extfieldsofstudy: Medicine Biology

text:
Genetic Diversity and Structure of Domestic Cavy (Cavia porcellus) Populations from Smallholder Farms in Southern Cameroon

Although domestic cavies are widely used in sub-Saharan Africa as a source of meat and income, there are only a few studies of their population structure and genetic relatedness. This seminal study was designed with the main objective to assess the genetic diversity and determine the population structure of cavy populations from Cameroon to guide the development of a cavy improvement program. Sixteen microsatellite markers were used to genotype 109 individuals from five cavy populations (Wouri, Moungo and Nkongsamba in the Littoral region, and Mémé and Fako in the Southwest region of Cameroon). Twelve markers worked in the five populations with a total of 17 alleles identified, with a range of 2.9 to 4.0 alleles per locus. Observed heterozygosity (from 0.022 to 0.277) among populations was lower than expected heterozygosity (from 0.42 to 0.54). Inbreeding rates between individuals of the populations and between individuals in each population were 59.3% and 57.2%, respectively, against a moderate differentiation rate of 4.9%. All the tested loci deviated from Hardy-Weinberg equilibrium, except for locus 3. Genetic distances between populations were small (from 0.008 to 0.277), with a high rate of variability among individuals within each population (54.4%). Three distinct genetic groups were structured. This study has shown that microsatellites are useful for the genetic characterization of cavy populations in Cameroon and that the populations investigated have sufficient genetic diversity that can be used to be deployed as a basis for weight, prolificacy and disease resistance improvement. The genetic of diversity in Southern Cameroon is wide and constitute an opportunity for cavy breeding program.

Introduction
Cavy culture has demonstrated its utility and effectiveness for poverty alleviation in Andean countries in South America (Chauca 1997). However, in numerous sub-Saharan African countries considerable populations of domestic cavies (Cavia porcellus L.) exist that serve for meat consumption, income generation and manure production in mixed crop-livestock systems (Ngou Ngoupayou et al. 1995;Kouakou et al. 2011;Matthiesen et al. 2011;Niba et al. 2012;Maass et al. 2014;Yiva et al. 2014).
It is not known, where, when and how often cavies have been introduced in Cameroon. However, it is speculated that cavy husbandry has been known at least since 1968 (Yiva et al. 2014). Except for two development projects (Hardouin 1995;Nuwanyakpa et al. 1997), there was no promotion of the animal, but farmers took up cavy culture by themselves across the country. To increase productivity, one project introduced descendants from a large body size line developed in Peru to Northwest Cameroon in 1996(Nuwanyakpa et al. 1997. Niba et al. (2012), however, presume that this breed has possibly been assimilated in the local gene pool.
Since 2001, the use of cavies has been promoted by the Ministry of Livestock, Fisheries and Animal Industries in Cameroon as part of short-cycle livestock species targeted to meet the national demand for animal proteins (Niba et al. 2012). Domestic cavies are widely reared and consumed in Cameroon (Manjeli et al. 1998;Ngou Ngoupayou et al. 1995;Niba et al. 2012;Yiva et al. 2014), contributing to income generation in poor households since their rearing does not require significant capital (Lammers et al. 2009;Niba et al. 2012).
Although cavies appear morphologically similar considering their coat color pattern, genetically they might represent different populations. Largest cavy populations are known to exist in Cameroon with high phenotypic viabilities ; this have been studied by molecular markers Wikondi et al. 2015) in some populations and did not cover the southern part of the country. Ayagirwe (2014) suggested that the main ecotypes in southern Cameroon are from Wouri, Moungo and Nkongsamba in the Littoral (coastal) region, and Fako and Mémé in the Southwest region.
Because of their polymorphism and their homogeneous distribution in the genome, molecular markers are powerful tools for describing animal populations (Mohamed et al. 2010). However, the use of molecular markers in domestic cavy to reveal diversity patterns is recent and limited to few populations from South America (Spotorno et al. 2004;Kanitz et al. 2009;Burgos et al. 2011) and, only lately, from Africa for the Côte d'Ivoire cavy population (Kouakou et al. 2015). It is, therefore, necessary to extend these studies to populations in other African countries for better understanding cavy genetic diversity to help to guide cavy breeding in sub-Saharan Africa.

Study area
This study was conducted in Wouri, Moungo and Nkongsamba in the Littoral (coastal) region, and Mémé and Fako in the Southwest region of Cameroon. The Littoral and Southwest administrative regions of Cameroon, a unimodal agro-ecological zone, are located between 2°6' and 6°12' north, 8°48' and 10°30' east, with elevations from 0 to 4095 m a.s.l. This region occupies an area of 4.5 million ha, of which 282,000 ha (6.3%) are cultivated with agricultural crops. The climate is typically equatorial: with an average annual rainfall of 2500 to 4000 mm, air humidity of 85 to 90%, and mean monthly temperature between 22 and 29°C (FAO 2008).

Sampling and collection of blood
A total of 109 blood samples were collected from adult cavies of about four months of age. To select within the five populations, the snowball method was used (Salganik and Heckathorn 2004), because of lack of information available on cavy keepers. Identified cavy-keeping household helped in identifying others. Usually, one animal was sampled if the cavy population size per household was below 30 animals, except if conspicuously unusual phenotypes were encountered. The animals were selected with the objective of obtaining the largest variability of phenotypic characteristics within each population following the method of Kosaki and Juo (1989).
Blood samples were collected from adult animals by pricking the disinfected ear vein with a 22 gauge needle. Blood was spotted onto Whatman FTA Classic Cards (Cambridge Bioscience, United Kingdom), according to the manufacturer's instructions and air dried.

DNA extraction
For the total DNA extraction, twenty 1.5 mm punches of the FTA Card blood spots were added to 1 ml of 100 mM Tris pH 8, 0.1% SDS and the supernatant was removed after 30 min of agitation. A total volume of five hundred µl of 1.5 M guanidinium thiocyanate was added, and after 10 min of vortexing all the supernatant was removed. The discs were then washed three times with 500 µl of triple distilled water, with 10 min of centrifugation between each wash. Then, 50 µl triple distilled water was added to the discs and the mixture placed in a water bath and heated to 90 °C for 20 min. After 30 min of cooling, DNA was recovered by centrifugation and stored at -20°C.

PCR amplification of microsatellites
Sixteen microsatellite markers previously reported for the species Cavia porcellus were evaluated (Spotorno et al. 2004;Kanitz et al. 2009;Burgos et al. 2011). PCR amplification was carried out in C1000 thermal cycler (Bio-Rad). The final volume of the reaction was 10 µl, containing 2 µl of 40 ng/µl template DNA, 1x DreamTaq buffer containing 2 mM MgCl2 (Fermentas), 200 M of each dNTP, and 0.4 U of DreamTaq DNA polymerase (Fermentas). PCR conditions were: 95°C for 3 min, followed by 35 cycles of 30 sec at 94°C for template denaturation, 30 sec at 60°C primer annealing and 60 sec at 72°C for primer extension, followed a final primer extension step at 72 °C for 20 min. PCR products were analyzed using 1.5% agarose gel electrophoresis.

Data analysis
The data was captured using the GenScan ® collection software (Applied Biosystems) and the allelic data analyzed using the GeneMapper ® software version 4.1 (Applied biosystems). A total 103 data points were achieved out of the expected 109 data point giving an overall success rate of 94.5 % .The data was compiled into a spreadsheet as a standard GeneMapper output file and used in subsequent analysis.
Allele scoring was done using GeneMapper software V4.1 (Timothy 2009). The observed heterozygosity (Ho) and unbiased expected heterozygosity (He) were calculated under the assumption of Hardy-Weinberg equilibrium with allele frequencies observed according to Nei (1978); the average number of alleles (Na) (Crow and Kimura 1970) were calculated using Arlequin software V3.1 (Excoffier et al. 2005). The test of Hardy-Weinberg equilibrium was conducted with Power Marker software V3.25 (Liu and Muse 2005).
Admixture were investigated. For grouping individuals into a K th number of population, Bayesian probabilistic group assignment was done using STRUCTURE software (Pritchard et al. 2000). K values analyzed ranged from to 1-5 and each one was simulated five times. Correlated allele frequency with mixing model was used for runs with 100,000 iteration following a 10,000 burn-in period. The Delta K method described by Evanno et al. (2005) was applied for inferring optimal k-values.

Indices of genetic diversity within and among populations
Mean number of alleles observed varied from 2.909 ± 1.221 for the population of Nkongsamba to 4.0 ± 1.549 for the population of Wouri, with an overall average of 3.455 ± 1.809 (Table 1). The lower number of alleles found for the population of Nkongsamba may partly reflect geographical differences observed between different areas, but also the smaller sample size compared to the other populations.
The observed and expected heterozygosity rates were calculated for each population under Hardy-Weinberg equilibrium. To estimate the importance of genetic polymorphism, observed and expected heterozygosity rates were compared. The expected rates were higher than those observed for all populations reflecting a heterozygote deficiency. The highest rate was 0.277 in the population of Wouri. These results were consistent with the rates of inbreeding observed for all loci studied, which were very high IN all studied populations (Table 1). Table 2 shows the results of the Hardy-Weinberg equilibrium test for all loci studied and for all populations. The population of Nkongsamba presented a high balance compared to other populations. Outside the locus cavy 3, which was in equilibrium in all populations, all other deviated from that equilibrium. However, considering each subpopulation, there were also loci in balance with the Hardy-Weinberg equilibrium. This was the case of loci cavy 2, 3, 8, 11 for Fako, cavy loci 2, 3, 8 for Wouri; cavy 3, 6, 8, 10 and 14 for Mémé; and loci cavy 3 and 7 for Moungo, whereas in the population of Nkongsamba, only loci cavy 2, 7 and 8 showed a deviation from the Hardy-Weinberg equilibrium. The equilibrium observed for locus cavy 3 confirmed the absence of inbreeding at this locus, while the other loci showed a high rate of inbreeding and violations of equilibrium conditions. Molecular variance assessed in the total study population is summarized in Table 3. A high variability was observed among individuals within each population (54.4%) and between individuals of the total population (40.6%), leaving only a small percentage (4.9%) of variation among populations.
Among the five populations studied, those from Nkongsamba and Moungo had the greatest genetic distance but also the lowest genetic identity, respectively, 0.227 and 0.758 (Table 4). Populations from Mémé and Fako, both from Southwest Region, on the other hand, were those closest as having the greatest genetic identity and the lowest genetic distance (0.992 and 0.008, respectively). Low genetic distances observed among populations show that they were close and probably had a common history.

Structure and phylogenetic relationship between populations
The number of genetic populations was equal to K = 3 (Figure 1), the peak obtained following Evanno et al. (2005). Thus, the overall study population was considered to be composed of three distinct genetic types. Populations from Wouri and Moungo were composed of one dominant subpopulation (green) despite the presence of introgression, whereas other populations consisted of two subpopulations. Observed introgression implies that much of the distribution of individuals is done regardless of their geographical origin and, probably, uncontrolled crosses take place between existing genetic types.

