Testing of 1701 water wells in northeastern Pennsylvania shows that methane is ubiquitous in groundwater, with higher concentrations observed in valleys vs. upland areas and in association with calcium-sodium-bicarbonate, sodium-bicarbonate, and sodium-chloride rich waters—indicating that, on a regional scale, methane concentrations are best correlated to topographic and hydrogeologic features, rather than shale-gas extraction. In addition, our assessment of isotopic and molecular analyses of hydrocarbon gases in the Dimock Township suggest that gases present in local water wells are most consistent with Middle and Upper Devonian gases sampled in the annular spaces of local gas wells, as opposed to Marcellus Production gas. Combined, these findings suggest that the methane concentrations in Susquehanna County water wells can be explained without the migration of Marcellus shale gas through fractures, an observation that has important implications for understanding the nature of risks associated with shale-gas extraction. Significant media attention has been focused on the potential for methane impacts in drinking water wells located within areas of hydraulic fracturing activities for shale-gas development. Distinguishing among the various sources of methane gas that may affect drinking water wells requires proper assessment of background conditions. In this study, we review the results of background methane and groundwater quality surveys, in conjunction with geologic and historical information, to develop a better understanding of the potential sources of methane levels in drinking water wells in Susquehanna County in northeastern Pennsylvania.Susquehanna County has experienced substantial gas extraction activities in the Marcellus shale since 2006. Prior to that time, there was not a significant history of oil and gas operations in this county, thereby providing a unique opportunity to evaluate the potential effects of shale-gas extraction on groundwater resources in the Appalachian basin. Other researchers have suggested that elevated methane concentrations in water wells in Susquehanna County are the result of regional impacts from shale-gas extraction activities e.g., . To testOur research indicates that shale-gas extraction has not resulted in regional impacts on groundwater quality in Susquehanna County, a finding which suggests that hydraulic fracturing is not responsible for the creation or enhancement of wide-spread pathways by which Marcellus shale gas can rapidly travel to the surface.Our study focused on characterizing the geologic and hydrogeologic context of methane occurrence in water wells in northeastern Pennsylvania. For this purpose, we have collected and reviewed the following types of information: (1) geologic data on regional structure and stratigraphy, (2) studies on aquifer dynamics and geochemical characteristics, and (3) historical documentation regarding the occurrence of hydrocarbon gases in the shallow and deep subsurface. Within this context, we have evaluated data collected from 1701 water wells in Susquehanna County to determine the prevalence and distribution of elevated methane concentrations and other groundwater parameters. We have also assessed isotopic and molecular analyses of gases from 15 water wells sampled as part of an ongoing stray gas investigation in the Dimock Township to determine the origin of methane concentrations above the Pennsylvania DEP action level.Geologic information discussed in this article was compiled from prior studies on the stratigraphy and structure of the northeastern portion of the Appalachian basin, as well as new data acquired during exploration activities associated with recent Marcellus shale gas development. Specifically, stratigraphic information recorded during the drilling of 24 shale-gas wells located in Susquehanna and Wyoming counties facilitated the development of the geologic cross-section presented in The 1701 water samples analyzed as part of an extensive predrill water well survey from 2008 through 2011 were collected in the field and analyzed in NELAC accredited laboratories in accordance with procedures detailed in the supporting information. Statistical analyses of predrill data were conducted using Statistical Software ProUCL 4.1.01, which was developed and provided to the public by the United States Environmental Protection Agency (US EPA). Molecular and isotopic analyses of gases in shale-gas wells and local water wells in the Dimock Township were collected as part of an ongoing stray gas investigation by the Pennsylvania DEP and Cabot Oil and Gas Corporation in 2009 through 2011 using procedures detailed in the supporting information. This article also reports molecular and isotopic data from dissolved gases in Susquehanna County water wells collected by microbial in origin, that is, formed from the microbial breakdown of organic material, and thermogenic in origin, that is, formed from the abiotic degradation of organic material in formations under high temperatures and pressures at depth. Microbial methane commonly occurs in shallow bedrock and surficial deposits of alluvium  and glacial drift   . In Susqoutwash) . In areaoutwash) .Surficial deposits in Susquehanna County are underlain by the Catskill and Lock Haven Formations, a series of interbedded sandstone, siltstone, and shale deposits formed during the Upper Devonian period (359 to 385 million years ago) b ICS 20. The CatThe Bradford Sands, a series of thick sandstone deposits, form the base of the Lock Haven Formation, and are underlain by the Brallier Formation  and the Trimmers Rock Formation, which are comprised of interbedded sandstone, siltstone, and shale stratum of Upper Devonian age . The TulAn extensive natural fracture network is present in both the Catskill and Lock Haven Formations, with penetrating north-south oriented vertical planar fractures bisected by multiple inferior fracture sets and bedding planes .On a larger scale, seismic data provides valuable information regarding the structural setting and presence and/or absence of large-scale faults that could serve as conduits for the transport of fluids or gases. An interpretation of the stratigraphic and structural framework beneath Susquehanna and Lackawanna Counties from a portion of a regional 2D seismic line that transects the area is depicted in The great majority of water wells in Susquehanna County penetrate the fractured Catskill Formation, in which groundwater flow in unweathered bedrock appears to be controlled by secondary permeability primarily through vertical to near vertical north-south oriented fractures  . Most ofHistorical documentation suggests that the presence of methane gases in the shallow subsurface has been observed for over 200 years in Susquehanna County, long before the expansion of shale-gas fracturing in this area in 2006. For example, there are several dozen instances of flammable effervescing springs and water wells dating back to the late 1700s , and there are numerous reports of gas shows between 80 and 800 feet during the drilling of oil and gas wells . For exaIn addition to researching the hydrogeology, geology, and history of Susquehanna County in an effort to better understand natural groundwater conditions, we have also collected and analyzed water samples from this area to assess the presence of methane, ethane, and other water quality parameters. The results of this analysis provide additional information with regards to the distribution of hydrocarbon gases, and their association with physical geography, gas production areas, and groundwater quality.2 area within Brooklyn, Harford, and Gibson Townships (southeast Susquehanna County), which did not have significant gas development operations at the time. These samples were intended to establish baseline methane concentrations in groundwater in areas of Susquehanna County without substantial gas extraction activities and were primarily analyzed for concentrations of dissolved gases .This article evaluates data from 1701 water wells sampled in Susquehanna County to characterize baseline groundwater conditions during the period of 2008 to 2011. Over 900 of these samples were collected in accordance with Pennsylvania DEP regulations (Oil and Gas Act 13), which stipulates that an operator must conduct a “predrill or prealteration” water survey prior to the drilling of each gas well in order to maintain the right to contest any subsequent claims of groundwater impact. These samples were analyzed for both dissolved gas concentrations and general water quality parameters related to primary and secondary drinking water standards. Several hundred additional water well samples were collected from an 80-mileproposed wells; however, several of the water wells sampled were located in close proximity to previously drilled gas wells. As a result, 322 of the water wells have been characterized as located in a “gas production area” (defined as an area within 1 km of gas wells drilled prior to the time of sampling), while 1379 of the water wells are considered to be located in a “nonproduction area” . The 1 km radius used here to define gas production areas is considered a conservative distance, consistent with the radius utilized by Collectively, these 1701 samples are referred to as “predrill” because they were sampled prior to the drilling of specific To evaluate the possibility of a relationship to topography, dissolved methane concentrations measured in water wells were plotted on a light detection and ranging (LiDAR) bare-earth elevation map overlain with the National Hydrography Dataset (NHD). As indicated in To support statistical analysis of this observed correlation, the water well population was subdivided into wells located in valleys (defined as the area within 1000 feet of a major NHD flowline or 500 feet of minor tributaries to NHD flowlines) vs. wells located in uplands . As shown in p-value = 0.007). A similar comparison of methane concentrations in upland water wells showed that methane concentrations in upland gas production areas were approximately equal  to those in upland nonproduction areas . Furthermore, as a whole, no statistically significant difference was observed between methane concentrations in water wells in gas production areas vs. water wells in nonproduction areas (regardless of topographic location) .In order to evaluate whether elevated methane concentrations in the data set exhibited a relationship to gas development activities, methane concentrations in water wells located in gas production areas were compared to those located in nonproduction areas. Of the water wells in gas production areas (322 wells), approximately 3.7% contained methane concentrations that exceeded 7000 µg/L, while 3.3% of water wells in nonproduction areas (1379 wells) contained methane concentrations above 7000 µg/L. This slight difference may be attributable to the fact that gas production areas contain a greater percentage of valley water wells (61% of 322 water wells) than nonproduction areas (49% of 1379 water wells). To evaluate whether the prevalence of valley water wells in gas-production areas was a factor, the methane concentrations of valley water wells in gas production areas were exclusively compared to those of valley water wells in nonproduction areas. Using a one-way Mann-Whitney U-test, methane concentrations in valley gas-production valley areas were found to be less than or equal to those in valley nonproduction valley areas  origin of methane concentrations in the predrill water well samples can narrow the list of potential sources. One conventional approach to assessing thermogenic vs. microbial origin relies upon the ratios of methane to ethane, where gases with ratios less than 100 have been characterized as thermogenic, while those with ratios greater than 1000 have been characterized as microbial  .13C > –50‰, δ2H > –250‰). This suggests that these water wells could contain a notable thermogenic component, despite their traditionally microbial methane to ethane ratio.However, comparison of methane to ethane ratios to available isotopic data in predrill water well samples in nearby Bradford County (located directly west of Susquehanna County) suggest that the conventional approach for characterizing thermogenic vs. microbial origin based on methane to ethane ratios may not apply in the geologic context of northeastern