Both effects are highly significant, and we estimate that 12±3% of all heritable variation in human gene expression is due to cis variants.Variation in gene expression is a fundamental aspect of human phenotypic variation. Several recent studies have analyzed gene expression levels in populations of different continental ancestry and reported population differences at a large number of genes. However, these differences could largely be due to non-genetic  effects. Here, we analyze gene expression levels in African American cell lines, which differ from previously analyzed cell lines in that individuals from this population inherit variable proportions of two continental ancestries. We first relate gene expression levels in individual African Americans to their genome-wide proportion of European ancestry. The results provide strong evidence of a genetic contribution to expression differences between European and African populations, validating previous findings. Second, we infer local ancestry  at each location in the genome and investigate the effects of ancestry proximal to the expressed gene ( cis) versus at distal locations (trans), we can use ancestry effects to quantify the relative contributions of cis and trans regulation to human gene expression. We estimate that 12±3% of all heritable variation in human gene expression is due to cis variants.Variation in gene expression is a fundamental aspect of human phenotypic variation, and understanding how this variation is apportioned among human populations is an important aim. Previous studies have compared gene expression levels between distinct populations, but it is unclear whether the differences that were observed have a genetic or nongenetic basis. Admixed populations, such as African Americans, offer a solution to this problem because individuals vary in their proportion of European ancestry while the analysis of a single population minimizes nongenetic factors. Here, we show that differences in gene expression among African Americans of different ancestry proportions validate gene expression differences between European and African populations. Furthermore, by drawing a distinction between an African American individual's ancestry at the location of a gene whose expression is being analyzed ( Admixed populations are uniquely useful for analyzing the genetic contribution to phenotypic differences among humans. Phenotypic differences that are observed among human populations may have systematic non-genetic causes, such as differences in environment Gene expression is a fundamental determinant of cellular phenotypes, and understanding how gene expression variation is apportioned among human populations is an important aspect of biomedical research, as has been true for apportionment of human genetic variation at the DNA level In this study, we analyzed lymphoblastoid cell lines from 89 African-American samples and investigated the relationship between expression levels of ∼4,200 genes and the proportion of European ancestry. We compared the results with those predicted from the differences in expression levels between 60 European samples  and 60 African samples (YRI from HapMap) cis or trans variants. Previous studies in humans have been successful in mapping both cis and trans effects, but the results they provide are far from complete, due to limited sample sizes cis vs. trans associations that were reported varies widely across these studies, perhaps due to differences in power or choices of significance thresholds cis vs. trans regulatory variation in human gene expression has not yet been established. Here, by measuring how gene expression levels across all genes vary with local ancestry  either proximal to the expressed gene (cis) or elsewhere in the genome (trans), we estimate that 12±3% of heritable variation in human gene expression is due to cis variants.Heritable variation in gene expression may be due to 100 African-American (AA) samples from the Coriell HD100AA panel were genotyped on the Affymetrix SNP 6.0 GeneChip. Genotyping was conducted at the Coriell Genotyping and Microarray Center, and the genotype data was obtained from the NIGMS Human Genetic Cell Repository at Coriell (see Web Resources). In addition, genotype data from 60 European (CEU), 60 African (YRI), 45 Chinese (CHB) and 44 Japanese (JPT) samples was obtained from Phase 2 HapMap r2 of 0.98 between inferred local ancestry and true local ancestry in simulated African-American data sets . The HAPMIX program inputs AA genotype data and phased CEU and YRI data from Phase II HapMap Local ancestry  at each location in the genome was estimated for each AA sample using the HAPMIX program, a haplotype-based approach that has been shown to attain an Lymphoblastoid cell lines for 60 HapMap CEU, 60 HapMap YRI and the Coriell HD100AA samples were obtained from Coriell Cell Repositories (see Web Resources). Gene expression was assayed using the Affymetrix Genome Focus Array, as described previously g, we normalized gene expression measurements for CEU and YRI to have mean 0 and variance 1 across 120 CEU+YRI samples, and normalized gene expression measurements for AA by applying the same normalization for consistency. We implicitly assume an additive genetic model in which gene expression has genetic and non-genetic components, with part of the genetic component predicted by ancestry. Let gse denote normalized gene expression of gene g for sample  s. Let sθ denote the genome-wide European ancestry proportion of sample s, so that sθ has value 1 for CEU samples and 0 for YRI samples as above, and fractional values for AA samples. We consider a model in which gse = gθsa+gsν for CEU and YRI samples and gse = gθsca+gsν for AA samples, where c is a global parameter and gsν represents the residual contribution to gene expression that is not predicted by ancestry. Thus, the parameter c represents a validation coefficient measuring the aggregate extent to which the observed gene expression differences ga between CEU and YRI (differences between sample means) are heritable.For each gene c and ga of this model: (1) Starting with the initial guess c = 1, we alternated computing maximum likelihood estimates for ga  conditional on c, and computing a maximum likelihood estimate for c conditional on ga , and iterated to convergence. In each case, the maximum likelihood estimates were obtained via linear regression . (2) For each g, we estimated values gã,CEU+YRI by regressing gse against sθ using CEU and YRI data only, and gã,AA by regressing gse against sθ using AA data only. We then regressed gã,AA against gã,CEU+YRI to obtain an estimate of c. In this computation, we scaled our estimates of gã,CEU+YRI using the sampling error correction ξ (described below in Computation of QST) to remove the effect of sampling error on the denominator Σg2 of our estimate of c. . We observed that approaches (1) and (2) produced identical estimates of c, indicating that both approaches are effective in finding the best fit to the model. We followed approach (2) to plot gã,AA vs. gã,CEU+YRI and to compute estimates of c specific to different values of |gã,CEU+YRI|.We implemented two different approaches for fitting the parameters We repeated the above computation using genotype data instead of gene expression data. We restricted the analysis to markers in which the average of CEU and YRI frequencies was between 0.05 and 0.95. Although AA genotypes at each marker were used twice in this computation—both for estimating genome-wide ancestry using all markers and for measuring the effect of genome-wide ancestry on genotype at a specific marker—we note that with hundreds of thousands of markers, our estimate of genome-wide ancestry is negligibly impacted by data from a specific marker.cis ancestry and trans ancestry on gene expression in AA. Roughly, we define cis ancestry as the local ancestry at the gene whose expression is being analyzed, and trans ancestry as the average ancestry at non-cis regions. We extended our above model by letting gse = cisagγgsc+transagθs+νgsc for AA samples, where gsγ denotes the estimated local ancestry of sample s at the SNP closest to the center of gene g . We note that although trans ancestry is theoretically defined as the average ancestry at non-cis regions, this quantity is in practice virtually identical to sθ because cis regions (regardless of the precise definition of cis) form an extremely small proportion of the genome. Because chromosomal segments of ancestry in AA typically span >10 Mb cis ancestry gsγ. The probabilistic estimates of local ancestry produced by HAPMIX are extremely accurate (see above), so that gsγ is typically close to 0.00, 0.50 or 1.00 . To avoid complications in local ancestry analyses on the X chromosome, we restricted this analysis to 4,015 autosomal genes.  We estimated the global parameters cisc and transc as above, accounting for the correlation between genome-wide and local ancestry by using residual values of gsγ (adjusted for sθ) to compute cisãg,AA, .We investigated the effects of F denote the proportion of total variance in gene expression that is attributable to population differences. For quantitative traits with an additive genetic basis, the quantity that is analogous to single-locus estimates of FST is not F, but rather QST = F/(2−F) . Due to sampling error, the observed difference gã in normalized gene expression between CEU and YRI samples (i.e. the coefficient obtained from a regression of gse on sθ) has variance 2F+(1–0.5F)/30, where 1/30 is the sum of reciprocals of CEU and YRI sample sizes. We thus estimated mean F as (Varg(gã) – 1/30)/(2 – 0.5/30). The ratio between mean F and Varg(gã)/2 represents a sampling error correction that we call ξ. We estimated median F as the median value of gã2/2 times ξ. The value of ξ was 0.93, indicating that the sampling error correction had only a minor effect on these computations. To account for differences between CEU and YRI due to non-genetic factors, we adjusted F by multiplying it by c.  We then computed QST = F/(2−F). We calculated the standard error of our estimate of F via jackknife, repeating the computation of F 120 times with one of the 120 CEU+YRI samples excluded in each computation, and estimating the standard error as the standard deviation of the 120 estimates times the square root of 120.Let http://ccr.coriell.org (Coriell Cell Repositories)http://ccr.coriell.org/Sections/Collections/NIGMS/GenotypeCopyData.aspx?PgId564&coll=GM (The NIGMS Human Genetic Cell Repository at Coriell)http://www.hapmap.org http://www.ncbi.nlm.nih.gov/geo (Gene Expression Omnibus)http://www.stats.ox.ac.uk/˜myers/software.html (HAPMIX program)FST values between the set of 89 AA samples and possible linear combinations αCEU+(1−α)YRI, adjusting for sample size. The lowest value of FST = 0.0009 was obtained at α = 0.21. Thus, the 89 AA samples are extremely well-modeled as a mix of CEU and YRI, with average ancestry proportions of 21% CEU and 79% YRI. Though this justifies our modeling approach using CEU and YRI, we caution against drawing historical inferences from this finding: because FST scales with the square of admixture proportion, it is possible that African Americans inherit a small percentage of their ancestry from a more diverse set of populations.We analyzed Affymetrix 6.0 genotype data from the African-American panel of 100 samples from Coriell Cell Repositories, together with HapMap samples see . We firsr2>0.99) with coordinates along the top principal component   .We measured gene expression in lymphoblastoid cell lines from 60 CEU and 60 YRI samples from HapMap and 89 AA samples from Coriell, using the Affymetrix Genome Focus Array see . Our basg is equal to ga per unit of European ancestry for CEU and YRI samples , and equal to gca per unit of European ancestry for AA samples, where c is constant across genes . Thus, gene expression differences among AA samples of varying ancestry strongly confirm that heritable differences contribute to observed gene expression differences between CEU and YRI. Performing the analogous computation with genotype data, we obtained c = 0.96, confirming that c is close to 1 for genetic effects  or to variants elsewhere in the genome (trans). We inferred the local ancestry of each AA sample at each location in the genome  and transc  were significantly different from zero. Thus, only a small fraction of the effect of ancestry on gene expression is due to ancestry at the cis locus. On the other hand, performing the analogous computation with genotype data, we obtained cisc = 0.99 and transc = –0.03, indicating as expected that the effect of ancestry on genotype is entirely due to ancestry at the cis locus, and confirming the high accuracy of our estimates of local ancestry.The effect of ancestry on gene expression in African Americans may be due either to variation in regulatory variants proximal to the gene (nome see . A descrrans see . We obtaonly see . Both cccisπ of heritable gene expression variation between Europeans and Africans that is due to cis variants as cisc/(cisc+transc) = 12%, with a standard error of 3%. An important question is whether our estimate of cisπ can be extended to all heritable variation in human gene expression. If the relative magnitude of cis vs. trans effects were different for all variation as compared to population variation—equivalently, if the relative magnitude of population variation relative to all variation were different for cis vs. trans effects—then the answer to this question would be no. To evaluate whether this is the case, we computed FST for ∼3,000 unique cis eQTL SNPs and ∼700 unique trans eQTL SNPs identified in a recent study of gene expression in human liver FST values of 0.158 for cis eQTLs and 0.154 for trans eQTLs, which were not significantly different from 0.159 for all HapMap SNPs , based on standard errors computed using the EIGENSOFT software cis vs. trans effects, implying that our estimate of cisπ = 12±3% applies to all heritable variation in human gene expression.We estimate the proportion F, and the quantity QST = F/(2−F) which is analogous to FST for genetic  data  between CEU and YRI in gene expression correspond, with overwhelming statistical significance, to differences among African Americans of varying ancestry, implying a substantial heritable component to the population differences. In reaching this conclusion via analysis of an admixed population, we eliminate confounding with non-genetic contributions to observed differences between the ancestral populations, which could result from differences in environment, differences in preparation of cell lines, or batch effects. The value of 0.43 for the “validation coefficient” c did not vary appreciably as a function of the magnitude of observed gene expression differences between CEU and YRI. This suggests that the effects of ancestry on gene expression are widespread across genes, as opposed to affecting only a fraction of genes. Although there exist genes for which the observed effect of ancestry on expression levels is close to zero  of segments of continental ancestry in African Americans, but this has little effect on our conclusions, since a 10 Mb region represents a proportion of the genome that is much smaller than the 12% proportion of heritable variation in gene expression that we attribute to variation at the cis locus. Comparing our results to results obtained in other species, we note that two recent studies of gene expression in Drosophila also reported that cis effects explain a small fraction of heritable variation Drosophila studies had suggested a larger role for cis effects In addition to validating the aggregate effects of ancestry on human gene expression, we were able to partition heritable variation into Text S1Supplementary Note.(0.02 MB PDF)Click here for additional data file.


