Early deafness leads to enhanced attention in the visual periphery. Yet, whether this enhancement confers advantages in everyday life remains unknown, as deaf individuals have been shown to be more distracted by irrelevant information in the periphery than their hearing peers. Here, we show that, in a complex attentional task, a performance advantage results for deaf individuals.We employed the Useful Field of View (UFOV) which requires central target identification concurrent with peripheral target localization in the presence of distractors – a divided, selective attention task. First, the comparison of deaf and hearing adults with or without sign language skills establishes that deafness and not sign language use drives UFOV enhancement. Second, UFOV performance was enhanced in deaf children, but only after 11 years of age.This work demonstrates that, following early auditory deprivation, visual attention resources toward the periphery slowly get augmented to eventually result in a clear behavioral advantage by pre-adolescence on a selective visual attention task. Several studies have demonstrated that early auditory deprivation (deafness) results in specific, compensatory changes in visual processing. In particular, deaf individuals exhibit enhanced performance for tasks performed in the visual periphery. Accordingly deaf individuals asked to make a key press in response to stimuli presented in the central or peripheral visual field, exhibit faster RTs than hearing individuals for peripheral targets but not for central ones advantages when it comes to more complex visual tasks is, as yet, unknown. We consider here the possibility that enhanced peripheral attention may result in better or worse performance depending upon the task demands. Deaf individuals are more distracted than their hearing peers by irrelevant information occurring in the visual periphery Whether this enhancement confers The performance of deaf and hearing individuals on a computerized adaptation of the Useful Field of View task UFOV;  was evalThe majority of studies reporting enhancement of visual attention to the periphery have recruited deaf individuals born deaf to deaf parents who learned American Sign Language (ASL) as a first language. This leaves open the possibility that enhancements in attention are restricted to this sub-population and do not generalize to the deaf population at large. This is of concern as most studies reporting deficient visual attention have focused on deaf non-signers. Therefore, in addition to recruiting deaf native signers, we included deaf individuals who experienced early auditory deprivation but did not learn a sign language. In addition, the impact of sign language was further evaluated using hearing subjects, born to deaf parents, who acquired ASL as a first language. Some of the studies referenced above have included hearing native signers, and have suggested that sign language use is not sufficient to induce enhanced peripheral attention This research was approved by the Research Subjects Review Board at the University of Rochester, NY.Each subject was assessed using a modification of the UFOV paradigm All three tasks were presented within a circular gray field subtending 21° of visual angle. Each stimulus display was followed by a visual noise mask presented on the whole screen and then a prompt appearing at fixation. Participants indicated their response (in speech or sign) for the central task, and the experimenter manually entered that response. For the peripheral localization response, participants touched the screen at the location where they believed the peripheral target to have appeared. Trials were classified as correct if the subject accurately identified both the central icon's identity and the location of the peripheral target . An adaptive staircase procedure was employed for all three tasks – after three consecutive correct responses, the stimulus duration was reduced by 1 frame (1/60 second); one incorrect response resulted in the stimulus duration being increased by 1 frame. Each task finished after twelve reversals, ten consecutive correct trials at ceiling (1 frame), or 72 trials, whichever occurred sooner. A threshold measure was calculated by averaging the stimulus duration of the last 10 correct trials. In the divided attention training and the UFOV tasks – which required both central and peripheral responses – trials where the central target was incorrectly identified were ignored .Stimuli were presented using Matlab software and the Psychophysics Toolbox installed on a Apple G4 Titanium laptop computer running OS 9.2.2. The laptop was connected to a 23″ Apple Cinema Display via an Apple ADC-DVI adaptor, with a 60 Hz refresh rate. The display was adapted to function as a touch screen using pressure-sensitive resistive (PSR-1®) technology, supplied and fitted by Troll Touch Touchscreens .Subjects were tested in a single experimental session lasting approximately 25–30 minutes. Subjects were in a chin rest, positioned 40 centimeters from the center of the touch screen. Instructions were given in sign or speech and clarified if necessary. Subjects