Pennsylvania. Specifically, detected methane and ethane, and of those samples, approximately 72% (156 samples) displayed ratios of methane to ethane greater than 1000 . Samples from three of these water wells exhibited classically microbial  methane to ethane ratios but traditionally thermogenic carbon and hydrogen isotopic signatures. Consequently, at this time, the thermogenic, microbial, mixed thermogenic/microbial, or microbially altered  origin of the predrill dissolved gas samples from water wells cannot be determined.In Susquehanna County, 1540 (91%) of the 1701 predrill water well samples were analyzed for both methane and ethane concentrations. Exactly 217 of these samples contained han 1000 . IsotopiTo assess potential differences in groundwater sources, predrill water well samples in Susquehanna County were classified with regard to the relative concentrations of major cations  and anions . The cation/anion composition of the groundwater is a product of the aquifer matrix , groundwater residence and/or rock-water interaction times, and the prominence of redox processes . Consequ3 (281 samples), Ca-Na-HCO3 (46 samples), Na-HCO3 (20 samples), Ca-HCO3-Cl (32 samples), and Ca-Na-HCO3-Cl (20 samples). To characterize the relationship between water type and dissolved methane concentrations, the distribution of methane concentrations has been determined for each of the five principal water types. As shown in 3 and Na-HCO3 water types, respectively, exceeding this level. By contrast, none of the 281 Ca-HCO3 water type samples exceed 1000 µg/L of methane. Only a limited number of groundwater samples matched the Na-HCO3-Cl and Na-Cl water types ; nevertheless, these samples exhibit several of the highest methane concentrations observed in the predrill dataset, with three of the four reported concentrations exceeding 10,000 µg/L.The Piper diagrams shown in Concentrations of individual groundwater quality parameters measured during predrill sampling were also evaluated to assess a possible correlation with dissolved methane concentrations. These data provide a comprehensive data set for the following parameters: alkalinity, barium, calcium, chloride, magnesium, manganese, pH, potassium, sodium, strontium, sulfate, and TDS. Data for an additional eight groundwater analytes  contained greater than 50% nondetect values and were therefore not amenable to this analysis .To detect possible water quality trends related to methane, the concentration distribution for each parameter was determined for four ranges of methane concentrations: nondetect, detected to 1000 mg/L, greater than 1000 µg/L to 7000 µg/L, and greater than 7000 µg/L. The median concentrations of the following parameters were found to increase with increasing methane levels: barium, chloride, manganese, pH, sodium, strontium, and TDS . Median Isotopic and molecular analyses of dissolved gases can provide a means to differentiate between: (1) microbial vs. thermogenic gas sources, (2) distinct sources of microbial gas, and (3) distinct sources of thermogenic gas e.g., . In thisIn 2009 and 2010, several water wells in the Dimock Township of Susquehanna County were reported to contain methane concentrations above the Pennsylvania DEP action level of 7000 µg/L PA DEP , 2010b.Shale-gas wells are constructed of a series of steel casing strings  of various diameters that are installed concentrically within the wellbore, in a telescoping fashion. These casing strings serve the dual purpose of providing structural integrity of the wellbore and isolating surrounding formations  from the underlying production zone. The space between the casing string and the wellbore is referred to as the “annular space,” which is commonly cemented in shallower casing strings, but may be left uncemented  in deeper sections, depending on site-specific conditions. The annular space of a gas well is in contact with surrounding formations, and can therefore contain gases from adjacent strata that may be present naturally above the targeted gas-producing shale formation.In the three Dimock area gas wells, gas samples were collected from annular spaces surrounding the (1) shallow casing string adjoining potential freshwater aquifers, (2) the intermediate casing string, which penetrates to a depth several thousand feet beneath surface, and (3) the production casing string, which terminates within the Marcellus shale. Within these gas wells, the annular space surrounding the production casing string was cemented from the bottom of the wellbore to a depth above the Marcellus shale. The cementing of this portion of annular space is designed to prevent Marcellus formation gases and fluids from entering the overlying section of wellbore via an open annulus. Consequently, gases sampled from annular spaces surrounding the casing strings in Dimock Township gas wells were considered to represent a mixture of those originating in formations above the Marcellus Shale , which were penetrated in some sections by open annular spaces. One gas sample was also collected from the well cellar, a sunken pit that provides access to the gas wellhead which serves as a functional interface for the casing strings and the gas production line at the surface. This sample is considered to be a composite of gases found within the annular spaces of the casing strings.The Pennsylvania DEP also collected Marcellus shale gas  from inside the production casing and production pipeline of a local gas well. These samples are supplemented by Marcellus shale gas  collected by Cabot (a primary operator in the area) from within the production casing of additional gas wells in the Dimock Township. Collectively, these analyses represent 12 samples of potential source gases from 7 shale-gas wells located within the greater Dimock Township area  gases. Values of δ13C-CH4 and δ2H-CH4 represent the difference between the 13C/12C and 2H/1H isotopic ratios of a sample, and that of a recognized standard. More negative values of δ13C-CH4 and δ2H-CH4 are said to be isotopically “depleted” in the heavier isotopes , whereas more positive values are said to be isotopically “enriched.”The use of δ13C-CH4 and δ2H-CH4 values of Marcellus shale gas and overlying Middle and Upper Devonian gases sampled from the annular spaces of casing strings in the Dimock Township plot along a continuous trend. Based on the well-documented occurrence of less thermally mature methane in Middle and Upper Devonian Formations throughout the Northern Appalachian basin . Such isotope reversals have commonly been observed in Marcellus and other Middle Devonian gases below the Tully limestone  and those from the production pipeline/production casing (5 to 7‰ difference) suggests that Marcellus production gases may be distinguished from Middle and Upper Devonian gases in the annular spaces/well cellar of local gas wells.The δbsurface . Of the and –36‰ b. All reimestone . ComprehIt is important to note that, because of local variations in thermal maturity and possible source material, the isotopic and molecular signatures of formation gases in the Dimock Township cannot be extrapolated to represent the signatures of gases from the similar stratigraphic units in different parts of Susquehanna County or Pennsylvania. Consequently, the signatures of gases from the Marcellus and overlying Middle and Upper Devonian Units, as presented in In addition to characterizing gases originating from the Marcellus shale and overlying deposits in the Dimock Township, the Pennsylvania DEP and Cabot analyzed groundwater samples from 15 local Dimock area water wells for this same suite of isotopic analyses  and δ2H values  that appear consistent with those of thermogenic Middle and Upper Devonian gases sampled from the annular spaces/well cellar, microbial gases, or a mixture of the two. Based on the δ13C-CH4 and δ2H-CH4 values alone, samples from at least 2 of the 15 water wells contain a clear microbial component .For 15 of the 23 water well samples, the ratio of methane to ethane is less than 100, suggesting thermogenic origin. The remaining eight water wells and one salt spring contain methane to ethane ratios ranging from 100 to over 6000. This wide spread of values could represent several distinct thermogenic and microbial gas sources, a mixture of different sources, and/or gases that have undergone alteration during migration. The δ13C value of ethane was measured in 13 of the water well samples (originating from 10 different water wells). Of these 13 samples, 11 exhibited a methane-ethane δ13C isotope reversal  in Susquehanna County. A map of sampling locations in Susquehanna County  from local Marcellus production gases. However, 3 of the 11 water wells contain methane with δ2H and δ13C values that plot within the elevated range of values that characterizes Marcellus production gas in the Dimock Township . On this basis alone, these dissolved gases appear to match production gas samples from the Marcellus shale. However, closer examination of the data from these three water wells suggests that microbial oxidation in the subsurface is likely responsible for the elevated δ2H and δ13C values.The USEPA also analyzed dissolved gases in 11 water wells in the Dimock Township in 2012 as part of follow-up sampling to the original Pennsylvania DEP stray-gas investigation (Table S6). Eight of the 11 water wells display combined δ13C-CH4 values that are slightly more enriched (δ13C-CH4 = –25 to –27‰) than those exhibited by Marcellus production gas samples (δ13C-CH4 = –28 to –30‰). However, these same samples display δ2H-CH4 values that are significantly more enriched (–122 and –140‰) than local Marcellus production gas (δ2H-CH4 = –161 to –157‰). The particularly elevated δ2H signatures of the methane in these two samples is suggestive of microbial oxidation of methane, whereby both the δ2H and δ13C values of methane are altered to be enriched, but the change in the δ2H value of methane is 8 to 14 times greater than the change in the δ13C value of methane  exhibit δ methane .2H and δ13C values  that plot in the depleted end of the isotopic range characterized by Marcellus shale gas. However, the sample from House 2 does not exhibit an isotope reversal between the δ13C values of methane and ethane that characterizes Marcellus shale gas samples.The sample from the third water well (House 2) exhibits δEvaluation of a more complete suite of geochemical analyses over time could help discern the degree to which mixing of different gas sources and alteration processes in the subsurface  have affected the signatures of water well gas samples collected by various parties in Susquehanna County.2H and δ13C values of gases in Dimock area water wells sampled by the Pennsylvania DEP, Cabot, Osborn et al., and the USEPA indicate that hydrocarbon gases present in the majority of the water wells are consistent with gases in the well cellar and annular spaces of casing strings that intersect Middle and Upper Devonian formations above the Marcellus. The presence of a δ13C methane-ethane isotope reversal in numerous water wells would suggest a source below the Tully Limestone in the Middle Devonian Mahantango Formation penetrated by several of the annular spaces sampled. These findings support the conclusion that the methane concentrations in these water wells can be explained with no necessary contribution from deeper Marcellus shale gas.The δIsotopic and molecular analyses of hydrocarbon gases can provide valuable information on the source of natural gases, but not necessarily the mechanism of migration. In this article, for the Dimock Township, we have evaluated available isotopic and molecular analyses of local water well samples with the primary goal of assessing gas origin. To identify gas migration pathways, geochemical data should be assessed in conjunction with other lines of evidence which are not within the scope of this article. These lines of evidence include details on the construction, completion, and integrity of local gas wells and water wells. In addition, the variation of methane concentrations and groundwater quality in potentially impacted water wells over time, as well as the spatial distribution of methane concentrations in groundwater relative to potential locations of gas sources, can provide crucial information on potential pathways of migration.The isotopic and molecular analyses presented in this paper primarily represent