While follitropin delta clearance from serum depended in part on the hepatic asialoglycoprotein receptor (ASGPR), follitropin alfa clearance was unaffected by ASGPR inhibition in rat or genetic ablation in mice. The distinct properties of follitropin delta and follitropin alfa are likely to contribute to the differing pharmacokinetic and pharmacodynamic profiles observed in women and to influence their efficacy in therapeutic protocols for the treatment of infertility.Recombinant FSH proteins are important therapeutic agents for the treatment of infertility, including follitropin alfa expressed in Chinese Hamster Ovary (CHO) cells and, more recently, follitropin delta expressed in the human cell line PER.C6. These recombinant FSH proteins have distinct glycosylation, and have distinct pharmacokinetic and pharmacodynamic profiles in women. Comparative experiments demonstrated that follitropin delta and follitropin alfa displayed the same  In addivenience . As a reFSH is composed of two extensively glycosylated protein subunits, and to express recombinant FSH (rFSH) for therapeutic use, the genes encoding the human FSH subunits are introduced into a mammalian cell line from which FSH protein is secreted and purified . Early rRecently follitropin delta has been described (FE 999049), the first recombinant FSH protein in clinical development that is expressed in a human cell line (PER.C6), with individualized dosing optimized based on each patient’s weight and anti-Mullerian hormone (AMH) level , 11, 12.in vitro at the human FSH receptor compared to more basic FSH isoforms, and also display lower serum clearance and longer circulating half-life , and diluted and tested as indicated in each experimental method. Follitropin delta samples produced in the human cell line PER.C6 were provided by Ferring Pharmaceuticals  and characterized and diluted for testing as indicated in each experimental procedure. The first international standard for recombinant FSH (NIBSC 92/642) was obtained from NIBSC .3H] FSH as the radiolabel . Incubation Buffer and Stop Buffer was 10 mM Tris, 25 mM MgCl2, 0.5% BSA, pH 7.5. Follitropin delta used in these experiments had a protein concentration of 0.62 mg/mL and an assumed molecular weight of 33,000 g/mol. A sample of follitropin alfa with an assumed molecular weight of 31,000 g/mol, with 5.5 µg or 75 IU powder dissolved in 1.2 mL of the incubation buffer and the stock solution at 0.15 µM was aliquoted and stocked at −20°C. For both compounds, all dilutions were performed in the incubation buffer.The binding displacement assay for the human FSH receptor was performed utilizing  FSH and the indicated quantity of follitropin alfa or follitropin delta. Unifilter GF/B filters  were pre-soaked with 10 mM Tris–HCl, 25 mM MgCl2, 0.5% BSA, pH 7.5. Incubated mixtures were filtered through pre-soaked filters using a Packard FilterMate Harvester, and washed 3–4 times using the same buffer. Filters were then dried, MICROSCINT 0 scintillation fluid (PerkinElmer) was added (30 µL/well) and scintillation counting was performed using a Packard Topcount NXT instrument (1 min per well).To initiate binding, FSH receptor membranes containing 180 µg protein were incubated one hour at 37°C with 0.4 nM of  FSH in this assay was 0.2 nM with a Bmax of 28 fmol/mg protein (data not shown). The average IC50 in three experiments for displacement of propionyl-3H FSH was virtually identical for the two rFSH proteins, 0.302 nM for follitropin alfa and 0.299 nM for follitropin delta, with the results from a representative experiment shown in Ki of approximately 0.1 nM for both proteins.FSH receptor binding affinity was measured by displacement of radiolabeled FSH from the human FSH receptor. Propionyl-in vitro bioactivity of follitropin delta and follitropin alfa, both rFSH proteins were incubated in vitro with human embryonic kidney cells (HEK-293 cells) engineered to stably express the human FSH receptor, measuring production of the second messenger cAMP. The potency of the two rFSH proteins was virtually identical in this assay .To compare the is assay , with foin vitro bioactivity of follitropin delta and follitropin alfa was then compared using luteinized granulosa cells isolated from follicles of IVF patients and expressing the endogenous human FSH receptor. These experiments examined the impact of rFSH proteins on granulosa cell production of estradiol, progesterone, inhibin A and inhibin B, and on the expression of genes including CYP19a1, 3B-HSD and Inh A. Granulosa cells were collected following triggering of final follicle maturation at oocyte pickup from a total of 13 women undergoing controlled ovarian stimulation prior to IVF. Both rFSH proteins induced progesterone production approximately 300% normalized to basal levels in the absence of rFSH (P > 0.1), and with similar potency (3B-HSD was induced approximately 200% by both rFSH proteins (CYP19a1 gene was induced 40–50% by both rFSH proteins (data not shown) and INHA expression was induced by both rFSH proteins 50–100% over basal levels (data not shown).The  potency . Inhibin potency , while eproteins , the CYPThe Steelman–Pohley bioassay concept was used to demonstrate the bioactivity of follitropin delta in rat compared to an international reference standard of recombinant FSH produced in CHO cells. When follitropin delta and follitropin alfa were compared by Steelman–Pohley assay based on dosing of a previously assessed bioactivity, the two compounds produce parallel curves and, thus, are very similar in their pharmacodynamic behavior in rat .Figure To examine the role of the ASPGR in clearance of follitropin delta and follitropin alfa, the rFSH proteins were injected intravenously in rats in the presence or absence of a saturating dose of asialofetuin (ASF), a ligand for the ASGPR that can compete and block the hepatic clearance of other ASGPR ligands. Co-injection of a saturating dose of ASF reduced the clearance of follitropin delta from serum, resulting in a 42% increase in the AUC compared to the absence of ASF  and a siTo further examine the role of ASGPR in clearance of follitropin alfa and follitropin delta, their pharmacokinetics were compared in wild-type and ASGPR knockout mice, injecting identical volumes and concentrations of protein subcutaneously . FollitrFollitropin delta and follitropin alfa display distinct pharmacokinetic and pharmacodynamic properties in healthy female volunteers, with follitropin delta displaying lower clearance from serum, producing greater exposure and a greater pharmacodynamic response . In addiin vitro. The two rFSH proteins displayed the same apparent binding affinity for the human FSH receptor, and virtually identical potency in the activation of the human FSH receptor in a HEK-293 cell-based assay, measuring the induction of cAMP production in response to agonist ligand. Differences in the clinical activity of the two rFSH proteins are unlikely to be caused by differences in their binding and activation of the FSH receptor.When compared directly it was found that follitropin delta and follitropin alfa elicit indistinguishable activity at the human FSH receptor in vitro responses in patient-derived luteinized granulosa cells, measuring FSH-induced hormone production and gene regulation. In addition to inducing progesterone production to similar levels, both rFSH proteins increased 3B-HSD expression, the enzyme responsible for the conversion of pregnenolone to progesterone , which mediates the rapid clearance from serum of glycoproteins containing terminal galactose, GalNac or alpha2,6 sialic acid, and GalNac with 2.6 sialic acid, the latter having a high affinity for the rat ASGPR , 34.Asgpr knockout mice compared to wild-type mice, further supporting the role of the hepatic ASGPR in clearance of follitropin delta but not follitropin alfa from serum. This difference in rFSH clearance from serum by the liver is likely to result in differing metabolic fates for the two rFSH proteins.Inhibition of ASGPR-dependent clearance in rats by co-injection of a saturating dose of ASF  reduced Other factors might also play a role in the distinct pharmacokinetics of follitropin delta. Human expression of hepatic ASGPR is lower than in mouse or rodent, suggesting less dependence on this clearance mechanism in humans . Renal cThe distinct properties of follitropin delta have important implications for its pharmacology and clinical use. The Steelman–Pohley assay for the determination of FSH bioactivity utilizes a recombinant reference standard  expressed in CHO cells. As a result, bioactivity in the Steelman–Pohley assay compared to the reference standard does not directly predict the pharmacodynamic response of follitropin delta in humans, as was observed. Dosing follitropin delta according to mass rather than bioactivity measured in rat is important as a result. This principle is being pursued in follitropin delta clinical development, with individualized dosing by mass optimized for each patient based on their AMH level and weight , 12. TheGlenn Croston was paid as a consultant by Ferring Pharmaceuticals. Daniel Plaksin is an employee of BTG Israel, a subsidiary of Ferring Pharmaceuticals. Claus Yding Andersen, Janni V Jeppesen and Kirsten T Macklon received funding from Ferring Pharmaceuticals. Wolfgang Koechling is an employee of Ferring Pharmaceuticals A/S.This research was funded by Ferring Pharmaceuticals and its subsidiaries.


