FTO) gene variants and metabolic traits in several populations; however, there are no gene-lifestyle interaction studies, to date, among Asian Indians living in India. In this study, we examined whether dietary factors and physical activity modified the association between two FTO single nucleotide polymorphisms (rs8050136 and rs11076023) (SNPs) and obesity traits and type 2 diabetes (T2D).Lifestyle factors such as diet and physical activity have been shown to modify the association between fat mass and obesity–associated  participants randomly selected from the urban component of the Chennai Urban Rural Epidemiology Study (CURES). Dietary intakes were assessed using a validated interviewer administered semi-quantitative food frequency questionnaire (FFQ). Physical activity was based upon the self-report. Interaction analyses were performed by including the interaction terms in the linear/logistic regression model.interaction = 0.04), where the ‘A’ allele carriers had 2.46 times increased risk of obesity than those with ‘CC’ genotype (P = 3.0 × 10−5) among individuals in the highest tertile of carbohydrate intake . A significant interaction was also observed between SNP rs11076023 and dietary fibre intake (Pinteraction = 0.0008), where individuals with AA genotype who are in the 3rd tertile of dietary fibre intake had 1.62 cm lower waist circumference than those with ‘T’ allele carriers (P = 0.02). Furthermore, among those who were physically inactive, the ‘A’ allele carriers of the SNP rs8050136 had 1.89 times increased risk of obesity than those with ‘CC’ genotype (P = 4.0 × 10−5).There was a significant interaction between SNP rs8050136 and carbohydrate intake (% energy)  contains supplementary material, which is available to authorized users. FTO) variants were found to be consistently associated with obesity-related traits in several populations and has been the strongest common genetic predictor of obesity known so far [FTO has shown the strongest association with BMI, where the FTO SNPs increased the risk of obesity by 1.20–1.32 fold in Europeans [FTO variant, rs9939609, increased the risk of obesity by 1.15 times, which is equivalent to BMI increase by 0.30 kg/m2 per effect allele [FTO SNPs (rs1588413 and rs8050136) increased obesity risk by 1.27 and 2.06 times, which is equivalent to BMI increase by 0.33 and 0.54 kg/m2 per effect allele, respectively [FTO gene is abundantly expressed in hypothalamus and has been shown to be involved in the control of satiety/appetite [FTO was considered as a candidate for our gene-diet/-physical activity interaction study. Several studies have examined the influence of lifestyle factors such as diet and physical activity on the association between FTO variants and obesity traits [Metabolic traits such as obesity and type 2 diabetes (T2D) are generally caused by overconsumption of energy, sedentary lifestyle and genetic susceptibility . Candidan so far –3, 6–9. uropeans  and by 1uropeans . A recenappetite . Given iappetite , 13–16, y traits ; howeverIndia has the second largest number of people with T2D in the world, where 62.4 million people in India have T2D according to the Indian Council of Medical Research-INdia DIABetes (ICMR-INDIAB) Study . Asian IFTO variant with obesity-related traits [FTO variant on obesity traits in several populations [FTO-diet interactions have shown conflicting results, where some studies found that high energy and fat intakes or low carbohydrate intake might strengthen the association of FTO variants with obesity [FTO single nucleotide polymorphisms (SNPs) with obesity-related traits and T2D in 1,618 individuals in the Chennai Urban Rural Epidemiology Study (CURES).Studies in European populations have shown that physical activity and dietary intake may modify the association of the d traits , 29. Inculations , 28; how obesity , 31, whi obesity . These in = 1529) were invited to visit the center for detailed studies. In addition, every 10th individual of the 26,001 individuals without known diabetes was invited to undergo oral glucose tolerance tests using a 75-g oral glucose load (dissolved in 250 ml of water) (Phase 3 of CURES). Those who were confirmed by oral glucose tolerance test to have 2-h plasma glucose value ≥ 11.1 mmol/l (200 mg/dl) based on World Health Organization (WHO) consulting group criteria were labelled as “newly detected diabetes” and those with 2-h plasma glucose value < 7.8 mmol/l (140 mg/dl) as being normal glucose tolerant (NGT) [CURES: The study participants were recruited from the urban component of the Chennai Urban Rural Epidemiology Study (CURES). CURES is a cross-sectional epidemiological study conducted on a representative sample of the population of Chennai (formerly Madras city) in Southern India . The metnt (NGT) . InformeAnthropometric measurements including weight, height, and waist were obtained using standardized techniques. The BMI was calculated as weight (in kg) divided by the square of height (in m). Biochemical analyses were done on a Hitachi-912 Auto Analyzer  using kits supplied by Roche Diagnostics (Mannheim). Fasting plasma glucose (glucose oxidase–peroxidase method), serum cholesterol (cholesterol oxidase-phenol-4-amino-antipyrene peroxidase method), serum triglycerides (glycerol phosphatase oxidase-phenol-4-amino-antipyrene peroxidase method), and high-density lipoprotein cholesterol  were measured. Low-density lipoprotein cholesterol was calculated using the Friedewald formula . GlycateDietary intakes were assessed using a previously validated and published  intervieDietary glycemic load of the individual serving was calculated by multiplying the carbohydrate content (grams per serving) of each food by its glycemic index value. This glycemic load value was then multiplied by the frequency of consumption and these products were summed to obtain average daily dietary glycemic load .Physical activity measurement: Physical activity was based upon a previously validated self-report questionnaire . Based oFTO SNPs (rs8050136 and rs11076023) were chosen based on their previous association with obesity and T2D in the European and South Asian populations [FTO gene which is highly conserved across species and is located in the putative transcriptional factor  binding site [FTO gene and hence it can be speculated that it might have an effect on the stability of mRNA and gene expression. The SNPs were genotyped by polymerase chain reaction on a GeneAmp PCR system 9700 thermal cycler . The PCR products were digested with MluCI (rs8050136) and HinfI (rs11076023) restriction enzymes  and the digested products were resolved by a 3 % agarose gel electrophoresis. Based on the analysis of 200 blind duplicates (20 %), there was 100 % concordance in the genotyping. Furthermore, a few variants were confirmed by direct sequencing with an ABI 310 genetic analyzer . The FTO SNPs were in Hardy Weinberg equilibrium (P > 0.05).The two commonly studied ulations , 17, 38.ing site . The rs12) and obese (BMI ≥ 25 kg/m2) [t test as appropriate was used to compare groups for continuous variables. Given the low frequency of the rare homozygotes, dominant model was used (comparing common homozygotes with the combined group of rare homozygotes and heterozygotes). The genetic associations with the continuous and categorical outcomes were examined using linear and logistic regression models, respectively, adjusting for age and gender. When obesity/BMI was an outcome, T2D status was adjusted and when T2D was an outcome, obesity was adjusted in the model, in addition to age and gender. Participants were divided into tertiles of carbohydrate intake (% energy) , glycemic load  and dietary fibres . Interactions between each SNP and dietary intake/physical activity were assessed using linear and logistic regressions and the likelihood ratio test. The interplay between FTO variants and dietary intake/physical activity was investigated by including an interaction term in the regression models. All analyses were carried out using STATA, version 13. A P value of less than 0.05 was considered to be statistically significant.Results from the descriptive analyses are presented as means and SD for continuous variables and as percentages for categorical variables. Generalized obesity was defined according to the World Health Organization Asia Pacific Guidelines for Asians as non-obese (BMI < 25 kg/m5 kg/m2) . A goodn2). As shown in Table P < 0.0001), waist circumference (P < 0.001), systolic and diastolic blood pressures (P < 0.0001), fasting plasma glucose and insulin levels (P < 0.0001), total cholesterol (P < 0.0001), triglycerides (P < 0.0001), high-density (P = 0.001) and low-density (P < 0.0001) lipoprotein cholesterols and HbA1c (P < 0.0001). FTO gene variants, rs8050136 and rs11076023, have been shown to be associated with obesity and T2D, respectively, in the CURES study [In the CURES population, 42 % of the individuals were overweight/obese (BMI > 25 kg/mFTO SNP rs8050136 and carbohydrate intake % energy) (Pinteraction = 0.04) on obesity, where the ‘A’ allele carriers of the SNP rs8050136 had 2.46 times increased risk of obesity than those with ‘CC’ genotype (P = 1.0 × 10−5) among individuals in the highest tertile of carbohydrate intake   on obesity .There was a significant interaction between interaction = 0.0008), where individuals with ‘AA’ genotype who are in the 3rd tertile of dietary fibre intake (44 g/d) had 1.62 cm decrease in waist circumference than those with ‘T’ allele carriers (P = 0.02) , where individuals with ‘AA’ genotype who are in the 3rd tertile of dietary fibre intake (44 g/d) had 0.50 kg/m2 decrease in BMI than those with ‘T’ allele carriers (P = 0.07)  was also seen, where the ‘A’ allele carriers of the SNP rs11076023 in the highest tertile of carbohydrate  had 1.57 times increased risk of T2D than those with ‘TT’ genotype (P = 0.002)  Fig. . A simil07) Fig. . A bordeFTO SNPs and fat and protein energy percentages, glycemic index and dietary fibre on obesity traits and T2D were statistically significant  (Tables None of the other interactions between the ) Tables .Table 2IP > 0.11 for all comparisons), sub-group analysis within each levels of physical activity revealed stronger associations of the SNP rs8050136 with obesity traits among those who were physically inactive. Among those who were physically inactive, the ‘A’ allele carriers of the SNP rs8050136 had 0.95 kg/m2 per minor allele increase in BMI (P = 0.002), 3.06 cm per minor allele increase in waist circumference (P = 2 × 10−4) and 1.89 times increased risk of obesity (P = 4.0 × 10−5), compared to those with ‘CC’ genotype. Given that the overall interaction was not significant and the effect was observed only after stratification based on physical activity levels, we tested for the association between the SNP and physical activity levels to avoid spurious interaction effects due to collider bias [P = 0.53) suggesting that the observed interaction is not spurious.We observed interactions between the SNP rs8050136 and physical activity on BMI, waist