s2fieldsofstudy: Biology Medicine

text:
Time course of pulmonary burden in mice exposed to residual oil fly ash

Residual oil fly ash (ROFA) is a common pollutant in areas where oil is burned. This particulate matter (PM) with a broad distribution of particle diameters can be inhaled by human beings and putatively damage their respiratory system. Although some studies deal with cultured cells, animals, and even epidemiological issues, so far a comprehensive analysis of respiratory outcomes as a function of the time elapsed after exposure to a low dose of ROFA is wanted. Thus, we aimed to investigate the time course of mechanical, histological, and inflammatory lung changes, as well as neutrophils in the blood, in mice exposed to ROFA until 5 days after exposure. BALB/c mice (25 ± 5 g) were randomly divided into 7 groups and intranasally instilled with either 10 μL of sterile saline solution (0.9% NaCl, CTRL) or ROFA (0.2 μg in 10 μL of saline solution). Pulmonary mechanics, histology (normal and collapsed alveoli, mononuclear and polymorphonuclear cells, and ultrastructure), neutrophils (in blood and bronchoalveolar lavage fluid) were determined at 6 h in CTRL and at 6, 24, 48, 72, 96, and 120 h after ROFA exposure. ROFA contained metal elements, especially iron, polycyclic aromatic hydrocarbons (PAHs), and organochlorines. Lung resistive pressure augmented early (6 h) in the course of lung injury and other mechanical, histological and inflammatory parameters increased at 24 h, returning to control values at 120 h. Blood neutrophilia was present only at 24 and 48 h after exposure. Swelling of endothelial cells with adherent neutrophils was detected after ROFA instillation. No neutrophils were present in the lavage fluid. In conclusion, the exposure to ROFA, even in low doses, induced early changes in pulmonary mechanics, lung histology and accumulation of neutrophils in blood of mice that lasted for 4 days and disappeared spontaneously.

INTRODUCTION
Many studies associate events of urban air pollution with significant health effects on the exposed population, including morbidity and mortality due to cardiopulmonary diseases or lung cancer (Dominici et al., 2006;Fajersztajn et al., 2013). These outcomes have been observed even at pollution levels below current national and international ambient air quality health standards (Lin et al., 1999).
Residual oil fly ash (ROFA) consists of PM produced by oilburning systems and is rich in transition metals. It has been used in murine models as a surrogate material to investigate the responses to PM inhalation Kodavanti et al., 1998). ROFA administration via intratracheal/intranasal instillation and aerosol inhalation disclosed functional and structural alterations such as acute lung injury, alveolar septal thickening, increased cellularity and lung inflammation Gavett et al., 1997Gavett et al., , 1999Ghio et al., 2002;Hamada et al., 2002;Kodavanti et al., 2002). Additionally, ROFA exposure has been studied in association with chronic allergic pulmonary inflammation, cigarette smoke, and lung infection (Gavett et al., 1999;Antonini et al., 2002;Arantes-Costa et al., 2008;Biselli et al., 2011). We previously reported that chronic allergic mice exposed to ROFA show even higher hyperresponsiveness, bronchoconstriction and mast cell infiltration after methacholine challenge than those not exposed (Avila et al., 2011). However, so far the timeline of the lung alterations following a single exposure to ROFA particles has not been reported.
Thus, we aimed to investigate the time-dependency of lung impairment in animals that underwent a single exposure to ROFA, simulating the situation of someone visiting a polluted place for a day. For such purpose, we analyzed ROFA composition, lung mechanics, alveolar collapse, inflammatory cells recruitment, and pulmonary ultrastructure in different time points after exposure.

ANIMALS
All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences, USA. The experiments were approved by the Ethics Committee on the Use of Animal, Health Sciences Center, Federal University of Rio de Janeiro (Protocol IBCCF 046).
Eighty-four BALB/c mice (6-7 weeks of age) obtained from the animal facilities of the Federal University of Rio de Janeiro, Brazil, were housed in plastic cages with absorbent bedding material and maintained on a 12-h daylight cycle. Food and water were provided ad libitum.

PREPARATION
Forty-two mice (25-30 g) were randomly divided into 7 groups intranasally instilled with: sterile saline solution (0.9% NaCl, CTRL, n = 6) or ROFA (0.2 μg of ROFA in 10 μL of saline solution, n = 36). In CTRL group the experiments were done at 6 h after instillation, whereas in ROFA groups the mice were studied at 6, 24, 48, 72, 96, and 120 h after exposure (n = 6/group). Right before the instillation, the mice were anesthetized with sevoflurane and either saline or ROFA were gently instilled into their snouts with the aid of a precision pipette. They rapidly recovered after instillation. These animals were used for the measurement of pulmonary mechanics and histology. In another group of 42 mice submitted to the same protocol, inflammatory cells were counted in the blood and in the broncho-alveolar lavage fluid (BALF).

ROFA COMPOSITION
The ROFA was obtained from an incinerator located at the University Hospital, University of São Paulo, Brazil. The particles were digested in an HNO 3 -HClO 4 mixture and then analyzed by flame atomic absorption spectroscopy (VARIAN AA1475, Varian, Inc., Palo Alto, CA, USA) to determine their elemental composition. ROFA was also analyzed by gas chromatography (GC-14B with automatic injector AOC-1400, Shimadzu Corp, Kyoto, Japan) and high performance liquid chromatography (RF-10 with fluorescence detectors, Shimadzu Corp, Kyoto, Japan) for organochlorine and polycyclic aromatic hydrocarbon (PAH) quantification, respectively. All analytical procedures above were determined as formerly described (Mazzoli-Rocha et al., 2008;Riva et al., 2011). The distribution of particle sizes was previously reported (Avila et al., 2011), and the average particle diameter amounted to 66.5 μm. It should be stressed that around 7.6% of ROFA particles presented an average diameter smaller than 10 μm, and about 2.1% were smaller than 2.5 μm (Avila et al., 2011).

PULMONARY MECHANICS
At the aforementioned experimental times after instillation the animals were sedated (diazepam, 1 mg i.p.), anesthetized (pentobarbital sodium, 20 mg/kg i.p.), tracheotomized, and a snugly fitting cannula (0.8 mm i.d.) was introduced into the trachea. Then, the animals were paralyzed with pancuronium bromide (0.1 mg/kg i.v.), and ventilated (frequency of 100 breaths/min, tidal volume of 0.2 ml, and flow of 1 ml/s) with a constant-flow ventilator (Samay VR15, Universidad de la Republica, Montevideo, Uruguay). A positive end-expiratory pressure amounting to 2 cmH 2 O (Saldiva et al., 1992b) was applied to the expiratory line of the ventilator and the anterior chest wall was surgically removed. For the determination of pulmonary mechanics a 5-s end-inspiratory pause could be generated by the ventilator when needed.
A pneumotachograph (1.5 mm ID, length = 4.2 cm, distance between side ports = 2.1 cm) was connected to the tracheal cannula for the measurements of airflow (V ). Lung volume (V T ) was determined by V signal integration. The equipment resistance (Req) including the tracheal cannula was calculated (Req = 0.12 cmH 2 O/mL/s) and found constant up to flow rates of 26 mL/s. The equipment resistive pressure (Pres,eq = Req·V ) was subtracted from pulmonary resistive pressure so that the present results represent intrinsic values. Transpulmonary pressure (PL) was measured proximally to the tracheal tube by a Validyne MP45-2 differential pressure transducer (Engineering Corp., Northridge, CA, USA). All signals were conditioned and amplified in a Beckman type R Dynograph (Schiller Park, IL, USA). Flow and pressure signals were also passed through lowpass 8-pole Bessel filters (902LPF, Frequency Devices, Haverhill, MA, USA) with the corner frequency set at 100 Hz, sampled at 200 Hz with a 12-bit analog-to-digital converter (DT2801A, Data Translation, Marlboro, MA, USA), and stored on a microcomputer. All data were collected using LABDAT software (RHT-InfoData Inc., Montreal, QC, Canada).
Lung resistive ( P1) and viscoelastic/inhomogeneous ( P2) pressures, total pressure drop ( Ptot = P1 + P2), static elastance (Est), and elastic component of viscoelasticity ( E) were computed by the end-inflation occlusion method (Bates et al., 1985(Bates et al., , 1988. Briefly, after end-inspiratory occlusion, there is an initial fast drop in PL ( P1) from the pre-occlusion value down to an inflection point (Pi) followed by a slow pressure decay ( P2), until an apparent plateau is reached. This plateau corresponds to the elastic recoil pressure of the lung (Pel). P1 selectively reflects airway resistance in normal animals and humans and P2 reflects stress relaxation or viscoelastic properties of the lung, together with a small contribution of time constant inequalities (Bates et al., 1988;Saldiva et al., 1992b). Lung static (Est) and dynamic elastances (Edyn) were calculated by dividing Pel and Pi by V T , respectively. E was calculated as Est-Edyn (Bates et al., 1985(Bates et al., , 1988. Pulmonary mechanics was measured 10-15 times in each animal. All data were analyzed using ANADAT data analysis software (RHT-InfoData Inc., Montreal, QC, Canada). The duration of the experiments approximated 30 min.