Failure of ventilation in response to different stressors  was associated with a disturbance of CNS ion homeostasis that shares the characteristics of CSD and SD-like events in vertebrates. Hyperthermic failure was preconditioned by prior heat shock  and induced-thermotolerance was associated with an increase in the rate of clearance of extracellular K+ that was not linked to changes in ATP levels or total Na+/K+ ATPase activity. Our findings suggest that SD-like events in locusts are adaptive to terminate neural network operation and conserve energy during stress and that they can be preconditioned by experience. We propose that they share mechanisms with CSD in mammals suggesting a common evolutionary origin.Cortical spreading depression (CSD) is closely associated with important pathologies including stroke, seizures and migraine. The mechanisms underlying SD in its various forms are still incompletely understood. Here we describe SD-like events in an invertebrate model, the ventilatory central pattern generator (CPG) of locusts. Using K Hyperthermic failure of neural operation is associated with a loss of ion homeostasis and a major redistribution of ions, including Ksion CSD . In mamm2+ conductances and increases activity of the Na+/K+ ATPase + currents in locust metathoracic neurons + homeostasis.Ventilation in locusts is under the control of a reliable central pattern generator (CPG) located in the metathoracic ganglion (MTG) +]o during failure of motor pattern generation induced by different stressors  and used ouabain (a specific Na+/K+ ATPase toxin) both to induce repetitive, propagating waves of ionic disturbance and to test the role of the Na+/K+ ATPase in HS-mediated thermoprotection. We demonstrate that the surges of [K+]o associated with cessation of ventilatory motor patterning represent SD-like phenomena whose occurrence can be interpreted as an adaptive response to conserve energy in the locust, rather than the culmination of cellular collapse. Using TTX and TEA we show that SD-like behaviour is not activity-dependent and that some part of the [K+]o surge is via TEA-sensitive K+ channels. Finally we show that the hyperthermic SD-like event can be preconditioned by a prior HS acting to increase the rate of [K+]o clearance, and that the effect of HS is not obviously mediated by the Na+/K+ ATPase.We compared the dynamics of [K+]o, which occurred in 100% of CON preparations . When the temperature was allowed to return to baseline levels, [K+]o remained elevated before gradually decreasing  and anoxia (100% N2) (+]o reached during surges induced by hyperthermia (52±2 mM), NaN3 (85±2 mM) and anoxia (79±3 mM) were different  = 43.634). The peak [K+]o induced by hyperthermia was lower than the [K+]o peaks induced by NaN3 and anoxia . The recovered motor pattern was robust but not identical to the pre-stress motor pattern (see e.g. i and ii).Ventilatory motor pattern activity and extracellular potassium ion concentrations in the MTG were measured during treatment with various cellular stressors in order to establish a relationship between stress-induced circuit failure and an extracellular build-up of potassium ions 2. Hyper+]o were reliably triggered by Na+/K+ ATPase inhibition using 10−4 M ouabain and by locally increased [K+]o within the ganglion  was sufficient to bring [K+]o to threshold for induction of an abrupt surge  was evident without recovery  .To determine if the peak [K+ and K+ channel blockers  to determine if the hyperthermic SD-like event in the locust is dependent on neural activity and if K+ efflux is at least in part via TEA-sensitive K+ channels. Bath application of 10−6 M TTX quickly abolished electrical activity from ventilatory muscle 161 while [K+]o remained stable at ∼10 mM for 15 minutes prior to the temperature ramp . The abrupt rise in [K+]o was delayed in TTX-treated locusts, occurring at higher temperatures during the continuous ramp increase than in CON locusts  (−1 M) was required to successfully abolish motor pattern generation, although electrical activity was still detected from muscle 161. The hyperthermic [K+]o event occurred at 38.9±0.6°C in locusts treated with 10−1 M TEA compared to 39.3±0.9°C (no significant difference) in CON locusts  (+]o reached a higher second plateau in TEA-treated locusts (109±9 mM) that was not different from the second plateau in CON locusts .We used voltage-gated Na+]o event induced by various cellular stressors. ATP levels in the MTG taken from locusts subjected to hyperthermia before and after heat shock preconditioning (CON and HS), 10−4 M ouabain, 10−3 M sodium azide, and anoxia (100% N2) were compared prior to stress, at failure and at subsequent recovery of the motor pattern . Stress treatment and the state of motor pattern generation, i.e. failure vs. recovery, both affected ATP levels  = 17.435; P = 0.002, F = 10.193). At failure of the motor pattern, NaN3-treated locusts had lower ganglionic ATP levels than all other groups. Locusts subjected to anoxia and hyperthermia (CON) had lower ATP levels than locusts treated with 10−4 M ouabain . At recovery of the motor pattern NaN3-treated locusts had lower ganglionic ATP levels than locusts subjected to 10−4 M ouabain and hyperthermia (HS) suggesting that recovery was independent of metabolic state . ATP levels in ganglia at failure and recovery of the motor pattern were not different except in NaN3-treated locusts . Notably, HS preconditioning did not increase ATP levels at hyperthermic failure or subsequent recovery of the motor pattern compared to CON-hyperthermia locusts  = 0.531). Anoxia exposure affected ganglionic ATP  = 11.418). Anoxia (15 minutes) did not affect ATP levels in either CON or HS locusts, even though locusts entered anoxic coma within 1 minute. ATP levels dropped after 30 minutes of anoxia and remained stable for 90 minutes in CON and HS locusts . Following 2 hours in an anoxic environment, ATP increased after 60 minutes of normoxia in CON and HS locusts .Cell energetics may play a role in the initiation of, and recovery from, the [K−5 M ouabain to test whether increased Na+/K+ ATPase activity could underlie the increased thermotolerance conferred by HS pre-treatment −5 M ouabain is not normally sufficient to induce the SD-like events of +/K+ ATPase (see below). HS pre-treatment and 10−5 M ouabain bath application affected [K+]o before and during a temperature ramp  were examined after 15 minutes of ouabain treatment at room temperature and in response to the initial 5°C increase of the temperature ramp to determine the effects of HS pre-treatment and 10−5 M ouabain treatment on K+ homeostasis  = 0.703; P<0.001, F = 42.772) . HS pre-treatment affected Δ[K+]o after a 5°C increase in temperature, however there was no effect of 10−5 M ouabain treatment  = 15.584; P = 0.988, F = 0.000223) .We used 10ure ramp Figure 6+]o event and subsequently hastened recovery and re-establishment of normal [K+]o (+]o event at failure (+]o in C and HS locusts (data not shown) indicating that the effect of HS on rate of recovery was not due to a decrease in the initial ionic disturbance. There was a strong positive correlation between time to recovery and failure temperature in control locusts that was shifted by HS and by 10−5 M ouabain   = 11.777; P = 0.007, F = 8.140) . HS pre-treatment and 10−5 M ouabain treatment also affected time to recovery of the motor pattern with a similar lack of interaction  = 15.302; P = 0.005, F = 8.823) .HS pre-treatment increased the upper temperature limit and shortened time to recovery when these measures were examined independently  = 2.846; P = 0.482, F = 0.502)  = 14.395; P = 0.024, F = 5.478) . Thus the proportion of the [K+]o clearance rate sensitive to the 10−5 M ouabain treatment was halved by HS from ∼40% in control preparations to ∼20% after HS.Rates of [K+]o clearance rates we also measured steady-state Na+/K+ ATPase activity within the MTG. Na+/K+ ATPase activity  was 144±12 nmol ATP/min/mg protein in CON ganglia compared to 133±9 nmol ATP/min/mg protein in HS ganglia . In the presence of phosphatase inhibitors, Na+/K+ ATPase activity was 120±7 nmol ATP/min/mg protein in CON ganglia compared to 116±10 nmol ATP/min/mg protein in HS ganglia . This suggests that total Na+/K+ ATPase activity is not a target for protection by HS preconditioning.To complement the results obtained by ouabain treatment on [K+]o and its subsequent restoration when temperature is allowed to return to normal. Anoxia and ATP depletion evoked similar tissue responses, which could also be induced when we disturbed the ionic equilibrium by impairing Na+/K+ ATPase activity using ouabain or by local neuropile injections of high [K+] saline. The fact that a prior anoxia-induced [K+]o surge occluded the hyperthermic event suggests that these different stressors converged on the same mechanism. In addition, the nature of these events as large ionic disturbances propagating throughout the MTG neuropile makes it unlikely that different stressors would have additive effects. The amplitude of the hyperthermic [K+]o surge was smaller than those induced by either anoxia or ATP depletion and we attribute this partly to the confounding effect of its occurrence at high temperatures (>40°C). This is supported by further analysis that shows a significant negative correlation between the failure temperature during hyperthermia  and [K+]o at its peak , and may be due to the fact that at the point of hyperthermic failure [K+]o increased from its room temperature level  thereby reducing the driving force on K+ ions. We conclude that the different stressors converged on a single tissue mechanism.In a central pattern generator controlling ventilation in the locust, we have shown that hyperthermic failure and recovery of neural function are tightly correlated with an abrupt rise in [K+]o+]o+]o), 2) were induced by the same stimuli, 3) were not activity-dependent, 4) occurred as repetitive waves with ouabain, 5) propagated at around 2 mm/min, 6) did not cross between ganglia through the connectives  and 7) were associated with complete depression of neural activity manifest by failure of ventilatory pattern generation. Thus the locust tissue response, reflected in the all-or-none surge of [K+]o, shares most, if not all, of the essential characteristics of CSD.CSD occurs in vertebrate grey matter and is characterized by a rapid and nearly complete depolarization of neurons that propagates at 2–5 mm/min as a wave across the cortex. This propagating wave is due to a disordered regulation of ionic homeostasis evident as a massive redistribution of ions across neuronal membranes, including large increases of [K+/K+ ATPase (10−4 M ouabain) and by local injection of high [K+] saline into the neuropile. Moreover there was no correlation between the occurrence of motor pattern failure and recovery with levels of ATP in the MTG. Energy status of a cell  can change and activate signaling pathways without measureable effects on ATP +]o to near normal levels and thus the time taken to recover was related to the rate of clearance of extracellular K+ ions. Treatment with 10−5 M ouabain rendered preparations more thermosensitive by decreasing failure temperature and increasing recovery time