prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: Is the ISR necessary for transgene reactivation?

Read the following text and answer briefly.

Context 1: Current address: Division of Brain Sciences, Department of Medicine, Imperial College London, London, United Kingdom 
In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan  However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap  We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation  Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome  Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs  The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated  Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2  Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease 
Copyright: © 2018 De Vito et al  This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited 
Data Availability: All relevant data are within the paper and its Supporting Information files  RNAseq data are available in the ArrayExpress database under the accession number E-MTAB-6452 
Funding: This study was funded by the Ajinomoto Innovation Alliance Program, (AIAP; https://www ajinomoto com/en/rd/AIAP/index html#aiap) (to M V S and D G), which is a joint research initiative of Ajinomoto Co , Inc , Japan  One of the authors [M B ] is an employee of Ajinomoto Co , and his specific roles are articulated in the ‘author contributions’ section  The commercial funder provided support in the form of salary for author [M B ] and some of the necessary research materials (medium for cell culture), but did not have any additional role in the study design, data collection and analysis, or preparation of the manuscript, and the authors had unrestricted access to the data  Due to a confidentiality agreement, the commercial funder participated only in the decision to publish the data obtained during the study, without any restriction 
Competing interests: This study was funded by Ajinomoto Co , Inc , Japan and one of the authors [M B ] is an employee of this commercial funder  No other employment or consultancy relationships exist with the commercial funder, and no patents, products in development, or marketed products result from this study  The authors declare that no competing interests exist and that the commercial affiliation of one of the authors does not alter the adherence of authors to all PLOS ONE policies on sharing data and materials 
In animals, excessive, insufficient, or imbalanced nutrient availability is known to strongly impact on phenotype and health, both short and long-term, and across generations [1, 2]  In particular, studies in yeast, animal models and humans have shown that reduced food intake, reducing either overall calories, or only sugars, proteins, or even single amino acids (AA), such as Methionine (Met), may extend lifespan and healthspan, and reduce the risk of cancer and other age-related diseases [3–9]  In addition, fasting or specific AA deprivation have shown potential therapeutic applications, owing to their ability to directly reduce the growth of some tumor types [10, 11], sensitize cancer cells to chemo- or immunotherapy [12, 13], and allow efficient hematopoietic stem cell engraftment   However, little is known about the specific processes and molecular mechanisms mediating the roles of nutrient restriction in human health and longevity 
A properly balanced diet in metazoans contains optimal amounts of a subset of AA, which cannot be synthetized de novo and are therefore named essential amino acids (EAAs)  In humans these include Met, Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Phenylalanine (Phe), Threonine (Thr), Tryptophan (Trp), and Valine (Val), while a few others are considered as semi-essential, such as Glutamine (Gln) and Tyrosine (Tyr) [15, 16]  Consistently, EAA deprivation triggers a cell-autonomous adaptive response, characterized by extensive metabolic and gene expression modifications, implementing biosynthetic, catabolic, and plasma membrane transport processes, aimed at reconstituting the full AA complement [17, 18]  The best known and conserved pathways responding to AA deprivation are triggered by mechanistic Target of Rapamycin Complex 1 (mTORC1) and General amino acid Control Non-derepressible 2 (GCN2) protein kinases [15, 19, 20]  Activation of mTORC1 requires in particular the presence of Gln, Arg and Leu, but also Met , which activate the kinase through sensors mainly acting upstream of Rag GTPases at lysosomal membranes   In turn, mTORC1 promotes cell growth, proliferation and anabolism upon activation, and translational attenuation and autophagy upon inhibition [19, 20] 
By contrast, GCN2 is activated by deprivation of any individual EAA, by means of its histidyl-tRNA synthetase-related domain, which binds uncharged tRNAs accumulating during AA limitation [23, 24]  Upon activation, GCN2 phosphorylates and inhibits its only known downstream target, namely the eukaryotic Initiation Factor 2 α (eIF2α), thereby initiating the Integrated Stress Response (ISR)  This leads to attenuation of general translation, and induction of a transcriptional/translational program, aimed at increasing stress resistance and restoring cell homeostasis, by upregulating a specific subset of genes, including Activating Transcription Factor 4 (ATF4) and C/EBP-Homologous Protein (CHOP) [25–27]  Thus, inhibition of mTORC1 and activation of GCN2 by AA restriction cooperate to attenuate general translation at the initiation step, increase catabolism and turnover, and enhance stress resistance to promote adaptation   However, how these processes eventually induce protective mechanisms against the alterations associated with aging, which include pervasive epigenetic and transcriptional changes [28, 29], remains largely unknown 
We previously reported the unexpected observation that prolonged deprivation of either Tyr, or of both Methionine and Cysteine (Met/Cys), triggers the selective and reversible reactivation of exogenous transcriptional units, including plasmids, retroviral vectors and proviruses, integrated into the genome and transcriptionally repressed by defensive mechanisms against non-native DNA sequences [30, 31]  This phenomenon was observed both in HeLa epithelial and ACH-2 lymphocytic human cells, and was independent of the transgene or provirus (Ocular Albinism type 1, OA1; Green Fluorescent Protein, GFP; Lysosomal-Associated Membrane Protein 1, LAMP1; Human Immunodeficiency Virus-1, HIV-1), or of the exogenous promoter driving their transcription, either viral (cytomegalovirus, CMV; Long Terminal Repeat, LTR) or human (Phospho-Glycerate Kinase 1, PGK1; Elongation Factor-1α, EF-1α)   Furthermore, this transgene reactivation response was not reproduced by serum starvation, activation of p38, or pharmacological inhibitors of mTOR (PP242 or rapamycin), sirtuins and DNA methylation  By contrast, it was induced by pan histone deacetylase (HDAC) inhibitors, and by selective inhibitors of class II HDACs   Consistently, we found that the mechanism responsible involves epigenetic modifications at the transgene promoter, including reduced nucleosome occupancy and increased histone acetylation, and is mediated in part by reduced expression of a class II HDAC, namely HDAC4  
These findings indicate that AA deprivation induces a specific epigenetic and transcriptional response, affecting the expression of newly-integrated exogenous transgenes and proviruses, and suggesting that endogenous sequences sharing similar structural and functional features may represent a transcriptional target as well [30, 31]  In particular, transposable elements, such as LTR-retrotransposons (or endogenous retroviruses, ERVs), are genomic “parasites” anciently-integrated into the genome, and silenced by epigenetic mechanisms of mammalian cells against the spreading of mobile elements, eventually becoming "endogenized" during evolution [32, 33]  This raises the question of whether their expression is also sensitive to AA restriction  In addition, it remains unclear whether or not the transgene reactivation response is related to specific AA deprivations, and most importantly which is the AA sensing/signaling pathway involved, in particular whether the GCN2 kinase is implicated  Thus, here we used the reactivation of silenced transgenes in cultured cells, as a model to investigate a novel molecular pathway induced by imbalanced EAA starvation, implicated in the epigenetic/transcriptional regulation of exogenous non-native DNA sequences and possibly of other endogenous anciently-integrated genomic elements 
HeLa human epithelial carcinoma, HepG2 human hepatocellular carcinoma and C2C12 mouse skeletal muscle cells were maintained in DMEM containing glutaMAX (Invitrogen) and supplemented with 10% FBS (Sigma), 100 U/ml penicillin G (Invitrogen), 100 mg/ml streptomycin (Invitrogen), at 37°C in a 5% CO2 humidified atmosphere  Cell lines carrying integrated and partially silenced transgenes were also maintained in 600–1000 μg/ml G418 
