Citrus maxima (Burm) Merr.) fruits are interesting materials for studying the mechanisms of carotenoid accumulation. In this study, particularly high contents of linear carotenes were observed in the albedo tissue, segment membranes and juice sacs of ‘CH’. However, carotenoids, especially β-carotene and xanthophylls, accumulated more in the flavedo tissue of ‘FC’ than in that of ‘CH’. Additionally, the contents of other terpenoids such as limonin, nomilin and abscisic acid significantly differed in the juice sacs at 150 days postanthesis. A dramatic increase in carotenoid production was observed at 45 to 75 days postanthesis in the segment membranes and juice sacs of ‘CH’. Different expression levels of carotenogenesis genes, especially the ζ-carotene desaturase (CmZDS), β-carotenoid hydroxylase (CmBCH) and zeaxanthin epoxidase (CmZEP) genes, in combination are directly responsible for the largely different carotenoid profiles between these two pummelo fruits. The sequences of eleven genes involved in carotenoid synthesis were investigated; different alleles of seven of eleven genes might also explain the largely different carotenogenesis observed between ‘CH’ and ‘FC’. These results enhance our understanding of carotenogenesis in pummelo fruits.Carotenoids in citrus fruits have health benefits and make the fruits visually attractive. Red-fleshed ‘Chuhong’ (‘CH’) and pale green-fleshed ‘Feicui’ (‘FC’) pummelo ( Solanum lycopersicum) [Carica papaya) [Daucus carota) [Citrullus lanatus) [Psidium guajava) [Momordica cochinchinensis) [Citrus) [Carotenoids, especially lycopene, contribute to the red-fleshed color of many plants, such as tomato (ersicum) , papaya  papaya) , 3, red  carota) , watermelanatus) , pink guguajava) , gac (Moinensis) , and red[Citrus) . These r[Citrus) , carrot [Citrus)  and toma[Citrus) . Recentl[Citrus) , 12, 13.[Citrus) , the mec[Citrus) . The alt[Citrus) . Abscisi[Citrus) . In addi[Citrus) .cis-violaxanthin, violaxanthin, lutein, β-cryptoxanthin, α-carotene, β-carotene, phytofluene and phytoene have been detected in the flavedo of citrus fruits [Citrus reticulata) mainly accumulate 9-cis-violaxanthin, β-cryptoxanthin and β-carotene [Citrus maxima) [Citrus paradisi) [Citrus sinensis) [Citrus maxima) pummelo fruits accumulate an abundance of limonin and nomilin, while the low-carotenoid JS of pale green-fleshed ‘Feicui’ (‘FC’) pummelo fruits are associated with low limonin and nomilin accumulations [Carotenoids accumulate in almost all the tissues of citrus fruits. Specifically, carotenoid accumulation in the flavedo and juice sacs (JS) of fruits has been studied extensively , 21, 22.s fruits , 21, 22.carotene , 23. Int maxima) , 20, 24,inensis) ; colorleulations .DXS) gene and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene are critical in the methylerythritol 4-phosphate (MEP) biosynthesis pathway, which is a well-known pathway that is upstream of the carotenoid biosynthesis pathway [PSY) gene has been demonstrated to be a rate-limiting gene involved in carotenoid accumulation [trans-lycopene is synthesized from phytoene via four steps, and the enzymes coded by the phytoene desaturase (PDS) gene, ζ-carotene isomerase (ZISO) gene, ζ-carotene desaturase (ZDS) gene and carotenoid isomerase (CRTISO) gene together with light-mediated isomerization are involved in the process [LCYb) gene was thought to be an important enzyme affecting the accumulation of lycopene [LCYe) gene cyclizes lycopene twice to form α-carotene and is the first enzyme of the α-branch. The β-carotenoid hydroxylase (BCH), zeaxanthin epoxidase (ZEP) and neoxanthin synthase (NSY) genes are located downstream of the carotenoid pathway (Citrus sinensis cv. Anliu) sweet orange, the accumulation of lycopene in its red-fleshed mutant ‘Hong Anliu’ has been attributed to the upregulation of upstream genes and the downregulation of downstream genes [The 1-deoxy-D-xylulose-5-phosphate synthase  and JS of ‘CH’ and different alleles of 7 genes in the JS might be directly responsible for the largely different carotenogenesis between the ‘CH’ and ‘FC’ pummelo fruits.‘CH’ and ‘FC’ are excellent pummelo cultivars selected from local sources in Lishui, Zhejiang Province, but their origins are unknown. Their fruits are similar in size and shape but present different flesh colors . We prev10.17504/protocols.io.qsjdwcn.All protocols in this manuscript share the same protocols, Citrus maxima) fruits exhibiting typical characters at each corresponding fruit development stage were collected from the scientific research orchard of the Forestry Science Academy of Lishui, Zhejiang Province, in 2011. The fruits were collected at 45, 75, 100, 125 and 150 days postanthesis (DPA) (‘CH’ and ‘FC’ pummelo (is (DPA) . The fruAll the samples were treated with liquid nitrogen and then stored at –80°C. Some of them were lyophilized in a Heto Lyolab 3000  and homogenized to a powder in liquid nitrogen for the extraction of metabolites.et al. [trans-lutein, all-trans-violaxanthin and phytoene were purchased from CaroteNature , while those of β-carotene and all-trans-lycopene together with standards of limonin and nomilin were purchased from Sigma Co. Ltd. .Carotenoids were extracted from 1.0 g of lyophilized samples and then measured in accordance with the methods of Liu et al.  and Lee et al. . The caret al. [In accordance with the methods of Li et al. , 3.0 g o18 HPLC column  was used for limonin and nomilin separation. For quantification, an isocratic elution model was applied with acetonitrile:10% methanol  at an elution speed of 1 mL/min and an injection volume of 20 μL . Both limonoids were detected at a wavelength of 210 nm under ambient temperature.With respect to HPLC analyses, the same instrument was used to analyze the carotenoids, while a Cet al. [et al. [In accordance with the methods of Pan et al.  and Ding [et al. , phytohoet al. [et al. [In accordance with the methods of Ma et al. , the phy [et al. .Authentic standards of IAA, ABA, SA and JA were purchased from OlChemIm .et al. [CmDXS (encoding 1-deoxy-D-xylulose-5-phosphate synthase), CmDXR (encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase), CmPSY (encoding phytoene synthase), CmPDS (encoding phytoene desaturase), CmZISO (encoding ζ-carotene isomerase), CmZDS (encoding ζ-carotene desaturase), CmCRTISO (encoding carotenoids isomerase), CmLCYb1 (encoding lycopene β-cyclase gene1), CmLCYB2 (encoding lycopene β-cyclase gene2), CmLCYe (encoding lycopene ε-cyclase gene), CmBCH (encoding β-carotene hydroxylase), CmZEP (encoding zeaxanthin epoxidase) and CmNSY (encoding neoxanthin synthase) were examined via quantitative real-time PCR (qRT-PCR) analysis. The primer sequences of the 8 genes above and of an endogenous control gene (Actin) were designed in accordance with the methods of Liu et al. [CmDXS, CmDXR, CmPSY and CmZISO, which are listed in In accordance with the methods of Gao et al. , total Ru et al. , while tet al. [et al. [Ct) and the standard deviation (SD) for each sample were obtained in accordance with the methods of Gao et al. [Reverse transcription was performed as described by Liu et al. , while q [et al. . For eaco et al. .et al. [et al. [CmDXS alleles could not be amplified, possibly due to their low expression levels in all fruit tissues. The whole coding sequences of at least 10 clones for each carotenogenesis gene were sequenced from the cDNA mainly from the JS in each pummelo fruit. In particular, with respect to CmPSY, CmLCYb1, CmLCYb2, CmLYCe and CmNSY, nearly 50 clones of each gene in each pummelo fruit were obtained. However, due to the difficulties in obtaining new clones, only 15 clones of CmZEP were obtained for ‘FC’.To amplify the whole coding sequences of the eleven genes identified mainly from the JS of both pummelo fruits, primers were designed in accordance with the methods of Liu et al.  and Gao  [et al.  or via P® Max Super-Fidelity DNA Polymerase Kit  was used for allele amplification. The PCR products were electrophoresed and then screened on 1.5% agarose gels, after which they were recovered using an EZNA® Gel Extraction Kit  in accordance with the manufacturer’s instructions.A PhantaA vector was connected to the PCR amplification products using a Zero Background pTOPO-Blunt Simple Cloning Kit  in accordance with the manufacturer’s instructions. For the selection of positive clones, 100 ng/μL ampicillin and universal M13 primers were used, and the positive clones were sent to Tshingke Biological Technology Co.  for sequencing.http://multalin.toulouse.inra.fr/multalin/multalin.html). The amino acid sequences were deduced using Primer Premier 5, after which sequence alignment was carried out by ClustalW online  and GeneDoc software.The nucleotide sequences were aligned using MultAlin (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Their phylogenetic relationships were analyzed using MEGA 7.0.14. The maximum likelihood method was used to construct phylogenetic trees with 1000 replicate bootstrap supports, and values greater than 50% are shown at nodes.Homologous gene sequences of each carotenogenesis gene were obtained from the NCBI database .The developmental stages of ‘CH’ and ‘FC’  were pro‘CH’ and ‘FC’ pummelo fruits at five developmental stages were selected for analyses. The red-colored pigmentation in ‘CH’ started from the albedo at 75 DPA, followed by the SMs and JS at 100 DPA; this pigmentation ultimately peaked at 150 DPA in the SMs and JS. No red color was observed in any tissue of ‘FC’ at any stage . CombineIn the flavedo, lutein, violaxanthin, α-carotene and β-carotene were found in both pummelo fruits during development. Except at the first stage, the total carotenoid content in ‘FC’ was significantly higher than that in ‘CH’, which is in accordance with our previous report concerning fruits at the commercially mature stage . During In the albedo tissue, after the first stage (45 DPA), lycopene was the dominant carotenoid in ‘CH’, followed by phytoene and β-carotene. A dramatic accumulation of lycopene content in ‘CH’ was observed: it increased from trace levels at 45 DPA to the highest level at 125 DPA, after which it decreased beginning at 150 DPA. In contrast, a small amount of carotenoids were synthesized in ‘FC’. Only low levels of lutein at 75 DPA and α-carotene at both 75 DPA and 100 DPA were observed; all other carotenoids were at trace or nondetectable levels throughout the fruit development of ‘FC’ .In the SMs, the production of total carotenoids rapidly increased in ‘CH’ after 75 DPA. A large amount of lycopene accumulated, followed by β-carotene and phytoene. A dramatic accumulation of lycopene in ‘CH’ was observed from 45 DPA to 75 DPA. Afterward, the content increased by nearly 4-fold at 100 DPA and peaked at 125 DPA . However, lycopene and the total carotenoids both decreased dramatically at full maturity (150 DPA) . The contents of β-carotene and phytoene increased, exhibiting a trend similar to that of lycopene. In addition, the two carotenoids accounted for 6.2% of the total carotenoids after 45 DPA. However, low carotenoid biosynthesis was observed in ‘FC’, with only small amounts of violaxanthin and α-carotene detected at 75 DPA and 100 DPA, respectively .Likewise, in the JS of the ‘CH’ pummelo fruits, a large amount of lycopene was observed, followed by β-carotene and phytoene. A dramatic accumulation of lycopene in ‘CH’ was observed from 45 DPA to 75 DPA. Afterward, the level increased by nearly 3-fold at 100 DPA and peaked at 150 DPA, accounting for 89.5% of the total carotenoids. The contents of β-carotene and phytoene increased and