Light microscopy
A lower longitudinal laparotomy was done immediately after the determination of pulmonary mechanics, and heparin (1000 IU) was injected into the abdominal vena cava. Three minutes later the abdominal aorta and vena cava were sectioned, yielding a massive hemorrhage that quickly euthanized the animal. The trachea was clamped at end-expiration and the lungs were removed en bloc.
The left lung was quick-frozen by immersion in liquid nitrogen, fixed with Carnoy's solution (Nagase et al., 1992), and embedded in paraffin. Four-μm-thick slices were obtained by means of a microtome and stained with hematoxylin and eosin. Morphometry and cellularity index were evaluated with an integrating eyepiece with a coherent system with 100 points and 50 lines coupled to a conventional light microscope (Axioplan, Zeiss, Oberkochen, Germany). The point-counting technique was used across 10 random non-coincident microscopic fields to evaluate the fraction area of normal and collapsed airspaces and the amount of mononuclear (MN) and polymorphonuclear cells (PMN). Points falling on normal alveoli and collapsed airspaces were counted and divided by the total number of points in each microscopic field (200×). Points falling on MN and PMN cells were counted and divided by the total number of points falling on tissue area in each microscopic field (1000×) (Weibel et al., 1966). Two investigators, who were unaware of the origin of the coded material, examined the samples microscopically.

Transmission electron microscopy
To obtain a stratified random sample, three slices of 2 × 2 mm were cut from three different segments of the right lung (cranial, middle, and caudal lobes) and then fixed in 2.5% glutaraldehyde and 0.1 M phosphate buffer (pH = 7.4) for 60 min at −4 • C. The slices were then rinsed in phosphate buffer, postfixed in 1% osmic tetroxide in phosphate buffer for 30 min, and rewashed three times in phosphate buffer. Finally, the slices were dehydrated in an acetone series and then placed in a mixture of 1:1 acetone:Epon overnight before embedding in Epon for 6 h. After fixation, the material was kept for 48 h at 60 • C before undergoing ultramicrotomy for transmission electron microscopy (JEOL 1010, Tokyo, Japan).

EVALUATION OF NEUTROPHILS IN THE BLOOD AND IN THE BRONCHOALVEOLAR LAVAGE FLUID
At each experimental time, the animals were anesthetized with isoflurane and the tip of their tails were cut off to produce a blood smear. Neutrophil counts were determined in a Neubauer chamber by means of an optical microscope after dilution of blood samples in 2% acetic acid solution. The number of circulating neutrophils (100 cells counted/slide, 1000× magnification) was determined after differential cell counts on May-Grunwald-Giemsa stained blood smears. After blood sampling the mice were euthanized in a CO 2 chamber and the alveolar lavage done. For such purpose, the trachea was cannulated and the lungs gently washed twice with 1 ml of phosphate buffered saline (pH = 7.4). Neutrophils were counted after cytocentrifugation (Shandon, East Grinstead, UK) and staining with Diff-Quick (Baxter Dade AG, Dunding, Germany). At least 100 cells were counted and the results expressed as number of cells/mL.

STATISTICAL ANALYSIS
SigmaStat 11.0 statistical software (SYSTAT, Chicago, IL, USA) was used. When percentage values were to be tested, they firstly underwent arcsine transformation. The normality of the data (Kolmogorov-Smirnov test with Lilliefors' correction) and the homogeneity of variances (Levene median test) were tested. Since in all instances both conditions were satisfied, One-Way ANOVA followed by Bonferroni post hoc test was used (when required) to assess differences between ROFA groups and CTRL mice. The significance level was set at 5% (p < 0.05).
The fraction area of alveolar collapse and PMN cell influx into the lung parenchyma were higher in ROFA than in CTRL at 24, 48, 72, and 96 h. Similarly, normal alveolar spaces and MN cells were significantly lower in the FIGURE 2 | Elastic components of lung mechanics in mice. Est, static elastance and E, elastic component of viscoelasticity. CTRL, mice instilled with saline solution (0.9% NaCl, measurements were done 6 h after exposure), and ROFA, animals that received residual oil fly ash (0.2 μg in 10 μL of saline solution). Measurements were done 6, 24, 48, 72, 96, and 120 h after exposure. Columns represent the average of 6 mice in each group, 10-15 determinations per animal. Bars represent SEM. * Significantly different from CTRL (p < 0.05).
latter groups than in CTRL. ROFA120 showed values similar to CTRL for histological parameters (Figures 3, 4). ROFA particle was observed in alveolar space of ROFA6, 24, 48, 72, and 96 groups and not in ROFA120 (Figure 3, insert B).
Electron microscopy of lung parenchyma in CTRL mice showed preserved types I and II pneumocytes, endothelial cells, alveolar interstitial wall, and components of the extracellular matrix. At 24 h, ROFA group showed endothelial damage as represented by swelling, vacuolization and neutrophils adhered to the pulmonary capillary wall (Figure 5).
The amount of neutrophils in the blood was higher in ROFA24 and ROFA48 than in the CTRL mice. Thereafter they did not differ from CTRL animals. No statistically significant difference was detected for neutrophils in the BALF (Figure 6).
The survival rate was 100% in all groups throughout the experiments.

DISCUSSION
The time course of lung functional and histological impairment induced by ROFA dust has not been reported so far. Intranasal instillation of a low dose of ROFA (0.2 μg in 10 μL) induced a significant increase in resistive pressure, followed by an increment in viscoelastic/inhomogeneous pressures and elastances, accompanied by increased alveolar collapse, influx of PMN cells, ultrastructural alterations in lung parenchyma and increased number of neutrophils in the blood. These outcomes returned to control values at 120 h after exposure.
PM is a heterogeneous mixture of gas, liquid, and solid particles of different origins and sizes in suspension in the air, displaying close physical and chemical interactions. PM is classified, according to its aerodynamic diameter, as coarse (2.5-10 μm; PM10), fine (0.1-2.5 μm; PM2.5), and ultrafine (≤0.1 μm) (Donaldson et al., 2001). The different profiles of size and composition may influence particle toxicity and, consequently, the magnitude of adverse health effects (Saldiva et al., 2002). In human beings, toxicity becomes very important when aerodynamic diameter of the particles is 10 μm, which enables them to reach the pulmonary alveoli. In rats and mice, this value approximates 2 μm for intratracheally instilled silica (Wiessner et al., 1989;Takayoshi et al., 2007). The ROFA used in the present study was mainly composed of particles bigger than 10 μm (Avila et al., 2011), which would be less harmful than the smaller ones (Donaldson et al., 2001). However, around 7.6% of ROFA particles presented an average diameter less than 10 μm, and about 2.1% were smaller than 2.5 μm. Another concomitant study of our group (Avila et al., 2011), using the same dose and PM, reported lung impairment at 24 h after exposure. It should be stressed that the fine and ultrafine particles are known as "breathable" and are able to penetrate the airways, reaching the alveoli (Dusseldorp et al., 1995;Peters et al., 1997;Brown et al., 2002;Tao et al., 2003). Indeed, in ROFA6, 24, 48, 72, and 96 groups the administered pollutant was detected in the alveolar space (Figure 3).
A recent study analyzed the composition, sources and toxicity of PM2.5 collected in different cities in the United States and observed an association between its toxicity and the number of vehicles and industries (Seagrave et al., 2006). São Paulo is the most industrialized center of Latin America and has about 20,000,000 inhabitants. There are about 7,000,000 vehicles in the area using three types of fuel: gasoline, diesel and alcohol (CETESB, 2013). Because of its geographical characteristics, São Paulo presents thermal inversions, resulting in significant increases in air pollution. Thus, São Paulo represents an excellent place to assess the effects of air pollution on health.
In order to avoid the consequences of particle overload, we administered a low dose of PM to the mice, reflecting more precisely the adverse pulmonary consequences of ambient particle concentrations. The mean daily concentrations of PM2.5 and PM10 in São Paulo, where ROFA was collected, amount to 60 and 120 μg/m 3 , respectively (CETESB, 2013). Considering that a mouse inspires 0.03 m 3 of air in 24 h, it represents 6.7 μg/m 3 of ROFA dust in the present work. Particles were administered by intranasal instillation to the anesthetized animal, which is a useful and well-accepted model of exposure to PM FIGURE 3 | Photomicrographs of lung parenchyma stained with hematoxylin-eosin (x200). CTRL, mice instilled with saline solution (0.9% NaCl, measurements were done 6 h after exposure). ROFA, animals that received residual oil fly ash (0.2 μg in 10μL of saline solution). Measurements were done 6, 24, 48, 72, 96, and 120 h after exposure. Arrows show representative thickened septa and circles indicate collapsed alveoli. In each panel insert A shows alveolar walls and inflammatory cells therein (×1000 magnification) and insert B displays ROFA particle in the alveolar space when present (×400 magnification). Bar: 100 μm.

FIGURE 4 | Collapsed and normal areas, and influx of polymorphonuclear (PMN) and mononuclear (MN) cells. CTRL, mice
instilled with saline solution (0.9% NaCl, measurements were done 6 h after exposure), and ROFA, animals that received residual oil fly ash (0.2 μg in 10 μL of saline solution). Measurements were done 6, 24, 48, 72, 96, and 120 h after exposure. Columns represent the average of 6 mice in each group. Bars represent SEM. Data were gathered from ten random, non-coincident fields per mouse. * Significantly different from CTRL (p < 0.05).