Discussion
This study reports, a study on genetic diversity and population structure of domestic cavies from Cameroon at molecular level. Seventeen alleles were observed in the total population with an average of 3.5 alleles per locus in five populations studied. The lowest number of alleles where observed in Nkongsamba ecotype (2.909) while Wouri had the greatest number (4.00). Poutougnigni et al. (2015) and Wikondji et al. (2015) obtained respectively 5.1 and 5.9 alleles per locus in population from forest and highland regions in Cameroun. Compared to Southwest region, number of alleles per ecotype from littoral region seems to be more variable. The overall number of alleles observed in this study was lower than that determined by Kouakou et al.  *** ns *** *** *** Cavy14 *** ns *** ns *** Cavy15 ** ns *** *** *** Cavy16 *** ns *** ** *** ns, not significant; *, **, and ***, significant at p ≤ 0.05, p ≤ 0.01 and p ≤ 0.001, respectively  (2015) who obtained on average 5.98 ± 0.37 alleles from Cote d'Ivoire cavy populations using the same markers; however, these authors sampled from large parts of the country, while this study focused on a much narrower geographic area in Cameroon. Burgos-Paz et al. (2011) in three lines of domestic cavy from Colombia/South America, recorded on average 6.8 alleles per locus with a total of 34 alleles, when using 5 microsatellites; however, this number is close to that obtained in the tame line (4.8 alleles). Unsurprisingly, Kanitz et al. (2009) found an even higher average number of alleles of 8.5 and 6.3, respectively, for the wild species Cavia magna and Cavia aperea.
The relatively low number of alleles observed in the current study could be due to the small sample size, assuming that an increase of the sampled population would result in an increase of the probability of finding new alleles (Foulley et al. 2006). In addition, the low number of alleles observed could also be related to mutations, deletions and a high level of inbreeding in the population. Finally, it is to be expected that only a fraction of genetic diversity available in South American domestic cavies has been introduced to Africa, whenever and how often this introduction has taken place.
In the whole studied populations, the expected heterozygosity was high than the observed heterozygosity. The same observation have been done by Wikondi et al. (2015) and Poutougnigni et al. (2015) in highland and forest cavy populations from Cameroon. Populations from Southwest region had the high deviation from expected heterozygosity (0.499 for Mémé and 0.302 for Fako) compared to those form littoral. Both the expected and observed heterozygosity values obtained by Kouakou et al. (2015), respectively, 0.619 ± 0.03 and 0.528 ±0.04 for expected and observed heterozygosity are higher than those reported in this study. The same conclusions are found considering the results from Kanitz et al. (2009) studying two wild cavies; Cavia magna and Cavia aperea (0.681 and 0.648, and 0.656 and 0.573, respectively). The high rate of inbreeding (0.60) observed in the studied populations could help explain the low rates of observed and expected heterozygosity. On the other hand, the largest deviation from expected heterozygosity (0.499 across all loci for Mémé) was lower than that reported by Burgos-Paz et al. (2011).
Except for locus cavy 3, all studied loci deviated from Hardy-Weinberg equilibrium in the general population. These results differ from those obtained by Kanitz et al. (2009), who observe a balance for all markers in C. aperea, but deviation in the loci cavy1, 5 and 10 in C. magna. Burgos-Paz et al. (2011) record a deviation from Hardy-Weinberg equilibrium at 4 out of 5 loci in three lines of domestic cavy in Colombia (native cavy, improved and tame cavy). These results show that, as long as there is restricted flock size, Hardy-Weinberg equilibrium is deviated in some loci.
The deviation from the equilibrium in this study can be explained by the fact that in Cameroon, the average size of a cavy population is 18 per household (Ngou Ngoupayou et al. 1995;Yiva et al. 2014), and this small flock size cannot lead to random mating. In addition, there is often exchange of animals among farmers within a region and even among regions.
The high rate of molecular variance observed between individuals from different populations and between individuals of the same population have been already reported as well by Wikondi et al. (2015) and Poutougnigni et al. (2015) in highland and forest cavy populations from Cameroon and would suggest there are opportunities for intra-population selection before crossing individuals from the different populations for possible genetic improvement of the species in Cameroon. Similar results have been found by Kouakou et al. (2015) who also recorded a small molecular variation among populations (2.6%), while within populations and individuals it was intermediate (22.0%) and large (75.4%) respectively. This could be related to low exchange of animals within Côte d'Ivoire compared to Cameroon.
The five populations in this study were structured into three distinct genetic groups. Evolutionary theory of populations can help explain these groups. According to Mohamed et al. (2010), the genetic constitution of a population has a possibility of variation over time, and there are forces that determine genetic variations of populations. The result of these evolutionary forces is to vary the rate of heterozygosity of the population compared to the Hardy-Weinberg equilibrium. In addition, two populations of the same species may be subject to different evolutionary factors, resulting in genetic divergence from one another over time, which can produce an effect on both allele frequencies and the relationship between observed and expected heterozygosity. Chauca (1997) noted that, in general, the population of cavy has preserved significant genetic and phenotypic variability.
A low level of genetic structure as observed in this study and a high rate of inbreeding has been reported from several production systems. This could be attributed to nonrandom mating and selection (e.g., Granevitze et al. 2007 for chicken; Kumar et al. 2006 for buffalo;Serrano et al. 2009 for goats;Wu et al. 2009 for ducks). As a short-cycle small livestock species, cavy is not different when considering its rapid growth, high reproductive rate and the traditional farming system in Cameroon. The species is susceptible to very high inbreeding rates as found in this study.

Conclusion
The results of this study show that existing microsatellites developed for cavy populations in South America are useful tools for the genetic characterization of cavy populations in sub-Saharan Africa. They indicate that the studied populations from southern Cameroon have a satisfactory genetic diversity that can be used as a basis for conservation of and selection from this small livestock species to improve productivity. Future studies should expand to include cavy populations from northern and northwestern regions of Cameroon as well as those from other African countries. This may help not only to identify useful pockets of less related genetic diversity for improvement but also clarify the possible evolution of domestic cavy on the African continent.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Atypical protein disulfide isomerases (PDI): Comparison of the molecular and catalytic properties of poplar PDI-A and PDI-M with PDI-L1A

Protein disulfide isomerases are overwhelmingly multi-modular redox catalysts able to perform the formation, reduction or isomerisation of disulfide bonds. We present here the biochemical characterization of three different poplar PDI isoforms. PDI-A is characterized by a single catalytic Trx module, the so-called a domain, whereas PDI-L1a and PDI-M display an a-b-b’-a’ and a°-a-b organisation respectively. Their activities have been tested in vitro using purified recombinant proteins and a series of model substrates as insulin, NADPH thioredoxin reductase, NADP malate dehydrogenase (NADP-MDH), peroxiredoxins or RNase A. We demonstrated that PDI-A exhibited none of the usually reported activities, although the cysteines of the WCKHC active site signature are able to form a disulfide with a redox midpoint potential of -170 mV at pH 7.0. The fact that it is able to bind a [Fe2S2] cluster upon Escherichia coli expression and anaerobic purification might indicate that it does not have a function in dithiol-disulfide exchange reactions. The two other proteins were able to catalyze oxidation or reduction reactions, PDI-L1a being more efficient in most cases, except that it was unable to activate the non-physiological substrate NADP-MDH, in contrast to PDI-M. To further evaluate the contribution of the catalytic domains of PDI-M, the dicysteinic motifs have been independently mutated in each a domain. The results indicated that the two a domains seem interconnected and that the a° module preferentially catalyzed oxidation reactions whereas the a module catalyzed reduction reactions, in line with the respective redox potentials of -170 mV and -190 mV at pH 7.0. Overall, these in vitro results illustrate that the number and position of a and b domains influence the redox properties and substrate recognition (both electron donors and acceptors) of PDI which contributes to understand why this protein family expanded along evolution.

Introduction
Oxidative protein folding is an essential process, occurring generally in oxidizing sub-cellular compartments, and which is required, in particular, for the maturation and assembly of newly synthesized secreted and membrane proteins. In the periplasm of prokaryotes, the proteins responsible for the formation and isomerisation of disulfide bonds belong to the Dsb (DiSulfide Bonds) protein family. This protein family is composed of several members referred to as DsbA to DsbL [1,2]. The major actors, conserved in many bacteria, are the DsbA-DsbB couple which is involved in oxidation reactions, the DsbC-DsbD couple in isomerisation reactions, DsbE or CcmG in cytochrome c maturation and DsbG in the reduction of sulfenic acids [2][3][4]. In eukaryotes, the proteins responsible of the oxidative protein folding are essentially found in the endoplasmic reticulum (ER) and in the intermembrane mitochondrial space. In the ER, the proteins involved belong mostly to the endoplasmic reticulum oxidase (ERO) and protein disulfide isomerase (PDI) families [5]. In addition, proteins named quiescin sulfhydryl oxidases (QSOX) displaying oxidase activity in vitro are also present in this compartment and various physiological functions were proposed for QSOX [6][7][8].
In the mitochondrial intermembrane space, the oxidation of cysteines is ensured by the Mia40-Erv1 disulfide relay [9].
All PDI, as well as some Dsb members, belong to the thioredoxin (Trx) superfamily displaying a common structural fold named the Trx fold [10,11]. All PDI possess at least one Trx domain referred to as a or b domain which consists of about 100 amino acids and adopts a Trx fold. The difference is that a modules exhibit a catalytic active site (most frequently WCGHC) whereas b modules do not possess the typical CxxC catalytic motifs but instead are thought to be important for substrate recognition [12,13]. The isoforms usually differ by the number and the positions of a and b domains and by the active site signature of a domains. The classical PDI isoforms, which are present in all eukaryotic organisms, possess four modules with an ab-b'-a' organisation. Several PDI isoforms possess a more limited number of domains as well as additional domains that have been evolutionary added and which likely confer specific functions to the isoforms possessing them [5].
PDI constitute a multigenic family composed of 5 genes in yeast and up to 20 genes in human [14,15]. In photosynthetic organisms, PDI cluster into 9 classes (A, B, C, D, E, F, L, M and S), 3 are specific to algae (D, E, F), 1 is specific to land plants (A) and the 5 others are present in both phyla [5,[16][17][18][19]. Land plants possess around 10 members [5]. Some soybean PDI isoforms belonging to classes L, M and S play essential roles in seed and pollen maturation and development [17][18][19][20][21][22]. The plastidial form of a Chlamydomonas reinhardtii PDI-L is involved in the redox-dependent transcriptional regulation in the chloroplast [23]. A maize PDI-A protein is accumulated in response to an ER stress, but, in contrast to classical PDI, it might not reside in the ER or only transiently [16]. It was also recently proposed that the Hordeum vulgare PDI5.1 isoform, a PDI-A, is involved in the susceptibility toward various bymoviruses and particularly the barley yellow mosaic virus [24]. However, very little is known concerning the biochemical and physiological properties of many plant PDI.
Thus, the aim of this study was to compare the biochemical properties of three poplar PDI (PDI-A, PDI-L1a and PDI-M) exhibiting different domain organisations and belonging to three different classes, by examining in particular their in vitro reductase and oxidase/isomerase activities using both physiological and non-physiological substrates. For poplar PDI-M, a deeper analysis was performed using proteins with mutated active site cysteines in order to investigate the importance of each catalytic domain for the oxidation or reduction of disulfide bonds. The data indicated that, although the redox potentials of the two modules of PDI-M are very close, each Trx module preferentially performed specific reactions, the a°domain being rather competent for oxidation and the a domain for reduction.

