtapose them with RNA. The additional contacts serve to increase the total interaction surface, thus they enhance the binding affinity and also tune specificity of the interaction.
One such example is the RRM domain of splicing factor Tra2-β1 in complex with RNA (PDB IDs 2CQC, bound; and 2RRA, unbound) ( Fig 4A). Here, both the N-and C-terminal regions of the RRM are disordered in the free state, but adopt a folded structure in the complex with RNA, forming extensive contacts [30]. Therefore, the complete interface consists not only of the canonical β-sheet residues of the RRM, but also encompasses the terminal residues flanking the RRM. Interaction with the N-and C-terminal extensions not only increases binding affinity, but it is also important for specificity, as recognition of the RNA involves hydrogen bonding with several of the RRM flanking residues [31]. It is very likely that the structural ordering of the two termini of the RRM in the complex serves yet another important purpose. The build-up of Tra2-β1 is unique in the sense that its RRM is located between two RS (arginine-serine) domains, and the folding of the RRM-flanking linkers upon RNA binding also induces the correct positioning of the RS domains. Furthermore, the folding of the disordered termini might also assist in the formation of functional protein-protein interactions of human Tra2-β with other splicing factors. Direct interactions of human Tra2-β with two such novel splicing factors, hnRNP-G and SRp30c modulate the inclusion of exon7 of the survival motor neuron gene (SMN2) in the final transcript, preventing the lethal condition of proximal spinal muscular atrophy (SMA) [32,33]. In accordance with the functional importance of the terminal residues of the RRM in Tra2-β1, our analysis of sequence and disorder conservation ( Fig  4A, bottom) indicates that both the N-and C-terminus of this domain are segments of 'constrained' disorder, underlining that the functional importance of their interactions with RNA and additional protein partners are manifested in the conservation of both their sequence and  [13] score pair corresponds to a specific position in a multiple sequence alignment. The score pairs are binned, and the bins are colour coded: from light orange (few) to dark blue (many). Disorder is more conserved in RNA-binding protein chain (C) and especially in the RNA-binding interface residues (D) than in protein-or DNA-binding protein chains (A and B respectively). disorder. The sequence of the RRM itself is highly conserved, and in addition the sequences of the flanking segments are also strongly conserved. Disorder within RRM, a well-structured fold, is low; the flanking regions, however, are disordered and, importantly, this disordered nature is consistent across all the aligned sequences.
Another biologically relevant example of the role of disordered N-and C-terminal extensions of RRMs for RNA recognition is provided by the conservation of sequence and disorder in the case of mRNA 3'UTR recognition by the nuclear polyadenylated RNA-binding protein 4 (Hrp1) (Fig 4B). In this instance, RNA recognition and binding occurs via tandem RRM modules. In addition to the primary RNA binding surfaces offered by the beta-sheets of both RRMs and their C-and N-terminal flexible residues, the inter-domain linker (connecting RRMs 1 and 2) also plays a critical role in the interaction. While the linker forms a short alpha-helix in the crystal structure of the protein-RNA complex (PDB 2CJK, bound) [34], it is disordered in the unbound state and by NMR chemical shift differences it undergoes significant structural changes. The helix contains a large number of charged residues which make it disordered in the isolated form, and are important in stabilizing the complex with RNA through salt-bridge interactions. In accordance with its importance in binding to the RNA, the linker region is predicted to be of 'constrained' disorder ( Fig 4B). This mode of RNA recognition, which involves active participation of the linker, is also seen in the crystal structures of Sex-lethal [35], PABP [36], HuD [37] and nucleolin [38]: in all these cases, the linker connecting the two RRM domains is disordered in the free protein, and becomes folded in the complex with RNA.

Structural Consequences of Conformational Flexibility
We predicted that a significant fraction of the residues in RNA-binding proteins are intrinsically disordered, and showed that their disorder is evolutionarily conserved, especially in the regions that constitute the binding interfaces. Such a strong enrichment of flexible residues should have significant effects on the conformational stability of these proteins. To test this, we calculated the conformational energies of each protein chain in our PDB dataset using the energy scoring function of FoldX [20] (Fig 5A). According to two-sample Welch t-tests, the structures of RNA-binding chains are significantly less stable (higher energies), in comparison to DNA-binding or protein-binding protein chains. This indicates that most of the RNAbound protein structures found in PDB are likely to be unstable in the unbound form, and are only stabilized by binding to RNA. Indeed, when the normalized accessible surface area and the normalized interface area of each chain are displayed (Fig 5B), RNA-binding protein chains often occupy the area of the plot that is specific to disordered proteins that fold upon binding according to Nussinov et al [21], forming relatively large interaction interfaces. It appears that RNA-binding proteins are more disordered, and make larger interfaces than DNA-and protein-binding proteins. In fact, there is a positive correlation (Pearson correlation coefficient of 0.44) between the ratio of disordered residues and the normalized size of interaction interfaces in this class of proteins. Fig 5C and 5D provide examples of this correlation. The formation of the interface in the protein-RNA complex of the Levi coat domain (PDB ID 1AQ3) includes approximately 10% of the domain residues, and has a disorder content of 24% (Fig 5C). In contrast, nearly 90% of the residues are in interaction with RNA in the complex of the ribosomal L37 domain, which has a disorder content of 43% (Fig 5D).