Frontiers in Physiology | Respiratory Physiology
September 2014 | Volume 5 | Article 366 | 6 FIGURE 5 | Electron microscopy of lung parenchyma. Upper panel: CTRL (6625×), mouse instilled with saline solution. Note the preserved type II pneumocyte (PII) and the alveolar interstitial wall. * Red blood cell. Lower panel: ROFA (8400×), animal that received residual oil fly ash (0.2 μg in 10 μL of saline solution). N, adherent neutrophils; C, capillary; A, alveolar space; EC, endothelial cell. Measurements were done 6 (CTRL) and 24 h (ROFA) after exposure. (Southam et al., 2002). In experimental models similar to ours, which used ROFA instilled intranasally in mice, doses amounting to 25 times (Biselli et al., 2011;Magnani et al., 2013;Marchini et al., 2014) and up to 60 times greater (Arantes-Costa et al., 2008) than ours were used. ROFA is a suspension of the material produced after oil burning, which was used in some experimental models of exposure to air pollution (Ghio et al., 2002;Arantes-Costa et al., 2008;Avila et al., 2011;Marchini et al., 2014). Although ROFA exposure does not exactly mimic the overall environmental pollution, this PM contains high concentrations of many components of air pollution. Previous studies report that PMs from different sources, including ROFA (Kodavanti et al., 1998), are able to induce inflammatory processes (Sørensen et al., 2003;Park et al., 2006). Animal studies demonstrate that the bioavailability of soluble transition metals is responsible for the pulmonary injury and inflammation observed after ROFA exposure Kodavanti et al., 1998). The ROFA used in the present work contains predominantly iron and nickel (Table 1), in line with other studies. Metals, including iron, vanadium, and nickel, are present in high concentrations as water-soluble salts in fly ash (Schroeder et al., 1987) and largely reproduce the lung injury induced by ROFA. Without those metals pulmonary toxicity decreases . ROFA containing iron, aluminum, vanadium and nickel induced more pronounced cellular oxidative imbalance and lung injury (Lewis et al., 2003). Iron is deeply linked to the generation of reactive oxygen species (ROS) (Park et al., 2006), surfactant dysfunction (Chauhan and Misra, 1991), epithelial damage, increased vascular permeability and inflammatory response followed by impaired pulmonary function (Soukup et al., 2000;Dye et al., 2001).
In the present study, PM concentration of PAH, particularly naphthalene, acenaphthylene, acenaphthene and other elements with potential carcinogenic risk (benzo[a]antracene, benzo[a]pyrene, Dbenzo[ah]antracene and ind[123cd]pyrene) were detected ( Table 2). Another study of our group, analyzing particles produced by traffic, detected benzo[a]pyrene and benzo[a]anthracene in samples of total suspended PM from São Paulo, confirming the high levels of PAH in this city (Mazzoli-Rocha et al., 2008). Washing the diesel particles with hexane removed a large amount of PAHs and improved respiratory outcomes in mice (Laks et al., 2008). Finally, the presence of PAH in ROFA has been associated with the triggering of inflammation, generation of ROS, and lipid peroxidation (Sørensen et al., 2003), especially in alveolar macrophages and epithelial cells (Li et al., 2002).
ROFA was tested for the presence of some organochlorine compounds, and a few were found: op -DDE, pp -DDT, op DDT, G-HCH, endosulfan and dieldrin (Table 3). These substances are pesticides and constitute a family of persistent, lipophilic compounds whose use was banned because they cause a variety of diseases in humans and wildlife (Androutsopoulos et al., 2013). The chlorinated pesticides may be absorbed into the body through the skin, respiratory and digestive tracts (Yohannes et al., 2014). Organochlorines cause neurotoxic, hormonal, immunomodulating, and tumorigenic effects (Androutsopoulos et al., 2013). However, to our knowledge, no study evaluated the association between exposure to organochlorines and pulmonary impairment.
Although most of lung changes in mechanical properties, histology and inflammatory response occured at time point of 24 h, our control group was studied at 6 h after exposure. To support our approach, we compared our CTRL group with that previously reported and measured at 24 h after exposure to ROFA (Avila et al., 2011), Their results are very similar to ours, thus allowing the use of a 6-h CTRL group. Furthermore, we coped with Russel and Burch's (1959) principle of the 3Rs (reduction, refinement, and replacement of the animal use) to minimize the number of experimental animals. A higher resistive pressure (that reflects Newtonian or ohmic resistance) was the first response to ROFA, as found in ROFA6 group (Figure 1). It can be possibly explained anatomically, since central airways are the first lung structure to be exposed to ROFA. In a previous study, we also found increased central airway resistance 24 h after exposure to ROFA; it should be noted that the authors did not perform any measurements before that time point (Avila et al., 2011). Viscoelastic and total pressures, static elastance and elastic component of viscoelasticity increased significantly in ROFA24 group and remained elevated until 4 days after exposure (ROFA96), when compared to CTRL (Figures 1, 2). We also detected higher Est and mechanical parameters related to the lung periphery 24 h after exposure to ROFA (Avila et al., 2011). These results could be explained by the concomitant increase in alveolar collapse and lung PMN content (Figures 3, 4). At the same time neutrophils adhered to the swollen pulmonary capillary wall (Figure 5), indicating activation of the endothelium and of leukocyte integrins (Langer and Chavakis, 2009), as a result of a local proinflammatory stimulus presumably triggered by ROFA. These neutrophils would migrate through the endothelium and reach the pulmonary interstitial space (Figure 4). ROFA24 also presented a higher count of PMN in the blood (Figure 6), suggesting a systemic inflammatory status. All these findings were also present in ROFA48 mice. In line with our results in ROFA24 mice, some authors observed impaired lung mechanics, alveolar collapse, influx of inflammatory cells to the lung (Avila et al., 2011), inflammatory process in the perivascular area, and inflammatory infiltration in the interstitial space (Medeiros et al., 2004). Interestingly, a recent study demonstrated increased TNF-α and IL-6 plasma levels and PMN leukocytes activation at 1, 3, and 5 h after ROFA exposure (Marchini et al., 2014), but they used doses 25 times larger than ours. Finally, it should be mentioned that surfactant secretion by type II pneumocytes is impaired after inhalation of air pollutants (Müler et al., 1998) and exposure to fly ash modifies surfactant composition (Srivastava and Misra, 1986;Chauhan and Misra, 1991) and rheology (Anseth et al., 2005), yielding alveolar instability and collapse. At 72 h after exposure blood PMN count returned to control values, but the pulmonary parameters remained higher than CTRL, suggesting that the overall process started to recede in the organism. ROFA96 presented similar results. At 120 h all measured parameters returned to baseline values.
Neutrophils count in the BALF resulted negative (Figure 6). The method may be not sensitive enough to detect the inflammatory changes after the nasal instillation of ROFA or the cells indeed did not cross the airway epithelium. In accordance with our findings, the intranasal instillation of ROFA did not disclose inflammatory alterations in mice BALF, even taking into consideration the use of a dose 500 times larger than ours (Medeiros et al., 2004). On the other hand, the intratracheal instillation of ROFA in a dose 60 times larger than that in this study triggered inflammatory alterations in BALF (Gavett et al., 1999). The difference between these two apparently discrepant results could be the local of administration of the pollutant.
Epidemiological studies can add translational information to our findings. Dose-dependent decreased indexes of pulmonary function, including diminished forced vital capacity, forced expiratory volume in 1 s, and forced expiratory flows were described in boilermakers 24 h after exposure to ROFA (Hauser et al., 1995(Hauser et al., , 1996. In a 2-year longitudinal study a significant association between working at oil-fired industries and reduced lung function was detected . Finally, ROFA-exposed individuals presented impaired pulmonary function, which was resolved 4 weeks after they were removed from their working stations in an oil-fired electricity generating plant (Lees, 1980).
Our study presents some limitations: (1) the animals were exposed intranasally rather than directly to environmental air. On one hand they received only ROFA, but on the other one the results do not represent exactly what would be found around the sampling site; (2) we did not measure levels of inflammatory cytokines that could have been modified by exposure to ROFA.
In conclusion, we demonstrated that the exposure to low doses of ROFA rapidly compromised pulmonary mechanics and histology, triggered the influx of polymorphonuclear cells into the lung, and increased the neutrophil count in the blood of mice. These pathophysiological findings resolved 5 days after exposure.

AUTHOR CONTRIBUTIONS
Giovanna Marcella Cavalcante Carvalho-interpretation of data for the work; drafting the work and revising it for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Lilian Katiê da Silva Nagato-interpretation of data for the work, experimental design and organization, data analyses; revised the work for important intellectual content; final approval of the version to be published, agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Sheila da Silva Fagundes-data acquisition; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Flávia Brandão dos Santos-data acquisition; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Andrea Surrage Calheiros-data acquisition; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Olaf Malm-chemical analyses; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Patricia Torres Bozza-data analyses; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Paulo Hilário N. Saldiva-data analyses; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Débora Souza Faffe-experimental design and organization; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Patricia Rieken Macedo Rocco-experimental design and organization; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Walter Araujo Zin-experimental design and organization, hypotheses, interpretation of data for the work; drafting the work, revised the work for important intellectual content; final approval of the version to be published, agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

SUPPORT
Centers of Excellence Program (PRONEX/FAPERJ), The Brazilian Council for Scientific and Technological Development (CNPq), Carlos Chagas Filho Rio de Janeiro State Research Supporting Foundation (FAPERJ), Brazilian Ministry of Science, Technology and Innovation (MCTI), and Financing for Studies and Projects (FINEP). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.


extfieldsofstudy: Biology Medicine

text:
Epigenetic Regulation of Individual Modules of the immunoglobulin heavy chain locus 3′ Regulatory Region

The Igh locus undergoes an amazing array of DNA rearrangements and modifications during B cell development. During early stages, the variable region gene is constructed from constituent variable (V), diversity (D), and joining (J) segments (VDJ joining). B cells that successfully express an antibody can be activated, leading to somatic hypermutation (SHM) focused on the variable region, and class switch recombination (CSR), which substitutes downstream constant region genes for the originally used Cμ constant region gene. Many investigators, ourselves included, have sought to understand how these processes specifically target the Igh locus and avoid other loci and potential deleterious consequences of malignant transformation. Our laboratory has concentrated on a complex regulatory region (RR) that is located downstream of Cα, the most 3′ of the Igh constant region genes. The ~40 kb 3′ RR, which is predicted to serve as a downstream major regulator of the Igh locus, contains two distinct segments: an ~28 kb region comprising four enhancers, and an adjacent ~12 kb region containing multiple CTCF and Pax5 binding sites. Analysis of targeted mutations in mice by a number of investigators has concluded that the entire 3′ RR enhancer region is essential for SHM and CSR (but not for VDJ joining) and for high levels of expression of multiple isotypes. The CTCF/Pax5 binding region is a candidate for influencing VDJ joining early in B cell development and serving as a potential insulator of the Igh locus. Components of the 3′ RR are subject to a variety of epigenetic changes during B cell development, i.e., DNAse I hypersensitivity, histone modifications, and DNA methylation, in association with transcription factor binding. I propose that these changes provide a foundation by which regulatory elements in modules of the 3′ RR function by interacting with each other and with target sequences of the Igh locus.

