text:
SKLB023 Blocks Joint Inflammation and Cartilage Destruction in Arthritis Models via Suppression of Nuclear Factor-Kappa B Activation in Macrophage

Rheumatoid arthritis (RA) is the most common arthritis and is mainly characterized by symmetric polyarticular joint disorders. Our previous study demonstrated a novel small molecule compound (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-ylidene) methyl) phenoxy) acet-amide (SKLB023) showed potently anti-arthritic effects in a rat arthritis model, however, the underlying mechanisms for this are largely unknown. Both NF-κB and macrophages were reported to play important roles in the pathologic processes of RA. The purposes of this study were to indicate whether NF-κB and macrophages contributed to anti-arthritic effects of SKLB023 in two experimental arthritis models. Our results showed that SKLB023 could significantly improve joint inflammation and cartilage destruction both in adjuvant induced arthritis (AIA) and collagen-induced arthritis (CIA) models. We further found that the binding activation of NF-κB to DNA in joint tissues and RAW264.7 macrophages were suppressed by SKLB023. SKLB023 also inhibited the NF-κB activity in peritoneal macrophages by luciferase assay. Furthermore, the number of macrophages in synovial tissues was decreased after the treatment of different doses of SKLB023. The levels of TNF-α, IL-1β, and IL-6 in plasma, and the levels of TNF-α, NO, and IL-1β in peritoneal macrophages were down-regulated by SKLB023. Finally, SKLB023 attenuated the expression of iNOS and COX-2 in vivo and suppressed the phosphorylations of components of the mitogen-activated protein kinases (MAPKs). These observations identify a novel function for SKLB023 as an inhibitor of NF-κB in macrophages of RA, highlighting that SKLB023 was a potential therapeutic strategy for RA.

Introduction
Rheumatoid arthritis (RA), which afflicts about 1% of the world's population, is a systemic, chronic, autoimmune inflammatory disease that preferentially attacks the synovial lining cells of the joints, destroys local articular structures, and further affects related tissues and organ systems [1,2]. Therefore, therapy to joint inflammation and cartilage destruction is a logical strategy for preventing the progression of RA [3,4].
In RA, the joint synovial tissue is abnormally infiltrated by a variety of specific cells such as macrophage, T cell, B cell, granulocyte, and monocyte [5]. Among these inflammatory cells, macrophages are abundantly present in the inflamed synovial membrane and at the cartilage-pannus junction, contributing to joint inflammation and cartilage destruction in the acute and chronic progression of RA [6][7][8]. The infiltration/activation of macrophages stimulates the over-expression of several proinflammatory cytokines including TNF-a, IL-1b, and IL-6 which are, at least in part, dependent on the activation of NF-kB [9]. Additionally, large numbers of synovial lining macrophages in RA represent a source of nitric oxide (NO) that promote the highly expression of TNF-a production and further induce the incidence of synovitis [10], and the transcription of proinflammatory cytokine genes in lipopolysaccharide (LPS)-stimulated macrophages is usually regulated by the activation of NF-kB signaling pathway [11].
The activation of members of NF-kB transcription family is proven to be a crucial factor for contributing to chronic inflammatory responses in the pathogenesis of RA [12][13][14]. NF-kB is highly activated in synovial tissue of patient with RA [15,16] and NF-kB binds to DNA sequence and further induces the transcription and production of cytokines, chemokines, and inducible nitric oxide [17,18]. Besides, activation of NF-kB usually has great effect on other signal transduction proteins, many of which are relevant to RA, including iNOS, COX-2 and components of the mitogen-activated protein kinase (MAPK) [19,20]. Thus, the modulation of NF-kB pathways may be effective approaches for the treatment of RA and other chronic inflammatory disorders.
In the present study, we demonstrate that systemic administration of SKLB023 effectively ameliorates the chronicity and progression of RA both in AIA and CIA experimental models. The potential mechanisms of SKLB023 in RA may be inhibiting the activation of NF-kB signaling pathways in macrophages.

Ethics and Statement
Animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by our Institutional Animal Care and Use Committee of the Sichuan University in China (IACUC number: 20100318). All surgery was performed under chloral hydrate anesthesia, and all efforts were made to minimize suffering.
Reagents, Cell Line and Cell Culture SKLB023 was prepared as previously described [21]. Indomethacin, LPS (Escherichia coli, O111: B4), and other analytical grade chemicals were purchased from Sigma. RAW264.7 cell line was obtained from the American Type Culture Collection (TIB 71, Rockville, MD, USA). Dulbecco's modified Eagle medium (DMEM), penicillin, streptomycin, and fetal bovine serum were purchased from Gibco Life Technologies (Rockville, MD, USA). RAW264.7 cells were grown in DMEM with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37uC in a humidified atmosphere of 5% CO2 in air.

AIA Model in Lewis Female Rats
Seven-week-old female Lewis rats were purchased from Tengxin Laboratory Animal Co. Ltd. (Chongqing, China). Freund's complete adjuvant (CFA, Chondrex) was prepared at 10 mg/ml by suspending heat-killed Mycobacterium tuberculosis (Mt) H37Ra (Difco, Detroit, USA) in incomplete Freund's adjuvant (IFA, Chondrex). Lewis female rats were injected subcutaneously at the tail base with a 100 ml of CFA emulsion, and randomly divided into five groups with each containing 8 animals. Treatment was initiated on day 14 after the immunization. Different dosages of SKLB023 suspended in 1.0% methylcellulose/PBS (10, 20, 50 mg/kg) were daily administrated by oral gavage (o.g.). The clinical score for the progression of AIA was examined every three day and the scoring for each limb ranged from 0 to 4 (0 = no arthritis; 1 = redness or swelling of one toe/ finger joint; 2 = redness and swelling of more than one toe/finger joints; 3 = involvement of the ankle and tarsal-metatarsal joints; 4 = redness or swelling of the entire paw). The arthritis score was calculated by summing the scores from all paws [22]. Animals were sacrificed on day 28, and the plasma samples were collected and the concentration of TNF-a, IL-1b, and IL-6 was determined using commercial ELISA kits (R&D Systems).

CIA Model in DBA/1J Male Mice
DBA/1J male mice (6-7 weeks old) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). CIA was induced by subcutaneous injection with a 100 mL emulsion which contained 100 mg of bovine CII (Chondrex) and 100 mg of CFA (2 mg/ml) at the base of the tail. On day 21, 50 ml of booster emulsion containing 50 mg of bovine CII and IFA was injected subcutaneously at the tail, but at a different location from the site of first injection. Mice were randomly divided into four groups with each containing 8 animals and then treated with 2.5 mg/kg Indomethacin (Indo), 10 mg/kg, and 20 mg/kg SKLB023, respectively. The treatment continued for 27 days, during which the arthritis index was measured every three days. To determine the arthritis index, each paw was graded on a scale of 0-4 (0 = no visible signs; 1 = edema and erythema of a single joint or digit; 2 = 2 joints; 3 = more than 2 joints; and 4 = severe arthritis of the entire paw). The arthritis score was calculated by summing the scores from all paws [23]. Animals were all sacrificed on day 67, and the plasma samples were collected. The serum concentration of TNF-a, IL-1b, and IL-6 was analyzed using ELISA kits (R&D System).

Histologic Examination and Immunohistochemical Analysis
Both legs and hind paws of Lewis rats and DBA/1J mice were removed, fixed with 4% paraformaldehyde in PBS, decalcified for 15 days with EDTA, and then embedded in paraffin. The paraffin sections were stained with H&E and Safranin O-fast green. For the immunohistochemical analysis, the ankle joints were cut into 5 mm sections and fixed in cold acetone for 20 min. Endogenous peroxidase was quenched with 3% H 2 O 2 for 5 min. Sections were pretreated with 3% goat serum for 1 h at 37uC before the application of primary antibody. Indirect immunoperoxidase staining was performed at 37uC for 1 h. Specimen was treated with anti-CD68 monoclonal antibodies (Cell Signaling Technology, Beverly, MA) to identify the synovial macrophages.
All sections were evaluated histologically by two independent observers. For H&E, the gradation of arthritis was scored from 0 to 4 according to the intensity of lining layer hyperplasia, mononuclear cell infiltration, and pannus formation, as described previously [24]: 0, normal ankle joint; 1, normal synovium with occasional mononuclear cells; 2, definite arthritis, a few layers of flat to rounded synovial lining cells and scattered mononuclear cells; 3, clear hyperplasia of the synovium with three or more layers of loosely arranged lining cells and dense infiltration with mononuclear cells; 4, severe synovitis with pannus and erosions of articular cartilage and subchondral bone. Safranin O staining was scored with a semiquantitative scoring system (0-3), where 0 represents no loss of proteoglycans and 3 indicates complete loss of staining for proteoglycans [25]. For immunohistochemical staining, expression of the CD68 in the synovial tissue of all ankle joints present was scored semiquantitatively on a 5-point scale Significantly less loss of safranin O staining was observed in the animals treated with SKLB023 than in controls, indicating inhibition of cartilage destruction. (D) CD68 expression was measured by immunohistochemistry in rats with AIA. It was scored in a blinded manner on a scale of 0 to 4 as described in Materials and Methods. A statistically significant reduction of CD68 expression was observed in the rats treated with different doses of SKLB023. Error bars represented SEM, **P,0.01, and ***P,0.001 indicated significant differences from the control group. doi:10.1371/journal.pone.0056349.g002 [26]. A score of 0 represented minimal expression, while a score of 4 represented abundant expression of a marker.

NF-kB luciferase Reporter Assay
NF-kB-luc transgenic mice on a Balb/c background (Xenogen Corp., Alameda, CA) were injected with 2-3 ml of 3% thioglycollate and peritoneal exudated cells were harvested after 3 days. Peritoneal macrophages were adhered onto six-well plates (2610 6 cells) for 3 h. Non-adherent cells were removed, and then the attached cells were cultured with LPS (1 mg/ml) alone or with 20 mM of SKLB023 for 10, 30, and 60 min [29]. Activated macrophages were harvested and lysed in lysis buffer, and then lysate was used in accordance with luciferase assay kit (E1500, Promega, Madison, WI).