The C2C12 cell line was provided by ATCC  HeLa and HepG2 cells were obtained by Drs  F  Blasi and G  Tonon at San Raffaele Scientific Institute, Milan, Italy, respectively, and were authenticated by Short Tandem Repeat (STR) profiling, using the Cell ID System kit (Promega), according to the manufacturer’s instructions  Briefly, STR-based multiplex PCR was carried out in a final volume of 25 μL/reaction, including 5 μL Cell ID Enzyme Mix 5X, 2 5 μL Cell ID Primer Mix 10X and 3 ng of template DNA  The thermal cycling conditions were: 1 cycle at 96°C for 2 min, followed by 32 cycles at 94°C for 30 sec, 62°C for 90 sec, and 72°C for 90 sec, and 1 cycle at 60°C for 45 sec  The following STR loci were amplified: AMEL, CSF1PO, D13S317, D16S539, D21S11, D5S818, D7S820, TH01, TPOX, vWA  Fragment length analysis of STR-PCR products was performed by Eurofins Genomics, using standard procedures of capillary electrophoresis on the Applied Biosystems 3130 XL sequencing machine, and assessment of the STR profile was performed at the online STR matching analysis service provided at http://www dsmz de/fp/cgi-bin/str html 
Stable cell clones, expressing myc-tagged human OA1 (GPR143) or GFP transcripts, were generated using pcDNA3 1/OA1myc-His or pcDNA3 1/EGFP vectors   Briefly, HeLa, HepG2 and C2C12 cells were transfected using FuGENE 6 (Roche) and selected with 800, 1000, and 650 μg/ml of G418 (Sigma), respectively, which was maintained thereafter to avoid loss of plasmid integration  G418-resistant clones were isolated and analyzed for protein expression by epifluorescence and/or immunoblotting 
Full DMEM-based medium, carrying the entire AA complement, and media deprived of Met/Cys (both AAs), Met (only), Cys (only), Alanine (Ala), Thr, Gln, Val, Leu, Tyr, Trp, Lys and His were prepared using the Nutrition free DMEM (cat #09077–05, from Nacalai Tesque, Inc , Kyoto, Japan), by adding Glucose, NaHCO3, and either all 20 AAs (for full medium) or 18–19 AAs only (for deprivations of two-one AAs)  Single AAs, Glucose, and NaHCO3 were from Sigma  Further details and amounts utilized are indicated in S1 Table  All media were supplemented with 10% dialyzed FBS (Invitrogen), 100 U/ml penicillin G (Invitrogen), 100 mg/ml streptomycin (Invitrogen), and G418 as required  HBSS was from Invitrogen  Cells were seeded at 10–30% of confluency; cells to be starved for 48 h were plated 2–3 times more confluent compared to the control  The following day, cells were washed and cultured in the appropriate medium, with or without EAA, for 24–48 h 
L-Histidinol (HisOH), PP242, Integrated Stress Response Inhibitor (ISRIB), SP600125, Cycloheximide (CHX) were from Sigma; Salubrinal was from Tocris Bioscience; U0126 was from Promega  Drugs were used at the following final concentrations: HisOH at 4–16 mM; PP242 at 1–3 μM; ISRIB at 100 nM; SP600125 at 20 μM in HepG2 cells and 50 μM in HeLa cells; Cycloheximide (CHX) at 50 ug/ml in HepG2 cells and 100 ug/ml in HeLa cells; Salubrinal at 75 μM; U0126 at 50 μM  Vehicle was used as mock control  Treatments with drugs to be tested for their ability to inhibit transgene reactivation (ISRIB, SP600125 and U0126) were initiated 1h before the subsequent addition of L-Histidinol (ISRIB) or the subsequent depletion of Met/Cys (SP600125 and U0126) 
Total RNA was purified using the RNeasy Mini kit (Qiagen), according to manufacturer’s instructions  RNA concentration was determined by Nanodrop 8000 Spectrophotometer (Thermo Scientific)  Equal amount (1 μg) of RNA from HeLa, HepG2 and C2C12 cells was reverse transcribed using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) using oligo-dT as primers, and diluted to 5 ng/μl  The cDNA (2 μl) was amplified by real-time PCR using SYBR green Master Mix on a Light Cycler 480 (Roche), according to manufacturer’s instructions  The thermal cycling conditions were: 1 cycle at 95°C for 5 min, followed by 40–45 cycles at 95° for 20 sec, 56° for 20 sec and 72° for 20 sec  The sequences, efficiencies and annealing temperatures of the primers are provided in S2 Table  Data were analyzed with Microsoft Excel using the formula EtargetΔct target (control-sample) /EreferenceΔct reference (control-sample)   Reference genes for normalizations were ARPC2 (actin-related protein 2/3 complex, subunit 2) for HeLa and HepG2 cells; and Actb (actin beta) for C2C12 cells, unless otherwise indicated 
siRNA (Mission esiRNA, 200 ng/μL; Sigma) against ATF4 and GCN2 were designed against the targeted sequences NM_001675 and NM_001013703, respectively  Cells seeded in 6-well plates were transfected with 1 μg of siRNAs and 5 μL of Lipofectamine 2000 (Invitrogen), following manufacturer’s instructions, at day 1 post-plating for ATF4 and at day 1 and 2 post-plating for GCN2  At day 2 (ATF4) or 3 (GCN2) post-plating, cells were washed and cultured in medium in the absence or presence of HisOH 4 mM for 6 h  siRNAs against RLuc (Sigma), targeting Renilla Luciferase, were used as negative control  For CRISPR/Cas9 experiments, we used the “all-in-one Cas9-reporter” vector, expressing GFP (Sigma), which is characterized by a single vector format including the Cas9 protein expression cassette and gRNA (guide RNA)  GFP is co-expressed from the same mRNA as the Cas9 protein, enabling tracking of transfection efficiency and enrichment of transfected cells by fluorescence activated cell sorting (FACS)  The human U6 promoter drives gRNA expression, and the CMV promoter drives Cas9 and GFP expression  The oligonucleotide sequences for the three gRNAs targeting GCN2 exon 1 or 6 are listed in S2 Table  We transfected HeLa and HepG2 cells with these plasmids individually (one plasmid one guide) and sorted the GFP-positive, transfected cells by FACS  Screening GCN2-KO clones was performed by western blotting  In the case of HepG2-OA1 cells, two rounds of selection were necessary to obtain three GCN2-KO clones by using a guide RNA against exon 1  Compared to the original HepG2-OA1 cell line and to the clone resulting from the first round of selection (185#27), the selected clones E23, F22 and F27 showed a very low amount—if any—of residual GCN2 protein (see results) 
Genomic DNA of HeLa and HepG2 cells was purified using DNeasy Blood and Tissue kit (Qiagen), according to the manufacturer’s instructions  DNA concentration was determined by Nanodrop 8000 Spectrophotometer (Thermo Scientific)  PCR conditions for amplification of GCN2 exon 1 and 6 were as follows: 1 cycle at 94°C for 5 min, followed by 35 cycles at 94°C for 40 sec, 56°C for 40 sec, and 72°C for 40 sec; and a final extension step of 5 min at 72°C  The primer sequences are provided in S2 Table 
For OA1, western immunoblotting was carried out as described   For GCN2, cells were lysed in RIPA buffer, boiled at 95°C for 5 min and resolved on a 7 5% polyacrylamide gel; immunoblotting was then performed following standard procedures  Primary Abs were as follows: anti-human OA1, previously developed by our group in rabbits ; anti-GCN2 (Cell Signaling, Cat  #3302) 
Statistical analyses were performed using Microsoft Excel for Mac (version 15 32, Microsoft) for Student’s t-test; or GraphPad Prism (version 5 0d for Mac, GraphPad Software, Inc ) for one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s multiple comparisons post-tests  T-test was used when only two means, typically sample versus control, were compared, as specified in the figure legends  One way ANOVA was used for multiple comparisons, followed by either a Dunnett’s (to compare every mean to a control mean), or a Tukey’s (to compare every mean with every other mean) post-test, by setting the significance level at 0 05 (95% confidence intervals)  Both tests compare the difference between means to the amount of scatter, quantified using information from all the groups  Specifically, Prism computes the Tukey-Kramer test, allowing unequal sample sizes  P values in Figures are generally referred to comparison between a sample and the control (full medium/mock), and are indicated as follows: *P<0 05, **P<0 01, ***P<0 001  Comparisons not involving the control are similarly indicated, by a horizontal line at the top of the graphs, encompassing the two samples under analysis  Additional details regarding the specific experiments are reported in the Figure Legends 