exhibited a continuously increasing trend until the last development stage as did lycopene. On average, the two carotenoids accounted for 6.5% of the total carotenoids after 45 DPA, which is quite similar to the results in the SMs. As in the SMs, small amounts of carotenoids accumulated in the JS of ‘FC’; the highest levels were detected for violaxanthin at 125 DPA. Moreover, less contents of lutein, α-carotene and β-carotene were observed before 100 DPA, after which point the contents decreased to nondetectable or trace levels. In addition, undetectable or trace levels of phytoene and lycopene were observed at all stages of ‘FC’ .The contents of limonin and nomilin in the flavedo of ‘CH’ fruits were significantly lower than those in the ‘FC’ fruits from 100 DPA to 150 DPA. However, in the albedo, the contents of both limonoid aglycones in ‘CH’ were significantly lower than those in ‘FC’ at the two early stages, after which the contents dramatically increased at the last stage. However, both limonin and nomilin in the albedo of ‘FC’ continued to decrease to the lowest observed levels at 150 DPA. Interestingly, the highest limonin and nomilin production was observed in the SMs of both pummelo fruits; the limonin and nomilin contents in the SMs were 25.0-, 7.5-, 9.5-fold and 29.5-, 12.5-, 8.5-fold greater than those in the flavedo, albedo and JS of ‘CH’, respectively, and 5.5-, 3.0-, 7.0-fold and 2.0-, 2.5-, 6.5-fold greater than those in the flavedo, albedo and JS of ‘FC’, respectively. Notably, in SMs and JS, the limonin content was significantly higher in ‘CH’ than in ‘FC’ at both 100 DPA and 150 DPA. Additionally, at 150 DPA, the nomilin content in ‘CH’ was markedly higher than that in ‘FC’. In contrast, both the limonin and nomilin contents were significantly lower in ‘CH’ than in ‘FC’ at 125 DPA .Owing to the large differences in the contents of carotenoids and limonoid aglycones in the JS at the last developmental stage, we measured the concentrations of phytohormones in the JS to determine whether such differences were caused by the regulation of phytohormones. ABA was lower in ‘CH’ than in ‘FC’, which was consistent with the results for Star Ruby (red-fleshed) and Marsh (white-fleshed) grapefruits , while ICmDXS, CmDXR and CmZISO were significantly higher in ‘FC’ than in ‘CH’ before 125 DPA, while the expression levels of CmPSY, lycopene β-cyclase gene 1 (CmLCYb1), CmBCH and CmZEP were constantly higher in ‘CH’ throughout all developmental stages or during most developmental stages. At 150 DPA, the expression levels of only CmZISO and CmLCYe were downregulated in ‘CH’, and the expression level of only CmZDS was the same in both ‘CH’ and ‘FC’, whereas the expression levels of the other 8 genes were significantly upregulated in ‘CH’. Throughout all the stages, the largest fold change in gene expression was observed for CmBCH, followed by CmPSY, CmPDS and CmZDS, while the expression of CmLCYb1 remained relatively stable . However, compared with those in ‘CH’, the expression levels of CmPSY, CmLCYb1 and CmLCYe in ‘FC’ were higher or the same at three stages. Notably, a generally increasing trend was observed in the expression levels of CmPDS, CmZDS, CmZISO, CmLCYe and CmZEP in both pummelo fruits, although larger fold changes occurred in ‘FC’. Compared with that in ‘FC’, the expression level of CmLCYb1 in ‘CH’ was quite stable, but it drastically increased after 125 DPA , CmLCYe, CmBCH and CmZEP were detected in ‘CH’ than in ‘FC’ (if not at the same level). The expression levels of CmDXS, CmDXR, CmPSY, CmPDS, CmZISO and CmCRTISO were higher in ‘CH’ than in ‘FC’ in at least three stages. Notably, throughout all stages, the expression levels of CmLCYe, CmBCH and CmZEP were significantly higher in ‘CH’ than in ‘FC’. Interestingly, with the exceptions of the upregulation of CmDXR, CmPSY and CmLCYb1, the expression levels of the other nine genes were downregulated at 150 DPA in ‘FC’. Compared with that of the other genes, the expression of CmLCYb1 in ‘CH’ was relatively steady. The expression levels of CmLCYb2 and CmNSY were lower in ‘FC’ than in ‘CH’ at four of the five stages, while in ‘CH’ alone, the levels remained comparatively stable but were elevated at 125 DPA or 150 DPA ; the same results were obtained for CmDXS, CmDXR, CmZDS, CmZISO and CmLCYb1. Upregulated expression levels of CmPSY, CmPDS and CmCRTISO were observed at the last stage in ‘FC’. However, relatively steady expression levels of CmDXS, CmLCYb1 and CmLCYe were observed in ‘CH’ throughout the fruit developmental stages  and one different nonsynonymous site , are present in ‘CH’ and ‘FC’, respectively  were investigated  in the albedo, SMs and JS of ‘CH’, the sequences of 11 carotenogenesis genes were therefore checked for possible mutations. ach gene . The difach gene –S11 Figsectively . Howeverstigated ; S2 Fig.o fruits , whereasach gene –S8 Figs.https://blast.ncbi.nlm.nih.gov/Blast.cgi); we named them the same as the closest previously reported genes in phylogenetic trees. The CmDXR and CmZISO genes were first cloned in citrus regardless of genome sequencing  were significantly higher in the JS of ‘CH’ than in those of ‘FC’, implying that the biosynthesis of the initial carotenoid metabolites in the carotenogenesis pathway was largely induced in ‘CH’. With respect to the rate-limiting gene CmPSY in carotenogenesis, we observed differences in two sites between CmPSYa and CmPSYb  were identified in the Arabidopsis PSY gene; these variants resulted in different translation rates for regulating AtPSY enzyme activity [Based on our results of carotenogenesis, it might not be appropriate to simply attribute the accumulation of lycopene, phytoene or β-carotene in ‘CH’ to the upregulated expression of upstream genes and the downregulated expression of downstream genes, as Liu et al.  reportedd CmPSYb . Consided CmPSYb . Both Cms colors , suggestactivity .LCYb1 [LCYb2 [LCYe [NSY [β-LCY2a was shown to have higher expression levels in navel oranges and white-fleshed marsh grapefruits, whereas nonfunctional β-LCY2b was expressed at much higher levels in red-fleshed Star Ruby fruits [CmLCYb1 and CmLCYb2 in the JS of ‘CH’ might explain the higher β-carotene content throughout the fruit development stages.The LCYb1 , 44, 50,1 [LCYb2 , LCYe [5CYe [NSY  genes fuy fruits , 31. Siny fruits , 21, 31,y fruits , 52. In CmBCH and CmZEP was constantly significantly higher in the JS of ‘CH’ than in those of ‘FC’, which is similar to results reported in Star Ruby grapefruit [et al. reported that maize crtRB1 alleles that encode an enzyme that exhibits BCH function are associated with reduced transcript expression and higher β-carotene concentrations [CmBCH and the β-carotene accumulation were higher in the albedo, SMs and JS of ‘CH’ than in those of ‘FC’ at most development stages  gene was accompanied by increased contents of lycopene and β-carotene in tomato fruits [Actinidia fruit development [Notably, the expression of the downstream genes apefruit  but oppoapefruit . Yan et trations . Howeverges Figs  and 5. Icontents . Furthero fruits . The carelopment .LCYb1, PSY, PDS and CCD1 and upregulating their expression [Citrus reticulata), CrMYB68 downregulated the expression of two carotenoid pathway genes, CrBCH and CrNCED5, which resulted in total flavedo carotenoid contents that were lower than those in the wild type [Mimulus lewisii R2R3 MYB transcription factor mutant rcp1 contains low amounts of carotenoids exhibits low expression levels of almost all carotenoid biosynthesis pathway genes [et al. reported that a stay-green protein, SlSGR1, dramatically reduced lycopene biosynthesis via the direct inhibition of SlPSY1 activity [SlPSY1 [PSY promoter, which suppresses its expression and reduces carotenoid accumulation in Arabidopsis [Transcription factors also play important roles in carotenoid accumulation. Overexpression of CsMADS6 resulted in higher lycopene contents and higher β-carotene contents in the sepals and fruits of tomato, respectively, by binding the promoters of pression . In a stild type . The Mimay genes . Luo et activity . However [SlPSY1 . Phytochbidopsis . OverallCmZDS, CmBCH and CmZEP, in combination might be directly responsible for the largely different carotenoid profiles between these two pummelo fruits. The lycopene accumulation in specific ‘CH’ fruit tissues might strongly be caused by the different alleles of the CmDXR, CmPSY, CmCRTISO, CmLCYb1, CmLCYb2, CmLCYe, and CmZEP genes. The different numbers of ‘AAT’ microsatellite repeat units in PSY transcripts were not related to lycopene accumulation in red-flesh pummelo fruits.The development stage in which dramatic carotenoid production in the albedo, SMs and JS of ‘CH’ occurred was from 45 DPA to 75 DPA, and the total carotenoids increased by more than 39-, 40- and 14-fold, respectively. Different expression levels of carotenogenesis genes, especially S1 FigA: CmDXRa and CmDXRb were detected in ‘CH’ and ‘FC’, respectively. Two amino acid differences in sequences were observed between CmDXRa and CmDXRb. B: Phylogenetic analysis of CmDXR.(DOC)Click here for additional data file.S2 FigA: Six CmPSY alleles were detected in ten pummelo fruits. The orange framework means ‘N’ repeats. B: Phylogenetic analysis of CmPSY.(DOC)Click here for additional data file.S3 FigA: CmPDS was identical between ‘CH’ and ‘FC’. B: Phylogenetic analysis of CmPDS.(DOC)Click here for additional data file.S4 FigA: CmZISO was identical between ‘CH’ and ‘FC’. B: Phylogenetic analysis of CmZISO.(DOC)Click here for additional data file.S5 FigA: CmCRTISOa and CmCRTISOb were detected in ‘CH’ and ‘FC’, respectively. The red frameworks show different sites between CmCRTISOa and CmCRTISOb. Two amino acid differences in sequences were observed between ‘CH’ and ‘FC’. B: Phylogenetic analysis of CmCRTISO.(DOC)Click here for additional data file.S6 FigA: CmLCYb1a and CmLCYb1b were detected in ‘CH’ and ‘FC’, respectively. Note that single amino acid differences in sequences were observed between CmLCYb1a and CmLCYb1b. B: Phylogenetic analysis of CmLCYb1.(DOC)Click here for additional data file.S7 FigA: CmLCYb2a and CmLCYb2b were detected mainly in ‘CH’ and ‘FC’, respectively. Note that a single amino acid difference in sequences was observed between CmLCYb2a and CmLCYb2b. B: Phylogenetic analysis of CmLCYb2.(DOC)Click here for additional data file.S8 FigA: CmLCYea and CmLCYeb were detected in ‘CH’ and ‘FC’, respectively. A single amino acid difference in sequences was observed between CmLCYea and CmLCYeb. B: Phylogenetic analysis of CmLCYe.(DOC)Click here for additional data file.S9 FigA: CmBCH was identical between ‘CH’ and ‘FC’. B: Phylogenetic analysis of CmBCH.(DOC)Click here for additional data file.S10 FigA: CmZEPa and CmZEPb were detected in ‘CH’ and ‘FC’, respectively. A single amino acid difference in sequences was observed between CmZEPa and CmZEPb. B: Phylogenetic analysis of CmZEP.(DOC)Click here for additional data file.S11 FigA: CmNSY was identical between ‘CH’ and ‘FC’. B: Phylogenetic analysis of CmNSY.(DOC)Click here for additional data file.S1 TableCitrus sinensis  annotation project (http://citrus.hzau.edu.cn/orange/).The accession number was obtained from the (DOC)Click here for additional data file.S2 Tablehttp://www.ncbi.nlm.nih.gov/). The superscript ‘b’ genome accession means that the accession number was obtained from the Citrus sinensis  annotation project (http://citrus.hzau.edu.cn/orange/).The superscript ‘a’ gene accession means that the accession number was obtained from the NCBI database ((DOC)Click here for additional data file.