were given the correct answer on the first 2–3 trials if they still appeared to be confused.diff) are reported alongside statistical test results and estimates of effect size .All statistical tests were conducted with an α = .05. Confidence intervals for differences between group means  were recruited at a school in Austin (TX) and at a camp in Madison (SD), as well as from participant pools at RIT/NTID (NY) and Gallaudet University (DC). All were deaf native signers who reported being born with severe-profound auditory deprivation  to deaf parents from whom they learned ASL as a first language. In the absence of a reliable and easily administered ASL proficiency test, subjects were asked to rate their ASL comprehension and production proficiency on a scale from 1 = perfect to 4 = hardly. All deaf signers gave themselves a rating of 1.0 in ASL comprehension and 1.0 in ASL production.Deaf adult signers  were students recruited at the National Technical Institute for the Deaf (NTID) in Rochester, NY. All reported being born with severe-profound auditory deprivation . Although most reported knowing some ASL, their first regular exposure to ASL had been at NTID where they were recruited for this study during their first quarter in order to limit that exposure. Accordingly, they reported an inability to communicate clearly in ASL (on average rating themselves 3.3 in ASL comprehension and 3.2 in ASL production). All deaf non-signing subjects preferred testing to be conducted using spoken English.Deaf adult non-signers  were recruited from a summer camp for KODAs (‘kids of deaf adults’) in Eagle Bay, NY. All reported learning ASL from their parents as infants, and expressed competence in ASL (on average rating themselves 1.8 in ASL comprehension and 1.8 in ASL production). None reported any hearing loss, and all testing was conducted in ASL.Hearing adult signers born to deaf parents  were recruited from a participant pool at the University of Rochester, NY. All reported normal hearing and no knowledge of any sign language.Hearing adult non-signers  it was important to establish that the central task was attentionally demanding in this context, and thus in competition with the peripheral target for attentional resources. While this task provided no independent, concurrent measure of central task performance, identification accuracy was calculated for the last 1/3 of trials for all subjects see . Due to 2 = .24, CI95diff = 8–30 ms). Deaf subjects demonstrated a clear advantage over hearing subjects, requiring less time to concurrently discriminate a central target and localize a peripheral target embedded within a field of distractors. An effect of sign language use was not predicted, and although a trend can be seen for sign language users to have lower thresholds than non-signers, the effect was much smaller and not statistically significant  = 3.02, p = .091, partial η2 = .08, CI95diff = 2–21 ms). There was no significant interaction between auditory deprivation and signing  = 0.18, p = .677, partial η2 = .01) confirming the primary role of auditory deprivation in the advantage noted in the deaf population.UFOV thresholds (i.e. with distractors present) were entered into a two-way ANOVA with auditory deprivation  and signing  as between subjects factors see . This re2 = .26, CI95diff = 2–6 ms; effect of signing: F = 9.52, p = .004, partial η2 = .21, CI95diff = 1–6 ms; interaction between auditory deprivation and signing: F = 6.94, p = .012, partial η2 = .16; divided attention task: effect of auditory deprivation: F = 41.40, p<.001, partial η2 = .54, CI95diff = 3–7 ms; effect of signing:  = 31.25, p<.001, partial η2 = .47, CI95diff = 3–6 ms; interaction between auditory deprivation and signing: F = 33.65, p<.001, partial η2 = .48). Deaf non-signers performed significantly worse on both of these tasks, albeit still requiring less than 33 milliseconds of presentation. This is in accord with reports from Quittner and colleagues Although the two other tasks  were included for training purposes, deaf non-signer participants differed from the other groups in a manner worthy of note . On both2 = .15, CI95diff = 4–39 ms).UFOV thresholds were reanalyzed with each subject's performance on these training tasks as covariates. The pattern of findings did not change, with the main effect of auditory deprivation remaining the sole significant effect  = 6.21, p = .018, partial ηThis first experiment establishes the role of auditory deprivation in the enhancement of peripheral visual attention noted in the deaf population. Both deaf signing and deaf non-signing adults excelled at the UFOV task. This shows that the enhancement is not limited to the use of isolated targets but generalizes to complex tasks such as the UFOV, which combines selective visual attention with the requirements of performing two tasks . Although deaf non-signers displayed better performance on the UFOV task than their hearing peers, they showed worse performance on the central stimulus identification and divided attention tasks. This result is surprising in the face of their enhanced performance on the UFOV task. The two