Material and methods
Cloning, site-directed mutagenesis, production and purification of recombinant proteins The sequences encoding poplar PDI-L1a (Potri.002G082100.1), PDI-M (Potri.014G160000.1), PDI-A (Potri.009G004500.2) were amplified from leaf cDNAs of Populus trichocarpa x Populus deltoides for PDI-L1a and P. trichocarpa for PDI-M and PDI-A, and cloned into pET3d for PDI-L1a or into pET-15b for PDI-M and PDI-A. In order to express proteins in their mature forms, the 21, 23 and 26 N-terminal amino acids corresponding to the putative ER targeting sequences in PDI-L1a, PDI-M and PDI-A respectively were removed. The primers used for cloning and site-directed mutagenesis (PDI-A K56G, PDI-M C36/C39S and C165/C168S) are listed in Table 1. Recombinant plasmids were used to transform the Escherichia coli BL21 (DE3) pSBET strain. The culture conditions were as described in [25]. The purification of PDI-L1a consisted in a succession of ammonium sulfate precipitation, ACA44 gel filtration and DEAE Sephacel chromatography as described [25]. The purification of His-tagged PDI-M and PDI-A variants was performed by affinity chromatography on IMAC columns (Sigma Aldrich) from the soluble fraction obtained after a 30 min centrifugation (27,000 x g) of cells lysed by sonication. The washing buffer was 30 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole and the elution buffer was 30 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole. All proteins were finally dialyzed against a 30 mM Tris-HCl pH 8.0, 1 mM EDTA, 200 mM NaCl buffer by ultrafiltration on YM10 membranes and concentrated. The same procedure was applied for the anaerobic purification of PDI-A in the glove box except that imidazole was removed by desalting on a G25 column and the protein was concentrated using 500 μL centricon centrifugal filters omitting EDTA in the buffer. Protein concentrations were determined using molar extinction coefficients at 280 nm of 39,100 M −1 cm −1 for PDI-L1a, 55,265 M −1 cm −1 for PDI-M, 55,140 M −1 cm −1 for PDI-M C36/C39S and PDI-M C165/C168S, 21,220 M −1 cm −1 for PDI-A and PDI-A K56G.

Oligomerization state determination of PDI-A
The oligomerization state of PtPDI-A was analyzed using an Ä KTA Purifier system equipped with a Sephadex75 10-300 column (GE Healthcare). 100 μg of fresh anaerobically-purified proteins reduced in the presence of a 10-fold DTT excess at room temperature was loaded on the column at a flow rate 0.5 ml min-1, and detection was recorded at 280 and 420 nm. The columns were calibrated using the 29-700 kDa molecular weight standards (Sigma).

Determination of the redox midpoint potential
Oxidation-reduction titrations using the fluorescence of adducts formed between the protein and monobromobimane (mBBr) were carried out at ambient temperature as described previously [26,27]. The 500 μl reaction mixtures contained 100 μg protein in 100 mM HEPES-NaOH buffer pH 7.0, together with defined mixtures of oxidized and reduced glutathione to set the ambient potential (E h ) with an overall concentration in glutathione of 2 mM.

PDI-mediated recycling of 2-Cys Peroxiredoxin (Prx)
The PDI-dependent recycling of A. thaliana 2-Cys Prx was measured using a NADPH-coupled spectrophotometric method at 25˚C as described previously [30]. The assays were carried out in a total volume of 500 μl containing 30 mM Tris-HCl pH 8.0, 1 mM EDTA, 200 μM NADPH, 1 μM AtNTRB, 5 μM At2-Cys Prx, 100 μM H 2 O 2 and 2.5 μM PDI. The decrease in absorbance was followed at 340 nm. The peroxidase activity was determined after subtracting the spontaneous reduction rate observed in the absence of PDI.

NADP-Malate Dehydrogenase (NADP-MDH) activation
The purification and activation test of NADP-MDH from Sorghum bicolor are as described in

Quantification of iron and sulfide contents
Protein concentrations of PDI-A have been estimated using the bicinchoninic acid (BCA) protein assay (Interchim) using bovine serum albumin as a standard. For iron measurements, known concentrations of PDI-A prepared in 130 μl are mixed with 90 μl of 1 M perchloric acid (PCA). After 15 min incubation at room temperature, the reaction mixtures are centrifuged at 10,000 x g for 5 min. Then, 144 μl of 3.15 mM bathophenantroline-disulfonic acid, 72 μl of 192 mM sodium ascorbate and 152 μl of 6.2 M ammonium acetate are sequentially added to 180 μl of the supernatant. The reaction mixtures are incubated 30 min at room temperature and centrifuged at 10,000 x g for 5 min. The absorbance ratio between 535 and 680 nm is determined for each sample including the standard curve (from 0 to 150 μM) made using a 500 μM ammonium iron(II) sulfate solution prepared by a 1/20 dilution from a 10 mM solution prepared in 1 N HCl. For sulfide measurements, known concentrations of PDI-A prepared in 100 μl are mixed with 300 μl of 1% zinc acetate, immediately followed by the addition of 15 μl of NaOH 3N. The solution is vigorously mixed and incubated at room temperature for 10 min. Then, 75 μl of a 0.1% N,N-dimethyl-p-phenylenediamine (DMPD) solution prepared in HCl 5N are added, immediately followed by the addition of 16 μl of a 23 mM iron chloride solution prepared in HCl 1.2 N, and the mixture is vigorously mixed. The reaction mixture is then incubated for 30 min (standard curve) or 3h (samples) at 4˚C. All samples are centrifuged 5 min at 10,000 x g and the sulfide concentration is determined by measuring the absorbance at 670 nm. The standard curve (0 to 100 μM) is prepared from a 200 μM Li 2 S solution prepared by a 1/100e dilution from a 20 mM stock solution prepared in 0.3 N NaOH.

The three selected poplar PDI have particular sequence characteristics and domain organisations
In order to analyze the biochemical properties of PDI with different domain organisations, three poplar isoforms referred to as PDI-L1a, PDI-M and PDI-A have been selected for an indepth analysis [5]. PDI-L1a and PDI-M are multi-modular proteins composed of 4 and 3 domains respectively but both proteins possess two a domains containing the conventional WCGHC active site signature (Fig 1A and 1B). On the contrary, PDI-A exhibits a single a domain with a modified WCKHC signature, which is also found in land plant orthologs. It is interesting to note that ancestral PDI-A relatives found in Selaginella moellendorffii and Physcomitrella patens have the regular WCGHC signature.
Besides active site residues, there are 5 additional strictly conserved residues, a Phe and a Leu at positions -5 and +6 compared to the catalytic Cys, the cis-Pro conserved in all oxidoreductases having a Trx fold, and a Tyr and an Arg in the C-terminal part. This Arg was proposed to modulate the pKa of the C-terminal active site Cys, necessary for the PDI reoxidation step, by moving into and out of the active site, depending on the catalytic step [32]. Two other residues, a Glu and a Lys, highlighted in Fig 1A and 1B, are involved in the modulation of the N-terminal active site cysteine pKa at least in E. coli Trx1 [33]. Whereas they are strictly conserved in poplar PDI-L1a (E 52 /K 86 and E 397 /K 431 ), only the Glu is conserved in the two a domains of PDI-M, the Lys being replaced by an Ala (A 85 ) or a His (H 213 ) in the a°and a modules respectively. None of them is conserved in PDI-A. All these observations raise the

Among atypical PDI, PDI-A is inactive and PDI-M is less efficient than the regular PDI-L1a
After producing in E. coli all three proteins devoid of their putative N-terminal ER targeting sequences and purifying them to homogeneity, the classical test used for measuring PDI activity i.e., the oxidative refolding of a reduced and denatured RNase A, has been used for an initial comparison of their respective activity. PDI-L1a was the most efficient protein, as, after an incubation time of 15 min, it allowed the recovery of ca 90% of the activity relatively to the one observed with native RNase A (Fig 2A). At the same time, RNase A activity recovery was only 60% in the presence of PDI-M whereas the results obtained with PDI-A were similar to the reaction achieved by omitting PDI, indicating that the latter did not promote oxidation reactions, at least with RNase A.
Then, the capacity of all these PDI to catalyze the reduction of disulfide bridges instead of promoting their formation was analyzed by measuring insulin reduction, which is accompanied by its precipitation as measured by recording absorbance at 650 nm. In this test, PDI-L1a was the most efficient protein, even more than poplar Trx h1, with the reduction starting after 0.5 and 4 min respectively at concentrations of 5 μM (Fig 2B). PDI-M can also perform insulin reduction but, as in the previous assay, it was less efficient than PDI-L1a or Trx h1. PDI-A was not able to reduce insulin at all. Considering the presence of two catalytic domains in PDI-L1a and PDI-M, the better reducing activity of PDI-L1a compared to PDI-M could suggest that both catalytic domains of PDI-L1a possess reductase activity whereas only one PDI-M domain may be efficient. Moreover, independently of the rate of the reaction, the fact that the turbidity was lower is the case of both PDI compared to Trx h1 might indicate that not all insulin disulfide bonds are reduced by PDI.
Finally, since a variety of PDI can be efficiently reduced by a NADPH/NADPH thioredoxin reductase (NTR) system, we examined whether PDI can be reduced by the A. thaliana NTRB isoform using DTNB as an electron acceptor. Although this is presumably not the physiological reductant of ER-targeted PDI, PDI-A does not have the typical ER-retention signal contrary to PDI-L1a and PDI-M and such a system would be extremely convenient afterwards for measuring the reducing capacity of PDI by using usual Trx targets. As in the other assays, PDI-L1a was more efficient than PDI-M and PDI-A was inactive ( Fig 2C).