G. sulphuraria, which is 14°C below its optimal growth temperature of 42°C. Cold stress was applied for more than 100 generations to identify components that are critical for conferring thermal adaptation. After cold exposure for more than 100 generations, the cold-adapted samples grew ∼30% faster than the starting population. Whole-genome sequencing revealed 757 variants located on 429 genes (6.1% of the transcriptome) encoding molecular functions involved in cell cycle regulation, gene regulation, signaling, morphogenesis, microtubule nucleation, and transmembrane transport. CpG islands located in the intergenic region accumulated a significant number of variants, which is likely a sign of epigenetic remodeling. We present 20 candidate genes and three putative cis-regulatory elements with various functions most affected by temperature. Our work shows that natural selection toward temperature tolerance is a complex systems biology problem that involves gradual reprogramming of an intricate gene network and deeply nested regulators.Rapid fluctuation of environmental conditions can impose severe stress upon living organisms. Surviving such episodes of stress requires a rapid acclimation response, e.g., by transcriptional and post-transcriptional mechanisms. Persistent change of the environmental context, however, requires longer-term adaptation at the genetic level. Fast-growing unicellular aquatic eukaryotes enable analysis of adaptive responses at the genetic level in a laboratory setting. In this study, we applied continuous cold stress (28°C) to the thermoacidophile red alga  In thisG. sulphuraria strain RT22 adapted to growth at 42°C was split into two batches, which were grown separately at 42°C (control condition) and 28°C (temperature stress) for a period spanning 8 months. Bacteria were cultured on agar plates under non-photosynthetic conditions, with glucose (50 mM) as the sole carbon source. To select for fast-growing populations, the five largest colonies of each generation were picked. The samples were propagated across generations by iteratively picking the five biggest colonies from each plate and transferring them to a new plate. The picked colonies were diluted in 1 ml Allen Medium containing 25 mmol glucose. The OD750 of the cell suspensions was measured at each re-plating step using a spectrophotometer. Approximately 1,000 cells were streaked on new plates to start the new generation. The remaining cell material was stored at -80°C until DNA extraction. This process was reiterated whenever new colonies with a diameter of 3–5 mm became visible. During the 240 days of this experiment, a total of 181 generations of G. sulphuraria RT22 were obtained for the culture grown at 42°C, whereas 102 generations were obtained for G. sulphuraria RT22 grown at 28°C.A starting culture of DNA from each sample was extracted using the Genomic-tip 20/G column , following the steps of the yeast DNA extraction protocol provided by the manufacturer. DNA size and quality were assessed via gel electrophoresis and Nanodrop spectrophotometry . TruSeq DNA PCR-Free libraries (insert size = 350 bp) were generated. The samples were quantified using the KAPA library quantification kit, quality controlled using a 2100 Bioanalyzer , and sequenced on an Illumina  HiSeq 3000 in paired-end mode (1 × 150 bp) at the Genomics and Transcriptomics Laboratory of the Biologisch-Medizinisches Forschungszentrum in Düsseldorf, Germany. The raw sequence reads are retrievable from the NCBI’s Small Read Archive (SRA) database (Project ID: PRJNA513153).G. sulphuraria RT22  using the BWA aligner . The bootstrapping process was iterated three times until convergence was reached . Finally, the recalibration model was built upon the final samples to capture the maximum number of variable sites. Variants were called using the haplotype caller in discovery mode with -ploidy set to 1  and –mbq set to 20  and annotated using snpEff v4.3i (Single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) were called separately on the dataset using the GATK software version 3.6-0-g89b7209 . The ana aligner  with theff v4.3i . The cal0 and T1 in the samples taken at 28°C, which was lost in T3 and regained after T5. The binary sequence represents the ten samples, six “cold” and four “warm,” according to their condition (“cold | warm”) and time point of sampling . Hence, the first six digits denote samples taken at 28°C, the latter four digits those taken at 42°C; “000000| 0101” would represent a mutation in the T2 sample taken at 42°C that was lost in T3 and regained in T4, and “011010| 0101” would represent a variant that does neither with respect to the sampling succession (repeated gain and loss) nor the growth condition of the samples (mutation occurs at both temperatures). By contrast, variants that were gained and fixed in the subsequent samples of a certain growth condition were considered as “non-random variants” that may reflect significant evolutionary patterns. Thus, “111111| 0000” would indicate that a mutation between T0 and T1 in the samples taken at 28°C was fixed over the measured period. Similarly, “000000| 0111” would indicate a mutation between T2 and T3 in the samples taken at 42°C that was fixed throughout the generations. As such, it was possible to determine all possible pattern combinations for non-random evolutionary patterns. The binary sequence “111111| 1111” represented the case where all ten samples contained a different haplotype when compared to the reference genome. In this specific case, systematic discrepancies between the reference genome and the DNA-Seq reads are the cause of this pattern. Variants following the “111111| 1111” pattern were removed from the dataset.A main goal of this analysis was implementation of a method that enabled discrimination between random variants and variants that may be connected to temperature stress (non-random variants). The following logic was implemented: All variants were transformed to binary code with regards to their haplotype toward the reference genome. When the haplotype was identical to the reference genome, “0” was assigned. Variant haplotypes were assigned “1.” Random variants were gained and lost without respect to the sampling succession along the timeline and the different temperature conditions. Consequently, a “fuzzy” pattern of, e.g., “110011| 0000,” would indicate a mutation between TPRJNA513153 and BioSamples SAMN10697271 - SAMN10697280.The DNA sequencing results are described in n = 6) and at 42°C (n = 10). Both datasets failed the Shapiro–Wilk normality test (p > 0.05) and showed a visible trend over time. The difference in growth between the populations was tested using the Wilcoxon rank sum test. Further, timepoints along a timeline constitute a dependent sampling approach by which the growth performance of an earlier timepoint is likely to influence the growth performance of a later timepoint.Various statistical methods were applied for the different analyses performed in this project. Culture growth was measured for at 28°C  (p > 0.05). By contrast, samples grown at 28°C gradually adapted to the colder environment and significantly  decreased their doubling time by ∼30% during the measured period.Samples grown at 42°C were re-plated 10 times during the 7 months of the experiment due to faster colony growth, whereas cultures growing at 28°C were re-plated only six times . Culture 0.0002) . The gro 0.0002) . AlthougA total of 470,680,304 paired-end DNA-Seq reads were generated on an Illumina HiSeq 3000 sequencer. Of these, 462,869,014 were aligned to the genome (98.30%) using BWA . The aveG. sulphuraria RT22), of which 1116 were found at least twice in the variant dataset. Of those, 234 of the GOs were significantly enriched .The vast majority of the 757 genic variants was not fixed over time and did not follow consistent evolutionary patterns . HoweverTo contextualize the function in broader categories, we manually sorted all significantly enriched GOs into the following ten categories: “Cell Cycle,” “Cytoskeleton,” “Gene Regulation,” “Membrane,” “Metabolism,” “Photosynthesis,” “Stress/Signaling,” “Transport,” “Other,” and “NA” . GO termp = 0.0024; “re-entry into mitotic cell cycle,” GO:0000320, p = 0.0405), DNA replication , prophase , metaphase , anaphase , and telophase . Mutations also accumulated in genes controlling cell cycle checkpoints of mitosis .Functions related to the cell cycle accounted for 79/234 (33.8%) of the enriched GOs. Mitosis was affected at every stage: initiation , cell polarity , and subcellular compartmentalization and localization . Interestingly, some transcriptional regulators of cell growth seem to be conserved across the eukaryotic kingdom. GOs such as “branching involved in open tracheal system development”  and “eye photoreceptor cell development”  were also found, indicating high amino acid sequence similarity within this category. Further, temperature stress altered genes with functions involved in cell death .Genes with functions involved in cell differentiation and maturation of p = 0.0118). Also, we found a significant proportion of mutations affecting genes linked to the epigenetic control of gene expression, which can occur through methylation of DNA , as well as modulation of chromatin density and histone interactions that change the accessibility of whole genomic regions to transcription  (p = 0.0020), mRNA processing , post-transcriptional silencing  as well as alteration of ribosome structure components  and rRNA methylation components . In this regard, GO terms linked to posttranslational protein modification were also enriched .Maintenance of steady and balanced reaction rates across cellular systems is essential for cell survival and poses a major challenge when an organism is confronted with changes in temperature . In this 0.0040) . Furtherp = 0.0039) and disassembly are mostly driven by tubulin concentrations at the beginning and the end of microtubules once a critical microtubule size is reached (p = 0.0031). The reaction equilibrium between tubulin polymerization and monomerization is temperature-dependent and requires accurate regulation . Shifting temperatures from 37°C to 25°C leads to massive microtubular dissociation in homoeothermic species  as well as membrane-associated proteins , transporters , and transport functions . Furthermore, temperature imposes significant restrictions to vesicles, which play a central role in molecule trafficking between organelles and in endocytosis. Vesicle formation in particular appears to be affected by temperature .Another major component that is also influenced by temperature is cell integrity with regards to membrane fluidity  and transport . Cell membranes are selectively permeable and vital for compartmentation and electric potential maintenance. In this context, membrane . Membranp = 0.0051; “hyperosmotic response,” GO:0006972, p = 0.0039; and “ER overload response,” GO:0006983, p = 0. 0040). Here, we found a total of 18/234 GOs (7.7%) derived from genes involved in cell signaling upon which temperature changes appeared to exhibit significant evolutionary pressure driving the accumulation of variants. A broad array of receptors  performs signal transduction through phosphorylation of other proteins and molecules. The signal acceptors, in turn, influence second messengers and further signaling components that affect gene regulation and protein interactions. GO annotations indicate involvement of temperature in genes coding for receptors  as well as in genes coding for the signal acceptors .Cell signaling comprises the transformation of information, such as environmental stress, to chemical signals that are propagated and amplified through the system where they contribute to the regulation of various processes  in both anabolism , and catabolism . In spite of pronounced changes in gene expression of metabolic enzymes during short-term cold stress in G. sulphuraria 074W , they are able to actively adjust their metabolic rates by regulating the amount of available enzyme (“Gene Expression”). Passively, mutations can alter enzyme structure, thereby adjusting the affinity of enzymes toward ligands. Variants affecting the genetic code of genes attributed to this category influence a broad variety of metabolic pathways , likely because the experiment was performed under heterotrophic conditions in continuous darkness.The majority of photosynthetic light reactions are catalyzed by enzymes located in the photosynthetic thylakoid membranes. Hence, photosynthesis is based upon temperature-dependent proteins located in temperature-dependent membranes . AbnormaG. sulphuraria RT22, we selected candidate genes for closer analysis using two different approaches. First, we assumed that high mutation rates in a specific gene reflect increased selective force upon its function and regulation. To identify potential targets of temperature-dependent microevolution, we searched for “variant hotspots,” here defined as the 99th percentile of genes most affected by variants. We computed variant number-dependent Z-scores for each gene and extracted genes with a Z-Score > 2.575. This procedure led to identification of seven genes, so-called “variant hotspots,” containing at least seven independent variants per gene. Next, we extracted 41 variants that followed non-random evolutionary patterns, here defined as the gain of a variant and its fixation in the subsequent samples that was exclusive to either 28°C or 42°C (To further investigate the temperature adaptation of  or 42°C . Eighteecis-trans isomerase (Gsulp_RT22_64_G1844). Other candidate genes were involved in transcription and translation, e.g., a NAB3/HDMI transcription termination factor (Gsulp_RT22_83_G3136), or in ribosomal biogenesis  and required cochaperones . Three candidate genes were solute transporters . Most interestingly, two genes connected to temperature stress were