training tasks differ from the main UFOV task along several dimensions preventing us from drawing firm conclusions. The central identification task focuses entirely on central processing, rather than peripheral processing in the context of an additional central task like in the UFOV task. The divided attention task requires both peripheral and central processing in the same manner as for the UFOV task, but it differs from the UFOV task in terms of its very low attentional load There are two alternative mechanisms that can be ruled out by the overall pattern of data reported. The first is that any deficits observed for deaf individuals stem from the need to make sequential manual responses (sign SHORT or LONG and then touch the screen) whereas hearing individuals can make a simultaneous oral-manual response (say “short” or “long” while touching the screen at the same time). If this were the case, then there should be a deficit for deaf signers across all tasks requiring two responses, which is clearly not the case. Despite the need to execute sequential responses for the two tasks, deaf signers outperform hearing subjects on the UFOV task, and show comparable performance on the divided attention task. Indeed, the deaf non-signers who performed poorly on the divided attention task made simultaneous oral and manual responses to the targets. The second alternative is a perceptual enhancement in the peripheral visual field of deaf individuals. Such an enhancement would predict enhanced performance on the divided attention task for all deaf individuals. To the contrary, deaf non-signers showed impaired performance on the divided attention task and deaf signers showed similar performance as their hearing peers. This pattern of finding reinforces the view that peripheral processing enhancements in deaf individuals result from changes in selective attention, and not perceptual modifications In Experiment 2 we ask at what age such a redistribution of attention becomes apparent in a sample of deaf children compared to a group of hearing peers 7 to 17 years of age. Deaf children were recruited from a camp and deaf school where ASL was the primary means of communication. The experimental design, apparatus and procedure were the same as those employed in Experiment 1. Previous studies suggest that visual selective attention skills are relatively stable in hearing subjects by 7–10 years of age Written informed consent was obtained from all children and a parent or legal guardian. All children were rewarded with a $15 gift card. As in Experiment 1, action video game players were tested but their data are not reported here.AGE = 9;1, 16 males), 11–13 year old middle school students , and 14–17 year old high school students .Hearing children were recruited from a school district in the Rochester NY area. Mailings were sent from the school district to parents of all children aged 7 to 17 years. The response rate was approximately 15%. All children had normal or corrected-to-normal vision and no known history of neurological or cognitive impairment. They were screened to ensure none required an Individualized Educational Program (IEP) indicating the need for accommodations due to learning or language impairment. Children were divided into three age categories: 7–10 year old elementary/primary students , 11–13 year olds , and 14–17 year olds . All had an unaided hearing loss >70 dB in their better ear and used ASL on a daily basis as their primary means of communication. None had undergone cochlear implant surgery. Sixteen (33%) had hearing parents, although all of these children had started to learn ASL in pre-K classes. Parental hearing status had no effect on the measures used, and is not considered further. Given their background, this group is more similar to the native signers adults described above, and differs in aetiology from the children typically considered in the literature on deafness, visual attention and cochlear implants Deaf children were recruited from deaf schools in Rochester NY and Austin TX, and a camp for deaf children in Madison, SD. School or camp administrators mailed letters to the parents of all children aged between 7 and 17 years, resulting in a 10% response rate. All children had normal or corrected-to-normal vision and no known history of neurological or cognitive impairment. Although most of the deaf children had IEPs as a result of their deafness, none had reported attentional problems or learning disabilities. The deaf children divided into the same age categories as hearing children: 7–10 year olds  and age group  = 6.49, p = .002, partial η2 = .11), and a significant two-way interaction between auditory deprivation and age group  = 7.10, p = .001, partial η2 = .11). This interaction led us to assess the effects of age group separately for deaf and hearing children. As predicted, for hearing children the UFOV thresholds did not vary as a function of age group  = 0.12, p = .884, partial η2<.01), whereas they did for deaf children  = 25.43, p<.001, partial η2 = .53). While deaf 7–10 year olds performed equivalently to hearing 7–10 year olds, older deaf children