To examine the expression behavior of genomic repeats upon AA starvation, we performed a transcriptomic analysis taking advantage of an intramural sequencing facility  HeLa-OA1 cells were cultured in normal medium (for 6-30-120 hours) or in absence of Met/Cys (for 6-15-30-72-120 hours)  Total RNA was prepared using Trizol (Sigma) to preserve transcripts of both small and long sizes (from Alu, of about 0 3 kb, to Long Interspersed Nuclear Elements, LINEs, and ERVs, up to 6–8 kb long), DNase treated to avoid contamination of genomic DNA, and processed for NGS sequencing by Ovation RNA-Seq System V2 protocol and HiSeq 2000 apparatus  Raw sequence data (10–20 M reads/sample) were aligned to the human genome (build hg19) with SOAPSplice   Read counts over repeated regions, defined by RepeatMasker track from UCSC genome browser , were obtained using bedtools suite   Normalization factors and read dispersion (d) were estimated with edgeR , variation of abundance during time was analyzed using maSigPro package , fitting with a negative binomial distribution (Θ = 1/d, Q = 0 01), with a cutoff on stepwise regression fit r2 = 0 7  Read counts were transformed to RPKM for visualization purposes  The expression of the OA1 transgene and HDAC4, which are progressively up- and down-regulated during starvation, respectively , were used as internal controls 
For genomic repeat analysis, reads belonging to repetitive elements were classified according to RepeatMasker and assigned to repeat classes (total number in the genome = 21), families (total number in the genome = 56) and finally subfamilies (total number in the genome = 1396), each including a variable number of genomic loci (from a few hundred for endogenous retroviruses, up to several thousand for Alu)  Repeat subfamilies were then clustered according to their expression pattern in starved vs control cells, by maSigPro using default parameters, and repeats classes or families that are significantly enriched in each cluster, compared to all genomic repeats, were identified by applying a Fisher Exact test (using scipy stats, a statistical module of Python)  Alternatively, differentially expressed repeat subfamilies were identified by averaging three time points of starvation (15-30-72 h) and controls  Repeats significantly up- or downregulated (104 and 77, respectively) were selected based on a P value <0 05 (unpaired two-tailed Student’s t-test, assuming equal variance), and analyzed for their class enrichment by a Fisher Exact test as described above 
For gene set enrichment analysis of Met/Cys deprived vs control HeLa cells, differentially expressed genes were selected considering three time points of starvation (15-30-72 h) and controls, based on a P value <0 05 (unpaired two-tailed Student’s t-test, assuming equal variance) and a fold change >2  This led to a total of 2033 differentially expressed genes, 996 upregulated and 1037 downregulated  The enrichment analysis was performed separately for up and down regulated genes, or with all differentially expressed genes together (both), using the KEGG database  The analysis was performed with correction for the background of all expressed genes (about 13600 genes showing an average expression over 3 starvation and 3 control samples of at least 5 counts) and by using default parameters (adjusted P value and q-value cut-off of <0 05 and 0 2, respectively)  Differentially expressed genes were also selected considering all starvation time points, as with genomic repeats, by maSigPro using default parameters, and a fold change of at least 1 5, leading to similar enrichment results (not shown)  RNAseq gene expression data are available in the ArrayExpress database under the accession number E-MTAB-6452 
To provide proof-of-principle that AA starvation may affect the expression of transposable elements, we performed an RNAseq analysis of the previously described HeLa-OA1 cells, carrying an integrated and partially silenced OA1 transgene   Since the reactivation of the transgene by starvation is a progressive phenomenon , we performed a time-course experiment, where each time point represents one biological sample, rather than a biological triplicate of a single time point  To this aim, cells were cultured either in normal medium, or in absence of Met/Cys for different time points (6-15-30-72-120 hours), resulting in the progressive upregulation of the OA1 transgene during starvation (Fig 1A and 1B), consistent with previously published results   The expression of genomic repeats was determined according to RepeatMasker annotation and classification into classes, families, and subfamilies  Repeat species were then subjected to differential expression and enrichment analyses in starved vs control conditions  Out of 1396 annotated repeat subfamilies, 172 species displayed a differential expression profile during starvation 
Fig 1  Exogenous transgene and endogenous retroviruses are upregulated in Met/Cys-deprived HeLa cells 
(A,B) Exogenous integrated transgene (OA1) mRNA abundance in HeLa-OA1 cells, cultured in Met/Cys-deprived medium for the indicated time points, and analyzed by RNAseq (A), or RT-qPCR (B), compared to full medium  Data represent RPKM (A), or mean ± SD of 2 technical replicates, expressed as fold change vs  control (full medium at 6 h = 1) (B)  (C) Clustering of 172 genomic repeat subfamilies, differentially expressed upon starvation, according to their expression profile  (D) Class distribution of repeat subfamilies belonging to differential expression clusters, compared to all genomic repeat subfamilies (first column)  Class DNA includes DNA transposons; SINE includes Alu; LINE includes L1 an L2; LTR includes endogenous retroviruses and solitary LTRs; Satellite includes centromeric acrosomal and telomeric satellites; Others includes SVA, simple repeats, snRNA, and tRNAs  LTR-retroelements are significantly enriched among repeats that are upregulated upon starvation, while LINEs are significantly enriched among repeats that are downregulated  *P<0 05, ***P<0 001 (Fisher exact test) 
As shown in Fig 1C, the clustering of differentially expressed repeats, according to their expression pattern, reveals profiles comparable to the behavior of the transgene in the same conditions, i e  upregulation upon starvation and no change in regular medium (Cluster 1 and 2)  In particular, Cluster 1 contains sequences that, similarly to the OA1 transgene, are progressively upregulated upon starvation (Fig 1A and 1C) , while Cluster 2 contains sequences that are upregulated at early time points  Interestingly, repeat families that are significantly enriched in these two clusters belong mostly to the group of LTR-retrotransposons, including ERV1, ERVK, ERVL, ERVL-MaLR and other LTR sequences (Fig 1D; S1A and S2A Figs)  By contrast, DNA transposons (such as TcMar-Tigger) and L1 non-LTR retrotransposons are enriched among repeats that are downregulated during starvation, particularly at late time points (Clusters 3 and 4) (Fig 1D; S1A and S2A Figs)  Consistent results were obtained by selecting significantly up- or downregulated genomic repeats (overall 181 species), based on their average expression out of three time points of starvation (15-30-72 h, when the transgene upregulation is more homogeneous) and controls, and on a P value <0 05 (S1B and S2B Figs)  These findings suggest that EAA starvation induces genome-wide effects involving repetitive elements, and that—among major repeat classes—it upregulates in particular the expression of ERVs 
In addition, to obtain a general overview of main gene pathways changing their expression together with the transgene during AA starvation, we performed gene expression and enrichment analyses of regular genes, by considering three time points of starvation (15-30-72 h) and controls  Differentially expressed genes were selected based on a P value <0 05 and a fold change between means of at least 2, and analyzed with the EnrichR tool   As shown in Fig 2 and S1 File, enrichment analyses against the KEGG and Reactome databases reveals a predominance of downregulated pathways, namely ribosome and translation, proteasome, AA metabolism, oxidative phosphorylation and other pathways related to mitochondrial functions, which are affected in Huntington, Alzheimer and Parkinson diseases (http://www genome jp/kegg/pathway html)  In particular, a large fraction of ribosomal protein mRNAs is downregulated upon Met/Cys starvation (Fig 2A and 2C; S1 File), consistent with the notion that their genes–despite being scattered throughout the genome—are coordinately expressed in a variety of conditions   This reduced expression may depend on multiple pathways that control ribosome biogenesis in response to external stimuli, including the downregulation of Myc activity , the downregulation of mTORC1 [42, 44], or possibly the activation of the ISR, as described in yeast   By contrast, upregulated genes show a significant enrichment for transcription and gene expression (Fig 2B)  Similar results were obtained by the Gene Ontology Biological Process (GO-BP) database (S1 File), overall indicating a general downregulation of translation and metabolism, and upregulation of transcription, during the time interval of Met/Cys starvation corresponding to the transgene upregulation 