As shown by genomic studies, colorectal cancer (CRC) is a highly heterogeneous disease, where copy number alterations (CNAs) may greatly vary among different patients. To explore whether CNAs may be present also in histologically normal tissues from patients affected by CRC, we performed CGH + SNP Microarray on 15 paired tumoral and normal samples. Here, we report for the first time the occurrence of CNAs as a common feature of the histologically normal tissue from CRC patients, particularly CNAs affecting different oncogenes and tumor-suppressor genes, including some not previously reported in CRC and others known as being involved in tumor progression. Moreover, from the comparison of normal vs paired tumoral tissue, we were able to identify three groups: samples with an increased number of CNAs in tumoral vs normal tissue, samples with a similar number of CNAs in both tissues, and samples with a decrease of CNAs in tumoral vs normal tissue, which may be likely due to a selection of the cell population within the tumor. In conclusion, our approach allowed us to uncover for the first time an unexpected frequency of genetic alteration in normal tissue, suggesting that tumorigenic genetic lesions are already present in histologically normal colonic tissue and that the use in array comparative genomic hybridization (CGH) studies of normal samples as reference for the paired tumors can lead to misrepresented genomic data, which may be incomplete or limited, especially if used for the research of target molecules for personalized therapy and for the possible correlation with clinical outcome.The online version of this article (doi:10.1007/s13277-016-5181-0) contains supplementary material, which is available to authorized users. Colorectal cancer (CRC) is a heterogeneous disease defined by distinct molecular signatures, distinct pathological features, and distinct natural histories. There are at least three major molecular pathways to CRC genetically identified by chromosomal instability (CIN), microsatellite instability (MSI), and the CpG island methylator phenotype (CIMP). The CIN pathway is characterized by structural rearrangements, amplifications, deletions, and copy number alterations (CNAs). The MSI pathway’s main feature is the mutation or aberrant methylation of DNA mismatch repair genes, whereas in the CIMP pathway, excessive promoter methylation occurs .Several groups have studied chromosomal aberrations in CRC by means of array comparative genomic hybridization (array-CGH) being the most common reported aberration gains on chromosomes 20q, 13q, 7p, and 8q and losses on chromosomes 18q, 17p, and 8p .More recently, the increase in genomic studies lead to the identification of copy number aberrations linked to adenoma-carcinoma progression or linked to metastases. For example, gains on chromosomes 7p, 13q, and 20p/q and loss on 18q were reported as early DNA alterations . Gains aCopy numbers of 3q, 10q, 11q, and 15q and Xp regions were reported as being linked to node metastases , whereasWhile several array-CGH analyses have been performed on colonic tumoral tissues, genetic analysis of histologically normal tissues is still lacking. In fact, in all the studies reported so far, colon normal tissues were used individually or pooled as reference for tumor but were never analyzed in itself, comparing different individual samples. Only one report shows small gains at 19q and 20q in pooled normal tissues , suggestTo address this very important issue, we analyzed by CGH + SNP Microarray the genome of 15 paired tumoral and histologically normal colonic tissues. Here, we report for the first time the occurrence of CNAs as a common feature of the normal tissue from CRC cancer patients and discuss its significance.A total of 15 paired samples of sporadic tumoral and surrounding histologically normal tissue (taken at a distance of 10 cm from the tumor) were obtained from patients undergoing surgery in three different centers. University of Milano-Bicocca Ethics Committee approval and written informed consent was obtained before tissue collection, but blood samples were not included. Staging and grading were done according to the World Health Organization Consensus Classification.For the array-CGH analysis, genomic DNA was extracted from fresh biopsies after enzymatic digestion with trypsin/EDTA 1X (EuroClone) at 37 °C for 60–90 min and proteinase K (Roche) at 37 °C for 30 min and purified using phenol/chloroform (Carlo Erba). Sample preparation, slide hybridization, and analysis were performed using CytoSure ISCA + SNP array 4x180K and CytoSure Cancer + SNP array 4x180K  according to the manufacturer’s instructions. Sex-matched commercial DNA samples (Promega) were used as reference DNA for normal samples, while normal paratumoral DNAs were used as reference for tumoral tissues. Tumoral samples were checked for the presence of at least 70 % of tumor epithelial cells by an expert pathologist on a slide of neighboring tissue. We collected fresh, consecutive, and unselected samples to perform array-CGH immediately after the histological diagnosis, without other information.DNA extraction from normal and tumoral paired fresh samples received from the different hospitals was carried out in our laboratory, where all the DNA quality control checks required by array-CGH protocol were performed at every step: after the purification with phenol/chloroform, after enzymatic digestion, and after labeling. No differences were observed among samples from different hospitals. All samples showed a suitable quality for the protocol standards.Cot-1 DNA, hybridization was performed at 65 °C with rotation for 40 h at 20 rpm.Briefly, the test and reference DNAs were denatured and labeled by random priming with Cy3-dUTP and Cy5-dUTP, respectively. Then, both samples were concentrated and then coupled according to the efficiency of labeling . Following probe denaturation and preannealing with The arrays were scanned at 2-μm resolution using an Agilent microarray scanner and analyzed using CytoSure Interpret Software.Experiments showed excellent DLRS values , so mosaic CNAs were also included in the study with a good confidence. To calculate the estimated percentage of mosaicism, we used the formula determined by Cheung et al. .We applied a filtering option of a minimum of four aberrant consecutive probes and a minimum absolute average log2 ratio that differs among all samples and depends to DLRS values, so it is related to the quality of experiment. In particular, non mosaic gains and losses are identified by standard log2 ratio values for all samples: values over 0.6, which correspond to three copies, identify non-mosaic gains; values under −1, which correspond to 1 copy, identify non-mosaic losses. Accordingly, log2 ratio values for mosaic gains range between the DLRS value and 0.6 and for mosaic losses between the DLRS value and −1. The loss of heterozygosity (LOH) regions were selected with a score above 200 by CytoSure Interpret Software.To identify the most represented ontology classes among the genes contained in normal gained regions, we used the GOstat software . The GO We performed array-CGH analysis on 15 paired tumoral and histologically normal samples obtained from patients affected by CRC. Males and females were equally represented: eight females and seven males. The mean age was 71.9 (range 52–85 years) and the majority of samples were adenocarcinomas (12 of 15). Tumors were mostly located in the ascending colon and hepatic flexure (40 %), following by sigmoid colon, descending colon, and rectum. The samples were almost equally distributed between stage II and stage III and between grade 2 and grade 3 , which was not present when samples were divided by grade or by tumor location (data not shown).The CNAs of histologically normal samples greatly varied among different patients ranging from 26 to 345 (average 160). Comparing the number of CNAs detected in normal tissues vs reference DNA, a statistically significant difference was evident between stage II and stage III samples , X (7 CNAs), 5, 7, 8, 10, and 17 (6 CNAs each). No shared CNAs were found on chromosomes 6, 18, and Y Table . NotablyShared CNAs could be divided into three types: (1) CNAs containing at least one tumor-associated gene Fig. , (2) CNARUNX1, a gene involved in translocations in different types of leukemia and generally considered a tumor suppressor in myeloid neoplasms. Among the CNAs shared by the great majority of the samples, there are also two gains that were reported as polymorphic in the Database of Genomic Variants (DGV), hence devoid of a pathogenetic role: a non-mosaic gain, localized on 14q32.33, containing KIAA0125 and ADAM6 genes, shared by 14/15 samples and one containing IGKV present in 12 cases. A large group of gains, shared by approximately half of the samples, included the oncogenes MPL , TRIB2 , ERBB2 , and ARAF  and other genes implicated in oncogenesis such as TERT , FGFR1 , and RECQL4 . Tumor suppressor genes such as PTCH1, DAB2IP, and WT1, usually lost in tumors, were also included in the gained regions of approximately half of the samples. Also, RET , CTSB , and CDKN2A  copy number gains were found, but they are included in DGV as benign variants.Interestingly, 13/15 samples showed a gain on 21q22.12 containing MYC oncogene and one on chromosome Xp21.3 containing the MAGEB18, MAGEB6, and MAGEB5 genes.To note, the only two normal samples, whose paired tumoral tissue has an undifferentiated histotype, shared two gains not present in any other normal sample, one on chromosome 8q24.21 including ERBB2, RECQL4, and MYC oncogene and MAGEB family has been frequently reported in colorectal carcinoma, as shown in a recent review [The copy number gain of t review . Twelve p < 0.05) GO terms. The most represented category was regulation of the biological process which comprises regulation of transcription, of gene expression, of growth, of apoptosis and of cellular metabolic process.Next, in order to find statistically overrepresented GO categories among the genes included in the gained regions Fig. , we perfp < 0.05, Student’s t test), but it was not present when the samples were divided by grade or by tumor location (data not shown). CNA distribution within the chromosomes was variable, being chr17, chr1, and chr2 those with a higher average number of CNAs . Comparing the number of CNAs in different tumoral samples (excluding the two samples with a higher number of CNAs), a statistically significant difference was evident between stage II and stage III samples and between stage IV and stage III samples  samples with an increased number of CNAs in tumoral vs normal tissue (five cases); (2) samples with a similar number of CNAs in both tissues (two cases); (3) samples with a decrease of CNAs in tumoral vs