Atypical PDI have redox midpoint potentials adequate for an oxidoreductase activity
Besides structural considerations, the redox properties of oxidoreductases are also governed by the pKa of the catalytic cysteines and the redox potential of the catalytic disulfides. Since PDI-A was inactive in all assays and possessed a particular active site signature and differences are observed among PDI, we sought to evaluate the redox potentials of both atypical PDI, PDI-A and PDI-M. It is well documented that the nature of the spacing residues between the two cysteine residues influences the redox potential values of proteins of the Trx superfamily [34,35]. Hence, for PDI-A, a K56G variant, the active site sequence of which was changed from WCKHC to the regular WCGHC signature, has been produced and characterized. The E m value of PDI-A was determined to be -170 mV ± 5 mV whereas the one measured for PDI-A K56G was -150 mV ± 5 mV ( Fig 3A). However, the observed 20 mV increase for the PDI-A K56G variant did not allow acquiring oxidoreductase activity since it was also inactive in all assays used previously (RNase A refolding, insulin and DTNB reduction) (data not shown). For PDI-M, the redox potentials of the disulfide formed by the cysteine residues found in both a domains was measured after producing two variants corresponding to the full-length protein but where the dicysteinic WCGHC motif of each a domain was substituted by a WSGHS motif. Using PDI-M C165/168S and PDI-M C36/39S variants, E m values of -170 mV ± 5 mV and of -190 mV ± 5 mV have been determined for the catalytic disulfides found in the a°and a domains respectively (Fig 3B). The PDI-M C165/168S variant will be thereafter

PDI-A incorporates a [Fe 2 S 2 ] cluster upon expression in E. coli
The absence of activity for PDI-A was intriguing. By comparing E. coli cell pellets after centrifugation, we observed a darker colour for the one expressing PDI-A. For this reason, the protein was purified under anaerobic conditions. The UV-visible absorption spectrum of the purified brownish protein exhibited two peaks around 320 and 420 nm and a shoulder at 460 nm which are likely characteristic of the presence of an iron-sulfur (Fe-S) cluster of the [Fe 2 S 2 ] type ( Fig 4A). The analytical measurement of iron and sulfide contents revealed 1.28 ± 0.13 Fe atoms and 1.10 ± 0.05 S atoms per monomer. Analytical gel filtration experiments showed a small part of aggregated protein and two major, not well separated, peaks with elution volumes at 7.91, 12.65 and 15.5 ml respectively (Fig 4B). Considering the theoretical molecular mass of PDI-A of ca 15 kDa, the peak at 12.65 ml corresponding to an apparent volume of ca 115 kDa may be interpreted as an octameric form and the one at 15.5 ml corresponding to an apparent volume of ca 27 kDa may be interpreted as a dimeric form. Both peaks contained PDI-A with Fe-S cluster as attested by the presence of the absorbance at 420 nm. Altogether, although, PDI-A seems to exist under various oligomeric forms, these results could be consistent with PDI-A binding a [Fe 2 S 2 ] cluster into a dimer. The association of dimers may lead to this higher oligomeric form. Further spectroscopic analyses are however needed for a definitive assessment of the type(s) of cluster that PDI-A can incorporate.

The two catalytic domains of PDI-M do not possess equivalent oxidoreductase properties
In subsequent experiments, we focused our effort on the atypical PDI-M. Using PDI-M(a°) and PDI-M(a) variants, we have investigated the contribution of each catalytic domain to the PDI-M redox properties using the same assays i.e., RNase A oxidative refolding, DTNB and insulin reduction, but also by testing activities typical of Trx i.e., the capacity to activate the chloroplastic NADP-MDH, a well-characterized redox-regulated chloroplastic protein catalyzing the conversion of oxaloacetate to malate, and to sustain the peroxidase activity of a chloroplastic Prx belonging to the 2-Cys Prx subgroup by regenerating the reduced active form of the protein.
In the RNase A refolding assay, PDI-M(a°) presented an efficiency comparable to the one of PDI-M whereas PDI-M(a) exhibited only 50 to 60% of this activity after 15 minutes (Fig 5). This suggested that the a°module is likely the major contributor for the oxidase activity measured with the intact protein and that there may be a partial compensation by the second domain in the absence of the catalytic cysteines of the a°module.
Then, the reductase activity of the respective PDI-M domains was tested by assessing their capacity to reduce insulin and to activate NADP-MDH in the presence of DTT. In the insulin reduction assay, PDI-M(a) was more efficient than PDI-M(a°) (Fig 6A). In this case, the fact that the activity of both domains was decreased compared to the activity of PDI-M suggests that both domains contribute to the reductase activity of PDI-M although with different efficiencies. In the NADP-MDH activation assay, we observed that PDI-M was the only PDI representative able to activate the enzyme since PDI-A (data not shown) and PDI-L1a were unable to do that (Fig 6B). This was quite surprising taking into account the unfavourable thermodynamic barrier, NADP-MDH possesses disulfides with very negative redox potential, around -300 mV at pH 7.0 [36]. However, the observed activation was less efficient compared to the one observed at identical concentrations with Trx h1 used as a reference. The two PDI-M domains presented different reactivities toward oxidized NADP-MDH, PDI-M(a) was even slightly more active than PDI-M whereas PDI-M(a°) was less active (Fig 6B). Altogether, these data indicated that the a module is a more efficient reductant than the a°module.
The properties of PDI-M domains have been further examined using the DTNB assay and AtNTRB as an electron donor. The PDI-M(a°) domain was as efficient as PDI-M whereas PDI-M(a) was only very poorly able to promote DTNB reduction (Fig 7A). To check whether this was due to the inability to be reduced by NTR or to reduce DTNB, insulin reduction assays have been performed with the NADPH/NTR system as the electron supplier instead of DTT ( Fig 7B). The absence of activity for PDI-M(a), which was in fact the most efficient for insulin reduction when DTT was used as a reductant, obviously indicates that the a domain is not or only poorly reduced by NTR and that the catalytic disulfides of PDI-M are asymmetrically targeted by NTR. On the other hand, insulin reduction by PDI-M(a°) was less efficient than by PDI-M (Fig 7B). Since PDI-M(a°) and PDI-M had comparable efficiency for DTNB reduction, these results indicate that mutating the active site cysteines of the a domain may somehow affect the redox properties of the a°module (possibly at the level of the 3D structure or of substrate recognition (here insulin)) which incidentally suggests that these two adjacent domains are connected.
Finally, we have taken advantage of the NADPH-coupled NTR reduction system to examine the reduction of Prx by PDI-M. Indeed, it was shown recently that an ER-targeted human 2-Cys Prx (PrxIV) can accept electrons from two PDI isoforms, P5 and ERp46 [37, 38]. Although there is no evidence yet that a 2-Cys Prx is targeted to the ER in plants, we tested the biochemical capability of PDI-M to reduce the chloroplastic Arabidopsis 2-Cys Prx (At2-Cys Prx). As shown in Fig 8, PDI-M promoted the regeneration of At2-Cys Prx in the presence of the NADPH-NTR system as an electron donor. As expected from its poor reduction by AtNTRB, the PDI-M(a) was almost inefficient, only ca 10% activity was retained compared to PDI-M. The PDI-M(a°) retained about 50% activity. Such a decrease may again indicate that having mutated the active site cysteines of the a module impacted the a°module reactivity.

Discussion
Protein disulfide isomerases are versatile proteins catalyzing the reduction, formation and/or isomerisation of disulfide bonds in vitro and also possibly in vivo depending on the substrates, on their subcellular localizations and/or on the cellular and subcellular redox potentials. In connection with these elements and with the variety of substrates, there are multiple PDI with variable module arrangements which provide the necessary flexibility in the PDI activity pattern [39][40][41]. For multi-modular proteins, each domain could ensure a "specific" function. As an example, the b' module of mammalian ERp57 or PDI proteins, which is redox inactive, is essential for substrate or chaperone partner recruitment [13,15,42,43]. By comparing the biochemical properties of plant PDI belonging to three different classes and with different modular organisation, we also brought elements indicating that the catalytic domains within a given PDI or between PDI do not have the same redox properties.