DISCOVERY OF 3 RR ENHANCERS
The Igh locus spans~3 Mb, beginning near the telomere on murine chromosome 12 with the component variable (V ), diversity (D), and joining (J ) segments of the variable region, followed by the multiple constant region (C H ) genes (Figure 1). My laboratory has been interested in the regulation of the Igh locus's multiple recombination and mutation processes that generate a diverse antigenrecognition repertoire. The entire 3 Igh regulatory region (RR) (enhancers and insulators) has been shown by others to potentially contribute to regulation of variable region formation (VDJ joining) (1). Importantly, it is definitively essential for class switch recombination (CSR) (2), and somatic hypermutation (SHM) (3). This review focuses on our studies on the structure and epigenetic regulation of the 3 RR as it contributes to those antibody diversification processes.
The first transcriptional enhancer identified in mammalian cells was the intronic enhancer of the Igh locus (Eµ), positioned between the 3 -most J H segment and the 5 -most C H region, Cµ [reviewed in Ref. (6)]. Eµ was found to confer expression upon Igh genes when transfected into B cells, and was generally considered to be of critical importance in enabling B cell-specific expression of the Igh locus.
Not surprisingly, perhaps, Eµ was not the only B cell-specific enhancer in the Igh locus. When B cell lines that had deleted Eµ were found to retain the ability to express the Igh gene (7,8), the questions of gene regulation of Igh genes became increasingly provocative. What exactly was Eµ's role? Was Eµ required to initiate Igh expression but not to maintain it? Were there additional enhancers that compensated for the absence of Eµ, and where in the Igh locus might they be found? Examining a rat cosmid, the Neuberger group identified a DNA sequence with B cell-specific enhancer activity that was located~25 kb downstream of Cα, the most 3 of the C H genes (9): this was the first of the 3 enhancers to be identified.
This newly identified enhancer was satisfyingly predicted to account for the aberrant expression of myc in various B cell malignancies when myc was activated as an oncogene via chromosomal translocation with the Igh locus. The translocation breakpoints in switch sequences upstream of C H genes divorced the intronic enhancer from the oncogenic transcription unit, leaving myc www.frontiersin.org FIGURE

| Schematic representation of mouse (A) and human (B)
Igh gene loci with emphasis on the 3 RR. The depicted orientation reflects the centromere (proximal) to telomere (distal) orientation of the genome sequences. DNase I hypersensitive sites hs3A, hs1.2, hs3B, and hs4 (mouse) (red line); and hs3, hs1.2, and hs4 (human) are enhancers of the 3 RR. (A) Hs1.2 is the center of a palindromic region (double-headed arrow) (see text). The 3 RR hs5-8 region (gray hexagon) downstream of hs4 contains CTCF sites interspersed with Pax5 sites, and has insulator activity (blue line). Each of the downstream neighboring non-Igh genes, hole (Tmem121), crip1 and 2, and mta-1, has the same transcriptional orientation (demarcated by purple arrows), which is opposite to that of all the immunoglobulin heavy chain genes. Arcs indicate examples of physical interactions that occur in B cells among the elements of the 3 RR, and between these elements and regulators of germline transcripts (GT) that are located upstream of switch sequences associated with each C H gene, or with the expressed VDJ gene. Transcription factor binding sites for Pax5, NFκB, octamer-binding proteins, and a G-rich DNA binding protein are present in each of the 3 RR enhancers (4) (red line). The CTCF-binding region (blue line) has binding sites for Pax5 and cohesin in addition to CTCF (5). (B) The human 3 Igh enhancers and other features are shown to scale under the scheme of the locus. Numbers represent the actual location within human chromosome 14 (NT_026437.10). This figure has been used with permission from its original publication in Molecular Immunology. Some annotations and modifications have been added.
apparently under the control of the 3 enhancer [Ref. (9) and reviewed by Vincent-Fabert et al. (10)]. A murine homolog of this enhancer was isolated and named hs1.2 for its two DNAse I hypersensitive (hs) sites (11).
Once more than one Igh enhancer was known, i.e., hs1.2 and Eµ, it was natural to ask whether there were additional enhancers. Potential enhancers were identified by DNase I hypersensitivity assays that marked DNA sites that were accessible to transcription factors, and enhancer activity was generally analyzed using transient transfection assays in B cell lines reflecting different stages of B cell development. In a series of experiments by various investigators, using genomic sequences that were only then becoming identified, additional DNase I hypersensitive sites 3 of Cα were detected, primarily using the mouse locus as a model (12)(13)(14)(15). A mouse BAC sequence identified by Roy Riblet was found to encompass the entire 3 RR and the nearest downstream non-Igh genes (AF450245) (16). Similar experiments identified analogous enhancers of the human Igh locus (17)(18)(19), with the enhancer-containing segment of the human 3 RR fully characterized by the Max laboratory (20). Figure 1 shows the general features of the enhancer-containing segments of the 3 RR in both mouse and human. A CTCF/Pax5 binding region with insulator activity is located within the 3 RR downstream of the 3 RR enhancers (hs5-8), and will be discussed further below.

STRUCTURAL FEATURES OF THE 3 RR ENHANCER-CONTAINING REGIONS
There are noteworthy structural features of the 3 RR (note that this region is sometimes referred to as 3 Eα) [reviewed in Ref. (21)(22)(23)(24) (Figure 1)]. (1) Multiple DNase I HS sites with enhancer activity are dispersed in relatively large DNA segments (~28 kb in mouse) -a total of four enhancers in mouse (hs3a, hs1.2, hs3b, and hs4) and three in humans (hs1.2, hs3, and hs4). (2) In humans, there are two individual 3 RRs, one each downstream of Cα1 and Cα2, respectively. They are quite similar to each other in sequence. The orientation of hs1.2 with respect to upstream and downstream sequences is reversed in the two 3 RRs in human, and also between rat and mouse 3 RRs (18). (3) A conserved palindrome feature, although not its specific sequences, flanks the central enhancer -hs1.2 (25). In mouse, the palindrome extends in both directions from hs1.2 to terminate at two virtually identical enhancers, hs3a and hs3b (26). Compared to mouse, the hs1.2 palindromic region in humans is shorter (27). Other species also have a conserved palindrome (28). (4) The hs4 enhancer is located outside and downstream of the palindrome. (5) Individual 3 RR enhancers in a given species, like hs1.2, hs3, and hs4, differ in sequence from each other (except for the virtually identical hs3a and hs3b in rodents). (6) There are limited homologies in enhancer sequence between species (e.g., hs1.2 in human and hs1.2 in mouse) (27).
Other than revealing a conserved palindromic structure, the regions between the 3 RR enhancers show virtually no homology between rodents and humans (27). Nonetheless, other particular sequence features stand out, as identified through genomic Southern analysis and percentage identity (dot-plot) analysis (29). In mouse (and rat), the "palindromic" sequences that separate hs1.2 from each of the terminal enhancers at the end of the palindrome, hs3a and hs3b, contain families of direct and inverted repeats (26) while the human (and chimpanzee) 3 RR revealed several regions of repetitive switch-like sequences (27). More recently, the Cogne laboratory specifically sought and identified multiple switch-gamma 1-like repeats in the mouse and human 3 RRs that were situated close to each of the four enhancers, as well as less distinct although evident, similar sequences in other species, like rabbit and dog (30). In mouse, the 3 RR is highly polymorphic (26,31), showing variations in the lengths of the sequences between the enhancers and the number of repeats in these regions. The hs1.2 region in humans is polymorphic, with varying frequency of alleles in different populations (32). Polymorphic patterns of human hs1.2, i.e., alleles, are associated with different autoimmune disorders, such as lupus (33).
In summary, the 3 RR contains several enhancers located in two structurally distinctive modules -(1) a palindromic region (mouse hs3a-hs1.2-hs3b) and (2) a separate structural unit (hs4). Interenhancer regions reveal repetitive, switch-like sequences potentially of functional significance for the Igh locus. Downstream of the enhancer-containing segment of the 3 RR is additional DNase I hypersensitive sites (hs5-8), which contain CTCF and Pax5 sites and have insulator activity. This hs5-8 region is discussed more fully later.