Fig 2  Gene set enrichment analysis of Met/Cys-deprived HeLa cells 
Differentially expressed genes between three time points of starvation (15-30-72 h) and controls were selected based on a P value <0 05 and a fold change of at least 2, leading to a total of 996 upregulated, and 1037 downregulated genes  The enrichment analysis was performed separately for up and down regulated genes, using the EnrichR tool and the KEGG (A) and REACTOME (B, C) databases  Ranking is based on the combined score provided by EnrichR, and categories are displayed up to 20 items with an Adjusted P value <0 05  No significant categories were found with upregulated genes against the KEGG database  All data are shown in S1 File  The enrichment analysis using all differentially expressed genes together did not reveal any additional enriched process 
To characterize the pathway leading to the reactivation of silenced transgenes, we used HeLa-OA1 and HeLa-GFP cells, as described   In addition, to test cell types relevant for AA metabolism, such as liver and muscle, we generated clones of HepG2 human hepatoma and C2C12 mouse skeletal muscle cells, stably transfected with plasmids for OA1 and GFP transgenes, respectively (HepG2-OA1 and C2C12-GFP cells; endogenous OA1 is not expressed in any of these cell types)  In all cases, the integrated transgenes are under the control of the CMV promoter in the context of a pcDNA3 1 plasmid, are partially silenced, and can be efficiently upregulated by HDAC inhibitors (trichostatin A, TSA; ref   and S3A, S3B and S4A Figs), indicating that their expression is controlled at least in part by epigenetic mechanisms, as previously described  
To establish whether the reactivation response results from the shortage of specific AAs only, such as Met/Cys, or it is triggered by any AA deprivations, we cultured HeLa-OA1, HeLa-GFP, HepG2-OA1 and C2C12-GFP cells for 24–48 hours with a battery of media deprived of EAAs or semi-EAAs, including Met/Cys, Thr, Gln, Val, Leu, Tyr, Trp, Lys, and His  As negative controls, cells were cultured in full medium, carrying the entire AA complement, and in a medium deprived of Ala, a non-essential AA  The expression of the transgene transcript was then evaluated by RT-qPCR  As shown in Fig 3, and in S3C and S4B Figs, most EAA-deficiencies induced reactivation of the OA1 or GFP transgenes in all four cell lines, with the notable exception of Trp deprivation, which consistently resulted in no or minimal reactivation of the transgenes  Indeed, despite some variability, Met/Cys deficiency, but also Thr, Val, Tyr, and His deprivation always gave an efficient response, while Leu, Gln and Lys elicited evident responses in some cases, but not in others  Depletion of Phe gave results comparable to Tyr deprivation, however it significantly altered multiple reference genes used for normalization and therefore was eventually omitted from the analysis (not shown)  Finally, in the above experiments we used a combined Met/Cys deficiency, to avoid the potential sparing of Met by Cys  and for consistency with our previous studies   Nevertheless, the analysis of single Met or Cys starvation, both at the protein and transcript levels, revealed an exclusive role of Met deprivation in transgene reactivation, consistent with the notion that Cys is not an EAA (S3D and S3E Fig) 
Fig 3  EAA deprivation induces reactivation of silent transgenes in HeLa and HepG2 cells 
Relative transgene (OA1) and CHOP mRNA abundance in HeLa-OA1 (A) and HepG2-OA1 (B) cells, cultured in various AA-deprived media for 48 h and 24 h, respectively, compared to full medium  Mean ± SEM of 3 independent experiments  Data are expressed as fold change vs  control (full medium = 1)  *P<0 05, **P<0 01, ***P<0 001 (one way ANOVA, followed by Dunnett’s post-test vs  full medium) 
Collectively, these results indicate that transgene reactivation by EAA starvation is reproducible with most EAAs, shared by different cell types (epithelium, liver, and skeletal muscle), and conserved in different mammalian species (human, mouse) 
mTORC1 inhibition and GCN2 activation trigger the best-known signaling pathways responding to AA starvation   We previously showed that inhibition of mTORC1 is not sufficient to reproduce transgene reactivation in HeLa cells   By contrast, the involvement of GCN2 and the ISR, including the downstream effectors ATF4 and CHOP, has never been tested  In addition, this pathway has been typically assessed in transient assays, lasting for a few hours, which may not be comparable with the prolonged starvation conditions necessary to reactivate the transgene expression (at least 15–24 h)  Thus, we tested whether CHOP expression was upregulated upon incubation of HeLa-OA1, HepG2-OA1 and C2C12-GFP cells in media deprived of different EAAs for 24–48 h 
As shown in Fig 3 and S4B Fig, we found that CHOP expression is increased in all EAA-starvation conditions, but not in the absence of Ala, in all tested cell lines  Similar, yet less pronounced, results were obtained with ATF4, consistent with the notion that activation of this transcription factor is mainly mediated by translational upregulation (not shown) [15, 26]  However, the upregulation of CHOP does not parallel quantitatively that of the transgene, neither appears sufficient to induce it  In fact, CHOP is highly upregulated even upon Trp starvation, which consistently results in no or minimal reactivation of the transgenes (compare CHOP with OA1 or GFP expression; Fig 3 and S4B Fig)  Thus, while the ISR appears widely activated upon EAA starvation, the upregulation of its downstream effector CHOP only partly correlates with transgene reactivation and may not be sufficient to induce it 
The activation of the ISR upon AA starvation suggests that GCN2 may be involved in the transgene reactivation response  Therefore, we tested whether direct pharmacological activation of this kinase is sufficient to trigger the transgene reactivation similarly to starvation  In addition, we used pharmacological inhibitors of mTOR to corroborate previous negative results in HeLa cells  in the other cell lines under study  To this aim, HeLa-OA1 or GFP, HepG2-OA1 and C2C12-GFP cells were cultured in the presence of different concentrations of PP242 (mTOR inhibitor) or L-Histidinol (GCN2 activator, inhibiting tRNAHis charging by histidyl-tRNA synthetase), either alone or in combination for 24 h, compared to Met/Cys-deprived and full medium  As shown in Fig 4 and S5 Fig, while inhibition of mTORC1 consistently leads to minor or no effects, in agreement with previous findings , treatment with L-Histidinol results in efficient reactivation of the transgene in HepG2-OA1 and C2C12-GFP cells, but not in HeLa cells 
Fig 4  mTOR inhibition and GCN2 activation differently affect transgene expression in HeLa and HepG2 cells 
Relative transgene (OA1) and CHOP mRNA abundance in HeLa-OA1 (A) and HepG2-OA1 (B) cells, cultured in Met/Cys-deprived medium, or in the presence of PP242 (mTOR inhibitor; 1–3 μM) or L-Histidinol (HisOH, GCN2 activator; 4–16 mM), either alone or in combination for 24–48 h, compared to full medium  Mean ± SEM of 4 (A) or 3 (B) independent experiments  Data are expressed as fold change vs  control (full medium = 1)  *P<0 05, **P<0 01, ***P<0 001 (one way ANOVA, followed by Dunnett’s post-test vs  full medium)  PP-1 and PP-3, PP242 at 1 and 3 μM, respectively; HisOH-4 and HisOH-16, L-Histidinol at 4 and 16 mM, respectively 
Specifically, L-Histidinol is not effective in HeLa-OA1 and HeLa-GFP cells, either alone or in combination with PP242 (Fig 4A and S5A Fig), or by using different concentrations of the drug, with or without serum (not shown)  In these cells, L-Histidinol appears also unable to trigger the ISR, as indicated by lack of CHOP upregulation, possibly due to their different sensitivity to the drug  These findings are consistent with previous reports, describing the use of L-Histidinol in HeLa cells in conditions of low His concentration in the culture medium , which would resemble AA starvation in our system and therefore may not be applicable  Thus, even though the amount of the amino alcohol was adapted to exceed 20 to 80 times that of the amino acid, as described , HeLa cells may be resistant or able to compensate 