circumference and obesity  .We also observed a statistically significant interaction of the SNP rs8050136 with physical activity on T2D (P3) Table . None ofFTO SNPs, rs8050136 and rs11076023, with obesity traits, with the effect of the SNP being more pronounced among those who consumed high levels of carbohydrate and dietary fibre. The study also provides evidence that low levels of physical activity may accentuate the risk of obesity by the FTO SNP rs8050136. Given the high prevalence of T2D in South Asia and as 28–44 % of Asians carry at least one copy of the FTO risk allele [Our study provides the first gene-diet interaction data on obesity-related traits and T2D in an Asian Indian population. The present study shows that carbohydrate and dietary fibre intake may modify the association of the FTO SNP rs8050136 and carbohydrate energy percentage on obesity, where the minor allele carriers had higher risk of obesity among those in the highest tertile of carbohydrate energy percentage. While our findings are in contrast to the large European studies which failed to find an interaction between FTO and dietary carbohydrate intake on obesity [interaction = 0.0004); however, the effect allele of the FTO SNP rs9939609 (strong LD with rs8050136) increased the risk of obesity among those with low carbohydrate and high fat intakes. In the present study, the total carbohydrate intake was much higher  compared with that reported in the Western population  [While carbohydrates do not constitute the bulk of the energy in the West ~45 %) , the die obesity , 44, 45, % , the  obesity  showed a238 g/d)  and in a238 g/d) . This miThe positive interaction that was observed between SNP rs11076023 and dietary fibre intake on BMI and waist circumference in our study  is consistent with the previous report from the Finnish Diabetes Prevention Study  , where hFTO and physical inactivity on obesity has been reported in several studies from Europe and Asia [FTO SNP rs8050136 had higher risk of obesity. Our finding is in accordance with the previous large studies among Europeans [FTO risk allele on obesity.The prevalence of physical inactivity among South East Asians 17 %) has been shown to be lower than that of Europeans (34.8 %) . Howeverand Asia , 17; how % has beuropeans , 17 wherFTO variants and dietary intake and physical activity on obesity traits in this Asian Indian population. The small sample size might be one of the reasons why there was no consistency in the gene–diet interactions, where SNP rs8050136 showed an interaction with carbohydrate intake (energy %) while SNP rs11076023 showed an interaction with dietary fibre. However, we cannot rule out the fact that complex traits such as obesity are caused by multiple gene variants and lifestyle factors and hence, it is not necessary for all the variants in the gene to show interaction with the same dietary factor. Given the questionnaire based assessment of physical activity levels, measurement error cannot be completely excluded. Finally, even though we adjusted for age in all the analyses, it is possible that the unmatched age in cases and controls can introduce a bias in the study. Two main strengths of our study is the validated FFQ, which has shown high reproducibility and validity for total carbohydrates, dietary fibre, glycemic index and glycemic load, and the analysis of interactions in a well characterised population.Our study has several limitations. We performed a cross-sectional study and hence, we are unable to infer causality between the consumption of carbohydrate-rich foods/physical inactivity and development of obesity. Although confounders were adjusted in all our analyses, residual confounding due to unknown factors cannot be excluded. Interactions were significant only with total carbohydrate intake (energy %) but not when split as total and added sugars and refined and whole cereals (data not shown). Even though our study has a small sample size compared to the large European cohorts, we were still able to confirm the previously reported interactions between FTO SNPs, rs8050136 and rs11076023, on obesity is influenced by high carbohydrate and dietary fibre intakes and low physical activity levels. Given that India leads the world in diabetes [FTO gene influences obesity and T2D through dietary and exercise interventions is warranted to advance the development of behavioral intervention and personalised lifestyle strategies, which could reduce the risk of metabolic diseases in this Asian Indian population.In summary, this is the first study to provide evidence for a gene-diet and gene-physical activity interaction on obesity and T2D in an Asian Indian population. Our findings suggest that the effect of the diabetes , our stu


Drosophila melanogaster embryos and examined the underlying genomic variation within and between species. We found that divergence along the D. melanogaster lineage is periodic across nucleosome regions with base changes following preferred nucleotides, providing new evidence for systematic evolutionary forces in the generation and maintenance of nucleosome-associated dinucleotide periodicities. Further, Single Nucleotide Polymorphism (SNP) frequency spectra show striking periodicities across nucleosomal regions, paralleling divergence patterns. Preferred alleles occur at higher frequencies in natural populations, consistent with a central role for natural selection. These patterns are stronger for nucleosomes in introns than in intergenic regions, suggesting selection is stronger in transcribed regions where nucleosomes undergo more displacement, remodeling and functional modification. In addition, we observe a large-scale (∼180 bp) periodic enrichment of AA/TT dinucleotides associated with nucleosome occupancy, while GC dinucleotide frequency peaks in linker regions. Divergence and polymorphism data also support a role for natural selection in the generation and maintenance of these super-nucleosomal patterns. Our results demonstrate that nucleosome-associated sequence periodicities are under selective pressure, implying that structural interactions between nucleosomes and DNA sequence shape sequence evolution, particularly in introns.An estimated 80% of genomic DNA in eukaryotes is packaged as nucleosomes, which, together with the remaining interstitial linker regions, generate higher order chromatin structures  D. melanogaster by examining the inter- and intra-species genomic variation relative to these fundamental chromatin building blocks. The sequence changes across nucleosome-bound regions on the melanogaster lineage mirror the observed nucleosome dinucleotide periodicities. Importantly, we show that the frequencies of polymorphisms in natural populations vary across these regions, paralleling divergence, with higher frequencies of preferred alleles. These patterns are most evident for intronic regions and indicate that non-protein coding regions are evolving toward sequences that facilitate the canonical association with the histone core. This result is consistent with the hypothesis that interactions between DNA and the core have systematic impacts on function that are subject to natural selection and are not solely due to mutational bias. These ubiquitous interactions with the histone core partially account for the evolutionary constraint observed in unannotated genomic regions, and may drive broad changes in base composition.In eukaryotic cells, the majority of DNA is packaged in nucleosomes comprised of ∼147 bp of DNA wound tightly around the highly conserved histone octamer. Nucleosomal DNA from diverse organisms shows an anti-correlated ∼10 bp periodicity of AT-rich and GC-rich dinucleotides. These sequence features influence DNA bending and shape, facilitating structural interactions. We asked whether natural selection mediated through the periodic sequence preferences of nucleosomes shapes the evolution of non-protein-coding regions of  Sequence-dependent differences in the physical properties of DNA influence its associations with the histone core, as well as the kinetics of nucleosome assembly and stability e.g. RNA polymerase II, transcription factors and ATP-dependent remodelers) are central to establishing nucleosome positions along genomic DNA , particularly in genic regions, with sequence providing a weaker contribution Although these broadly conserved dinucleotide patterns have been cited as evidence for a genomic “code” for nucleosome positioning Dinucleotides are instead thought to play a distinct but integrally connected role in directing and preserving the ‘rotational positioning’ of nucleosomal DNA within species) and divergence (variation between species). Individual mutations influencing histone-DNA interactions may have only slight, undetectable phenotypic effects in the laboratory; in contrast, the associated fitness consequences in large natural populations can strongly shape rates of divergence and levels of polymorphism over many generations One approach to discovering function is to look for evidence of natural selection in sequence polymorphism , we identify clear signals associated with natural selection, which parallel the observed periodicities in divergence. This selection is strongest in intronic regions, where nucleosome assembly and positioning are expected to have greater functional impacts. These findings support the hypothesis that the widely observed sequence affinities of the core octamer have functional consequences that are subject to natural selection. Given the dominant role of nucleosomes in the packaging of the genome and their conserved sequence preferences, their interactions may broadly shape the sequence of melanogaster and other genomes.The whole-nucleosome-resolution analyses considered above cannot leverage the specific structural predictions of dinucleotide interactions with the core and their strong mechanistic implications. Examination of polymorphism and divergence at each base pair position across the nucleosomal DNA opens a rich and precise view, as well as powerful tests of alternative mechanisms such as biased mutation and natural selection. We report the discovery of fine-scale periodicities in inter- or intra-species sequence variation relative to nucleosomes and discuss their implications for the role of natural selection mediated through nucleosome function. Our analysis of DNA sequence polymorphism and divergence across isolated nucleosomal fragments from D. melanogaster embryos, performed Micrococcal nuclease (MNase) digestion, and used paired-end sequencing to position fragments on the genome  and the proximate outgroup  and C→T  substitutions compared to A→G and T→C  at which these patterns evolve is unknown. Lineage-specific or “polarized” divergence is the proportion of nucleotide sites that are different in  and T→C . This isGiven the substantial variation in individual substitution rates, we next examined specific pathways to determine their relative contributions. Of all pathways, G→A, C→T, A→G and T→C exhibited the most obvious periodicities