TRANSCRIPTIONAL REGULATION OF 3 RR ENHANCERS
Relatively coincident with these studies on the Igh locus were studies of genes of the β-globin locus, which, like the Igh genes, are subject to developmental regulation. Multiple DNase I hypersensitive sites, each with enhancer activity, are located upstream of the β-globin genes. This enhancer-containing region is referred to as the locus control region because endogenous deletions here are found to affect expression of distally situated globin genes. Experimental questions on Igh genes have paralleled experiments carried out in the β-globin locus and in other loci [recent review in Ref. (34)]: (1) what are the protein factors that bind to and regulate these Igh enhancers? Can they account for B cell-specific regulation? (2) Are the different enhancers similar in their function, their relative "strength" and their activity on the target Igh locus? How do these enhancers work together? Can these questions be answered not only for in vitro, cellular conditions but also within the animal context itself?
Electrophoretic mobility shift assays (EMSA) provided a tool to identify proteins with the potential to bind enhancers. Using nuclear extracts, we identified a B cell-specific binding protein with sites throughout the Igh locus, including hs1.2 (35,36). Based on its cellular expression pattern, we predicted that this protein was B cell-specific activating protein (BSAP), now called Pax5, as originally identified by the Busslinger laboratory (37); and various observations were consistent with that prediction (36). Additional 3 RR binding factors were identified leading to recognition of a quartet of proteins -Pax5, octamer-binding proteins, NFκB, and a G-rich DNA binding protein -that worked together on each of the murine 3 enhancers (4) (Figure 1).
Our experiments revealed that BSAP/Pax5 bound to each of the mouse 3 RR enhancers, where it could act as a repressor or activator (4). For example, mutational inactivation of a BSAP/Pax5 binding site of hs1.2 resulted in an increase in hs1.2 enhancer activity upon transfection into B cell lines that expressed endogenous BSAP/Pax5 (36). This finding showed that "BSAP" could be a repressor of hs1.2. The enhancer activity resulting from mutation of the BSAP/Pax5 binding site depended on the binding of the remaining transcription factors (38). A similar outcome applied not only to BSAP/Pax5 but also to each individual component of this quartet, as individual mutation of other binding sites each resulted in an increase in hs1.2 enhancer activity (38). Collectively, then, this quartet worked in concert to repress hs1.2, while it activated hs4, revealing that individual 3 RR enhancers had different B cell-specific activities (4). Interestingly, human 3 enhancers do not have Pax5 binding sites, suggesting that humans and mice have different modes of 3 RR regulation. However, it is not known how or whether the differences in Pax5 binding affect the function of the 3 RR in human and mouse. Human hs4 showed binding to octamer-binding proteins, NFκB, and YY1 under some circumstances, and human hs1.2 to octamer-binding proteins and Spi1, and to NFκB for some of the polymorphic hs1.2 variants (33,(39)(40)(41). Other 3 enhancer binding proteins have also been identified (40,42).

ADDITIONAL MODULE DOWNSTREAM OF 3 RR ENHANCERS: THE CTCF-BINDING REGION OF THE 3 RR
Had we identified all the regulators of the 3 RR [reviewed in Ref. (43)]? Various observations suggested that additional functional motifs were present beyond hs4. For example, the nearest non-Igh genes downstream of hs4, i.e., hole (Tmem121), Crip, and mta-1, each had a transcriptional orientation that was opposite to that of all of the V, D, J, and C H elements of the Igh locus. This backto-back orientation led us to predict that a terminus of the Igh locus might be located in this segment (16). In fact, we found additional DNase I hypersensitive sites downstream of hs4, which included hs5, 6, and 7, and has now been extended to include a CTCF-binding site, named hs8 (44,45). Discussions via Sandy Morse with Victor Lobanenkov introduced us to CTCF as a mammalian insulator (46), and we predicted that CTCF sites might be present in this region. EMSA of 50 overlapping DNA sequences with recombinant CTCF revealed a CTCF-binding module of the 3 RR [recently referred to as 3 CBE, CTCF-binding elements (47)], and transient transfection assays confirmed functional insulator activity in the absence of any enhancer activity (44). The CTCF sites are interspersed with Pax5 binding sites within the hs5-8 region (5).

FUNCTIONAL ANALYSIS OF 3 RR REGULATORY ELEMENTS THROUGH TARGETED DELETIONS 3 RR ENHANCERS
Analysis of the function of the endogenous 3 RR began with the description of spontaneous 3 RR deletion mutants identified in cell lines. For example, a low-producing variant (LP1.2) of a mouse plasmacytoma cell line was shown to have sustained a deletion of the entire 3 RR (15,48). This suggested that the 3 RR supported high levels of Igh expression in plasma cells. With the development of both transgenic (49,50) and endogenous models, the 3 RR has been over many years the focus of multiple targeted deletions [reviewed in Ref. (24)]. Although the efficiency of targeting of this 3 RR region has been hampered, perhaps because of its complex structure, there has been gradual, ongoing success. Deletion of individual enhancers had no significant phenotypic consequence implying that the remaining elements, each constellation of which is different, can provide 3 RR function. Deletion of two or more enhancers gave phenotypic consequences of varying degrees, e.g., deletion of hs3b and hs4 together eliminated class switching to all isotypes except for IgG1 (51). Now, there are mice from which the entire~28 kb 3 enhancer region has been deleted (2), and these have provided a clear demonstration of the potency of the complete 3 RR enhancer region. Without 3 RR enhancers, mice are able to express only reduced levels of IgM at the plasma cell stage, they lack class switch recombination to all isotypes (2) and they are deficient in SHM (3). There is no impairment of VDJ joining (52). Studies by the Cogne laboratory showed that 3 RR enhancers hs1.2 and hs4 were transcriptionally active in B cells, and hs1.2 could be targeted by AID, revealed by detectable although relatively low levels of SHM (30). These AID-dependent mutational and recombination processes involving the 3 RR with Sµ resulted in deletion of the entire IgC H region and B cell death (30). This revealed an ongoing competition between generation of live class switched mutated B cells and dead B cells, termed "locus suicide" by the authors. In all, these data strongly show that the 3 RR enhancer region (hs3a-hs4) is critical for CSR and SHM and functions through synergy among the multiple 3 RR enhancers.

CTCF/Pax5 BINDING REGION
Similar to the analysis of the 3 RR enhancers, we used targeted deletion to examine the effect of the CTCF/Pax5 binding region of the 3 RR on Igh expression (53). We were surprised to find that deletion of the 8 kb hs5-7 region resulted only in a mild phenotype. There was an increase in recombination of the most proximal D gene, DQ52, to J H 3, a reduction in contraction between distal V H J558 and proximal V H 7183 genes, and an~2-fold increase in V H 7183 gene usage-all suggesting a modest contribution of the CTCF/Pax5 region of the 3 RR to steps in VDJ joining. Nonetheless, upon targeted deletion of hs5-7, there were essentially normal levels of Igh recombination for V H formation and CSR, normal levels of Igh expression and allelic exclusion, and B cell development was unaffected. One possibility to account for these observations was our finding that two CTCF sites remained downstream of the seven sites that had been deleted, in the segment called "38" in the manuscript and now termed hs8. In addition, CTCF sites are associated with each of the downstream non-Igh genes. This suggests that a full deletion of CTCF sites in this region might reveal a more extensive phenotype.

PHYSICAL INTERACTION OF THE 3 RR WITH ITS TARGET SITES IN THE Igh LOCUS
V H promoters and I promoters that drive germline transcription (GT) for CSR are situated quite far in a linear distance from the 3 RR; yet it is implied that they all function together through physical interaction (Figure 1). In fact, our finding of an inversion of the Igh locus in a variant of the MPC11 plasma cell line that extended from the V H through to the 3 RR (54) was indicative of a loop formed by interactions between DNA sequences at V H and 3 RR inversion breakpoints (55). Chromosome conformation capture (3C) technology has been important in documenting interactions that occur in a cellular context, by fixing these by formaldehyde crosslinking, cutting away intervening DNA stretches with restriction enzymes, ligating remaining neighboring fragments, and documenting these interactions by PCR with selected primer pairs. Using 3C, we sought to confirm the dependence of H chain expression in plasma cells on an intact 3 RR (55): indeed, we found that the 3 RR interacted with the J H -Eµ region. This interaction took place even in cells in which Eµ was deleted. Not only was there interaction between the 3 RR and its target sequences, but there was also interaction among component 3 RR enhancers and insulators, including the CTCF/Pax5 binding unit (hs5-8) (3 CBE). Notably, substitution of hs1.2 by the NeoR gene in a variant of the MPC11 plasma cell line resulted in loss of Igh expression (56) and abrogation of the 3 RR loop structure; i.e., looping was essential for Igh expression. Collectively, these experiments show that the entire 3 RR, including enhancers and insulators, works as a physical unit.
The Kenter laboratory focused on normal spleen cells stimulated to undergo switching for their 3C experiments (57). They reported that in resting B cells, but not in T cells, the 3 RR interacted with the VDJ -Eµ region. Upon LPS ± IL4 stimulation of splenic B cells, they found that specific I/switch regions that drive GT were brought into the VDJ -3 RR loop. Splenic B cells from mice that were unable to carry out GT and CSR as a result of the combined deletion of the hs3b and hs4 3 RR enhancers failed to show these interactions.
Interestingly, mice bearing the combined deletion of hs3a and hs3b (58) had no defects in GT or CSR, but interactions between the 3 RR and I/switch regions that ordinarily were cytokinedependent were already at an induced level in the hs3a/hs3b deleted mouse. Collectively, these data provided support for a loop interaction model by which H chain expression and CSR are dependent on physical interaction of the 3 RR with target Igh sequences.
Presuming that the CTCF/Pax5 region (3 CBE) of the 3 RR interacts with other Igh-associated CTCF sites, such candidate CTCF target sites have been defined by colleagues using array analysis and genome-wide ChIP (59,60). Moving 3 -5 upstream of the 3 RR, there are no CTCF sites in C H and J H genes until those detected in the 5 D H segment (1, 61). The V H region contains multiple CTCF sites, some associated with specific Pax5 binding sites, termed PAIR (62). Recent studies showed that the 3 RR CTCF/Pax5 binding region interacts with the two D H -associated CTCF sites (IGCR1), targeted deletion of which showed their critical role in appropriate regulation of VDJ joining (1). We might, therefore, predict that the deletion of the complete 3 RR CTCFbinding region with which IGCR1 interacts would have a major influence on VDJ joining.
Genome-wide analyses have been used to identify interactions between 3 RR elements, e.g., hs3b and hs8, and the rest of the Igh locus (47,63). Studies with 4C (47) have identified Pax5dependent interactions in Rag −/− pro-B cells where V H genes are poised to contract prior to VDJ joining. These 3 RR interactions are maintained even when individual regulatory elements, such as Eµ, IGCR1, and the entire 3 RR enhancer region from hs3A to hs4, are deleted. This implies some independent means of interaction, perhaps involving retained 3 RR CTCF-binding sites, or synergy among regulatory elements that enables continued interactions even when single elements are deleted. Notably, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) of long-range chromatin interactions has revealed interactions of the 3 RR with transcribed Igh genes in B cells activated by LPS + IL4 that are not detected in embryonic stem (ES) cells (63), in accord with developmental differences in 3 RR interactions.