also affected by mutations. An error-prone iota DNA-directed DNA polymerase (01_Gsulp_RT22_79_G2795), which promotes adaptive point mutation as part of the coordinated cellular response to environmental stress, was affected at 28°C , an armadillo/beta-catenin repeat family protein (Gsulp_RT22_107_G5273), the GTPase-activating ADP-ribosylation factor ArfGAP2/3 (Gsulp_RT22_82_G3036), and a peptidylprolyl  at 28°C , as well at 28°C .Galdieria and other microorganisms by providing adaptive advantages (G. sulphuraria RT22 gained variants (p < 0.05). Of the HGT candidates, only Gsulp_RT22_67_G2013, a bacterial/archaeal APC family amino acid permease potentially involved in the saprophytic lifestyle of G. sulphuraria, accumulated a significant number of mutations (12 variants).Horizontal gene transfer has facilitated the niche adaptation of vantages . Five ofvariants . We testG. sulphuraria 074W, where gene expression was measured at 28°C and 42°C ; 342 were orthologs to a variant-containing gene, representing 79.7% of all genes containing variants in G. sulphuraria RT22. The dataset is representative . Based on this result, 36.3% of the variant-containing genes were differentially expressed. By contrast, 40.1% of the genes unaffected by variants were differentially expressed (p < 0.05). Hence, genes affected by variance during this microevolution experiment did not react more, or less, pronouncedly to fluctuating temperature.We tested if the 6.1% of genes that gained variants were also differentially expressed during a temperature-sensitive RNA-Seq experiment in and 42°C . Of the xpressed . The difcis-regulatory elements, such as promoters and enhancers, that constitute the binding sites of transcription factors and, thus, affect activation and transcriptional rate of genes. Promoters are required for transcriptional initiation but their presence alone results in minimal levels of downstream sequence transcription. Enhancers, which can be located either upstream, downstream, or distant from the genes they regulate, are the main drivers of gene transcription intensity and are often thought to be the critical factors of cis-regulatory divergence  to k-mer length 10  to identify intergenic sequence patterns prone to variant accumulation   , RE1 (Reg motif) , and 3-Ag motif) .G. sulphuraria RT22 to a temperature-dependent microevolution experiment for 7 months. One culture was grown at 28°C, representing cold stress, and a control culture was grown at 42°C. This experiment aimed to uncover the genetic acclimation response to persistent stress, rather than the short-term acclimation response of G. sulphuraria to cold stress , may be able to thrive at high temperatures, but not for such a prolonged period.In this study, we subjected two populations of d stress . We perfht occur . A similperiment . While f-6/base/generation for samples grown at 28°C and 1.10 × 10-6/base/generation for samples grown at 42°C, which we interpret as an indication of greater evolutionary stress at 28°C. Hence, suboptimal growth temperatures constitute a significant stress condition and promote the accumulation of mutations. In comparison, mutation rates in other microevolution experiments were 1.53 × 10-8/base/generation–6.67 × 10-11/base/generation for the unicellular green freshwater alga Chlamydomonas reinhardtii and 5.9 × 10-9/base/generation in the green plant Arabidopsis thaliana (C. reinhardtii might result from the selective strategy employed in this experiment (only the five biggest colonies were selected to start the next generations). Although the cold-stressed samples accumulated twice as many mutations per generation in comparison to the control condition, the number of gained variants over the same period was higher in the 42°C cultures due to faster growth rates.We identified 1243 filtered variants (966 SNPs + 277 InDels), of which 757 (63.5%) were located on the coding sequence of 429 genes and 486 (36.5%) in the intergenic region. The mutation rate was estimated to be 2.17 × 10thaliana . The 100G. sulphuraria RT22 was analyzed using GO enrichment analysis. More than 75% of the 234 significantly enriched GOs affected genes functions involved in the processes of cell division, cell structure, gene regulation, and signaling. In short, the cellular life cycle appears to be targeted by variation at any stage starting with mitosis, morphogenesis, and finishing with programmed cell death. By contrast, genes directly affecting metabolic processes were less affected by mutation and made up only 10% of the enriched GOs. These observations were also confirmed through the functional annotation of the seven genes most affected by variants (“variant hotspots”) as well as the 13 genes carrying non-synonymous variants with non-random evolutionary patterns.The impact of temperature-driven microevolution on the cellular functions of cis-regulatory elements are a common source of evolutionary change . Our results also emphasize the difference between short-term acclimation and long-term adaptation with regard to temperature stress, highlighting the multiple facets of adaptation that can be measured using different technologies. The short-term stress response of G. sulphuraria and the long-term stress response in C. merolae were quantified using transcriptomic and proteomic approaches, respectively (Galdieria, or the impact of non-coding RNA elements, both of which may provide additional layers for adaptive evolution (We show here that the significant growth enhancement of samples grown at 28°C over more than 100 generations was driven mainly by mutations in genes involved in the cell cycle, gene regulation, and signal transfer, as well as mutations that occurred in the intergenic regions, possibly changing the epigenetic methylation pattern and altering the binding specificity to ectively . At the ectively  apparatuvolution .The datasets generated for this study can be found in NCBI Small Read Archive, PRJNA513153.AR and AW conceived the study and developed the experimental design, edited and drafted the final version of the manuscript. AR did all the experimental work, data analysis, and drafted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To assess the association between exposure to physical workload throughout working life and risk of mobility limitations in midlife in a population-based Danish cohort.The study was cross-sectional with a retrospective exposure assessment, and data were from a questionnaire used in the Copenhagen Aging and Biobank. Cumulative physical workload was estimated by combining information about the participants’ employments and data from a job exposure matrix. Daily amount of lifting was standardised in ton-years (lifting 1000 kg/day/year) and grouped in 5 exposure groups (no/minor (1–2 ton-years)/low (3–10 ton-years)/moderate (11–20 ton-years)/high exposure (> 20 ton-years)). The outcome was self-reports of mobility limitations  in midlife. The association between exposure and outcome was analysed using logistic regression models.We included 4996 men and 2247 women, mean age 56 years. 21% of men and 10% of women were in the highest exposure-group (> 20 ton-years). Higher cumulative exposure was associated with higher odds for mobility limitations. Exposure to more than 20 ton-years compared to no exposure increased the odds for limitations in walking, age-adjusted odds ratio (OR) 3.2 (95% CI: 2.4–4.3) for men, 2.3 (1.4–3.8) for women. Corresponding results for running: 2.5 (2.2–3.0) for men, 1.6 (1.2–2.2) for women, and for limitations in climbing stairs: 4.2 (3.3–5.2) for men, 1.7 (1.2–2.4) for women. Results were attenuated when confounders were added.Exposure to physical workload throughout working life is associated with higher odds for mobility limitations in midlife. In this way, workload was standardized as ton-years (lifting 1000 kg each working day in 1 year).From our previous work  without resting?” with the response categories: “Easily”, “With little difficulty”, “With much difficulty”, “Not at all”. , The National Institute of Public Health, University of Southern Denmark). We categorised the answers in: (1) no limitations (“Easily”) or (2) limitations . The second and third questions were: “Does your health now limit you in the following two activities: (a) Running 100 meters, (b) Climbing stairs to the 2nd floor?” with response categories: “Yes, limits a lot”, “Yes, limits a little”, “No, does not limit at all”. We dichotomized the answers to: (1) no limitations  and (2) limitations (“limits a little”/“a lot”). These questions are modified from SF-36 Health Survey . The CAMB questionnaire provided information about the number of chronic diseases among participants, and these were grouped in three categories: “no”, “one”, and “two or more” chronic diseases. The diseases counted were asthma, diabetes, hypertension, angina pectoris, stroke, myocardial infarction, bronchitis, emphysema, rheumatoid arthritis, osteoarthritis, cancer, anxiety, depression/other psychiatric diseases and back pain. Unfortunately, we had no information about severity of diseases. Pain in nine regions of the body was summarized ; the minimum score was 9 (no pain in any of the regions) and the maximum was 81 . In the questionnaire, we have information about lifestyle factors which can be confounders or mediators in the association between physical workload and mobility limitation. High physical workload can lead to less energy or motivation for leisure-time physical activity (LTPA) which is known to influence mobility. However, LTPA could also be a confounder influencing the capacity of physical work and mobility. LTPA was categorised as “active” (≥ 4 h a week), “moderately active” (< 4 h a week) and “sedentary” (0 h). Smoking and alcohol consumption was seen as confounders. Smoking history was calculated as pack-years , and current alcohol consumption was categorized in units of alcohol per week.n = 4996 in model 1 and n = 4514 in model 3 (men) and n = 2247 in model 1 and n = 1993 in model 3 (women)]. All analyses were performed in PROC LOGISTIC (SAS 9.4). The level of statistical significance was 5%.The effect of physical workload was assumed to be gender-specific, and all analyses were performed separately for each sex, as suggested by Silverstein et al.  using gamma coefficients.Mean age for men was 57.6 years (range 53–59 years), and for women 55.0 (range 52–63 years). Mean duration of working life was 30 years for men and 26 years for women based on summation of the five longest held employments listed in the questionnaire. Descriptive data are presented in Table In unadjusted analyses, cumulative physical workload was associated with risk of limitations in mobility Table .In all models, men had higher odds of mobility limitations with higher level of physical workload. Among women, more exposure-years were associated with higher odds of mobility limitations except from women with high exposure. Women with high exposure had lower odds of mobility limitations compared to women with low or moderate exposure. Introduction of confounders and mediators in the analysis attenuated the associations for both men and women (Models 2 and 3).Men had higher odds of mobility limitations with higher level of physical workload and a exposure–response relationship was observed Fig. . IntroduMen had a strong exposure–response relationship between level of physical workload and odds of limitations climbing stairs in unadjusted analyses. Including age increased the associations slightly. As seen in the two previous analyses, introduction of confounders and mediators attenuated the associations, but the associations were still statistically significant, when lifestyle factors were included in model 3, except in the lowest exposure-group. Among women, the results were comparable to the other outcomes, and there was no statistically significant association between exposure and limitations in climbing stairs after adjustments in Model 2 and 3.n = 4996): 0.399 (p < 0.0001) and women (n = 2247): 0.395 (p < 0.0001). Walking and climbing stairs: men (n = 4996): 0.513 (p < 0.0001) and women (n = 2247): 0.506 (p < 0.0001). Running and climbing stairs: men (n = 4996): 0.637 (p < 0.0001) and women (n = 2247): 0.681 (p < 0.0001). The statistical association between self-reports and objective measures of physical performance was high, calculated between performance in chair-rise test and mobility limitations (gamma coefficient 0.58 for men and 0.63 for women).The outcome measures are associated, and Pearson correlation coefficients have been calculated: walking and running: men  has been included in the analysis. Including participants from CPC increased the sample size and especially the number of women compared to our previous studies in the CAMB cohort, though tests of the effect of cohort as a variable in the analyses had no effect on the associations (results not shown). A limitation in this study is the inclusion of all participants at baseline, also participants out of work. However, participants out of work in midlife could have had up to 20 or 25 years of work despite being retired, and therefore they were included in the analyses. To study this problem, we repeated the analyses with participants employed at baseline, and the associations were attenuated. However, the trends were similar indication higher odds for mobility limitations among men with increasing physical workload. The odds for mobility limitations among women were further attenuated.The study design is also a limitation. To study the genuine exposure–response pattern, a cohort study following participants over time should be performed including objective measures of outcome.In conclusion, exposure to physical workload throughout working life is associated with increased odds of mobility limitations in both men and women. However, the associations are attenuated when possible confounders are added in the statistical models.