Isolation of Peritoneal Macrophages and Cytokine Analyses in vitro
BALB/c mice were injected with 2-3 ml of 3% thioglycollate and peritoneal exudated cells were harvested after 3 days. Peritoneal macrophages were adhered onto six-well plates (2610 6 cells) for 3 h. Non-adherent cells were removed, and then the attached cells were cultured with DMEM medium alone or activated with LPS (1 mg/ml) and 20 mM of SKLB023 for18 h. Culture supernatants were analyzed for NO using the Greiss reagent, and for TNF-a, IL-1b, and IL-6 using ELISA kits (R&D Systems, Minneapolis, MN) as recommended by the manufacturer.
Western Blot Analysis RAW264.7 macrophages (ATCC), were cultured in 6-well plates (1610 6 cells/well) overnight, and then incubated with LPS (1 mg/ml) and 20 mM of SKLB023 for 10, 30, and 60 min. Cells were washed with PBS, and lysed in lysis buffer containing 0.1% Triton X-100, 20 mM Tris (pH 8.0), 100 mM KCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail. Total cell lysates were electrophoresed, and transferred to PVDF membrane. The membrane was incubated with antibodies overnight at 4uC, and 1 h at room temperature with anti-rabbit or anti-mouse secondary antibodies linked to horseradish peroxidase. Proteins were visualized using the chemiluminescence system (Bio-Rad) and autoradiography. Proteins extracted from the ankles of AIA and CIA was subjected to

Statistical Analysis
Data are presented as means 6 s.d. for each group. The Mann-Whitney U test was used for comparison of medians from

SKLB023 Ameliorates AIA
To assess different doses of SKLB023 in the development of arthritis, the AIA model in Lewis female rats was initially performed. After the oral administration of SKLB023 at a dosage of 50 mg/kg for two weeks, the arthritic signs characterized by erythematous oedema appearing in limbs and joint swelling were markedly improved ( Figure 1A). The treatment with SKLB023 contributed to a gradual suppression of these histopathological changes, and inhibited the mononuclear cell infiltration and pannus formation in synovial tissues. There was a statistically significant difference in scores for H&E staining between the SKLB023-treated group and control group ( Figure 1B, 1C). Importantly, these treated rats access SKLB023 displayed delayed onset, lower incidence and decreased severity of AIA compared to untreated rats ( Figure 1D). The articular scores, especially at a dose of 20 and 50 mg/kg, were both # 4 at the end point of treatment (both P,0.01).
The degradation and destruction of articular cartilage tissues was assessed by Safranin O-fast green staining, a method to detect the matrix proteoglycan depletion. Proteoglycan loss in the ankle joints of groups treated with SKLB023 was significantly decreased than that of control groups (Figure 2A, 2C), stating that the cartilage profiles in RA have been significantly improved after the daily administration of SKLB023 as illustrated by the increased deep red Safranin O staining in cartilage tissues (Figure 2A).
CD68 is the major phenotype of macrophage-like synovial cells which played a critical role in the progression of RA. Representative analysis of the infiltration of CD68 positive cells in synovial tissue of AIA rats were displayed using immunohistochemical methods ( Figure 2B). Large numbers of CD68 positive cells in the control groups were detected in the lining layers of synovium. In contrast, the number of CD68 positive cells in SKLB023-treated groups was markedly decreased to 52.0% (P,0.01), 43.4% (P,0.001), and 24.9% (P,0.001) at a dose of 10, 20, and 50 mg/kg, respectively ( Figure 2D).

SKLB023 Ameliorates CIA
Previously, 20 mg/kg of SKLB023 exhibited similar activity to 50 mg/kg of that in AIA model. And then, the CIA model in DBA/1J male mice was further evaluated for the effects of SKLB023 on preventing autoimmune arthritis at the dosages of 10, and 20 mg/kg. Representative photographs of the tarsotibial joint swelling of the hind paws from DBA/1J mice were shown in figure 3A. 20 mg/kg/day of SKLB023 inhibited the erythematous oedema in CIA mice, which was comparable to indomethacin at a dose of 2.5 mg/kg/day. H&E staining of the ankle joints indicated that administration of SKLB023 at a dose of 20 mg/kg resulted in a significant suppression of these histopathological changes compared to control group ( Figure 3B, 3C). These SKLB023-treated mice (20 mg/kg) exerted similar therapeutic results to the ones treated with indomethacin (2.5 mg/kg). However, a 10 mg/kg/day dose of SKLB023 reduced the arthritic severity, but did not block the arthritic onset ( Figure 3D). Additionally, the SKLB023-treated CIA mice at a dose of 20 mg/ Representative analysis of the infiltration of CD68 positive cells in synovial tissue of CIA mice were presented in figure 4A. Mice treated with 10, and 20 mg/kg SKLB023 decreased the infiltration of CD68 positive cells to respective 51, and 19% compared to untreated mice, while mice treated with indomethacin only decreased to 62%.

SKLB023 Inhibited Proinflammatory Cytokines Production in vivo
As shown in figure 5A, the serum levels of TNF-a, IL-1b, and IL-6 in AIA rats were significantly increased compared to the control ones while the levels (especially IL-1b) were decreased in a dose-dependent manner after the treatment with SKLB023 (10, 20, and 50 mg/kg). In addition, the plasma concentrations of these proinflammatory cytokines were also effectively down-regulated by SKLB023 and close to the ones of indomethacin in CIA model ( Figure 5B).

SKLB023 Suppressed the Activation of NF-kB Both in vivo and in vitro
To confirm that SKLB023 blocked NF-kB activity in vivo, we carried out electrophoretic mobility shift assay (EMSA) using tissue samples prepared from the ankle joints of the AIA mice. NF-kB DNA-binding activity was decreased in the joints of rats treated with different dosages of SKLB023, in contrast to untreated rats ( Figure 6A).
To determine the effects of NF-kB inhibition by SKLB023 on RA in vitro, we obtained the nuclear extracts from LPS-stimulated RAW264.7 cells in the presence or absence of SKLB023. The EMSA results showed that LPS could significantly increase the NF-kB-DNA binding affinity at 10, 30, and 60 min, whereas the binding activity was suppressed by the pretreatment of SKLB023 at the concentration of 20 mM ( Figure 6B). The densitometry data for both in vivo and in vitro NF-kB-DNA binding affinity were showed in Figure S1. Furthermore, SKLB023 dosedependently inhibited LPS-induced NF-kB transcriptional activity in peritoneal macrophage derived from NF-kB-luciferase transgenic mice ( Figure 6C).

SKLB023 Decreased Proinflammatory Cytokines Production in vitro
Our previous studies indicated that SKLB023 inhibited the production of NO in RAW264.7 cells. In present investigation, we evaluated the in vitro anti-inflammatory effects of SKLB023 on the activation of peritoneal macrophage by ELISA analysis ( Figure 6D). We demonstrated that the inducible production of several important proinflammatory cytokines such as TNF-a, IL-1b, and NO were reduced by SKLB023 in a dose-dependent manner, whereas IL-6 seemed to be not sensitive to SKLB023 in vitro.

SKLB023 Attenuated the Expression of iNOS and COX-2 and the Phosphorylations of MAPKs
As the activation of NF-kB plays a crucial role in LPS-induced transcriptional regulation of iNOS and COX-2 gene expressions in macrophages, we determined whether SKLB023 inhibited the expressions of iNOS and COX-2 proteins in AIA rats. In normal Lewis rats, iNOS and COX-2 proteins were rare and even undetectable. Importantly, SKLB023 ( Figure 7A) dose-dependently blocked the iNOS and COX-2 protein expressions ( Figure 7B), stating that the down-regulation of iNOS and COX-2 proteins by SKLB023 were favorable for the treatment of RA in rats.
Activation of MAPKs pathway is widely involved in aberrant inflammatory responses. Thus, the total expression of MAPKs proteins (ERK1/2, p38 MAPK, and JNK) and their corresponding phosphoproteins (p-ERK1/2, p-p38 MAPK, and p-JNK) were subsequently analyzed by Western blot. Previous report demonstrated that the maximal levels of MAPKs phosphoproteins expressed during 30 min after the stimulation of LPS in human and murine monocytes/macrophages [30]. In our study, RAW264.7 cells were pretreated with SKLB023 at a concentration of 20 mM for 30 min, stimulated by LPS (1 mg/ml, for 0, 5, 10, and 30 min), and then performed for the analysis of MAPKs activation. As depicted in figure 7C, the phosphorylated effects of ERK1/2, p38 MAPK, and JNK were gradually up-regulated by the induction of LPS. However, SKLB023 attenuated the phosphorylations of ERK1/2, p38 MAPK at 5, 10, and 30 min, and of JNK at 10 min, suggesting that SKLB023 attenuated the expression of the phosphorylations of MAPKs.