demonstrated better thresholds, outperforming their hearing peers and the youngest deaf children  and age group  as between subjects factors revealed significant main effects of auditory deprivation  = 17.85, p<.001, partial ηdren see .2 = .07), but a significant effect for deaf children  = 11.87, p<.001, partial η2 = .34). Deaf 7–10 year olds had significantly worse thresholds than both 11–13 year olds  and 14–17 year olds . Similarly, post-hoc analyses for the divided attention task showed no significant main effect of age group for hearing children  = 2.09, p = .132, partial η2 = .06), whereas it did significantly affect the performance of deaf children  = 7.17, p = .002, partial η2 = .24). As observed for the central identification task, deaf 7–10 year olds had significantly worse thresholds than 11–13  and 14–17 year olds . Using performance on the two training tasks as covariates did not change the qualitative pattern of findings for the experimental UFOV task.Interestingly, the training tasks indicated worse performance in the youngest deaf group compared to the other groups see . Post-hoand visual selection. Deaf adults required 43–58 ms (CI95%) of display presentation to perform at 79% correct, whereas hearing adults required significantly more time (CI95% = 60–79 ms). This enhancement reflects early, severe-profound loss of audition rather than use of a visual-spatial language – the effect was seen in both deaf signers and non-signers, with little-or-no contribution from signing.The UFOV task requires subjects to divide attention between central and peripheral locations, while also selecting a target from amongst distractors. It is an attention-demanding task, requiring not only central attention but also attention to the periphery Although a tendency for more effective visual search in deaf than in hearing individuals has been reported Data from children revealed that this attentional enhancement is not observed until after 7–10 years of age, although the precise point within this age group could not be determined due to sample size limitations. Nevertheless, there is the suggestion that a robust cross-modal enhancement in visual selective attention is not observed until after several years of auditory loss. Further study is required to identify exactly when and how this delayed enhancement is brought about. The lack of improvement observed in hearing children suggests that maturational factors are unlikely to contribute. Rather, it may be the duration of auditory deprivation that plays the key role, with over 7–10 years of auditory deprivation required for effects to be manifested behaviorally. Alternatively, it may reflect a ‘sleeper effect’ There is some evidence that the youngest deaf children found the central stimulus identification task to be more difficult than did their hearing peers, with this difficulty extending to the divided attention task. Data from deaf adults suggest that any such deficit is no longer apparent by adulthood, at least for those who have early and full access to a first language (deaf native signers). All of the young deaf children in this study had early language access through ASL, although the extent of their social and linguistic interactions with caregivers during early infancy cannot be assessed post hoc. It is important to note that studies reporting deficiencies in visual attention skills have typically used central visual field tasks employing rapid stimulus presentations with young deaf children This work establishes that auditory deprivation is not a causal factor for attentional difficulties. All deaf individuals tested performed at least as well and often significantly better than their hearing peers on the UFOV measure, an attentionally-demanding task Worse performance in the youngest deaf children and those deaf adults with limited access to a natural language early in development was noted under some conditions. While these results are in line with previous work documenting attentional deficits in deaf children, the present study makes it clear that such challenges early in childhood are not predictive of deficient functioning as development proceeds.Table S1Central task performance in selective UFOV task. For the UFOV selective attention task, mean central identification accuracies and mean stimulus presentation durations were calculated based upon the last 1/3 of trials for each subject. Accuracy levels indicate that the central task is attentionally demanding for all subject groups. However, accuracy cannot be compared directly across groups, as presentation durations differed. After normalizing accuracies as a function of presentation duration, performance did not significantly differ as a result of deafness or sign language use.(0.04 MB DOC)Click here for additional data file.Movie S1Two-alternative forced choice (2-AFC) discrimination task at the center of the visual field - a face icon was presented in the center of the screen and participants had to decide whether it had long or short hair.(0.42 MB MOV)Click here for additional data file.Movie S2The same 2-AFC central discrimination task as in (0.62 MB MOV)Click here for additional data file.Movie S3The 2-AFC central discrimination task with localization of a peripheral target presented at 20° of visual angle at one of eight possible cardinal/intercardinal locations and embedded in a field of distractors. The distractors appeared along the eight possible cardinal/intercardinal axes at 6.67, 13.33 and 20 degrees of visual angle.(0.52 MB MOV)Click here for additional data file.