measurements of hydrocarbon gases in local strata and water wells in the Dimock Township. Without additional analyses of samples throughout the greater Susquehanna County area, the signatures of hydrocarbon gases in local area gas and water wells cannot be extrapolated to represent those of methane or ethane that may be found in strata or water wells throughout the region. However, the evaluation of Dimock area isotope and molecular analyses of methane and ethane provide valuable insight into the complexity associated with using geochemical fingerprinting to identify the origin of hydrocarbon gases in regions where multiple thermogenic gas sources are present naturally.Methane has been present naturally throughout Susquehanna County in the shallow subsurface for at least 200 years, as indicated by the well-documented history of hydrocarbon gas shows during water well and gas well drilling, as well as natural seeps of hydrocarbon gases observed in the form of bubbling springs, ponds, and water wells. Our evaluation of 1701 recent predrill water well samples from Susquehanna County confirms that methane is common in drinking water aquifers today, with approximately 78% of sampled water wells exhibiting detected methane concentrations, and 3.4% exceeding the 7000 µg/L Pennsylvania DEP action level. Elevated methane concentrations show a clear relation to topography, with levels above the Pennsylvania DEP action level disproportionately found in valleys.Furthermore, methane concentrations in valley water wells in gas production areas  versus valley water wells in nonproduction areas  show no statistical difference, indicating that the regional presence of elevated methane concentrations in valleys is a natural phenomenon. This is not an original finding, as the presence of elevated methane levels in valley water wells in the Appalachian basin has also been observed in a study in West Virginia by The correlation of methane concentrations with topography, rather than areas of active shale gas extraction, indicates that the use of hydraulic fracturing for shale gas in northeastern Pennsylvania has not resulted in widespread gas migration into the shallow subsurface. Certainly, as described in the 1980s by 3 or Na-HCO3 water types, which in combination comprise only 16% of the sample population, yet represent 69% of the methane concentrations greater than 1000 µg/L. In addition, although only four water samples match a Na-HCO3-Cl or Na-Cl water type, three of these samples exhibit methane concentrations exceeding 10,000 µg/L. Samples with methane concentrations greater than 1000 µg/L also exhibit relatively elevated levels of barium, chloride, manganese, pH, sodium, strontium, and TDS, and relatively lower levels of sulfate and calcium. Similar geochemical relationships with elevated methane concentrations were previously reported in a study by Valley water wells in Susquehanna County exhibit median methane concentrations similar to that of upland water wells; however, the 90th percentile concentrations of methane in valley wells is significantly elevated relative to upland wells. This observation suggests that some valley water wells access natural sources of elevated methane via interconnection with specific groundwater units and/or enhanced pathways of methane migration. As shown in 3 type water to Na-HCO3 or Na-Cl-HCO3 type water as a result of (1) the mixing of circulating freshwater with sodium-chloride water and/or brine that occurs in deeper bedrock and aquifers, and/or (2) progressive cation exchange, whereby calcium, magnesium  ions replace sodium on mineral exchange sites, thereby liberating sodium in groundwater , and/or are in contact with sodium-chloride rich waters that occur in deeper bedrock and aquifers. Specifically, water in the deeper parts of sandstone aquifers that contain carbonate components  commonly transitions from Ca-HCOundwater . It follundwater .Longer groundwater residence times are also associated with anaerobic groundwater conditions related to microbial consumption of oxygen and other electron acceptors  in the presence of an organic substrate. Evidence for the occurrence of these redox processes can include increased concentrations of manganese and iron from the reduction of iron and manganese oxides, as well as decreased sulfate concentrations (and associated sulfide odors) related to sulfate reduction .In Susquehanna County, deeper groundwater with longer residence times may be preferentially accessed by water wells that penetrate to the depth of deeper groundwater units or are in communication with fault and fracture networks. Comprehensive information on the depth of water wells sampled in our predrill dataset is not available. However, available information on aquifer characteristics in northeastern Pennsylvania indicates that the Lock Haven Formation contains discrete saline groundwater zones, which have been noted to produce methane gas and/or hydrogen-sulfide . AnalysiValley formation in Susquehanna County has been associated with a greater density of fractures and lineaments, which likely serve as enhanced pathways for both water and gas migration. In West Virginia, 4 oxidation) concentrations, and a decrease in oxidation-reduction potential , an increase in pH and bicarbonate  in northeastern Pennsylvania suggests that methane naturally present in many Susquehanna County water wells contains thermogenic components from the Catskill and Lock Haven formations. In addition, microbial methane, produced by methanogenesis in anaerobic groundwater units, may also be a strong component of the methane migrating into local drinking water wells. As discussed earlier, the present-day migration of deep thermogenic gases from the Mahantango and Marcellus Formations into the shallow subsurface in Susquehanna County appears to be limited by the presence of a regional structural detachment above the Tully stratigraphic level, which has been noted in local seismic interpretations. The data presented in this paper show that elevated methane concentrations exhibit a common association with (1) topographic lows and (2) groundwater geochemistry that reflects longer residence times and/or mixing with deeper NaCl waters. This observation suggests that elevated sources of either thermogenic or microbial methane from the Catskill and Lock Haven Formations are accruing in deep groundwater units, and/or within long discrete groundwater flow paths to lowland discharge points, that are preferentially accessed by certain valley water wells.This evaluation represents a regional-scale assessment of trends observed in groundwater geochemistry in a large dataset during a 3-year period. However, for an individual water well, it is important to remember that factors such as proximity to roads  can result in localized variations in chloride, total dissolved solids, and other water quality parameters. In addition, seasonal fluctuations in aquifer recharge , residential water use, changing weather patterns, and local well construction can also affect site-specific water quality.Our evaluation of 1701 groundwater quality analyses shows that methane is common in Susquehanna county water wells and is best correlated with topography and groundwater geochemistry, rather than shale-gas extraction activities. This finding suggests that shale-gas extraction in northeastern Pennsylvania has not resulted in regional gas impacts on drinking water resources and that, in turn, the hydraulic fracturing process has not created extensive pathways by which gas from the Marcellus shale could rapidly migrate into the shallow subsurface.Our evaluation of site-specific isotopic and molecular data from water wells in the Dimock Township suggests that hydrocarbon gases present in these water wells are consistent with Middle and Upper Devonian gases above the Marcellus sampled in the annular spaces of local gas wells. This evaluation also emphasizes the complexity associated with differentiating between multiple thermogenic gas sources that may exhibit subtle variations. Isotopic analyses were not performed on the 1701 predrill water well samples to determine the origin of elevated methane concentrations observed throughout Susquehanna County. However, consideration of regional geology, historical publications, structural data, water well completion records, and water quality data suggest that methane naturally migrating into Susquehanna County water wells is either thermogenic, likely originating from Upper Devonian deposits overlying the Marcellus shale, or microbial, originating from anaerobic groundwater units with long residence times.


While the question of why organisms reproduce sexually is still a matter of controversy, it is clear that the foundation of sexual reproduction is the formation of gametes with half the genomic DNA content of a somatic cell. This reduction in genomic content is accomplished through meiosis that, in contrast to mitosis, comprises two subsequent chromosome segregation steps without an intervening S phase. In addition, meiosis generates new allele combinations through the compilation of new sets of homologous chromosomes and the reciprocal exchange of chromatid segments between homologues. Progression through meiosis relies on many of the same, or at least homologous, cell cycle regulators that act in mitosis, e.g., cyclin-dependent kinases and the anaphase-promoting complex/cyclosome. However, these mitotic control factors are often differentially regulated in meiosis. In addition, several meiosis-specific cell cycle genes have been identified. We here review the increasing knowledge on meiotic cell cycle control in plants. Interestingly, plants appear to have relaxed cell cycle checkpoints in meiosis in comparison with animals and yeast and many cell cycle mutants are viable. This makes plants powerful models to study meiotic progression and allows unique modifications to their meiotic program to develop new plant-breeding strategies. While the reasons why meiosis and sex have evolved are under debate (see review and hypothesis by Hoerandl and Hadacek in this issue of Plant Reproduction), it is widely accepted that meiosis was derived from mitosis   und WIH2 that encode novel peptides whose absence leads to the loss of a morphological distinguishable functional megaspore mother cell  family in Arabidopsis, maize and rice implicated small RNAs in regulating meiotic progression, the repression of germ cell fate in somatic tissues, or, as was shown in rice, by repressing a somatic fate in germ cells  and of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6)  in rice that encodes for protein with a RNA recognition motif. Loss of MEL2 function results in a failure of most meiocytes to enter meiotic S phase. The few cells that proceed to prophase arrest at early stages and show perturbed meiotic characteristics  encodes an unknown protein from maize, and most am1 alleles lack all indications of meiotic prophase, suggesting a key role in establishing a meiotic program  complete a meiotic program and give rise to viable gametes /MALE STERILE 5 (MS5)/POLLENLESS 3 that encodes a protein with a yet unknown function undergo a third meiotic division without intervening S phase, indicating a failure in shutting down the meiotic program  argues that TDM1 may also act in some way to restrict meiotic CDK activity  up to many mitotic divisions (in moss) during the gametophytic life phase. Remarkably, mutants for the Arabidopsis gene Progression through the mitotic cell cycle has been found to rely on quantitative and qualitative aspects of CDK-cyclin complexes Fig. a. On theCyclin-dependent kinases are regulated at multiple levels, and a key determinant of CDK activity is the amount and type of cyclin partners that are available Pines  Fig. 2)2). ThereIn plants, clear homologues for A- and B-type cyclins have been found next to a cyclin class that has been named D-type cyclins but is equidistant to animal D- and E-type cyclins , there are more than 30 cyclins in Arabidopsis Fig. . Thus, acdka;1 mutants are viable but severely compromised, precluding clear developmental analyses , was proposed to be an epiallele of a wheat CDKA;1 homolog. The presence of Ph1 prevents the recombination between the three homoeologous genomes  that are present in bred wheat and thus is a crucial determinant of wheat fertility . Even if so, it is not at all understood how