Discussion
In this study, we have shown that SKLB023 improved the severity of RA in a dose-dependent manner in two animal models of arthritis. We found that SKLB023 decreased the number of macrophages in the synovial tissues and suppressed the activity of NF-kB in AIA rats. We also demonstrated that SKLB023 blocks joint inflammation and cartilage destruction in arthritis models via suppression of nuclear factor-kappa B activation in macrophage.
Activation of macrophages plays a pivotal role in the development and progression of joint inflammation and cartilage destruction in RA. And further these activated cells secreted high levels of proinflammatory cytokines (NO, IL-1b, TNF-a, and IL-6) in the joint synovial tissues of RA [31]. Our studies have shown that SKLB023 suppressed the infiltration of CD68 (macrophage marker) positive cells in ankle joints of AIA rats and CIA mice. Besides, all the serum levels of IL-1b, TNF-a and IL-6 were decreased by the treatment with different doses of SKLB023 compared to those of the control groups in vivo. Thus, SKLB023 could protect joints from inflammation through its influence on macrophages. However, SKLB023 significantly inhibited the expression of IL-1b, TNF-a, NO but not IL-6 in peritoneal macrophages in vitro. This may indicate that SKLB023 inhibited the IL-6 production in a different pathway responsible for the in vivo effects.
NF-kB could regulate cell proliferation, apoptosis, cytokine expression, and metalloproteinase production. In addition, they modulate various genes through both transcriptional and posttranscriptional mechanisms in RA and play key roles in adaptive immune inflammatory responses [32,33]. The functions of NF-kB suggest that the inhibition of NF-kB activity would reduce the production of proinflammatory cytokines and further modulate the related inflammatory reactions [34,35]. Therefore, an alternative targeting to the signal pathways of NF-kB was proposed [36]. In the present study, we found that SKLB023 suppressed the DNA-binding activity to NF-kB in ankle joints of AIA rats. In addition, we demonstrated that the activity of NF-kB also be inhibited by SKLB023 in peritoneal macrophages and macrophage cell line, RAW264.7. Furthermore, Additional activation of p38 and extracellular-signal-regulated kinase mitogen-activated protein kinases (MAPKs) was necessary to trigger destructive inflammation on top of the already active NF-kB [37]. It is interesting that we observed SKLB023 attenuated the phosphorylations of ERK1/2, p38 MAPK, and JNK in RAW264.7. NF-kB is activated in the synovium of patients with RA and regulates genes that contribute to inflammation, including iNOS and COX-2. In our previous study, SKLB023 inhibited the expression of iNOS and COX-2 in vitro. Accordingly, in our present study, we further demonstrated that SKLB023 decreased the expression of iNOS and COX-2 in vivo. These results indicated that SKLB023 not only affected the binding activity of NF-kB to DNA, also the expression of proteins related with NF-kB signaling pathways in RA, which warrant the amelioration of SKLB023 on the RA and other inflammatory diseases.
During the past 15 years, the improved understanding of the pathophysiology of RA has led to a series of dramatic changes in the therapeutic strategies [38]. However, severe side effects have been observed in the long-term administration of many drugs for the treatment of RA [39]. Recently, a better understanding of the signal transduction systems and gene regulation by transcription factors involved in cytokine production has opened the way for the discovery of novel therapeutic compounds useful in treating patients with RA [40,41]. Our experiments shown that inhibition of transcription factor NF-kB in the specific cell type macrophage improved the condition of RA decreased the production of cytokines and the expression of RA related proteins.
In conclusion, inhibition of NF-kB in macrophages by SKLB023 ameliorated the joint inflammation and cartilage destruction in the progression of RA, and it serves as a novel therapeutic strategy for rheumatoid arthritis. Future works are required to identify how SKLB023 inhibits the NF-kB signaling cascade, and how SKLB023 decrease the production of IL-6 in vivo. Figure S1 The densitometry data for NF-kB-DNA binding affinity. (A) The densitometry data for NF-kB-DNA binding affinity in AIA rats. Error bars represented SEM, **P,0.01, and ***P,0.001 indicated significant differences from the AIA rats group. (B) The densitometry data for NF-kB-DNA binding affinity in RAW264.7 cells. Error bars represented SEM, *P,0.05, and ***P,0.001 indicated significant differences from the group treated with LPS alone. (TIF)


extfieldsofstudy: Chemistry Medicine

text:
Identification of Free Nitric Oxide Radicals in Rat Bone Marrow: Implications for Progenitor Cell Mobilization in Hypertension

Nitric oxide (NO) has been implicated in matrix metallopeptidase 9 (MMP9)-dependent mobilization of hematopoietic stem and progenitor cells from bone marrow (BM). However, direct measurement of NO in the BM remained elusive due to its low in situ concentration and short lifetime. Using NO spin trapping and electron paramagnetic resonance (EPR) spectroscopy we give the first experimental confirmation of free NO radicals in rodent BM. NO production was quantified and attributed to enzymatic activity of NO synthases (NOS). Although endothelial NOS (eNOS) accounts for most (66%) of basal NO, we identified a significant contribution (23%) from inducible NOS (iNOS). Basal NO levels closely correlate with MMP9 bioavailability in BM of both hypertensive and control rats. Our observations support the hypothesis that inadequate mobilization of BM-derived stem and progenitor cells in hypertension results from impaired NOS/NO/MMP9 signalling in BM, a condition that may be corrected with pharmacological intervention.

Introduction
Proper endothelial function is crucial for vascular homeostasis. Onset and progression of cardiovascular disease is characterized by an imbalance in the endothelial cell (EC) damage and repair [1]. Lost EC are replaced either by mitotic proliferation of local mature EC and vessel wall-resident stem cells [2], or by the recruitment of endothelial progenitor cells (EPC) from the bone marrow (BM) [3,4]. Given the fact that proliferation of mature EC is fundamentally limited by telomere shortening and cellular senescence [5,6], endothelial monolayer requires constant rejuvenation by EPC. In man, EPC have been defined as circulating CD34 + progenitor cells (PC) expressing the kinase insert domain receptor, a minor cell population that constitutes less than 0.004% of the circulating mononuclear cell fraction [7]. Numerous reports have shown an inverse relation between the number and/or function of circulating CD34 + cells and adverse metabolic and hemodynamic risk factors for cardiovascular disease [8][9][10].
The process of (E)PC mobilization is tightly controlled by activation of matrix metallopeptidase 9 (MMP9; EC 3.4.24.35) via the eNOS/NO/cGMP/MMP9 signaling pathway in BM [11][12][13][14]. Although the relevance of upstream endothelial nitric oxide synthase (eNOS; EC 1.14.13.39) activity for EPC mobilization and function is well-established [15][16][17], unequivocal detection and quantification of basal nitric oxide (NO) production in freshly-isolated BM cells has not yet been reported presumably due to the low concentration (nM range) and short lifespan (,1 ms) of NO radicals in vivo [18]. Furthermore, BM cell suspensions are heterogeneous mixtures of different cell types at various stages of differentiation, of which but a small fraction are capable to produce NO. In the present study, we developed a protocol for electron paramagnetic resonance (EPR) spin trapping of free NO radicals in rat BM cell suspensions. With this technique we confirmed that eNOS is the dominant source of NO in BM. Since hypertension is closely associated with endothelial dysfunction and impaired (E)PC mobilization [19], we compared NO level in the BM of normotensive and hypertensive rats. Further, with an adapted assay for human MMP9 activity [20], we quantified MMP9 bioavailability in these samples. Several publications [16,17] have reported improved (E)PC mobilization and function after prolonged pharmacological enhancement of eNOS expression with AVE9488. The authors proposed that impaired function and mobilization of (E)PC correlates with NO levels in the BM.
Here we confirm such close correlation between NO production and MMP9 levels in the BM of hypertensive as well as normotensive rats.

Materials and Methods
Deoxycorticosterone acetate-salt model of hypertension The experimental protocol was conducted in accordance with institutional guidelines and approved by the Ethics Committee on Experimental Animal Welfare of Maastricht University (The Netherlands). To examine the effect of hypertension on NO production in BM we used 8 male and 4 female outbred Wistar rats purchased from Charles River (The Netherlands). Upon arrival, 9 weeks old animals with a body weight ranging from 226 to 250 gram were housed in groups of 2 animals per cage. Before experimentation, rats were allowed to acclimatize to the animal room for 7 days under controlled temperature and humidity conditions with an alternate 12:12 hr light-dark cycle. During the experiments rats had free access to standard rat chow ssniff R/M-H (ssniff Spezialdiä ten GmbH, Germany).
All rats underwent left unilateral nephrectomy and, after 1 week of recovery, a silicon pellet (Sylgard 184 silicone elastomer base; Dow Corning, Midland, MI, US) containing 100 mg of 11-Deoxycorticosterone acetate (DOCA; Sigma, Germany) was implanted subcutaneously. Both procedures were performed under 1.5-4.0% isoflurane (IsoFlo; Abbott) anesthesia. The rats had ad libitum access to high-salt drinking water containing 0.9% NaCl (Sigma, Germany) and 0.2% KCl (Sigma, Germany) throughout the course of the experiment. Corresponding control rats were implanted with a subcutaneous silicon pellet without DOCA and these rats received regular tap water to drink. To record mean arterial pressure (MAP), after 6 weeks, heparinized (5 U/ml; Leo pharmaceuticals, Denmark) indwelling polyethylene catheters were introduced in a femoral artery and advanced into the lower abdominal aorta under 3-4% isoflurane anesthesia. After 2 days of recovery, the arterial catheter was connected to a pressure transducer (Micro Switch 150 PC) and its output was sampled at 2.5 kHz. MAP was derived from these signals using the IDEEQ data-acquisition system (instrument services, Maastricht University). Afterwards animals were sacrificed under 3-4% isoflurane anesthesia by exsanguination and the femurs, humeri and tibias were taken from each rat to isolate BM cell suspensions for analyses of NO and MMP9.

BM cell isolation and treatment
BM cells were harvested at atmospheric oxygen level (ca 21%) by flushing isolated rat limb bones with warm (37uC) 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Sigma, Germany) buffer (pH 7.4) supplemented with 5 mM glucose (LUMC pharmacy, the Netherlands) and 2.5 mM CaCl2 (Sigma, Germany) using a syringe. The six limb bones of a single rat typically delivered 400 (3846169) million white blood cells (WBC) as counted with a Beckmann Coulter Ac T system. The BM cell suspension from every rat was split into several different samples and used for MMP9 and NO detection after cell treatment with or without NOS activators and/or inhibitors.
Identification of NOS isoforms that contribute to NO production in the BM of Lewis rats The experimental protocol was conducted in accordance with institutional guidelines and approved by the Ethics Committee on Experimental Animal Welfare of Leiden University Medical Center (The Netherlands). To identify the contribution of the different NOS isoforms to BM-derived NO production we used inbred Lewis rats (Harlan Laboratories, Boxmeer, The Netherlands). Animal characteristics, routine housing conditions and the harvesting of BM cells were performed as described above.
We applied the selective NOS inhibitor (N-(3-aminomethyl)benzyl)acetamidine (1400 W, Alexis Biochemicals, USA) at 2 concentrations: 0.1 mM -for inhibition of inducible NOS (iNOS) only, 10 mM -for inhibition of iNOS and neuronal NOS (nNOS) simultaneously. The effect of 1400 W on NO production in the BM was investigated in triplicates.
In appropriate cases, constitutive NOS isoforms (eNOS and nNOS) were activated with 10 mM calcium ionophore A23187. Alternatively, 500 mM L-NAME was used to inhibit all NOS isoforms.