in divergence . In someWhen mapped onto the DNA from the nucleosome structure Divergence patterns should also reflect the observed nucleosome-scale periodicities in base and dinucleotide frequencies  and S3. melanogaster lineage are fixed at higher rates across nucleosomes relative to linkers, mirroring underlying AA/TT dinucleotide frequencies. This is in apparent contrast to the report that the cytosine deamination mutational pathway (a major source of G→A:C→T transitions) and associated divergence is suppressed by nucleosome occupancy D. melanogaster embryos are generally more AT-rich and have higher rates of G→A:C→T substitution than their adjacent “linker” regions, inconsistent with the fundamental claim in Chen, et al.These large-scale patterns allow us to resolve general trends in divergence relative to nucleosome occupancy. We find that, on average, G→A:C→T changes along the The divergence patterns we observe are consistent with known nucleosomal dinucleotide preferences per-n147 frequencies of G-A and C-T SNPs in a sample of 36 D. melanogaster genomes from Raleigh (North Carolina) exhibit nucleosomal patterns paralleling those observed for dinucleotides and polarized divergence , exhibit clear periodicities that generally parallel divergence and nucleosomal dinucleotide frequencies .The hypothesis that nucleosome structure and function impose natural selection on genomic sequence variation predicts periodicities in the frequency spectra. Indeed, the average vergence  and S9. vergence . A allelvergence . Partitiπ, (closely related to Tajima's Dp is the frequency of a SNP in the sample and π as the average (per SNP) deviation in p is equivalent to (1−p) mitigates the impacts of errors in the inference of the ancestral state p. Weak positive selection is predicted to skew π), while weak negative selection leads to more negative Δπ. Thus, systematic differences in selective forces at different positions across n147 regions should yield a pattern in Δπ that parallels that observed for divergence. These patterns of Δπ are superimposed on the observed genome-wide average negative skew To systematically assess the periodicity in the frequency spectra we calculated a new index, Δeles see . The “foπ for G→GA polymorphisms, we discovered a clear ∼10 bp periodic skew in frequency across nucleosomal regions, mirroring G→A divergence , with peaks aligning with regions of high TT frequency; while intergenic n147 share a subset of these peaks  also show a periodic skew in allele frequencies. Introns display a striking periodicity in A→AG Δπ aligned with GC frequency  that are discordant with the GC dinucleotide periodicity. Like underlying GC frequency, intronic A→AG Δπ has a concave upward shape. We observe weaker but interesting indications of continued periodicity in linker regions, consistent with selection for the preservation of rotational positioning in association with translational repositioning. The patterns of Δπ for 5 non-overlapping subsets of n147 regions were similar  population π values. This African sample is assumed to represent a larger, more stable population from the center of the species distribution, while the Raleigh sample represents the serial diasporas out-of-Africa and into North America. Notwithstanding differences in average Δπ, these strong and predicted periodicities in nucleosomal in Δπ support our hypothesis that direct interactions between the histone core and DNA sequence polymorphisms yield functional effects with fitness consequences.The periodicity of nucleosomal Δin vivo positions of our isolated nucleosomes reflect the innate preferred rotational positions of the particular genome used. Derived SNPs detected in a single strain are likely to be in high frequency, and thus we might observe periodicity in the frequency spectra at such SNPs in the absence of natural selection. To test for the impact of this hypothesized ascertainment bias on the periodicity of Δπ, we filtered the n147 for those in which the source genome bore the ancestral alleles. Despite the unavoidable thinning of the data, we observed clearly periodic polarized Δπ for those pathways with the strongest initial signals, e.g. intronic G→GA, C→CT and A→AG , G→GA and C→CT are the most common polymorphisms and have among the most positive Δπ, indicating weak positive selection, in addition to being the most rapidly diverging bases  and rotational positioning of functional elements, then we can further hypothesize that transcribed (intronic) nucleosomal sequences should exhibit stronger periodicity than untranscribed (intergenic) nucleosomal sequences. Consistent with this hypothesis, correlations of lineage specific divergence and Δπ), since strictly neutral mutations should display the same frequency distribution across the genome While natural selection is the most direct interpretation of these results, interactions between chromatin proteins and DNA damage and repair are well documented π. Biased gene conversion (BGC), a process where heteroduplex regions formed between homologs are repaired in a direction favoring one base, can create SNP dynamics analogous to those of directional selection π and its periodicities for G→GA and C→CT SNPs are comparable to those of A→AG, any explanation of our results invoking BGC would have to involve multiple distinct gene conversional biases that depend on nucleosome position. While this is conceivable and worthy of further investigation, we conclude that the canonical GC-biased gene conversion is not a significant component of the evolutionary dynamics leading to these intricate nucleosomal patterns of polymorphism and divergence.There is, however, one potential “selectively neutral” mechanism to explain the observed periodic patterns in Δmelanogaster lineage, ∼0.006, is about one tenth of that for synonymous divergence corresponding to 9 out of 10 mutations being selected against Whether these periodic patterns are the product of natural selection or BGC, the magnitude of the average force shaping the dynamics of nucleosomal SNPs must be small compared to that affecting the evolution of nonsynonymous variants. The shifts in G→A divergence between peaks and valleys in the n147 are ∼0.001 against a background average of ∼0.01, suggesting relatively weak constraint of 1 in 10 mutations. The non-synonymous rate of divergence on the Evidence of natural selection supporting nucleosome-associated sequence periodicities and the implication of their biological impact casts the potential functions of non-protein-coding regions in a new light. Substantial portions of Drosophila, human and other genomes appear to be under evolutionary constraint, yet lack any functional annotation π patterns supporting them) demonstrate that sequences that facilitate rotational positioning are specifically enriched relative to adjacent nucleosomes. So, while periodic sequence patterns are considered more relevant to rotational positioning, they clearly interact with the translational positioning of arrayed nucleosomes in Drosophila. Going forward, deeper and more detailed population genomic analyses should provide a unique window into the complex in vivo interactions between DNA sequence and nucleosome function.Our results suggest dinucleotide periodicities and the rotational positioning that they guide have significant biological consequences. Sequences affecting rotational positioning can influence the binding of transcriptional activators and participate in regulation of expression or gene splicing The significance of these periodic patterns of polymorphism and divergence is amplified in light of the substantial proportion of the eukaryotic genome packaged in nucleosomes (four-fold greater than that of coding sequence in Drosophila) and the broad conservation of dinucleotide interactions with the histone core. Indeed, no other DNA-protein interaction remotely approaches the genomic density or structural impact of nucleosomes. The striking periodic variation we observe relative to nucleosomes fundamentally changes expectations about divergence and SNP frequency, particularly in non-protein-coding regions. Our results point to a layer of evolutionary forces across entire genomes, emanating from the interactions of DNA sequence variation with the structure and function of the histone core.Embryos were collected from population cages Pelleted frozen nuclei were resuspended in CIB+PI, and chromatin was digested with 0.5 U/ml Micrococcal nuclease (Sigma) for 37°C for 15 min. MNase treated nuclei were pelleted, resuspended in 0.1% NP-40 PBS+PI, and incubated at 4°C for 3 hrs to release (primarily) mononucleosomes. Nuclei were re-pelleted, and chromatin from the supernatant was phenol-chloroform extracted. Digestion was analyzed on an agarose gel.Approximately 882 ng of DNA was used as starting material for paired-end sequencing library construction following the Illumina protocol (PE-102-1001). 10 µl of paired end adapter oligos were ligated to the end-repaired, A-tailed fragments in a 50 µl reaction. The adapter-ligation product was gel-purified to select molecules approximately 150–700 bp in length and re-suspended in 30 µl total volume. 1 µl of size-selected ligation product was used as template for 12 cycles of library enrichment PCR in a 50 µl reaction volume. The enriched library was purified using QIAGEN MinElute columns and sequenced (2×36 cycles) on one lane of a flow cell (FC42JB8) with an Illumina GAIIx running the Illumina software SCS v2.4.135/Pipeline v1.4.0.Subsequently, 8 µl of the same size-selected ligation product was used as a template for 10 cycles of library enrichment PCR in a 100 µl reaction volume. To enrich for 147 bp fragments, the library was purified using QIAGEN MinElute columns, then size selected on an agarose gel to recover fragments approximately 273 bp in length . This size-selected library was sequenced (2×36 cycles) on four lanes of a flow cell (FC61BGN) with an Illumina GAIIx running the Illumina software SCS v2.5.38/Pipeline v1.5.0.Berkeley Drosophila Genome Project's Release 5 reference sequence maq map –a 1000 -e 300 command. Otherwise, default map parameters were used.Reads that passed the Illumina pipeline's quality filters were then aligned to the Release 5.16 FlyBase Annotation of the D. melanogaster genome and classified as intronic or intergenic. For classification, all bases, including the flanking ±50 bp, were required to map entirely to a contiguous intronic or intergenic region. Heterochromatic reads were removed using cytogenomically-defined boundaries Mapped paired end clones of length 147 bp were then filtered based on π across n147 regions , both positional and average calculations took the dyad symmetry into account. Substitutional pathways were switched at the dyad axis, such that positions −73 to −1 were joined to the reverse complement of bases 1–73. Where included, flanking regions (±50 bp) were treated similarly. For larger scale positional divergence and genomic averages, data from complementary substitutional pathways were combined.In calculating