What is the biochemical function of the single module PDI-A?
Using the battery of substrates tested, we did not detect any oxidase or reductase activity for the atypical mono-module PDI-A, which raises the question of its physiological role and partners. First, considering the DKDL C-terminal sequence, divergent from classical ER retention signals (very often KDEL or KEEL), the question of its subcellular localization is still open. It is worth mentioning that human ERp18, a small mono-domain PDI was shown to exhibit an oxidase activity on synthetic peptides [44]. However, despite their comparable modular organisation, PDI-A and ERp18 might not be true orthologs, exhibiting only ca 15% sequence identity. Proteins involved in oxidation reactions usually exhibit redox potentials ranging from less than -100 mV for some prokaryotic DsbA proteins to -180 mV for the a modules of classical PDI [41,45,46]. Hence, the E m value of -170 mV determined for PDI-A is clearly compatible with oxidation reactions and should not be the reason for the absence of activity. So, why is PDI-A inactive? A first possible explanation could be that the transit peptide has been cleaved at an unfavourable position, eliminating some N-terminal residues essential for activity. However, this seems unlikely since we have cleaved only 26 amino acids in a region which is not conserved and without predicted secondary structure and the protein is fully soluble. Hence, the absence of reactivity is more likely linked to specific differences in the sequence of PDI-A compared to other PDI. First, the CVPWCKHC active site signature which is conserved in PDI-A orthologs replaces the usual [Y/F]APWCGHC (Fig 1). We initially thought that the presence of a charged residue between the two cysteines might be strongly unfavourable. However, we have observed that the atypical WCKHC, although influencing the redox midpoint potential, was not responsible for the absence of activity. In fact, there are many other sequence variations that would explain the lack of oxidoreductase activity. First, the FA or YA pair, found in the above-mentioned signature and usually quite conserved in PDI, is replaced by a CV pair. Then, among the three residues (Glu, Lys, Arg, highlighted in Fig 1) susceptible of contributing to the lowering the pKa of the catalytic cysteines in some Trxs, only the Arg is present in PDI-A [32, 33, 47]. The two other residues (Glu, Lys) are replaced by residues with an opposite charge (Lys, Glu) in PDI-A (Fig 1A). From its absence in PDI-M and the work performed on E. coli Trx1, the Lys does not seem to be as important as the Glu and might thus not be absolutely required [33]. Besides these sequence variations, it is still possible that we have not used proper substrates yet in our in vitro assays and that this protein possesses highly specific physiological partners. Alternatively, it may have evolved to play a completely different function, unrelated to dithiol-disulfide exchanges. The observation that PDI-A can incorporate a [Fe 2 S 2 ] cluster was totally unexpected because this has to our knowledge never been observed for a PDI member. It appears a bit less surprising when taking into account that proteins sharing the same thioredoxin fold, i.e. some thioredoxins or glutaredoxins, including the ER-targeted GRX6 isoform of S. cerevisiae, are able to do this [48][49][50][51]. However, the physiological relevance of this observation is unclear and it remains to be investigated whether such cluster is not an artefact linked to E. coli expression and can indeed be formed in plant cells. It would also be important to determine the subcellular localization of the protein, because Fe-S cluster maturation systems are only found in organelles and in the cytosol, not in the ER [52]. Does the domain organisation of PDI confer them different biochemical properties?
The second aim of the study was the biochemical characterization of a poplar PDI-M isoform possessing an a°-a-b domain organisation quite different from the a-b-b'-a' of the classical and well-characterized PDI-L. In particular, the organisation of the catalytic domains in tandem raises the question of their respective contribution to protein reactivity and of their possible connectivity. The latter point is in fact one of the unsolved questions for most PDI, although such cooperativity has been proposed based on the U shape arrangement of the four Trx domains of the yeast PDI1p, very similar to DsbC or DsbG [41]. We have compared the PDI-L1a and PDI-M capacity to reduce insulin and NADP-MDH or to oxidize NTR and RNase-A. It was previously reported that some PDI can act as oxidants of NTR or in other words that NTR can reduce oxidized PDI [53].
Except for the NADP-MDH activation assay, where it was not active at all, PDI-L1a was always more efficient than PDI-M in all other assays measuring reductase or oxidase activities. This difference of activity observed with poplar proteins is similar to what was recently observed with rice orthologs, indicating that this may be a general feature of these two classes [40]. The better efficiency of PDI-L1a, in particular in reduction assays, may be explained by the strict conservation of the charged amino acids required for decreasing the pKa of the N-terminal catalytic cysteine. Indeed, both a modules of PDI-L1a possess the usual Glu and Lys residues, whereas both domains of PDI-M contain the Glu but an Ala and a His in the a°and a modules respectively (Fig 1). That PDI-L1a was inactive towards NADP-MDH whereas PDI-M exhibited noticeable activities could possibly derive from a natural tendency to interact with non-folded or folded proteins respectively. Indeed, it was shown for mammalian enzymes that PDI-L interact directly with misfolded or unfolded proteins via the mediation of a hydrophobic patch in the b' module, whereas P5 proteins, the mammalian PDI-M orthologs, are partners of the BiP chaperone, a folded protein, which is thought to bring mis-oxidized/misfolded proteins to P5 for their correct oxidative folding [54]. Hence, by analogy and despite a lower "intrinsic" reductase activity compared to PDI-L1a, PDI-M could interact with and promote the activation of native proteins such as NADP-MDH. In agreement with the difference observed in their in vitro biochemical properties, it was shown by genetic studies that a rice PDI-M ortholog, OsPDI2.3, and a rice PDI-L ortholog, PDIL1.1 do not have redundant functions in the ER, having for instance different storage proteins as substrates in the endosperm [21].
These dual and opposite oxidase/reductase activities observed in vitro raise the question of their physiological relevance and occurrence. The in vivo measurement of the redox state of PDI in HEK-293 cells showed for instance a mixture of fully reduced (50%), fully oxidized (16%) and partially oxidized forms of the a or a' modules (18/15%) [55]. These results indicate first that neither of the two domains of this human PDI might exclusively catalyze substrate oxidation or reduction, but also that the protein redox state may be adequate for both reactions. This potential dual function has been nicely illustrated recently by demonstrating that several PDI can reduce ER-resident proteins as 2-Cys Prx (PrxIV) or vitamin K epoxide reductase [37, [56][57][58]. This alternative route of PDI oxidation which would occur independently of ERO1 protein i.e., without producing H 2 O 2 , should clearly decrease reactive oxygen species formation in the ER [59].
Are there specific contributions and/or connections between the catalytic domains in multi-domain PDI?
It is documented that the a' module (the C-terminal one) of a human regular PDI-L, which presents the highest redox potential (ca -150 mV), possesses a preponderant oxidase activity toward reduced and scrambled RNase A [41]. Also, it was shown that only one of the catalytic disulfides of this PDI is directly oxidized by Ero1α and with a slow turnover rate [39]. With the above-mentioned evidence showing the complexity of assessing and interpreting the in vivo redox state [55], these elements suggest that the presence of two catalytic a domains might be important for the formation of native disulfides in proteins entering the secretory pathway by ensuring either oxidase or isomerase reactions.
Although experiments have not been achieved with poplar PDI-L1a, we have observed such a duality among the a domains in PDI-M. The a module, having the cysteine pairs with the lowest redox potential (-170 mV) is the most efficient for reduction reactions (insulin, NADP-MDH) (Figs 6 and 7). On the contrary, the a°module, having the cysteine pairs with the highest redox potential (-150 mV), is more efficient for oxidation reaction than the a module, promoting the recovery of RNase A activity similar to PDI-M levels. Accordingly, the a°m odule of orthologs (rice PDI2,3 and mammalian P5) is also the major contributor in the oxidative refolding of reduced or scrambled RNase A [40,60]. In line with this observation, the a°m odule can efficiently oxidize NTR contrary to the a module. The incapacity of the a module to oxidize NTR or RNase A may relate to steric hindrance or unfavourable protein/protein interactions rather than to thermodynamic factors, the redox potentials of both domains being still rather close. The structure of the E. coli NTR-Trx complex indicated that Trx interacts with NTR mostly via hydrophobic residues (W33, I60, G74 and I75) and via R73 [61]. By comparing all a domains of PDI-L1a and PDI-M, it is interesting to note that two of these five residues differ in the a domain of PDI-M compared to the three other a domains analyzed where they are all strictly similar (not shown). Hence, it is tempting to attribute the inability of the a domain of PDI-M to interact with NTR to these changes. Another possible explanation could rely on steric factors. In PDI-L1a, if it adopts a regular structure, the two a domains should be found at the extremity of the U-shape structure and thus be very likely accessible to NTR. On the contrary, the fact that the two a domains in PDI-M are adjacent and that the a domain is sandwiched between the a°and b domains may be problematic for the accessibility of NTR which is an homodimer of two identical 35 kDa subunits.
In the course of these experiments and taking advantage of the fact that the a°module of PDI-M is reduced by AtNTRB whereas the a module is not or very poorly, we have observed some sort of cross-talk or interference between both domains. Indeed, PDI-M(a°) is as efficient as PDI-M in the assay with the small DTNB molecule (Fig 7A) whereas it is much less efficient with insulin and 2-Cys Prx, retaining about 50% activity (Fig 7B and 7C). Thus, it may be that the presence of a redox active a module is needed for a maximal efficiency of the a°module with some substrates. Alternatively, introducing cysteine substitutions in the a module may have generated subtle conformational changes which hamper recognition of large substrates by the a°module. These substitutions may have more impact in PDI-M because the two catalytic domains are adjacent.


Leopoldia neumannii sp. nov. (Asparagaceae, Scilloideae): a new species of Muscari sensu lato from Greece

Abstract: Leopoldia neumannii, a new species from Greece (regions of Ipeiros and Peloponnisos) is described and compared with other species of the genus. Aspects regarding the taxonomic classification of the Leopoldia group are discussed in light of an unresolved taxonomic situation within the Muscari sensu lato group. Citation: Böhnert T. & Lobin W. 2017: Leopoldia neumannii sp. nov. (Asparagaceae, Scilloideae): a new species of Muscari sensu lato from Greece. — Willdenowia 47: 179–185. doi: https://doi.org/10.3372/wi.47.47210 Version of record first published online on 5 September 2017 ahead of inclusion in September 2017 issue.

During the last centuries, several taxonomic concepts of Muscari s.l. have been proposed (for reviews see Bentzer 1973;Speta 1982Speta , 1998. one of the more recent proposals was made by Garbari & Greuter (1970), who divided the group into four genera. Those authors, as well as recognizing Muscari s.str., raised M. subg. Leopoldia (Parl.) Peterm. and M. subg. Muscarimia Kostel. ex los. to generic rank and described Pseudomuscari as a new genus based on karyological evidence, although they included only a handful of species. in addition, morphological characters such as the prominent bluish to violet sterile flowers as well as the more or less zygomorphic fertile flowers with apophyses on the shoulders were used to separate Leopoldia from the other genera (Garbari & Greuter 1970). however, beside those results as a major argument for a multi-generic treatment, Garbari & Greuter (1970) argued that Muscari s.l. would be comparable to the linnaean sense of the genus Hyacinthus l., which is nowadays widely accepted as split into several genera (e.g. Bellevalia lapeyr., Brimeura Salisb., Hya cinthella Schur, etc.).
in contrast, Speta (1982Speta ( , 1989Speta ( , 1998 and other authors (cf. Davis & Stuart 1980, 1984 favoured treating the group as a single genus (Muscari s.l.), accepting four subgenera: M. subg. Muscari, M. subg. Leopoldia, M. subg. Moscharia (Baker) Chouard (≡ M. subg. Muscarimia) and M. subg. Pseudomuscari (losinsk.) D. C. Stuart. Speta (1982, 1989) favoured the "one genus concept", with a view on several morphological characters shared among all species, e.g. the degree of fusion of the tepals, the alternate arrangement of the anthers, a uniform placentation, as well as the occurrence of sterile flowers. furthermore, Karlén (1984) found conflicting results in his work on the karyotypes of different Muscari species from Greece, compared to Garbari & Greuter (1970), which Speta (1989) interpreted as further evidence for his one genus concept. Some years earlier, Davis & Stuart (1980) stated in their Flora europaea treatment of Muscari that "the differences between the subgenera are considered to be of a lower order of importance than those that separate Muscari s.l. from Bellevalia and Hyacinthella".
however, since the late 1980s no further taxonomic treatments of the genus have been proposed. Speta (1982) discussed the typification of the genus Muscari s.l. and also presented a typification of the four subgenera, but his concept was not applied by others, possibly because he published in German. While other genera, such as the closely related genus Hyacinthoides heist. ex fabr., have been solved based on molecular phylogenetic methods (e.g. Grundmann & al. 2010), only the systematic position of the genus Muscari within Asparagaceae has been assessed so far by a handful of weakly supported phylogenies (cf. Pfosser & Speta 1999;Pfosser & al. 2003).
nevertheless, according to Govaerts (2016), 14 Leo poldia species are accepted, although at least seven more species currently treated in Muscari should be considered as belonging to Leopoldia (Böhnert unpubl.). Until a full taxonomic and systematic revision can be presented (ongoing own work), we apply the classification of Aspara gaceae according to Govaerts (2016).

Material and methods
assessment of the new species was made based on fresh and herbarium material taken from cultivated plants propagated vegetatively from plants collected by m. neumann at the holotype locality in august 2003 (permit: 608/12.05.2003) and the paratype locality in november 2011. line drawings were made following the digital drawing technique protocol by Coleman (2003). for scanning electron microscope (Sem) images, pollen material was mounted on aluminium stubs and sputtercoated with palladium (SCD 040, Balzers, liechtenstein). Samples were observed in a Stereoscan 200 Sem Böhnert 409 (Bonn001673!). in addition, measurements from different flora treatments (Chernyakovskaya & al. 1935;Bentzer 1973;Davis & Stuart 1980, 1984 were used to supplement our own measurements, shown in Table 1. Diagnosis -Leopoldia neumannii is distinguished from all other Muscari (s.l., including Leopoldia) species in its overall size of more than 50 cm tall. it differs from other Leopoldia species in its remarkably larger inflorescence as well as in its bulb size. it differs from L. comosa (the morphologically closest species) in having bulbs nearly twice as large and in forming many bulbils, whereas L. comosa never forms bulbils. in L. neumannii the hanging leaves are longer and the hooded apex is always wilted before reaching maturity, whereas in L. comosa the more erect leaves are shorter and usually do not wilt at the apex. in L. neumannii the fertile flowers are cylindric and cream-brown, whereas in L. comosa they are rather bellshaped and comparatively darker brown on the perigon tube. in L. neumannii the pedicels are shorter and slightly hanging, forming a cylindric raceme, whereas in L. co mosa the longer pedicels of the lower sterile flowers form a pseudo-umbellate termination of the raceme.