Spin trapping of NO in BM suspensions
NO trapping in BM cell suspensions was done according to the protocol previously published [21]. It involves addition of Fe 2+citrate (10 mM final; FeSO 4 and trisodium citrate, Sigma Aldrich, Germany) and sodium diethyldithiocarbamate (DETC, 2 mM final, Sigma Aldrich, Germany) to produce the lipophilic Fe-DETC complex that traps the free NO radicals.
After 30 min incubation at 37uC under an atmospheric oxygen level and 5% CO 2 , the suspension was placed on ice to terminate enzymatic NOS activity. The paramagnetic trapping adducts are mono-nitrosyl-iron complexes (MNIC, or NO-Fe 2+ -DETC) and located in the apolar cellular fraction that was harvested by 10 minutes of centrifugation at 4uC and 3006g. The pellet with the cellular fraction was resuspended in strong HEPES buffer (150 mM, pH 7.4), topped up to a final volume of ca 400620 ml and snap frozen in liquid nitrogen as a small frozen column of 4.8 mm diameter for EPR assay.
The EPR spectra were measured on a modified X-band ESP300 spectrometer (Bruker BioSpin, Rheinstetten, Germany) operating with 20 mW microwave power. These frozen samples were carefully centered in a Bruker ER4103TM cylindrical cavity operating in TM 110 mode with unloaded Q,10.000. The sample temperature was kept at 77uK with a quartz liquid finger Dewar filled with liquid nitrogen. The magnetic field was modulated at a frequency of 100 kHz with 5 G amplitude. The detector gain was 2610 5 , time constant 82 ms, and ADC conversion time 82 ms. Up to four field sweeps were accumulated to improve signal to noise. With these spectrometer settings, the detection limit was ca 10 pmol MNIC [21]. The MNIC yields in the tissue samples were quantified by comparing the EPR intensity of the triplet structure with that of frozen reference samples of paramagnetic NO-Fe 2+ -MGD complexes (10 mM) in PBS buffer. This comparison was done at identical temperature using the same hardware, microwave power and spectrometer settings (except for amplifier gain). This procedure affords an accuracy of the MNIC yields of ca 10%.
The sample reduction with dithionite (50 mM) was required [22] to suppress the signals from Cu 2+ -DETC complexes, that are usually visible in EPR spectra of biological samples. In unreduced state, these signals dominate and overlap with the EPR spectrum of the true MNIC, thereby obscuring its observation [23]. The reduced tissues show undistorted spectra of NO-Fe 2+ -DETC with their characteristic triplet, which is possible to quantify. Given our modest MNIC yields, reduction with dithionite was absolutely required for quantification of MNIC in BM.

MMP9 activity assay
To measure MMP9 activity in the supernatant of BM cell suspension we developed a rat-specific assay that allows us to quantify total as well as active MMP9. The assay was adapted from a human-specific MMP9 immunocapture activity assay [20] by modifying the MMP9 standard and optimization of its activation by p-aminophenylmercuric acetate (APMA). It has a detection range from 4 to 16.000 pg MMP9 per ml.
In short, rat BM supernatants were incubated overnight with anti-MMP9 antibodies (S22-2, QuickZyme Biosciences, Leiden, The Netherlands), coated to a high binding 96-well plate (polystyrene stripwell plate; Corning B.V. Life Sciences, The Netherlands). To detect total MMP9 activity, bound MMP9 was activated with 0.5 mM APMA at 37uC for 4 hours. As a standard, serial dilutions ranging from 0 to 16 ng/ml of recombinant rat pro-MMP9 (R&D systems, Minneapolis, USA) were used and activated in parallel with rat BM samples. Active MMP9 was detected with a colorimetric enzymatic assay using the modified enzyme Ukcol and the chromogenic peptide substrate S-2444 [20]. The resultant color was read at 405 nm in a microplate spectrophotometer immediately and every 10-20 minutes during the next 4 hours, while the plate was incubated at 37uC. The absolute concentration of MMP9 in a sample was determined by interpolation from a calibration curve.

Statistical analysis
All values are presented as mean with standard deviation. Treated and untreated groups were compared using the nonparametric Mann-Whitney U test, as appropriate for skewed data. Correlations were analyzed by Spearman's correlation method. All statistical calculations were performed using GraphPad Prism software (La Jolla, California, USA). P#0.05 was considered to denote statistical significance.

Enzymatic generation of NO is detectable in rat BM
NO radicals are implicated in many (patho)physiological processes, but hard to detect in living tissues due to low concentration and short lifespan. Therefore, most studies use downstream metabolites like NO 2 2 (Griess assay), NO 2 (chemiluminescent assay) or N 2 O 3 (diaminofluorescein assay) as experimental proxies for 'true' NO [24]. In biological systems the latter may be detected with EPR spectroscopy [25] and spin trapping with Fe-DETC complexes, as this affords selectivity for detection of free NO radicals.
We used this method to quantify NO levels at 37uC in buffered neutral solution (pH = 7.4) open to ambient air. Such conditions preclude potential artefactual NO release from S-nitrosothiols, endogenous nitrite anions and organic nitrates [21,26]. After 30 minutes of trapping in freshly isolated BM cell suspensions, samples were snap frozen and the trapping yield of MNIC was quantified with EPR spectroscopy at 77uK. Figure 1A shows the X-band EPR spectrum of a BM sample (100610 6 WBC) collected from a DOCA-treated Wistar male rat and stimulated for NO production with Ca-ionophore A23187. This EPR spectrum is quantified as 60 pmol MNIC and unambiguously identified from its characteristic triplet lineshape, g-factor (g = 2.035) and triplethyperfine coupling constant A N 14 = 12.6G. Figure 1B shows the spectrum of a strong calibration sample containing circa 4 nmol MNIC.
To confirm that detected NO is a result of activity of NOS enzymes, we quantified MNIC in BM samples of Wistar rats (5362610 6 WBC per sample) in the presence and/or absence of NOS activators and inhibitors ( Figure 2). In absence of any stimulus, a basal yield of 2162 pmol MNIC is visible above the instrumental noise with a S/N ratio of 2-3. Upon stimulation with 10 mM ionomycin or 10 mM A23187, that increase intracellular calcium levels and activate the constitutive eNOS and nNOS isoforms [27], MNIC yields were raised to 4564 and 4965 pmol respectively. Moreover, in presence of 500 mM L-NAME, a nonselective NOS inhibitor, MNIC yields remained below the detection threshold of 10 pmol both in the absence and presence of Ca-ionophore. These experiments demonstrate conclusively that the formation of MNIC adducts in rat BM arises from enzymatic arginine-citrulline conversion by NOS. The specificity of Fe-DETC complexes for free NO radicals affords the first direct confirmation of the production of NO radicals per se in BM.

Endothelial NOS is the dominant source of NO in BM
Next, we aimed to define the identity of the NOS isoforms that contribute to the basal NO production in rat BM. Along with eNOS, at least two other NOS isoforms could potentially contribute NO in the BM-iNOS and nNOS. Unfortunately there are no 100% isoform-specific NOS inhibitors. Among others, 1400 W stands out as highly isoform-selective NOS inhibitor that can be used in vitro and in vivo, with K i values of 0.007 mM, 2 mM, and 50 mM for purified human iNOS, nNOS, and eNOS respectively [28]. In rat aortic rings, 1400 W has been found to be at least 1,000-fold more potent against iNOS compared to eNOS [28]. Therefore, we used 1400 W for an identification of active NOS isoforms in rat BM.
Since the MNIC yields in BM cell suspension of 5362610 6 WBC had S/N ratios of 2-3, we decided to increase the sample size to the equivalent of 100610 6 WBC per sample and as a result, the MNIC spectra became more apparent. Also, given the smaller standard deviation for an inbred over outbred rat strain, we used Lewis rats to investigate the NOS isotype-specific contribution to NO production in BM.
We examined MNIC yields in BM suspensions of Lewis rats at [1400 W] = 0.0 (basal), 0.1 and 10.0 mM. The results of three independent experiments show that without stimulation or inhibition, the basal NO production was 3866 pmol MNIC per 10 8 cells. We note that this Lewis yield is in good agreement with 2162 pmol MNIC per 5362610 6 cells previously found in Wistar rats. This basal yield was taken as 100% ( Figure 3). Pretreatment of BM cells with 0.1 mM 1400 W reduced MNIC yield to 7761% of basal. As only iNOS is inhibited by this low concentration of 1400 W, we estimate that iNOS contributes to approximately 23% of baseline NO levels. At [1400 W] = 10 mM, we find a further loss of 1162% in MNIC yield, attributable to nNOS. Finally, the addition of 500 mM L-NAME completely abolished MNIC. Based on these yields, we conclude that the majority (circa 66%) of basal NO derives from eNOS, with smaller but nonnegligible contributions from iNOS and nNOS.

NO production in BM is affected by peripheral hypertension
Hypertension is strongly associated with chronic inflammation, oxidative stress and endothelial dysfunction in both humans and rats [29,30]. Moreover, in both these species, circulating levels of EPC are reduced suggesting impaired BM (mobilization) function [19]. Therefore we tested whether peripheral hypertension might affect the eNOS and iNOS activity in BM and modify the NO bioavailability there. To this end, we induced hypertension in Wistar rats by unilateral nephrectomy followed by chronic leaching of DOCA-salt from an implanted silicon pellet [31,32]. After 6 weeks of DOCA-treatment, MAP was increased to levels seen in severe peripheral endothelial dysfunction [33,34] (table 1). In all control rats, the MNIC yields in the BM were above 20 pmol and clearly detectable with an average value of 38616 pmol MNIC per 10 8 cells. The MNIC yields in the BM of DOCA-hypertensive rats were lower and harder to quantify. In two out of six cases MNIC yields remained below the detection limit of 10 pmol. In other BM samples of hypertensive rats MNIC yield exceeded 20 pmol with a mean value circa 2967 pmol per 10 8 cells. With MNIC yield near the EPR detection limit and considerable variation between individual Wistar rats (an outbred Figure 2. The effect of NOS inhibitors and/or activators on NO level in BM of Wistar rats. X-band EPR spectra at 77uK of frozen BM cell suspensions from Wistar rats after 30 min of NO trapping with Fe 2+ -DETC. The samples (5362610 6 BM cells) were reduced by 50 mM dithionite to remove the overlapping signal from paramagnetic Cu 2+ -DETC. A basal yield of 2162 pmol MNIC is obtained in absence of stimulus (top), clearly recognizable by its characteristic hyperfine triplet structure at g = 2.035. Stimulation with ionomycin or A23187 calcium ionophores raises the MNIC yields to 4564 and 4965 pmol. In presence of the NOS inhibitor L-NAME, the MNIC yields remained below the detection threshold of ca 10 pmol. The EPR spectra show significant background signals not related to MNIC, as visible in the bottom spectra. Such background signals derive from paramagnetic iron centers, and some residual Cu 2+ -DETC as commonly found in biological samples [18]. doi:10.1371/journal.pone.0057761.g002 Figure 3. Relative contribution of NOS isoforms to basal NO production in BM of Lewis rats. The basal NO level that was obtained in absence of a stimulus was taken as 100%. Contribution of the NOS isotypes was determined using two concentrations of the NOS isoform-specific inhibitor 1400 W. 1400 W was used at final concentrations of 0.1 mM for iNOS inhibition and 10 mM for inhibition of both iNOS and nNOS. With L-NAME (500 mM, final column), the signal intensity was below the detection threshold. Values represent biological triplicates. doi:10.1371/journal.pone.0057761.g003 strain), the averaged NO yields present with large error margins. These large margins make NO levels in BM of DOCA group not significantly different from those in the control group.
As shown in table 2, basal MNIC yields from inbred Lewis and outbred Wistar rats are in full agreement, with smaller standard deviation for the genetically homogenous inbred strain. This result confirms that EPR dosimetry can be used for the quantification of NO provided MNIC yields exceed ca 20 pmol.
Interestingly, pre-incubation with Ca-ionophore A23187 raises BM-derived NO levels about twofold in both strains (Wistar DOCA 60624 pmol MNIC per 10 8 cells; Wistar controls 61627 pmol MNIC per 10 8 cells; Lewis 6567 pmol MNIC per 10 8 cells; tables 1 and 2). Taken together, these data suggest that hypertensive NO deficiency may reflect differences in posttranslational regulation of NOS rather than differences in NOS expression per se.