A number of studies have demonstrated an association of neuropathic pain and chronic low back pain (CLBP), but the outcome difference in each medical management is poorly understood. This study is aimed to investigate treatment patterns of neuropathic pain in CLBP patients and to explore patient-reported outcomes (PROs) including quality of life (QoL) and functional disability by treatment patterns.Data were extracted from the neuropathic low back pain (NLBP) outcomes research. It was a multicenter and cross-sectional study in which 1200 patients were enrolled at 27 general hospitals, from 2014 to 2015. Of total, 478 patients classified as neuropathic pain were used for this subgroup analysis. The patients were divided into 2 groups according to treatment patterns (with vs. without the targeted therapy [TT] of neuropathic pain). Demographic and clinical features were collected by chart reviews and PROs were measured by patient's survey. QoL was assessed by EuroQoL 5-dimension (EQ-5D) questionnaire. Functional disability was measured by the Quebec Back Pain Disability Scale (QBPDS). Multiple linear regression analyses were conducted to compare the PROs between TT group and non-targeted therapy (nTT) group.P < 0.01) and EQ-VAS  than the nTT group. The TT group's QBPDS score was lower than the nTT group, although its statistical significance still has not been reached .Among the NLBP patients , EQ-5D index, EuroQoL-Visual Analog Scale (EQ-VAS), and QBPDS Scores (mean ± standard deviation) were 0.40 ± 0.28, 54.98 ± 19.98, and 46.03 ± 21.24, respectively. Only 142 (29.7%) patients had pharmacological TT of neuropathic pain. Univariate analyses revealed no significant mean differences between TT group and nTT group in the EQ-5D index (0.41 ± 0.27 and 0.39 ± 0.28), EQ-VAS (56.43 ± 18.17 and 54.37 ± 20.69), and QBPDS (45.31 ± 21.32 and 46.31 ± 21.24). After adjustment with covariates, TT group had higher scores of EQ-5D index (β = 0.07; We found that considerable proportion of the NLBP patients remains untreated or undertreated. Although TT group had significantly better QoL than nTT group, only 29.7% of NLBP patients had pharmacological TT. Therefore, clinicians should consider using TT for better QoL of neuropathic pain patients. Thus, NLBP may require to be considered as an important clinical problem.Low back pain (LBP) is one of the most common musculoskeletal disorders, resulting in significant personal, social, and economic burden. Mechanical conditions of the spine, including disk disease, disk herniation, spondylosis, spinal stenosis, and fractures, account for up to 98% of LBP cases. Neuropathic pain in chronic LBP was reported to be highly prevalent and neuropathic pain affects the social and psychological well-being of LBP patients. Hiyama et al reported that NLBP patients had significantly higher visual analog scale (VAS) scores and lower the scores of short form 36 (SF-36) and Japanese Orthopedic Association Back Pain Evaluation Questionnaire than LBP patients with nociceptive pain. This result suggests that NLBP affects the physical, social, and psychological well-being compared to nociceptive LBP patients. Hence, it is significant to identify the involvement of neuropathic pain in LBP patients and to effectively manage NLBP.Neuropathic pain profoundly decreased the quality of life (QoL).However, there was no multicenter cohort study not only for the prevalence of NLBP patients but also for the treatment pattern in Korean NLBP patients. Although treatment pattern and outcome could be different in each different country and health care system, it would be valuable to understand relationship between the pattern and the outcome of NLBP treatment. Serotonin noradrenaline reuptake inhibitors and topical lidocaine were recommended as second-line therapies. Third-line therapies included tramadol and controlled release opioid analgesics. Recommended forth-line treatments were cannabinoids, methadone, and anticonvulsants. The special interest group of the international association for the study of neuropathic pain recently suggested guidelines of pharmacological management of neuropathic pain. According to their guidelines, tricyclic antidepressants (TCAs), gabapentin, pregabalin, and topical lidocaine were recommended as first-line treatment options and second-line treatments included opioid analgesics and tramadol.Pharmacotherapy is the primary clinical approach for managing NLBP. Canadian pain society provided guidelines of pharmacological management of chronic neuropathic pain as follows: first-line treatments were specific antidepressants (tricyclics) and anticonvulsants .Although these guidelines for neuropathic pain management were provided, there was still a lack of available data on treatment patterns of NLBP patients and outcome by treatment pattern.Therefore, the purpose of this study was to investigate the treatment patterns of NLBP in Korea and to explore the patient-reported outcomes (PROs) including QoL and functional disability by the treatment patterns.22.1This was a subgroup analysis of chronic low back pain (CLBP) patients with neuropathic pain derived from the NLBP outcome research (OR) that was multicentered, cross-sectional study. Data were collected between December 2014 and May 2015 from 27 nationwide general hospitals of South Korea. This study was approved by the all participated centers’ Institutional Review Board. We included CLBP patients who have moderate degree of LBP at least (VAS > 4) and received “minimally adequate treatment” with a medication trial lasting at least 4 weeks. Patients judged by physicians to meet the following criteria were included: age 20 years; CLBP at least 3 months; patients diagnosed with LBP owing to herniated disc, stenosis, spondylosis, spondylolysis, spondylolisthesis, or degenerative disc disease, according to magnetic resonance imaging or computed tomography findings; VAS at least 4; pain medication at least 4 weeks before the enrollment; and patients who were able to understand and willing to complete the subject information sheet and informed consent form. If the patients had following criteria, they were excluded: cancer, sprain, infection, fracture, ankylosing spondylitis, myofascial pain, or sacroiliitis; surgery within 3 months; current participation in other interventional studies; or patients with a critical or unstable health condition. The patients were clearly informed about the aim of our study, and their informed consents were obtained. With a significance level of 0.05 and an estimated error rate of 2.8%, the required number of patients to be enrolled was calculated to be approximately 1200: A target sample size was estimated based on the assumption that the prevalence of NLBP is 37%.Among the total 1200 patients enrolled in NLBP OR, NLBP patients whose scores were at least 4/10 in Douleur Neuropathique 4 (DN4) questionnaire were included in this subgroup analysis  were measured by the patient's survey. Age, sex, height, and body weight were included in demographic information. Clinical variables contained diagnosis of LBP, comorbidities, symptom period, pain VAS scores, DN4 score, Quebec Task Force Classification for Spinal Disorders (QTFC-SD), and pain control state (pharmacotherapy and surgery). As one of clinical characteristics, QTFC-SD was used to categorize patients’ spinal disorders on the basis of clinical examination and pain localization.2.2.2,27Treatment patterns were divided into 2 groups according to whether patients received targeted therapy (TT) of neuropathic pain or not. The TT group included anticonvulsants, TCAs, and topical lidocaine, whereas non-targeted therapy (nTT) group included opioid analgesics, nonopioid analgesics, and muscle relaxants.2.2.3 = 1 – (0.165 + 0.003 × M2 + 0.274 × M3 + 0.058 × SC2 + 0.078 × SC3 + 0.045 × UA2 + 0.133 × UA3 + 0.048 × PD2 + 0.130 × PD3 + 0.043 × AD2 + 0.103 × AD3 + 0.347 × N3 + 0.014 × I2sq)Final EQ-5D index's scoreQoL was assessed by generic EuroQoL 5-dimension (EQ-5D) questionnaire. EQ-5D is made up of 2 parts: a descriptive system, which could be converted into a single index, and a EuroQoL Visual Analogue Scale (EQ-VAS). The descriptive measurement consists of 5 dimensions: “mobility,” “self-care,” “usual activities,” “pain/discomfort,” and “anxiety/depression.” Each dimension has 3 levels: no problems, some problems, and severe problems, coded as numbers of 1, 2, and 3 in order. These scores, measured by descriptive part, were converted into scores of a single index (EQ-5D index) with a range from −0.229 to 1 point by applying the equation as follows:The EQ-VAS with a range from 0 to 100 points was used for subjective evaluation of patient's current health state. The ends of the scale were marked “best imaginable health state” and “worst imaginable health.” In these 2 measurements, a higher score means a higher QoL.Quebec Back Pain Disability Scale (QBPDS) was used for measuring functional disability. The QBPDS consists of 20 questions and has a scale of 0 to 5 for each question with a range from 0 to 100 points. A higher score of this tool indicates a more severe disability of function.2.3This investigation was designed as multicenter cross-sectional observational study of LBP patients. The patients  were provided with all study-related information, and they signed an informed consent form. All participating medical institutions obtained approval from their respective institutional review boards.2.4χ2 test, as appropriate. Mean differences of patient's QoL measured by EQ-5D index and EQ-VAS scores as well as their functional disability measured by QBPDS were also examined using the independent t test. An appropriateness of a use of the test was examined by histogram and normal probability plot for each of numerical variables. For variables with positively skewed data, a logarithmic transformation was performed before the t test. Multiple linear regression analyses were performed to compare scores of EQ-5D index, EQ-VAS, and QBPDS between 