Phenology -The new species flowers in may and fruits in June and July.
Distribution and ecology -Leopoldia neumannii is documented so far only from two localities, both in Greece: the Vikos Gorge in ipeiros (the holotype locality) and mt lirkio in Peloponnisos (the paratype locality). The species occurs above 1000 m altitude at both localities. in the Vikos Gorge it grows on ledges of steep cliffs with small depressions forming pot-like structures where sediments and to a minor degree humus accumulate. Eponymy -Leopoldia neumannii is named in honour of its original collector, michael neumann, horticulturist responsible for the geophyte collection at the Bonn Botanic Gardens.
Remarks -individuals of the new species cultivated outside of a pot can reach significantly larger bulb sizes, up to 70 × 75 mm, and single individuals with bulbs more than 100 mm in diameter have been observed. larger bulb size appears always to be correlated with the formation of many bulbils. Leopoldia neumannii differs in many characters compared to the morphologically closest species, L. caucasica, L. comosa and L. tenuiflora. Whereas bulbs of L. neuman nii can reach sizes of nearly 50 mm long or more and produce many bulbils per growing season, the other species always have smaller bulbs and no or almost no bulbils. furthermore, the scape is on average about twice as long as that of L. comosa and even longer compared with the other two species (although in some Greek populations of L. comosa plants with scapes up to 100 cm long have been documented; cf. Bentzer 1973). a common character in Leopoldia is the extended and ascending pedicels of the sterile flowers forming a pseudo-umbellate termination of the raceme, which is not the case in L. neumannii and only to a minor degree in L. tenuiflora. Differences between the four species were also found in pollen size. The pollen of L. neumannii appears >10 µm shorter compared to the measurements for L. caucasica, L. comosa and L. tenuiflora by Pehlivan & özler (2003). however, only two pollen grains were measured for the new species, so this result might not be representative. further morphological differences between L. neumannii and the morphologically related species are summarized in Table 1. at the Vikos Gorge locality, plants of Leopoldia co mosa have not been found on the cliffs, but occur on the ground in open forest at altitudes below 1000 m, where L. neumannii is always absent (m. neumann pers. com.). an additional collection of L. neumannii from above 1000 m on mt lirkio in ne Peloponnisos (see below) indicates a wider distribution of the new species at a similar altitude to the Vikos Gorge locality. according to observations by the collector (m. neumann pers. comm.), L. neumannii seems to replace L. comosa at altitudes above 1000 m, where the latter is always absent, indicating allopatric speciation. it can be assumed that, due to some superficial similarities between L. comosa and L. neumannii, the new species has been hitherto overlooked as distinct. nevertheless, further field observations and herbarium studies are needed to understand the distribution patterns and conservation status as well as the systematic situation in this group and in Muscari s.l. in general.


extfieldsofstudy: Biology Medicine

text:
DDAH1 mediates gastric cancer cell invasion and metastasis via Wnt/β‐catenin signaling pathway

Gastric cancer (GC) represents the fourth most common malignant neoplasm and the second leading cause of cancer death. Despite therapeutic advances in recent decades, the clinical outcome remains dismal owing to the fact that most patients with GC show advanced disease at diagnosis and current chemotherapy only confers a modest survival advantage. Identification of key molecular signaling pathways involved in gastric carcinogenesis and progression would aid in early diagnosis and provide a rational design for targeted therapies in selected patients with advanced GC, to improve their outcome. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is the main enzyme that can degrade asymmetric dimethylarginine, an endogenous nitric oxide synthase (NOS) inhibitor. Increased DDAH1 expression and NO production have been linked to multiple pathological conditions including cancer. However, the prognostic significance of DDAH1 in patients with GC and its function in GC progression remain undefined. In this study, we found that downregulation of DDAH1 was frequently detected in GC tissues and strongly correlated with more aggressive phenotypes and poor prognosis. Functional assays confirmed that forced expression of DDAH1 in the GC cells suppressed cell migration and invasion in vitro, as well as metastatic potential in vivo. DDAH1 overexpression inhibited the epithelial–mesenchymal transition process by increasing β‐catenin degradation through the attenuation of Wnt/GSK‐3β signaling. In contrast, knockdown of DDAH1 produced the opposite effect. These findings suggest that DDAH1 functions as a tumor suppressor in GC and may be exploited as a diagnostic and prognostic biomarker for GC.

Gastric cancer (GC) represents the fourth most common malignant neoplasm and the second leading cause of cancer death. Despite therapeutic advances in recent decades, the clinical outcome remains dismal owing to the fact that most patients with GC show advanced disease at diagnosis and current chemotherapy only confers a modest survival advantage. Identification of key molecular signaling pathways involved in gastric carcinogenesis and progression would aid in early diagnosis and provide a rational design for targeted therapies in selected patients with advanced GC, to improve their outcome. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is the main enzyme that can degrade asymmetric dimethylarginine, an endogenous nitric oxide synthase (NOS) inhibitor. Increased DDAH1 expression and NO production have been linked to multiple pathological conditions including cancer. However, the prognostic significance of DDAH1 in patients with GC and its function in GC progression remain undefined. In this study, we found that downregulation of DDAH1 was frequently detected in GC tissues and strongly correlated with more aggressive phenotypes and poor prognosis. Functional assays confirmed that forced expression of DDAH1 in the GC cells suppressed cell migration and invasion in vitro, as well as metastatic potential in vivo. DDAH1 overexpression inhibited the epithelial-mesenchymal transition process by increasing b-catenin degradation through the attenuation of Wnt/GSK-3b signaling. In contrast, knockdown of DDAH1 produced the opposite effect. These findings suggest that DDAH1 functions as a tumor suppressor in GC and may be exploited as a diagnostic and prognostic biomarker for GC.

Introduction
Gastric cancer (GC) is one of the most prevalent cancer types and the second leading cause of oncologic deaths worldwide. About seventy percentage of these cases occur in developing countries, especially in East Asia (Ferlay et al., 2015;Hartgrink et al., 2009;Parkin et al., 2005). Recently, it has become possible to cure GC if it can be detected early. However, the majority of patients with GC are usually diagnosed at an advanced stage leading to poor clinical outcomes (Hohenberger and Gretschel, 2003). Despite the rapid therapeutic advances, the 5-year survival rate of GC is still low (Karimi et al., 2014;Sehdev and Catenacci, 2013). Therefore, identification and better understanding of novel biomolecules and signaling pathways involved in the gastric carcinogenesis and progression remain of great importance.
Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is the main enzyme that metabolizes asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase (NOS; Kostourou et al., 2002). Consequently, DDAH1 could indirectly increase NO. Numerous studies have demonstrated that overexpression of DDAH1 promotes angiogenesis in vitro and increases glioma growth in vivo through enhanced expression of NO and VEGF (Kostourou et al., 2002;Smith et al., 2003;Wojciak-Stothard et al., 2007). DDAH1 also plays a vital role in early embryogenesis (Breckenridge et al., 2010;Leiper et al., 2007). More recently, a study has shown that DDAH1 can inhibit the renal fibrosis process via the Wnt/b-catenin pathway (Liu et al., 2016), a well-established crucial signaling event associated with a number of human diseases (Miao et al., 2013;Miki et al., 2011;de Sousa et al., 2011) including cancer (Ramachandran et al., 2012). Wnt signaling regulates various functions of the cancer cell such as proliferation, cell fate decision, motility and invasion through activating b-catenin (Clevers, 2006). A large body of evidences have suggested that the canonical Wnt/b-catenin pathway plays an important role in inducing epithelial cancer cells to undergo epithelial-mesenchymal transition (EMT; Brabletz et al., 2005;Taki et al., 2003), which facilitates migration through the extracellular matrix and distant metastasis.
However, the role of DDAH1 in GC development and progression is unclear, and the mechanisms underlying DDAH regulation of malignant transformation remain to be elucidated. In this study, we evaluated the clinical significance of the expression of DDAH1 in a cohort of 150 human GC specimens and analyzed its correlation with clinicopathological features and survival of the patients. We further investigated the functional impact and underlying molecular mechanisms of DDAH1 on GC progression in a series of in vitro and in vivo assays.

Cell lines and clinical samples
Seven human GC cell lines (NCI-N87, MKN74, AGS, NUGC-3, MKN45, MGC803, and HGC-27) were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cell lines were maintained in RPMI-1640 (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) except AGS in Ham's F12 medium (Cellgro, Manassas, VA, USA) and incubated at an atmosphere containing 5% CO 2 at 37°C. Human GC samples and their corresponding nontumorous gastric tissues were collected at the time of surgical resection at The First Affiliated Hospital of Fujian Medical University (Fuzhou, China) from 2008 to 2010. The tissues were immediately frozen in liquid nitrogen and stored at À80°C freezer or fixed in 10% formalin for paraffin embedding. All samples were collected with patients' informed consent, and the study was approved by the institutional review board and regulatory authorities of Fujian Medical University. Clinicopathological classification and staging were determined according to American Joint Committee on Cancer seventh edition of GC TNM staging (Wittekind, 2010). No patients had received chemotherapy or radiotherapy before surgery.

Tissue microarray and immunohistochemistry
A tissue microarray was constructed using two cores of 1 mm in diameter per sample from the 150 patients with GC. Immunohistochemistry (IHC) studies were performed on formalin-fixed, paraffin-embedded tissue microarrays using human anti-DDAH1 antibody (1 : 200; Abcam, Cambridge, UK) and b-catenin antibody (1 : 100; Cell Signaling, Danvers, MA, USA). The degree of DDAH1 staining was quantified according to the following calculation: the score of stained tumor cells (0, ≤ 5% positive cells; 1, 5-25% positive cells; 2, 26-50% positive cells; 3, 51-75% positive cells; 4, ≥ 75% positive cells) multiplied with the score of staining intensity (0, no staining; 1, weak staining, light yellow; 2, moderate staining, yellow brown; 3, strong staining, brown) to obtain a final score ranging from 0 to 12. A final score of 3 or less was classified as low-expression group, while 4-12 as high-expression group. b-Catenin staining was considered positive if > 10% of the tumor cells showed yellow or brown staining.

Western blot analysis
Tissues or cells were lysed in Western and IP cell lysis buffer (Beyotime, Shanghai, China) with PMSF (Amresco, Solon, Ohio, USA) for 30 min on ice at 4°C, followed by centrifugation at 12 000 g for 10 min at 4°C. The supernatants were collected as total proteins and then measured using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). The same amount of proteins in each well were separated with 10% SDS/PAGE and transferred to a 0.45-lm PVDF membrane (Amersham Hybond; GE Healthcare, M€ unchen, Germany). Then, the membrane was blocked in 0.5% albumin from bovine serum . After three washes for 10 min each in TBST, the membrane was further incubated with the secondary antibodies for 1 h at room temperature, and the blots were developed using enhanced chemiluminescence (Lulong Biotech, Xiamen, China).

RNA extraction and real-time quantitative PCR
Total RNA was isolated from cell lines or frozen tissues with Qiagen RNeasy kit according to the manufacturer's instruction. 1 mg RNA was reverse-transcribed using miScript Reverse Transcription Kit (Qiagen, Hilden, Germany) for the first-strand complementary DNA synthesis. Quantitative PCR was performed using SYBR Premix EX Taq kit (Takara, Shiga, Japan). The specific primers were used to detect the relative mRNA expression of DDAH1, b-catenin, E-cadherin, ZO-1, vimentin, N-cadherin, and Snail by the 2 ÀDDCt method. The expression level was normalized against endogenous b-actin. All the primers were designed by BioSune Biotechnology Co., Ltd (Shanghai, China; Table S1).