normal tissue (eight cases). Samples falling in the first group, such as those from patients 2 and 6, are characterized by increased genomic instability. Instead, in samples of the third group, a decreased genomic instability was evident , probably due to a selection of the cell population in tumoral tissues. From this last group, we analyzed CNAs present only in the tumoral tissue (unique) and disomic in the paired normal sample, assuming a possible role in tumor progression; only two CNAs shared by at least three of eight samples met this parameter: one comprising TRIB2 gene), 3q29 (MFI2), 10q11.21 (RET), 12q13.12 , 21q22.12 (RUNX1), Xq21.1 (BRWD3), Xq25 (GRIA3), Xq25 (XIAP). Most of these genes were reported to be involved in several types of cancer , we evaluated the distribution of the chromosome probes and identified the aneuploidies, also at mosaic Fig. .The most frequent aneuploidies in tumoral samples were as follows: loss of 18q ; loss of whole chromosome 18 ; gain of 20q ; gain of whole chromosome 20 ; gain of 8q ; gain of 13q ; loss of 17p ; gain of whole chromosome 7   and both gains of chr13 and chr20 (4 cases). One case showed chromothripsis on chromosome 13 and 15. No aneuploidies were found in the normal samples.http://www.ogt.co.uk).The slides used in our analysis combine array-CGH based CNA detection with fully research-validated SNP content, allowing confident and cost-effective CNA and loss of heterozygosity (LOH) identification using a single array , we found a copy-neutral LOH on region Xp21.3 which contains the LOH corresponding to the copy number loss are not reported because the concordance confirms deletion of interested region.Next, we analyzed a possible correlation between the age of patients and CNAs. In normal samples, a decrease of CNAs in patients less than 65 years and a small increase in those over 65 years was found, similar to those reported in literature for tumoral samples Fig. . At variMoreover, we analyzed the origin of the samples and noticed that normal and tumoral samples coming from one single hospital showed a higher  number of CNAs when compared to those coming from the other centers Fig. .Colorectal cancer is a highly heterogeneous disease and for this reason, several genomic studies have been performed, always analyzing in detail tumoral tissues using paired normal tissues as reference for the tumoral counterpart. Only one paper so far reported small gains at 19q and 20q in pooled normal tissues . In stroAccordingly, a very recent paper reported that transcriptional profiles of paired normal samples could be of great interest to better understand tumorigenesis mechanisms and progression and identify potential therapeutic targets .Our approach of performing CGH + SNP Microarray on 15 paired histologically normal and tumoral samples allowed us to find a high number of CNAs (average: 160) also in normal colonic tissues. Our experiments showed excellent DLRS values, so also mosaic CNAs were included in the study with a good confidence. The possibility to detect mosaic CNAs allows obtaining a representation of all sample subpopulations, not only the most represented. For this reason, we found a higher number of CNAs compared to some previous studies.Mosaicism is defined as the occurrence of more than one genetically diverse cell population in an organism arising from a single fertilized egg . SomaticRUNX1 gene is considered a tumor suppressor gene in myeloid neoplasm and was recently implicated in gastrointestinal tumorigenesis, as a novel tumor-suppressor gene, which maintains the balance between the intestinal stem/progenitor cell population and epithelial differentiation [RUNX1 were associated with colorectal cancer risk [In normal tissues many of affected genes involved or claimed to be related to the tumorigenic process. For example, the ntiation . Moreovecer risk .PTCH1, DAB2IP, and WT1 were included in the gained regions. Lower levels of PTCH1 mRNA were associated to high-risk for metastasis [PTCH1 promoter might be an early event of colon carcinogenesis [WT1 may act both as a tumor suppressor or oncogene in different context [Surprisingly, other tumor-suppressor genes, such as tastasis , and abeogenesis , thus le context . In colo context  and its  context .TRIB2, resulting from gene amplification, has been described in lung cancer with a potential role in tumorigenesis [RET promoter CpG island methylation is associated with a poor prognosis in stage II colorectal cancer [RET fusion kinases has also been identified [RET CNAs has been reported in colorectal cancer [MPL, TRIB2, and ARAF copy number gains have not previously been identified in colon samples.Traditionally, histologically normal tissue has been considered to be appropriate control for copy number studies as it was not thought to have copy number aberrations: unexpectedly, instead, our data showed several oncogenes with copy number gain. Overexpression of igenesis . RET prol cancer , but RETentified . Moreovel cancer . ConversMYC oncogene was found in two normal tissues, whose matched tumors have an undifferentiated histotype. Since this gain has not been found in other normal samples, we put forward the hypothesis that it may be considered as an early alteration for this tumor histotype.Finally, a mosaic gain on 8q24.21 containing RET gene, both in gain and in loss, as previously reported [APP gene was both in gain and in loss in different samples. Recently, increased expression of APP in several types of cancer has been reported and it has been implicated in proliferation and motility of advanced breast cancer [APP CNAs is shown in cancer.As for normal tissues, also in tumoral samples, we found CNAs containing reported . Also, at cancer . To our RBFOX1 and PCDH11Y genes. RBFOX1 belongs to a family of evolutionarily conserved RNA-binding proteins and regulates tissue-specific alternative splicing: deletion of RBFOX1 has been already reported in colon tumors [PCDH11Y instead plays a role during development of the central nervous system, but it has also been reported to be involved in prostate cancer progression [PCDH11Y can be present in an altered copy number in CRC.We speculated that CNAs present only in tumors and disomic in normal samples might have a possible role in tumor progression. For example, in tumoral tissues, we found two gained regions shared among at least three samples and containing n tumors  whereas n tumors . PCDH11Ygression . Ours isAs already noted, the use in array-CGH analysis of normal tissue as a reference for the corresponding tumor allows uncovering only alterations not equally represented in both tissue, leaving equal ones undetected and implying that CNAs present in both tissues are more amplified in the tumor; otherwise, they would have been scored as disomic.TRIB2, MFI2, RET, WNT1, DDN, PRKAG1, KMT2D, RUNX1, BRWD3, GRIA3, and XIAP. All these genes are known to be involved, at least to a certain degree, in different types of cancer: based on our findings, we suggest that they might have a critical role also in colorectal cancer onset and progression. Moreover, some of these genes are target of current clinical trials for other types of cancer, such as RET for non-small cell lung cancer, RUNX1 for leukemia, and XIAP for pancreatic and breast cancers , so they could be interesting targets also for colorectal cancer therapy.The identified gained regions contain Tumor copy number changes were also compared with data of the Cancer Genomic Atlas (TCGA) database, where all tumors were paired with normal tissue specimen which could be blood/blood components, adjacent normal tissue, or previously extracted germline DNA from blood . In the Loss of heterozygosity (LOH) occurs in a variety of tumor types and is considered important for tumor progression. After the loss of one allele, the remaining allele can be affected by somatic mutation or harbor a disease-prone polymorphic variant . It may Analysis of homozygote calls for each chromosome revealed a strong difference in LOH regions between normal and tumoral samples.MAGE family genes and interestingly, it is observed in copy number gain in two other normal samples. It is possible to assume that this region may be subject to genomic alterations in normal samples.Normal samples showed a lower number of LOH compared to tumoral samples, being only two copy-neutral LOHs shared between two different samples and located in Xp21.3 and Xq26.2. The Xp21.3 region contains TP53 mutation [APC gene. APC LOH was found in four samples accompanied by copy number loss in two cases and by the presence of two copies of the same regions in the other two samples; this identifies a copy neutral LOH. According to these data, a recent paper reported that most of sporadic colorectal carcinomas with LOH for APC return to copy number neutrality with the duplication of the remaining allele [Copy-neutral LOH was found on chromosome 5q for four tumoral cases. LOH of 5q was reported in ovarian cancer, frequently accompanied by mutation . It was mutation . In 5q rg allele . The copCNAs were also compared with age of patients and samples’ origin. It was reported that humans are commonly affected by somatic mosaicism for stochastic CNAs. The rate of CNA occurrence may differ between cell types or may be related to the age of individual cells . We founIn conclusion, our approach to perform CGH + SNP Microarray on paired normal and tumoral samples, allowed us to uncover for the first time an unexpected frequency of genetic alteration in normal colonic tissue from colon cancer patients, suggesting that (1) tumorigenic genetic lesions are already present also when the colonic tissue appear histologically normal; (2) the use in array-CGH studies of normal samples as reference for the paired tumors can lead to misrepresented genomic data, due the loss of identical CNAs between the two samples; and (3) even available array-CGH data may be incomplete or limited, especially if used for the research of target molecules for personalized therapy and for the possible correlation with clinical outcome.Table S1Shared copy number alterations in normal samples. (XLSX 16 kb)Table S2Statistically over-represented GO terms in gained genes. (DOC 64 kb)Fig S1Statistically over-represented GO terms in gained genes of normal samples. The most represented classes of GO terms are reported, divided into three categories: biological process, molecular function and cellular component. (GIF 34 kb)High resolution image (TIFF 247 kb)Fig S2Examples of graphical representation of log2 ratio averages and standard deviations of chromosome probes (case 1). This figure was automatically produced by CytoSure Interpret Software and showed log2 ratio averages and standard deviations for all chromosomes in patient 1 normal and tumoral samples. (GIF 231 kb)High resolution image (TIFF 2302 kb)Fig S3Distribution of CNAs according to patients’ age. Blu dots indicate the number of CNAs in normal samples, pink dots in tumoral samples and green dots in tumoral samples reported in the literature. Lines indicate the trend line. (GIF 27 kb)High resolution image (TIFF 363 kb)Fig S4Distribution of CNAs according to different hospitals. Box plots representation of copy number alterations in different hospitals. The first and third quartiles are at the ends of the box, the median is indicated with an inside line of the box, and the maximum and minimum value are indicated with dots. (GIF 17 kb)High resolution image (TIFF 287 kb)