Even though most of the disordered regions of RNA-binding proteins appear to obtain a more rigid conformation upon binding, only a fraction of the regions adopt regular secondary structural elements. Overall, around a quarter of every disordered residues adopts either helical (16.2%) or strand (11.1%) conformations. This ratio is even smaller on the binding interfaces, where only every fifth residue occurs within regular secondary structural elements. This indicates that while the global conformation of the RNA-binding proteins tends to be compact, the disordered regions generally remain more extended even in the bound form, and often wrap around RNA-segments, making large interfaces.

Functional Implications of Intrinsic Disorder
Disordered regions are enriched in disorder-promoting amino acids, such as glycine, proline or arginine, and are depleted in order-promoting hydrophobic residues that could form a stable hydrophobic core [39]. Ribosomal proteins are known to be enriched in positively charged residues [40], and this feature is generally true for RNA-binding proteins. Compared to the background amino acid composition of the complete PDB dataset, we found that there is a 40% increase in the relative amount of arginines and a 33% increase in lysines. When considering only those residues that are in direct contact with RNA, these numbers further increase tremendously: 180% in the relative amount of arginines and 116% in lysines. Such significant biases in the amino acid composition clearly indicate the importance of electrostatic interactions in RNA-binding, and could also account for the prevalence of intrinsic disorder. Upon examining the sequence conservation of each residue type on the RNA-binding interfaces, arginines were found to be slightly more conserved than the average, while the most conserved residues in disordered regions were cysteins, glycines and tryptophanes (Fig 6). This conservation pattern indicates the functional importance of those residues that do not actively participate in establishing electrostatic interactions, but may offer additional features, such as unusual aspects of the polypeptide backbone or hydrophobic interactions with bases of the nucleotides. A further benefit may be provided by 'fly-casting', i.e. a binding rate acceleration as a result of a relatively large capture radius of IDPs compared to structured protein segments [41].
Besides supporting a high number of favourable electrostatic interactions and increasing the capture radius of the protein, there is yet another major advantage of conformational flexibility, which is its multi-functionality or 'moonlighting' [42]. It has been shown that arginine-rich RNA-binding motifs (ARMs) may bind, different RNAs by adapting to different binding surfaces [43,44]. One such classical example is the Tat protein of the Jembrana disease virus (JDV), which can bind different TAR RNA sites [43]. These proteins are unique transcription factors, which bind mRNA transcripts rather than DNA. The RNA-recognition site of JDV Tat was found to bind not only to its native TAR site, but also HIV and BIV TAR RNAs. The conformation of the bound ARM of Tat is context dependent and unique in each interaction. It has been speculated that while arginines play a key role in establishing electrostatic interactions, the other residues are responsible for providing specificity via negative steric and electrostatic effects [44], which could explain the conservation of additional amino acids found in IDRs of RNA-binding proteins.
Additionally, conformational flexibility could be favourable in allowing a multi-domain protein chain to act as a scaffold by binding multiple protein and nucleic acid partners at the Amino acid specific conservation scores on disordered regions. Position-specific conservation score for each amino acid across the disordered regions of RNA-binding proteins. Negatively charged residues are less conserved, while arginines are more conserved than the average (blue dashed line). However, additional residues are also significantly more conserved than the average. Residues with mean sequence conservation scores significantly higher than that of the overall dataset are darker orange, while significantly less conserved residues are lighter orange. doi:10.1371/journal.pone.0139731.g006 Functions of Intrinsic Disorder in RNA-Binding Proteins same time. In order to test this hypothesis, we investigated the correlation of distinct features of the RNA-binding chains with the number of their partners, with focus on the possible effects of intrinsic disorder (Fig 7). Apparently, the number of partners does not increase with higher flexibility of the protein chain; on the contrary, there is a slight negative correlation (Kendall's tau -0.131). Of the examined parameters, only the area of the interface (Pearson 0.458) and the length of the sequence (Kendall's tau 0.178) show positive correlation with the number of bound partners in the complex. Based on these findings it seems unlikely that the increase of disorder content in RNA-binding proteins was driven by an optimization towards functioning as molecular scaffolds.