concomitant with a decrease in the rate of extracellular K+ ion clearance during the recovery period. This indicates an important role for the Na+/K+ ATPase pump, as in vertebrate SD-like phenomena. The [K+]o disturbance induced by injection of high [K+] saline propagated within the locust MTG at a similar rate as the depolarizing wave in mammalian brain slices during CSD. Studies suggest opening of intercellular gap junctions as a mechanism of SD propagation in vertebrates The biophysical mechanism of the extensive redistribution of ions causing CSD is incompletely understood. It nevertheless seems clear that SD-like events in mammals are triggered by positive feedback processes involving an initial ionic disturbance reaching a threshold and caused by neuronal overexcitation or by treatments or conditions that impair ionic homeostasis . In a model hippocampal pyramidal neuron, SD-like events can be triggered by manipulating ion clearance mechanisms or levels of excitation, causing the net current in the apical dendrites to turn inward +]o levels reached a high plateau and motor pattern recovery was precluded.Our findings show that the events we recorded in locust ganglia are SD-like ionic disturbances similar to CSD and possibly induced by common mechanisms, which in theory would suggest that these mechanisms have been evolutionarily conserved. Moreover in the locust there is evidence that this is an adaptive event to conserve energy and prevent complete cellular collapse. In the case of hyperthermia this was evident when temperature was increased to the point at which o would yield different results suggesting a role for the pump. In addition, this result does not rule out the possibility that HS affected the trafficking of Na+/K+ ATPase complexes in neuronal and glial membranes. Nevertheless an effective concentration of ouabain (10−5 M) did not prevent the protective effects of HS on K+ clearance in the MTG. Indeed the ouabain-sensitive proportion of extracellular K+ ion clearance was reduced by HS suggesting either that HS somehow rendered membrane-bound Na+/K+ ATPase less sensitive to ouabain or that HS was acting via a different mechanism such as glial buffering via inwardly rectifying K+ channels. This issue is currently unresolved.We found that prior HS preconditioned neural function in the metathoracic ganglion, allowing the ventilatory pattern generator to operate at higher temperatures and to recover more rapidly from hyperthermic failure on return to normal temperatures. Such preconditioning significantly protects neural function and has been well established in insect preparations In summary we conclude that in locust CNS different stressors converge on an adaptive SD-like event that can be preconditioned by prior HS to delay its occurrence until higher temperatures. We believe that the mechanisms underlying this event are much like those underlying CSD and other SD-like phenomena in mammals and a conserved genetic component to this thermotolerance pathway in insects has recently been demonstrated Locusta migratoria migratorioides (R. and F.), 4–6 weeks past the imaginal ecdysis, were randomly chosen from a crowded colony maintained in the Department of Biology at Queen's University. The animals were housed in cages within the colony under a 12 h:12 h light:dark cycle. Each cage was individually lit with a 40 W fluorescent light bulb. Room temperature was maintained at 25±1°C, with a constant humidity of 23±1%. Animals were provided with wheat seedlings and carrot slices once daily and an ad libitum mixture of 1 part skim milk powder, 1 part torula yeast, and 13 parts bran, by volume. All animals were collected from the colony at the same time each morning and were placed in a two litre ventilated plastic container for 4–8 hours prior to experimentation.Adult male locusts, Locusts designated for control (CON) and HS pre-treatments were separated into different containers. HS locusts were placed in a humid incubator (45°C) for 3 hours followed by 1–5 hours of recovery at room temperature (21±2°C). CON animals were held at room temperature for 4–8 hours.+]o was measured with K+ -sensitive microelectrodes. The K+ -sensitive microelectrodes were made from unfilamented glass pipettes  that were cleaned with methanol (99.9%) and dried on a hot plate, then pulled to form a low resistance (5–7 MΩ) tip. The microelectrodes were silanized by exposure to dichlorodimethylsilane (99%) (Sigma-Aldrich) vapor while baking on a hot plate (100°C) for one hour. After cooling, the microelectrodes were filled at the tip with Potassium Ionophore I-Cocktail B  to form an artificial membrane and then back-filled with 500 mM KCl. The tips of the K+ -sensitive microelectrodes were suspended in distilled water until experimentation. Reference electrodes were made by pulling a filamented pipette  to form a low resistance (5–7 MΩ) tip and were filled with 3 M KCl.o (mM) using the Nernst equation. Electrode sensitivities ranged from 54 to 58 mV for a 10-fold change in [K+]. A reference and K+ -sensitive electrode pair were discarded if the slope did not fall within this range or if the voltage reading exhibited atypical sensitivities. The K+ -sensitive microelectrode was inserted through the sheath of the MTG adjacent to the reference electrode in the area of the ventilatory CPG.K+ -sensitive electrode is logarithmically related to the potassium concentration which was obtained by transforming the [K+]o trace in mV to mM using the Nernst equation:K is the membrane potential at which K+ is at equilibrium, R is the gas constant (8.315 J · K−1 · mol−1), T is the temperature in ° Kelvin (TKelvin = 273.16 + TCelsius), F is Faraday's constant , z is the valence of the ion, and [K+]o and [K+]i are the concentrations of K+ outside and inside the cell, respectively. For a membrane permeable only to K+ at room temperature (20°C), substituting the appropriate numbers and converting natural log (ln) into log base 10 (log10) results in the equation:+-sensitive electrode voltage recording was set to zero in a stock solution of 15mM KCl and the following conversion of the above equation was used for determination of extracellular potassium concentrations (corrections for temperature differences were made when appropriate):The voltage of the K+]o to settle at around 10mM. Saline passed through a glass pipette wrapped in NichromeTM wire, and the temperature of the saline was controlled by varying the amount of current passed through the wire. Temperature at the ganglion was monitored using a thermocouple connected to a digital thermometer . Internal temperature was raised in a ramped manner 5°C per minute from room temperature (∼20°C) until failure of the motor pattern (+]o was observed (+]o plateau (not necessarily in a ramped manner) until a second plateau was reached  was first bubbled into the superfusing saline for 5 minutes then blown over the preparation within the chamber. 100% N2 was maintained until 1 minute post-failure of the motor pattern at which point the preparation was re-oxygenated.The locust preparation was enclosed in a sealed chamber. Nitrogen gas (N−3 M sodium azide (NaN3), an electron transport chain inhibitor that prevents cells from using oxygen, which was bath-applied until 1 minute post-failure. 10−4 M ouabain, a Na+/K+ ATPase toxin, was bath-applied continuously for 1 hour. 10−6 M tetrodotoxin (TTX), a Na+ channel blocker, and 10−1 M tetraethylammonium chloride (TEA), a voltage-gated K+ channel blocker, were bath-applied for 15 minutes prior to a ramped increase in temperature (5°C per minute). For TTX experiments, the heater was turned off once the [K+]o surge was observed and the temperature at the [K+]o surge was taken as the temperature at half the maximum amplitude of the [K+]o increase. For TEA experiments, the temperature ramp was continued beyond the first [K+]o plateau until a second plateau was reached, at which point the heater was turned off.All drugs were dissolved in standard locust saline and bath-applied in semi-intact preparations as described above . Preparations were bathed in standard locust saline for a 10 minute stabilization period prior to pharmacological treatment. Drugs were bath-applied continuously for the full duration of each experiment with the exception of 10−5 M ouabain to determine the role of ouabain-sensitive Na+/K+ ATPase activity in thermotolerance and [K+]o homeostasis. Animals were divided into four groups: 1) CON, N = 17; 2) HS, N = 18; 3) CON locusts treated with 10−5 M ouabain (CON-OUA), N = 10; 4) HS locusts treated with 10−5 M ouabain (HS-OUA), N = 8. Experiments on animals receiving different (pre-) treatments were interspersed over time with each other. All preparations were bathed for 25 minutes with saline before starting the temperature ramp. For 10−5 M ouabain treatment, the ganglion was bathed with standard locust saline for 10 minutes, and then with 10−5 M ouabain for 15 minutes prior to the temperature ramp. At 25 minutes, internal temperature was raised in a ramped manner 5°C per minute until failure of the motor pattern. Failure temperature (°C) and time to recovery (s) were scored as the temperature at which rhythmical ventilator activity ceased and the length of time taken to recover motor pattern function, respectively. In order to determine the nature of the relationships between time to recovery and failure temperature for each group, values were log-transformed to make the relationships linear, which allowed accurate statistical analyses of the data. The correlations are shown in +]o increase and decrease associated with hyperthermic failure and recovery of the motor pattern were also determined. Rate of [K+]o increase (mM/s) was calculated as the slope between the baseline at the inflection point of the [K+]o increase and the maximum peak [K+]o. Rate of [K+]o decrease (mM/s) was calculated as the slope between the maximum peak [K+]o and the baseline [K+]o inflection point. The sample sizes for each analysis varied depending on the ability to measure parameters in some or all locusts within each group.CON and HS locusts were treated with 10+] in the MTG neuropile we pressure-injected using a PicoSpritzer III  a small volume (35 nl) of locust saline containing a 15-fold higher [K+] . Two K+ -sensitive microelectrodes were inserted into the MTG, one next to the injection point and another in a different region of the ganglion. The distance between the recording electrodes (∼0.4 to 0.6 mm) was determined as well as the time to onset of the [K+]o surge measured by each electrode. Using these values the speed of propagation (mm/s) of the [K+]o disturbance between the two recording electrodes was calculated.To locally increase o). Injection of very small volumes of high K+ saline triggered a tissue response, however ignition of the all-or-none [K+]o event occurred only when a threshold was reached.Gradual increases in the volume of K(0.89 MB TIF)Click here for additional data file.Figure S2+]o disturbance was the same in CON and HS locusts. There were no main effects of HS pre-treatment or 10−5 M ouabain treatment on the peak [K+]o associated with failure of the motor pattern .The degree of the [K(0.76 MB TIF)Click here for additional data file.