The pain caused by surgical procedures performed routinely for managing livestock husbandry is recognised as a significant animal welfare issue for food security. In recent years, there has been progress encouraging the uptake of pain relief in extensively managed livestock operations, with research and development offering options for the practical delivery of anaesthetics and analgesics during these procedures. In Australia, topical anaesthetic and buccal meloxicam treatments are now commercially available for use during routine surgical husbandry procedures of lambs and calves. A study to assess the effect of these treatments on weight gain and behavioural variables following concurrent surgical castration and amputation dehorning in beef calves is reported. Results showed that a combination of topical anaesthetic and buccal meloxicam appeared to reduce pain following castration and dehorning, with improved weight gain and increased lying activity in the first few days following the procedures. In addition, some individual behaviours expressed by the calves on the day of treatment suggested pain was relieved by topical anaesthetic and buccal meloxicam, although further clarification of this observation is required. These findings demonstrate that provision of topical anaesthetic and buccal meloxicam to beef calves undergoing surgical castration and amputation dehorning can result in improved animal welfare and production.p < 0.001). CONN calves spent less time lying compared to BMTA calves on all days (p < 0.001). All dehorned and castrated calves spent more time walking (p = 0.024) and less time eating (p < 0.001) compared to CONP calves. There was a trend for CONP calves to spend the most time standing and CONN calves to spend the least time standing (p = 0.059). There were also trends for the frequency of head turns to be lowest in CONP and BMTA calves (p = 0.098) and tail flicks to be highest in CONN and BM calves (p = 0.061). The findings of this study suggest that TA and MEL can potentially improve welfare and production of calves following surgical castration and amputation dehorning.The use of pain relief during castration and dehorning of calves on commercial beef operations can be limited by constraints associated with the delivery of analgesic agents. As topical anaesthetic (TA) and buccal meloxicam (MEL) are now available in Australia, offering practical analgesic treatments for concurrent castration and dehorning of beef calves, a study was conducted to determine their efficacy in providing pain relief when applied separately or in combination. Weaner calves were randomly allocated to; (1) no castration and dehorning/positive control (CONP); (2) castration and dehorning/negative control (CONN); (3) castration and dehorning with buccal meloxicam (BM); (4) castration and dehorning with topical anaesthetic (TA); and (5) castration and dehorning with buccal meloxicam and topical anaesthetic (BMTA). Weight gain, paddock utilisation, lying activity and individual behaviours following treatment were measured. CONP and BMTA calves had significantly greater weight gain than CONN calves ( Bos indicus breeds are dominant and herds are managed on extensive areas of land [Bos indicus breeds [Dehorning and castration are routine husbandry procedures that are performed in the global cattle industries. These procedures are of particular importance to northern Australian beef properties, where  of land . Dehornis breeds . Castrats breeds . On thess breeds . The infs breeds . Pain ass breeds . Althougs breeds . ® , containing lignocaine and bupivacaine, is now commercially available for use during various livestock husbandry procedures, including application to mulesing and tail docking wounds in lambs [® Buccalgesic OTM , is registered for use during surgical castration of lambs [The need for provision of a practical method of delivering pain relief in livestock systems has been recognised for over a decade in Australia. Following extensive research, the topical anaesthetic gel Tri-Solfenin lambs ,4,5 and in lambs ,6,7,8. Sof lambs  and calvof lambs . The topof lambs . The TA Bos indicus weaner calves in an extensively managed system. We hypothesised that TA and BM would improve weight gain and reduce pain-related behaviours following concurrent surgical castration and amputation dehorning, especially when administered in combination. This study aimed to assess the effects of TA and MEL, separately and in combination, on weight gain and behavioural variables following concurrent surgical castration and amputation dehorning of Bos indicus or Bos indicus crossbred weaner bull calves (approximately 6–8 months of age). All animals were sourced from a commercial beef herd in QLD, Australia and were undergoing routine weaning and marking (as previously described). One week prior to commencement of experiment 1, all calves were mustered, separated from their mothers and held in a set of ‘weaning yards’ with ad libitum access to water and lucerne hay, as is commonly practiced on northern Australian beef herds.The experimental protocol was approved by the Animal Ethics Committee of the University of Sydney  and was conducted in accordance with the guidelines of the ‘Australian code for the care and use of animals for scientific purposes’ . Two expFor both experiments, calves were randomly allocated to one of five treatments in the order that they were processed by use of random numbers generated in Microsoft Excel 2007 : (1) no castration or dehorning/positive control (CONP); (2) castration and dehorning/negative control (CONN); (3) castration and dehorning with pre-operative buccal meloxicam (BM); (4) castration and dehorning with intra-operative topical anaesthetic (TA); and (5) castration and dehorning with pre-operative buccal meloxicam and intra-operative topical anaesthetic (BMTA). There were 50 calves per treatment group for experiment 1. A subset of these calves (20 per treatment group) was fitted with global positioning system (GPS) units and a further subset of these calves (10 per treatment group) was fitted with accelerometers. In experiment 2, there were 12 calves in the CONP and BMTA treatment groups and 11 calves in the CONN, BM and TA treatment groups.Experiment 1 was performed over 7 days, from the day of treatment (day 0) to 6 days post-treatment (day 6). On day 0, calves were processed through a race where they were weighed using cattle scales, Livestock Manager TSi 2  within the cattle crush, Ultimate Crush . They were restrained in a head bale for ear tagging and ear notching. BM and BMTA calves were treated with buccal meloxicam (MEL) at this point. Calves were then moved through a separate race to a weaner cradle  where they were restrained in left lateral recumbency for treatment and attachment of GPS and accelerometer units. TM GPS units (17 × 25 × 5 mm) , designed for use on domestic cats and their attached battery packs (17 × 20 × 49 mm), were placed in acrylonitrile butadiene styrene plastic Jiffy enclosure boxes (130 × 68 × 44 mm), Jiffy box  and secured in place with Styrofoam. The boxes were then enclosed with their supplied lids and fixing screws and attached to Gripwell luggage straps (25 mm × 2 m) , using zip ties. On day 0, a single luggage strap was secured around the neck of each animal with the plastic box positioned on the upper right side of the neck to ensure the GPS antenna was unobstructed from satellite signals. In addition, the plastic boxes were fixed in place on the neck of the animal with Parfix® fast grip contact adhesive . On day 6, the luggage straps and boxes were quickly and carefully removed from the cattle by cutting the straps using a knife. These GPS units were used as they are lightweight (22 g) and low-cost and therefore practical and cost-effective for tracking greater numbers of individual animals [Commercially produced CatLog animals .HOBO Pendant G Acceleration Data Loggers , were inserted into pieces of foam sponge and secured on each calf to the lateral aspect of the right hind leg proximal to the fetlock using 3M™ VetRap™ Bandaging Tape  and Norton Bear 50 mm × 15 m Silver Cloth Tape . The units were positioned such that the x-axis was perpendicular to the ground and pointing ventrally, the y-axis was parallel to the ground and pointing cranially and the z-axis was parallel to the ground and pointing toward the midplane. 2) where they remained until the last animal was processed. All calves except CONP calves were castrated and dehorned and TA and BMTA calves were treated with topical anaesthetic (TA), as described below, whilst still restrained in the cradle. Calves were released into another holding yard (300 m2) where they remained until 06:00:00 a.m. the following day when they were moved to a large paddock (619 ha) for a further 6 days. During this time, calves had ad libitum access to pasture and water. On day 6 at 06:00:00 a.m., calves were mustered back into the holding yards adjacent to the handling facilities and processed through the first race. Whilst in the race, GPS and accelerometer units were removed, then the calves were weighed in the cattle crush and released.The marking process commenced at 07:30:00 a.m. and was concluded by 05:00:00 p.m. hours. When all calves had been processed, they were moved into a laneway (700 m2) for behavioural recording, as described below. This process commenced at 07:30:00 a.m. and concluded by 08:30:00 a.m.Experiment 2 was conducted over 3 days , with 17 calves (3–4 per treatment group) treated on day A and 20 calves (4 per treatment group) treated on days B and C. Each day, calves were processed as per experiment 1. Calves were individually numbered on both sides and the back of the body with Dy-Mark Spray & Mark 350 g Spray Paint  while in the race. Following treatment, calves were released into a holding yard . Dehorning was conducted by opening the cup, placing it over the horn, applying downward pressure and closing the handles to remove the horn tissue and immediate surrounding skin. The scalpel blade and the cup dehorner were chemically sterilised between use on each animal.