Discussion
RNA-binding proteins have often been associated with intrinsic disorder, yet the functional advantages of flexibility remain unclear [5]. Intrinsically disordered regions that make contacts with protein or nucleic acid partners undergo induced folding or disorder-to-order transitions to make a more structured conformational state [6]. Such transitions have an inherent entropic cost, which may make the interaction weaker than between rigid partners [5]. In this work, we provide a comprehensive and detailed overview on the prevalence and role of structural disorder RNA-binding proteins.
We show that RNA-binding proteins are significantly enriched in disorder, and that a major fraction of the disordered residues are found within the binding interfaces that are in direct contact with RNA. Disordered interface residues tend to fall into the category 'Constrained' disorder, since both their amino acid sequence and their disorder feature are highly conserved. In contrast, in the full length RNA-binding protein chains, the conservation of disorder is twice as high, as the conservation of the underlying sequence. In this latter case the function of the protein segment relies more upon the overall structural flexibility than on particular amino acid residues, which is in line with the general idea that disordered regions face less stringent evolutionary pressure [45], as for example demonstrated by the HIV Rev motif. Rev is an ARM, much like Tat, and it has been shown that this disordered segment is robust against substitutions [46]. Based on the distinct differences in the conservation profiles of de facto binding residues and those that mainly function as flexible linkers, it would seem likely Fig 7. Parameters that are correlated with the number of interacting partners. In order to test the hypothesis that disorder is favourable for allowing the protein chains to act as molecular scaffolds, we investigated several parameters in correlation with the number of bound partners in RNA-protein complexes. While the number of partners is positively correlated with the area of the interaction interface (A), and slightly with the length of the sequence, it is weakly and negatively correlated with the ratio of disordered residues. All three parameters were normalized. doi:10.1371/journal.pone.0139731.g007 Functions of Intrinsic Disorder in RNA-Binding Proteins that tools, such as DisCons [13]that investigate and quantify the conservation of both the amino acid sequence and the disordered nature of a protein may offer an additional layer of information that can complement and enhance the performance of RNA-binding site prediction software, such as RNABindR [47], NAPS [48] or RNAProB [49], even though some of these methods already take evolutionary information into account. While the accuracy of these tools is progressively getting higher, it was shown recently that different methods sometimes yield conflicting predictions [50]. Evaluating the conservation profiles in case of such conflicts could serve as cross-validation and may provide additional support for the validity of a specific prediction.
The disordered and conserved residues that interact with RNA are significantly biased in their amino acid composition, having more than twice as much positively charged residues as the average in PDB. These disordered and positively charged chains form extended segments that maximize the interfaces between RNA and protein, supporting the notion that disordered regions are able to establish well-fitted and larger interaction interfaces than their folded counterparts [51,52]. Additionally, the stability of the structure of RNA-binding protein chains is affected drastically by the abundance of intrinsically disordered residues.
All together, these findings demonstrate that the main functional contribution of intrinsic structural disorder in RNA-binding proteins is that it allows the formation of large, extended interaction interfaces dominated by electrostatic interactions. Another important role of conformational flexibility is to support multi-functional regions, such as the ARM sites, which can target different RNA partners via context dependent binding-induced folding [7,43]. Such multi-functionality is especially favourable for viruses, in which new functional protein-RNA interactions may evolve rapidly, without non-functional intermediates [44], coupled with the additional advantage of genome compaction [29].

Conclusions
In this study we presented a comprehensive analysis on the enrichment of structural disorder in RNA-binding proteins, and look for possible explanations of this phenomenon in terms of the functioning of IDRs through disorder-to-order transitions. Since such conformational changes are entropically expensive, the functional advantages of excessive conformational flexibility is questionable. We suggest that intrinsic disorder provides for two major advantages: First, these proteins establish large, extended electrostatic interaction interfaces dominated by positively charged, conserved disorder-promoting residues. Tight contacts within these large interfaces is a result of induced fit [52], which in combination with the 'fly-casting' effect can accelerate and optimize molecular recognition. Second, conformational flexibility makes multi-functionality (i.e. 'moonlighting') feasible by targeting different RNA partners with the same disordered protein segment, by acquiring conformations in a context-dependent manner [7,43]. While positively charged residues within these IDRs contribute to electrostatic interactions, other residues provide specificity, mostly by negative steric effects. Such multi-functionality also supports genome compaction and the rapid evolution of new interactions without the disadvantage of non-functional intermediates.