NO levels in rat BM are physiologically relevant
Given the importance of the eNOS/NO/MMP9 signalling cascade for (E)PC mobilization from BM [11,13,35], we raised the question whether NO deficiency in the BM of hypertensive rats reflects in activity of downstream regulatory proteins like MMP9, the presumptive rate limiting factor for (E)PC mobilization. Adapting a human MMP9 activity assay to rodent biological fluids/materials, we quantified MMP9 in BM of hypertensive and control rats as 0.960.4 mg per 10 8 cells and 1.160.6 mg per 10 8 cells, respectively. Given the large errors, these averages appear not statistically different (p = 0.41). However, the very tight correlation between MMP9 and MNIC in individual rats (Figure 4, Spearman rho = 1.000 and p,0.05) corroborates our hypothesis that inadequate (E)PC mobilization in hypertension results from compromised NOS activity.

Discussion
Here, we give the first confirmation of the enzymatic release of free NO radicals in rat BM. Our data show that in a healthy state eNOS is a dominant source of NO in BM. Additionally, in BM of experimental hypertensive rats the NO level is lower than in healthy controls and directly relate to the production of MMP9, supporting observations that eNOS deficiency impairs the (E)PC mobilization.
EPR is widely used in biosystems for detection and quantification of paramagnetic complexes with trapped NO radicals. At the same time, the EPR spectra of frozen mammalian tissues are usually dominated by Cu 2+ -DETC complexes, which overlap with MNIC, obscuring the latter [23]. Reduction with dithionite allowed us to suppress the Cu 2+ -DETC signal and make MNIC spectra visible [22]. In addition to removing the overlapping Cu-DETC signal, reduction with dithionite is known to enhance MNIC yields in vivo and in vitro, thereby enhancing the sensitivity of the NO trapping considerably [36,37].  A paramagnetic MNIC adducts in living tissues is usually taken as evidence for free NO radicals. This is not strictly true, since endogenous S-nitrosothiols, nitrite and organic nitrates could be an alternative non-enzymatic sources of NO [18].
There are several mechanisms of free NO release from endogenous S-nitrosothiols that can occur in vitro. For example, decomposition of S-nitrosothiols into NO and a thiyl radical occurs spontaneously by thermolysis or photolysis, or by the catalytic activity of reduced transition metal ions like Cu + or Fe 2+ [18]. Alternatively, high concentrations of low molecular weight Snitrosothiols may even nitrosylate iron complexes like the Fe-DETC traps used here, by direct transfer of the NO-moiety [38]. However, at physiological S-nitroso levels, the reaction rate of such transnitrosation or decomposition is six orders of magnitude smaller than the trapping rate of free NO radicals and it makes this alternative pathway of MNIC formation negligible in living tissues [18].
Again, endogenous nitrite anions are prone to be reduced to free NO under hypoxic conditions by a plethora of ubiquitous mammalian enzymes like xanthine oxidase or cytochrome P450 [26]. Therefore, we should carefully consider such alternative pathways for release of NO and formation of MNIC.
It is known that DETC ligands may cause confounding artefacts by inhibiting NFkB and Cu-Zn-SOD. As discussed at length [36], such artefacts are not significant under the conditions used in this work.
In our case it is justified to attribute the observed MNIC yields to free NO radicals deriving from enzymatic NOS activity in the BM suspension. First, the NO trapping experiments were carried out under normoxic conditions, so that the hypoxic pathways described above cannot operate. Second, our experiments were performed in buffered neutral solutions (pH = 7.4), where spontaneous acidic decomposition of nitrite cannot occur. In addition, the complete abolition of MNIC by NOS inhibitors confirms that such artefactual pathways do not generate significant quantities of MNIC in our experiments. Therefore, we attribute the formation of detected MNIC adducts unequivocally to arginine-citrulline conversion by NOS.
Such sensitivity and specificity of MNIC for NO makes the EPR trapping technique uniquely suited to assess endogenous NO levels in vivo or in freshly isolated tissue samples as BM.
As a cautionary note, we apprise that the MNIC should not be equaled with NO production, since only a minor fraction of NO is trapped by Fe-DETC and the majority of NO is lost via other reaction pathways. Therefore, the MNIC yield is only a firm lower bound for the true NO yield. In practical biological settings of laboratory animals or cell cultures, only about 10% to 20% of NO gets trapped into MNIC [18].
Next to a first direct confirmation and quantification of NO production in freshly isolated BM, we found that the basal, i.e. unstimulated, NO levels in inbred Lewis rats (n = 3, 3866 pmol MNIC per 10 8 cells) are identical to those in outbred Wistar rats (n = 6, 38616 pmol MNIC per 10 8 cells), with a smaller standard deviation for the genetically identical Lewis rat strain. In the presence of a Ca-ionophore, NO production almost doubled in both rat strains (Table 2). These findings appear plausible and attest to the reliability of NO trapping.
To better appreciate the magnitude of MNIC production in BM suspensions, we compared it with MNIC yields in two types of cultured EC. As shown in Table 2, non-stimulated murine microvascular brain EC (bEnd.3 cells) release circa 14706150 pmol MNIC per 10 8 cells [21]. We find that freshly isolated and cultured primary human umbilical vein EC (HUVEC) release fourfold less NO. This is consistent with previous observations that the bEnd.3 cell line expresses relatively high levels of eNOS and displays a copious basal release of NO [21]. Nevertheless, the fact that the BM suspensions, although heterogeneous mixtures of WBC, hematopoietic cells at various stages of differentiation, adipocytes, fibroblasts and EC, still achieve nearly 10% of HUVEC yields, indicates that basal BMderived NO levels are quite significant.
Based on the calcium dependence and the selective sensitivity for the isotype-specific NOS inhibitor 1400 W, we conclude that eNOS is the major contributor (66%) to the NO production of BM cell suspensions.
Inducible NOS produces NO at a high rate in a calciumindependent manner and is conventionally thought to be expressed in response to inflammatory mediators [39]. However, we found that iNOS is responsible for nearly one quarter (23%) of basal NO production in rat BM. This serendipitous finding might surprise at first, however a substantial basal iNOS activity in BM is not implausible since previous studies identified iNOS RNA in bone and in BM-derived PC even in the absence of inflammation [40,41]. It was reported that normal osteoblast maintenance requires some degree of iNOS activity [42] and therefore expression of iNOS in BM cell suspension is expected. Our experiments confirm that iNOS contributes significantly to basal NO levels in BM, even in the absence of inflammation.
Given the dominant contribution of eNOS to basal NO production in rodent BM and the reduced amount and/or function of (E)PC in hypertension or other cardio-vascular disease [8,10,16,17] we expected to detect reduced eNOS derived NO production in the BM of DOCA-hypertensive rats. On the other hand, hypertension is known to be associated with a chronic inflammatory state [43] inducing an oxidative stress that might affect the intracellular redox environment [8] and production of iNOS-derived NO. Our data confirmed that the BM of DOCAhypertensive rats produces lower MNIC yields than the BM of control rats. This supports the hypothesis that, in patients at risk for cardiovascular disease such as hypertension, peripheral endothelial dysfunction extends to the bone marrow and may result in reduced NO bioavailability and, consequently, impaired MMP9-dependent (E)PC mobilization [11,19]. Indeed, in the individual BM samples we found a tight correlation between NO and MMP9 (Figure 4), the upstream and downstream mediators of BM cell mobilization, respectively.
Finally, the fact that Ca-ionophores elevate NO levels even in the BM of hypertensive rats provides proof of principle that eNOS activity may be recovered by pharmacological intervention.
We strongly believe that detection of NO by EPR may serve as a highly valuable tool to assess the effect of therapeutic strategies aimed at correcting NOS function and (E)PC mobilization from BM in experimental animal models of cardiovascular disease. Furthermore, our approach for NO detection may be more broadly applicable as the NOS/NO/sGC/cGMP/PKG/MMP9 pathway is active in other organs and tissues and involved in (patho)physiological processes such as ischemia/reperfusion injury, organ fibrosis or synaptic plasticity in the nervous system.

Innovation
With NO spin trapping, we demonstrate significant release of NO radicals in BM suspensions of rats. The NO production reflects enzymatic activity of NO synthases, with significant contributions from iNOS even under non-inflammatory conditions. We observe a close correlation between NO and MMP9 in BM, proving the principle that MMP9 levels may be modified by upstream pharmacological intervention in the NOS/NO/MMP9 pathway. Given the role of MMP9 in mobilization of hematopoi-etic stem and (endothelial) progenitor cells, our findings confirm NO levels in the BM as a potential modifiable upstream target to promote E(PC) mobilization in cardiovascular disease.


extfieldsofstudy: Biology Medicine

text:
Revolving SEM images visualising 3D taxonomic characters: application to six species of the millipede genus Ommatoiulus Latzel, 1884, with description of seven new species and an interactive key to the Tunisian members of the genus (Diplopoda, Julida, Julidae)

Abstract A novel illustration technique based on scanning electron microscopy is used for the first time to enhance taxonomic descriptions. The male genitalia (gonopods) of six species of millipedes are used for construction of interactive imaging models. Each model is a compilation of a number of SEM images taken consecutively while rotating the SEM stage 360°, which allows the structure in question to be seen from all angles of view in one plane. Seven new species of the genus Ommatoiulus collected in Tunisia are described: Ommatoiulus chambiensis, Ommatoiulus crassinigripes, Ommatoiulus kefi, Ommatoiulus khroumiriensis, Ommatoiulus xerophilus, Ommatoiulus xenos, and Ommatoiulus zaghouani spp. n. Size differences between syntopic adult males of Ommatoiulus chambiensis and Ommatoiulus xerophilus spp. n. from Châambi Mountain are illustrated using scatter diagrams. A similar diagram is used to illustrate size differences in Ommatoiulus crassinigripes, Ommatoiulus khroumiriensis spp. n. and Ommatoiulus punicus (Brölemann, 1894). In addition to morphological differences, the latter three species display allopatric distribution and different habitat preferences. A dichotomous interactive key with a high visual impact and an intuitive user interface is presented to serve identification of the 12 Ommatoiulus species so far known from Tunisia. Updates on the North African Ommatoiulus fauna in general are presented.