Plasmids and establishment of stably transduced GC cell lines
The open reading frame of human DDAH1 (GenBankNM_012137.3) was constructed into the eukaryotic expression vector pCDH (Invitrogen, Carlsbad, CA, USA). The recombinant plasmid pCDH-DDAH1, lentivirus plasmids pMDL, p-VSV-G, and p-REV (Invitrogen) were cotransfected in 293T at 30% confluence in 10-cm culture dishes. The medium was collected after filtration with 0.45-lm filters and added into the culture of GC cells (AGS and MGC803) in 6cm culture dishes. After incubation for 48 h, puromycin at 2 lgÁmL À1 (Sigma-Aldrich) was used to select stably transduced cells for 7-14 days. Stable DDAH1-overexpressing clones (AGS-pDDAH1 or MGC803-pDDAH1) and the empty vector-transfected cells (AGS-pCDH or MGC803-pCDH) were used for further study. For the generation of DDAH1-knockdown clones, two shRNA sequences targeting different sites of DDAH1 were synthesized and cloned into pSuper-retro-puro plasmid (Oligoengine, Seattle, WA, USA). The recombinant plasmid or empty vector was cotransfected with packaging plasmids pIK (Invitrogen) into 293T cells. The supernatants were collected and used to infect AGS or MGC803 cells. The puromycin-resistant clones were expanded into cell lines as DDAH1-knockdown cells (AGS-psh1DDAH1, AGS-psh2DDAH1, MGC803-psh1DDAH1, MGC803-psh2DDAH1) or the control cells (AGS-pSuper or MGC803-pSuper). To rescue DDAH1 expression in the DDAH1-knockdown AGS and MGC803 cells, six synonymous point mutations were introduced into the shRNA target regions in the full-length DDAH1 cDNA. The shRNA-targeted sequence in DDAH1 was CGC TAC GAC AAA CTC ACT; the mutations introduced in the shRNA-targeted sequence of DDAH1 were CGA TAT GAT AAG CTA ACC. The shRNA-resistant cDNA was subcloned into pcDNA3.1 vector for transfection. The oligonucleotides and primers used are listed in Table S1.

Cell proliferation assay
Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo, Kuma-moto, Japan). Cells were seeded at a density of 1000 cells per well in 96well plates and incubated at 37°C, 5% CO 2 for 24, 48, 72, 96 or 120 h. 10 lL of CCK-8 solution was added into each well and incubated at 37°C for 4 h. The absorbance at 450 nm was measured using a microplate reader (Bio-Tek, Winooski, VT, USA).

Colony formation assay
5 9 10 2 cells were plated in 60-mm plates with Ham's F12 or RPMI-1640 containing 10% FBS and cultured for 14 days. Colonies were stained with crystal violet for 5 min, and the number of colonies containing 50 or more cells was counted. For soft agar colony formation assay, 2 9Ham's F12 or RPMI-1640 containing 20% FBS and 2 9 10 3 cells was mixed with equal volume of 0.7% agarose and immediately plated in six-well plates containing an underlayer of 0.5% agarose made in 19Ham's F12 or RPMI-1640 supplemented with 10% FBS. The plates were cultured at 37°C under 5% CO 2 for 14-21 days. Surviving colonies (> 50 cells per colony) were counted and photographed with a Qimaging Micropublisher 5.0 RTV microscope camera (Olympus, Tokyo, Japan).

Cell migration and invasion assay
For the migration assay, 6 9 10 4 cells in serum-free media were plated into the upper chamber of Transwell insert (8-mm pore size; BD Biosciences, San Jose, CA, USA). For the invasion assay, the Transwell insert was coated with Matrigel (BD Biosciences) and 1 9 10 5 cells were plated onto the top of the coated insert. The medium containing 20% FBS in the lower chamber served as chemoattractant. After 24 h of incubation at 37°C in a 5% CO 2 humidified incubator, the cells that did not migrate or invade through the pores in the upper chambers were removed with a cotton swabs and then the lower surface of the filter was stained with 0.1% crystal violet in 20% methanol for 10 min, imaged, and counted using a Qimaging Micropublisher 5.0 RTV microscope camera (Olympus).

Wound-healing assay
Cells were plated into six-well plates and grown to nearly 100% confluence. A same size scratch was made through the cell monolayer using a 200-lL disposable pipette tip. After washing with PBS, fresh culture medium was added and the cells were incubated at 37°C in 5% CO 2 . Wound closure was photographed at 0, 36 or 48 h.

Animal studies
All work performed with animals was approved by the Institutional Animal Care and Use Committee at the Fujian Medical University. For in vivo tumor growth study, five BALB/c nude mice at the age of 4-6 weeks were subcutaneously inoculated with 2 9 10 6 AGS-pDDAH1 and AGS-pCDH cells bilaterally into the left and right flank region. After the tumors were palpable, the tumor volume was determined every 4 days by the formula: length 9 width 2 /2, and then plotted as a function of time to generate the in vivo growth curves. On day 24 after implantation, all animals were euthanized and the tumors were removed, photographed, and weighed. Bold values indicate statistical significance with P < 0.05. For the in vivo metastasis study, 2 9 10 6 AGS-pDDAH1 or AGS-pCDH cells were resuspended in 0.1 mL serum-free Ham's F12 and injected into the tail veins of ten BALB/c nude mice. At 8 weeks postinjection, 5 mice in each group were euthanized and the number of tumor nodules formed on the lung and liver surfaces was counted. Lungs and livers were excised and embedded in paraffin for hematoxylin and eosin staining. The remaining five mice were continuously observed until death was recorded for all the five animals.

Dual luciferase reporter assay
The TCF-responsive luciferase constructs, Top-Flash and its mutant Fop-Flash (Addgene, Cambridge, CA, USA), were used to study b-catenin transcriptional activity. Twelve hours after GC cells were seeded into 24-well plates, the cells were then cotransfected with Top-Flash and pRL-TK (Addgene) report vector or Fop-Flash and pRL-TK report vector. The relative luciferase activity was determined using a dual luciferase reporter assay kit (Promega, Madison, WI, USA). The PRL-TK report vector was used as an internal control.

Statistical analysis
Statistical analysis was performed using SPSS 22.0 for Windows. Student's t-test was used to determine the statistical differences in each two-group comparison. All data were presented as means AE SD from three independent assays. Associations between DDAH1 expression and the clinicopathological parameters of the patients with GC were analyzed using the Pearson v 2 test. The survival curves were plotted using Kaplan-Meier analysis. Differences were considered significant when P < 0.05.

DDAH1 is downregulated in human GC and its low expression correlates with the patients' poor clinical outcome
First, we investigated DDAH1 expression in 150 human GC tissues and the corresponding adjacent normal tissues (NT) using IHC. As shown in Fig. 1A and Table 1, expression of DDAH1 was significantly lower in GC tumors compared with their corresponding adjacent normal tissue. Among 150 pairs of samples, 67.3% of GC tissues showed low DDAH1 expression, whereas only 18.7% of the corresponding adjacent normal tissue expressed the low level of DDAH1. Western blot analysis and qRT-PCR also confirmed a lower expression of both DDAH1 protein (Fig. 1B) and mRNA level (Fig. 1C) in the gastric cancer tissues as compared to the adjacent normal tissues. Correlation analysis of DDAH1 expression with patients' clinicopathological characteristics (Table 2) demonstrated that DDAH1 expression was negatively associated with the presence of lymph node metastasis (P = 0.034) and diffuse classification (P = 0.013), but positively with differentiation status (P = 0.003, Fig. 1D). The rest of pathologic variables including gender, age, tumor size, depth of tumor invasion, clinical stage, lymphovascular invasion and nerve invasion exhibited no significant association with DDAH1 expression. Kaplan-Meier survival analysis showed that patients with low DDAH1-expressing tumors had significantly shorter survival than patients with high DDAH1-expressing tumors (Fig. 1E, median survival time 24.6 months versus 59.4 months). To further ascertain the downregulation of DDAH1 in GC, expression of DDAH1 in seven GC cell lines was examined by western blot analysis. Consistent with the results obtained from primary GC tissues, DDAH1 was downregulated in all the GC cell lines examined (Fig. 1F). Notably, DDAH1 expression level was also positively correlated with the differentiation status of the cells. Taken together, these results indicate that DDAH1 may function as a tumor suppressor and downregulation of it may promote the development and progression of GC.

Effect of DDAH1 on GC cell proliferation, migration, invasion, and metastatic potential
Given the evidence that DDAH1 expression was of prognostic relevance in GC, next we sought to determine the functional role of DDAH1 in GC malignant behaviors both in vitro and in vivo. We used both gene silencing and overexpression strategies to specifically knock down or overexpress DDAH1 in the GC cell lines AGS and MGC803. Stable overexpression or knockdown of DDAH1 in these cells was confirmed by western blot analysis ( Fig. 2A). Overexpression or silencing of DDAH1 did not affect the proliferation rate of AGS or MGC803 cells as evaluated by CCK-8 assay (Fig. 2B,C), either by colony formation assay (Fig. 2D,E) or by soft agar colony formation assay (Fig. 2F,G). Similar to the results obtained from the in vitro growth assays, DDAH1 did not affect the growth of GC xenografts in vivo (Fig. 2H-J).
The effect of DDAH1 on cell migration was compared using a Boyden two-chamber assay in which the cells were induced to migrate through a filter by the presence of FBS on the opposite side. As shown in Fig. 3A, overexpression of DDAH1 suppressed the cell migratory ability, whereas DDAH1 knockdown enhanced the migration. A wound-healing/scratch assay was also used to confirm the changes in cell migration as the extent of wound closure can be taken as a direct measure of cell motility. As shown in Fig. 3B,C, upregulation of DDAH1 remarkably attenuated the wound closure, whereas DDAH1-knockdown cells closed the wound more rapidly than the control cells. The invasive potential of the GC cells was assessed by a modified Boyden chamber invasion assay. Likewise, overexpression of DDAH1 significantly decreased the number of cells that invaded through Matrigel compared with the empty vectortransfected pCDH cells. In contrast, knockdown of DDAH1 in AGS and MGC803 cells led to a significant increase in their invasive ability (Fig. 3D). From the functional studies, we identified that sh2DDAH1 had greater effects than sh1DDAH1 on producing the phenotypic changes; therefore, it was chosen for subsequent experiments.
To determine whether changes in the cell migration and invasiveness detected in vitro could translate into differences in metastatic potential in vivo, 2 9 10 6 AGS-pDDAH1 or AGS-pCDH cells were injected into the tail vein of BALB/c nude mice and evidence of metastasis to the liver and lungs was examined 8 weeks after the injection. As shown in Fig. 3E, the number of nodules formed in the liver of the mice injected with DDAH1-overexpressing AGS-pDDAH1 cells was remarkably less compared to the mice injected with the control AGS-pCDH cells. Although no visible metastatic nodules were observed on the surface of the lungs, the micrometastases in the lungs can be detected by hematoxylin and eosin staining that showed a significant reduction in the lung metastases in the mice injected with AGS-pDDAH1 cells compared with the mice injected with the control AGS-pCDH cells (Fig. 3F). Consistent with the in vivo metastasis data, all the five mice injected with AGS-pCDH cells died within 95 days, whereas 60% of the mice injected with AGS-pDDAH1 survived for that duration (Fig. 3G). Collectively, these results provide compelling evidence for an important role of DDAH1 in GC progression.