The protein provides a powerful model for exploration of the structural molecular mechanisms of fibril formation from a full-length protein in vitro. Fibrils have been assembled from β2m under both low pH conditions, where the precursor is disordered, and at neutral pH where the protein is initially natively folded. Here we discuss the roles of sequence and structure in amyloid formation, the current understanding of the structural mechanisms of the early stages of aggregation of β2m at both low and neutral pH, and the common and distinct features of these assembly pathways.β This protein folds natively into a β-sandwich fold consisting of two β-sheets, one containing four strands (ABED) and the other three (CFG) . In vivo, β2m is the non-covalently attached light chain of the human major histocompatibility complex 1 (MHC1) 2m is shed into the serum and degraded by the kidneys, maintaining the serum β2m concentrations at 0.09–0.17 μM in healthy individuals 2m, resulting in increases in the serum concentration by up to 60-fold 2m as amyloid fibres in the synovium of joints.To comprehend the process of amyloid fibril formation in atomistic detail much work has been performed on a range of proteins in vitro. Here, we review some of the techniques employed and the results obtained in the study of the structural and molecular mechanisms of fibril formation of the protein βee (CFG) B [5]. A 2m fibril formation in vitro. We discuss the fibril formation pathways under different conditions, how they may be related to one another and to the mechanism occurring in vivo; and how the amyloid forming potential of β2m is modulated by sequence and structural changes.Here we consider the current state of research into the mechanism of β22m is highly amyloidogenic  2m in the MHC class I complex illustrates why the protein may contain such a highly aggregation-prone sequence. Aromatic residues including Phe56, Trp60, Phe62 and Tyr63 (as well as the hydrophobic residue Leu65), which all lie in the aggregation-determining sequence, make important contacts with the MHC I heavy chain (At low pH (<pH 3.0), low ionic strength (⩽50 mM) and encouraged by agitation, βvy chain D [5]. In32m fibril formation proceeds rapidly from a highly dynamic state with lag-dependent kinetics 2m polypeptide chains. Elongation then proceeds rapidly to form amyloid-like fibres by monomer addition At low pH β2m fibrils was gained in a study at pH 2.5 by total internal reflection fluorescence microscopy, which indicated that this process is unidirectional when the fibrils are fixed on a glass surface, implying a polarised aggregation mechanism Information towards understanding the elongation of β42m at neutral pH, since the protein is initially natively folded at pH 7. Biological components, including glycosaminoglycans, proteoglycans, collagen and Cu2+ (the latter in the presence of 1 M urea) have all been used to promote, or induce, fibril formation at neutral pH in vitro 2m at, or close to, neutral pH 2m, allowing the aggregation-prone regions of the polypeptide sequence to be exposed and to participate in intermolecular interactions.To mimic conditions that are more physiologically relevant, studies in vitro have adopted a number of methods to drive amyloid formation of β2m variants, P32G and P32V, have been used to show that wild-type β2m populates a native-like folding intermediate  en route to the native state that contains a non-native trans Pro32 bond 2m fibril seeds trans Pro32 residue is important for β2m to nucleate amyloid fibrils at neutral pH, as both P32G and P32V β2m which form trans Gly32 or Val32 cannot form amyloid-like fibrils spontaneously, although P32G can elongate stabilised seeds more efficiently than wild-type β2m under the conditions employed T and enabling fibril nucleation at pH 7.0 2m has been shown to form oligomers and fibrils at neutral pH by addition of Cu2+ and 1 M urea ∗, that appears identical to IT by size exclusion chromatography (SEC) The βT (or M∗), have provided molecular insights into the possible aggregation mechanisms of β2m at neutral pH. Burst-phase amplitudes from real-time NMR studies of β2m folding indicate that residues distal to Pro32 maintain native-like structure in the intermediate, while regions proximal to Pro32 in the B/C loop are disordered until trans to cis isomerisation of the Pro32 peptide bond takes place T , adjacent to the aromatic-rich, aggregation-prone region (∼60–70), and retains a native fold in vitro 2m that is highly unstable and frequently visits an unfolded state (such as V37A) does not engender amyloidogenicity alone The possibility that partial or global unfolding of the initially folded monomer of β72m aggregation both under