Recently, with the purpose of enhancing the potency of epidermal growth factor receptor (EGFR)-based therapies, we designed a novel strategy termed ‘Cascade-release targeting’ that seeks to develop molecules capable of degrading to multiple tyrosine kinase (TK) inhibitors and highly reactive electrophiles, in a stepwise fashion. Here we report on the first prototype of this model, RB24, a masked methyltriazene, that in addition to being an inhibitor on its own was designed to degrade to RB14, ZR08, RB10+a DNA alkylating methyldiazonium species. The cascade degradation of RB24 requires the generation of two reactive electrophiles: (a) an iminium ion and (b) a methyldiazonium ion. Thus, we surmise that these species could alkylate the active site of EGFR, thereby irreversibly blocking its action and that DNA damage could be induced by the methyldiazonium. Using the EGFR-overexpressing human epidermoid carcinoma of the vulva cell line, A431, we demonstrate herein that (a) RB24 and its derived species  irreversibly inhibit EGFR autophosphorylation, (b) RB24 induced significant levels of DNA strand breaks, (c) sustained inhibition of EGFR by RB24 was associated with blockade of MAPK activation and c-fos gene expression, (d) RB24 induced irreversible cell growth inhibition with a 100-fold greater potency than Temodal™, a clinical methyltriazene. The pronounced growth inhibitory potency of RB24 was attributed to its ability to simultaneously damage DNA and irreversibly block EGFR TK activity. Overexpression of the epidermal growth factor receptor (EGFR) family and its cognate ligand has been correlated with aggressiveness and poor prognosis in various tumours such as breast, ovarian and prostate cancer . SeveralThe lack of sustainability of the antitumour action of reversible inhibitors has stimulated the design of new irreversible inhibitors of EGFR tyrosine kinase (TK). One such compound, PD183805, bearing a 6-acrylamido group designed to alkylate cysteine 773 in the active site of the ATP-binding pocket irreversibly blocks EGFR TK and is now in Phase I development in patients with head and neck, breast and non-small cell lung carcinoma . Other aα , a DNA repair enzyme that by repairing the O6-alkylguanine DNA adduct confers significant resistance to AGT+ cells  to generate three inhibitors of EGFR and at the final stage of degradation a methyldiazonium species capable of damaging DNA . The A431 cell line was maintained in RPMI-1640 supplemented with 10% FBS and antibiotics as described previously  assay. Briefly, following drug treatment and recovery, cells were fixed using 50 μl of cold trichloroacetic acid (50%) for 60 min at 4°C, washed five times with tap water and stained for 30 min at room temperature with SRB (0.4%) dissolved in acetic acid (0.5%). The plates were rinsed five times with 1% acetic acid and allowed to air dry. The resulting coloured residue was dissolved in 200 μl of Tris base (10 mM), and optical density was read for each well at 492 nm using a Bio-Rad microplate reader (model 2550). Each point represents the average of at least two independent experiments run in triplicate.To study the irreversible effects of our compounds on cell proliferation, 200 cells well6) were preincubated in a six-well plate with 10% serum at 37°C for 48 h and starved overnight for 24 h, after which they were exposed to a dose range of each drug for 2 h and subsequently treated with 50 ng ml−1 EGF for 15 min at 37°C. Cells were washed with PBS and resuspended in cold lysis buffer ). The lysates were kept on ice for 30 min and collected by centrifugation at 10 000 rpm for 20 min at 4°C. The concentrations of protein were determined using the Bio-Rad protein assay kit . Equal amounts of protein were added to a 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene difluoride membrane . Nonspecific binding on the membrane was minimised with a blocking buffer containing nonfat dry milk (5%) in PBST. Thereafter, the membranes were incubated with primary antibodies  for the detection of phosphotyrosine, or anti-EGFR ) for determination of corresponding receptor levels. Blots were incubated with HRP-goat anti-mouse antibody (Bio-Rad Laboratories) and the bands visualised with an enhanced chemiluminescence system . Band intensities were measured using the SynGene GeneTools software package.A431 cells , protein lysates were obtained as described above and Western blot was performed as previously reported . The memμM of each compound for 90 min. One set of cells was then stimulated with EGF (50 ng ml−1) for 15 min and extracts were made as described under the Western blotting procedure above. The other set of cells was washed free of the compound with warm serum-free media and incubated for 2 h. Thereafter, the cells were washed, incubated for another 2 h, washed again and then incubated for a further 4 h. This set of cells was then stimulated with EGF and extracts were prepared as for the first set.This assay was performed as previously described (−1) for 30 min. Total RNA was isolated using the High Pure RNA Isolation Kit of Roche Molecular Bochemicals (Germany), following the manufacturer's instructions. Quantitative analysis of c-fos mRNA and G3PDH mRNA (2 μg of RNA for each sample) was preformed by Titan One Tube RT–PCR Kit , following the manufacturer's instructions and using the following primers: 5′ATGATGTTCTCGGGCTTC3′ (sense), 5′CTCCTGCCAATGCT CTGC3′ (antisense) for c-fos and 5′CCATGGAGAAGGCTGGGG3′ (sense), 5′CAAA GTTGTCATGGATGACC3′ (antisense) for G3PDH.A431 cells were grown to confluence in six-well plates and then incubated in serum-free medium for 24 h. Cells were exposed to the indicated concentrations of the drug prior to stimulation with EGF (50 ng ml6 cells and mixed with agarose (1%) in PBS at 37°C in a 1 : 10 dilution. The gels were cast on Gelbond strips  using gel-casting chambers, as previously described  N-lauryl sarcosine, 10% (v/v) DMSO and 1% (v/v) Triton X-100, pH 10.0). After being kept on ice for 30 min, the gels were gently rinsed with distilled water and immersed in a second lysis buffer  containing 1 mg ml−1 proteinase K for 60 min at 37°C. Thereafter, they were rinsed with distilled water, incubated in alkaline electrophoresis buffer for 30 min at 37°C and electrophoresed at 300 mA for 20 min. The gels were subsequently rinsed with distilled water and placed in 1 M ammonium acetate for 30 min. Thereafter, they were soaked in 100% ethanol for 2 h, dried overnight and stained with SYBR Gold  for 20 min. Comets were visualised at × 330 magnification and DNA damage was quantitated using the Tail Moment parameter (i.e. the distance between the barycentre of the head and the tail of the comet multiplied by the percentage of DNA within the tail of the comet). A minimum of 50 cell comets were analysed for each sample, using ALKOMET version 3.1 image analysis software.The alkaline comet assay was performed as previously described  . Thus, the marked irreversibility of the action of RB24 may be partially imputed to additive antiproliferative contribution of species generated during the degradation steps that precede the formation of ZR08. This antiproliferative contribution may result from their ability to block EGFR-mediated growth signalling, since as previously mentioned the transient iminium ion species generated during these degradation steps may not damage DNA. The partially reversible growth inhibitory potency of ZR08 (third step of the cascade) may perhaps be due to the dependence of its antiproliferative activity on the ultimately released reversible inhibitor RB10, the concentrations of which might have been drastically depleted by multiple washouts. This indicates that the ultimate antiproliferative effect of ZR08 is mediated by a combination of DNA damage inflicted by the methyldiazonium species and EGFR TK inhibitory activity induced by RB10.A synergistic interaction between the strong EGFR TK inhibitory potency and the marked DNA-damaging potential of RB24 may also partly account for its remarkable potency against the A431 cells. Indeed, blockade of EGFR in these cells was accompanied by potent inactivation of Erk1,2 kinase and inhibition of c-fos gene expression. We now know, based on previous experiments, that blockade of EGFR-mediated signalling may not affect the levels or function of the AGT enzymes. However, in vivo.In conclusion, we have demonstrated that a molecule engineered to possess complex EGFR inhibitory properties and capable of further releasing DNA-damaging species could induce significantly more sustained antiproliferative activity than a reversible inhibitor of the same class (e.g. RB10). Moreover, its multiple properties have conferred antiproliferative activity in a cell line in which a classical methylating agent of the same class does not show any detectable activity. Thus this model may well represent a new strategy to ameliorate the chemotherapy of EGF-dependent refractory tumours. Further studies are ongoing to demonstrate the potency of this novel drug


We conducted lifespan studies using 40 mutant and 40 wild type (WT) littermates of equal gender numbers and found that both the median and maximum lifespans were significantly increased in mutant males compared to WT littermates. The median lifespan was increased from 884 days to 1005 days (p = 0.006 as determined by the log rank test) and the 80% lifespan (defined here as 80% deaths) was increased from 941 days to 1073 days (p = 0.004 as determined by the Wang-Allison test). There was no difference in either median or 80% lifespan in female genotypes. WT mice of both genders became increasingly obese with age, while mutant mice maintained their lean phenotype into old age. Adiposity was found to correlate with lifespan for males only. 50% of male mice between 30 and 35 g, corresponding to about 5% body fat, for either genotype lived over 1000 days. No male mouse outside of this weight range achieved this lifespan. During their last month of life, WT mice began losing weight , but RIIβ−/− male mice maintained their lean body mass to end of life. This attenuation of decline was not seen in female mutant mice. Old male mutant mice were insulin sensitive throughout their life. Both genders showed modestly lower blood glucose levels in old mutants compared to WT. Male mutants were also resistant to age-induced fatty liver. Pathological assessment of tissues from end of life male mutant mice showed a decrease in tumor incidence, decreased severity of renal lesions, and a trend towards a decrease in age-related cardiac pathology. These findings help establish the highly conserved nature of PKA and suggest that disruption of PKA affects physiological mechanisms known to be associated with healthy aging.Mutations that cause a reduction in protein kinase A (PKA) activity have been shown to extend lifespan in yeast. Loss of function of mammalian RIIβ, a regulatory subunit of PKA expressed in brain and adipose tissue, results in mice that are lean and insulin sensitive. It was therefore hypothesized that RIIB null (RIIβ It mediates signal transduction and transcription for many cellular processes, and plays a major role in the control of triglyceride storage and metabolism in response to nutrient status. PKA activity is triggered by G protein-coupled receptors in response to numerous neurotransmitters and hormones, and is mediated by increasing intracellular cAMP levels through the modulation of adenylyl cyclase (AC) activity −/−) mice are lean in comparison to their wild type littermates, have increased resting metabolic activity, body temperature, uncoupling protein 1 (UCP1) concentrations, and lipid hydrolysis Mammalian PKA consists of two regulatory and two catalytic subunits −/− mice would be characterized by anti-aging phenotypes based on data showing that disruption of PKA extends lifespan in yeast, as well as the anti-obesity and anti-diabetic phenotypes of these mice. Our preliminary findings help establish the highly conserved nature of PKA