DDAH1 suppresses epithelial-mesenchymal transition
EMT is considered as a crucial event in cancer metastasis. To determine whether the observed phenotypic changes in malignant behaviors of the GC cells as a result of DDAH1 alteration were related to EMT, expression of markers associated with EMT was quantified in the GC cell lines by western blot analysis and qRT-PCR. As shown in Fig. 4A-F, overexpression of DDAH1 in AGS and MGC803 cells upregulated the expression of epithelial markers (E-cadherin and ZO-1) but downregulated the expression of both mesenchymal markers (vimentin and N-cadherin) and EMT repressor Snail at both protein and mRNA levels. In contrast, knockdown of DDAH1 produced the opposite effect, while E-cadherin protein level was not detectable in the MGC803 cells. The observation that knockdown of DDAH1 increased both protein and mRNA levels of b-catenin as well as Snail 1 mRNA level further supports the notion that b-catenin and Snail are likely to be downstream targets of DDAH1. To confirm the specificity of the effect of DDAH1 knockdown on the expression of b-catenin and other EMT markers, DDAH1 expression was restored in the AGS-pshDDAH1 and MGC803-pshDDAH1 cells by transfecting them with a pcDN3.1 vector expressing DDAH1 mRNA that had been mutated to prevent the shRNA from causing degradation. As expected, reexpression of DDAH1 in the AGS-pshDDAH1 and MGC803-pshDDAH1 cells reversed the expression of b-catenin and EMT markers to a pattern similar to what was observed in the control cells (Fig. 4B). To extend the findings from DDAH1 knockdown, DDAH1-specific inhibitor PD404182 was also applied to examine the effect of enzymatic inhibition of DDAH1 on the expression of b-catenin and EMT markers. As anticipated, pharmacological inhibition of DDAH1 activity produced essentially the same effects as DDAH1 knockdown (Fig. 4C). Furthermore, immunofluorescent study confirmed that DDAH1 was positively correlated with E-cadherin expression but negatively with vimentin (Fig. 4G). Bright field images of the morphology of DDAH1-overexpressing or DDAH1-knockdown cells demonstrated that knockdown of DDAH1 expression induced morphological changes reminiscent of EMT as the cells lost their epithelial cobblestone-like morphology to acquire a more elongated fibroblast-like shape. In contrast, overexpression of DDAH1 endowed the cells with more characteristic epithelial morphological features (Fig. 4H). These data strongly implicate that EMT process contributes to GC cell migration and invasion upon the loss of DDAH1 expression.

DDAH1 inversely regulates b-catenin expression in GC and mediates EMT through bcatenin
Wnt/b-catenin pathway is well known to play an important role in inducing EMT. To investigate whether DDAH1 affected b-catenin transcriptional activity in GC, Top-Flash reporter assays were used to assess the effect of DDAH1 on b-catenin activity. DDAH1 overexpression significantly reduced the transcriptional activity of b-catenin, while knockdown of DDAH1 increased the activity (Fig. 5A). Then, we used fluorescent microscopy to detect the intracellular localization of both DDAH1 and b-catenin. As shown in Fig. 5B, DDAH1 is distributed diffusely in the cytoplasm only, whereas b-catenin could be found both in the cytoplasmic and in nuclear area. Similarly, overexpression of DDAH1 was accompanied by reduction in nuclear and cytoplasmic b-catenin, whereas silencing of DDAH1 increased nuclear and cytoplasmic b-catenin protein level. Fractionation experiment also confirmed a decrease in b-catenin in the cytoplasm and nuclei of DDAH1-overexpressing cells but an increase in nuclear and cytoplasmic b-catenin in the DDAH1-knockdown cells (Fig. 5C). To further confirm the negative regulation of DDAH1 on b-catenin expression, immunohistochemical staining was performed on the GC tissues. As shown in Fig. 5D, DDAH1 expression was reversely correlated with the expression of b-catenin. Given the firm evidence that DDAH1 reversely regulated b-catenin expression in GC, it is tempting to speculate that one route by which loss of DDAH1 promotes EMT may be through the activation of b-catenin. To explore this, we transiently knocked down b-catenin in the AGS-pshDDAH1 cells and looked for expression pattern changes in those EMT-associated markers. Figure 5E shows that treatment of AGS-pshDDAH1 with b-catenin siRNA effectively restored the expression of epithelial markers (E-cadherin and ZO-1), but markedly reduced the expression of both mesenchymal markers (vimentin and N-cadherin) and EMT repressor Snail as compared with the scrambled siRNAtransfected control cells (Lane 4 versus Lane 3). Such acquisition of epithelial characteristics also translated into less aggressive phenotypes as reflected by decreased cell migration and invasion (Fig. 5F). These data suggest that the effect of DDAH1 knockdown on EMT induction is mediated primarily by the enhanced b-catenin. b-catenin activity in TOP-Flash reporter assay. Expression was normalized with Renilla luciferase activity. The results was performed in three independent experiments (*P < 0.05). (B) Coimmunofluorescence staining showing that nuclear and cytoplasmic b-catenin was negatively correlated with DDAH1 expression. Expression of DDAH1 was shown in green and b-catenin in red. DAPI was used as a nuclear counterstain. Scale bar, 25 lm. (C) Fractionation experiments demonstrated that upregulation of DDAH1 decreased the protein levels of nuclear and cytoplasmic b-catenin in AGS and MGC803, while downexpression of DDAH1 enhanced the nuclear and cytoplasmic b-catenin expression. Nuclear and cytoplasmic protein loading was normalized for equal levels of laminB and b-actin. (D) Relationship between DDAH1 expression and b-catenin cytoplasmic/nuclear accumulation in the 150 GC samples using IHC. Scale bar, 50 lm. In the GC samples with high DDAH1 expression, the percentage of b-catenin-positive expression was 36.7%, significantly lower than those with low DDAH1 expression (63.4%). (E) The DDAH1-silenced stable cells and negative control cells were transfected with b-catenin siRNA or control siRNA. Western blot analysis showed the effect of b-catenin on the expression of EMT markers and Snail in the cells. (F) The stimulatory effect of shDDAH1 on the GC cell migration and invasion was blocked by b-catenin siRNA. The representative images were shown, and the data were expressed as mean AE SD (*P < 0.05; NS, no statistical significance).

DDAH1 regulates b-catenin expression via WNT/GSK-3b pathway
Glycogen synthase kinase-3b (GSK-3b)-dependent control of b-catenin stability or degradation is thought to be the central regulatory mechanism of the canonical Wnt signaling pathway (Clevers, 2006). In the presence of Wnt, GSK-3b is phosphorylated and inhibited, thus preventing targeting of b-catenin for degradation. More intriguingly, Wnt signal is found to be able to inhibit GSK-3b-mediated phosphorylation and consequently enhances Snail protein level leading to EMT in certain cell types (Yook et al., 2005(Yook et al., , 2006Zhou et al., 2004). Therefore, we were interested to determine whether Wnt/GSK-3b pathway is involved in the regulation of b-catenin induced by the loss of DDAH1. We found that knockdown of DDAH1 in the GC cells increased the levels of Wnt1, enhanced phosphorylation of GSK-3b at Ser9, and reduced the phosphorylated forms of b-catenin (Ser33/37/Thr41). Overexpression of DDAH1 displayed the opposite results (Fig. 6A). An important contribution of Wnt/ GSK-3b signaling to regulate b-catenin expression was further supported by the observation that the addition of XAV939 (a WNT inhibitor) into the DDAH1knockdown AGS-pshDDAH1 cells decreased GSK-3b phosphorylation at Ser9, thus increasing the phosphorylated form of b-catenin for degradation to reduce the total b-catenin level (Fig. 6B), which was in accordance with the diminished ability of cells to migrate and invade (Fig. 6C). The aforementioned changes in the signaling molecules and cell migratory and invasive abilities were further confirmed when the AGS cells were treated with DDAH1-specific inhibitor PD404182 rather than shRNAi-knockdown cells (Fig. 6D,E). Conversely, treatment of the DDAH1-overexpressing AGS-pDDAH1 cells with CHIR99021 (a WNT agonist or GSK-3b inhibitor) inactivated GSK-3b by binding to it and subsequently disabled its phosphorylation, which led to diminished b-catenin phosphorylation, therefore restoring the expression of total b-catenin initially inhibited by DDAH1 overexpression (Fig, 6F). In line with this, the migratory and invasive capabilities of the AGS-pDDAH1 cells in the presence of CHIR99021 substantially increased (Fig, 6G). Collectively, these results suggest that the effect of DDAH1 on b-catenin stabilization or degradation is mediated, at least in part, through Wnt/GSK-3b signaling.

Discussion
This study demonstrates that DDAH1 is frequently downregulated in clinical GC samples and its low expression is closely associated with more lymph node metastasis, lower histological differentiation, and poorer clinical outcome. In the experimental systems, forced expression of DDAH1 in the GC cells suppressed cell migration and invasion in vitro as well as metastatic potential in vivo. DDAH1 overexpression inhibited EMT process by increasing b-catenin degradation through the attenuation of Wnt/GSK-3b signaling. In contrast, knockdown of DDAH1 produces the opposite effects. These findings clearly implicate that DDAH1 functions as a tumor suppressor in GC and may be exploited as a prognostic biomarker in GC.
We found that overexpression or knockdown of DDAH1 in AGS or MGC803 cells did not cause any changes in the rate of proliferation in vitro nor the growth rate in vivo. Kostourou et al. (2002) showed that DDAH1 did not influence glioma cell proliferation in vitro. However, there are a few reports demonstrating that DDAH1 could promote glioma growth by angiogenesis in vivo (Boult et al., 2011;Kostourou et al., 2002;Wojciak-Stothard et al., 2007). The discrepancy may be attributed to the different cell types.
EMT is associated with dissemination of cancer, thereby facilitating tumor progression (Brabletz, 2012;Iwatsuki et al., 2010). During EMT, cancer cells decrease cell-cell junctions and gain the mesenchymal characteristics, thus enhancing their abilities of migration and invasiveness (Thiery, 2002;Thiery et al., 2009). In our work, we found that DDAH1 expression was positively correlated with the epithelial markers (E-cadherin and ZO-1) and reversely with the mesenchymal markers (vimentin and N-cadherin). In agreement with this result, DDAH1 has been reported to increase expression of E-cadherin as well as to decrease the levels of a-SMA, collagen I, and that Wnt1, p-GSK-3b (Ser9), and b-catenin inversely correlated with DDAH1 expression, whereas p-b-catenin (Ser33/37/Thr41) positively correlated with DDAH1 expression. The histograms indicate the levels of the Wnt1 protein determined from three independent experiments after normalization to b-actin, and the levels of phosphorylated GSK-3b and b-catenin were normalized to their relative total proteins (*P < 0.05). (B) The stimulatory effect of DDAH1 knockdown on b-catenin expression was inhibited by XAV939 (a Wnt inhibitor). (C) The stimulatory effect of DDAH1 knockdown on cell migration and invasion was diminished by XAV939. The representative images were shown, and the data were expressed as mean AE SD (*P < 0.05; NS, no statistical significance). (D) The stimulatory effect of pharmacological inhibition of DDAH1 by PD404182 on b-catenin expression was suppressed by XAV939 (a Wnt inhibitor). (E) The stimulatory effect of pharmacological inhibition of DDAH1 on cell migration and invasion was diminished by XAV939. The representative images were shown, and the data were expressed as mean AE SD (*P < 0.05; NS, no statistical significance). (F) The inhibitory effect of DDAH1 overexpression on b-catenin expression was reversed by CHIR99021 (a Wnt agonist). (G) The inhibitory effect of DDAH1 overexpression on cell migration and invasion was reversed by CHIR99021. The representative images were shown, and the data were expressed as mean AE SD (*P < 0.05; NS, no statistical significance).