2 groups after adjusting potential confounders. Variables with P < .1 from bivariate analyses were selected as the potential confounders. Degree of performance of the regression model was measured by its coefficient of determination (R2). Collinearity among explanatory variables has also been checked, and no noticeable problem found in the multiple regression models used in this study. All statistical analyses were performed using the SAS software, version 9.4 , and a 2-tailed P value <.05 was considered as statistically significant.Patient's demographic and clinical characteristics were summarized as mean ± standard deviation (SD) for continuous variables or frequency (percentage) for categorical variables. Comparisons of patient's characteristics between the TT group and nTT group were made by Student 2 independent sample t test or 33.1P < .0001). In the TT group, the most frequent pain type was pain with distal extremity radiation (49.3%) followed by spinal stenosis (16.2%) and spinal nerve root compression (14.8%). pain with distal extremity radiation (50.0%) showed the highest proportion in the nTT group as in the TT group. However, unlike the TT group, pain with proximal extremity radiation (17.6%) was the secondly highest, followed by pain with radiation and neurologic finding (11.3%).Demographic and clinical features of NLBP patients are presented in Table P < .0001).Our data also showed that the subject's severity of LBP measured by QBPDS was associated with their anxiety/depression which was the fifth dimension of EQ-5D index measured as “none,” “some/moderate,” and “extreme.” Specifically, patient having more severe anxiety/depression showed significantly higher mean QBPDS score , there were no differences in the EQ-5D index (0.41 ± 0.27 and 0.39 ± 0.28), EQ-VAS (56.43 ± 18.17 and 54.37 ± 20.69), and QBPDS (45.31 ± 21.32 and 46.31 ± 21.24)    , the patients managed by the TT showed higher scores of EQ-5D index (1) Table  and EQ-V5) Table  than the4neuropathic pain is often undertreated or treated with ineffective or untested modalities.This study showed the mismatch between diagnosis and treatment pattern of NLBP patients. Although 39.8% of LBP patients met the DN4 criteria for neuropathic pain, only 29.7% were receiving pharmacological treatment with proven efficacy in neuropathic pain. The literature reveals that  NLBP results from a primary lesion or a malfunction within the somatosensory system, whereas nociceptive LBP is caused by tissue injury and/or inflammatory process. The general clinical diagnosis of neuropathic pain was based on the evidence of a lesion or a disease of the nervous system, which was ascertained by interviewing the patients and performing clinical examinations. However, it is complex and difficult methods to detect neuropathic pain in LBP patients.A reason for the low proportion of patients having TT would be difficulty in distinguishing the certain clinical difference between NLBP and nociceptive LBP. Identifying the underlying mechanism of chronic pain allows the use of pharmacological agents targeting specific pain mechanisms.Some clinicians used screening tools of neuropathic pain as a simpler means. However, there were various standardized screening measurements incurring wide variation in diagnosis of neuropathic pain and no consensus on the diagnosis of neuropathic pain.In addition, although there were many evidence-based guidelines in the pharmacological management of neuropathic pain due to the attempts for developing a therapeutic approach of related societies, neuropathic pain treatment guideline for LBP was absent. Serotonin noradrenaline reuptake inhibitors and topical lidocaine were recommended as second-line therapies. Third-line therapies included tramadol and controlled release opioid analgesics. Recommended forth-line treatments were cannabinoids, methadone, and anticonvulsants. The special interest group of the international association for the study of neuropathic pain recently suggested guidelines of pharmacological management of neuropathic pain. According to their guidelines, TCAs, gabapentin, pregabalin, and topical lidocaine were recommended as first-line treatment options and second-line treatments included opioid analgesics and tramadol.Canadian pain society provided guidelines of pharmacological management of chronic neuropathic pain as follows: first-line treatments were specific antidepressants (tricyclics) and anticonvulsants .We defined the TT and nTT based on these 2 references. Target therapy included TCAs, gabapentin, pregabalin, and topical lidocaine, which are recommended as first- or second-line medical treatment because their efficacy in neuropathic pain has been established in multiple randomized controlled trials (RCTs).,32 QoL measured by EQ-5D scores of patients with neuropathic pain (0.40 ± 0.28) was considerably lower in this study than those of other chronic diseases reported in the previous study , which showed the negative impact of the neuropathic pain in CLBP patients. After adjustment with covariates, the patients managed by pharmacological TT showed higher EQ-5D index and EQ-VAS scores than the nTT group, which suggested that the neuropathic pain patients who received the TT showed significantly better outcome in terms of pain control and QoL than the nTT group. In many RCTs, medications included in TT group of our study were proven in terms of efficacy of neuropathic pain therapy. Two studies of antidepressants in neuropathic pain reported that TCAs provided the identical efficacy in the neuropathic pain management.,40 Gabapentin and pregabalin have shown the efficacy in the studies on comparison between anticonvulsants and placebo in patients with several neuropathic pain conditions.,42 The 5% lidocaine patch has shown excellent efficacy and tolerability in allodynia patients due to various types of peripheral neuropathic pain.,42Neuropathic pain patients had severe and chronic symptoms that impaired their QoL.This study has several limitations. First, some of the well-known risk factors for NLBP include advanced age, female sex, and diabetes with or without hypertension, obesity, smoking, and psychological factors such as depression. Although these data demonstrated that neuropathic LBP and depression are significantly correlated with each other, the relationship between neuropathic LBP and other risk factors was not examined in this study because the aim of this study was to investigate treatment patterns of NLBP patients and to explore PRO including QoL and functional disability by treatment patterns.Second limitation of this study stems from the differential diagnosis of neuropathic pain. This study used DN4 questionnaire to diagnose a neuropathic pain in LBP patients based on a more reliable identification and qualification of a neuropathic pain. Despite the good sensitivity and specificity of the DN4 questionnaire, the question remains whether the distinction between neuropathic and nociceptive symptom profiles truly represents the biological background of pain, or whether it may be an artificial effect. Moreover, these categories of pain overlap to some degree. Although screening tools may give guidance to clinicians by selecting patients that need further diagnostic evaluation and pain management by specialists, they clearly do not replace clinical judgment. In this regard, the evaluation method using the questionnaire that was employed in this study is also a limitation.In addition, it should be noted that because other pain measures were not part of the study, no comparisons can be made between the DN4 questionnaire and other neuropathic pain screening scales.The third limitation of this study results from the subject population because it was mostly performed at a tertiary care university hospital. The spectrum of presenting patients obviously differs between primary care clinics and community hospitals. Fourth, this subgroup analysis has the limitation on the sample size, not calculated to investigate this subgroup. Thus, it is difficult to generalize the study results. Lastly, we could use only measured variables in this study as the potential confounders.Despite these limitations, there have been no other similar studies to compare the treatment outcome between TT and nTT in the NLBP population and this study showed that TT could be associated with a better QoL of NLBP patients. There were, however, relatively low numbers of patients having pharmacological TT of neuropathic pain in this study. These results could suggest that in cases with neuropathic pain, appropriate pharmacological treatments for the neuropathic component should be considered to have better QoL. Further studies are required to explore effects of TT on the improvement of QoL and functional ability in larger population with NLBP.Conceptualization: Bo-Jeong Seo, Young-Joo Kim.Data curation: Jae Taek Hong, Jin-Hwan Kim, Keun-Su Kim, Chong-Suh Lee, Hyun-Chul Shin, Woo-Kyung Kim, Joo-Han Kim, Jung-Kil Lee, In-Soo Kim, Yoon Ha, Soo-Bin Im, Sang Woo Kim, In-Ho Han, Jun-Jae Shin, ByeongCheol Rim, Kyung-Soo Suk, Jin-Hyok Kim, Ye-Soo Park Park, Bong-Soon Chang, Deuk Soo Jun, Young-Hoon Kim, Jung-Hee Lee, Woo-Kie Min, Jung Sub Lee, Si-Young Park, In-Soo Oh, Jae-Young Hong.Formal analysis: Juneyoung Lee.Investigation: Jae Taek Hong, Hyun-Chul Shin, Woo-Kyung Kim, Joo-Han Kim, Jung-Kil Lee, In-Soo Kim, Yoon Ha, Soo-Bin Im, In-Ho Han, Jun-Jae Shin, ByeongCheol Rim, Kyung-Soo Suk, Jin-Hyok Kim, Ye-Soo Park Park, Bong-Soon Chang, Deuk Soo Jun, Young-Hoon Kim, Jung-Hee Lee, Woo-Kie Min, Jung Sub Lee, Si-Young Park, In-Soo Oh, Jae-Young Hong.Methodology: Jae Taek Hong, Bo-Jeong Seo.Project administration: Bo-Jeong Seo.Supervision: Keun-Su Kim, Chong-Suh Lee, Bo-Jeong Seo, Young-Joo Kim.Validation: Jae Taek Hong, Jin-Hwan Kim, Bo-Jeong Seo.Visualization: Bo-Jeong Seo.Writing – original draft: Jae Taek Hong, Jin-Hwan Kim.Writing – review & editing: Keun-Su Kim, Chong-Suh Lee, Bo-Jeong Seo, Young-Joo Kim, Juneyoung Lee.