dinucleotide frequencies, divergence, π and ΔThe n147 dinucleotide frequencies are the averages (in the reference sequence) over all n147 fragments for each position. Genomic over/underrepresentations of dinucleotides were calculated by dividing the difference between observed and expected frequencies by the expected frequency. Estimates of expected intergenic and intronic dinucleotide frequencies were calculated based on underlying base frequencies. Observed frequencies were computed directly from the reference sequence.D. melanogaster genomes from Raleigh, North Carolina were released by DPGP (http://www.dpgp.org/1K_50genomes.html - Reference_Release_1.0). The sequencing, alignment and assignment of estimated quality scores are described in Langley. et al., 2012 D. melanogaster genomes from Rwanda, Africa were released by DPGP (http://www.dpgp.org/dpgp2/update_20Jan2012/dpgp2_v2_rg.ID5.nohets.fastq.bz2). The sequencing, alignment and assignment of estimated quality scores are described in Pool et al., 2012 The sequences of the euchromatic portions of 36 melanogaster lineage, frequency, expected heterozygosity (π) and the index of skew in the frequency spectrum (Δπ) were based solely on sites that could be polarized using a multiple alignment of D. melanogaster, D. simulans, D. yakuba and D. erecta genomes i.e., simulans and yakuba and/or erecta have the identical base. For consideration of the potential impact of ascertainment bias association with the isolation of nucleosomes, sites were subjected to a more stringent polarization  and calculations were done only on sites where the experimental genome had the ancestral allele. These statistics  were estimated with two alternative weightings. The first, per-n147, gave weight to genomic sites proportional to their occurrence at nucleosome positions among the n147. Thus, in instances where a particular site was found multiple times in the n147 set, the divergences or SNPs at that site were give proportionally more weight. This in effect weights the signal by nucleosome site “occupancy.” The second, per-site, counted normalized the weighting such that each site  contributed equally, independent of its recurrence in the n147. The per-n147 estimates reflect those nucleosomal sequences that were readily isolated, while the per-site method treats each population genomic variant equally.Calculations of divergence on the π consists of random non-overlapping regions sampled from the n147. These analyses are presented to simply address whether the periodicities arise solely from a ∼10 bp periodicity in the overlap of n147 fragments. For divergence, non-overlapping n147 sets  were generated by random sampling of intergenic and intronic n147 without replacement. Newly drawn sequences were added to the non-overlapping subset only if they did not share any positions with prior sampled sequences. These non-overlapping subsets together cover 53% of the target intergenic and intronic regions in the euchromatic autosomes (chr2 and chr3) covered by the full n147. For non-overlapping Δπ, intergenic and intronic n147 (not including the flanking ±50 bp) were first filtered for only those nucleosomal regions containing the relevant SNP (taking dyad symmetry into account). This produced non-overlapping intergenic and intronic sets of ∼90,000 regions each for G→GA and C→CT and ∼55,000 regions for A→AG. These sets were then subjected to random sampling without replacement. A new n147 was added to the set if it did not overlap any already in the set. Non-overlapping G→GA and C→CT intronic and intergenic sets contained ∼46,000 regions each and A→AG sets contained ∼30,000.A third representation of the mapping of divergence and Δπ yielded similarly periodic patterns reflecting the fact that while the genomic coverage of the n147 is not deep, it is also relatively uniform (cv 0.65) and the periodicities are not arising from a small subset of the n147 or interactions from overlapping n147 sequences.All three of these methods for calculating divergence and Δe.g. number of G→A) divided by the number of polarized sites inferred to have been the specific ancestral state . For n147 average divergence, 5 bp at each end of the fragments were trimmed from the analysis to minimize the influence of sequence bias at the enzyme cleavage site (Divergence was based on the species reference sequences (see above). Polarized divergence estimates were calculated as the number of specific substitutions  with sample size between nmin and nmax. jx is the frequency of an allele at the thj of iS sites with sample size i. These SNPs can be categorized simply by state  or also as derived from an inferred ancestral state . For n147 average All estimates of frequency and expected heterozygosity, π were calculated over sample sizes between D, a normalization of d, the difference between two estimates of the same population parameter, 4Nμ, where μ is the mutation rate to selectively neutral alleles and N is the population size. These estimates aren, where ix is the frequency of one of the two alleles at each of the S segregating sites in an arbitrary genomic segment of length L base pairs. Thus 4Nμ.We require a sample-based index of the skew in the site frequency spectrum. Tajima test statistic for a genomic segment, but an index of the same deviation, that can be aggregated across heterogeneously sampled data and compared across classes of genomic annotation.But here we seek not a little) d,S segregating sites with frequency i/n in the sample, n(i)/SG. n(i)G can not be used to compute properties of a sample unless one can argue that the sites evolved independently and are sampled independently. If we choose a set of S segregating sites , rather than a genomic segment, we have the expected heterozygosity in a sample of size n,per segregating site from its predicted value under the assumptions of equilibrium between selectively neutral mutation and genetic drift in a Wright-Fisher population.To that end considerTo estimate this deviation across sites with different sample sizes, we can calculate the weighted average, weighting by the reciprocal of the variance.n under the assumptions of the neutral model (above).The variance of π(n) for S sites with sampling depth n, simply as Var[Δπ(n)]/S.If the frequencies at different sites are independent then we can estimate the sample variance of ΔΔπ can be estimated by the weighted averagenmin>3 and nmax = largest sample size.Assuming again that these SNPs are sampled independently both within and over sample sizes , the average π for each position in the n147 regions (and its average across positions) was calculated over polarized sites with sampling sizes (n) between 32 and 34 in the Raleigh π the n147 regions were trimmed as described above for divergence. Data from complementary pathways were merged for larger scale positional Δπ.ΔD. melanogaster reference sequence from bases 6 to 141 of the n147 regions . For density plots and Spearman's ρ, only n147 regions with at least 50 (out of 147) intergenic bases polarizable were included in the analyses. As above, the interior 5 bp on each end of n147 regions were trimmed from the analysis. For correlations between divergence and genomic intergenic GC frequencies, Spearman's ρ were reported for non-overlapping 500 bp windows in which at least 166 intergenic bases were polarizable and no more than 250 bp in the reference were “N”.n142-152 coverage of intergenic n147 regions was defined as the sum of the coverage of the region by the larger set of 142–152 bp nucleosomal fragments. This corresponds to what some authors call “occupancy.” GC frequency of intergenic n147 regions was calculated based on the nucleotide frequencies in the π plots  divergence on DNA from the structure of the nucleosome we colored-coded values of each base pair in a schematic rendering of the DNA strands of Figure S1A) Electrophoretic separation of DNA isolated from digestions of embryonic chromatin with three dilutions of MNase (at top). (B) Distributions of sizes of sequenced nucleosomal fragments. The proportion of paired-end sequenced fragments of each size is plotted against the size (bp). The sizes of 8,124,351 fragments that mapped to the 96.6 Mbp of euchromatic arms from a single GA2x lane of the initial library prepared from DNA isolated from the 1× digestion in panel A is shown in black. Almost completely superimposed are the distributions of sizes of fragments from four GA2x lanes of a size-selected fraction of that initial library . Also shown in light green is the position of the n147 fraction used in the most of the analyses.Paired end sequencing of MNase released mononucleosomal fragments produces a range of fragment sizes. ((EPS)Click here for additional data file.Figure S2Dinucleotide frequencies across intergenic (black) and intronic (red) n147 regions display a ∼10 bp periodicity. Dinucleotide frequencies are represented for bases 0 to −73 across n147 and −50 bp of flanking sequence.(EPS)Click here for additional data file.Figure S3Nucleotide frequencies surrounding intergenic and intronic n147 regions show a nucleosome-scale periodicity. Single nucleotide frequencies surrounding n147 regions (±600 bp).(EPS)Click here for additional data file.Figure S4Per-site divergence on the melanogaster lineage for the substitutional pathways displays ∼10 bp periodicity, consistent with per-n147 results. Smoothed average per-site polarized divergence across intergenic and intronic n147 regions (±50 bp) for specific substitutions is shown. Indicated dinucleotide frequencies are plotted above for reference.(EPS)Click here for additional data file.Figure S5melanogaster lineage is periodic across n147 regions. (A) Smoothed average polarized divergence for specific substitutions across regions defined by non-overlapping subsets of intergenic and intronic n147 (±50 bp). Intergenic dinucleotide frequencies are represented above for reference. (B) Smoothed intergenic A→G divergence mapped on to bases +73 to −6 from the nucleosome structure C) Smoothed average combined A→G:T→C polarized divergence surrounding intergenic (black) and intronic (red) n147 regions (±1 kb). Read depth of intronic (light grey) and intergenic (grey) n142-152 is represented below.Polarized divergence on the (EPS)Click here for additional data file.Figure S6MNase cleavage sites show consistent sequence bias at the boundaries of intergenic and intronic n147 fragments. Average 5′-3′nucleotide frequencies surrounding the MNase cleavage sites for intergenic and intronic n147 fragments (±30 bp).