and suggest that disruption of PKA affects physiological mechanisms known to be associated with healthy aging.We therefore hypothesized that old RIIβWe show that both the median and 80% lifespans (age at which 80% of cohort is dead) are significantly increased in PKA RIIβ mutant males  P = 0.0. Note th−/− littermates maintained low leptin levels with age , although there was not a sufficient sample size for statistical analysis.At 2 to 5 months of age, both male and female mutants had body weights approximately 10% less than those of WT littermates (data not shown). By 19 to 20 months of age, both male and female WT littermates were approximately 25% heavier than the respective RIIβ mutant mice . When olwith age . Serum l−/− male mice had maximal body weights between 30 and 35 g . It was found that with age, WT mice developed livers up to twice the size of a younger WT liver . We injected insulin into a separate cohort of young (2 to 5 month) and old (18 month) RIIβ mutant males and WT littermates. Both age groups of mutant mice were more insulin sensitive than WT littermates (p≤0.05)  . Howevers of age . Serum i(p≤0.05)  suggesti−/− mice also showed a trend toward better both Ea/Aa ratios and myocardial performance indices compared to WT littermates . In a cross-sectional cohort of six male mice per genotype at 24 months of age, RIIβ mutant mice were significantly protected from age-related left ventricular (LV) hypertrophy compared with WT littermates p<0.001, . Becausetermates .When weights of individual mice at 19 to 20 months of age were compared to measurements taken during the last month of life, WT males showed an average loss of several grams, while RIIβ mutant littermates maintained their weight (p≤0.05) . This ph−/− males compared to WT (−/−) was also examined. While different types of heart lesions from both genotypes were mild on average, it was found that there was a trend for the mutants to have better scores. The differences, however, were not significant  precluded extensive histopathological assessment. Tissues, collected from old mice that were euthanized because they were determined to die within several hours, were immediately fixed in 10% buffered formalin, stained with hematoxylin/eosin and read by a board certified veterinary pathologist with expertise in mouse pathology (P. Treuting). Evaluations were done on tissues from 6 mutant and 6 WT males at end of life. We were able to determine significant differences between genotypes in renal scores for 3 of 10 different types of graded renal lesions. Tubular degeneration and both interstitial and pelvic inflammation showed significantly better scores in RIIβed to WT . Liver aed to WT . Heart tnificant . LookingWe have shown that disruption of PKA RIIβ in mice extends lifespan and protects against age-related obesity, weight loss at end of life, cardiac hypertrophy, incidence and severity of age-related pathology, and insulin resistance. Although the mechanisms for the extended lifespan are not yet known, the fact that it is observed only in males provides a uinique paradigm for further investigation of this interesting gene pathway. In this regard, we show preliminary evidence to suggest that the lifespan phenotype seen in the absence of RIIβ is directly associated with decreased body fat but not insulin sensitivity. We also show that RIIβ null males have less age-related fatty liver, cardiac hypertrophy, cancer and renal pathology.−/− males weighed less, and had less body fat than their WT littermates. In contrast, there was no correlation between adiposity and lifespan in −/−RIIβ females. Gender-related phenotypic differences have been found in other long-lived mouse models such as the Ames dwarf mouse which shows a greater lifespan increase in females During maturity, many of the WT mice of both genders showed a gradual but significant increase in body weight, found to be associated with an increase in fat mass. Long-lived RIIβ−/− mutants remains to be investigated.Another factor which may contribute to the observed gender specificity of the longevity phenotype is differences in fat distribution between genders. Less important to health than overall adiposity is the type of fat. Sex differences in body fat distribution are well recognized, with males storing more fat in the visceral area than females −/− mice were found to have lower serum levels of leptin than WT mice. In humans, plasma leptin levels, which increase with obesity, are a predictor of heart disease, stroke and cardiovascular mortality in males only −/− mice play a role in their longevity, or are merely an indirect result of their reduced adiposity.Not surprisingly, given their reduced adiposity, old RIIβ−/− males showed a decrease in liver fat that paralleled body weight and hence also correlated with lifespan. Hepatic adiposity is related to obesity. Male RIIβ−/− mice were found to be resistant to age-associated fatty livers. Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver disease from steatosis to cirrhosis and is defined by the American Association for the Study of Liver Diseases as fat accumulation in the liver exceeding 5% to 10% by weight −/− males.RIIβ−/− mice were found to be insulin sensitive compared to WT mice, regardless of age. It is, however, unlikely that the increase in lifespan of male RIIβ−/− mice bears any relation to their increased insulin sensitivity, as we did not find differences in insulin sensitivity between young and old WT mice, and the modestly lower blood glucose levels found in the mutants did not show gender specificity. Therefore, while RIIβ−/− mice may have enjoyed health benefits due to chronic insulin sensitivity over the course of their lifespan, insulin sensitivity in old RIIβ−/− mice did not reflect attenuation of an age-related dysfunction, and the insulin sensitivity seen in the male mutants is probably not the cause of their extended lifespan. Interestingly, while the insulin resistance test did not show significant differences between young and old either WT or RIIβ−/− mice, and while neither genotype became hyperglycemic with age, old WT mice did develop hyperinsulinemia. While the attenuation of age-induced obesity in RIIβ−/− mutants does not seem to promote longevity and healthful aging by its effects on peripheral or systemic action of insulin on glucose homeostasis, it may do so by a reduction in insulin-related intracellular signaling through the IGF-1 pathway.Studies on different long-lived mouse models suggest that the role of insulin in longevity is complex. Mutants such as the Ames and Snell dwarf mice show both extended lifespan as well as increased insulin sensitivity −/− mice had attenuated age-related cardiac hypertrophy as well as better myocardial performance indices and Ea/Aa ratios. These data are in agreement with cardiac sparing in adenylyl cyclase (AC5) null mice, which demonstrate an upregulation of both the Raf/MEK/ERK signaling pathway and cell protective molecules such as superoxide dismutase −/− mice, AC5 null mice are long-lived, suggesting an association of the PKA pathway with cardiac protection and extended lifespan.It is well documented in human studies that aging is accompanied by slow progressive and irreversible structural changes and functional declines in the heart. Echocardiography in healthy populations from the Framingham Study and Baltimore Longitudinal Study on Aging showed an age-dependent increase in the prevalence of left ventricular hypertrophy, decline in diastolic function, and relatively preserved systolic function at rest but a decline in exercise capacity, as well as an increase in the prevalence of atrial fibrillation  consisted of 20% protein, 5% fat, and 53% carbohydrate. All mice had access to automatically- delivered, reverse osmosis-treated water. Mice were examined daily until their natural death, defined as being found dead in their cage or being unresponsive to touch and euthanized. Kaplan-Meir survival curves were constructed using the known date of birth and date of death for each mouse. The log-rank test was then used to determine statistical differences between cohorts. Maximum lifespan is defined as the mean age of the oldest 20% of mice from each genotype and gender. A total of 20 mice per genotype per gender  was used to asses survival, with no mice remaining alive at the end of the study. All procedures were performed with the approval of the University of Washington Institutional Animal Care and Use Committee.Mice were assigned to cohorts at the time of weaning based on genotype and gender. Mice of both genders on the C57BL/6 background were allowed to live to the end of their natural life. All mice were maintained in the same specific pathogen free housing environment and fed a standard rodent chow ad lib, with no difference in food intake between any of the genotypes. They were housed in gender-specific groups of three to five in individually ventilated cages . The housing environment was a specific pathogen free barrier facility, and the room kept at a constant temperature of 22 degrees C, with a 12-hr light/dark cycle. Sentinel animal screening consisted of quarterly testing for infectious bacterial, viral and parasitic pathogens, with all tests being negative throughout the study. The −/−) were also measured, and their body composition analyzed in vivo using quantitative magnetic resonance (QMR) methods . For QMR measurements, conscious rodents were placed in the sample holder and the sample holder then inserted into the center of the magnetic resonance machine. Each animal underwent 3 replicate measurements. The measurement procedure itself causes no harm to the animal. Serum leptin was measured for the following male, WT and RIIβ−/− littermates: 2 month-old (5 WT and 4 RIIβ−/−) and 16 month-old (8 WT and 9 RIIβ−/−) and the following female WT and RIIβ−/− littermates: 2 month-old (6 WT and 3 RIIβ−/−) and 16 month-old (3 heterozygous RIIβ+/− and 2 RIIβ−/−). For serum leptin measurements, food was removed from mice 12 h before the collection of blood from the retro-orbital sinus into serum separator tubes ; after separation, plasma was either used immediately or stored at −80°C until analysis. Plasma leptin was measured using an ELISA kit  as per manufacturer's instructions.Body weights were measured weekly beginning at 19 to 24 months of age and until end of life. Body weights of an additional cohort of 18 month-old male mice (5 WT and 6 RIIβ−/− littermates). Mice were euthanized by CO2, and livers were dissected out and weighed. A small (approximately 30 mg) piece of the large lobe was set aside for QNMR composition measurements. Composition measurements were measured using quantitative nuclear magnetic resonance imaging (QNMR), with a EchoMRI™ 3-in-1 Animal Tissue Composition Analyzer, at the University of Washington Mouse Metabolic Phenotyping Center (MMPC).Liver composition analyses were performed on 18 month-old, male mice (5 WT and 6 RIIβ−/− littermates: 2 month-old (5 WT and 4 RIIβ−/−) and 16 month-old (8 WT and 9 RIIβ−/−) and the following female WT and RIIβ−/− littermates: 2 month-old (6 WT and 3 RIIβ−/−) and 16 month-old (3 heterozygous RIIβ+/− and 2 RIIβ−/−). For serum insulin measurements, food was removed from mice 12 h before the collection of blood from the retro-orbital sinus into serum separator tubes ; after separation, plasma was either used immediately or stored at −80°C until analysis. Plasma insulin was measured using an ELISA kit  as per manufacturer's instructions. Insulin resistance assays were performed on additional cohorts of 6 mice per genotype, for young (2 to 5 month) and old (18 month) males and young (2 to 5 month) females. For insulin resistance assays, mice were first fasted overnight for 16 hours. Mice were then injected intraperitoneally with human insulin  diluted with sterile diluent  at a dose of 1.0 units insulin/kg body wt for the insulin resistance assay. Blood glucose measurements were performed as previously described, at 0, 30, 60 and 90 