WHAT PROTEINS SUPPORT LOOP FORMATION INVOLVING THE 3 RR?
To tackle this question, we used lentiviral delivery of shRNA directed against expression of CTCF, Oct-2, and a coactivator of Oct-2, namely Pou2af1 [i.e., OCAB, OBF-1, BOB1, each of which, as proteins that bound to the 3 RR, was a candidate for loop promotion (64)]. Despite reduced levels of these proteins in response to shRNA, no alterations in loop formation or Igh expression were observed in the mouse MPC11 plasmacytoma cell line we examined. Interestingly, in contrast to our observations, there was a report that reduction of Pou2af1 expression in the same plasma cell line using step-wise selection of a cell line containing two independent shRNA's led to reduction in Igh expression and 3C interactions (65). Accordingly, we concluded (64) that there are likely some conditions under which Pou2af1 can facilitate 3C interactions involving the 3 RR, among them the possibility that this approach had selected a variant cell line that was dependent on Pou2af1. Potentially, under other conditions, 3C interactions depend on a protein other than Pou2af1, or on synergy involving more than one protein, or on the low levels of individual proteins remaining from inefficient knock down.

EPIGENETIC REGULATION OF 3 RR DURING B CELL DEVELOPMENT
Over several years, we have worked to know how components of the 3 RR are individually regulated, enabling them, in turn, to act together as a unit in CSR and SHM, and potentially also for VDJ joining. "Active" DNA segments are generally associated with DNase I hypersensitivity, specific histone marks, and DNA demethylation, which will be discussed individually below.

HISTONE MARKS
Non-B cells that were studied had varying profiles of histone acetylation of the 3 RR (66). For example, a macrophage cell line had active marks of AcH3 and AcH4, while T cells lacked both AcH3 and AcH4. In mouse erythroleukemic (MEL) cells, the CTCFbinding region, but not 3 RR enhancers, was associated with acetylated histones. Therefore, active histone marks of the 3 RR were not necessarily B cell-specific. In B cells, modules of the 3 RR sequentially acquire active histone marks during development (44). The CTCF-rich region first acquires these marks, followed progressively 5 to hs4 and then to the palindromic enhancers. ChIP experiments of the 3 RR showed that in pro-B cells, hs5 and hs6 of the CTCF-binding region were associated with AcH4 and low levels of AcH3, while hs4 was also associated with AcH4 but not with AcH3. In pre-B cells, the entire hs4-8 region was associated with both acetylated marks; and then in B cells, the palindromic enhancers also acquired these marks. These observations suggest step-wise activation of different modules of the 3 RR during B cell development, raising the possibility that specific combinations of 3 RR modules, involving palindromic enhancers, hs4, and the CTCF-binding region, have specific functional contributions.

DNA DEMETHYLATION
Early studies had bluntly monitored DNAse I hypersensitivity and DNA demethylation in the region now shown to contain the entire 3 RR (12), but as the complete 3 RR structure became known, a finer analysis was made possible (66). The CTCFbinding region was generally constitutively demethylated in all cell types analyzed. In several sources of non-B cells, the 3 RR's palindromic region was demethylated without a corresponding association with active histone marks. However, in B cells, three epigenetic marks -DNase I hypersensitivity, active histone marks, and DNA demethylation -were collectively engaged; and progressive demethylation paralleled acquisition of active histone marks. Hs4 and downstream CTCF-binding sites were DNase I hypersensitive and demethylated, as assessed by relative sensitivity to the methylation-sensitive isoschizomers HpaII and MspI, beginning in pro-B cells and extending throughout B cell development. The palindromic region became hypersensitive and partially demethylated only later in B cell development. We found upon comparison www.frontiersin.org of wild-type with Pax5-deficient pro-B cells in which Pax5 expression could be reinitiated, that in the absence of Pax5, there was scattered demethylation of the palindromic region. Re-expression of Pax5 could promote methylation of the palindromic region. These findings suggested that Pax5 was a critical factor in over-all B cell-specific epigenetic regulation of the 3 RR. In other studies involving targeted deletion of linker histone H1 genes, we found that linker histone H1 was also important for the methylation of hs4-hs8 in wild-type ES cells.

EPIGENETIC REGULATION OF 3 RR DURING CSR
Despite the critical role of the 3 RR in CSR (and SHM), there are no apparent changes in histone marks in the 3 RR during switching in cultured cells (44). This implies that the 3 RR in resting B cells is already epigenetically poised for its activity for CSR. Instead, we have observed dynamic changes in Pax5 interaction over time in response to LPS stimulation (5). In resting B cells, Pax5 bound hs4 and the 3 RR was mostly methylated. When GT was at a peak at~48 h after commencement of LPS stimulation, Pax5 binding had shifted from hs4 upstream to hs1.2 and downstream to hs7. By 72 h, when CSR was essentially complete, Pax5 had resumed its beginning position at hs4. ChIP analysis of cell sources that were deficient in GT or in CSR showed differences in these Pax5 binding patterns, in accord with the notion that shifts in Pax5 binding reflected mechanisms by which the 3 RR supported GT and CSR.
We have proposed a model by which mouse 3 RR enhancers form a scaffold through which Pax5 can interact. Deletion of any individual enhancer leaves residual Pax5 sites in each of the remaining enhancers and in the CTCF/Pax5 binding region, which allows the 3 RR to remain functional.

ON THE HORIZON: EXPERIMENTS ON THE 3 RR HOW DOES THE 3 RR FUNCTION?
The 3 RR enhancer region is critical for GT and CSR, and SHM, and the CTCF/Pax5 binding region could contribute to VDJ joining. We predict that a scaffold formed by modules of the 3 RR supports physical interactions with target sequences required to accomplish these various activities. With deletions of individual 3 RR enhancers having little phenotypic consequence, one can ask how many different structural solutions are there to 3 RR activity? Why are there multiple modules? Do the changes in epigenetic alterations of 3 RR modules that occur during development indicate specific activities for individual modules? Could the 3 RR help target DNA repair proteins involved in SHM or CSR? What roles does the 3 RR share with other cis acting sequences that are critical for SHM, such as those in the light chain loci? What is distinctive about CSR, which is specific to the Igh locus? What is the role of the conserved palindrome? How did the 3 RR evolve? What are the species-specific aspects of 3 RR regulation?
Recent experiments on the β-globin LCR have identified hierarchical regulation by multiple transcription factors (67). Binding of individual factors can provide a foundation for subsequent binding of other factors. Experiments of these types on the 3 RR could be equally informative in answering how this region functions. Indeed, more complete deletion of 3 RR CTCF-binding sites, and targeted deletions and mutations in 3 RR modules would also be informative. The new CRISPR technology (68) should facilitate these constructions and provide answers to many questions.

IS THE CTCF/Pax5 BINDING REGION THE TERMINUS OF B CELL-SPECIFIC REGULATORS OF THE Igh LOCUS?
A persuasive set of experiments says "yes" to the role of the 3 RR as a terminus of Igh regulation via chromatin accessible marks. These experiments have shown that active chromatin marks extend unilaterally 3 -5 from the 3 RR as far as 450 kb when Igh-myc translocations are assayed in endemic Burkitt lymphoma samples (69). This supports the identification of the CTCF/Pax5 region as a functional insulator of the Igh locus. In addition, 4C studies have implicated hs8 as a 3 boundary for Igh locus interactions (47). Yet, as described below, there is a replicative terminus further downstream, raising the possibility of additional Igh locus regulators.

ROLE OF REPLICATIVE TERMINUS DOWNSTREAM OF CTCF/Pax5 REGION
Our experiments in collaboration with the Schildkraut laboratory identified an origin of an~500 kb Igh temporal replicative transition region in MEL (non-B) cells. DNA replication initiates~45 kb downstream of the CTCF/Pax5 module of the 3 RR between crip1 and Tmem121 (Figure 1) and extends progressively 3 -5 throughout S phase to replicate the 3 RR, C H , J H , D H , and most proximal V regions (70,71). All V H genes replicate late in S phase. In pre-B cells, the entire Igh locus replicates early in S phase, indicating the firing of multiple origins that are ordinarily quiescent in non-B cells (71). In B cells, a temporal transition region is again apparent, but origins appear to be closer to or within the 3 RR, suggesting that the replication landmark is flexible (72). These data implied that Igh replication is under B cell-specific developmental control. In MEL cells, the downstream origin, which is located beyond the limits of the 3 RR, may be a terminus for Igh locus regulation. It is of interest that changes in Igh DNA replication are associated with changes in nuclear location of the Igh locus (71, 73) but can be independently regulated (74). In pro-and pre-B cells, the Igh locus is located away from the nuclear periphery, while in MEL and ES cells, and in B and plasma cells, the Igh locus is located at the nuclear periphery. These observations raise the question of whether there are finer demarcations of nuclear subcompartments generally associated with the Igh locus and the 3 RR? What regulates the movement of the locus from one position to another?