down-regulation of CDK activity could accomplish the complex phenotypes seen in Ph1.Interestingly, an important chromosome pairing regulator of wheat, cdkb1;1 and cdkb1;2 indicates only a minor or redundant role of B1-type CDKs in meiosis. B2-type CDKs were found to accumulate specifically in M phase  and TDM1 (see sections on meiotic entry and meiotic checkpoints). While loss of SMG7 results in arrest at anaphase II, the double mutant tam smg7 also progresses till this arrest point, i.e., the effect of tam can be suppressed by mutations in SMG7 , an atypical cyclin that shows similarities with A- and B-type cyclins  and presumably spindle formation  and second in contributing to general kinase levels preventing the formation of premature cytokinesis.Unexpectedly, given its localization to the spindle in late meiosis I and II, mutants in CYCA2;3 and CYCA2;4 were not found to be present in meiosis, and mutants in CYCA2;1 and CYCA2;2 were fully fertile. However, the triple mutant cyca2;2 cyca2;3 cyca2;4 shows defects in chromosome condensation and segregation, arguing either for low expression/masked detection of CYCA2;3 and/or CYCA2;4 or for a complex compensatory interaction in which normally not expressed A2-type cyclins become up-regulated in a single-mutant background  in plants /GIGAS CELL (GIG) exit meiosis after the first division , meiotic recombination 11 (mre11), rad50, and rad51  would all have caused checkpoint-induced meiotic arrest in mouse (Romanienko and Camerini-Otero The absence/low stringency of meiotic cell cycle checkpoints in plants offers an unprecedented possibility in breeding. Two novel breeding methods were recently shown to be feasible in plants (Arabidopsis) that make use of specific meiotic mutant situations to engineer new inheritance patterns. Notably, these mutant situations . In this triple mutant, no recombination occurs, sister chromatids segregate at meiosis I and the second meiotic division is omitted. Consequently, these plants execute a mitosis-like meiotic cell division that produces viable diploid spores with a genotype identical to the parent. Since in Arabidopsis, haploid or diploid gametes can directly be grown into seeds and subsequently into plants (Ravi and Chan Marimuthu et al.  describeA second proposed breeding method, reverse breeding, aimed at developing a technique to dramatically reduce the complexity of meiotic recombination, in which not alleles, but only non-recombinant parental chromosomes segregate (Wijnker et al. smg7, tam and tdm. The list of meiotic cell cycle regulators in plants is rapidly growing with major accomplishments just over the last few years. A main challenge in the future will be to untangle the specific from the redundant functions of the different CDK-cyclin complexes apparently at work in meiosis. Of key importance is the identification of the targets of these complexes and how their differential phospho-status will then promote the coordinated progression of the complex meiotic events. An immediate application of a deeper understanding of plant meiosis is the development of new plant-breeding strategies that may allow the propagation of hybrids and show the possibility to reconstruct complex allelic combinations. Thus, further insights into the regulation of the meiotic division program hold the promise for yet new possibilities in breeding to meet the challenges of our agriculture in the twenty-first century.A detailed understanding of meiotic progression in plants is not only important for basic insights into one of the largest classes of living organisms and crucial components in most ecosystems on earth but also for emerging questions in speciation and genomic dynamics for which plants provide powerful model systems. The viability of many plant cell cycle and meiotic mutants furthermore allows the analysis of double mutants and the untangling of epistatic interactions, as was nicely illustrated through the experiments on the interactions between


Gastrointestinal stromal tumours (GISTs) are the most common primary mesenchymal neoplasia in the gastrointestinal tract, although they represent only a small fraction of total gastrointestinal malignancies in adults (<2%). GISTs can be located at any level of the gastrointestinal tract; the stomach is the most common location (60-70%), in contrast to the rectum, which is most rare (4%). When a GIST invades into the adjacent prostate tissue, it can simulate prostate cancer. In this study, we report on a case comprising the unexpected collision between a rectal GIST tumour and a prostatic adenocarcinoma.We describe the complexity of the clinical, endoscopic and radiological diagnosis, of the differential diagnosis based on tumour biopsy, and of the role of neoadjuvant therapy using imatinib prior to surgical treatment.Although isolated cases of coexisting GISTs and prostatic adenocarcinomas have previously been described, this is the first reported case in the medical literature of a collision tumour involving a rectal GIST and prostatic adenocarcinoma components.http://www.diagnosticpathology.diagnomx.eu/vs/1238437468776331.The virtual slide(s) for this article can be found here:  The term gastrointestinal stromal tumor (GIST) was first introduced in 1983 by Mazur and Clark as a neutral term between smooth muscle tumours and schwannomas. GIST is currently used to refer to a heterogeneous group of mesenchymal tumour lesions, acquired or congenital, with common morphological profiles that usually settle within the digestive tract.Although GISTs are the most common primary mesenchymal tumours of the gastrointestinal tract (70%), they represent only a small fraction of all gastrointestinal malignancies in adults (<2%).GISTs usually affect adults between 6th and 8th decades of life without specific gender prevalence M:F, 1.1:1) [.1:1 [1].In 1998, Hirota et al. showed that the majority of GISTs exhibit activating mutations in the KIT protooncogene. Subsequently, in 2003, a new mutation in PDGFRA and the sensitivity of GISTs to tyrosine kinase inhibitors such as imatinib were demonstrated. These findings represent an important advance in the clinical management and our understanding of the biology of this tumour [GISTs can be located at any level of the gastrointestinal tract. The stomach is the most common location 60-70%), whereas rectal GIST represents only 4% of all GISTs . When th0%, whereThe patient examined in this study was a 52-year-old male with no relevant personal or family history of disease, who presented with rectal tenesmus, rectal bleeding, and obstructive urinary symptoms. A digital rectal exam revealed a fixed submucosal mass in the anterior rectal wall, attached to the prostate and located 2–3 cm from the anal margin. The prostate specific antigen (PSA) value was 3.16 mg/ml, with a pathologic PSA ratio of 0.0937. Rectoscopic and echo-endoscopic evaluations revealed a rectal neoplasia with prostatic infiltration. A computed tomography (CT) scan performed at 5 months prior to the surgical intervention revealed a relatively well-defined heterogeneous mass of 60 mm × 36 mm × 45 mm affecting the anterior wall of the rectum in contact with the prostate, with effacement of the fat separating the two organs.Magnetic resonance imaging (MRI) performed at the same time revealed a large (7–8 cm) tumour in the distal rectum, affecting the adjacent prostate and causing stenosis of the rectal lumen. The CT scan and the MRI results both revealed an absence of metastasis in the local and distant lymph nodes.A biopsy of the tumour revealed a profile that was consistent with a mesenchymal neoplasia of uncertain malignancy that was positive for CD117 (cytoplasmic and membranous staining), the expression of which is characteristic of GISTs.The treatment protocol consisted of neoadjuvant treatment with imatinib abdominoperineal rectal amputation with a radical cystectomy and prostatectomy. A preoperative CT scan demonstrated an apparent reduction in the tumour size, measuring 50 mm × 30 mm, where the separation between the rectum and prostate was difficult to delineate.Grossly, the surgical specimen was an excrescent mass located within the anterior rectal wall that exhibited a 7-cm maximum diameter and focally ulcerated mucosa Figure : A. An iThe prostate tissue adjacent to the infiltrative front of the spindle cell tumour exhibited diffuse proliferation of microglandules and well-defined cribriform structures with focal intraluminal mucin secretion and atypical cells with prominent nucleoli Figure .Histopathological diagnosis identified the collision tumour involving a high-risk GIST of the spindle cell variant that originated in the anterior rectal wall and that exhibited perirectal extension and wide infiltration of the adjacent peripheral prostate lobules, as well as a prostatic acinar adenocarcinoma with a combined Gleason score of 6 (3+3) and confined involvement to the left and right prostate lobules (pT2c). The seminal vesicles and urinary bladder exhibited no evidence of neoplasia.Currently, after 8 months of postoperative follow-up and neoadjuvant treatment with imatinib, the patient is asymptomatic and cancer-free.GIST is the most common type of non-epithelial neoplasia diagnosed in the gastrointestinal tract  with an incidence rate of 10–20 cases per million population . HoweverThe immunohistochemical profile of the tumour was similar to gastric GISTs. As many as 90-100% of tumour cells exhibited strong immunopositivity for CD117, DOG-1, and CD34 and were generally immunonegative for SMA and S-100. These tumours behave in an aggressive fashion, exhibiting a high rate of recurrence, pelvic extension, and hepatic and bone metastases [The differential diagnosis of rectal GIST with prostatic involvement in the biopsy specimens is primarily based on immunohistochemical evaluation and includes different stromal neoplasias with spindle cell patterns that can affect the rectum and prostate. These tumours include primarily smooth muscle tumours , schwannomas, fibromatosis, a solitary fibrous tumour and a malignant melanoma. The differential histological and immunological characteristics of the tumours localised in this region are summarised in Table .To confirm the diagnosis of a GIST, a KIT or PDGFRA mutational analysis can be performed, although this was not a possible option in our patient.GIST is known to coexist with certain neoplasias, including pulmonary chondromas and paragangliomas (Carney’s triad). For the diagnostic of CT (Carney´s triad) are necessary two of the three components. The association of GIST and pulmonary chondroma is the most common combination . SporadiHowever, these cases have been described as synchronous or metachronous association between sporadic GISTs and other neoplasia ,12, withRadical cystoprostatectomy performed due to bladder carcinoma showed prostatic acinar adenocarcinoma in the 49.6% of the cases , where 8Rectal GISTs are rare tumours, whereas prostatic adenocarcinoma is the most prevalent type of non-cutaneous neoplasia in elderly men. On this basis, rectal and prostate tumours are generally initially considered as prostatic adenocarcinomas or prostatic infiltrations of rectal adenocarcinomas. Only 6 cases have been described of prostatic infiltration by rectal GISTs -7, all oUntil recently, the treatment of giant rectal GISTs has been limited to surgical resection, except for cases of inoperable tumours or metastases that contraindicated the surgical treatment (indicating that the tumour was incurable under those specific circumstances). The use of imatinib as a neoadjuvant treatment offers the possibility of a partial tumour regression and stabilisation of the disease . In geneIn our study, neoadjuvant therapy with imatinib did not substantially reduce the tumour size, and histologically, we only observed extensive haemorrhagic foci without a decrease in the number of tumour cells or the presence of myxohyaline stroma, necrosis, or cystic degeneration, as previously described in cases that exhibited an initial response to treatment . HoweverThe authors obtained the rights for publication of this report and any accompanying images.GIST: Gastrointestinal stromal tumours; PSA: Prostate specific antigen; CT: Computed Tomography; MRI: Including Magnetic Resonance; HPF: High-Power Field.The authors declare that they have no competing interests.All authors read and approved the final manuscript.