(EPS)Click here for additional data file.Figure S7melanogaster lineage correlate with GC content and nucleosome “occupancy”. A) Plots of the rate of divergence for intergenic n147 sequences for six substitutional pathways versus the log10 of coverage by n142-152 (“occupancy”). Correlation coefficients are reported in B) Plots of the rate of divergence for intergenic n147 sequences for six substitutional pathways versus their fraction GC. Correlation coefficients are reported in Divergence of six substitutional pathways along the (EPS)Click here for additional data file.Figure S8melanogaster lineage and AA/TT frequency varies with n147 GC content and nucleosome “occupancy.” (A) Average frequency of AA/TT dinucleotides (green) and rate of G→A:C→T divergence (orange) for intergenic n147 (and 1 kb flanking) regions binned by nucleosome “occupancy”, i.e., coverage of the n147 by the larger set of n142-152 nucleosomal fragments. n147 regions were ranked by coverage and separated into 5 bins (top is lowest and bottom is highest). Average divergence and AA/TT frequencies are shown for each bin. Intergenic n142-152 coverage is represented in grey . Although the axis limits for coverage vary, the scale of these axes is consistent. (B) Average G→A:C→T divergence across intergenic n147 regions for 5 bins based on n142-152 coverage . (C) Average G→A:C→T divergence across intergenic n147 regions for 5 bins based on ranked n147 GC content . (D) Scatter plot of intergenic n147 GC frequency against log10 n142-152 coverage. Spearman's ρ is reported.G→A:C→T divergence on the (EPS)Click here for additional data file.Figure S9Per-n147 SNP frequencies display a ∼10 bp periodic variation across intergenic and intronic n147 regions. Smoothed average per-n147 frequency of A (A) or T (B) alleles for G-A or C-T polymorphic sites respectively in the Raleigh sample (purple). Frequencies after removal of singleton classes are plotted in blue.(EPS)Click here for additional data file.Figure S10per-n147 SNP frequencies across intergenic n147 regions show evidence of ∼10 bp periodicities aligning with dinucleotide periodicities. (A) Average smoothed (black) and unsmoothed (grey) per-n147 frequencies of derived alleles for ancestrally G to G-A polymorphic, ancestrally C to C-T polymorphic and ancestrally A to A-G polymorphic sites in intergenic regions. (B) The same as A but with singleton classes  removed.Polarized (EPS)Click here for additional data file.Figure S11per-n147 SNP frequencies across intronic n147 regions show evidence of ∼10 bp periodicities aligning with dinucleotide periodicities. (A) Average smoothed (black) and unsmoothed (grey) per-n147 frequencies of derived alleles for ancestrally G to G-A polymorphic, ancestrally C to C-T polymorphic and ancestrally A to A-G polymorphic sites in introns. (B) The same as A but with singleton classes  removed.Polarized (EPS)Click here for additional data file.Figure S12π has little impact on the ∼10 bp periodic patterns for per-site Δπ and per-n147 Δπ. Average smoothed (black) and unsmoothed (grey) per-site Δπ across n147 regions (±50 bp) for G→GA, C→CT, and A→AG polymorphic sites. Color-coded dinucleotide frequencies are included for reference.The weighting of estimates of Δ(EPS)Click here for additional data file.Figure S13per-n147 Δπ across five independently sampled non-overlapping subsets of intergenic and intronic n147 sequences retain much of the ∼10 bp periodic signal. Average smoothed (black) and unsmoothed (grey) Δπ  for G→GA, C→CT, and A→AG polymorphic sites across regions defined by non-overlapping subsets (see The patterns of sets see  of intro(EPS)Click here for additional data file.Figure S14Per-n147 Δπ across intergenic and intronic n147 for Rwandan lines shows evidence of nucleosome-associated SNP periodicities in an independent sample. Average smoothed (black) and unsmoothed (grey) per-n147 Δπ for Rwandan lines for G→GA, C→CT, and A→AG polymorphic sites across regions defined by intronic and intergenic n147 (±50 bp). Frequencies of the indicated dinucleotides are plotted for reference.(EPS)Click here for additional data file.Figure S15π. Average smoothed (black) and unsmoothed (grey) intronic per-n147 Δπ across n147 regions (±50 bp) for G→GA, C→CT, and A→AG polymorphic sites in the Raleigh sample where the experimental strain had the ancestral allele.Ascertainment bias resulting from SNPs in the experimental strain does not explain the ∼10 bp periodic patterns of Δ(EPS)Click here for additional data file.Figure S16π in the regions flanking n147 sequences. Average combined A→G:T→C divergence (green) and A→AG:T→TC Δπ (black) surrounding intergenic and intronic n147 (±1 kb). Read depth of intronic or intergenic n142-152 is represented in grey below (axis label in blue).A→G:T→C Δ(EPS)Click here for additional data file.Figure S17π follow trends seen for n147 regions. Average scaled genome-wide π, Δπ and divergence for specific substitutions, partitioned by intergenic versus intronic Click here for additional data file.Table S1The genomic coordinates (Release 5) for intergenic autosomal 147 bp nucleosomal fragments ±50 flanking bp.(TXT)Click here for additional data file.Table S2The genomic coordinates (Release 5) for intronic autosomal 147 bp nucleosomal fragments ±50 flanking bp.(TXT)Click here for additional data file.Table S3Correlations between the rate of divergence of the indicated substitutional path and GC content or nucleosome “occupancy”.(TXT)Click here for additional data file.Table S4per-n147) of π, Δπ and divergence for intronic and intergenic nucleosomal regions (n147), a non-overlapping subset of n147 and the euchromatic genome (see text).Estimates ((TXT)Click here for additional data file.


Here atom probe tomography, transmission electron microscopy and electron beam-induced current are used to clarify the dependence of Se content on the photoactive properties of CdTexSex1− alloy layers in bandgap-graded CdTe solar cells. Four solar cells were prepared with 50, 100, 200 and 400 nm-thick CdSe layers to reveal the formation, growth, composition, structure and photoactivity of the CdTexSex1− alloy with respect to the degree of Se diffusion. The results show that the CdTexSex1− layer photoactivity is highly dependent on the crystalline structure of the alloy (zincblende versus wurtzite), which is also dependent on the Se and Te concentrations.The published external quantum efficiency data of the world-record CdTe solar cell suggests that the device uses bandgap engineering, most likely with a CdTe Using a CdTexSex1− alloy in CdTe solar cells leads to better performances. Here, Poplawsky et al. show that the photoactive properties of the alloy depend on its chemical composition and structural properties, and derive a phase diagram to further optimize alloy growth for photovoltaic devices. Jsc)1et al.Jsc increases of the world-record cell over the past 4 years13et al. showed that replacing the traditionally used CdS window layer with CdSe increases the Jsc by increasing the cell photo-response for short- and long-wavelength photons. In addition, Yang et al.5xSex1− alloy layer formed when CdTe is deposited on CdSe is necessary to optimize the fabrication of Se-induced bandgap-graded CdTe solar cells and further increase solar property parameters, such as the open-circuit voltage (Voc), fill factor (FF) and the overall PCE of CdTe solar cells using Se diffusion.Thin-film CdTe-based solar modules are relatively easy to produce at a low cost, making them one of the most competitive commercially available solar technologies in terms of price per watt, even though the world-record cell has a power conversion efficiency (PCE) of 22.1%, which is well below the theoretical PCE ∼33%)3%Jsc)1etxSex1− layer that reduces the bandgap of CdTe due to bowing effects, which explains the increased photo-response for long-wavelength photons. The reported increase of photocurrent in the short-wavelength regime suggests that the deposited CdSe layer completely diffuses into the CdTe because residual CdSe, or CdS for CdS window layer CdTe solar cells, absorb photons without contributing to the photocurrent31112The solubility of Se into CdTe is much higher than S, and, therefore, can easily diffuse from the CdSe layer into the CdTe during the CdTe growth and post-processing treatments56789x≤1, the CdTexSex1− alloy has a cubic zincblende structure and for 0≥x>0.3, the CdTexSex1− alloy has a wurtzite structure141516xSex1− alloys must be understood to improve devices using Se diffusion in CdTe solar cells.Previous reports have shown that for 0.6≤xSex1− diffusion layers formed during the CdTe growth processes are directly correlated with the photoactivity and junction position to better understand the photovoltaic properties of the resultant CdTexSex1− alloy layer. The carrier separation properties of CdSe/CdTe solar cells prepared with varied CdSe layer thicknesses  are measured with 30–50 nm resolution using scanning electron microscopy (SEM)-based electron beam-induced current (EBIC), while the crystallography and Se concentration are measured at the nanoscale using transmission electron microscopy selected area diffraction (TEM-SAD) and atom probe tomography (APT), respectively. These complementary techniques reveal the dependence of the Se content and crystalline structure of the CdTexSex1− layer during the CdTe growth processes on the ability of the CdTexSex1− layer to convert photons into usable electrical power.In this study, the Se content and crystalline structures (zincblende versus wurtzite) of the CdTeJ–V curves acquired for the CdTe solar cells prepared with 50, 100, 200 and 400 nm CdSe window layers used in the microscopy studies along with data acquired for a 130 nm-thick CdS layer CdTe solar cell are shown in Voc, Jsc, FF and PCE) of the representative solar cells can be found in et al.Voc, Jsc and total PCE among the four fabricated CdSe window layer cells. Compared with the standard CdS/CdTe solar cell, the CdSe/CdTe cells showed an enhanced photo-response in the short- and long-wavelength regions when CdSe window layers were limited to <200 nm in thickness. However, the EQE in the short-wavelength regime drops markedly when the CdSe window layer thickness increases to 400 nm  substrate is not photoactive, which would mean that the CdSe layer is not photoactive. However, the samples with 50–200 nm CdSe window layers do not exhibit drastic current losses for short-wavelength photons.The decreased EQE for the short-wavelength photons for the 400 nm CdSe layer cell suggests that a large part of the CdSe window layer interfacing the fluorine-doped-SnOVoc that is low1819Voc, Jsc and so on, with a window-less CdTe solar cell (CdTe directly deposited on FTO)Voc. The CdSe/CdTe interface does not show an enhanced EBIC signal as the initial CdSe layer thickens -SEM images of the magnified FTO/CdSe interfaces and the associated EBIC maps are shown in The most marked reduction in the overall PCE occurs for the 400 nm-thick CdSe layer , where JxSex1− interface, such that the EBIC measurements do not discern whether the pn-junction occurs directly at the FTO/CdTexSex1− interface or slightly within the CdTexSex1− alloy layer close to the interface. The increased Voc of the cell with the 100 nm CdSe layer compared with the 50 nm CdSe layer suggests that less recombination is occurring at the FTO/CdTexSex1− interface, such that the additional CdSe better passivates the surface between the FTO and CdTexSex1− layers. The EBIC average line profiles clearly show that the junction is