denaturing and native conditions have provided important new insights into the structural changes associated with fibril formation for this protein that may have relevance for understanding amyloid in general. The sequence of β2m has a high amyloid forming potential as inferred from aggregation prediction algorithms and peptide studies Studies of the molecular mechanisms of β2m can form similar long-straight amyloid-like fibrils commencing from two very different structural states reinforces the view of a generic assembly mechanism. Under each set of conditions similar hydrophobic and aromatic regions of the sequence appear to be key to the aggregation mechanism. While reducing the stability of the native fold and increasing global or subglobal unfolding events is important in initiating amyloid formation, some structural preference, for instance the presence of a non-native trans Pro32, is clearly linked to the ability to aggregate. Even in the acid unfolded state it appears that specifically structured species presenting aggregation-prone regions are required for amyloid formation, whilst other transiently structured regions may modulate aggregation by sequestering sequences with high amyloid forming potential.The fact that many different proteins with unrelated sequences and native folds can all form amyloid-like fibrils with nucleation-dependent kinetics implies the possibility of a common mechanism. The finding that β2m amyloid fibrils will be required to provide unequivocal evidence for the convergence of the assembly pathways at pH 2.5 and 7.0 and to provide detailed information as to whether the protein is completely refolded in the fibril structure or retains remnants of its native fold. Towards this goal recent cryo-EM studies have shown a remarkably complex architecture for the fibrils formed from β2m at pH 2.5 2m have helped to confirm, the amyloid-forming potential of a protein is clearly a function of how structure modulates the intrinsic β-aggregation propensity of the amino acid sequence, not just for the natively folded protein but also for proteins that aggregate from highly unfolded initial precursor states.It is currently unclear whether the same oligomeric species are populated during assembly at acidic and at neutral pH and future endeavours will need to focus on the structural properties of the oligomers that form as aggregation proceeds. Such studies may provide clues as to how fibrillation can be impeded therapeutically and how fibril formation proceeds mechanistically. Indeed, whilst oligomers are commonly observed during fibril formation, there is as yet no definitive proof that such species are on pathway to the fibrillar form. Furthermore, atomistic insights into the structure of β


Saccharomyces cerevisiae chromosome III lacking all efficient origins, the 5ORIΔ-ΔR fragment, as a model for chromosomes with large inter-origin gaps. We used this construct in a modified synthetic genetic array screen to identify genes whose products facilitate replication of long inter-origin gaps. Genes identified are enriched in components of the DNA damage and replication stress signaling pathways. Mrc1p is activated by replication stress and mediates transduction of the replication stress signal to downstream proteins; however, the response-defective AQmrc1 allele did not affect 5ORIΔ-ΔR fragment maintenance, indicating that this pathway does not contribute to its stability. Deletions of genes encoding the DNA-damage-specific mediator, Rad9p, and several components shared between the two signaling pathways preferentially destabilized the 5ORIΔ-ΔR fragment, implicating the DNA damage response pathway in its maintenance. We found unexpected differences between contributions of components of the DNA damage response pathway to maintenance of ORIΔ chromosome derivatives and their contributions to DNA repair. Of the effector kinases encoded by RAD53 and CHK1, Chk1p appears to be more important in wild-type cells for reducing chromosomal instability caused by origin depletion, while Rad53p becomes important in the absence of Chk1p. In contrast, RAD53 plays a more important role than CHK1 in cell survival and replication fork stability following treatment with DNA damaging agents and hydroxyurea. Maintenance of ORIΔ chromosomes does not depend on homologous recombination. These observations suggest that a DNA-damage-independent mechanism enhances ORIΔ chromosome stability. Thus, components of the DNA damage response pathway contribute to genome stability, not simply by detecting and responding to DNA template damage, but also by facilitating replication of large inter-origin gaps.In eukaryotic chromosomes, DNA replication initiates at multiple origins. Large inter-origin gaps arise when several adjacent origins fail to fire. Little is known about how cells cope with this situation. We created a derivative of  Loss of genome integrity underlies aspects of aging and human disease. During DNA replication, two parallel signaling pathways play important roles in the maintenance of genome integrity. One pathway detects DNA damage, while the other senses replication stress. Both