The clinicopathologic association of tumor immune response is largely unknown. We systematically investigated this matter in human cancers.Different cancer types exhibited distinct immune gene profiling. Four cancer types exhibited a significant and positive correlation of immune response with patient age. Significant but inconsistent correlation of immune response was observed with gender, surgical stage, and TNM stage in a small number of cancer types. In contrast, the histological grade appears to have much stronger and more consistent association with immune response as compared to the other clinicopathologic factors. Specifically, patients with high grade had significantly higher immune responses than those with low grade in 5 out of 12 analyzed cancer types. In addition, both histological and molecular classifications had a significant and strong association with tumor immune response.t-test was used to examine the association of patient age with immune response. Multiple tests with the Benjamini-Hochberg correction also were performed.t-distributed stochastic neighbor embedding was used to assess similarity of immune gene profiling in human cancers. The Mann-Whitney or Kruskal-Wallis test was, respectively, used to compare the tumor immune response in two or more groups that were stratified by patient clinicopathological characteristics, such as gender, grade, stage , histology, and molecular subtypes. Spearman correlation with student’s Tumor grade should be taken into account in selection of patient candidates for immunotherapy. Prospective verification is needed before use of the findings for clinical practice. Cancer immunotherapy is an attractive treatment strategy and associated with improved clinical outcomes in multiple cancer types , 2. ImmuHowever, the clinicopathological association of immune response is largely unknown in many other cancer types, and it has not been systematically investigated in a wide array of human cancers. Moreover, many other clinicopathological features besides tumor stage or metastasis are not yet used as selection criteria for cancer immunotherapy. We recently devised an mRNA-based metric of preexisting immune conditions from the global immune gene signature  and systt-distributed stochastic neighbor embedding (t-SNE) on the 10,062 PanCanAtlas tumor samples and expression data of the 382 global immune signature genes [To assess the dissimilarity of immune gene expression profiling across human cancers, we carried out re genes  ; kidney renal clear cell carcinoma ; brain lower grade glioma ; bladder urothelial carcinoma ; and head and neck squamous cell carcinoma   . MoreoveP = 0.0016, adjusted), while lung adenocarcinoma (LUAD) patients showed the opposite trend   exhibited significant correlation of immune response with surgical stage before and after multiple testing corrections, respectively. Different from histological grade, the association of surgical stage with immune response is not consistent among human cancers. In particular, kidney renal clear cell carcinoma (KIRC) patients with advanced-stage disease had significantly higher immune response than those with early-stage disease (djusted) . CompareP = 0.0044, adjusted) and skin cutaneous melanoma , patients with T3/4 had significantly lower immune response than those with T1/2, while kidney renal clear cell carcinoma  showed the opposite trend. The colon adenocarcinoma  patients with M1 had significantly lower immune response than those with M0, while kidney renal clear cell carcinoma  showed the opposite trend.Next we examined the relationship between TNM stage and tumor immune response. After multiple testing correction, we found that three cancer types exhibited significant correlation of immune response with tumor T stage, two cancer types with tumor M stage, and none with tumor N stage . SimilarP = 4.09 × 10-05, Mann-Whitney test, P = 0.09, P = 1.02 × 10-05, P = 3.33 × 10-09, P = 3.84 × 10-09, Kruskal Wallis test, Next we examined the relationship between tumor immune response and histological or molecular classifications in human cancers that have been used in clinical practice . ApproxiP = 7.59 × 10-07, Kruskal-Wallis test, P = 1.52 × 10-31, P = 1.04 × 10-06, P = 2.01 × 10-11, P = 0.0001, We next correlated tumor immune response with well-established molecular subtypes and found a significant association in several cancer types. The breast invasive carcinoma (BRCA) patients were mainly categorized into four subtypes  following the PAM50 classification, which exhibited a statistically significant difference in tumor immune response , and no targeted therapies are available, due to the molecular characteristics of this disease. Our results showed that the breast cancer basal group (highly concordant with TNBC) had significantly higher immune response than LumA/B breast cancer patients, suggesting that these patients  might be good candidates for currently emerging immune checkpoint inhibitor therapy. Consistent with this finding, it was recently reported that atezolizumab (antibody against PD-L1 protein) was shown to prolong progression-free survival; the drug was recently approved by FDA for treatment of metastatic TNBC patients .Our results further demonstrated that the hypermutated (HM) subtype in colon adenocarcinoma (COAD) had a significantly higher immune response than either the chromosomal instability (CIN) or genome stable (GS) subtypes, making such patients potential candidates for immunotherapeutic treatments. Given that the majority of these hypermutated (HM) cases were MSI-high (MHS-H) tumors, these data were well consistent with previous reports that the colon adenocarcinoma (COAD) cases with MSI-H were typically excellent candidates for immunotherapy . In addiP = 0.07), suggesting that early-stage LIHC patients were likely favorable candidates for currently emerging immunotherapy [Our results showed that liver hepatocellular carcinoma (LIHC) had distinct immune expression profiling  but therotherapy . In contotherapy  who needotherapy . Accumulotherapy  but alsootherapy . It’s thotherapy .The present study has several limitations. First, it was a retrospective study. Second no patients in this study received immunotherapy, for which we cannot evaluate the clinical benefit of immune response in terms of responsivity. Still, these analyses provide useful insights into the relationship between clinicopathologic characteristics and tumor immune response and can guide further clinical trial development.A total of 10,062 PanCanAtlas tumor samples covering 32 different cancer types were used in this study, with more details as described in our previous publication . The corTo visualize the grouping of cancer samples, we performed t-Distributed Stochastic Neighbor Embedding (t-SNE) on the 10,062 TCGA PanCanAtlas tumor samples and the 382 immune genes we recently identified . The expIn this study, patient age was treated as a continuous variable and Spearman rank-order correlation was used to examine the relationship between tumor immune response and age. The difference in immune response between male and female patients was examined by using the Mann-Whitney test to assess the association of tumor immune response with gender.To examine the relationship between tumor immune response and histological grade, we grouped patient samples annotated as either “G1”, “G2”, or “Low Grade” in the clinical file, categorized as “Low Grade.” Those annotated as either “G3”, “G4”, or “High Grade” are categorized as “High Grade.” All the other samples were excluded from the analysis. As a result, only 12 tumor types remained for the grade-immune response correlation analysis. Then the median difference in immune response between low-grade and high-grade group was compared.Similarly, to correlate tumor immune response with surgical stage, we grouped patients samples annotated as either “Stage I”, “Stage IA”, “Stage IB”, “Stage II”, “Stage IIA”, “Stage IIB”, or “Stage IIC” in the clinical file, categorized as “Early Stage”. Those annotated as “Stage III” or “Stage IV” were categorized as “Advanced Stage”. All the other samples were excluded from the analysis. As a result, a total of 21 cancer types had surgical stage information. The median difference in immune response between the Early-Stage and Advanced-Stage group was compared.To correlate tumor immune response with TNM stage, we first combined T1 and T2 together as the T1/2 group, and T3 and T4 together as the T3/4 group. Then the median difference in immune response between the T1/2 and T3/4 groups was compared. Similarly, we combined N0 and N1 together as the N0/1 group, and N2 and N3 together as the N2/3 group. Then the median difference in immune response between the N0/1 and N2/3 groups was compared. Finally, we compared the median difference in immune response between M0 and M1 groups.In this study, we only chose those histological or molecular subtypes that have already been used in clinical practice  for the t-test was used to assess the statistical significance in the Spearman correlation. In all cases, multiple testing was performed with Benjamini-Hochberg (BH) correction. All statistical tests were two-sided, and a P value of less than 0.05 was considered significant. The calculations and graphs were made with GraphPad Prism, version 7.03 .The Mann-Whitney test was used to assess statistical significance in immune response between the dichotic groups stratified by gender, grade, surgical stage, TNM stage, and histology . The Kruskal-Wallis test was used to assess statistical significance in immune response among more than two groups, such as the PAM50 subtype in BRCA, molecular subtypes in gastrointestinal cancer and uterine corpus endometrial carcinoma (UCEC). The Student’s


Among the various forms of natural energies, heat is the most prevalent and least harvested energy. Scavenging and detecting stray thermal energy for conversion into electrical energy can provide a cost-effective and reliable energy source for modern electrical appliances and sensor applications. Along with this, flexible devices have attracted considerable attention in scientific and industrial communities as wearable and implantable harvesters in addition to traditional thermal sensor applications. This review mainly discusses thermal energy conversion through pyroelectric phenomena in various lead-free as well as lead-based ceramics and polymers for flexible pyroelectric energy harvesting and sensor applications. The corresponding thermodynamic heat cycles and figures of merit of the pyroelectric materials for energy harvesting and heat sensing applications are also briefly discussed. Moreover, this study provides guidance on designing pyroelectric materials for flexible pyroelectric and hybrid energy harvesting. Energy has been a prime concern of the scientific community and industrial areas worldwide. As the demand for self-powered autonomous electronics with low power consumption has grown drastically in the electronics industry, energy harvesters have received immense focus and are being widely studied. Solid-state batteries, which are commonly used as an energy source in devices, require frequent and periodic maintenance such as recharging or replacement. Thus, various scavenging methods (harvesting the energy from stray energy sources) have been proposed to provide sustainable energy supplies for small electronic devices ,7,8,9,10Among the above forms of stray energy, heat is ubiquitous and serves as a low-grade waste . To convThe typical application arena of pyroelectric energy conversion is a thermal sensor, which can detect thermal signals at the moment. However, it can also be used as a thermal energy harvester if the conversion efficiency and total converted energy are sufficiently high to charge electrical energy storage devices, such as a supercapacitor or battery. Although the PyEH concept was introduced in the 1960s, it still remains a comparatively less studied area ,21,22,23−3 to mW∙cm−3) using pyroelectric energy conversion, which can be used to drive devices, such as liquid color displays (LED), light-emitting diodes (LED), and wireless devices O3, and BZT-50BCT, i.e., 0.5Ba(Zr0.2Ti0.8)O3-0.5(Ba0.7Ca0.3)TiO3, in which the maximum harvestable energy density 629 kJ∙m−3∙cycle−1 was obtained using the Olsen cycle [0.5Bi0.5TiO3 (NBT)-based ceramics were also explored for the electrocaloric effect (ECE) and PyEH applications [x)Na0.5Bi0.5TiO3-0.06BaTiO3-xSrTiO3 (NBBSTx) where x = 0.10, 0.15, 0.20, and 0.25 of SrTiO3 (ST) insertion. The composition of the insertion of ST into the NBT ceramic strongly affects both the ECE and PyEH characteristics because of the decrease in the ferroelectric-relaxor transition temperature. Here, the PyEH calculations were performed using the Olsen cycle, as shown in h and Th while maintaining El and Tl constant at 0 kV∙cm−1 and 303 K, respectively, as shown in −3∙cycle−1 for the EH and TH as 40 kV∙cm−1 and 423 K, respectively. This study demonstrates how the tuning of the pyroelectric thermal energy density of the ceramics can be performed with