minutes after injection.Blood glucose was measured on the same day as the initial body weights on all mice from the lifespan cohort at 19 to 24 months of age. For blood glucose measurements, food was removed from mice 6 h before blood was drawn by tail pricking. Analyses were performed using a glucometer and Comfort Curve test strips . Serum insulin was measured for the following male, WT and RIIβ−/−) were analyzed. The mice were anesthetized by placing them in a flow-through system containing 3–4% isoflurane in a 100% oxygen mix. Following loss of consciousness, the mice were placed on a modified mask assembly that allowed a continuous flow of 1–2% isoflurane in an oxygen mix. The mice could breathe spontaneously, and the depth of anesthesia was monitored by continuous recording of heart rate. Heart rates were not allowed to fall below 400 . The mice were taped to an isothermal pad that maintained their body temperature at 37°C. EKG and heart rate were monitored throughout the imaging procedure. Heart rate was determined from a surface electrocardiogram. From a transthoracic approach, two-dimensional targeted M-mode echocardiographic recordings were obtained.Male mice 24 months of age were used for non-invasive echocardiography. Six mice of each genotype (WT and RIIβ−/−) male mice were also examined as described above. Cardiac lesions were graded on a severity scale from 0–4 and were examined for interstitial fibrosis, cytomegaly, inflammation, mineralization, valvular changes, arteriosclerosis and amyloidosis End of life (EOL) mice were processed for histochemical analyses. Selected tissues collected at necropsy were fixed in 10% phosphate buffered formalin and processed routinely, sectioned at 4 um and examined by a board-certified veterinary pathologist (PT) blinded to mouse genotype. Morphological diagnoses were assigned for all tissues examined with detailed histopathological evaluation of the kidney and contributing causes of moribundity/death (COD) were assigned as described (56). Renal lesions were scored on a 0–4 severity scale (as described in


There is an urgent need for an improved understanding of the sources, distributions and properties of atmospheric aerosol in order to control the atmospheric pollution over northeastern Himalayas where rising anthropogenic interferences from rapid urbanization and development is becoming an increasing concern.−3 and 19.6±11.1 µg m−3 respectively. Fine mode aerosol dominated during dry seasons and coarse mode aerosol dominated during monsoon. Nitrate existed as NH4NO3 in fine mode aerosol during winter and as NaNO3 in coarse mode aerosol during monsoon. Gas phase photochemical oxidation of SO2 during premonsoon and aqueous phase oxidation during winter and postmonsoon were the major pathways for the formation of SO42− in the atmosphere. Long range transport of dust aerosol from arid regions of western India was observed during premonsoon. The acidity of fine mode aerosol was higher in dry seasons compared to monsoon whereas the coarse mode acidity was higher in monsoon compared to dry seasons. Biomass burning, vehicular emissions and dust particles were the major types of aerosol from local and continental regions whereas sea salt particles were the major types of aerosol from marine source regions.An extensive aerosol sampling program was conducted in Darjeeling , latitude 27°01′N and longitude 88°15′E), a high altitude station in northeastern Himalayas, during January–December 2005. Samples were collected using a respirable dust sampler and a fine dust sampler simultaneously. Ion chromatograph was used to analyze the water soluble ionic species of aerosol. The average concentrations of fine and coarse mode aerosol were found to be 29.5±20.8 µg mThe year-long data presented in this paper provide substantial improvements to the heretofore poor knowledge regarding aerosol chemistry over northeastern Himalayas, and should be useful to policy makers in making control strategies. Anthropogenic contributions to aerosols  together produce a cooling effect, with a total direct radiative forcing of −0.5 Wm−2 and an indirect cloud albedo forcing of −0.7 Wm−2Atmospheric aerosol is linked to visibility reduction, adverse health effects and heat balance of the Earth, directly by reflecting and absorbing solar radiation and indirectly by influencing the properties and cloud processes and, possibly, by changing the heterogeneous chemistry of reactive greenhouse gases Increasing pollutant emissions associated with the fast-growing economies of southeastern Asian countries have led to the progressive increase of aerosol concentrations above the natural background The transport of optically-active aerosol to the higher Himalayas is a matter of concern, since most of the glaciers in the region have been retreating since 1850 A short-term sampling program in the Nguzompa glacier basin near Mt. Everest A strong seasonal variation in aerosol chemistry is expected in this northeastern Himalayan region. During premonsoon and summer months, due to enhanced convection, aerosols are lofted to elevated altitudes in the troposphere. Together with the westerly premonsoon winds, enhanced convection and the steep pressure gradient across the Himalayan-Gangetic region steer aerosols aloft. With the onset of rainy season , the heavy dust loading significantly diminishes due to aerosol washout from the atmosphere and enhances the loading of sea salt aerosols to a significant level. During winter and postmonsoon, northeasterly winds from the subcontinent bring anthropogenic aerosols over the Himalayan region. In addition to that, massive biomass burning during winter also plays a role in the loading of anthropogenic aerosols over northeastern Himalayas. These distinctly different seasonal behaviors of aerosol in northeastern Himalayas prompted us to make a year-long study on the formation and distribution of atmospheric aerosols over Darjeeling, a high altitude station in northeastern Himalayas.In order to understand the seasonal nature of the predominant water soluble ionic species in fine (aerodynamic diameter less than 2.5 µm) and coarse mode (aerodynamic diameter more than 2.5 µm) aerosol a year-long aerosol sampling was done during January–December 2005. This study presents the possible formation mechanisms of secondary ionic species in different seasons, distribution of primary ionic species, long range transport of dust aerosol and the interaction between transported marine aerosol and locally generated anthropogenic aerosol. Finally, an attempt was made to find out the possible types of aerosols from different source regions.The primary focus of this study was to determine the relative contribution of natural and anthropogenic components on the total aerosol loading and their distribution between fine and coarse mode aerosols at a high altitude hill station in northeastern Himalayas which could provide the scientific basis for controlling atmospheric pollution over this geo-politically and environmentally important region.Darjeeling is one of the most popular tourist hill-stations in eastern India with a population of ∼100,000. The overall areas of the Darjeeling district and Darjeeling Township are about 1200 and 11.44 squared kilometers, respectively. Darjeeling Township is located at an average altitude of ∼2000 meter above sea level (masl) and surrounded by different types of topography of the lower-eastern-Himalayas. The southern region comprises the marshy low-lying area at an average height of ∼100–300 masl. The apex is formed by the Phalut ridge  at the border between Nepal and India. The eastern frontier lies along two rivers, locally called Tista and Rangeet.The sampling site shown in  is locat−1) along with total rainfall (mm) over the entire study period (Jan–Dec) are shown in −1 and during monsoon it was mainly southwesterly with an average speed of 1.18 m s−1. In order to know the wind pattern variations in different seasons, the monthly mean wind vectors (at 850 hPa level) for four different seasons; winter (Dec–Feb), premonsoon (Mar–May), monsoon (Jun–Sep) and postmonsoon (Oct–Nov) are shown in The monthly variations of surface meteorological parameters like temperature (°C), relative humidity (%), wind speed  while particulate matter of aerodynamic diameter more than 10 µm was collected in a cup placed under the cyclone.For the collection of total respirable suspended particulate matter (aerodynamic diameter less than 10 µm) from ambient air, a respirable dust sampler was used. The sampler (model APM 460BL) was manufactured by Envirotech Instrument Pvt Ltd, which pioneered the development of indigenous air monitoring instruments all over India. The sampler collected the samples with a flow rate of 1.4 m3 hr−1. After entering the air particles through the inlet of the sampler the coarse particulate matter (aerodynamic diameter more than 2.5 µm) was removed using a GF/A (Glass Fiber) filter paper of 37 mm diameter immersed in silicone oil, used as an impaction surface. The impaction surface was placed above the main aerosol collection filter. The fine particles were collected on a PTFE filter paper of 47 mm diameter.For collection of fine particulate matter or fine mode aerosol, a fine dust sampler was used from the same company (model APM 550). The flow rate was 1 mrd day during dry seasons  and every 4th day during monsoon (Jun–Sep). Thus a total of 111 samples were collected (81 during dry season and 30 during monsoon). Although collection of samples on daily basis would be better in carrying out this kind of aerosol study, collection of samples of more than 111 were beyond our scope. Each sampling was started at 0900 hrs  and run for ∼24 hrs. Both the samplers were placed on the terrace of a three-storied building (∼15 m above ground level) on our institute premises.On average, an aerosol sample  was collected on every 3−3) was determined dividing the aerosol mass by total volume of air (m3).The mass concentration of aerosol was determined by the gravimetric measurement. The filters were placed in desiccators for ∼24 hrs before and after the sampling to remove the absorbed water and weighed in a controlled environment chamber after taking the filters out of the desiccators before and after the sampling using a semi-micro balance . The aerosol mass (µg) was determined by the differences between initial and final weight of the filter and the concentration  and all the data were run by LYNX-software of version V0007. The weather station was run continuously and the data were recorded at the interval of half an hour throughout the year covering all the sampling events. The weather station was equipped with a tower and all the sensors of wind speed and its direction, relative humidity (RH) and temperature were fitted with that tower at a height of 15 m from the ground level. The rainfall data was obtained from Indian Meteorological Department, India.