In contrast, in other cell types, L-Histidinol has been utilized in regular DMEM, to mimic the AA response triggered by DMEM lacking His [48, 49]  Consistently, in HepG2-OA1 cells, L-Histidinol is sufficient to elicit extremely high levels of transgene reactivation, and its combination with PP242 results in additive or even synergistic effects, possibly due to an indirect effect of mTOR inhibition on GCN2 activity (Fig 4B) [50, 51]  Similarly, C2C12-GFP cells efficiently reactivate the transgene upon treatment with L-Histidinol, but not PP242 (S5B Fig)  However, differently from HepG2-OA1 cells, simultaneous treatment of C2C12-GFP cells with L-Histidinol and PP242 does not lead to synergistic effects  Consistent with stimulation of the ISR, CHOP and to a minor extent ATF4 are upregulated by L-Histidinol in both cell lines, yet their expression levels show only an incomplete correlation with those of the transgene (Fig 4B, S5B Fig, and not shown) 
The finding that GCN2 activation by L-Histidinol is sufficient to reactivate the transgenes in both HepG2-OA1 and C2C12-GFP cells pointed to this kinase, and to the downstream ISR, as the pathway possibly involved in the EAA starvation response  Thus, we investigated whether the ISR is sufficient to trigger upregulation of the OA1 transgene in HepG2-OA1 cells by pharmacological means  As CHOP expression does not correspond quantitatively and is not sufficient to induce transgene reactivation, we tested the role of the core upstream event of the ISR, namely the phosphorylation of eIF2α , which can be induced by pharmacological treatments, independent of GCN2 (Fig 5A)  To this aim, we used Salubrinal, a specific phosphatase inhibitor that blocks both constitutive and ER stress-induced phosphatase complexes against eIF2α, thereby increasing its phosphorylation   We found that, while the ISR is activated upon Salubrinal treatment, as shown by increased CHOP expression, it does not induce OA1 transgene reactivation (Fig 5B) 
Fig 5  The ISR is neither sufficient nor necessary to induce transgene reactivation in HepG2 cells 
(A) Schematic representation of GCN2 activation by AA starvation, resulting in phosphorylation of eIF2a and initiation of the downstream ISR  In addition to GCN2, the ISR may be activated by other eIF2a kinases (PKR, HRI and PERK; not shown in the picture)  (B) Relative transgene (OA1) and CHOP mRNA abundance in HepG2-OA1 cells treated for 24 h with Salubrinal (a drug that induces the ISR by inhibiting the dephosphorylation of eIF2α; 75 μM), compared to full medium  Mean ± range of two experiments  Data are expressed as fold change vs  control (DMEM = 1)  *P<0 05 (paired two-tailed Student’s t-test vs  control)  (C) Relative transgene (OA1) and CHOP mRNA abundance in HepG2-OA1 cells treated for 6 h with L-Histidinol (HisOH, GCN2 activator; 4 mM), in the absence or presence of ISRIB (a drug that bypasses the phosphorylation of eIF2α, inhibiting triggering of the ISR; 100 nM)  Mean ± range of two experiments  Data are expressed as fold change vs  control (DMEM = 1)  **P<0 01, ***P<0 001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs  control, unless otherwise indicated)  (D) Relative transgene (OA1) and ATF4 mRNA abundance in HepG2-OA1 cells transfected with control (CTRL) or anti-ATF4 siRNAs, and incubated in the presence or absence of L-Histidinol (HisOH, GCN2 activator; 4 mM) for 6 h  Mean ± range of two experiments  Data are expressed as fold change vs  control (w/o HisOH = 1, top; control siRNA = 1, bottom)  *P<0 05 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs  control, unless otherwise indicated) 
To test whether the ISR is necessary to trigger the transgene response to L-Histidinol, we used the chemical compound ISRIB, which inhibits the activation of the ISR, even in the presence of phosphorylated eIF2α, likely by boosting the activity of the guanine-nucleotide exchange factor (GEF) for eIF2α, namely eIF2B [53, 54]  HepG2-OA1 cells were stimulated with L-Histidinol, either in the presence or absence of ISRIB  As shown in Fig 5C, while the expression of CHOP is inhibited by ISRIB, as expected, the reactivation of the OA1 transgene is not affected  In addition, knockdown of the closest eIF2α downstream effector ATF4 by siRNAs does not interfere with the reactivation of the OA1 transgene by L-Histidinol (Fig 5D)  Together, these data suggest that eIF2α phosphorylation and the downstream ISR pathway are neither sufficient nor necessary to induce transgene reactivation 
To definitively establish if GCN2 is necessary to trigger the transgene reactivation response to EAA starvation, we directly suppressed its expression by CRISPR/Cas9-mediated knock-out (KO)  We generated three independent GCN2-KO clones from the parental HeLa-OA1 cell line, by using three different guide RNAs, two against exon 1 (clones 183#11 and 185#5), and one against exon 6 (clone 239#1) of the GCN2 gene  Genomic characterization confirmed the presence of mutations on both alleles of exon 1 of the GCN2 gene in clone 183#11, and on both alleles of exon 6 in clone 239#1; by contrast, clone 185#5 showed multiple alleles in exon 1, consistent with the presence of two cell populations, and was not characterized further at the genomic level (S6 Fig)  None of these clones express GCN2 at the protein level, as shown by immunoblotting (Fig 6A)  To test the GCN2-KO cells for their ability to respond to EAA starvation, parental HeLa-OA1 cells and the three GCN2-KO clones were cultured in media deprived of Met/Cys or Thr (corresponding to the most effective treatments in this cell line; see Fig 3A) for 24–48 h and transgene expression was assessed by RT-qPCR  We found that the reactivation of the OA1 transgene is neither abolished, nor reduced by KO of GCN2, thus excluding that this kinase is necessary for the response to EAA starvation in HeLa-OA1 cells (Fig 6B and 6C) 
Fig 6  GCN2 knockout does not interfere with transgene reactivation in HeLa cells 
(A) Immunoblotting of protein extracts from the HeLa-OA1 parental cell line and GCN2-KO clones 183#11, 185#5 and 239#1, immunodecorated with anti-GCN2 antibody  Arrow, GCN2 specific band  Ponceau staining was used as loading control  (B, C) Relative transgene (OA1) mRNA abundance in HeLa-OA1 cells and GCN2-KO clones, cultured in Met/Cys (B) or Thr (C) deprived medium for 24 h or 48 h, respectively, compared to full medium  Mean ± SD of 3 technical replicates from 1 experiment  Data are expressed as fold change vs  control (full medium = 1)  Since independent clones may display variable reactivation responses (e g  due to different levels of transgene expression in basal conditions), the results are not shown as means of the three clones, but as separate replicates 
Similarly, we generated GCN2-KO clones from the parental HepG2-OA1 cell line by the same strategy  By using a guide RNA against exon 1 of the GCN2 gene, we obtained three independent GCN2-KO clones, namely E23, F22 and F27  Genomic characterization confirmed the presence of mutations on both alleles of exon 1 of the GCN2 gene in clone F27 (S7 Fig) and all three clones showed a very low amount—if any—of residual GCN2 protein, compared to the original HepG2-OA1 cell line (Fig 7A)  To assess the ability of GCN2-KO cells to reactivate the transgene upon starvation, we cultured parental HepG2-OA1 cells and the three GCN2-KO clones in media deprived of Met/Cys or His (corresponding to the most effective treatments in this cell line; see Fig 3B) for 24 h, and evaluated the transgene expression by RT-qPCR  As shown in Fig 7B and 7C, we found that the reactivation of the OA1 transgene is neither abolished, nor reduced by KO of GCN2, as in HeLa cells  To further confirm this result, we knocked-down GCN2 by RNA interference (RNAi), and incubated the cells with or without L-Histidinol for 6 h  As shown in Fig 8, treatment of HepG2-OA1 cells with L-Histidinol results in efficient transgene reactivation, even upon significant GCN2 downregulation, both at the mRNA and protein levels  Taken together, these data strongly support the conclusion that GCN2 is not necessary for transgene reactivation in response to EAA starvation, either in HeLa or in HepG2 cells 
Fig 7  GCN2 knockout does not interfere with transgene reactivation in HepG2 cells 
(A) Immunoblotting of protein extracts from the HepG2-OA1 parental cell line and GCN2-KO clones 185#27, E23, F22, F27, immunodecorated with anti-GCN2 antibody  Clone 185#27 results from the first round of selection, and was used to generate clones E23, F22, F27  Arrow, GCN2 specific band  For GCN2 protein quantification, Ponceau staining was used as loading control and data are expressed as fold change vs  parental cell line (= 1)  (B, C) Relative transgene (OA1) mRNA abundance in HepG2-OA1 cells and GCN2-KO clones, cultured in Met/Cys (B) or His (C) deprived medium for 24 h, compared to full medium  Mean ± SD of 3 technical replicates from 1 experiment 
Context 2: McPherson County (standard abbreviation: MP) is a county located in the U S  state of Kansas   As of the 2020 census, the county population was 30,223  The largest city and county seat is McPherson  The county is named for Civil War General James B  McPherson 