beyond the FTO/CdTexSex1− interface, within the CdTexSex1− layer, for the 200 and 400 nm samples, and that the final junction position  is dependent on the original CdSe layer thickness.Average EBIC line profiles of the four samples prepared with different CdSe thicknesses are shown in xSex1− interface for the cells with 100 and 400 nm-thick CdSe layers.The grains interfacing the FTO layer resemble large columnar and twinned CdTe grains, as shown by the SE-SEM images in xSex1− interfaces. A bright-field (BF)-TEM image of the 400 nm CdSe layer is shown in dd(0001)=7.179 Å). The measured lattice parameters for this CdSe grain are a0=4.3 Å and c0=7.179 Å, compared with the literature values that range between a0=4.3 Å–4.4 Å and c0=7.01 Å–7.2 Å for CdSe with a relatively high Se content (>35%)22To elucidate the origin of the change in photoactive properties for the cells with 100 and 400 nm CdSe layers, focused ion beam (FIB) cross-sections were prepared for TEM analysis across the FTO/CdTexSex1− zincblende structure. The measured lattice parameter for this CdTexSex1− grain was a0=6.480 Å, which is consistent with the 6.480–6.483 Å literature values2425xSex1− alloys indicates that a threshold value of x ranging from 0.4 to 0.55 enables a phase transformation from the CdSe wurtzite structure to the CdTexSex1− zincblende structure16xSex1− alloy. The Se content of this alloy must have reduced to below 35 at. % to enable the phase transition from photoinactive wurtzite CdSe to the photoactive CdTexSex1− zincblende phase. During the phase transformation as a result of the Se/Te interdiffusion, the originally deposited CdSe grains recrystallized with the same orientation as the deposited CdTe grain, forming columnar grains extending from the FTO to the back contact. Such a phase transformation allows for the 100 nm sample to be photoactive across the entire CdTexSex1− layer, while the large columnar grains extending from the back contact are terminated by small non-photoactive wurtzite CdTexSex1− grains for the 400 nm-thick CdSe layer. It can be expected that the 400 nm CdSe layer retained a higher Se content than the 100 nm CdSe layer if the same amount of Te and Se diffusion occurred at the CdSe/CdTe interface during the CdTe CSS growth process; APT was used to verify this hypothesis.A BF-TEM image of the 100 nm CdSe layer is shown in xSex1− interfacial regions for the cells with 100 and 400 nm-thick CdSe layers.APT is a popular technique for detecting compositional variations at the nanoscale for CdTe-based solar cells due to its high elemental sensitivity, spatial resolution and relative ease of running CdTe materials in APT since the introduction of laser pulsing27282930313233xSex1− alloy in both samples exhibit graded Se and Te concentrations with the Se-rich region at the front of the cell. This is direct evidence that these solar cells are bandgap graded because the bandgap of the CdTexSex1− alloy is dependent on the value of x, where the bandgap of CdTe can decrease by as much as 8%, when x=0.6. A detailed review of how bandgap energies change with respect to the CdTexSex1− alloy composition can be found in ref. xSex1− layer and ended at the FTO layer, while the 400 nm sample fractured after passing through the interface separating the Se- and Te-rich phases. Unlike the data acquired for the sample with a 400 nm-thick CdSe layer, there is no distinct/rapid change in composition between the original CdSe and CdTe layers for the sample with the 100 nm CdSe layer. Rather, the Se and Te contents remain constant at ∼25 at. % between the FTO and 100 nm into the CdTexSex1− alloy. Isoconcentration surfaces for 27.5 at. % Te (green) and 27.5 at. % Se (purple) are also included in the APT reconstructions  may be a result of varied interdiffusion dynamics during the reaction between CdSe and CdTe, and the rather small CdSe grain size (<50 nm) and large network of GBs within the CdSe layer before CdTe deposition.While TEM-SAD was used to verify that the large columnar grains adjacent to the FTO layer shown in xSex1− grains with significantly different Te and Se contents. A proximity histogram Jsc, long-wavelength absorption and short-wavelength absorption of CdTe solar cells. The nature of Se diffusion from a deposited CdSe layer during CSS growth of CdTe and subsequent heat treatments, and its effect on the photovoltaic properties of CdTe devices, have been studied using a combination of SEM-EBIC, TEM-SAD and APT. APT results show that Se diffuses from the CdSe layer into the CdTe layer, forming a CdTexSex1− alloy and a Se concentration gradient within the CdTe. When the original CdSe layer is thin enough to significantly react with CdTe within the FTO/CdTe interface, it fully transitions into a zincblende-structured CdTexSex1− alloy that forms as large columnar grains that extend from the FTO to the back contact (shown by TEM and SEM imaging and TEM-SAD). SEM-EBIC measurements confirms that the zincblende CdTexSex1− alloy layer is photoactive. For thicker CdSe layers (400 nm), the original CdSe layer does not incorporate a sufficient amount of Te for the complete wurtzite-to-zincblende phase transformation during CdTe growth and subsequent heat treatments, such that the CdTexSex1− alloy layer contains both wurtzite and zincblende grains, with the higher Se content wurtzite grains interfacing with the FTO layer. SEM-EBIC data confirm that the wurtzite phase is photoinactive. EBIC average line profiles for all the devices with varying CdSe layer thicknesses show that the junction position is dependent on the original CdSe layer thickness. Overall, a photoactive layer containing a bandgap gradient, with a reduced bandgap close to the FTO/CdTexSe1x− interface, can be formed by diffusing an average of 25 at. % Se into the CdTe layer. This gradient increases the Jsc by increasing the absorption of long-wavelength photons. A bandgap-graded absorber layer is one critical component for high PCE CdTe-based solar cells. Therefore, the optimization of Se diffusion into the CdTe absorber layer is crucial for improving the state of the art of CdTe-based solar cells.Overall, the incorporation of a CdSe window layer increases the 2 heat treatment at 390 °C for 30 min, a Cu diffusion step and application of the back contact. Additional details of the growth procedure can be found elsewhereFour CdSe/CdTe cell structures with different starting CdSe layer thicknesses were fabricated. Each cell was grown on a commercial FTO soda lime glass supplied by NSG North America. The CdSe layers were magnetron sputtered on the FTO glass substrates at 250 °C using a 50 W radio frequency power in a 10 mTorr Ar purged chamber with CdSe layer thicknesses of 50, 100, 200 and 400 nm. Following the CdSe deposition on the FTO, each cell was exposed to the same growth and post-deposition procedures: a 4 μm CdTe layer was grown on the CdSe by CSS with a 610 °C substrate temperature, followed by a CdClJ–V curves were generated at room temperature in ambient air (25–30 °C) using a calibrated Xenon-arc lamp purchased from Newport with a reverse to forward bias (−0.2 to +1.0 V), 10 mV step size and 300 ms dwell time. National Renewable Energy Laboratory (NREL)-certified GaAs and Si reference cells were used to match the solar spectrum and 1-sun intensity. The cells were 0.08 cm2 and were isolated using a shadow mask (0.08 cm2) and scribing. The size of the shadow mask did not change the Jsc. The current density was also verified by the EQE measurements.All + beam acceleration voltage of 5 kV allowed for less than 10 nm of surface damage, as calculated from Stopping and Range of Ion in Matter simulations38The SEM-EBIC measurements were performed in a Hitachi S4800 cold field emission gun SEM equipped with a Gatan SmartEBIC system. All EBIC specimens were cross-section argon ion milled at liquid nitrogen temperatures using a Gatan Ilion+ ion polishing system to ensure a smooth, relatively damage-free surface. An ArAn FEI Nova 200 dual-beam FIB-SEM instrument was used to perform site-specific lift-outs of specimen ROIs and to perform annular milling to fabricate the needle-shaped APT specimens. A wedge lift-out geometry was used to mount multiple samples on a Si microtip coupon to enable the fabrication of multiple APT needles from one wedge lift-outThe APT data were acquired using a local electrode atom probe (CAMECA Instruments LEAP 4000X HR) equipped with an energy compensated reflectron lens and a 36% detection efficiency. The APT experiments were conducted at a specimen base temperature of 30 K by applying 3 pJ, 355 nm wavelength, 10 ps laser pulses at a repetition rate of 100 kHz, and a detection rate of 0.5–1.0%. Data reconstruction and analyses were performed using CAMECA IVAS 3.6.8 software.in situ lift-out method, which was followed by low-kV argon ion-milling using a Fischione 1040 Nanomill. An FEI Tecnai T20 TEM operated at 200 kV was used for BF-TEM imaging and SAD observations. STEM-EDS spectrum images were acquired using a Hitachi HF3300 TEM-STEM equipped with a Bruker X-Flash silicon drift detector EDS system and operated at 300 kV.TEM specimens less than 100 nm-thick were prepared using a Hitachi NB5000 FIB-SEM via a standard The data that support the findings of this study are available from the corresponding author on request.How to cite this article: Poplawsky, J. D. et al. Structural and compositional dependence of the CdTexSex1− alloy layer photoactivity in CdTe-based solar cells. Nat. Commun. 7:12537 doi: 10.1038/ncomms12537 (2016).Supplementary Figures 1-4 and Supplementary Tables 1-2