−, NO3− and SO42−) and cations  were quantitatively determined by Ion Chromatograph  using analytical column IonPac ® AS15 with micro-membrane suppressor ASRS ultra II 2mm, 38 mM. KOH as eluent and triple distilled water as regenerator for anions. Similarly, the IonPac ® CS17 column with micro-membrane suppressor CSRS ultra II 2mm, 6 mM methansulfonic acid as eluent and triple distilled water as regenerator were used for cations. For calibration purpose, the standards were procured from Dionex for cations and anions. Detection limits of the ionic species, concentrations corresponding to three times the standard deviation of five replicate blank level measurements for Na+, K+, Ca2+, Mg2+, NH4+, Cl−, NO3− and SO42− were 0.009, 0.0013, 0.003, 0.0015, 0.0024, 0.009, 0.005 and 0.008 µg m−3 respectively. The precision estimated from the standard deviation of repeat measurements of standard and samples was 2% for Na+, K+ and Ca2+; 3% for Mg2+; 5% for NH4+; 2% for Cl−, SO42− and 4% for NO3−. Trace gas SO2 was measured using an on-line SO2 analyzer  throughout all the sampling events at five minute interval.For the analysis of water soluble ions, chromatographic separation method was used −3 varying between 3.6 µg m−3 and 61 µg m−3, whereas coarse mode aerosol ranged between 5.4 µg m−3 and 32 µg m−3 with an average of 19.6±11.1 µg m−3. The large variation in concentrations of both fine and coarse mode aerosol  could enhance the aerosol loading in the atmosphere during winter. The sharp fall in fine mode aerosol concentrations and high precipitation amount (1783 mm) during monsoon indicates the wash out effect of aerosol and its components. On the other hand, the coarse mode aerosol did not show a sharp decrease in concentrations during monsoon due to the contribution of sea salt aerosol . Non-sea-sulphate and nitrate were also found to enhance the coarse mode aerosol concentrations during monsoon (discussed later in detail). The ratios of fine to coarse mode aerosol concentrations were found to widely vary between 0.36 (during August) and 4.4 (during January) with an average of 1.4±1.1. On an average, the average ratio of 1.72 was observed during dry seasons and 0.76 during monsoon. Thus, fine mode aerosol dominated over coarse mode aerosol during dry seasons whereas coarse mode aerosol dominated over fine mode aerosol during monsoon. A peak was observed in aerosol concentrations both in fine and coarse mode during two premonsoon months, April and May. This could be attributed to the long range transport of dust aerosol from arid regions of western India and even from Arabian deserts (discussed later in detail) during premonsoon. According to the elevated heat pump (EHP) mechanism proposed in The average concentration of fine mode aerosol in Darjeeling was found to be 29.5±20.8 µg m aerosol  during t+, NH4+, K+, Ca2+, Mg2+, Cl−, NO3−, SO42−) in fine and coarse mode aerosol over Darjeeling have been compared with the data reported in other high altitude stations in India and Nepal and other sites in Indian subcontinent.Water-soluble inorganic ions , located between 1900  and 5079  masl. Therefore, Ca2+ seems to be more homogeneously distributed vertically in Himalayan regions compared to other ionic species. The loading of dust aerosol enriched with Ca2+ in Himalayan region is mostly due to the long-range transport of dust aerosol originated in the western part of India (discussed later). However, the Ca2+ concentration over Mt. Abu in western India shows the highest concentration which is due to the widespread marble queries and stone-crushing mills in plain lands along the Aravali range in western India 2+ in the present study shows the highest concentration compared to the other sites. But if we look at the fine mode Mg2+ only, we find that our data do not significantly differ from the other sites (varying between 0.03 and 0.12 µg m−3) indicating homogeneous vertical distribution of Mg2+, like Ca2+, in Himalayan regions.For comparison with other high altitude stations, seven such stations  have been chosen . It is o+ and Cl− are higher in concentrations than Pune and Ahmedabad and comparable to Agra, but the most interesting feature is that the concentrations of Na+ and Cl− in Darjeeling are found to be higher than at Mumbai, a coastal city in western India. At this stage, we could not find a fool-proof explanation for this most interesting observation; further, long-term study is required to understand this phenomenon. NH4+ and NO3− in Darjeeling are found to show lower concentrations than at Mumbai, Pune or Agra. K+ and SO42− show higher concentrations in Darjeeling than at Pune and Ahmedabad whereas we observed a similarity in SO42− concentrations between Darjeeling and Mumbai. The concentrations of Ca2+ over all the urban sites are found to be higher than that over Darjeeling. The very high Ca2+ concentration over Ahmedabad is due to its close proximity to Thar deserts. On the other hand, the concentration of Mg2+ in Darjeeling is found to be higher than Pune and Ahmedabad and lower than Mumbai and Agra.The comparison of the data of aerosol ionic species of the present study with four urban sites  in India is shown in Conclusively, we observed that Darjeeling in northeastern Himalayas is strongly influenced by the sea salt and anthropogenic aerosol which are higher than all the high altitude stations in Indian and Nepal Himalayan region and even higher than most of the urban regions in India, compared with the present study. On the other hand, dust aerosol is found to be homogeneously distributed vertically between all the Himalayan regions but is significantly lower than urban regions in India.4+, NO3− and SO42−, the secondary components of aerosol, constituted 67.8±5.9% fine mode and 36.4±10.07% coarse mode aerosol in Darjeeling. The average concentrations of NH4+, NO3− and SO42− in fine and coarse mode aerosol were 0.88±0.76 µg m−3, 3.31±2.25 µg m−3, 3.8±2.9 µg m−3 and 0.05±0.04 µg m−3, 0.95±0.17 µgm−3, 2.5±2.1 µg m−3, respectively.NH4+ and NO3− in fine mode aerosol showed higher enrichment  compared to coarse mode aerosol. This is due to the fact that gas to particle conversion of gas phase HNO3 and NH3 to particulate NO3− and NH4+ are more feasible in nucleation (<0.1µm) and accumulation mode (>0.1 µm <2.5 µm) particles 4+ and NO3− show good correlation    between 4NO3 is given by the following equilibrium4NO3 volatilizes when temperature increases 2, then sulphate acts as a sink for ammonia. Once ammonia becomes ammonium bisulphate or ammonium sulphate, ammonium becomes unavailable for nitrate 2 oxidation by OH radical (discussed in section 4.2) during premonsoon. While fine mode nitrate shows minimum concentrations during monsoon due to the wash out effect or below cloud scavenging, the coarse mode NO3− shows higher concentrations during monsoon compared to the dry seasons. The presence of sea salt aerosol may favor the formation of coarse mode NO3− as NaNO3 by up taking of nitrogen oxides and HNO3 on the surface of sea salt aerosol − from sea salt aerosol and the simultaneous occurrence is particularly pronounced in coarse particle, typical of sea salt aerosol The higher concentration of particulate ammonium and nitrate in winter was due to the shifting from the gas phase of nitric acid to the particulate phase of nitrate at lower temperature. The formation of particulate NH2 in the atmosphere 2 followed by the condensation and absorption into the particle phase was the most important pathway for the production of fine mode SO42− in the atmosphere during premonsoon (SOR>0.1). The gas phase oxidation of SO2 to SO42− by OH radical is a strong function of temperature 2, aqueous phase H2O2/O3 oxidation of SO2 etc. Similar observation was also made in 2 by the soil dust particles at higher relative humidity during monsoon to form coarse mode sulphate of crustal origin 2 = 0.68 and number of samples, n = 30) non-sea-SO42− and non-sea-Ca2+ and between non-sea-SO42− and non-sea-Mg2+  in coarse mode aerosol during monsoon.The sulfur oxidation ratio defined as + and Cl− constituted 16.6±5.46% fine mode and 46.2±11.14% coarse mode aerosol. The average concentrations of Na+ and Cl− in fine and coarse mode aerosol were found to be 0.66±0.43 µg m−3, 1.21±1.00 µg m−3 and 2.16±2.01 µg m−3, 2.35±1.5 µg m−3 respectively. The seasonal distribution of Na+ and Cl− in coarse mode aerosol is similar in nature with the higher concentrations during monsoon. But the concentration of fine mode Cl− was found to be higher during the winter months and minimum during monsoon   is aligned at a rather northerly position during the monsoon season. The air masses are vertically raised by convective motion and transported horizontally by the upper air southerly monsoon flow to the Himalayas − compared to Na+ during the dry seasons  is believed to be associated with biomass and coal burning (domestic and railway). We also observed strong correlations between non-sea-K+ and Cl−  and between non-sea-SO42− and Cl−  during dry seasons  . The mon and Cl−  for both seasons  indicati+ in fine and coarse mode aerosol were 1.17±0.83 µg m−3 and 0.31±0.17 µg m−3, respectively. Biomass burning could have the highest abundance of fine mode K+ of all source emissions. The fine mode K+ could be released in the atmosphere by the burning of vegetative scrap + during winter was much higher (2.12±0.4 µg m−3) than premonsoon (1.37±0.8 µg m−3), monsoon (1.15±0.7 µg m−3) and post-monsoon (1.08±0.62 µg m−3) which was due to the massive biomass burning around Darjeeling especially during night times in winter. + which is similar to fine mode non-sea-SO42− variations. A strong correlation  between non-sea-K+ and non-sea-SO42− was also observed in fine mode aerosol during dry seasons  against the foothills of the Himalayas due to the enhanced convection and steep pressure gradient across the Himalayan-Gangetic region 2+ at two different altitudes, 800 and 3920 masl at two stations in Nepal Himalayan region during premonsoon was also observed showing the long-range transport from southwestern Asia and northern Africa 2+ over a station near Thar deserts was also observed The average concentrations of Casea-Mg2+  during dwww.arl.noaa.gov/ready/hysplit4.html) model developed by NOAA/ARL, back trajectories were computed for all sampling events at 0500 UTC. Two distinct source regions are identified, arid and semi-arid regions of western India including Thar deserts (45%) and upwind regions of Arabian deserts (32%), which are shown in 2+ and Mg2+ over Darjeeling from Thar deserts was found to be higher than the Arabian deserts. The correlation of Ca2+ and Mg2+ with trace metals like Fe, Al, Si during premonsoon would be useful to put the evidence towards the transport of dust aerosol from distant regions. However the studies on the effect of dust aerosol on the total aerosol loading over Himalayan region and its effect on the optical properties of aerosol during premonsoon are under development.Using HYSPLIT_4  compared to Darjeeling and other Himalayan regions (2+ and non-sea-Mg2+ both show minimum concentrations indicating the below cloud scavenging of dust aerosol due to heavy rain during monsoon (1783 mm). However, the negative concentration of coarse mode non-sea-Mg2+ during monsoon indicates that the entire coarse mode Mg2+ was from the marine source during monsoon.The higher concentrations of Ca regions  and 2 in− in the form of HCl gas. NOx transforms into gaseous nitrous and nitric acid, which react with NaCl in sea-salt aerosol to form NaNO3 and HCl. SO2 oxidation and to a lesser extent H2SO4 vapor condensation on sea salt aerosols can also lead to chloride depletion. The reaction pathways are given as follows − depletion (Cl−dep) can be calculated as follows + concentration in aerosol is assumed to be sea water only. In this study, the average depletion of Cl− in fine mode aerosol (61.2±13.3%) during monsoon was higher than coarse mode aerosol (31.02±9.6%). It was suggested that the surface reaction mechanism is the principal explanation for higher depletion of smaller particles − depletion decreases from 98% to 10% as particle size increases from 1.8 µm to 18 µm. Chloride depletion results when the acidic species, mainly nitrate, sulphate and some organic acids react with sea-salt particles and replace Cl+ concentrations in aerosol to the estimated original Cl− concentration in sea salt was used to determine the contribution of nitrate and non-sea-sulphate to chloride depletion. According to +] to measured [Cl−] in aerosol. The ratio, [Na+]/[Cl−] in unreacted original sea water is 0.85.An attempt was made to estimate the contribution of nitrate and non-sea-sulphate to the chloride depletion in fine and coarse mode aerosol. A ratio of the measured Na+]/[Cl−], in fine and coarse mode aerosol were higher than 0.85 because of chloride depletion. Interestingly, the ratios [Na+]/([Cl−]+[NO3−]) for coarse mode aerosol were found to be close to 0.85. This indicates that the measured coarse mode nitrate during monsoon was involved in chloride depletion. A strong