IS THE 3 RR INVOLVED IN INTER-CHROMOSOMAL INTERACTIONS?
The mechanism by which recurrent translocations involving the Igh locus take place and the role of the 3 RR are under close scrutiny (75,76). Epner and colleagues have reported a role for the 3 RR in transvection involving allelic interactions (77). Further, our studies have identified a region between hs4 and hs5 that has a methylation signature indicative of allelic expression (66). The Skok laboratory has observed allelic interactions in Igh genes, which are evident during steps of VDJ joining (78). The various 3C technologies and their broader counterparts, as noted in part above (47,63), should be very informative about the contribution of the 3 RR to genetic domains of interaction.

IS THERE A ROLE OF THE 3 RR AS A SUPER ENHANCER?
Recent genome-wide studies have reported "super enhancers" (79,80), DNA segments substantially larger than other "enhancer" regions and identified as having strong binding sites for BRD4, a member of the bromodomain and extraterminal (BET) subfamily of human bromodomain proteins, and for the Mediator complex with which BRD4 interacts. By these criteria, the 3 RR was predicted to be a super enhancer in multiple myeloma cells (79), where it upregulates expression of the myc oncogene to which it is juxtaposed as a result of a chromosomal translocation. An inhibitor of BRD4, JQ1, can lead to downregulation of myc expression in multiple myeloma cells. However, myc appears to be suppressed by JQ1 regardless of whether it is associated with Igh sequences through translocation (81), potentially via B cellspecific enhancers of myc (47,63)


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Recently published Streptomyces genome sequences

Many readers of this journal will need no introduction to the bacterial genus Streptomyces, which includes several hundred species, many of which produce biotechnologically useful secondary metabolites. The last 2 years have seen numerous publications describing Streptomyces genome sequences (Table (Table1),1), mostly as short genome announcements restricted to just 500 words and therefore allowing little description and analysis. Our aim in this current manuscript is to survey these recent publications and to dig a little deeper where appropriate. The genus Streptomyces is now one of the most highly sequenced, with 19 finished genomic sequences (Table (Table2)2) and a further 125 draft assemblies available in the GenBank database as of 3rd of May 2014; by the time this is published, no doubt there will be more. The reasons given for sequencing this latest crop of Streptomyces include production of industrially important enzymes, degradation of lignin, antibiotic production, rapid growth and halo-tolerance and an endophytic lifestyle (Table (Table11).

Table 1

Recent genome publications (2013 and 2014) for Streptomyces species

Table 2

Completely sequenced Streptomyces species genome sequences available in GenBank as of 29 April 2014

Mining genomes for secondary metabolism gene clusters
Given the strong emphasis on secondary metabolism in Streptomyces genomics research, it is timely that version 2.0 of antiSMASH has been released and published (Blin et al., 2013). This computational tool has become a de facto standard for mining secondary metabolism gene clusters in genome sequences. Version 2.0 is completely revamped and, significantly, can now be used with highly fragmented draft-quality genome sequences whereas the previous version only worked well with finished genomes. Clearly, this is of immense importance to the discovery of novel metabolites in the ever-expanding database of streptomycete draft-quality genome sequences. For example, antiSMASH 2.0 analysis of the Streptomyces roseochromogenes subsp. oscitans DS 12.976 genome sequence revealed 43 new gene clusters in addition to recovering the already known clorobiocin gene cluster (Ruckert et al., 2014).

The genome sequence of Streptomyces gancidicus strain BKS 13–15 was published before antiSMASH 2.0 became available. The authors state that seven genes mapped on to the streptomycin biosynthesis pathway based on gene-by-gene sequence similarities (Kumar et al., 2013) against homologues of genes in KEGG pathways (Kanehisa et al., 2012). However, we found no bioinformatic evidence for a streptomycin biosynthesis pathway encoded in this genome, although our antiSMASH 2.0 search did find 38 putative gene clusters. In common with many other pathways for secondary metabolism, genes for production of the aminoglycoside streptomycin are organized into a cluster of contiguous genes. The nucleotide sequences of at least two such clusters are available (GenBank accessions {"type":"entrez-nucleotide","attrs":{"text":"GU384160","term_id":"288549237","term_text":"GU384160"}}GU384160 and {"type":"entrez-nucleotide","attrs":{"text":"AJ862840","term_id":"62896300","term_text":"AJ862840"}}AJ862840 from Streptomyces platensis and Streptomyces griseus respectively). Our blastn searches (using these two cluster sequences as queries) failed to detect a complete streptomycin gene cluster in the S. gancidicus genome, but there were some regions of sequence similarity on a 111 kb contig (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"AOHP01000057","term_id":"455649599","term_text":"AOHP01000057"}}AOHP01000057). An antiSMASH 2.0 search failed to find any aminoglycoside biosynthetic cluster in this genome. We are not aware of any experimental evidence that this strain produces the aminoglycoside streptomycin and conclude that these seven genes highlighted by the authors (Kumar et al., 2013) most probably encode components of another, perhaps novel, pathway. This illustrates the value of the antiSMASH 2.0 tool, which has the potential to discover new pathways, rather than relying on similarity to the pathways already represented in the KEGG database (and therefore, by definition, not novel).

The case of Streptomyces species strain Mg1 (Hoefler et al., 2013) illustrates another consideration when mining bacterial genome sequences for secondary metabolism gene clusters. Many of the recently published Streptomyces genome sequences are assembled from massively parallel sequencing platforms such as 454 GS-FLX and Illumina HiSeq. The short sequence reads (typically less than 450 bp) and relatively high error rates associated with these platforms can lead to rather fragmented and/or incomplete genome assemblies. The situation is not helped by the biased sequence composition (approximately 70% G + C) of Streptomyces DNA. Furthermore, non-ribosomal peptide synthases (NRPS) and polyketide synthetases (PK) are long, modular proteins made up of many repeated domain units. This means that the genes encoding these key enzymes can be particularly difficult to assemble accurately from short sequence reads. To overcome this issue, the authors of the Mg1 genome project (Hoefler et al., 2013) exploited the PacBio SMRT sequencing technology, which provides sequences reads of several Kb in length, meaning that an entire PK or NRPS gene could be represented on a single sequence read, thus avoiding the difficulties of assembling repetitive sequence from short fragments. They also generated an assembly of the same genome based on 454 GS-FLX and Illumina HiSeq. The results were striking: more than 90% of the genome was represented in a single contig of 7.8 Mb in the PacBio-based assembly and the PacBio-based assembly was 19.9% longer than the 454/Illumina-based one (8 705 754 versus 7 260 368 bp). As the authors point out, this implies that more than 1 Mb of sequence in the PacBio-based assembly is missing from the 454/Illumina-based one, as can be seen in Fig. Fig.1A.1A. However, the 454/Illumina-based assembly is not simply a subset of the PacBio-based one; as illustrated in Fig. Fig.1B,1B, a substantial portion of the 454/Illumina-based assembly is missing from the PacBio assembly. Although it is by no means certain which assembly is more ‘correct’, it might be possible to generate a more complete genome assembly by reconciling the two different assemblies.

Figure 1

Comparison of two different genome assemblies for Streptomyces strain Mg1, one based on PacBio sequence data and the other based on 454 and Illumina sequence data. A illustrates alignment of both the assemblies against the PacBio-based assembly. B illustrates ...

Fragmentation and incompleteness of a genome assembly has implications for discovery of secondary metabolism gene clusters. In Fig. Fig.1C,1C, we show a putative NRPS gene cluster detected apparently intact in a single contig of the PacBio-based sequence assembly identified by antiSMASH 2.0. Searching the 454/Illumina-based assembly reveals two incomplete fragments of the gene cluster, lying on two different contigs, and with part of the cluster apparently absent. Although we should be cautious about extrapolating too much from this single anecdotal example, the evidence suggests that longer read lengths can be very valuable in genome mining for secondary metabolism clusters.

Introduction
Many readers of this journal will need no introduction to the bacterial genus Streptomyces, which includes several hundred species, many of which produce biotechnologically useful secondary metabolites. The last 2 years have seen numerous publications describing Streptomyces genome sequences (Table 1), mostly as short genome announcements restricted to just 500 words and therefore allowing little description and analysis. Our aim in this current manuscript is to survey these recent publications and to dig a little deeper where appropriate. The genus Streptomyces is now one of the most highly sequenced, with 19 finished genomic sequences (Table 2) and a further 125 draft assemblies available in the GenBank database as of 3rd of May 2014; by the time this is published, no doubt there will be more. The reasons given for sequencing this latest crop of Streptomyces include production of industrially important enzymes, degradation of lignin, antibiotic production, rapid growth and halo-tolerance and an endophytic lifestyle (Table 1).