®  was administered  by a single experienced technician via a hook nozzle into the buccal pouch for absorption through the oral mucosa. Buccal meloxicam was administered 1 to 2 and 0.5 to 1 h prior to castration and dehorning, for experiments 1 and 2, respectively.For the MEL, a gel formulation of meloxicam (10 mg/mL) as Ilium Buccalgesic®  was administered by a single, experienced technician via a spray applicator, where approximately 4 mL was applied for castration and another 4 mL for dehorning. For castration, TA was applied following extrusion of the testes and prior to severing the spermatic cords, by inserting the nozzle into the tunica vaginalis and delivering the product into the inguinal canal. For dehorning, it was applied directly onto the wounds immediately following the procedure. The method of application aimed to cover all injured tissue, including the spermatic cords which retract into the inguinal canal following the procedure.For the TA, a gel formulation containing lignocaine (40.6 g/L), bupivacaine (4.2 g/L), cetrimide (5 g/L) and adrenaline (24.8 mg/L) as Tri-SolfenWeight gain was calculated for each calf using the difference of the pre-treatment weight collected on day 0 and the post-treatment weight collected on day 6.TM software  to record a positional fix every 10 s using the Navstar global positioning system from 10:00:00 a.m. on day 0 for the entire experimental period (7 days). Location information was downloaded using the CatLogTM software and exported into Microsoft Excel 2007. Only positional fixes recorded whilst all animals were in the paddock were included. Hence, all positional fixes before 08:00:00 a.m. on day 1 and after 11:59:59 p.m. on day 5 were disregarded. Positional fixes that were located outside the paddock boundary, which included a 40 m buffer to accommodate for possible large location errors associated with down antennas, short-fix intervals and sky obstructions [GPS units were programmed using CatLogructions , were reructions  were remructions  using thructions .x-, y- and z-axes every 10 s from 10:00:00 a.m. on day 0. The loggers recorded until the memory was filled at 22:13:00 h on day 2. Following removal of the loggers, the data was downloaded using the Onset HOBOware software which converted the g-force readings into degrees of tilt. The data was then exported into Microsoft Excel 2007 and the degree of tilt on the x-axis was used to determine whether or not the calves were in a lying position at each 10-s reading. All data points prior to 12:00:00 p.m. on day 0 were removed as the last accelerometer unit was attached at 11:45:00 a.m. Tilt values >120° were interpreted as standing and tilt values ≤120° were interpreted as lying. These thresholds were based on values used in previous studies on dairy cows [The accelerometer loggers were pre-programmed using Onset HOBOware software  to record the g-force on the iry cows ,17 and aad libitum access to water and lucerne hay. Six HD 1080p Sports Action Cam video cameras , were attached at various points along the fence of the yard to capture video footage of the calves. Cameras were placed strategically to capture footage from all angles of the yard. This footage was later used to continuously record the frequency or duration of certain specified behaviours displayed by each animal in 5-min focal samples at 6 time points . The frequency and duration of behaviours were recorded by a single, trained observer using. The Observer® XT 12 observational data software package . The observer was blinded to treatment, although it was clear which calves were CONP calves due to the presence of intact horns. An ethogram was designed using The Observer® XT software whereby behaviours were categorised as states or points . For weight gain, outliers within treatment groups were identified using the boxplot procedure of Genstat®. A linear mixed models procedure was used to analyse data on weight gain, paddock utilisation and observed state behaviours. A generalised linear mixed models (GLMM) procedure with a binomial distribution was used to analyse data on lying activity generated from accelerometer readings. A macro was used in Microsoft Excel 2007 to calculate the frequency of lying bouts and average duration of lying bouts. A GLMM procedure with a Poisson distribution was used to analyse data on frequency of lying bouts and a linear mixed models procedure was used to analyse data on average duration of lying bouts. A generalised linear mixed models (GLMM) procedure with a Poisson distribution was used to analyse data on observed point behaviours. For weight gain (experiment 1), the fixed effect of the model was treatment . For paddock utilisation (experiment 1) the fixed effects of the model were treatment  × day . For total lying activity (experiment 1), frequency of lying bouts and average duration of lying bouts, the fixed effects of the model were treatment  × day  + BW (variate). For each observed behaviour  were missing upon the second weighing and 7  were identified as outliers within their treatment groups using the boxplot procedure of Genstatp < 0.001). CONP and BMTA calves had significantly greater weight gain values than CONN calves. CONP calves also had significantly greater weight gain values than BM and TA calves . While there was a significant effect of day on paddock utilisation (p < 0.001), this is not presented nor discussed further due to acknowledged logging time and duration differences across days.As part of the data ‘cleaning’ procedures, 8.4% of the total data points were removed; 16.5%, 4.1%, 4.6%, 11.9% and 3.7% of the data points were removed for treatment groups CONP, CONN, BM, TA and BMTA, respectively. There was no significant effect of treatment on paddock utilisation (p = 0.724). There was a significant interaction between treatment and day (p < 0.001) on total lying activity. CONN calves spent the least proportion of time lying and BMTA calves spent the greatest proportion of time lying on all days. All other calves spent an intermediate proportion of time lying compared to CONN and BMTA calves on all days. The proportion of time spent lying increased from day 0 to day 1 for all calves and again from day 1 to day 2 for all calves except CONP calves . There was no significant effect of treatment on the frequency of lying bouts or the average duration of lying bouts . While there was a significant effect of day on the average frequency of lying bouts and the average duration of lying bouts , this is not presented nor discussed further due to acknowledged logging time and duration differences across days.There was no significant effect of BW on the frequency of lying bouts or the average duration of lying bouts , one from time point 2  and 4 from time point 6 . Behaviours influenced by time only are neither presented nor discussed. As the behaviours ‘walk with a stiff gait,’ ‘walk with a limp,’ ‘stand statue’ and ‘lie abnormal’ occurred infrequently, it was decided to only analyse the behaviours ‘walk,’ ‘stand’ and ‘lie,’ instead of their modifiers . The behaviours head pawing and kicking occurred too infrequently for statistical analysis. p = 0.006) displayed by the calves. The frequency of ear flicks was significantly greater in TA calves than in CONP, CONN and BMTA calves at 120 min and significantly greater in BM calves than in TA calves at 240 min (There was a significant effect of treatment × time on the frequency of ear flicks ( 240 min . p = 0.049) and tail flicks (p = 0.04) displayed by calves. CONP calves displayed significantly less head turns than TA calves. CONP and TA calves displayed significantly less tail flicks than CONN and BM calves (p = 0.024), eating (p < 0.001) and drinking (p = 0.002). The duration of time spent walking was significantly less in CONP calves than in CONN and BMTA calves and significantly greater in BMTA calves than in BM and TA calves. The duration of time spent eating was significantly greater in CONP calves than in all other calves and significantly less in TA calves than in BMTA calves. The duration of time spent drinking was significantly greater in CONP calves than in BMTA calves . Calves treated on day 1 spent a greater duration of time drinking compared to calves treated on days 2 or 3 (p < 0.001), head turns (p < 0.001), ear flicks (p < 0.001), stamps (p = 0.022) and tail flicks (p < 0.001) displayed by calves. Calves treated on day 1 displayed more head shakes, head turns and ear flicks than those treated on days 2 and 3. Calves treated on days 1 and 2 exhibited more foot stamps on than those treated on day 3. The frequency of tail flicks decreased each day . The results presented demonstrate that a combination of TA and MEL can be a cost-effective addition to routine practice, whilst improving animal welfare . For exaIn cattle, GPS technology has mainly been used to monitor grazing behaviour ,39,40. TAccelerometers have been used to record activity of calves following surgical castration , disbuddBos Indicus beef calves [Bos Indicus calves following concurrent castration and dehorning.Observation of individual behaviours has previously been used to measure pain following castration ,21, dehof calves . In addif calves . TherefoBos indicus cattle which are usually unaccustomed to humans and handling [This study may be the first documented examination of the effects of TA and MEL following concurrent castration and dehorning of weaner calves. It should be considered that the stressful experiences of handling, weaning and concurrently performed surgical procedures may have had an effect on the results of this study, especially as these calves had none or very little prior interaction with humans and handling facilities. The importance of conducting studies that closely represent current industry practice and the possible changes to it has previously been acknowledged  and is ehandling ,23. In experiment 1, a significant improvement in weight gain was seen following castration and dehorning when a combination of TA and MEL had been administered at the time of marking, resulting in no difference between CONP and BMTA calves. This experiment also found a combination of TA and MEL increased lying activity in the first few days following treatment, suggesting a reduction in pain. In experiment 2, there were trends for TA and the combination of TA and MEL to reduce pain-related behaviours during a 6-h period following castration and dehorning that warrant further investigation. Overall, an improvement in weight gain, an increase in lying activity and behavioural trends indicative of efficacy demonstrate the potential for TA and MEL to improve welfare and production following castration and dehorning of beef calves. This is an important finding for large, extensive tropical beef production systems that are seeking practical options for improving animal welfare.