Hepatocellular carcinoma is the most common primary liver malignancy and is an international public health concern, constituting one of the most deadly cancers worldwide. Infection with hepatitis B virus and hepatitis C virus is a major risk factor for HCC in developed countries. Emerging evidence indicates that there are other important lifestyle factors that contribute to the international burden of HCC, such as alcohol consumption, diabetes, obesity, and the intake of aflotoxin-contaminated food. Obesity and diabetes are also likely to be risk factors for HCC, the most frequent subtype of liver cancer. The chief pathway by which obesity increases risk involves the association between obesity and nonalcoholic fatty liver disease (NAFLD). Coffee consumption has been studied extensively and appears to have a favorable effect on the prevention of liver diseases, including HCC. One hypothesis suggests that coffee intake lowers serum levels of gamma-glutamyltransferase (GGT), which is associated with a lower incidence of HCC. It is estimated that more than 80% of HCC cases are attributable to four principal causes that are avoidable. It is difficult to make dietary recommendations, because it is unknown whether consuming higher amounts of specific antioxidants will decrease the risk of developing hepatocellular carcinoma. A diet rich that is in polyunsaturated fatty acids and, possibly, B-carotene could reduce the risk of HCC, and high dietary GL is associated with an increased risk independently of cirrhosis or diabetes. Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and represents an international public health concern, constituting one of the most deadly cancers worldwide . InfectiEmerging evidence indicates that the etiology of many cases of HCC is multifactorial, comprising infectious origins, comorbid conditions, and environmental exposure. Accordingly, many cases are attributed to the growing prevalence of chronic infections with hepatitis B and hepatitis C viruses 2]3]4][3][4]2][[4][3][4][3][4]2]6]7]8][7][8]6][[8][7][8][7][8]6]. HoweverObesity is another risk factor for several chronic diseases, including hypertension, insulin resistance (IR), type 2 diabetes, dyslipidemia, and chronic heart disease . CollectAs the hepatic entity of metabolic syndrome, NAFLD/NASH is a risk factor for HCC, even in the absence of cirrhosis . A recenIn addition to increasing the prevalence of chronic liver disease, diabetes is an independent risk factor for the development of HCC. In a recent systematic review of 13 case control studies, 11 reports supported an association between diabetes and the development of HCC . Of the Coffee consumption has also been extensively studied and appears to have a favorable effect on the prevention of liver diseases, including HCC . There aIn a similar study, in the highest quartile of consumers of yogurt and milk, the intake of white meat and eggs correlated with a significantly lower likelihood of the development of HCC. This effect was observed in patients with and without viral hepatitis . Other sAnother important risk factor for the development of HCC is the contamination of foodstuffs with aflotoxin B1 (AFB1). AFB1 is a mycotoxin that is produced by the fungus Aspergillus, which grows readily on food when it is stored under warm, damp conditions 34]. Whe. Whe34].However, the hermetic effects of these phytochemicals must be considered, because at low doses they have stimulatory effects but are toxic at higher doses . VariousDue to conflicting results, it is difficult to make dietary recommendations, because it is unknown whether consuming higher amounts of specific antioxidants will decrease the risk of developing hepatocellular carcinoma. A diet that is rich in polyunsaturated fatty acids and, possibly, B-carotene can reduce the risk of HCC, and high dietary GL is associated with increased risk, independently of cirrhosis or diabetes. Coffee has favorable effects, as shown in many studies, especially in HCV infection.


It has now ascertained that the clinical manifestations of liver disease in the elderly population reflect both the cumulative effects of longevity on the liver and the generalized senescence of the organism ability to adjust to metabolic, infectious, and immunologic insults. Although liver tests are not significantly affected by age, the presentation of liver diseases such as viral hepatitis may be subtler in the elderly population than that of younger patients.Human immunosenescence is a situation in which the immune system, particularly T lymphocyte function, deteriorates with age, while innate immunity is negligibly affected and in some cases almost up-regulated.We here briefly review the relationships between the liver aging process and mast cells, the key effectors in a more complex range of innate immune responses than originally though. Nearly 13% of the inhabitants of the United States are aged more than 65 years; this percentage will increase substantially over the next 50 years[Although it can refer to any time-related process, the term “aging” is commonly used for post-maturational processes that lead to diminished homeostasis and increased vulnerability. The altered homeostasis in older organisms is probably the result of a genetic program that determines responses to exogenous influences and increases the predisposition to illness and death.1) increased mortality after maturation; 2) changes in the biochemical and physical properties of tissues; 3) a progressive decrease in physiological capacities; 4) a reduced ability to respond adaptively to environmental stimuli; and 5) increased susceptibility and vulnerability to disease[There are five main characteristics associated with aging in mammals: It is now recognized that human aging is a complex phenotype resulting from the continuous, lifelong adaptation of the body to unrepaired molecular and cellular damage to the organism caused by a variety of external and internal agents.immunosenescence occurs as the result of a chronic hyperstimulation of both adaptive and innate immune system components[immune status characterized by a global loss of efficiency  cells, and NKT lymphocytes, are affected by aging. In addiFunctional defects and altered frequencies of innate and adaptive immune cells impair local responses at the site of vaccine injection, hamper the generation of primary responses to neo-antigens, prevent the effective induction of memory lymphocytes, and decrease the effect of booster vaccination. As a result, antibody responses of elderly vaccines are weaker and decline faster, and long-term protective effects of vaccination cannot be taken for granted in elderly peoples,22-24. EIt is now accepted that aging is characterized by a peculiar state of chronic inflammation  under genetic control, which seems to be a consequence of a person’s life-long antigenic load-30 and lIt has also been shown that aging is accompanied by a 2-4-fold increase in the plasma levels of inflammatory mediators, cytokines and acute-phase proteins-37. AddiThe incidence of liver disease increases with age while the ability to withstand a hepatic insult falls with each decade. It is iOne of the most important age-related changes in liver function observed in animal models is a significant decrease in regenerative capacity of the liver, but not in the capacity to restore the organ to its original volume. Liver rOlder people have a higher incidence of acute liver failure and a higher mortality with acute hepatitis A,62. AlonPoynard et al. showed that age at HCV infection was a main risk factor for fibrosis,64. The Age is an independent risk factor for poor outcome in primary biliary cirrhosis (PBC) in addition to the presence of portal hypertension and impaired liver function-68. It hIt is well recognized that the incidence of HCC in patients with HCV correlates with progression of liver fibrosis. However, there is little information on the impact of aging on hepatocarcinogenesis. Furthermore, it is not known whether the putative etiologic factors and clinical and pathological features of HCC differ between young adults and older patients. The eldmaestung - a root of the English word mastication; the active form “measten” is still in use) because of their characteristic staining of proteoglycan and protease-rich cytoplasmic granules. Ehrlich also noted the tendency of MCs to be associated with blood vessels, nerves, and glandular ducts.Mast cells (MCs) have a rather unique position among cells of the immune response. Paul Ehrlich first described MCs in his 1878 doctoral thesis: he called them “mastzellen”  alone or tryptase and chymase together (MCTC). MCT are found mainly in mucosal sites whereas MCTC[Tryptases and chymases are the major proteases components of MC secretory granules. There are two well-known human MC classes that are distinguished by enzymatic immunostaining for mast cell proteases, tryptase .2) to initiate MCs degranulation changes in the aging animal, thus suggesting that aging induces reprogramming of MC degranulation[MCs and IgE have long been associated with the pathogenesis of the acute manifestations of the immediate hypersensitivity reaction, the pathophysiologic hallmark of allergic rhinitis, allergic asthma, and anaphylaxis. The central role of MCs in these disorders is now widely accepted. Additionally, MCs are considered to be critical effectors in many human inflammatory diseases, and the core of an immediate hypersensitive reaction: they have been incriminated in different diseases including allergy, asthma, rheumatoid arthritis, arteriosclerosis, chronic graft-versus-host disease, fibrotic disease, ischemic heart disease and malignancy, and contribute to the progression of chronic diseases-99. Any nulation.TC. Increased plasma histamine levels were also found in patients with PBC, in contrast to healthy patients, raising the possibility of a role for in vivo mast cell activation and mediator release in these diseases[The parenchyma of the liver is a complicated structure with numerous cell types having different functions in normal liver function and disease response. These include: Kupffer cells, hepatic stellate cells, sinusoidal endothelial cells, vascular endothelial cells, fibroblasts and pit cells. The majorities of these cells play definitive roles in liver pathophysiology and interact with hepatic MCs Figure . MC quandiseases. Hepaticdiseases.2, but rat livers have 1.8-12 MCs/mm2[It has been recognized that liver MCs are present under normal and pathological conditions in both humans and experimental animals: normal 2 MCs/mm2. Immunos2 MCs/mm2.MCs seem to be involved in the liver’s fibrotic response to chronic inflammation and paraMCs infiltrating portal tracts and surrounding damaged bile ducts have also been found in patients with chronic allograft rejection, thus suggesting that they may be important effector cells in the pathogenesis of chronic transplanted liver rejection. The livMCs have recently been investigated in order to study their involvement in the mechanisms leading to age-related chronic diseases. During fibrosis and hepatic disease progression (i.e. hepatitis), hepatocytes maintain a close interaction with other resident cell types as well as inflammatory cells. These populations come together to induce “sinusoidal capillarisation” which describes the events that cause the sinusoids to resemble capillaries. Capillarisation inhibits the normal processes of exchange between hepatocytes and plasma and has been found to be a condition that worsens liver function. It has been showed that liver sinusoidal endothelial cells become thicker and defenestrated, there is a sporadic deposition of collagen and basal lamina in the extra-cellular space of Disse, and the number of non-activated stellate or Ito cells increases. This psIn old age, there is a 30-50% reduction in the area of the