Mining genomes for secondary metabolism gene clusters
Given the strong emphasis on secondary metabolism in Streptomyces genomics research, it is timely that version 2.0 of antiSMASH has been released and published (Blin et al., 2013). This computational tool has become a de facto standard for mining secondary metabolism gene clusters in genome sequences. Version 2.0 is completely revamped and, significantly, can now be used with highly fragmented draft-quality genome sequences whereas the previous version only worked well with finished genomes. Clearly, this is of immense importance to the discovery of novel metabolites in the ever-expanding database of streptomycete draft-quality genome sequences. For example, antiSMASH 2.0 analysis of the Streptomyces roseochromogenes subsp. oscitans DS 12.976 genome sequence revealed 43 new gene clusters in addition to recovering the already known clorobiocin gene cluster .
The genome sequence of Streptomyces gancidicus strain BKS 13-15 was published before antiSMASH 2.0 became available. The authors state that seven genes mapped on to the streptomycin biosynthesis pathway based on gene-by-gene sequence similarities  against homologues of genes in KEGG pathways (Kanehisa et al., 2012). However, we found no bioinformatic evidence for a streptomycin biosynthesis pathway encoded in this genome, although our antiSMASH 2.0 search did find 38 putative gene clusters. In common with many other pathways for secondary metabolism, genes for production of the aminoglycoside streptomycin are organized into a cluster of contiguous genes. The nucleotide sequences of at least two such clusters are available (GenBank accessions GU384160 and AJ862840 from Streptomyces platensis and Streptomyces griseus respectively). Our BLASTN searches (using these two cluster sequences as queries) failed to detect a complete streptomycin gene cluster in the S. gancidicus genome, but there were some regions of sequence similarity on a 111 kb contig (GenBank: AOHP01000057). An antiSMASH 2.0 search failed to find any aminoglycoside biosynthetic cluster in this genome. We are not aware of any experimental evidence that this strain produces the aminoglycoside streptomycin and conclude that these seven genes highlighted by the authors  most probably encode components of another, perhaps novel, pathway. This illustrates the value of the antiSMASH 2.0 tool, which has the potential to discover new pathways, rather than relying on similarity to the pathways already represented in the KEGG database (and therefore, by definition, not novel).
The case of Streptomyces species strain Mg1 (Hoefler et al., 2013) illustrates another consideration when mining bacterial genome sequences for secondary metabolism gene clusters. Many of the recently published Streptomyces genome sequences are assembled from massively parallel sequencing platforms such as 454 GS-FLX and Illumina HiSeq. The short sequence reads (typically less than 450 bp) and relatively high error rates associated with these platforms can lead to rather fragmented and/or incomplete genome assemblies. The situation is not helped by the biased sequence composition (approximately 70% G + C) of Streptomyces DNA. Furthermore, non-ribosomal peptide synthases (NRPS) and polyketide synthetases (PK) are long, modular proteins made up of many repeated domain units. This means that the genes encoding these key enzymes can be particularly difficult to assemble accurately from short sequence reads. To overcome this issue, the authors of the Mg1 genome project (Hoefler et al., 2013) exploited the PacBio SMRT sequencing technology, which provides sequences reads of several Kb in length, meaning that an entire PK or NRPS gene could be represented on a single sequence read, thus avoiding the difficulties of assembling repetitive sequence from short fragments. They also generated an assembly of the same genome based on 454 GS-FLX and Illumina HiSeq. The results were striking: more than 90% of the genome was represented in a single contig of 7.8 Mb in the PacBio-based assembly and the PacBio-based assembly was 19.9% longer than the 454/Illumina-based one (8 705 754 versus 7 260 368 bp). As the authors point out, this implies that more than 1 Mb of sequence in the PacBio-based assembly is missing from the 454/Illumina-based one, as can be seen in Fig. 1A. However, the 454/Illumina-based

Digesting wood: Streptomyces viridosporus T7A
Streptomycetes may have important applications other than production of secondary metabolites, for example lignin degradation (Thomas and Crawford, 1998;Bugg et al., 2011;Brown and Chang, 2014). The aromatic polymer lignin is a major component of plant material and there is significant interest in organisms that can break down lignocellulose waste materials to generate useful products such as bioethanol (Bugg et al., 2011). Digestion of lignin is important not only because it can comprise up to 30% of plant biomass but also because its removal is necessary to facilitate degradation of Fig. 1. Comparison of two different genome assemblies for Streptomyces strain Mg1, one based on PacBio sequence data and the other based on 454 and Illumina sequence data. A illustrates alignment of both the assemblies against the PacBio-based assembly. B illustrates both the assemblies aligned against the 454/Illumina-based assembly. C illustrates a novel secondary-metabolism gene cluster identified by antiSMASH 2.0 (Blin et al., 2013) in both assemblies. The entire cluster is recovered intact in the PacBio-based assembly but it is split across two different contigs in the 454/Illumina-based assembly and part of the middle of the cluster is missing. Alignments in A and B were generated using Basic Local Alignment Search Tool Nucleotide tool BLASTN (Altschul et al., 1990) and visualized using the BLAST Ring Image Generator (BRIG) (Alikhan et al., 2011). The innermost ring indicates the genomic position. The next ring is a plot of G + C content. The remaining five concentric rings indicate the presence or absence of BLASTN hits at that position, with one ring corresponding to each of the five indicated genome assemblies. To aid clarity, each ring is represented in a different colour. Positions covered by BLASTN alignments are indicated with a solid colour; whitespace gaps represent genomic regions not covered by the BLASTN alignments. The graphics in C were cut and pasted directly from the antiSMASH output.
hemicellulose and cellulose. The enzymology of lignin degradation is best understood in fungi, but it has become apparent that a number of bacterial species also have this capability (Brown and Chang, 2014). For example, S. viridosporus T7A is able to solubilize lignin, probably via the action of at least one extracellular peroxidase (Thomas and Crawford, 1998). A complete genome sequence is now available for this strain (Davis et al., 2013), revealing a number of genes encoding candidate lignin-degrading enzymes (see Table 3). This species is closely related to Streptomyces ghanaensis for which a genome sequence is also available (GenBank: ABYA00000000) and which is notable for its production of the antibiotic moenomycin A (Subramaniam-Niehaus et al., 1997;Ostash et al., 2007;2009). Most of the candidate lignin metabolism genes in Table 3 are also conserved in S. ghanaensis. We are not aware of any published reports of S. ghanaensis being able to degrade lignin, but it would be interesting to experimentally test whether it has this capability; if it does not, then comparative genomics between these closely related strains might reveal novel genetic determinants of lignin degradation.

Genome size: Streptomyces violaceusniger
Among bacteria, streptomycetes have some of the largest genomes, typically within the range of 8.7 Mbp to 11.9 Mbp . However, the recently reported genome sequence of S. violaceusniger strain SP6 weighs in at just 6.4 Mb (Chen et al., 2013) and that of Streptomyces albus J1074 6.8 Mb (Zaburannyi et al., 2014). Although both sets of authors (Chen et al., 2013;Zaburannyi et al., 2014) claim theirs as the smallest reported genome of any streptomycete, in fact that record is held by the previously sequenced Streptomyces somaliensis strain DSM 40738, a pathogenic strain isolated from a human infection (Kirby et al., 2012). The assembly of this genome was just 5.18 Mbp in length; the authors of that study claim that this is consistent with results from pulsed-field gel electrophoresis. Our multilocus sequence analysis (data not shown) reveals that strain SPC6, also known as Streptomyces thermolilacinus SPC6, is not closely related to S. violaceusniger strain TU 4113 (GenBank: CP002994), which has an 11.14 Mbp genome. Rather, strains SPC6 and DSM 40736 are closely related and fall within a clade with several other strains for which draft genomes are available and with Streptomyces venezuelae for which a complete finished genome sequence is available (Pullan et al., 2011). Figure 2 shows the sizes of these genomes. It appears that genome reduction may have occurred at least twice in this clade: once in a common ancestor of SPC6 and DSM 40738, and also independently in an ancestor of strain CNT372 (GenBank: ARHT00000000). It is even possible that genome reduction has occurred independently in SPC6 and DSM 40738 as Fig. 2C reveals differences as well as similarities in gene conservation with respect the S. venezuelae reference sequence. Evidently, genome reduction has also occurred in S. albus strain J1074 (Zaburannyi et al., 2014), which is not closely related to this clade. In this strain, the  Fig. 2. Variation in genome size among Streptomyces somaliensis and its close relatives. A shows a section of a maximum-likelihood phylogenetic tree based on aligned sequences of five housekeeping genes (atpD, gyrB, recA, rpoB, trpB) extracted from draft genome sequence assemblies or, in the case of S. venezuelae, finished genome sequence, which is indicated by the black triangle. The tree was generated using MEGA6 (Tamura et al., 2013). B indicates the length of each genome assembly. C illustrates alignments of each genome assembly against the S. venezuelae reference genome, which consists of a single linear chromosome. Alignments were generated using Basic Local Alignment Search Tool Nucleotide tool BLASTN (Altschul et al., 1990) and visualized using the BLAST Ring Image Generator (BRIG) (Alikhan et al., 2011). The innermost ring indicates the genomic position. The next ring is a plot of G + C content. The remaining five concentric rings indicate the presence or absence of BLASTN hits at that position, with one ring corresponding to each of the five indicated genome assemblies.
To aid clarity, each ring is represented in a different colour. Positions covered by BLASTN alignments are indicated with a solid colour; whitespace gaps represent genomic regions not covered by the BLASTN alignments. reduction seems to have been achieved by deletion of duplicated genes. The evolutionary driver for genome reduction in streptomycetes is unclear, although it might not be mere coincidence that the smallest genome reported so far is from a pathogen, namely S. somaliensis (Kirby et al., 2012), and evolution of pathogenesis is often associated with genome reduction (Toft and Andersson, 2010).

The future of Streptomyces genomics
The availability of cheap sequencing has led to the generation of numerous genome sequences for Streptomyces and related species [e.g. ] with the objective of discovering novel metabolic products. However, sequencing the genome and discovering novel gene clusters is just the beginning; many of the metabolic products of these gene clusters are 'cryptic', not being expressed under normal laboratory conditions. Productive 'genome mining' requires either genetic modification of the cluster to force expression or cloning and expression of the cluster in a heterologous host (Gomez-Escribano and Bibb, 2014). The value of this approach, even starting from rather poor-quality draft genome sequences, has been demonstrated by the discovery of the gene cluster encoding cypemycin in Streptomyces sp. strain OH-4156, revealing an unusual class of post-translationally modified ribosomally synthesized peptides (Claesen and Bibb, 2010). There will inevitably be a lag between the initial frenzy of genome sequencing and the characterization of novel useful products as the biochemical investigations are more laborious than the sequencing. Another interesting emerging theme is the role of endophytic streptomycetes and the emerging picture that secondary metabolites contribute to the medicinal properties of their host plants [e.g. (Akshatha et al., 2014)]. The most recently published Streptomyces genome comes from strain PRh5, an endophyte of wild rice that produces nigericin, an antibiotic effective against mycobacteria (Yang et al., 2014).