As useful as gonopod illustrations are for taxonomic descriptions, they can sometimes be grossly misleading and result in misidentification and production of synonymic names, the Achilles' heel of descriptive taxonomy. There are several examples of this in the literature about millipedes, but most striking is perhaps the case presented by Hauser (2000) who demonstrated that amongst 11 subspecies and 100 varieties described for Craspedosoma alemannicum Verhoeff, 1910(see Schubart 1963a, based on differences in the length of podosternite (posterior gonopods) processes, only 9% are valid while the rest can be discarded. The reason for this is that the 'heterodactyly' on which Verhoeff (1915Verhoeff ( , 1916Verhoeff ( , 1917Verhoeff ( , 1939 based his infraspecific Craspedosoma taxonomy and which was subsequently adopted by most authors studying the taxonomy and ecology of the genus, was simply due to observation error. When the podosternite of Craspedosoma is viewed from varying angles, the relative lengths of its processes change (see Hauser 2000, figs 1, 2, 3).
Useful taxonomic characters in Ommatoiulus are almost exclusively derived from the gonopods. Differences between species are often subtle, and the pronouncedly "3D" nature of the gonopods makes recognition of the differences difficult. In many older papers dealing with Ommatoiulus taxonomy, authors have dissected the different parts of the gonopods and have illustrated them separately which has led not only to "angle-of-view" problems, but also to difficulties of relating the various gonopod components spatially to each other. By applying the novel imaging technique we have overcome these problems.

The study group: Ommatoiulus millipedes from Tunisia
Ommatoiulus is the dominant genus of julid millipedes in North Africa and the Iberian Peninsula. A total of 70 species have been described so far, and many more remain to be recognised and named. For example, Akkari and Enghoff (2012) recorded 19 Ommatoiulus species in the southernmost Spanish region, Andalusia, 10 of which they described as new. These authors further provided a historical overview tracing the general inconclusive taxonomic situation and gave an updated definition of the genus based on morphological characters and a key to the 19 Andalusian species which they estimated to constitute at most 1/6 of the total species richness for the genus. In spite of the wide distribution of a few species, e.g. O. sabulosus (Linnaeus, 1758) reaching 64°N in Fennoscandia, and O. moreleti (Lucas, 1860) with a near-cosmopolitan, synanthropic distribution, most Ommatoiulus species are confined to the Mediterranean region of North Africa and Iberia, and tend even to display small-scale endemism. For instance, of the 19 species recorded from Andalusia, only five were found in other areas (Akkari and Enghoff 2012).
North African species of the genus were examined in considerable detail by several authors, especially Brolemann (1921Brolemann ( , 1924Brolemann ( , 1925a and Schubart (1952Schubart ( , 1960Schubart ( , 1963b. Recent studies have mostly targeted the Tunisian fauna, describing new species (Akkari andVoigtländer 2007, Akkari and and in some cases detailing some aspects of developmental modalities (Akkari and Enghoff 2011). Akkari et al. (2009) presented detailed species accounts and new records from Tunisia in addition to a complete bibliographical review of the order Julida in North Africa, listing 24 Ommatoiulus species for the region.
Despite these contributions, the North African Ommatoiulus fauna is far from being thoroughly assessed, nor is its taxonomy close to being fully revised. Without doubt, numerous new species still await discovery, and several taxonomic questions still remain unsolved, such as the correct placement of the highly deviating species O. lapidarius (Lucas, 1846), type species of the subgenus Apareiulus Brölemann, 1897 (e.g., Attems 1952, Schubart 1963b. The same applies to the O. punicus species group established to facilitate understanding species affinities (Akkari and Voigtländer 2007) but which, in the light of recent revisionary work on the genus (Akkari and Enghoff 2012), might not reflect true relationships. To solve these questions an exhaustive revision of the genus in this area is needed.

Material and methods
Most specimens were hand collected during spring 2008 by N.A. and P.S. Supplementary material was obtained from museum collections. All studied specimens are preserved in 70% alcohol. Measurements were made using a Leica Wild M10 microscope equipped with an ocular micrometre. Photographs were taken using Visionary Digital's BK Plus Lab with a Canon EOS 7D. For scanning electron microscopy, parts of the specimens were cleaned with ultrasound, transferred to 96% ethanol then to acetone, air-dried, mounted on adhesive electrical tape attached to aluminium stubs, coated with platinum/palladium and studied in a JEOL JSM-6335F scanning electron microscope. Photographs were processed with a Leica Application Suite program and final stacking made with Zerene Stacker 1.04. The rotatable images were constructed from 18 SEM images taken at 20 degrees intervals starting from the mesal view and continuing until all 360 degrees were captured by rotating the SEM stage. The images were processed using Adobe Lightroom 4.3 by adjusting the black, highlight and white levels to achieve a uni-form background and contrast. Each image was then cropped to ensure a smooth transition between each frame during rotation. The images were imported into Adobe Flash CS5, where each image was made into a single frame and the series combined to form a rotating animation. The animation controls (moving from one frame to the next) were mapped to the mouse cursor using Actionscript 3.0. The html version available online was compiled using Magic 360. The interactive key was developed in Adobe Flash CS5.5 using Actionscript 2.0 to handle screen transitions and image swapping. Plates were assembled using Adobe Indesign CS 5.5. Respective image libraries of the interactive rSEM have been deposited in MorphBank. More details on the method of creation of the interactive models can be found in Cheung et al. (2013). The number of body rings is given as recommended by Enghoff et al. (1993): Number of podous rings (PR) + number of apodous rings (AR) + telson (T). The developmental stadium of a number of individuals was taken as being represented by the number of vertical rows of ocelli (RO). The real stadium number in julid millipedes has been shown to equal the number of RO+1 (Enghoff et al. 1993), e.g., a specimen with 9RO belongs to developmental stadium 10.
Diagnosis. Most similar to O. xerophilus sp. n., but easily distinguished by the shape of promerite and the presence of a distal notch and a small pointed process on solenomerite.
Etymology. Named after the type locality. Châambi Mountain is the highest mountain range in Tunisia, reaching 1550 m at peak Châambi.
Description. Males: L: 17-23 mm, H: 1.6-2 mm, 46-49 PR+1-2 AR+T; females: L: 18.5-32.2 mm, H: 2.4-3.6 mm, 44-50 PR+1-2 AR+T. General colour brownish with a black sputter, dorsally darker, with a black mid-dorsal line. Head dark brown to black with yellow spots in the occipital area, uniformly black frontally, with yellow spots at antennal level and labrum, the latter yellow and brighter; antennae dark brown. Prozonites covered with yellowish-brown spots on a blackish background, also laterally, interrupted by big black spots at ozopore level, dorsally black with a narrow transverse row of yellow spots anteriorly; metazonites glossy pale to whitish; legs light brown to yellowish. Telson: anal valves black, with a yellow sputter, preanal ring black, somewhat paler on the caudal projection; subanal scale light brown. Prozonites with fine oblique striae; metazonites with regular striation, laterally narrower; suture complete, strongly curving at ozopore level; ozopores small, rounded, situated on metazonites, at about their diameter from the suture. Anal valves with numerous submarginal and marginal setae, ca. 2 setae on the surface; subanal scale rounded and setose; preanal ring protruding in a caudal projection with ca. 3+3 setae on the tip and a small hyaline process.
Male sexual characters. Mandibular stipites expanded in well-rounded posteriorventral lobes, first pair of legs hook-shaped, remaining legs with postfemoral and tibial pads.
Gonopods. Promerite (P) bent anteriad (Figs 1, 2), proximally subrectangular, gradually narrowing in its distal third (Figs 1, 2); lateral margin with a moderately deep rounded incision (i). In a posterior view showing a distal process expanded in a subtriangular shape, bearing an apical blunt process (ap) and a lateral broad process pointing basad (lp); mesal ridge (M) apically broadened and delimiting a serrated lateral lamellar process; distal process laterally bearing a strong serrated ridge (se) marking a thickness on the posterior surface; telopodite (T) remnant ovoid located in the middle of the proximal part.
Posterior gonopod (Figs 3-6): Mesomerite (Ms) longer than the other processes of the gonopods, distally curved mesad and narrowing into an apical process folded and tapering toward the apex. Solenomerite (S) broad, slightly narrowing at midlength, proximally with several strong spikes (sp) on the posterior margin; anterior margin with a big, serrated process (pr) pointing distad, separated from the apical part by a rounded notch (n); apical part with an anterior marginally furrowed lamella (Figs 1, 4-6), and a setose wrinkled protruding lamella (wl) covering a protruding slender process (ds) (Figs 1, 3-6) housing the distal part and the opening of the seminal groove (g), the latter running from the fovea (F) at the base of the solenomerite up to process ds. Paracoxite (Px) broad and curved, emerging from a well-rounded coxite (Co), distal third broad then gradually narrowing in a rounded apex pointing mesad (Figs 3-6).
Distribution. Known only from the type locality, Châambi Mountain, Arid Bioclimatic zone, central Tunisia.
Habitat. Mixed forest with Quercus ilex and Pinus halepensis, under stones and in leaf litter.   To display the embedded multimedia object the use of Acrobat Reader (version 8 or higher) is required.
Diagnosis. Gonopods resembling those of O. punicus and O. khroumiriensis sp. n., but differing by the shape of promerite, a much broader and strongly serrated paracoxite, a broader mesomerite bearing subapical serrations on the mesal margin, and the apical processes on solenomerite.
Etymology. An adjunction of Latin words referring to the body size and leg colour, crassus meaning fat and nigripes, black leg.
Description. Males: L: 24.8-30 mm, H: 2.7-3.6 mm, 45-53 PR+1-2 AR+T. Females: 30-34 mm, H: 3.4-3.9 mm, 45-47 PR+1-2 AR+T. General colour grey, with alternating pale grey and golden brown, darker laterally, with a thin black mid-dorsal line. Head grey, with black sputter frontally, labral zone reddish-brown, brighter at the margin, antennae dark brown. Prozonites pale grey, with big black spots at the level of ozopores and below, a dense black sputter; metazonites whitish anteriorly and golden brown posteriorly, legs black. Telson: anal valves dark grey, bordered with black, preanal ring golden brown, darker on the tip of the caudal projection; subanal scale yellowish. Prozonites with scattered oblique striae; metazonites with regular striation becoming dense laterally; ozopores small and rounded, appearing as brown rounded spots located on metazonites, situated at about their diameter from the suture; the latter complete, only slightly curving at the level of ozopores. Anal valves mostly glabrous at the surface but bearing several submarginal and shorter marginal setae; subanal scale triangular, blunt and setose; preanal ring protruding in a caudal projection, with ca. 2+2 setae and a small hyaline process on the tip.
Male sexual characters. Mandibular stipites expanded in well-rounded posterior-ventral lobes, first pair of legs hook-shaped, remaining legs with postfemoral and tibial pads.
Gonopods. Promerite ( Fig. 7) broad, proximally almost rectilinear, bent 90 degrees at notch level; strongly narrowing in its distal third with a deep incision (i) on the lateral margin, latter almost rectilinear; mesal ridge (M) broad distally, protruding in a blunt process (mp) (Fig. 7); posterior surface irregular on the mesal side, bearing a number of strong setae aligned in front of the notch; distal process (ap) laterally broadened and rounded, showing a small mesal serrated process (ap1); remnant of telopodite (T) as a small bump located proximally.
Posterior gonopod (Figs 8-13). Mesomerite (Ms) long, sinuous, distal part asymmetrically enlarged mesolaterally, and showing in lateral view strong serrations at different levels on both margins (Figs 8,9,13), gradually narrowed apically in a hookshaped process (ho) curved and tapering toward the apex (Figs 11,12,13). Solenomerite (S) broadest at the base, narrowing at mid-length and bearing a number of strong setae near the posterior margin (Figs 10, 12, 13); in mesal view showing at mid-length a triangular process (pr) pointing distad (Figs 10, 13), latter separated from the apical part by a deep rounded notch (n); apically bearing a long curved process (ds) pointing mesad, housing the distal part and the opening of the seminal groove (g) and emerging between a posterior and an anterior folded hyaline processes (hp1, hp2) . Seminal groove running from the fovea (F) at the base of the solenomerite up to process ds (Figs 10,13). Paracoxite (Px) lamellar, broad and folded, emerging from a rounded coxite (Co), distally broadened, apical margin almost Distribution. Semi-arid and Arid bioclimatic zones in west central Tunisia, recorded from the governorates Kasserine, El Kef, Thala and Kairouan.
Habitat. Dry and open habitats, to 1500 m in Châambi Mountain. Diagnosis. Differing from all congeners by having a tri-lobate distal part of promerite and a bifurcated apical part of mesomerite, the latter divided in two short oppositely directed processes.