endothelium perforated by fenestrations (“porosity”). This is associated with increased endothelial thickness and extracellular matrix in the space of Disse, including collagen and basal lamina,110-112.It has been shown that MC density increases with age-120 but,4-treated young (2 months), middle-aged (6 and 12 months) and old male rats (19 months) as a quantitative index of acute toxic liver inflammation, and investigated whether the density is age-dependent[Recently it has been evaluated MC density in liver tissues taken from untreated and CClependent,122.4 intoxication showed hepatocyte necrosis and inflammatory cell infiltration in the perivenular areas, whereas the periportal areas were virtually unaffected.Histological examinations of the 12- and 19-month-old rats sacrificed two hours after CCl4 intoxication, with the 24-hour difference being statistically significant. The increase in MC density was less marked in the 19-month-old rats than in the young rats, but there were significant differences between the untreated rats and the treated rats two hours after CCl4 intoxication, and between the densities measured two and 24 hours after intoxication. The changes in the rats aged six and 12 months fell between those observed in the rats aged two and 19 months.It was found no statistical differences in MC density between the untreated rats of different ages, but MC density was considerably increased in the young rats both two and 24 hours after CClIt is well known that MCs are a primary cell population involved in inflammatory responses. By means of the rapid release of their pro-inflammatory molecules, MCs determine a series of events that lead to both immediate and late-phase responses.4 highlighted the fact that MC density is an important marker of an acute liver inflammatory reaction to toxic CCl4 damage: it considerably increased two hours after intoxication (which suggests that MC cells are recruited early in injured tissue) and the increase was even more evident after 24 hours, when the density was significantly greater than in the control animals[Quantitative analysis of MC density in untreated rats and rats receiving an intraperitoneal injection of CCl animals. As MC da) a decline in liver volume; b) an increase in the hepatic pigment (lipofuscin) deposition; c) a moderate decline in the Phase I metabolism of certain drugs; d) shifts in the expression of a variety of proteins and e) diminished hepatobiliary functions. Other more subtle changes may contribute to increased susceptibility to certain liver diseases in the elderly[e) Aging of the liver mainly affects the sinusoids. Pseudo-capillarization, manifested by reduced sinusoidal fenestration and subendothelial collagen deposition, causes a reduction in oxygen-dependent hepatocyte functions such as oxidative drug metabolism[f) Reduced hepatic blood flow in the elderly has been suggested to be the major effect of aging on the liver circulation. g) MCs can exert positive or negative immunomodulatory effects on immune cells by influencing the recruitment, survival, development, phenotype or function of immune cells and thereby enhance or suppress the initiation, magnitude and/or duration of immune responses[h) Quantitative analysis of MC density in liver specimens taken from untreated rats and rats receiving an intraperitoneal injection of CCl4 highlighted the fact that MC density is an important marker of acute liver inflammation. i) MC density two and 24 hours after CCl4 intoxication was greater in the young than in the oldest rats, which suggests that the latter have fewer MC recruiting stimuli and that young rats are characterized by a rapid response to toxic injury.It is indubitable that aging, being a complex process, involves an array of intertwined molecular pathways,124. Sim elderly. e) Agintabolism. f) Reduesponses. h) QuanIn conclusion, a better understanding of the mechanisms underlying the age-related liver changes may help to preserve hepatic function, improve morbidity and mortality, and hopefully reduce healthcare costs for the aging population. Additionally, it is likely that future studies of MCs in experimental models and human tissues will reveal more about their functions not only in the pathogenesis of liver and other diseases, but also in the immunosenescence process affecting human tissues.CCl4: Carbon tetrachloride; DCs: Dendritic cells; FcεRI: High-affinity IgE receptor; GM-CSF: Granulocyte macrophage colony stimulating factor; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus; hsp90: Heat-shock protein 90; ICAM-1: Intercellular adhesion molecule-1; IL: Interleukin; MCs: Mast cells; MCT: Tryptase; MCTC: Tryptase-chymase; MIP-1: Macrophage inflammatory protein alpha; NGF: Nerve growth factor; NK: Natural killer cells; NKT: Natural killer T-lymphocytes; PBC: Primary biliary cirrhosis; PGE2: Prostaglandin E2; PPAR: Peroxisome proliferator-activated receptor; PSC: Primary sclerosing cholangitis; SCF: Stem cell factor; TERC: Telomerase RNA component; TERT: Telomerase reverse transcriptase; TGF: Transforming growth factor; TLR: Toll-like receptor; TNF: Tumor necrosis factor; VEGF: Vascular endothelial growth factor.The authors declare that they have no competing interests.FG and MCI wrote the first draft; subsequent drafts were written by FG, GDC, LL, EC and MCI who had the overall supervision of the review processing. All authors read and approved the final manuscript.


Heme (iron-protoporphyrin IX) is an essential co-factor involved in multiple biological processes: oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction, and micro RNA processing. However, excess free-heme is highly toxic due to its ability to promote oxidative stress and lipid peroxidation, thus leading to membrane injury and, ultimately, apoptosis. Thus, heme metabolism needs to be finely regulated. Intracellular heme amount is controlled at multiple levels: synthesis, utilization by hemoproteins, degradation and both intracellular and intercellular trafficking. This review focuses on recent findings highlighting the importance of controlling intracellular heme levels to counteract heme-induced oxidative stress. The contributions of heme scavenging from the extracellular environment, heme synthesis and incorporation into hemoproteins, heme catabolism and heme transport in maintaining adequate intracellular heme content are discussed. Particular attention is put on the recently described mechanisms of heme trafficking through the plasma membrane mediated by specific heme importers and exporters. Finally, the involvement of genes orchestrating heme metabolism in several pathological conditions is illustrated and new therapeutic approaches aimed at controlling heme metabolism are discussed. Heme is an iron-containing porphyrin of vital importance since it constitutes the prosthetic moiety of several hemoproteins. It can interact with different inactive apo-heme proteins giving rise to active hemoproteins, whose function is ultimately determined by the properties of the polypeptide bound to it Dawson, . Thanks Other than acting as a prostetic group in hemoproteins, heme itself may influence the expression of many genes. In non erythroid cells, heme regulates its own production by down-regulating heme biosynthesis at the level of the rate-limiting enzyme 5-aminolevulinic acid (ALA) synthase 1 (ALAS1) and by up-regulating its metabolism , resulting in oxidative stress. Heme that is not bound to proteins is considered the labile heme pool; this portion of heme is derived from newly synthesized heme that has not yet been incorporated into hemoproteins, or heme that has been released from hemoproteins under oxidative conditions. “Free heme” is an abundant source of redox-active iron that can participate in the Fenton reaction to produce toxic free hydroxyl radicals. ROS damage lipid membranes, proteins and nucleic acids, activate cell signaling pathways and oxidant-sensitive, proinflammatory transcription factors, alter protein expression, and perturb membrane channels    Figure . WhetherThe control of intracellular heme content occurs at multiple levels. Here we focus on the regulation of heme synthesis, degradation and plasma membrane heme trafficking, which ensure the maintenance of appropriate intracellular heme concentration. Animal models discussed in this section are reported in Table Alas1 gene is located on chromosome 3 in humans and codes for an ubiquitously expressed protein whereas Alas2 gene is on the X chromosome and codes for an erythroid-specific protein  and the ATP-binding cassette transporter ABCB10 have been proposed to play this role.Yeast lacking YDL119c, the ortholog of SLC25A38, shows a defect in the biosynthesis of ALA . Then, the enzyme hydroxymethylbilane synthase (HMBS) catalyzes the head-to-tail synthesis of four porphobilinogen molecules to form the linear tetrapyrrole hydroxymethylbilane which is converted to uroporphyrinogen III by uroporphyrinogen synthase (UROS). The last cytoplasmic step, the synthesis of coproporphyrinogen III (CPgenIII), is catalyzed by uroporphyrinogen decarboxylase (UROD) , a homodimer weakly associated with the outside of the inner mitochondrial membrane. The following oxidation of protoporphyrinogen IX to protoporphyrin IX (PPIX) is catalyzed by protoporphyrinogen oxidase (PPOX), located on the outer surface of the inner mitochondrial membrane. Finally, ferrous iron is incorporated into PPIX to form heme in the mitochondrial matrix, a reaction catalyzed by the enzyme ferrochelatase (FECH)  gene. Two different isoforms of FLVCR1 exist, FLVCR1a and FLVCR1b, expressed on the plasma membrane and mitochondria respectively. FLVCR1b derives from an alternative transcription start site located in the first intron of the Flvcr1 gene thus resulting in the production of a shorter protein . The submitochondrial localization of FLVCR1b is still unknown as well as its ability to interact with other mitochondrial transporters. Further work is needed to definitively understand how heme is transported across the two mitochondrial membranes.Once synthesized, heme must be exported through the two mitochondrial membranes for incorporation into hemoproteins. The only mitochondrial heme exporter identified to date is the mitochondrial isoform of The importance of controlling the rate of heme synthesis is highlighted by the fact that mutations in many genes coding for enzymes involved in this pathway cause specific pathological conditions characterized by the accumulation of toxic heme precursors . The regulation of heme synthesis mainly occurs at the level of ALAS, the first and rate-limiting enzyme of the heme biosynthetic pathway. It has been demonstrated that heme plays an essential role in the regulation of its own synthesis by regulating the expression of ALAS.Alas1 mRNA. In addition, it has been shown that ALAS1 contains three heme regulatory motifs (HRMs). Heme binding to two of these HRMs, located in the mitochondrial targeting sequence of ALAS1, inhibits the transport of ALAS1 precursor protein in mitochondria  which interacts with iron regulatory protein (IRP) 1 and 2. The IRE binding activity of IRPs is regulated by cellular iron. In iron-deficient cells, the binding of IRPs to the 5'IRE inhibits the translation of Alas2 mRNA. In iron-repleted cells, IRPs are degraded thus stimulating the translation of Alas2 mRNA. This mechanism ensures the coordination of heme synthesis to the availability of iron thus avoiding the production of potentially toxic heme precursors when iron concentrations are limiting. The regulation of the IRE-binding activity of IRPs by cellular iron occurs through several mechanisms. The IRE binding activity of IRP1 is controlled by iron-sulfur clusters assembly while that of IRP2 by its oxidation, ubiquitination and degradation by the proteasome   and iron (Fe) Figure . BiliverHO-1 is highly inducible by