Macrophages have long been center stage in the host response to microbial infection, but only in the past 10–15 years has there been a growing appreciation for their role in helminth infection and the associated type 2 response. Through the actions of the IL-4 receptor α (IL-4Rα), type 2 cytokines result in the accumulation of macrophages with a distinctive activation phenotype. Although our knowledge of IL-4Rα-induced genes is growing rapidly, the specific functions of these macrophages have yet to be established in most disease settings. Understanding the interplay between IL-4Rα-activated macrophages and the other cellular players is confounded by the enormous transcriptional heterogeneity within the macrophage population and by their highly plastic nature. Another level of complexity is added by the new knowledge that tissue macrophages can be derived either from a resident prenatal population or from blood monocyte recruitment and that IL-4 can increase macrophage numbers through proliferative expansion. Here, we review current knowledge on the contribution of macrophages to helminth killing and wound repair, with specific attention paid to distinct cellular origins and plasticity potential. Immunological Reviews.This article is part of a series of reviews covering Monocytes and Macrophages appearing in Volume 262 of Infection with multicellular parasites represents one of the most significant challenges to both veterinary and public health. Medically relevant multicellular parasites are called ‘helminths’, a term that does not accurately reflect the vast phylogenetic diversity of this group, which includes the platyhelminths (flukes and tapeworms) and nematodes (roundworms). These animal phyla diverged over 1 billion years ago, predating the split between vertebrates and invertebrates In humans, helminths predominantly cause disease in developing countries but are a major veterinary problem throughout the world and often establish long-lasting infections that can last years and even decades. As a consequence, these infections are rarely lethal but can have devastating effects on life quality In this review, we focus on the role of macrophages in keeping the host fit in the context of a type 2 response induced by helminth infection. Macrophages have long been center stage as the target of Th1-type responses with a clear role in containment of microbial pathogens, particularly intracellular bacteria and protozoa. The function of macrophages activated by Th2 cytokines has been far more difficult to establish, despite their abundance in almost every helminth infection as well as other type 2 conditions such as allergy and asthma. While IL-4Rα ligation by either IL-4 or IL-13 has a dramatic effect on the macrophage transcriptional landscape Litomosoides sigmodontis, to understand the mechanisms of resistance and susceptibility to filarial nematode infection. Infective larvae (L3) invade via the skin, migrate through the lymphatics, and enter the pleural cavity, where they mature to adulthood. In susceptible BALB/c mice, the adult worms produce blood-circulating offspring (microfilariae) by day 50 postinfection. In resistant C57BL/6 mice the parasites are killed in the pleural cavity prior to reaching sexual maturity. This has proved a powerful model for the study of filariasis hi population was observed We have been using a murine model of filariasis, −/− mice et al. study and ours −/− mice but not in Stat-6−/− or IL-4Rα−/− animals.Macrophage expansion and survival had previously been associated with CSF1R signaling and differentiation of monocyte derived macrophages + WT mice are reconstituted with a 1:1 mix of Ly5.1+ WT and IL-4Rα−/− Ly5.2+ BM, we established that IL-4 acts directly on the macrophages and IL-4Rα expression is essential to initiate a program of proliferation L. sigmodontis infection were observed using these 50:50 WT:IL-4Rα−/− chimeras. No BrdU incorporation into IL-4Rαneg cells was observed following IL-4C injection, but during the early stages of L. sigmodontis infection, IL-4Rαneg cells did incorporate BrdU, albeit at significantly lower level than the WT cells. As discussed below, we subsequently identified CSF1-dependent mechanisms to be responsible for the residual proliferative expansion of these cells during infection. Critically, IL-4Rα positive macrophages have a competitive advantage, proliferating to a greater extent and eventually outnumbering their IL-4Rα negative counterparts −/− and IL-4Rα−/− mice in all infection models tested, emphasizing the central role of IL-4 in driving macrophage accumulation during infection. Because IL-13 can also signal through the IL-4Rα we addressed whether IL-13 was also capable of inducing macrophage proliferation. We generated an IL-13 complex and demonstrated that it was equivalently able to drive macrophage proliferation when delivered intraperitoneally IL-4C injection allowed us to take a reductionist approach to address the factors that contribute to helminth-driven macrophage proliferation. Using macrophage-intrinsic competitive bone marrow (BM) chimeras in which Ly5.1+ lymphocytes are required to generate significant macrophage numbers. In the absence of either RAG genes or class II, there is no expansion or alternative activation of macrophages in a peritoneal implant model of filarial infection L. sigmodontis infection model, proliferation does not occur until the onset of the adaptive immune response and does not occur in infected RAG-deficient mice The source of IL-4 or IL-13) that drives macrophage proliferation during helminth infection is likely to vary with tissue and stage of infection  that driOur discovery that IL-4 could drive expansion of resident macrophages occurred as a new paradigm in macrophage biology was unfolding. The established dogma had been that tissue-resident macrophages were derived from circulating bone marrow-derived monocytes. With new fate mapping technologies it became apparent that in most tissues including the serous cavities L. sigmodontis infection model, we used tissue-protected BM chimeras L. sigmodontis infection were derived from tissue-resident cells lamina propria of mice infected with the GI nematode, Heligmosomoides polygyrus  + cells via retinoic acid production. In contrast, some IL-4-induced gene products, such as Uncoupling protein 1, were seen only in the resident derived cells. These phenotypic differences between cells of embryonic origin versus bone marrow origin were verified in L. sigmodontis versus Schistosoma mansoni infections, respectively IL-4 has the capacity to induce alternative activation as well as proliferation in both resident and recruited settings L. sigmodontis model, we also observed that infection with the purely gastrointestinal nematode, H. polygyrus, caused expansion of peritoneal resident cells. This was somewhat surprising, as the parasite does not enter the peritoneal space. However, previous studies have shown the presence of Th2 cells in the peritoneal cavity of infected mice L. sigmodontis and H. polygyrus models, shielded bone marrow chimeras were used to verify the resident origin of the pleural and peritoneal macrophages, respectively. However, these infections may prove to be exceptions to the rule and in many, if not most, Th2 infections, there is likely to be recruitment of monocytes from the blood. Because the importance of macrophage origins has only recently become recognized, little information is available in different model systems, and thus we are relatively ignorant of what determines when a recruited population is dominant over resident expansion. Even in the L. sigmodontis model, over time there is increasing numbers of recruited monocytes (unpublished observation). We presume that ‘non-inflammatory’ entry of the infective larvae combined with local induction of IL-4 by innate cells means blood cell recruitment is initially avoided. It may be that only subsequent parasite death is sufficient to trigger monocyte recruitment. In settings in which bacteria are present, such as infection of the gastro-intestinal tract, monocyte recruitment will likely dominate Fig. In addition to the expansion of resident pleural macrophages in the S. mansoni infected mice S. mansoni infection, eggs become trapped in the liver and these induce a host-protective Th2-mediated granuloma, in which ΑΑΜΦ are a major component. Using intra-vital imaging and cell transfer experiments with Cx3Cr1GPF/+ mice, Girgis et al. et al. et al. lamina propria, and the lung in different Th2-inducing models  which show a high degree of immunomodulatory activity diverting potentially protective immune responses L. sigmodontis larvae exhibit limited migratory capacity in vaccinated mice H. polygyrus larvae become trapped inside the gut wall in resistant or immune mice This complexity is exemplified by the model filarial nematode, Nippostrongylus brasiliensis or H. polygyrusBrugia malayi and B. pahangi larvae in the peritoneal cavity of infected animals et al. in vitro macrophages and neutrophils work together to kill Strongyloides stercoralis larvae.The specific contribution of macrophages and, moreover, IL-4Rα-dependent activation in the killing and expulsion of helminths is still unclear. Macrophages make up a large proportion of the cells present at the site of various helminth infections H. polygyrus and depletion of macrophages using clodronate liposomes resulted in reduced resistance N. brasiliensis or Trichinella spiralis was unimpaired in LysMCre IL-4Rαflox/− mice relative to WT The evidence that macrophages are involved in worm killing is strong, but whether this requires alternative activation or is possibly hampered by it is still contentious. In a very recent study, expression of alternative activation markers positively correlated with resistance to infection with L. sigmodontis model suggest that there is a role for IL-4Rα-mediated macrophage activation in the control of this tissue nematode infection but also revealed a key difficulty with the LysMCre mice in the context of IL-4Rα expression. Infected LysMCre IL-4Rαflox/− mice showed increased number of circulating microfilaria at late stages of infection  or activating Fc receptors (FcRγ−/−), they demonstrate in a model of secondary H. polygyrus infection that antibodies activate macrophages to trap and immobilize infective larvae. Arginase was important for killing as discussed below. In this setting, the ability of antibodies to induce arginase was independent of the IL-4Rα, but in its absence macrophages fail to accumulate at the infection site A recent report by Esser-von Bieren It is therefore still early in our understanding of the mechanisms by which AAMΦ may contribute to parasite control. In the sections below, we discuss some of the findings about well-known markers of alternative activation and their role in resistance to helminth infection as well as more recent data from our laboratory identifying potential new effector molecules not previously associated with AAMΦ. Due to the fact that parasites can survive for very long periods of time in their respective hosts, it is reasonable to expect that a range and combination of different effector mechanisms need to work in concert to achieve effective parasite expulsion or killing. Of note, several AAMΦ products have been associated with an immune modulatory function rather than direct anti-parasitic effector responses. Thus, in many circumstances AAMΦ may not have a primary role in parasite killing but act to contain excessive inflammation and maintain tissue homeostasis.YM1 [chitinase-3-like-3 (Chi3l3)] is one of the most highly expressed genes in alternatively activated macrophages et al. Toxoplasma gondii. AMCase in humans and mice is strongly associated with Th2 responses AMCase and chitotriosidase, which actively degrade chitin, are thought to be important in the defense against fungal infections as well as in the degradation of inhaled chitin L. sigmodontis infection but only C57BL/6 mice are able to kill the parasites before patency in vitro and in vivo+ T-cell proliferation In contrast to the active chitinases, the function of YM1 and the other CLPs is still something of a mystery. This is despite a remarkable depth of knowledge on the structural biology of these proteins, due to the propensity for YM1 to form crystals in vitro leads to enhanced IL-10 production from T cells Another key molecule strongly associated with alternative macrophage activation and thus potentially important for the resistance