History

Early history

For many millennia, the Great Plains of North America was inhabited by nomadic Native Americans  From the 16th century to 18th century, the Kingdom of France claimed ownership of large parts of North America  In 1762, after the French and Indian War, France secretly ceded New France to Spain, per the Treaty of Fontainebleau  In 1802, Spain returned most of the land to France, but keeping title to about 7,500 square miles 

In 1803, most of the land for modern day Kansas was acquired by the United States from France as part of the 828,000 square mile Louisiana Purchase for 2 83 cents per acre  In 1848, after the Mexican–American War, the Treaty of Guadalupe Hidalgo with Spain brought into the United States all or part of land for ten future states, including southwest Kansas  In 1854, the Kansas Territory was organized, then in 1861 Kansas became the 34th U S  state 

19th century

From the 1820s to 1870s, the Santa Fe Trail passed through, what is now McPherson County  The trail entered the county, east of Canton, then south of Galva, then north of Inman, and west towards Lyons  In 1855, Charles O  Fuller established a ranch adjacent to the Running Turkey Creek Crossing about two miles south and one mile east of Galva  Fuller's Ranch provided accommodations for travelers on the Santa Fe Trail and was probably the first white settlement in McPherson County 

Peketon County was established in 1860, by the passage of a bill by S  N  Wood:  An act to establish Peketon County  Section 1  - That all that territory west of the sixth principal meridian and south of Township 16, in Kansas Territory, be and the same is hereby erected into a county, to be known by the name of Peketon County  On February 17, 1865, Peketon County was abolished, and McPherson County was made a part of Marion County, which extended from the west line of Chase County to the present western boundary of Kansas 

In 1868, Solomon Stephens and L  N  Holmberg were appointed Justices of the Peace—the first officers in what is now McPherson County  The next year (1869) occurred the first election for the township, now the county of McPherson  McPherson was regularly organized as a county in the spring of 1870, a mass meeting being held at Sweadal  Sweadal, the county seat thus selected, was located about one mile and a half southwest of the present site of Lindsborg  In September, however, the County Commissioners resolved to meet at the latter place, McPherson which had already been located some two years 

In April, 1873, a petition was filed for the county seat re-location  It was signed by 483 voters, and a special election was accordingly ordered for June 10  Upon that day, McPherson received 605 votes, New Gottland 325, King City 3 and Lindsborg 1; McPherson's majority over all, 276  In May the McPherson Town Company had offered, as an inducement for the location of the county seat at this point, the free use of rooms for ten years, and the donation of two squares of land on the town site  The offer was accepted the next month, the County Commissioners selecting blocks 56 and 65  Thus the county seat was established at McPherson and has remained since 

As early as 1875, city leaders of Marion held a meeting to consider a branch railroad from Florence  In 1878, Atchison, Topeka and Santa Fe Railway and parties from Marion County and McPherson County chartered the Marion and McPherson Railway Company   In 1879, a branch line was built from Florence to McPherson, in 1880 it was extended to Lyons, in 1881 it was extended to Ellinwood  The line was leased and operated by the Atchison, Topeka and Santa Fe Railway  The line from Florence to Marion, was abandoned in 1968  In 1992, the line from Marion to McPherson was sold to Central Kansas Railway  In 1993, after heavy flood damage, the line from Marion to McPherson was abandoned  The original branch line connected Florence, Marion, Canada, Hillsboro, Lehigh, Canton, Galva, McPherson, Conway, Windom, Little River, Mitchell, Lyons, Chase, then connected with the original AT&SF main line at Ellinwood 

In 1887, the Chicago, Kansas and Nebraska Railway extended its main line from Herington to Pratt   This main line connected Herington, Ramona, Tampa, Durham, Waldeck, Canton, Galva, McPherson, Groveland, Inman, Medora, Hutchinson, Whiteside, Partridge, Arlington,  Langdon, Turon, Preston, Natrona, Pratt   In 1888, this main line was extended to Liberal   Later, this line was extended to Tucumcari, New Mexico and Santa Rosa, New Mexico, where it made a connection with the Southern Pacific from El Paso, Texas   The Chicago, Kansas and Nebraska Railway was absorbed by the Chicago, Rock Island and Pacific Railway  This line is also called the "Golden State Route" 

20th century
The National Old Trails Road, also known as the Ocean-to-Ocean Highway, was established in 1912, and was routed through Windom, Conway, McPherson 

Geography

According to the U S  Census Bureau, the county has a total area of , of which  is land and  (0 3%) is water 

Adjacent counties
 Saline County (north)
 Dickinson County (northeast)
 Marion County (east)
 Harvey County (southeast)
 Reno County (southwest)
 Rice County (west)
 Ellsworth County (northwest)

Major highways
  Interstate 135
  U S  Route 56
  U S  Route 81
  K-4
  K-61
  K-153

Demographics

The McPherson Micropolitan Statistical Area includes all of McPherson County 

2000 census
As of the census of 2000, there were 29,554 people, 11,205 households, and 7,966 families residing in the county   The population density was 33 people per square mile (13/km2)   There were 11,830 housing units at an average density of 13 per square mile (5/km2)   The racial makeup of the county was 96 53% White, 0 81% Black or African American, 0 34% Native American, 0



ground_truth: No, it is not necessary.

prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: What was the name of the first white settlement in McPherson County?

Read the following text and answer briefly.

Context 2: 32% Asian, 0 06% Pacific Islander, 0 79% from other races, and 1 16% from two or more races   1 94% of the population were Hispanic or Latino of any race  37 1% were of German, 12 9% Swedish, 12 1% American, 6 7% English and 6 3% Irish ancestry according to Census 2000 

There were 11,205 households, out of which 33 00% had children under the age of 18 living with them, 62 50% were married couples living together, 6 00% had a female householder with no husband present, and 28 90% were non-families  25 50% of all households were made up of individuals, and 11 80% had someone living alone who was 65 years of age or older   The average household size was 2 49 and the average family size was 2 99 

In the county, the population was spread out, with 25 40% under the age of 18, 10 30% from 18 to 24, 25 20% from 25 to 44, 21 80% from 45 to 64, and 17 30% who were 65 years of age or older   The median age was 38 years  For every 100 females there were 95 90 males   For every 100 females age 18 and over, there were 92 90 males 

The median income for a household in the county was $41,138, and the median income for a family was $48,243  Males had a median income of $33,530 versus $21,175 for females  The per capita income for the county was $18,921   About 4 20% of families and 6 60% of the population were below the poverty line, including 5 20% of those under age 18 and 8 10% of those age 65 or over 

Government

Presidential elections
McPherson county is often carried by Republican candidates  The last time a Democratic candidate has carried this county was in 1964 by Lyndon B  Johnson 

Laws
Following amendment to the Kansas Constitution in 1986, the county remained a prohibition, or "dry", county until 1996, when voters approved the sale of alcoholic liquor by the individual drink with a 30 percent food sales requirement 

Education

Colleges
 McPherson College in McPherson
 Bethany College in Lindsborg
 Central Christian College in McPherson

Unified school districts
 Smoky Valley USD 400
 McPherson USD 418
 Canton-Galva USD 419
 Moundridge USD 423
 Inman USD 448

School district office in neighboring county
 Goessel USD 411
 Little River-Windom USD 444

Museums
 Birger Sandzén Memorial Gallery in Lindsborg
 McCormick-Deering Days Museum in Inman
 McPherson Museum in McPherson
 Lindsborg Old Mill & Swedish Heritage Museum in Lindsborg
 Kansas Motorcycle Museum in Marquette

Communities

Cities

 Canton
 Galva
 Inman
 Lindsborg
 Marquette
 McPherson (county seat) 
 Moundridge
 Windom

Unincorporated communities
† means a Census-Designated Place (CDP) by the United States Census Bureau 
 Conway
 Elyria†
 Groveland
 Johnstown
 New Gottland
 Roxbury†

Ghost towns
 Alta Mills
 Battle Hill
 Christian
 Doles Park
 Elivon
 King City
 Sweadal

Townships
McPherson County is divided into twenty-five townships   The cities of Lindsborg and McPherson are considered governmentally independent and are excluded from the census figures for the townships   In the following table, the population center is the largest city (or cities) included in that township's population total, if it is of a significant size 

See also
 List of people from McPherson County, Kansas
 National Register of Historic Places listings in McPherson County, Kansas
 McPherson Valley Wetlands
 Maxwell Wildlife Refuge

References

Notes

Further reading

 Wheeler, Wayne Leland  "An Analysis of Social Change in a Swedish-Immigrant Community: The Case of Lindsborg, Kansas " (PhD dissertation, University of Missouri-Columbia; ProQuest Dissertations Publishing, 1959  5905657) 