Ommatoiulus kefi
Etymology. Named after El Kef city, the type locality of the species. To display the embedded multimedia object the use of Acrobat Reader (version 8 or higher) is required. Description. Male: L: 26 mm, H: 2.7 mm, 53 PR+1 AR+T. General colour alternating whitish and blackish with a thin black mid-dorsal line. Head brown, lighter on the frontal part, with yellowish spots at antennal level, labral zone yellowish, becoming brighter at the margin, antennae brownish. Prozonites pale grey, dorsally scarcely sputtered with black; metazonites anteriorly dark, with a blackish background and a line of light brown spots below ozopores; legs whitish. Telson: anal valves and preanal ring blackish, paler towards caudal projection, subanal scale yellowish. Prozonites with scattered oblique striae; metazonites with regular striation becoming dense laterally; suture complete and rectilinear; ozopores small, rounded and located in metazonites, well apart from the suture. Anal valves with numerous submarginal and marginal setae and ca. 1-2 setae on the surface; subanal scale triangular, blunt and setose; preanal ring protruding in a caudal projection, with ca. 3+3 setae and a small hyaline process on the tip.
Male sexual characters. Mandibular stipites expanded in rounded posterior-ventral lobes, first pair of legs hook-shaped, remaining legs with postfemoral and tibial pads.
Gonopods. Promerite (Fig. 14) in posterior view subrectangular, mesal ridge (M) fairly broad, distally narrowing and protruding into a pointed apical lobe (al); apical margin protruding in a curved median lobe (me) pointing laterad and a shorter broad lateral lobe (lb); the three apical lobes separated by two rounded incisions; lateral margin almost rectilinear. Remnant of telopodite (T) ovoid, located at mid-length of promerite.
Posterior gonopod : Mesomerite (Ms) broadest at the base, distally protruding in a uniformly broad process, apically splitting into two short and curved To display the embedded multimedia object the use of Acrobat Reader (version 8 or higher) is required.
processes, pointing in opposite directions (m1, m2) (Fig 16, 17, 19); solenomerite (S) broad at the base, slightly narrowing at mid-length and showing a triangular process (pr) separated from the rest of the processes by a rounded (n) (Figs 17, 19), apical part of the solenomerite complex with a broad lamella (al1) extended latero-mesad, downturned and marginally furrowed (Fig. 16). Seminal groove (g) running from the fovea (F) at the base of solenomerite up to a slender and short conical process (ds) emerging on top of the median part of the apical lamella and pointing anteriad (Figs 15,17). Paracoxite (Px) stout, with smooth margins, emerging from a broad rounded coxite (Co) (Figs 18, 19). Distribution. Semi-arid bioclimatic zone in western Tunisia; hitherto known only from the type locality near El Kef city.
Comments. We have examined three females (MSNB) collected from the same locality but could not assign them with certainty to O. kefi as they show a different colour pattern. Diagnosis. Similar to O. punicus and O. crassinigripes sp. n. but readily distinguished by the shape of promerite having a deeper notch extended basad, much slen-derer processes of posterior gonopods, and a more sinuous mesomerite devoid of conspicuous serrations.

Ommatoiulus khroumiriensis
Etymology. The species name refers to the natural region of Khroumirie, NW Tunisia, to which the species seems confined.
Prozonites with scattered oblique striae; metazonites densely striated; suture complete, curving at ozopore level; ozopores small, rounded and located on metazonites, situated at about their diameter from the suture. Anal valves with 4-5 setae on the surface, a submarginal row of 12-13 setae and numerous short marginal ones; subanal scale triangular and setose; preanal ring protruding in a caudal projection with ca. 3+3 setae and a small hyaline process on the tip.
Male sexual characters. Mandibular stipites expanded in rounded posterior-ventral lobes, first pair of legs hook-shaped, remaining legs with postfemoral and tibial pads.
Gonopods. Promerite (Fig. 20) strongly narrowed distally with a deep lateral incision (i) extending meso-basad, distal process (ap) broad, subtriangular, with two pointed edges, the tip of apical process with a small pointed lobe (ap2); mesal ridge (M) distally protruding in a blunt small cylindrical process (mp), posterior surface of promerite with a row of strong setae emerging at the level of the notch, in close proximity to the mesal ridge.
Posterior gonopod (Figs 21-26). Mesomerite (Ms) large, longer than the other processes, uniformly broad, sinuous; distal third constricted to less than half breadth and apically protruding into a slender curved process, latter tapering and pointing mesad (Figs 21,23,24,26). Solenomerite (S) broadest at the base, narrowing at midlength, and bearing a number of strong setae near the posterior margin, distally with a broad, blunt triangular process (pr) separated from the apical part by a rounded notch (n), and with a long curved process (ds) protruding between two apical hyaline processes (hp3, hp4) and housing the apical part of seminal groove (g), the latter (g) running from the fovea (F) located at the base of the solenomerite (S) up to process ds. Paracoxite (Px) emerging from a depressed coxite (Co); Px curved, half as broad as in O. crassinigripes, gradually narrowing distad; lateral and apical margins, with a saw-like strongly jagged margin (Figs 22-26).
Habitat. Mixed forests dominated by Quercus faginea and Q. suber, or Pinus pinaster and Quercus suber. Diagnosis. Resembling O. chambiensis and O. xerophilus spp. n. in size and general shape of gonopods, but distinguished by the shape of promerite, a much more slender mesomerite and shorter and stouter paracoxite.
Etymology. The species name is a Greek noun meaning 'stranger', emphasising the fact that this species, found surprisingly in the collection of the MNHN shortly before completion of the manuscript, had remained unknown and out of the sight of a number of myriapodologists for more than 150 years.
Description. Male: L: 20.5 mm, H: 1.85 mm, 47 PR+2 AR+T; females: L: 18.5-21 mm, H: 2.26-2.46 mm, 42-48 PR+2-3 AR+T. General colour faded, generally greygreenish (very likely an artefact from the decomposition of the inserted label), somewhat lighter laterally. Head pale in the occipital and labral areas; antennae and legs darker. Prozonites with darker triangular spots laterally, latter situated along the ozopores line and forming two longitudinal dark bands, dorsally separated by a pale one; dorsum crossed by dark triangular spots and showing a thin black mid-dorsal line; metazonites mostly pale and glossy. Telson: anal valves and preanal ring dark, subanal scale pale. Prozonites with fine striae; metazonites with regular striae, denser on the sides, suture complete, curving at the level of ozopores; ozopores small, rounded, situated on metazonites situated at about their diameter from the suture. Anal valves setose; preanal ring with 3-4 setae on each lateral side, protruding in a short caudal projection with 1-4/5 setae and a small hyaline process on the tip. Subanal scale blunt to rounded and setose.
Male sexual characters. Mandibular stipites expanded in rounded posterior-ventral lobes, first pair of legs hook-shaped, remaining legs with postfemoral and tibial pads.
Gonopods. Promerite (Fig. 27) gradually narrowed distally, lateral margin with a shallow incision (i); apical process of promerite with a rounded margin pointing laterad; mesal ridge (M) narrow, distally protruding in a pointed apex (mp) separated from the apical process by a small apical incision; remnant of telopodite not very conspicuous.
Posterior gonopod (Figs 28-30): Mesomerite (Ms) uniformly broad proximally, strongly narrowed in its distal third and bent posteriad (Figs 28, 29); solenomerite (S) broad, with scattered setae on posterior margin, narrowing at mid-length, and bearing a large blunt process (pr); solenomerite apically with a broad folded lamella (Fl) and a small wrinkled lamella laying on the top of a slender and slightly protruding process (ds) housing the distal part of the seminal groove (g); seminal groove running from the fovea (F) and opening at the apex of process ds (Fig. 28). Paracoxite (Px) stout, distally curved mesad and narrowed into a slender apex pointing basad emerging from a broad and rounded coxite (Co) (Fig. 30).
Distribution. Exact locality unknown. The label mentions 'Tunis' which presumably refers to Tunisia in general.