against parasite infections is RELMα (Retnla) . Indeed, more than arginase or YM1, which can be induced by classical stimuli such as LPS A critical function for RELMα may prove to be in the regulation of host energy metabolism and glucose homeostasis during infection. RELMα is related to resistin, a hormone associated with insulin resistance The last of the ‘Big Three’ molecules associated with alternative activation of macrophages is arginase 1 H. polygyrus results in loss of a protective memory response and failure to expel the parasites H. polygyrus larvae in vitroN. brasiliensis larvae in the skin of infected animals S. mansoni-induced liver granulomas and therefore potentially important in restricting larval migration. Thus, the effects of arginase on parasite survival and resistance to infection may in part be mediated through its well-known role in tissue remodeling rather than immune modulation or arginine removal. Of note, the Wynn laboratory S. mansoni egg deposition, macrophage-derived arginase expression was central to immunosuppressive and regulatory but not pro-fibrotic functions. They propose that fibroblast derived arginase is more important for extracellular matrix deposition Depletion of macrophages or inhibition of arginase during secondary infection with in vitroL. sigmodontis worms in the pleural cavity of infected mice S. stercoralis larvae in a collaborative effort of alternatively activated macrophages and neutrophils Taenia crassiceps, type 1 responses and iNOS are essential for resistance at the early stages of infection As mentioned above, helminth infections are most commonly associated with a protective Th2 immune response and alternative activation of macrophages. However, anti-bacterial effector molecules such as reactive nitrogen or oxygen species due to enhanced activity of the corresponding enzymes  have been shown to effectively kill helminth parasites Mycobacterium tuberculosis infection and were thought to help prevent the spread of infectious mycobacteria in vitro will lead to fusion and MNGC-formation et al. B. malayi larvae lead to deformations/changes in the underlying cuticle of the parasite which allowed eosinophils to transmigrate underneath the cuticle and potentially damage the parasite. Partial encapsulation of parasites or simple attachment of immune cells alone, however, does not seem to be sufficient to efficiently kill adult parasites as we often observe healthy looking motile filarial nematodes in which a section of the parasite is encased in a granulomatous structure. Thus entrapment might just be the first step allowing the immune system enough time to deploy additional defense mechanisms.One characteristic of many helminth infections is the formation of dense granulomas around the invading parasite Attachment of macrophages to introduced foreign material (e.g. implants) creates a privileged space between the cell-membrane and the surface of the material into which degradative enzymes, acid, and reactive oxygen or nitrogen intermediates can safely be released without risk of deactivation or damage to the surrounding tissue B. malayi or exposure to thioglycollate To develop both a broader and more detailed picture of the function of the IL-4Rα on macrophages during nematode infection, we recently performed an in-depth RNAseq analysis of IL-4Rα-deficient and -sufficient macrophages elicited by implantation of the peritoneal cavity with the nematode In the context of filarial nematode infection, the absence of cellular recruitment might prevent the leakage of fluids and serum components from the vasculature that would normally occur during inflammatory processes Francisella tularensis by human macrophages but simultaneously reduces NF-κB signaling and pro-inflammatory cytokine secretion It may also be that complement components are contributing to the anti-inflammatory environment of helminth infection. C1q and C3, which are both highly upregulated in AAMΦ Another very prominent characteristic evident from our RNAseq data is the dramatic change in metabolic pathways utilized by macrophages following parasite infection IL-4Rα has a major impact on the expression of specific amino acid transporters in AAMΦ. In particular, there is increased expression of transporters associated with arginine uptake  and downregulation of a transporter involved in arginine efflux (Slc7a8) Macrophages are always present following tissue injury and regulate all aspects of repair, from the initial inflammatory phase, to cellular proliferation, angiogenesis, wound contraction, matrix deposition, and tissue remodeling and/or scar formation The evidence that IL-4Rα signaling on macrophages contributes to repair is mainly circumstantial although increasingly strong. The IL-4Rα dependent production of arginase is one of the earliest and best examples. Arginase contributes to tissue remodeling and repair because ornithine generated by arginase activity can be converted to polyamines and proline, supporting cell proliferation and collagen synthesis, respectively in vitroet al. In addition to arginase, RELMα and the CLP YM1 are highly but transiently upregulated in response to incisional wounding in vitroet al. Many other repair proteins are regulated by IL-4 or IL-13 in macrophages contributing to the evidence that tissue protection is a key function for AAMΦ. For example, transcriptionally, we observed that extracellular matrix degrading matrix metalloproteases (MMPs) are actively downregulated by IL-4Rα-mediated signaling in macrophages, while their inhibitors (TIMP1 and TIMP2) are upregulated Although more direct evidence is needed, there is little question that macrophages activated via the IL-4Rα contribute to repair. Wound repair, especially in adults, is often associated with scar tissue and when excessive leads to fibrosis. It has been proposed that wound healing is evolutionarily optimized for speed of healing under dirty conditions and thus scar tissue may be the price mammals must pay to close wounds sufficiently rapidly to avoid infection Because macrophages are rapidly recruited to the site of tissue injury, the predominant source of ‘wound’ macrophages are likely to be blood-circulating monocytes. The currently held view is that the early Ly6Chi monocytes are replaced by Ly6Clo ‘patrolling’ monocytes as repair progresses. Indeed, the inflammatory monocytes may only be needed to control potential infection, with the later stage macrophages more essential for full repair IL-4 has long been considered an anti-inflammatory cytokine The data supporting an anti-inflammatory role for AAMΦ have come from many laboratories and has been mostly based on the evidence that AAMΦ are important sources of immunosuppressive cytokines. These include TGF-β The ability of a single protein to be both anti-inflammatory and a central mediator of repair is demonstrated by TGF-β, which is known for both immune suppression and fibrosis in vivo, have made the story more complicated. Despite abundant expression of the IL-10 receptor, YM1, RELMα, arginase and many other AA markers, RNAseq analysis of nematode-elicited macrophages had sufficient depth of coverage to be able to say that IL-10 is not produced by F4/80hi macrophages in this context Although IL-10 is very strongly associated with a M2 phenotype and is often listed as a prototypic cytokine associated with alternative activation S. mansoni infection in mice that lack the IL-4Rα specifically on macrophages and neutrophils but have otherwise intact Th2 responses S. mansoni infection, these mice died from overwhelming inflammatory responses in the intestine and leakage of bacteria into the blood. Although not conclusive evidence, the data strongly suggest that in the absence of AAMΦ, these mice were unable to repair the damage caused by egg migration through the intestinal wall and the subsequent septic inflammation The combined anti-inflammatory/wound healing function of AAMΦ is strongly supported by a study of in vivo, are profoundly antiproliferative ex vivo. AAMΦ suppress proliferation of cells with which they are co-cultured ex vivo in an IL-4-dependent manner Fig. in vitro might at least in part be a side effect of nutrient sequestration to which T cells are highly sensitive A paradox that still needs to be resolved is that AAMΦ, which actively proliferate in vivo is not known. Although individual molecules expressed by AAMΦ  have been associated with immune suppression in vivoCritically, whether AAMΦ directly affect T-cell proliferation and polarization B. malayi infected animals and subjected to stimulation with LPS/IFNγ readily express pro-inflammatory cytokines  and are able to control intracellular replication of Leishmania mexicana parasites in vitro, is presumed to be important in the context of co-infections allowing macrophages to quickly adapt to new challenges.Classical macrophage activation, as found during bacterial infections, and IL-4Rα-mediated alternative activation are two extremes of a wide spectrum of overlapping activation phenotypes in vivo have been demonstrated in the setting of wound healing, where initially pro-inflammatory macrophages eventually convert to a more anti-inflammatory phenotype B. malayi infected mice early or late following parasite implantation, with an increase in pro-inflammatory cytokine production at later stages in vivo macrophage activation, especially in the context of infection, is rarely homogenous, and only a subset of macrophages will show signs of activation in vitro, macrophage plasticity during infection has to be re-evaluated. This is especially true in light of our new understanding of macrophage origins and proliferation et al. et al. B. malayi derived AAMΦ readily upregulated iNOS and pro-inflammatory cytokine-production following stimulation with LPS/IFNγ, we were never able to detect release of IL-12 from these cells, normally a key feature of classical macrophage activation Changes in macrophage activation phenotype in vitroB. malayi implant, as analyzed in our RNAseq analysis, are monocytic in origin (authors’ unpublished data). Despite clear evidence of proliferation and adoption of a resident-like phenotype  B. malayi implanted animals showed highly enhanced GATA6 expression as compared to thioglycollate elicited macrophages Nonetheless, bone marrow-derived macrophages can adopt a resident-like phenotype and re-populate the tissues after lethal irradiation We are entering a new era in macrophage biology, particularly in light of our new understanding of cellular origins. New questions are emerging daily, and we have raised many of them here in this review. For example, in the context of helminth infection, how do single molecules perform such diverse functions as parasite killing, immune suppression, and wound repair? More broadly, what is the extent of macrophage plasticity? Do recruited cells truly become fully functional resident cells? What are the epigenetic/transcriptional controls that regulate these functions both during development and locally in the tissues? New tools