County
 Through the Years: A Pictorial History of McPherson County; McPherson Sentinel' Heritage House Publishing Co; 1992 
 McPherson County First Courthouse Built About 1869 or 1870; Lindsborg News-Record; March 30, 1959 
 Pioneer Life and Lore of McPherson County, Kansas; Edna Nyquist; Democratic-Opinion Press; 1932 
 A History of the Church of the Brethren in Kansas (includes McPherson College history); Elmer LeRoy Craik; McPherson Daily; Republican Press; 397 pages; 1922 
 Portrait and Biographical Record of Dickinson, Saline, McPherson, and Marion Counties, Kansas; Chapman Bros; 614 pages; 1893 
 Standard Atlas of McPherson County, Kansas; Geo  A  Ogle & Co; 82 pages; 1921 
 Plat Book of McPherson County, Kansas; North West Publishing Co; 50 pages; 1903 
 Edwards' Atlas of McPherson County, Kansas; John P  Edwards; 51 pages; 1884 

Trails
 The Story of the Marking of the Santa Fe Trail by the Daughters of the American Revolution in Kansas and the State of Kansas; Almira Cordry; Crane Co; 164 pages; 1915  (Download 4MB PDF eBook)
 The National Old Trails Road To Southern California, Part 1 (LA to KC); Automobile Club Of Southern California; 64 pages; 1916  (Download 6 8MB PDF eBook)

Mennonite Settlements
 Impact of Mennonite settlement on the cultural landscape of Kansas; Brenda Martin; Kansas State University; 1985/1988  
 Mennonite settlement : the relationship between the physical and cultural environment; Susan Movle; University of Utah; 1975/1886 
 Status of Mennonite women in Kansas in their church and home relationships; Eva Harshbarger; Bluffton College; 1925/1945 

External links

County
 
 McPherson County - Directory of Public Officials
Historical
 , from Hatteberg's People'' on KAKE TV news
Maps
 McPherson County Maps: Current, Historic, KDOT
 Kansas Highway Maps: Current, Historic, KDOT
 Kansas Railroad Maps: Current, 1996, 1915, KDOT and Kansas Historical Society

 
Kansas counties
1867 establishments in Kansas

Context 3: \section{Introduction}

Spectral line surveys have revealed that high-mass star-forming
regions are rich reservoirs of molecules from simple diatomic species
to complex and larger molecules (e g ,
\citealt{schilke1997b,hatchell1998b,comito2005,bisschop2007}) 
However, there have been rarely studies undertaken to investigate the
chemical evolution during massive star formation from the earliest
evolutionary stages, i e , from High-Mass Starless Cores (HMSCs) and
High-Mass Cores with embedded low- to intermediate-mass protostars
destined to become massive stars, via High-Mass Protostellar Objects
(HMPOs) to the final stars that are able to produce Ultracompact H{\sc
  ii} regions (UCH{\sc ii}s, see \citealt{beuther2006b} for a recent
description of the evolutionary sequence)  The first two evolutionary
stages are found within so-called Infrared Dark Clouds (IRDCs)   While
for low-mass stars the chemical evolution from early molecular
freeze-out to more evolved protostellar cores is well studied (e g ,
\citealt{bergin1997,dutrey1997,pavlyuchenkov2006,joergensen2007}),
it is far from clear whether similar evolutionary patterns are present
during massive star formation 

To better understand the chemical evolution of high-mass star-forming
regions we initiated a program to investigate the chemical properties
from IRDCs to UCH{\sc ii}s from an observational and theoretical
perspective  We start with single-dish line surveys toward a large
sample obtaining their basic characteristics, and then perform
detailed studies of selected sources using interferometers on smaller
scales  These observations are accompanied by theoretical modeling of
the chemical processes   Long-term goals are the chemical
characterization of the evolutionary sequence in massive star
formation, the development of chemical clocks, and the identification
of molecules as astrophysical tools to study the physical processes
during different evolutionary stages  Here, we present an initial
study of the reactive radical ethynyl (C$_2$H) combining single-dish
and interferometer observations with chemical modeling   Although
C$_2$H was previously observed in low-mass cores and Photon Dominated
Regions (e g , \citealt{millar1984,jansen1995}), so far it was not
systematically investigated in the framework of high-mass star
formation 

\section{Observations}
\label{obs}

The 21 massive star-forming regions were observed with the Atacama
Pathfinder Experiment (APEX) in the 875\,$\mu$m window in fall 2006 
We observed 1\,GHz from 338 to 339\,GHz and 1\,GHz in the image
sideband from 349 to 350\,GHz   The spectral resolution was
0 1\,km\,s$^{-1}$, but we smoothed the data to
$\sim$0 9\,km\,s$^{-1}$  The average system temperatures were around
200\,K, each source had on-source integration times between 5 and 16
min  The data were converted to main-beam temperatures with forward
and beam efficiencies of 0 97 and 0 73, respectively
\citep{belloche2006}  The average $1\sigma$ rms was 0 4\,K   The main
spectral features of interest are the C$_2$H lines around 349 4\,GHz
with upper level excitation energies $E_u/k$ of 42\,K (line blends of
C$_2$H$(4_{5,5}-3_{4,4})$ \& C$_2$H$(4_{5,4}-3_{4,3})$ at
349 338\,GHz, and C$_2$H$(4_{4,4}-3_{3,3})$ \&
C$_2$H$(4_{4,3}-3_{3,2})$ at 349 399\,GHz)  The beam size was $\sim
18''$ 

The original Submillimeter Array (SMA) C$_2$H data toward the
HMPO\,18089-1732 were first presented in \citet{beuther2005c}  There
we used the compact and extended configurations resulting in good
images for all spectral lines except of C$_2$H  For this project, we
re-worked on these data only using the compact configuration  Because
the C$_2$H emission is distributed on larger scales (see
\S\ref{results}), we were now able to derive a C$_2$H image  The
integration range was from 32 to 35\,km\,s$^{-1}$, and the achieved
$1\sigma$ rms of the C$_2$H image was 450\,mJy\,beam$^{-1}$   For more
details on these observations see \citet{beuther2005c} 

\section{Results}
\label{results}

The sources were selected to cover all evolutionary stages from IRDCs
via HMPOs to UCH{\sc ii}s  We derived our target list from the samples
of \citet{klein2005,fontani2005,hill2005,beltran2006}   Table
\ref{sample} lists the observed sources, their coordinates, distances,
luminosities and a first order classification into the evolutionary
sub-groups IRDCs, HMPOs and UCH{\sc ii}s based on the previously
available data  Although this classification is only based on a
limited set of data, here we are just interested in general
evolutionary trends  Hence, the division into the three main classes
is sufficient 

Figure \ref{spectra} presents sample spectra toward one source of each
evolutionary group  While we see several CH$_3$OH lines as well as
SO$_2$ and H$_2$CS toward some of the HMPOs and UCH{\sc ii}s but not
toward the IRDCs, the surprising result of this comparison is the
presence of the C$_2$H lines around 349 4\,GHz toward all source types
from young IRDCs via the HMPOs to evolved UCH{\sc ii}s   Table
\ref{sample} lists the peak brightness temperatures, the integrated
intensities and the FWHM line-widths of the C$_2$H line blend at
349 399\,GHz  The separation of the two lines of 1 375\,MHz already
corresponds to a line-width of 1 2\,km\,s$^{-1}$  We have three C$_2$H
non-detections (2 IRDCs and 1 HMPO), however, with no clear trend with
respect to the distances or the luminosities (the latter comparison is
only possible for the HMPOs)  While IRDCs are on average colder than
more evolved sources, and have lower brightness temperatures, the
non-detections are more probable due to the relatively low sensitivity
of the short observations (\S\ref{obs})  Hence, the data indicate
that the C$_2$H lines are detected independent of the evolutionary
stage of the sources in contrast to the situation with other
molecules   When comparing the line-widths between the different
sub-groups, one finds only a marginal difference between the IRDCs and
the HMPOs (the average $\Delta v$ of the two groups are 2 8 and
3 1\,km\,s$^{-1}$)   However, the UCH{\sc ii}s exhibit significantly
broader line-widths with an average value of 5 5\,km\,s$^{-1}$ 

Intrigued by this finding, we wanted to understand the C$_2$H spatial
structure during the different evolutionary stages   Therefore, we
went back to a dataset obtained with the Submillimeter Array toward
the hypercompact H{\sc ii} region IRAS\,18089-1732 with a much higher
spatial resolution of $\sim 1''$ \citep{beuther2005c}   Albeit this
hypercompact H{\sc ii} region belongs to the class of HMPOs, it is
already in a relatively evolved stage and has formed a hot core with a
rich molecular spectrum   \citet{beuther2005c} showed the spectral
detection of the C$_2$H lines toward this source, but they did not
present any spatially resolved images  To recover large-scale
structure, we restricted the data to those from the compact SMA
configuration (\S\ref{obs})  With this refinement, we were able to
produce a spatially resolved C$_2$H map of the line blend at
349



ground_truth: The first white settlement in McPherson County was Fuller's Ranch, established by Charles O. Fuller.

prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: What molecule was the focus of the study?