pathways activate responses that include arrest of cell cycle progression, giving cells time to cope with the problem. These pathways have been defined by treating cells with compounds that induce either replication stress or DNA damage, but little is known about their roles during unperturbed DNA replication. They may be important when several adjacent replication origins fail to initiate and forks from flanking origins must replicate longer regions of DNA than normal to complete replication. We have used a derivative of budding yeast chromosome III lacking all efficient replication origins to identify mutants that preferentially destabilize this chromosome fragment, which mimics a chromosome with a large inter-origin gap. We found that the DNA damage response pathway, but not the replication stress response pathway, plays an important role in maintaining this fragment. The signal recognized in this case may be replisome failure rather than forks stalled at endogenous DNA damage. In the budding yeast, Saccharomyces cerevisiae, replicators are termed ARS elements and were identified by their ability to promote extrachromosomal maintenance of plasmids. Chromosomal replication origins coincide with ARS elements, which contain the binding site for the six-subunit initiator complex, ORC. During G1, ORC recruits additional proteins to form pre-replicative complexes (pre-RCs) that initiate replication during S phase S. cerevisiae is approximately 40 kb, based on both electron microscopic analysis of replicating DNA molecules In eukaryotic chromosomes, DNA replication initiates at multiple origins, specified by The presence of multiple origins on chromosomes raises the question of whether replicators are activated according to a fixed temporal program or whether their use is stochastic, i.e. different replicators are activated in different cells or in successive S phases. In budding yeast, 2D-gel analyses and replication timing studies suggested that replicators are activated according to a program, although some variability is inevitable because some replicators fire inefficiently Stochastic activation of replicators should occasionally produce large inter-origin gaps caused by failure of adjacent origins to initiate, referred to as the random gap problem One possibility is that ongoing replication activates a checkpoint response to prevent cells from undergoing mitosis prior to completion of S phase. Two intertwined checkpoints function during S phase . The DNARAD9, the mediator in the DNA damage response pathway cdc6 (+cdc18 in S. pombe), which encodes a pre-RC component, or of dbf4, the regulatory subunit of the Cdc7p kinase required for origin firing, proceed directly from G1 to mitosis despite failing to replicate any DNA Activation of an S phase response may occur in cells coping with long inter-origin gaps. Rad53p, the ortholog of the mammalian and fission yeast effector kinase, Chk2 , becomes−3OFM1-1, and two recessive, ofm6-1 and ofm14  rad9 mutation increased the loss rate of the 5ORIΔ-ΔR fragment, but did not cause the frequent rearrangement that was seen with the YAC We have created a derivative of yeast chromosome III lacking efficient replicators as a tool to detect mechanisms that contribute to replication of large inter-origin gaps (the 5ORIΔ-ΔR fragment - Here we report the results of a modified synthetic genetic array (SGA) screen S. cerevisiae viable deletion collection. One limitation of this screen is that essential genes could not be tested.Our previous visual screen for Ofm mutants was labor intensive, during both the initial visual screening of colonies grown from the mutagenized culture and in subsequent attempts to identify mutations responsible for the phenotype. Thus, we adapted synthetic genetic array (SGA) technology ade2Δ::natR xxxΔ::kanR haploid MATa progeny. We then used chromoduction ADE2 into each strain . The three highest scoring clusters among the Ofm mutants (P = 8×10−5–4×10−3) share many genes and correspond to the annotations “cell cycle checkpoint”, “DNA damage response, signal transduction”, and “DNA damage checkpoint”. The cell cycle checkpoint cluster  included all of the genes present in the other two clusters. When the possible/probable Ofm mutants were included in the analysis the highest scoring cluster was still “cell cycle checkpoint” (p = 8×10−7). In addition to the 7 genes above, the cluster included BIM1, BUB1, BUB2, BUB3, CSM3 and TOF1. RAD9, RAD17, and RAD24 function in the DNA damage response pathway while MAD2 and MAD3 function in the spindle checkpoint, though some results have suggested an additional role in the DNA damage checkpoint BFA1 and BUB2 are required to prevent mitotic exit in both the DNA damage and spindle checkpoint pathways −6) among the non-Ofm mutants corresponded to the annotation “mitotic cell cycle”. This cluster included all five mutants identified as non-Ofm mutants.A gene ontology (GO) analysis was performed on the 52 genes whose deletion caused Ofm phenotypes and on the 5 genes whose deletion caused non-Ofm phenotypes /(401−1)  = 1. This is the outcome we might expect for a mutation in a kinetochore