a variety of stimuli including oxidative stress, heat shock, hypoxia, ischemia-reperfusion, lipopolysaccharide, heavy metals, cytokines and its substrate heme. Heme is the most potent physiologic inducer of HO-1. The enzyme activity was shown to increase in many tissues, including the liver, kidney, adrenals, ovaries, lung, skin, intestine, heart, and peritoneal macrophages. HO-2 is ubiquitously expressed and participates in the normal heme capturing and metabolism. The isoenzymes HO-1 and HO-2 are products of two different genes. The two forms show only 45% aminoacid homology but they share a region with 100% secondary structure homology, that corresponds to the catalytic site Maines, . HO-3 haHerein, we focus on the inducible isoform HO-1.HO-1 plays a vital function in heme degradation and protects against heme-mediated oxidative injury. Overexpression of HO-1 is associated to the resolution of inflammation through the generation of beneficial molecules like CO, bilirubin, and ferritin resulting from catabolism of toxic heme   1  is a member of the ABC transporter family that was originally found to confer drug resistance in breast cancer cells , and other two proteins, MFSD7 (SLC49A3) and DIRC2 (SLC49A4) . Thus, the assumption that FLVCR2 is a heme importer is not definitive and further studies are needed to fully address the substrate specificity of this transporter.Brasier et al.  proposedHCP1/PCFT belongs to the SLC46 family of the MFS. The gene encoding HCP1/PCFT spans five exons and four introns and is located on chromosome 11B5 in mouse and 17q11 in human. The HCP1/PCFT protein is highly conserved among different species, sharing about 90% similarity between human and mouse or rat  ; the partial loss of UROS activity leads to the incomplete metabolism of hydroxymethylbilane and the accumulation of non-physiologic porphyrin isomers in the bone marrow, erythrocytes, urine and other organs. CEP is mainly characterized by cutaneous photosensitivity and hemolytic anemia , acute intermittent porphyria , hereditary coproporphyria  and porphyria variegate , respectively. These disorders are characterized by the accumulation of ALA, porphobilinogen and/or porphyrins primarily in the liver. The major manifestations of these disorders are the life-threatening acute neurologic attacks and abdominal pain ; impaired UROD activity leads to the accumulation of uroporphyrin and other highly carboxylated porphyrins in the skin, liver and erythrocytes. PCT is characterized by blistering skin lesions that appear most commonly on the backs of the hands (increased cutaneous photosensitivity) and liver disease whereas neurologic features are usually absent   is a zebrafish mutant characterized by a delay in erythroid differentiation, abnormal globin gene expression and heme deficiency. Using positional cloning strategies, it has been reported that the sau gene encodes for ALAS2, thus confirming that loss of ALAS2 leads to a microcytic, hypochromic anemia similar to XLSA  gene have been identified  n microsatellite polymorphism in the HO-1 promoter is thought to regulate the extension of HO-1 induction as well as its response to many stimuli. Individuals with a lower number of (GT)n repeats retain the ability to more efficiently induce HO-1 than individuals with a higher number of repeats. Multiple studies demonstrate that individuals with fewer repeats are less prone to certain pathologies. It is interesting to observe that most of the pathologies affected by this polymorphism in humans overlap with those in which the outcome is exacerbated by HO-1 deletion . Additionally, one study correlated the presence of the shorter (GT)n polymorphism with a longer life of the individual. Individuals who lived longer were more likely to have the “high transcriptional induction” genotype, associated with the short HO polymorphism , infectious diseases , cardiovascular diseases  and ischemia-reperfusion injury -10, some prostaglandins, VEGF , stromal cell-derived factor 1, nitric oxide (NO) and NGF (nerve growth factor) . Alternatively, in the last decade a great effort was made to design and study molecules able to bind and deliver CO in biological systems, known as CO-releasing molecules (CO-RMs)    , posterior column of the spinal cord, retina and retinal pigment epithelium. The highest levels of Flvcr1 mRNA have been found in the retina and spinal cord thus suggesting a correlation between the regional specificity of Flvcr1 mRNA expression and the selective degenerative pathology of PCARP  has been proposed due to the observation that the feline leukemia virus subgroup C causes a pure cell aplasia in cats by interfering with the expression of FLVCR1 and that mice lacking both FLVCR1 isoforms phenocopy the human disease   (Table ABCG2 gene that leads to a glutamine-to-lysine amino acid substitution (Q141K)  Table . ABCG2 e0) Table , the maiABCG2 gene have been identified and associated to altered drug resistance  (Table Mutations in the 0) Table . The FowFLVCR2 expression, functions or localization.Several kinds of mutations were identified in cases of Fowler syndrome , a rare autosomal recessive disorder first described in 1961 , a folate importer alternative to HCP1/PCFT, which is expressed in the upper small intestine. RFC1 shows a lower affinity for folates as compared with HCP1/PCFT, but can compensate for HCP1/PCFT loss in HFM patients treated with high doses of folates (Qiu et al., Hcp1/Pcft−/− mouse represents the murine model for this HFM syndrome (Salojin et al., Hcp1/Pcft−/− mice die by 10–12 weeks of age and show a haematological profile which closely resembles that observed in human HFM patients.The The excess of free-heme is toxic due to its high reactivity and its ability to promote oxidative stress on lipids, proteins and nucleic acids, eventually leading to cell death.The existence of multiple levels of heme control indicates that mammals specifically evolved systems able to limit heme amount outside and inside the cell to counteract heme potential toxicity.As a primary defence mechanism, Hx scavenges free circulating heme, thereby avoiding it to exert its pro-oxidant effects. Indeed, Hx saturation observed in hemolytic disorders leads to heme-driven tissue damage and oxidative stress.ALAS2 gene cause insufficient heme production, mitochondria iron overload, and oxidative stress, as observed in sideroblastic anemia (Sheftel et al., The control of heme synthesis and degradation has a key role in the regulation of intracellular heme levels and these two mechanisms are respectively inhibited and induced by heme itself. Alterations in these processes lead to pathological iron or heme accumulation. Mutations in FLVCR1 or FLVCR2 affect specific cell populations, sensory neurons and photoreceptors or vascular cells of the central nervous system, respectively. Polimorphisms in ABCG2 result in defective urate excretion while mutations in HCP1/PCFT cause folate malabsorption. These discrepancies may be related to the different kind of mutations taken into considerations, loss-of-function mutations in mice and point mutations resulting in proteins with altered function/localization in humans. It is possible that null mutations can affect heme metabolism also in humans or that specific polimorphisms in genes coding for heme transporters can differentially alter the import/export of specific substrates. The full comprehension of the role of FLVCR1-mediated heme export in human physiology and pathology is a challenge for future studies as well as the definition of the role of FLVCR2, ABCG2, and HCP1/PCFT in heme metabolism. Of course, the definition of protein structure and the identification of residues critical for heme transport in heme transporters are very important issues. In addition, the description of the regulation of these transporters in different tissues and following different stimuli will be of relevance to fully understand the multi-level system of heme control.The recent identification of plasma membrane heme transporters suggests that intracellular heme amount could be regulated not only at the level of its synthesis and catabolism but also at the level of heme export and import. Nevertheless, apart FLVCR1 for which studies in mouse models have provided compelling evidence that it exerts a physiologic role as a heme exporter, the experimental evidence that ABCG2, FLVCR2, and HCP1/PCFT are heme transporter with a physiologic role in heme/iron homeostasis are very poor. Thus, it would be more correct to refer to these molecules as secondary regulators of heme and/or porphyrin metabolism. This conclusion is corroborated by genetic studies in humans that failed in finding a clear association between mutations in genes coding for heme transporters and disorders of heme metabolism as was expected from studies on cellular and animal models. Mutations in In the future, the comprehension of these mechanisms could have important clinical implications. Targeting heme transporters could be therapeutically useful to prevent heme-driven tissue damage in pathologies characterized by enhanced oxidative-stress.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Colossoma macropomum, Piaractus mesopotamicus and Piaractus brachypomus) and their hybrids in different stocks purchased from three seed producers in Brazil. The main findings of this study were the detection of intergenus backcrossing between the hybrid ♀ patinga (P. mesopotamicus×P. brachypomus)×♂ C. macropomum and the occurrence of one hybrid triploid individual. This atypical specimen might result from automixis, a mechanism that produces unreduced gametes in some organisms. Moreover, molecular identification indicated that hybrid individuals are traded as pure species or other types of interspecific hybrids, particularly post-F1 individuals. These results show that serrasalmid fish genomes exhibit high genetic heterogeneity, and multi-approach methods and regulators could improve the surveillance of the production and trade of fish species and their hybrids, thereby facilitating the sustainable development of fish farming.Juvenile fish trade monitoring is an important task on Brazilian fish farms. However, the identification of juvenile fish through morphological analysis is not feasible, particularly between interspecific hybrids and pure species individuals, making the monitoring of these individuals difficult. Hybrids can be erroneously identified as pure species in breeding facilities, which might reduce production on farms and negatively affect native populations due to escapes or stocking practices. In the present study, we used a multi-approach analysis (molecular and cytogenetic markers) to identify juveniles of three serrasalmid species ( This inaccuracy primarily reflects the morphological similarity between species, particularly in the case of hybrids and parental species in the juvenile stage. Thus, the use of genetic markers is essential to monitor the production and management of fish hybrid, particularly for the trade between seed suppliers and fish farmers, which is a critical point in the production chain mt-co1) and Cytochrome b (mt-cyb), with nuclear genes, such as α-Tropomyosin (tpm1) and Recombination Activating Gene 2 (rag2) Currently, PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) and multiplex-PCR have been characterized as efficient methods for the rapid and inexpensive identification of hybrids in situ hybridization , chromosome markers have facilitated the precise identification of the hybrids tambacu and tambatinga, respectively Cytogenetic analysis methods have also been described for the identification of serrasalmid hybrids. Through C-banding and fluorescence Despite hybrid vigor in some cases, there are problems associated with the inadequate use of interspecific hybrids for aquaculture production. Occasionally, fish farmers have mistakenly