Ommatoiulus xerophilus
Diagnosis. Resembling O. chambiensis in the structure of mesomerite, paracoxite, but well distinguished from the latter by the characteristic globular apex of promerite and the shape of solenomerite devoid of a rounded notch.
Etymology. The species name is a Greek adjective referring to the affinity of the species for dry habitats.
Prozonites with fine striae; metazonites with regular striae, becoming denser laterally, suture complete, curving at the level of ozopores; latter small, rounded, situated on metazonites situated at about their diameter from the suture. Anal valves setose, with 6-7 setae on the surface and numerous submarginal and marginal setae; subanal scale rounded and setose; preanal ring with 1+1 setae on the sides, protruding in a caudal projection with 3+3 setae and a small hyaline process on the tip.
Male sexual characters. Mandibular stipites expanded in rounded posterior-ventral lobes, first pair of legs hook-shaped, remaining legs with postfemoral and tibial pads.
Gonopods. Promerite (P) (Figs 31, 32) bent anteriad, not very broad, with parallel margins, mesal ridge (M) broad, distally narrowing and truncate, bearing several serrations (se) and separated from the apical lobe (ap); by an incision; lateral margin mostly rectilinear, only slightly narrowing subapically at the level of the mesal incision; apical lobe globular, with rounded margin, curved laterad; posterior surface with a number of strong setae aligned distally; telopodite remnant inconspicuous.
Distribution. Arid bioclimatic zone, central Tunisia; hitherto known only from the type locality, Châambi Mountain.
Habitat. Open areas with scattered Pinus halepensis trees. Etymology. Named after Jebel Zaghouan, the type locality. Description. Male: L: 28.5 mm, H: 2.56 mm, 49 PR+1 AR+T. General colour alternate dark and light golden brown; dorsum with a thin black axial line. Head dark to blackish, with brownish spots on the frontal part and on the mandibular stipites, labral zone and mouth parts pale, marginally bright yellow; antennae brownish. Prozonites dark to blackish, covered with yellowish spots; metazonites pale brown to whitish laterally and golden brown dorsally, legs yellowish. Telson: anal valves black, preanal ring blackish, caudal projection yellowish, subanal scale yellowish. Prozonites with fine irregular striae; metazonites with regular striae, becoming dense laterally, suture complete, curving at the level of ozopores; ozopores small, rounded, situated in metazonites, situated at about their diameter from the suture. Anal valves setose, with 4-6 setae on the surface, ca 14 submarginal and numerous marginal setae; subanal scale rounded and setose; preanal ring with 2+2 setae on the sides, protruding in a caudal projection with (6-7)+(6-7) setae on the tip and bearing a small hyaline process.
Male sexual characters. Mandibular stipites expanded in rounded posterior-ventral lobes, first pair of legs hook-shaped, remaining legs with postfemoral and tibial pads.
Gonopods. Promerite (Fig. 36) proximally subrectangular, strongly narrowed distally by a deep incision (i) on the lateral margin; mesal ridge (M) broad, distally protruding in a serrated edge (se); apical part with a mesal triangular blunt process (rp) protruding mesodistad, and a lateral protruding process with two small apical bumps (bp1, bp2); posterior surface of promerite showing few scattered setae near the mesal margin; remnant of telopodite (T) large and ovoid, located at mid-length of the process.
Posterior gonopod (Figs 37-39): Mesomerite (Ms) large, and uniformly broad (Figs 37, 38) with a distal triangular pointed extension on the lateral margin (tp), distal third strongly curved mesoposteriad and narrowed in a long and slender apical process (Figs 37,38,39); solenomerite (S) broad, with scattered setae on posterior margin; anteriorly simply rounded devoid of processes; apically with a hyaline folded lamella (fl) and a slightly protruding process (ds) housing the distal part of the seminal groove (g); the latter running from the fovea (F) (Fig. 37) up to process ds. Paracoxite (Px) stout and curved apically slightly narrowing into a rounded apex directed mesad, coxite broad (Co) (Fig. 39).
Distribution. Semi-arid bioclimatic zone in northeastern Tunisia; known only from Zaghouan Mountain.
Habitat. Forest dominated by Pinus halepensis. Akkari et al. (2009) summarized all records of julidan millipedes, including Ommatoiulus species, from Tunisia and provided a complete list of the North African members of the order. A number of additions and corrections to the lists are presented here:

Updates to the list of Ommatoiulus species in North Africa
• We refute the presence of O. diplurus appendiculatus (Brolemann, 1925) in Algeria. This taxon is based on females and juveniles only, and Akkari and Enghoff (2012) already regarded the presence of O. diplurus (Attems, 1903) in North Africa uncertain. • The record of O. aumalensis (Brolemann, 1925) Manfredi 1939, Schubart 1952 with O. lapidarius, but re-examination of the holotype of J. rimosus (Museum für Naturkunde, Berlin) has shown that this is a valid species (Akkari 2013).

Discussion
Three of the new Tunisian Ommatoiulus species were found in Châambi Mountain, which is the highest mountain range in Tunisia. The mountain is located only a few kilometres from the Algerian border and in spite of its arid character is known to harbour a number of endemic species (cf. Kovařík 2006, Hartenberger 1986 (Fig. 40). Of special importance is the fact that the size difference is apparent within each developmental stadium. Thus, O. chambiensis males with 8RO have more podous rings and larger diameter than O. xerophilus males with 8RO. The same is true for specimens with 9RO although the studied material is much smaller. O. crassinigripes is the only schizophylline millipede collected at more than 1500 m elevation in Tunisia. However, it occurs also at lower elevation in central Tunisia (El Kef, Thala, Makthar, Kairouan) and is very unlikely to be an alticolous species like Ommatoiulus gravieri (Brolemann, 1924), which is known from 3000-3200 m altitude in Jebel Tachdirt of the High Atlas in Morrocco (Brolemann 1924, Akkari et al. 2009. O. crassinigripes is one of the largest Ommatoiulus species in Tunisia and, together with O. khroumiriensis, is the closest to O. punicus with regard to gonopod shape (Fig. 41). The three species, which were all treated under O. punicus by Akkari et al. (2009), have quite similar gonopod conformations, yet display a number of constant differences in both promerite and posterior gonopods (Table 1). Although resembling, differences in posterior gonopods can be clearly noticeable on the rotatable models (Figs 13,26,41), which, in addition to the external morphological features, indicate their separate taxonomic status. The external morphological differences concern colour pattern and size.  is mainly distributed around Tunis City, Cap Bon Peninsula and the eastern part of the Ridge viz. Zaghouan Mt. in the north (see Akkari et al. 2009). O. punicus was recorded from Aïn Draham, Tabarka and Nefza, as well as from Kairouan Plain by Akkari et al. (2009). These authors pointed out the variation observed in the material: "Ommatoiulus punicus as currently delimited is quite a variable taxon, and a detailed analysis may well necessitate splitting it into several (sub)species." Our renewed study has corroborated this assumption. Attems (1903Attems ( , 1926 (Attems 1926, figs 240-241) is O. khroumiriensis. On the other hand, the record of O. punicus from unspecified locality in Tunisia (Attems 1903: 144, figs 77-81) is here referred to as O. crassinigripes. Intercalary males are here recorded for the species Ommatoiulus khroumiriensis and O. crassinigripes. This is of little surprise considering that postembryonic development involving periodomorphosis (regression of secondary sexual characters followed by a return to a morphologically copulatory stadium following an additional moult) is considered as particularly prevalent for schizophylline species (Enghoff et al. 1993). Characteristic morphology of intercalary stadia was directly observed on reared specimens of three schizophyllines: O. moreleti (Lucas, 1860), O. sabulosus (Linnaeus, 1758) and Tachypodoiulus niger (Leach, 1814) while field-collected samples provided periodomorphic specimens belonging to no less than 8 species of genus Ommatoiulus, including O. punicus (see Akkari and Enghoff 2011 for species list). The same authors discussed the particular case of the species O. sempervirilis for which a large hand-collected sample revealed the complete absence of intercalary stadia and presence instead of four successive stadia of adult males implying a direct copulatory-copulatory succession (Akkari and Enghoff 2011).
Colour pattern and somatic characters cannot be used reliably to distinguish the Tunisian Ommatoiulus species, although we have provided information about these features above in order to point out fine differences between morphologically close or syntopic taxa. In the identification key (below) we prefer to include only gonopod characters. To display the embedded multimedia object the use of Acrobat Reader (version 8 or higher) is required.

Identification key to Tunisian Ommatoiulus species
The key is based on characters of the male gonopods.

11
Apical process of promerite (ap) bulgy, with a lobed process (ap1) on tip (Fig.  7); mesomerite (Ms) with strong subapical mesal serrations (Fig. 8); paracoxite (Px) broadened distally with truncated apex (Fig. 12) ........... O. crassinigripes -Apical process of promerite slender, without a lobed process; mesomerite (Ms) without mesal serrations (Fig. 52); paracoxite (Px) distally narrowed, with rounded apex (Fig. 53)  Interactive key to Tunisian Ommatoiulus species We provide an interactive key in Flash (SWF) format to the Ommatoiulus species known from Tunisia. The key is dichotomous and based on gonopod characters. These are illustrated with line drawings, light microscopy photographs and SEMs, and for some of the species with rotatable SEM animations. A species list and species pages are included in the key to provide additional information on species diagnostic characters, distribution and habitats. An introductory section for the first-time user provides background information of importance for applying the key. Adobe Flash Player (version 11.2 or higher) or a browser (e.g. Internet Explorer, Firefox and Chrome) with Flash Player plug-in is required to run the key.
To display the embedded multimedia object the use of Acrobat Reader (version 8 or higher) is required.