Drosophila melanogaster lacking functional cSOD exhibit a suite of phenotypes including decreased longevity, hypersensitivity to oxidative stress, impaired locomotion, and reduced NADP(H) enzyme activity in males. To date, cSOD-null phenotypes have primarily been characterized using males carrying one allele, n108redcSod, in a single genetic background. We used ANOVA, and the effect size partial eta squared, to partition the amount of variation attributable to cSOD activity, sex, and genetic background across a series of life history, locomotor, and biochemical phenotypes associated with the cSOD-null condition. Overall, the results demonstrate that the cSOD-null syndrome is largely consistent across sex and genetic background, but also significantly influenced by both. The sex-specific effects are particularly striking and our results support the idea that phenotypes cannot be considered to be fully defined if they are examined in limited genetic contexts.Mutations often have drastically different effects in different genetic backgrounds; understanding a gene’s biological function then requires an understanding of its interaction with genetic diversity. The antioxidant enzyme cytosolic copper/zinc superoxide dismutase (cSOD) catalyzes the dismutation of the superoxide radical, a molecule that can induce oxidative stress if its concentration exceeds cellular control. Accordingly,  We hypothesized that differences in the ROS state across cSOD activities would manifest as variation in response to sex and genetic background, with different genetic backgrounds potentially ameliorating or enhancing the phenotypic effects associated with the cSOD-null syndrome. Our results show that large-scale differences in cSOD activity result in pervasive phenotypic changes, and that these changes are significantly modified, both enhanced or suppressed, by differences in sex and genetic background. Higher levels of cSOD activity generally resulted in phenotypes closer to WT, which were more susceptible to modification by sex and genetic background.D. melanogaster Genetic Reference Panel (DGRP) lines established by the Mackay Lab  possesses the n108redcSod null allele on the 3rd chromosome (T5) possesses this same null allele on the 3rd chromosome in conjunction with a cSod transgene on the 2nd chromosome, under control of the native cSod promoter, creating a whole organism transgenic rescue line  and a middle range (30 and 80% of WT activity flies). Distinct schemes were designed to extract both 2nd and 3rd chromosomes from the T0 , where nTOTAL is the total number of flies, nTOP is the number of flies above the 5 cm line, and nBOTTOM is the number of flies below the 5 cm line (Adult negative geotaxis (hereafter simply “geotaxis”) was measured using an assay modified from i.e., animals possessing 30, 50, and 80% cSOD activity). Such a reduction in resolving power could lead us to detect no difference across backgrounds, even if small differences exist. Therefore, we conducted a second locomotor assay, the countercurrent climbing assay , the vials were tapped down, and the flies given 1 min to climb. Following 1 min, the top set of vials was shifted over, the vials tapped down, and the flies were allowed 1 min to climb . This process was repeated a total of seven times, with vials unopposed by other vials plugged to prevent flies from escaping . Flies were classified by climbing ability based on the vials they were in at the end of the trial: poor = vial 1, moderate = vials 2–4, or good = vials 5–8 /(n−1)ƩNk, where n = the number of climbing classes, Nk is the number of flies in the kth climbing class, and the climbing classes were assigned the k values poor (k = 1), moderate (k = 2), and good  as described by For all enzyme assays, adult flies were collected 48 hr post eclosion, aged for 3 d, and frozen at −80° in single-sex, single-genotype groups of four flies. Prior to homogenization, samples were weighed to the nearest 0.01 mg with a microbalance . All enzyme reactions were performed in a standard 96-well microtiter plate and absorbance measured with a microplate spectrophotometer .+), centrifuged at 13,000 rpm for 12 min at 4°, and the supernatant collected. Within each microplate well, the reaction mixture consisted of 10 μl of sample supernatant and 100 μl of the assay solution . NADP(H) produced was quantified as an increase in absorbance measured at 340 nm. For the MEN and IDH reactions, absorbance was measured at 25° every 9 sec for 3 min. For the G6PD reaction, absorbance was measured at 25° every 9 sec for 5 min. Enzyme activities for each genotype sample were calculated from the mean enzyme activity of three technical replicates. The total number of animals assayed was 1184 flies and 1248 flies, for the 0 and 50% comparison and the 30 and 80% comparison, respectively. Genes for MEN and IDH in D. melanogaster are located on the 3rd chromosome, while the gene for G6PD is located on the X chromosome. Therefore, differences in the 2nd chromosome genetic background will not change the MEN, IDH, and G6PD alleles present in the experimental flies.NADP(H) enzyme assays were performed as described by cSOD activity was quantified using a commercial assay kit  following the manufacturer’s suggested protocol with minimal modification. Briefly, fly samples were homogenized in chilled 20 mM HEPES buffer  at a ratio of 100 μl/fly and then diluted to a final ratio of 400 μl/fly. Fly homogenates were centrifuged at 4° for 10 min at 3000 rpm and the supernatant collected [samples are centrifuged at lower rpm than the NADP(H) samples to prevent MnSOD protein from pelleting]. Prior to assaying, the supernatant was diluted to a ratio of 1:4 supernatant to assay sample buffer. For each sample, two distinct reactions were performed to determine cSOD activity: one for total SOD activity and one for MnSOD activity. cSOD activity is then calculated as the difference between the two. cSOD activity is calculated this way because the cSOD assay cannot distinguish between the two cSOD isoforms, so an inhibitor is required to determine the activity of a single isoform. For total SOD activity (MnSOD and cSOD), the reaction mixture consisted of 10 μl of the diluted sample supernatant, 200 μl of radical detector, and 20 μl of xanthine oxidase. For MnSOD activity, each reaction consisted of 10 μl of the diluted sample supernatant, 190 μl of radical detector, 20 μl of xanthine oxidase, and 10 μl of 5 mM of sodium cyanide (which inactivates cSOD via destabilizing the Cu/Zn complex). All reactions were incubated at room temperature for 30 min and then endpoint absorbance was measured at 25° and 450 nm with a microplate spectrophotometer. Each sample was assayed twice for each reaction  and sample SOD activities were calculated using the equation generated from the SOD standard (bovine erythrocyte Cu/Zn SOD) curve. The total number of animals assayed was 636 flies and 624 flies, for the 0 and 50% comparison and the 30 and 80% comparison, respectively.Total protein concentration was quantified with a bicinchoninic acid assay using a commercial kit  with modifications following Post hoc comparisons were carried out using Tukey’s HSD test. All analyses were performed using JMP 12 statistical software. Separate comparisons were performed on data from crossing Scheme 1 (0 and 50% cSOD activity) and crossing Scheme 2 (30 and 80% cSOD activity) as they were conducted independently and the genotypes obtained from these crosses differed (due to the presence or absence of the cSOD transgene on the 2nd chromosome in the T0 and T5 lines). However, general trends across all four activity levels and the two comparisons were noted.ANOVA was used to partition phenotypic variation between cSOD activity, sex, genetic background, their interactions, and the error variance [y = cSOD + sex + genetic background + cSOD*sex + cSOD*genetic background + sex*genetic background + cSOD*sex*genetic background + Error; 2p) was used to estimate the magnitude of variation attributable to each factor and interaction. η2p measures the proportion of variation attributable to a particular factor, while removing variation that is explained by other predictor variables. η2p was determined using the equation η2p = SSFactor / SSFactor + SSError , using two separate crossing schemes (File S1). Large-scale differences in cSOD activity are thus a function of the overall cSOD genotype with only small, statistically insignificant variation across the genetic backgrounds. Hereafter, we refer to the different genotypes by their predicted (and observed) cSOD activities: 0, 50, 30, and 80% cSOD activity. Interestingly, in the 30–80% comparison there was a significant effect of sex on cSOD activity, with activity significantly lower in males than females, though sexual dimorphism in cSOD expression is not observed at the native cSOD locus , possibly reflecting the severity of the cSOD knockout condition. However, the lack of modifying effects on the cSOD-nulls is phenotype-specific as there were significant sex and genetic background effects in other phenotypes (below).As expected, scores of the life history phenotypes were lowest at 0% cSOD activity and higher at moderate cSOD activity . Scores File S1). Male and female 80% cSOD activity flies lived longer than 30% cSOD activity flies. As in the low-cSOD comparison, females lived longer than males across cSOD activities, with the magnitude of sexual dimorphism in longevity varying across genetic backgrounds . Male and female 50% cSOD activity flies had better locomotor ability than the cSOD-nulls, across both phenotypes . Interestingly, males had better geotaxic ability than females at both cSOD activities, though the magnitude of the sexual dimorphism varied across genetic backgrounds . While there was no significant effect of sex on countercurrent locomotion in the low cSOD comparison, we noted a similar trend to that observed in geotaxis; males had better climbing ability than females .As with the life history phenotypes, performance in both locomotor assays was lowest at 0% cSOD activity and higher at moderate cSOD activity . LocomotFile S1). This difference is consistent with earlier studies (File S1). Interestingly, genetic background had a larger effect on geotaxis in the 0–50% comparison than in the 30–80% comparison . In contrast, the opposite trend was observed in life history and countercurrent locomotion (even if the effect was not significant).In the 30–80% comparison, cSOD activity had a large effect on geotaxis and countercurrent locomotion, where overall locomotor ability in both phenotypes at 30% cSOD was lower than at 80% cSOD . However studies , and potIn calculating the CF for countercurrent locomotion we binned flies into three classes: poor, moderate, and good climbers, essentially grouping flies with “similar” performance. To determine whether this binning was masking sex or genetic background effects, we recalculated the CF values using the vials as single classes . This unbinned analysis also indicated no significant genetic background effects. The unbinned analysis indicated a significant effect of sex in the 0–50% comparison, and the trend of males having better climbing ability than females was observed in both analyses. Overall, binning seemed to have a limited effect on the analyses.File S1). Males tended to have higher NADP(H) enzyme activity than females, as long as some cSOD activity was present . Strikingly, only G6PD activity was modified by genetic background in the 0–50% comparison, with the effect of genetic background varying between males and females across cSOD activities .The NADP(H) enzymes also responded to cSOD activity levels, but in contrast to the life history and locomotor phenotypes, which require 50% cSOD activity to recover WT phenotypes, 30% cSOD activity was sufficient to restore the biochemical phenotypes to WT activities . ConsistFile S1). To determine if the biochemical results were simply a function of the covariate used (protein concentration), we repeated the biochemical analyses using fly mass and protein concentration as covariates and found essentially identical results to those using protein concentration alone (data not shown).In addition, and again comparable to life history and locomotor phenotypes, the effect of cSOD activity on the biochemical phenotypes tended to be smaller in the 30–80% comparison than the 0–50% comparison . ConsistD. melanogaster has shown that complete loss of cSOD activity modifies a broad suite of phenotypes, but that as little as 50% of normal cSOD activity is generally sufficient to fully restore WT phenotypes , consistent with other studies , and the life history and locomotor phenotypes as “distal” (phenotypes far from the mutation). As distal phenotypes are further downstream of the mutation, we expected them to be a function of a larger number of genetic and environmental interactions than the proximal phenotypes. However, interactions between MEN, IDH, and G6PD , and betThe strength of interaction between cSOD and each phenotype likely reflects the degree of connectedness between cSOD and the molecular mechanisms of each phenotype, presumably indirectly through the role of cSOD and NADP(H) in the antioxidant defense network  among females, potentially confounding analysis of such phenotypes. However, cSOD and associated phenotypes have primarily been studied in males . Fitness and longevity vary across the parent DGRP lines . Interestingly, countercurrent locomotion was less sensitive to genetic background or sex than was geotaxis . The countercurrent assay is more of an endurance, rather than reactive, assay than the geotaxis assay . While all these enzymes share the NADP(H) cofactor, each component of this network responds differently to genetic, metabolic, and environmental changes, and interactions across the network are complex and not simply compensatory  network, in which changes in activity of one enzyme cause changes in activities of the other enzymes, likely through pools of the shared NADP(H) cofactor , and the lines we created will only reveal dominant modifiers at 2nd chromosome loci. Extracting “high” and “low” line chromosomes from the parent lines altered their genetic context that may have modified the interactions that led to their high or low line classification. Further, we were constrained to only assaying 2nd chromosome heterozygotes, preventing us from observing any recessive interactions. One general conclusion across all the phenotypes is that genetic background effects were smallest in the cSOD-nulls, suggesting that this extreme condition limits these effects, but others [reviewed in Even with the caveat of the restriction to dominant modifiers, we were surprised that no background substantially suppressed or enhanced the cSOD-null phenotypes. Genetic variation at the 2nd chromosome background modified, but did not extensively change, the phenotypic variation that we observe. The fact that the parent DGRP lines show substantial variation for each phenotype indicates that the genetic variation present is capable of modifying the phenotypes. Thus, the lack of large-scale variation in our derived lines likely reflects a complex genomic architecture in background effects, suggesting that these effects are themselves complex and involve dominant and recessive interactions between loci across the genome.The cSOD-null syndrome is driven by the absence of cSOD activity, but is sensitive to, and modified by, sex and genetic background. While smaller in magnitude than the cSOD effects, background and sex did significantly affect phenotypes, including notable sex-specific effects, the magnitude of genetic background effects vary strikingly with the level of cSOD activity and phenotype. Consistent with this phenotype-specific sensitivity, the threshold amount of cSOD activity required to recover a phenotype to WT also differs, with life history and locomotor phenotypes having a higher threshold cSOD activity than the biochemical phenotypes. Surprisingly, no genetic backgrounds were found that resulted in large-scale enhancement or suppression of cSOD-dependent phenotypes. Finally, our results indicate that male and female flies substantially differ in their phenotypic responses to some factors, reinforcing the point that both male and female subjects, of any species, need to be assayed to understand biochemical and physiological processes.www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.043836/-/DC1.Supplemental material is available online at Click here for additional data file.