different insertion of ST into the system. In addition, the reported PyEH was higher than the reported PyEH for the conventional lead-based bulk ferroelectrics, such as PNZST  [−3∙cycle−1) [−3∙cycle−1).In both reports mentioned above, the BNT-BT-based lead-free ceramics exhibit a remarkable energy density, higher than that of lead-based ceramics reported up to now. This makes these lead-free ceramics strong candidates for application as thermal energy harvesters. In addition, some other lead-free ceramics composites were also studied for thermal energy harvesting by the same Vaish et al. group, such as KNTM, i.e., K[(Nben cycle . Apart fications . NBT cercycle−1) , PZN-0.4cycle−1) , and PMNcycle−1)  single-c2), were explored. Also, the thin films of these ceramics or polymers were extensively explored for PyEH as a result of nanoscale application such as a pyroelectric nanogenerator (PyNG). Yang et al. demonstrated that the PyNGs can harvest thermal energy more efficiently [−3∙cycle−1 were reported by Navid et al. [The aforementioned studies have demonstrated that the PyEH of lead-free ceramics are higher than that of the typical lead-based ceramics. However, the complex compositions and chemical formulas might hinder mass production from the application perspective. Therefore, ferroelectric materials with simple structures, such as ferroelectric polymers (PVDF-TrFE) and binary oxides  technique. Park et al. reported a colossal energy density in the nearly 9.2-nm-thick ALD-deposited HfxZr1-xO2 (where x is varied from 0.1 to 0.3) (HZO) on SiO2/Si substrate. The HfxZr1-xO2 with x = 0.2 and 0.3 exhibited values of 11500 (298–423 K and 0–3260 kV∙cm−1) and 5700 kJ∙m−3∙cycle−1 (298–423 K and 0–3260 kV∙cm−1), respectively, which was approximately 7.6 and 3.7 times higher than the largest value reported  in 5.6 mol% Si:HfO2 thin films [2 thin films exhibited the phase transition from a pure ferroelectric to field-induced ferroelectric as the temperature increases, which can be exploited for the PyEH and ECE as well. The 5.6 mol% composition exhibited the strongest constriction in P–E loop at room temperature and highest temperature as well. A very high pyroelectric coefficient in Si:HfO2 thin films leads to the excellent k2 values as compared to other materials. 0.2Zr0.8O2 thin films exhibited negligible pyroelectricity when no electric field is applied, hindering its application as sensors [2 thin films, pyroelectricity is exhibited, even at zero applied electric field. Along with k2, the Si:HfO2 thin films possess VF values comparable to those of previously reported lead-based ceramics, such as Pb(ZrxTi1-x)O3 (PZT) [1/3Nb2/3)O–0.25PbTiO3 (PMN-0.25PT) [iF) values were slightly lower.In another report from Hoffmann et al., a promising enhancement in the PyEH in HfOobtained . Here, tin films . The Si: sensors ,88. MeanO3 (PZT)  and sing-0.25PT) , while t0.97La0.02)(Zr0.55Sn0.32Ti0.13)O3 ceramics to enhance the PyEH ability. The authors obtained a maximum energy density of 270 kJ∙m−3∙cycle−1 from the ferroelectric material using the Olsen cycle with parameters ΔT = 298–453 K and ΔE = 10–80 kV∙cm−1 [3 single-crystals fabricated with a composition close to the morphotropic phase boundary (MPB) were studied by Zhuo et al. [As we have discussed in the previous section, although some of the lead-free materials have exhibited superior thermal energy harvesting performance to that of the lead-based pyroelectric materials, the lead-based materials show superior FoMs for applications such as infrared sensors, which make them an excellent choice for commercial sensor application. In the quest for excellent materials for PyEH application, a plethora of studies have been reported on PZT- and PMN-PT-based ceramics and their thin films ,120,121. kV∙cm−1 . Similaro et al. . Both af0.3Ti0.7)O3/PbOx buffer layer and <001>-oriented 0.67PMN-0.33PT thin films. In the calculation of the energy harvesting, the LT and LE are kept constant at 303 K and 0 kV∙cm−1. The obtained maximum energy densities for PMN-PT buffer-layered thin films and PMN-PT thin films were nearly 8000 kJ∙m−3∙cycle−1 (ΔT = 393 K and ΔE = 600 kV∙cm−1) and 6500 kJ∙m−3∙cycle−1 (ΔT = 393 K and ΔE = 600 kV∙cm−1), respectively. The notable point in this study was that there was a sharp jump in the energy density observed when the temperature range changes from 303 to 393 K to 303 to 423 K, which indicates that a further enhancement in the energy density can be achieved near its cT and MPB. The enhancement in the energy density (larger for PMN-PT buffered layer than the PMN-PT thin films) is attributed to the small difference in remnant polarization and low cT of the PMN-PT buffer layered thin films [The enhanced pyroelectric properties with high FoMs were reported by Mangalam et al. in PZT . In addiin films .−3∙cycle−1 (ΔT = 298–558 K and ΔE = 300–900 kV∙cm−1) in the 2-µm-thick Pb0.97La0.02(Zr0.75Sn0.18Ti0.07)O3 AFE, which was the largest reported value up to that date [P-E hysteresis loop, the energy density value decreased. The authors obtained the maximum energy density of 8400 kJ∙m−3∙cycle−1 at 100 Hz. Therefore, it can be inferred that the thermal-electrical energy harvesting ability of the films can be optimized by optimizing associated parameters such as the operating temperature, applied electric field, and its frequency. In another report from the very same group, the ECE and PyEH characteristics were reported, as the authors thoroughly investigated the effects of the phase structure on the ECE and PyEH capacities [0.97La0.02)(Zr0.95−xSnxTi0.05)O3 AFE films was varied with x = 0.08 (orthorhombic), 0.20 , and 0.38 (MPB) to control the phase structure, and maximum energy densities of 3800, 6800, and 4000 kJ∙m−3∙cycle−1 were achieved with x = 0.08, 0.20, and 0.38, respectively. The inference from the above two studies can be drawn that the PLZT-based AFE films yields a maximum energy density in the tetragonal phase.Although PLZT ceramics exhibit a lower pyroelectric coefficient than PMN-PT, the composition alterations of the antiferroelectric (AFE) PLZT ceramics exhibit promising harvestable energy density ,113. Thehat date . Here, tpacities . Sn-dopi−3∙cycle−1 in PbZr0.53Ti0.47O3/CoFe2O4 (PZT/CFO) multilayered nanostructures (MLN) using the Olsen cycle, which was nearly four times higher than the highest reported PyEH up to the date of publication [−3∙cycle−1 (ΔT = 100–300 K and ΔE = 0–400 kV∙cm−1), which are higher than any reported value, as shown in −1, respectively.Apart from the thin and thick film structures, multilayer structures have been also explored. Vats et al. reported a remarkably giant PyEH per cycle of 47372 kJ∙mlication , as showlication ,127. ThevF, iF, EF, and k2, as these have already been discussed earlier in the above sections. This section will primarily focus on the applications of pyroelectric materials as energy harvesters and sensors.Pyroelectric materials in bulk and thin-film forms have been discussed above for PyEH applications, such as heat sensors, thermal imaging or infrared sensors, fire alarms, and gas sensors. The selection of the materials is conducted based on the FoMs for these applications, i.e., Among the reported PyEH devices, the flexible energy harvesting devices have generated much interest owing to its unlimited potential for applications such as wearable and plantable devices on human body ,128,129.Zhang et al.  demonstr0.52Ti0.48)O3 films was examined by Ko et al. [3 as the bottom electrode, which enables the high temperature growth of the film. The flexible PZT film possesses high piezoelectric (140 pC/N) and pyroelectric coefficients (50 nC/cm2∙K) at room temperature. Among the aforementioned polymers and lead-based PyEHs, the lead-free material is also highly desired for flexible energy harvesting applications owing to its high-temperature stability and environmental compatibility. Yang et al. [3 nanowires-PDMS.Apart from the polymer-based energy harvesters, lead-based and lead-free ceramic thin films have also been explored owing to their excellent pyroelectric properties ,40,42,44PyEH efficiency can be enhanced by employing various energy sources. Basically, the hybrid energy harvesters are designed by employing two or more energy conversion mechanisms, such as pyroelectric, piezoelectric, photothermal, and thermoelectric processes , while eT is analogous to the stress Δσ for the piezoelectricity. Owing to the analogous symmetry of these two energy harvesting methods, much effort has been put into designing hybrid pyroelectric–piezoelectric energy harvesting systems [Because all the pyroelectric materials are also piezoelectric materials, there was great interest in the scientific community to attempt to combine both energy harvesting methods. In addition, there is a close similarity between the relevant equations of the pyroelectricity and piezoelectricity, as the temperature change Δ systems ,45,132. T) and mechanical stress (σ). Here, the cumulative change in the polarization can be expressed asd is the piezoelectric coefficient.Lee et al.  reportedWang et al.  demonstrSince the last few decades, small and wearable electronic devices have received enormous attention. Correspondingly, several pyroelectric materials have been studied and employed commercially for the sensor applications. A human body maintains its certain temperature and continuously emits infrared rays. Therefore, especially in the winter, there is a temperature gradient between human body and surrounding environment which can be further exploited to generate the electric potential. So, if PyNGs are placed in the human body in form of wearable devices, the temperature fluctuations can be converted into the electric potential between electrodes due to pyroelectric effect. Especially in winter, due to cold weather or air pollution, people prefer to wear a respirator; the human body temperature is ~37 °C, while for some regions with high altitude, the outdoor temperature in winter is as low as −20 to −30 °C. Xue et al. demonstrated the self-powered respiratory system installed with the proposed PyNG . SimilarvF and iF, are of importance while choosing the materials for sensors applications. The studies have suggested that triglycine sulfide (TGS) has great potential for pyroelectric energy conversion applications. Although the TGS has exhibited excellent FoMs, its application arena is limited because of its low Curie temperature (Tc = 49 °C) [High FoMs, such as = 49 °C) . A few l= 49 °C) ,136,137.= 49 °C) , barium = 49 °C) , potassi= 49 °C) ,141 have0.965Ti0.035)O3 + 1 wt.% Nb2O5, BaSn0.05Ti0.95O3, Ba0.67Sr0.33TiO3, and PbZr0.45Ti0.55O3 [To obtain the best performance from the pyroelectric material, there is a tradeoff between the pyroelectric coefficient, dielectric loss, dielectric constant, and material specific heat, which further limits the enhancement of the FoMs. Researchers have explored the alteration in the chemical and physical composition of ceramics, such as doping or introduction of pores into the ceramics ,143,144.Ti0.55O3 ,147,148.Ti0.55O3 .sP vs. temperature change (s/dTdP), a low piezoelectric coefficient (since higher coefficient causes piezoelectric noise in the device), the cT should be near to operating temperature, and the material should have low dielectric loss and a high dielectric constant. Also, the pyroelectric material must possess the high resistivity and low coercive field. As the pyroelectric materials have shown higher performance near the MPB, there have been several efforts to engineer the materials  to obtain the desired phase structure. Apart from these, the introduction of lower permittivity material into the pores of the pyroelectric material ceramic has helped significantly in enhancing its pyroelectric energy harvesting performance.In this review, the recent progress in pyroelectric ceramics/polymers, its thick and thin films for flexible energy harvesting, sensors, and as hybrid piezoelectric–pyroelectric applications in addition to the fundamental theories on pyroelectricity is systematically discussed. The discussion primarily was focused on the PyEHs where the measurement and calculations of the maximum harness-able energy density was done using Olsen cycle. The thermal energy harvesting performance of various lead-based, lead-free polymers and their prospects has been highlighted. The discussed studies in this article provide broad applications of the pyroelectric materials as PyEH and heat sensing devices. As the hybrid energy harvesters have a great potential as flexible and wearable energy harvesting devices (as shown in dT/dt). However, the more optimized paradigms of the hybrid PyEH might be quite helpful in enhancing the output power.The PyEH devices are currently limited by the inability to induce high frequency temperature fluctuations which further limits the harvestable amount of power. Similarly, in case of the hybrid pyroelectric–piezoelectric energy harvesters, the output power is largely restrained by the difference in the frequency of the mechanical stresses (bending or vibrations) and temperature changes (i.e.,