used hybrids as broodstock, as reported for tilapia, catfish, and carp In the present study, we used PCR-RFLP, multiplex-PCR, and cytogenetic methods to evaluate the juvenile fish trade between seed suppliers and fish farmers in Brazil, focusing on the genetic identification of F1 and post-F1 serrasalmid hybrids. The novelty of this study was the discovery of fertility in the hybrid patinga , and its use as broodstock in Brazilian aquaculture. Moreover, mistaken trade of hybrid tambacu was detected in fish farms and one post-F1 hybrid was characterized as triploid.We performed the genetic identification of 924 juvenile individuals from eight stocks of live fish purchased from three private Brazilian aquaculture seed producers  . All fisThis study was conducted in strict accordance with the recommendations of the National Council for Control of Animal Experimentation . The present study was performed under authorization N° 33435-1 issued through ICMBio . Fin fragments were collected from each fish under benzocaine anesthesia and all efforts were made to minimize suffering. DNA was extracted from the fin fragments using the Wizard Genomic DNA Purification Kit (Promega) according the manufacturer's protocol. The DNA concentration was assessed against a molecular marker standard  through electrophoresis on a 1% agarose gel.tpm1) and mitochondrial Cytochrome C Oxidase subunit I (mt-co1) genes; and 2) PCR-RFLP using the nuclear Recombination Activating Gene 2 (rag2) and mitochondrial Cytochrome b (mt-cyb) genes. Both methods provide diagnostic electrophoretic fragments for each parental species and their interspecific hybrids. Diagnostic sizes of the PCR products or restriction fragments are described in the The samples were genotyped using two methods and different genes, as previously described in situ hybridization (FISH) was performed using the method of Yang et al. Cytogenetic analysis was also performed to verify the ploidy level in individuals of the SPS2 stock. Chromosomal preparations were obtained according to the methods of Foresti et al. tpm1 , pure tambaqui (MGS1 and SES1), pacu (MGS2), and hybrid tambatinga (SES2), indicating that these species correspond to hybrid tambacu. The results of the multiplex-PCR analysis of the nuclear marker tpm1  revealed (mt-co1  showed tmt-co1 (fragment of 307 bp) and mt-cyb (fragments of 152 and 513 bp) genes, and segregating genotypes at the nuclear tpm1 and rag2 markers, as indicated below  with ♂ tambaqui. Consistent with this hypothesis, all the offspring showed the pacu genotypes for the mitochondrial ed below  and 3:tpm1 (fragments of 172 and 269 bp) and rag2 .Genotype A : pattern of hybrid tambacu for both nuclear markers tpm1 (131 and 172 bp) and rag2 .Genotype B : pattern of hybrid tambatinga for both nuclear markers tpm1 (172 and 269 bp) gene, and genotype of hybrid tambatinga for the rag2  gene.Genotype C : nuclear markers of hybrid tambacu for the i.e., genotype of hybrid tambatinga for the tpm1 (131 and 172 bp) gene, and genotype of hybrid tambacu for the rag2  gene.Genotype D : opposite nuclear patterns to the genotype C, tpm1  gene, and genotype of hybrid tambacu for the rag2  gene. This unexpected pattern is compatible with a triploid individual, consistent with the cytogenetic results shown below.Genotype E : atypical genotype comprising gene fragments from the three pure species  for the According to the Mendelian inheritance, the hypothesized backcross ♀ patinga (♀ pacu x ♂ pirapitinga) x ♂ tambaqui would produce each A–D genotype at 25% among the offspring , and thetpm1 gene locus and the centromere, but not in the rag2-bearing chromosomes, during female oogenesis, with the fusion/retention of the second polar body in the egg, resulting in a triploid individual , suggesting a polyploid individual with three chromosome sets (3n) , and cordividual .tpm1 and rag2, thus suggesting independent segregation between these markers.The FISH analysis of the specimens in the SPS2 stock revealed 5S rRNA clusters in the subcentromeric region of four chromosomes, some of which were non-homologous, as revealed through differences in morphology/size and the positions of the hybridization signals . In two Hybrids between tambaqui and pacu are popular in Brazil, as these hybrids combine the robustness and faster growth rate of tambaqui with the low temperature resistance of pacu Special attention should be given when hybrids are sold as pure species, as demonstrated in the present study for the stocks of tambaqui (MGS1 and SES1) and pacu (MGS2). The results showed that in addition to fraud, fish farmers are not aware of the potential biological and environmental risks represented by hybrids, whose impact could affect the aquaculture industry and threaten native species, as previously described in other species, such as tilapia, catfish, and trout In addition, the results of the molecular characterization in the present study provided clear evidence that individuals of female patinga are fertile, similar to the hybrids tambacu and tambatinga Pseudoplatystoma corruscans and Pseudoplatystoma reticulatumSerrasalmid hybrids have also been detected in the natural environment and some authors have suggested that these individuals are the consequence of aquaculture activities The combination of molecular markers and cytogenetic techniques was essential for the identification of a triploid individual in the present study and to confirm the hybridization processes. Cytogenetic information is considered a suitable tool to verify ploidy level and analyze parental chromosome sets in hybrids i.e., genetically identical to the parent without genetic variation of the resulting eggs Spontaneous triploid fish can be explained by several cytological mechanisms that produce unreduced diploid gametes Alternatively, these results are consistent with a cytological mechanism similar to automixis, in which meiosis is maintained and diploid gametes are generated after meiosis through the fusion/retention of the polar body. This phenomenon has been well documented in the triploid offspring of poeciliid interspecific hybrids Consistent with the data obtained in this study, the presence of 5S rRNA clusters in two pairs of chromosomes in diploid individuals is a common characteristic in fish genomes Oncorhynchus mykiss and Oncorhynchus clarkii lewisi.The molecular and cytogenetic data obtained herein are consistent with the hypothesis of the fertility of hybrid patinga. However, the presence of additional post-F1 hybrids (F2 or advanced backcrosses) cannot be ruled out at least for some specimens because of the small number of nuclear markers used in this study. According to Boecklen and Horward In Brazil, most aquaculture establishments are not licensed and there is little proposed legislation regulating fish breeding e.g., hybrid patinga) and hybrid tambacu can be erroneously traded as pure species. In conclusion, this multi-approach analysis  was useful for the detection of hybridization and the results provided new insights concerning the genome plasticity of serrasalmid species, including the occurrence of intergenus backcrossing between ♀ patinga (♀ pacu x ♂ pirapitinga) x ♂ tambaqui and the presence of an post-F1 hybrid triploid, likely derived from similar mechanisms of unreduced gametes in automixis described for other fish hybrids Consistent with Hashimoto et al.


Organ transplantation has evolved into a reliable life-saving procedure where good organ function is quickly restored by the transplant and, typically, the patient can return to an active lifestyle. Unfortunately, the human immune system reacts strongly against allogeneic tissues, and will destroy a transplanted organ within days or weeks after transplantation if the immune system is not depleted or inhibited by immunosuppressive drugs. Near elimination of the immune system through non-specific or speciThe ONE Study is the acronym for a large scale collaborative project funded by the European Community’s FP7[The ONE Study consortium explores the feasibility and potential of cell therapy in organ transplantation by bringing together experts to test different regulatory cell products composed of specific T cell , dendritic cell and macrophage subpopulations. While the primary objective of the study is to perform a stringent side-by-side comparison of these cell products using a single (ONE) standardized trial protocol, a secondary aim is to use this unique cooperative opportunity to experimentally examine the immunoregulatory characteristics of the cell products to be tested.The ONE Study. The first of these workshops has already taken place this year with an emphasis exclusively on producing and comparing suppressive populations of cells derived from monocytes, namely tolerogenic dendritic cells (tol DCs)[The ONE Study. While the final results of this first workshop are still pending, the assembled group of workshop participants decided it would be informative to perform a separate review of each of the monocyte-derived regulatory cell populations under investigation.To address the issue of comparing and contrasting suppressive cell populations with cell therapy potential, a series of investigative workshops have been planned within the context of tol DCs), myeloidtol DCs), IL-10-itol DCs),12. A ceTransplantation Research, workshop participants present literature and their perspectives on the specific cell population that they produced for comparison. It is important to keep in mind that the workshop investigators are studying the different monocyte-derived regulatory cells more from a cell production standpoint, versus taking a view towards normal physiological development. Indeed, the in vivo temporal and complex signals directing a blood monocyte or tissue macrophage to become activated, to take on professional antigen-presenting functions, to migrate to lymphoid structures, or to become suppressive, are not fully understood. However, the workshop participants primarily aim to distinguish or show similarities between suppressive monocyte-derived cell populations that can be generated under defined in vitro conditions. This is an important undertaking since cell therapy applications demand an ex vivo cultivation phase to manufacture a homogenous and reproducible cell product.In the current issue of etc.) of these closely related cells to each other, they are comparable to cells such as MDSCs, which can be generated under similar conditions, but in the presence of prostaglandin E2[2[ex vivo interferon gamma stimulation  tol DCs, (2) DC-10 cells, (3) rapamycin-conditioned DCs (rapa DCs), (4) MDSCs, (5) M regs and (6) monocytes conditioned with mesenchymal stem cells (MSCs). The variety of stimuli that are considered for cell production is especially evident with tol DCs. The workshop group concentrated on tol DCs generated under conditions including stimulation with low concentrations of GM-CSF, and witlandin E2 or otherndin E2[2; whether (M regs). While  (M regs, and hav (M regs, it remaONE Study workshop on monocyte-derived suppressor cells, the involved investigators deemed it useful to review the present literature on tolerogenic DCs and macrophage/myeloid derived suppressor cell populations. The authors of the reviews consider the similarities and differences of these closely related cell populations, and suggest how they may best be applied as a cell therapy meant to reduce organ transplant rejection. Indeed, cell therapy is entering a critical early phase of testing in transplantation, mandating that we take the necessary steps to determine which cell populations are most adaptable and effective for this purpose. The reviews in this issue of Transplantation Research offer a view into the future of cell therapy involving suppressive cells derived from readily available circulating monocytes.In preparing to assess the results of the first