Our data showed that the SML reflected the SSW during most parts of the year, with changes mainly responding to bloom formation and decay. OM composition during phytoplankton blooms clearly differed from periods of higher bacterial abundance. Of all components investigated, only the enrichment of total carbohydrates in the SML was inversely related to the wind speed indicating that wind-driven mixing also affected the accumulation of OM in the SML during our study.The sea surface microlayer (SML) is the uppermost layer of the water column that links the ocean and atmosphere. It accumulates a variety of biogenic surface-active and buoyant substances, including gelatinous material, such as transparent exopolymer particles (TEP) and Coomassie stainable particles (CSP), potentially affecting air–sea exchange processes. Here, we studied the influence of the annual cycle of phytoplankton production on organic matter (OM) accumulation in the SML relative to the subsurface water (SSW). Sampling was performed monthly from April 2012 to November 2013 at the Boknis Eck Time Series Station . For SML sampling, we used the Garrett screen, while SSW samples were collected by Niskin bottles at 1 m depth. Samples were analyzed for carbohydrates, amino acids, TEP, CSP, chlorophyll  The sea surface microlayer (SML) is the thin boundary layer between the ocean and the atmosphere and chemically and is physically different from the subsurface water (SSW). It is formed by a variety of surface-active and buoyant organic compounds that tend to accumulate at the air–sea interface . It is c−1  located near the entrance of the Eckernförde Bay  in the southwestern Baltic Sea, ~1 nautical mile off the coast  encountered over the year. The screen technique allows for collecting a relatively large amount of samples in a short period of time suitable even at the high wind speeds (Samples of the SML and SSW were collected monthly from April 2012 to November 2013 around the noontime from the weatherboard of the research vessel d speeds . BrieflyThe samples were placed cold (~4°C) and in the dark for ~3 h while carried to the home laboratory. Subsamples for dissolved carbohydrates and amino acids were filtered (see below) in the ship's laboratory immediately after sampling. All glass equipment involved in sample storage was previously cleaned by washing with 10% HCl and rinsing with ultrapure Milli-Q water (Elga).−1). The calibration factor changed three times during the study period, being 149 ± 22 from April to December 2012, 165 ± 24 from February to July 2013 and 158 ± 24 from August to November 2013. Stained TEP were examined under a compound light microscope equipped with a digital AxioCam MRc camera (Zeiss) at 200× magnification. About 30 images per filter were taken in a cross-section. Enumeration and sizing of gel particles (>0.2 μm2) was carried out with the aid of the image analysis software WCIF ImageJ.TEP were quantified both microscopically  and coloCSP were determined microscopically according to Both TEP and CSP filter blanks were prepared with Milli-Q water processed as seawater samples. The standard deviations between the replicate filters were generally <30% for the microscopic parameters , with higher values (50%) for few replicates. The replicates of the colorimetric method agreed within 25%.For bacterial cell counts, 4.5 mL subsamples were fixed with 200 μL glutaraldehyde 25% solution and then stored at −20°C until analysis. Bacterial cells were detected by flow cytometry following 2 atmosphere at 50°C and resuspended with ultrapure Milli-Q water. Determination of CHO monomers was carried out by high-performance anion exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) on a Dionex ICS 3000 system. Their separation was achieved by a Dionex CarboPac PA10 analytical column (2 × 250 mm) coupled to a Dionex CarboPac PA10 guard column (2 × 50 mm). This method allows measurement of fucose (Fuc), rhamnose (Rha), galactosamine , arabinose (Ara), glucosamine (GlcN), galactose , glucose (Glc), combined mannose and xylose (Man/Xyl), gluconic acid (GlcA), galacturonic acid  and glucuronic acid (Glc-URA). Due to coelution, mannose and xylose were measured as the sum of each other. The standard deviations of replicates were typically <20% for the majority of individual CHO, except galactosamine which diverged by 40% in total and by 30% in dissolved fractions.Carbohydrates (CHO) were determined by ion chromatography according to The ratio of the amino sugars glucosamine and galactosamine  was calculated as it may indicate the potential source of these compounds . Thus, a2, the ampoules were sealed and placed at 110°C for 24 h. The hydrolyzates were then dried under N2 atmosphere with the following resuspension in ultrapure Milli-Q water (repeated two times). Precolumn amino acids derivatizaton with o-phthaldialdehyde was used to obtain fluorescent derivatives of the individual amino acids, which were then separated on a Kinetex (Phenomenex) C18 150 mm-length column (2.7 μm particles). The eluent composed of the binary solvent system with (A) 0.01 M monosodium phosphate adjusted with NaOH to pH 7 and (B) acetonitrile. Asparagine and glutamine were deaminated by the hydrolysis and were quantified as aspartic acid (AsX) and glutamic acid (GlX). Glycine (Gly), serine (Ser), alanine (Ala), tyrosine (Tyr), valine , isoleucine (Iso), phenylalanine (Phe), AsX, GlX and leucine (Leu) replicates agreed within 10%, except threonine (Thr), arginine (Arg) and γ-aminobutyric acid (GABA), with the standard deviations <20%.Amino acids were determined by high-performance liquid chromatography (HPLC) following the approach described by a (Chl a) were collected as part of the monitoring program and measured using the fluorometric method of a were vacuum-filtered (<200 mbar) onto Whatman GF/F filters  whereupon Chl a was extracted in 90% acetone in the dark and analyzed on a Trilogy® fluorometer (Turner Designs). Prior to analysis, the fluorometer was calibrated with a Chl a standard .Samples for chlorophyll www.pangaea.de).All data are available at the PANGAEA database .The extent of the SML enrichment in organic material relative to the SSW, i.e. the enrichment factor (EF), was estimated by the ratio of the compound parameter in the microlayer to that in SSW.U-tests were applied to assess statistical differences between the SML and SSW components as well as to statistical comparison of EFs with unity. We considered that null hypotheses testing and correlations were significant at P < 0.05. Results are presented as mean value ±1 standard deviation (SD).Calculations and statistical tests were performed with Microsoft Office Excel 2010 equipped with statistics Add-In WinSTAT. Mann–Whitney Principal component analyses (PCAs) were done with NCSS 2004 software package . PCA is a multivariate regression analysis that allows the description of multivariable data by means of a few number of latent variables called principal components. This method is frequently used to determine compositional trends in seawater and sediment OM, in particular, to explore the extent of sample degradation . Wind speed varied from 1.0 to 15.6 m s−1, with an overall average of 7.0 ± 4.4 m s−1  occurred during sampling from April to August 2012, while calmer conditions (4.3 ± 2.7 m s−1) were met on most other sampling days, with periods of particularly low wind speed (<1.6 m s−1) in October 2012, March and August 2013.The temperature and salinity profiles Fig. a and b ia (Chl a) concentration in the SSW ranged from 1.5 to 9.0 μg L−1  and in April 2013 (up to 4.3 μg L−1), indicating the appearance of pronounced autumn and spring phytoplankton blooms. Bacterial abundance ranged from 1.9 to 31.7 × 105 cells mL−1 both in the SML and in the SSW. Increased values of bacterial abundance were observed throughout summer 2012 and spring–summer 2013  than that in 2013 (up to 31.7 × 105 cells mL−1).Chlorophyll L−1 Fig. d, with h3 to 39 × 103 mL−1, while their total area  and, to a lesser extent, also during the winter (November–February) periods. The abundance of TEP was significantly correlated with the total area in the SML  and in the SSW . For the SSW, TEP abundance also exhibited a significant correlation with bacterial abundance .Under the microscope, TEP determined in the SML and in the SSW appeared as small globules, strings or sheets of semitransparent material Fig. a and b. rea Fig. d ranged 3 to 41 × 103 mL−1and their total area . The total area of CSP revealed frequent oscillations, without long periods of higher and lower values. Correlation between the abundance and the total area of CSP was low but still significant . Similar correlations were also found between CSP and bacterial abundances . Furthermore, the abundance of CSP demonstrated a significant temporal coupling to that of TEP, with r2 = 0.57  in the SSW and r2 = 0.46  in the SML.Shapes of CSP were similar to those of TEP, however, some CSP clearly appeared as aggregates of smaller particles Fig. c and d. col) ranged from 102 to 223 μg Xeq. L−1. Both SML and SSW were enriched in TEP concentration seasonally (March–June), not repeating the winter extreme obtained by microscopy. In the SSW, TEPcol were significantly coupled to bacterial abundance throughout the year .TEP concentrations Fig. e determi−1. Dissolved fractions (DCCHO) comprised 29–86% and 45–95% of TCCHO in the SSW and the SML, respectively, with the lower percentages occurring in the course of the elevated carbohydrate abundance from July to December .TCCHO concentrations Fig. a varied The highest percentage among individual sugars had Glc with up to 81 Mol%, followed by the sum of Man and Xyl with up to 32 Mol%. Fuc, Gal, GlcN and Rha contributed up to 16 Mol% each, while all other sugars were generally less abundant (<4 Mol%). The sugar composition showed large variations among the samples collected at different seasons both in the SSW and in the SML. The most pronounced changes in carbohydrate composition were observed between November 2012 and August 2013, when Mol% of Glc decreased by a factor of 3 and Mol% of deoxysugars , Gal and GlcN increased by a factor of 4 to 7.Over the study period, the ratio of GlcN to GalN  varied between 11 and 762. On average, GlcN:GalN in TCCHO was slightly higher for the SSW (130 ± 169) than for the SML (110 ± 168) samples. For DCCHO, the difference between SML and SSW was even more pronounced with GlcN:GalN being 163 ± 238 in the SSW and 119 ± 238 in the SML. The most notable differences between SML and SSW were observed in June 2012, March and November 2013 (DCCHO only), when the ratios differed by a factor of 2–9 Fig. c and d.