correlation between coarse mode Na+ and coarse mode NO3− during monsoon  (+]/([Cl−]+[NO3−]) for fine mode aerosol were quite lower than 0.85 indicating the overestimation of nitrate, i.e. the measured nitrate was not totally the nitrate from sea-salt but also from some other sources. The ratios, [Na+]/([Cl−]+[non-sea-SO42−]) in fine mode aerosol were close to 0.85. This indicates that the measured fine mode non-sea-sulphate during monsoon was totally involved in chloride depletion. We observed a strong correlation between Na+ and fine mode non-sea-SO42−    added fu n = 30)  during mi.e. the interaction between marine  and urban (anthropogenic) aerosol has major implications not only in changing the chemical composition of aerosol in different size modes but also it governs the rate of dry atmospheric removal of the species involved in chloride depletion. For example, here in this study the chloride depletion from coarse mode sea salt aerosol was found to govern the dry atmospheric removal of nitrate from the atmosphere. The dry depositional flux of nitrate in fine and coarse mode aerosol was calculated as follows:−1 and that in coarse mode was considered to be 1 cm s−14NO3 during winter with the average deposition flux of 518±65 mg m−2 day−1. On the other hand, coarse mode nitrate was found to be enriched as NaNO3 after depleting chloride from sea salt aerosol during monsoon with a higher deposition flux of 1468±300 mg m−2 day−1. Thus we observed that the interaction between natural marine aerosol and anthropogenic urban aerosol enhanced the rate of dry atmospheric removal of nitrate.The chloride depletion from sea salt aerosol + per m3 of air is a parameter that characterizes the absolute acidity of atmospheric aerosols + derived from the aqueous extract of atmospheric aerosols and is estimated using an ionic balance of the inorganic ionic species. On the other hand, free acidity (pH) characterizes the relative acidity of atmospheric aerosols and depends on the water content and composition of aerosol and relative humidity + in nmol m−3) using all the ionic species according to +]total and the equivalent ratio of the sum of cations to the sum of anions (Σ+/Σ−) in fine and coarse mode aerosol. It was observed that fine mode aerosol acidity  was much higher during dry seasons (73 nmol m−3) compared to monsoon (−4.1 nmol m−3) whereas coarse mode aerosol acidity was higher during monsoon (19 nmol m−3) compared to dry seasons (−34.6 nmol m−3). The ratio, Σ+/Σ−, ranged between 0.46 to 1.43  in fine mode aerosol and between 0.9 to 1.95  in coarse mode aerosol. The cation deficiency in fine mode aerosol could be attributed to H+ whereas anion deficiency in coarse mode aerosol could be attributed to unanalyzed organic acid ions, carbonate, bicarbonate etc. The higher concentrations of nitrate, non-sea-sulphate and chloride during dry seasons increased [H+]total in fine mode aerosol whereas the lower concentrations of non-sea-magnesium and non-sea-calcium along with higher concentration of nitrate during monsoon increased [H+]total in coarse mode aerosol. Thus, the acidity of fine mode aerosol was mainly controlled by the species originated from fossil fuel and biomass burning whereas the acidity of coarse mode aerosol was mainly controlled by dust particles.Strong acidity in terms of nmol HIt is very important, and difficult as well, to identify the exact sources of the aerosol components in Darjeeling, the area under concern at the northeastern Himalayas, where composite anthropogenic activities including biomass burning and vehicular emission play a central role in loading of air pollutants in the atmosphere. Principal component analysis (PCA), a multivariate analysis technique +, Cl− and SO42− with wind speed having 41.5% variance of the data set. These species in PC1 correspond to the massive biomass burning throughout the year mainly during winter in Darjeeling. The coal engine which is used for the Darjeeling Himalayan Railways is also a major source of these species in the atmosphere. Thus, PC1 indicates the loading of K+, Cl− and SO42− as non-sea-K+, non-sea-Cl− and non-sea-SO42− respectively. The negative loading of wind speed in this PC indicates the dispersion of these fine mode species favored by higher wind speed. The second PC (PC2) shows the heavy loading of NH4+, NO3−, SO42− with 33.3% of the data variance. This PC is associated with the formation of secondary anthropogenic particles in the atmosphere. Different agricultural activities and usage of different ammoniated fertilizers in several tea gardens and also in tea processing plants, animal manure and human activities are the major sources for the emission of NH3 and/or NH4+ in the atmosphere. The third PC (PC3) is moderately loaded with NH4+ and highly loaded with NO3− along with temperature with 16.4% of the data variance. This indicates the emission of NOx from vehicular exhaust and its subsequent transformation to particulate nitrate mostly as ammonium nitrate. The negative loading of temperature in PC3 clearly indicates the inverse relation of formation of particulate nitrate with temperature as discussed earlier.For fine mode aerosol, three PCs were extracted . The fir+, Cl− and Mg2+ having 40.5% data variance. This indicates the loading of sea salt aerosol. The second PC (PC2) shows the heavy loading of Ca2+, Mg2+ along with wind speed with the moderate loading of K+. This explains 34.2% of the data variance and indicates the enrichment of calcium, magnesium and a fraction of potassium as non-sea-calcium, non-sea-magnesium and non-sea-potassium from soil dust particles. The loading of wind speed in this PC indicates the resuspension of soil dust in the atmosphere favored by the higher wind speed. The third PC (PC3) shows the loading of Na+ and NO3− with the data variance of 14.6%. This indicates the interaction of sea salt aerosol (NaCl) with nitrate (particulate or gaseous) and the production of coarse mode nitrate through the chloride displacement reactions discussed earlier. The fourth PC (PC4) shows the higher loading and association of SO42− only with 6.4% of data variance. The loading of sulphate singly in PC4 indicates the enrichment of non-sea-sulphate in coarse mode which is due to the aqueous phase transformation of SO2 to sulphate in high relative humidity during monsoon. The higher concentration of non-sea-sulphate during monsoon in coarse mode aerosol has already been discussed.For coarse mode aerosol, four PCs were extracted . The firIn order to find out the possibility of atmospheric transport of different species from outside of Darjeeling, backward trajectories for air parcels arriving at the sampling site during the sampling period were calculated with the HYSPLIT_4 model. Back trajectories were computed for all sampling events at 0500 UTC. The Final Run (FNL) meteorological data was used for the trajectory calculation.+]total (nmol m−3) and Σ+/Σ− ratio both in fine and coarse mode aerosol for three types of air masses.Different air masses travel through different regions and bring different chemical components with the aerosol, thus the distribution of chemical components among different air masses could shed some light on their possible sources. Based on the transport pathways of air masses, three typical air mass trajectories, representing local, continental , and marine were found in Darjeeling and have been shown in + and Cl− follow the same sequence and their coarse mode concentrations were found to be the highest for marine air parcel, indicating the strong influence from the sea. The higher concentrations of fine mode Cl− for continental and local sources indicate the influence of biomass and coal burning. The higher concentrations of coarse mode Ca2+ and Mg2+ from local and continental sectors indicate the resuspension of local and wind blown soil dust particles. The concentration of coarse mode Mg2+ from marine sectors shows the influence of sea salt Mg2+. Local and continental sectors show the higher concentrations of fine and coarse mode K+ indicating the strong influence of biomass burning and soil dust aerosol respectively. The higher concentrations of fine mode NH4+, NO3− and SO42− from local and continental sectors show a very strong influence of anthropogenic activities whereas coarse mode NO3− shows higher concentration from marine sectors. This coarse mode nitrate from marine sectors is not sea salt nitrate rather it indicates the interaction between sea salt aerosol and gas phase HNO3 and its association with sodium through chloride displacement reaction discussed earlier. Fine mode aerosol shows higher [H+]total and lower Σ+/Σ−  from the local compared to continental  and marine  source regions whereas coarse mode aerosol shows maximum [H+]total and minimum Σ+/Σ− ratio  from marine source compared to local  and continental sources . Thus, local and continental fine mode aerosols were more acidic than coarse mode aerosol whereas marine fine mode aerosols were less acidic compared to coarse mode aerosol.The higher contribution of local and continental sources to the fine mode aerosol was observed with a little influence of marine sources while the concentration of coarse mode aerosol was found to be higher from the marine source regions than the local and other continental sources. The concentrations of NaBased on the Principal Component Analysis (for source types) and HYSPLIT trajectory model (for source regions) the percentage distribution of several source types of water soluble ions from different source regions  in fine and coarse mode aerosol were determined and depicted in The major findings of this study can be summarized as:The average concentration of fine mode aerosol is found to be higher than coarse mode aerosol during dry seasons whereas monsoon shows higher loading of coarse mode aerosol compared to fine mode.2 is the major pathway for the formation of sulphate during premonsoon whereas some other aqueous phase transformation processes are important pathways during winter in sulphate formation.This study discusses the formation pathways of major secondary aerosol components. Nitrate is found to exist as ammonium nitrate in fine mode aerosol during winter whereas during monsoon nitrate exists as sodium nitrate in coarse mode aerosol. Photochemical oxidation of SOi.e. the interaction between marine and urban aerosol, is found to govern the dry removal of nitrate from the atmosphere. The nitrate which is found to be enriched mostly in fine mode during dry seasons gets enriched in coarse mode during monsoon because of the chloride displacement from coarse mode sea salt aerosol. Thus nitrate is found to be deposited  at a faster rate during monsoon compared to dry seasons.There is a major contribution of non-sea-sulphate and nitrate in the chloride depletion from fine and coarse mode sea salt aerosols, respectively. The chloride depletion, The acidity of fine mode aerosol is found to be higher in dry seasons compared to monsoon whereas the acidity of coarse mode acidity is found to be higher in monsoon compared to dry seasons. Non-sea-sulphate and nitrate are found to govern the acidity of fine mode aerosol whereas non-sea-calcium and non-sea-magnesium govern the coarse mode acidity.Three major source regions like local, continental (mainly from north-western part of India) and marine  sources are identified based on HYSPLIT backward trajectory model. Local and other continental source regions contribute 80% with high loading of secondary anthropogenic species whereas the contribution from marine source regions is 20% enriched with primary sea salt particles.Biomass burning and vehicular emissions are the major sources for fine mode local and continental aerosols with higher acidity whereas soil dust particles are the major source for coarse mode local and continental aerosols with lower acidity. On the other hand, the major source for fine and coarse mode marine aerosol is sea salt aerosols with higher acidity in coarse mode.