Read the following text and answer briefly.

Context 3: 338\,GHz with an angular resolution of $2 9''\times 1 4''$
(corresponding to an average linear resolution of 7700\,AU at the
given distance of 3 6\,kpc)  Figure \ref{18089} presents the
integrated C$_2$H emission with a contour overlay of the 860\,$\mu$m
continuum source outlining the position of the massive protostar  In
contrast to almost all other molecular lines that peak along with the
dust continuum \citep{beuther2005c}, the C$_2$H emission surrounds the
continuum peak in a shell-like fashion 

\section{Discussion and Conclusions}

To understand the observations, we conducted a simple chemical
modeling of massive star-forming regions  A 1D cloud model with a mass
of 1200\,M$_\sun$, an outer radius of 0 36\,pc and a power-law density
profile ($\rho\propto r^p$ with $p=-1 5$) is the initially assumed
configuration  Three cases are studied: (1) a cold isothermal cloud
with $T=10$\,K, (2) $T=50$\,K, and (3) a warm model with a temperature
profile $T\propto r^q$ with $q=-0 4$ and a temperature at the outer
radius of 44\,K  The cloud is illuminated by the interstellar UV
radiation field (IRSF, \citealt{draine1978}) and by cosmic ray
particles (CRP)  The ISRF attenuation by single-sized $0 1\mu$m
silicate grains at a given radius is calculated in a plane-parallel
geometry following \citet{vandishoeck1988}  The CRP ionization rate is
assumed to be $1 3\times 10^{-17}$~s$^{-1}$ \citep{spitzer1968}  The
gas-grain chemical model by \citet{vasyunin2008} with the desorption
energies and surface reactions from \citet{garrod2006} is used 
Gas-phase reaction rates are taken from RATE\,06 \citep{woodall2007},
initial abundances, were adopted from the ``low metal'' set of
\citet{lee1998} 

Figure \ref{model} presents the C$_2$H abundances for the three models
at two different time steps: (a) 100\,yr, and (b) in a more evolved
stage after $5\times10^4$\,yr  The C$_2$H abundance is high toward the
core center right from the beginning of the evolution, similar to
previous models (e g , \citealt{millar1985,herbst1986,turner1999}) 
During the evolution, the C$_2$H abundance stays approximately
constant at the outer core edges, whereas it decreases by more than
three orders of magnitude in the center, except for the cold $T=10$~K
model   The C$_2$H abundance profiles for all three models show
similar behavior 

The chemical evolution of ethynyl is determined by relative removal
rates of carbon and oxygen atoms or ions into molecules like CO, OH,
H$_2$O  Light ionized hydrocarbons CH$^+_{\rm n}$ (n=2  5) are quickly
formed by radiative association of C$^+$ with H$_2$ and hydrogen
addition reactions: C$^+$ $\rightarrow$ CH$_2^+$ $\rightarrow$
CH$_3^+$ $\rightarrow$ CH$_5^+$   The protonated methane reacts with
electrons, CO, C, OH, and more complex species at later stage and
forms methane   The CH$_4$ molecules undergo reactive collisions with
C$^+$, producing C$_2$H$_2^+$ and C$_2$H$_3^+$  An alternative way to
produce C$_2$H$_2^+$ is the dissociative recombination of CH$_5^+$
into CH$_3$ followed by reactions with C$^+$   Finally, C$_2$H$_2^+$
and C$_2$H$_3^+$ dissociatively recombine into CH, C$_2$H, and
C$_2$H$_2$  The major removal for C$_2$H is either the direct
neutral-neutral reaction with O that forms CO, or the same reaction
but with heavier carbon chain ions that are formed from C$_2$H by
subsequent insertion of carbon  At later times, depletion and
gas-phase reactions with more complex species may enter into this
cycle   At the cloud edge the interstellar UV radiation
instantaneously dissociates CO despite its self-shielding,
re-enriching the gas with elemental carbon 

The transformation of C$_2$H into CO and other species proceeds
efficiently in dense regions, in particular in the ``warm'' model
where endothermic reactions result in rich molecular complexity of the
gas (see Fig ~\ref{model})   In contrast, in the ``cold'' 10\,K model
gas-grain interactions and surface reactions become important   As a
result, a large fraction of oxygen is locked in water ice that is hard
to desorb ($E_{\rm des} \sim 5500$~K), while half of the elemental
carbon goes to volatile methane ice ($E_{\rm des} \sim 1300$~K)  Upon
CRP heating of dust grains, this leads to much higher gas-phase
abundance of C$_2$H in the cloud core for the cold model compared to
the warm model  The effect is not that strong for less dense regions
at larger radii from the center 

Since the C$_2$H emission is anti-correlated with the dust continuum
emission in the case of IRAS\,18089-1732 (Fig \,\ref{18089}), we do
not have the H$_2$ column densities to quantitatively compare the
abundance profiles of IRAS\,18089-1732 with our model  However, data
and model allow a qualitative comparison of the spatial structures 
Estimating an exact evolutionary time for IRAS\,18089-1732 is hardly
possible, but based on the strong molecular line emission, its high
central gas temperatures and the observed outflow-disk system
\citep{beuther2004a,beuther2004b,beuther2005c}, an approximate age of
$5\times10^4$\,yr appears reasonable   Although dynamical and chemical
times are not necessarily exactly the same, in high-mass star
formation they should not differ to much: Following the models by
\citet{mckee2003} or \citet{krumholz2006b}, the luminosity rises
strongly right from the onset of collapse which can be considered as a
starting point for the chemical evolution  At the same time disks and
outflows evolve, which should hence have similar time-scales   The
diameter of the shell-like C$_2$H structure in IRAS\,18089-1732 is
$\sim 5''$ (Fig \,\ref{18089}), or $\sim$9000\,AU in radius at the
given distance of 3 6\,kpc   This value is well matched by the modeled
region with decreased C$_2$H abundance (Fig \,\ref{model})   Although
in principle optical depths and/or excitation effects could mimic the
C$_2$H morphology, we consider this as unlikely because the other
observed molecules with many different transitions all peak toward the
central submm continuum emission in IRAS\,18089-1732
\citep{beuther2005c}  Since C$_2$H is the only exception in that rich
dataset, chemical effects appear the more plausible explanation 

The fact that we see C$_2$H at the earliest and the later evolutionary
stages can be explained by the reactive nature of C$_2$H: it is
produced quickly early on and gets replenished at the core edges by
the UV photodissociation of CO  The inner ``chemical'' hole observed
toward IRAS\,18089-1732 can be explained by C$_2$H being consumed in
the chemical network forming CO and more complex molecules like larger
carbon-hydrogen complexes and/or depletion 

The data show that C$_2$H is not suited to investigate the central gas
cores in more evolved sources, however, our analysis indicates that
C$_2$H may be a suitable tracer of the earliest stages of (massive)
star formation, like N$_2$H$^+$ or NH$_3$ (e g ,
\citealt{bergin2002,tafalla2004,beuther2005a,pillai2006})  While a
spatial analysis of the line emission will give insights into the
kinematics of the gas and also the evolutionary stage from chemical
models, multiple C$_2$H lines will even allow a temperature
characterization  With its lowest $J=1-0$ transitions around 87\,GHz,
C$_2$H has easily accessible spectral lines in several bands between
the 3\,mm and 850\,$\mu$m   Furthermore, even the 349\,GHz lines
presented here have still relatively low upper level excitation
energies ($E_u/k\sim42$\,K), hence allowing to study cold cores even
at sub-millimeter wavelengths   This prediction can further be proved
via high spectral and spatial resolution observations of different
C$_2$H lines toward young IRDCs 

\acknowledgments{H B  acknowledges financial support
  by the Emmy-Noether-Programm of the Deutsche Forschungsgemeinschaft
  (DFG, grant BE2578)  }






ground_truth: The focus of the study was on the reactive radical ethynyl (C$_2$H).