−1 .THAA concentrations ranged from 1.0 to 4.4 μmol L−1 Fig e. In gen−1 Fig f. The miThe composition of amino acids was characterized by the dominance of Gly, which contributed up to 35 Mol%. Less abundant were AsX, GlX, Ser and Ala, with the contribution up to 19 Mol% each. Thr comprised up to 11 Mol%, and the other amino acids were more scarce (<8 Mol%). The amino acid composition varied with seasons; however, changes were not as pronounced as those of sugars. Generally, variations in Mol% of the most abundant amino acids, including Thr, varied <2-fold (data not shown).col concentrations (data not shown). The highest EF was determined for abundances and total areas of gel particles with maximum values of 2.6 and 6.9, respectively, for TEP, and 1.5 and 2.8 for CSP. The results of Mann–Whitney U-tests, however, showed that none of the measured parameters was generally enriched in the SML .About 50% of the samples for carbohydrates and DHAA were not enriched significantly in the SML relative to the SSW, having EF between 0.8 and 1.2 Fig. . The othr2 = 0.38 . We also tested for an exponential relationship between the enrichment of TCCHO and the wind speed. The results indicated a slightly better correlation with r2 = 0.42 .No correlation between EF values of all organic components, excluding TCCHO, and wind speed was determined during this study. For TCCHO, EF showed a significant negative linear correlation, albeit with a relatively low coefficient of determination, The enrichment of each group of compounds, including TCCHO, varied during all three dates of low wind speed detected. Thus, EFs for amino acids and carbohydrates were higher during the calm sampling day in October 2012, but were around unity during the other calm events (March and August 2013). The abundance of TEP conversely showed elevated EF in March and August 2013, whereas it was less than unity in October 2012. Enrichment in CSP was observed only in March 2013.r > 0.70) were determined particularly for TCCHO and TEPcol concentrations as well as for the abundances of CSP.Table U-tests showed statistically insignificant difference .The pairs of the SML and the SSW data sets compared by Mann–Whitney a.Figure Loadings of individual sugars of TCCHO on PC1 in SML and SSW are shown in Fig. The PC1s in DCCHO were mainly related to a fewer number of individual sugars, as compared with TCCHO Fig. e. These The overall picture given by PC1 shows the temporal variations in carbohydrate composition, reflecting bloom development. In the course of the phytoplankton blooms, carbohydrates contained higher Mol% of Glc and lower Mol% of Fuc and Ara. In contrast, lower Mol% of Glc and higher Mol% of Fuc and Ara corresponded to the bacterial blooms Fig. g. Each oAlthough, the PC1 scores for THAA in SML and SSW differed from those in TCCHO, the same division of the data set in two parts remained Fig. b. The fiFive individual amino acids Gly, Leu, Iso, Phe and Tyr showed high loadings on the PC1s Fig. d in THAAContrary to THAA, the PC1 scores for DHAA did not reveal a clear division of the data set based on the association with the microorganism's abundances Fig. f. FurtheIt has previously been shown that different techniques used for SML collection yielded samples that differed in chemical composition and concentration , OM seems to be mixed well between the SML and SSW demonstrating no response to a further increase of wind. −1, similar to our observation.Wind speed is thought to be an important factor controlling the enrichment of particulate material in the SML; it was previously shown that enrichment of both particulate organic carbon and particulate organic nitrogen was negatively correlated with wind speed indicating that increased turbulence can remove particulate material from the SML  and by EWithout turbulent mixing, the enrichment of SML is generally determined by the surface activity of organic material depending, amongst others, on water pH, temperature, irradiance and bacterial degradation  and dinoflagellates species (mainly Ceratium spp.) make up the third stage of plankton community growth. This stage resembles the first one, with similar levels of phytoplankton biomass and rates of sedimentation. Finally, winter dormancy (January–February) with low phytoplankton biomass represents the fourth stage of the cycle.Seasonal fluctuation of biomass and phytoplankton species composition is an annually recurring feature in most temperate coastal systems, including Boknis Eck, where it has been described in detail . The firltic Sea . Over tha concentration and bacterial abundance data, we assume that the sequence of bloom stages similar to those described above occurred during the present study . Previous studies showed that the percentages of extracellular release during phytoplankton growth ranged from <14% for diatoms to <43% for dinoflagellates . Biddandfeeding” .Both grazing and bacterial degradation can lead to particulate and dissolved OM that is compositionally different from the phytoplankton source. The most common differences were reported to be among the relative abundances of Glc and Gly . In phytExudates released during phytoplankton blooms can also cause at least transient effects on composition of accumulated OM. It is known that exudates are complex exopolymeric substances (EPS) composed of carbohydrates and proteins, with carbohydrates representing a substantial fraction of the total release . SeveralCompositional variations associated with the vernal development of the different bloom stages explained the largest variance in TCCHO, DCCHO and THAA data sets, indicating that both production and consumption processes modify the molecular composition of OM. Examining the first principal component (PC1), we found that such modifications led to compositions included the higher Mol% of Glc and amino acids Leu, Iso, Phe and Tyr on the one side and the higher Mol% of Gly and sugars Fuc, Rha, Ara and Gal on the other side. As the former of these compositions occurred over the first and the third stages, it was primarily characterized by new production. In contrast, the latter of these compositions was generally represented over the second and the fourth stages indicating its association with degradation by consumption. Similar results, derived from OM in sediments , suggesting that TEP contributed to material comprised of these sugars. Alternatively, the increase in mole percentages of deoxysugars and Ara could also be caused by the presence of large amounts of degraded material corresponding to a notable bacterial abundance.Higher mole percentages of deoxysugars and Ara coincided with increased concentrations of TEPcol Fig. g, as wel−1, ≤3.8·108 particles mL−1, Euphausia pacifica and copepod Calanus pacificus can partially contain TEP  suggesting that TEP and CSP dynamics were coupled. A similar finding has previously been reported by The abundance of CSP was generally similar to that of TEP Fig. a and c, In the present study, both concentration and composition of OM was clearly controlled by the seasonal cycle of pelagic production and consumption. Consequently, the dynamics of OM in the SML reflected seasonal changes in SSW. The varying OM quality may influence the ability to form surface films at the air–sea interface with seasonal change. Future studies are needed to understand if and to what extent OM variations in the SML affect air–sea exchange processes.SML collected at the Boknis Eck Time Series Station in most cases showed slight enrichment in carbohydrates, amino acids and gelatinous material, TEP and CSP. The enrichment of total carbohydrates decreased with the wind speed, supporting the idea that the wind could partially control OM distribution during this study. Higher enrichment of amino acids occasionally coincided with calm wind periods, while some calm periods did not reveal significant enrichment in OM indicating that factors other than wind influenced the SML. Wind induced turbulent mixing, creating conditions for rapid exchange between SML and SSW, is however an important process to balance the loss of OM from the SML. Changes in OM concentration and composition in the SML mainly responded to the annual cycle of plankton production as observed for the water column, originating from both production and consumption processes. Thereby, phytoplankton derived OM was characterized by higher fractions of Glc in the pool of combined carbohydrates and by Leu, Iso, Phe and Tyr in the pool of combined amino acids. More degraded organic material had higher fractions of the amino acid Gly, of the deoxysugars Fuc and Rha, as well as of the neutral sugars Ara and Gal. Apart from degraded material, the higher fractions of deoxysugars and Ara may reflect increased TEP concentrations. CSP occurrence was closely associated with that of TEP, suggesting that temporal and spatial dynamics of both types of gels were coupled at our study site. So far, consequences of compositional change in the SML on air–sea exchange processes, such as gas-exchange, are far from being understood. This study suggests that compositional changes in OM at the air–sea interface are closely related to seasonal variations in the ecology of the underlying water column. Thus, for a better understanding of feed-backs between the ocean and the atmosphere ecological processes and ecosystem changes need to be considered.