component, and we would not consider the mutant to be an Ofm mutant. In another case, a mutation causes the 0ORIΔ-ΔR derivative to sustain 10 additional loss events and the 5ORIΔ-ΔR derivative to sustain an additional 900 loss events. In this case the Ofm index  =  (1000−100)/(11−1)  = 90. We would consider this high Ofm index to indicate a specific defect in maintenance of the 5ORIΔ-ΔR fragment.Our premise in undertaking this screen is that problems with the replication of the 5ORIΔ-ΔR derivative may be qualitatively different than the problems sustained by the 0ORIΔ-ΔR derivative by virtue of the presence of a long inter-origin gap. Therefore, we wanted to be able to make a quantitative comparison of loss rates that are very different. We reasoned that a comparison of the number of additional loss events sustained by the 5ORIΔ-ΔR and 0ORIΔ-ΔR derivatives in a given mutant would provide a measure of the strength of the Ofm phenotype. We define the “Ofm index” as the number of additional loss events per 10rivative . Two exarad9 allele and the rad9Δ allele cause Ofm phenotypes, with mutants strains having Ofm indices of 81 and 65, respectively  caused a similar loss rate of the 5ORIΔ-ΔR fragment, but the 0ORIΔ-ΔR loss rate was about 10-fold lower than in the complete ORF deletion strain, suggesting that the N-terminus of Mrc1p may contribute to maintenance of the 0ORIΔ-ΔR fragment (data not shown).The corresponding mediator in the replication stress response pathway is Mrc1p, a homolog of mammalian claspin. Mrc1p plays roles in both the replication stress response and normal replication fork progression fm index ; the mrcAQmrc1, made by mutating six consensus Mec1p phosphorylation sites MRC1, but retains the fork progression function. Plasmids carrying either wild type MRC1 or AQmrc1 complemented the high loss rate of the 5ORIΔ-ΔR fragment in the mrc1Δ strain . Thus, activation of dormant origins is a general phenomenon in mrc1Δ strains that most likely reflects slow fork progression.We further tested the role of RAD17, which encodes a subunit of a PCNA-like clamp, and RAD24, which encodes the large subunit of its clamp loader  because the rad53Δ mutation caused an increase in the loss rate of the 0ORIΔ-ΔR fragment  all showed wild type sectoring in our primary screen , and ARS304 on the full-length 5ORIΔ fragment by 2D gel analysis . One possible mechanism for the production of these stable derivatives is that replication forks collapse and are processed into double-strand breaks that are repaired by break-induced replication Unlike the 5ORIΔ-ΔR fragment and the full-length 5ORIΔ chromosome, the ΔL-6ORIΔ fragment was structurally unstable. Stable derivatives that had lost the cloNAT-resistance marker present at left-hand end of the fragment  arose in strains . Twelve et al. ctf) mutants identified in these screens, 14 were also identified in our screen. As expected, given that the ctf mutants were identified using chromosome fragments carrying a normal complement of replication origins, the majority of the ctf mutants we re-identified were found in the non-Ofm or possible/probable Ofm classes. The two scored as Ofm mutants are ctf18Δ and mad2. It seems likely that many ctf mutants were not identified in our screen because they caused only small increases in the rate of loss of the 5ORIΔ-ΔR fragment. Approximately 60% of the loss rates measured for chromosome fragments in ctf mutants are less than or equal to the loss rate of the 5ORIΔ-ΔR fragment; increases of that magnitude would not have been detected in our visual screen.We employed a novel modification of the SGA method to screen for mutations that preferentially destabilize a chromosome III derivative lacking efficient replication origins. The modification utilized a single chromosome transfer technique, chromoduction, to transfer the 5ORIΔ-ΔR fragment into an ordered array of the viable ORF deletion collection. Yuen Our results indicate that the DNA damage signaling pathway, but not the replication stress signaling pathway, contributes to the maintenance of the 5ORIΔ-ΔR fragment. While the DNA damage and replication stress response pathways share many components , mutatioRAD53 than on CHK1. Based on the results presented here, the DNA damage signaling pathway also contributes to the replication of large inter-origin gaps, which can arise when several adjacent origins fail to fire. Such gaps appear commonly during the replication of the rDNA array CHK1 with a contribution from RAD53 evident in the absence of CHK1.We found unexpected differences in the contributions that the DNA damage signaling pathway makes to maintenance of ORIΔ chromosome derivatives and the contributions that it makes to DNA damage resistance. First, the DNA damage signaling pathway detects and stabilizes forks stalled at sites of damage and facilitates repair or bypass of the damage; studies with DNA damaging agents mec1Δ and mrc1Δ mutations have different effects than rad9Δ and rad24Δ mutations on the stabilities of the ΔL-6ORIΔ and full-length 5ORIΔ derivatives. Dormant origins near the left end of chromosome III are more strongly activated in a mec1Δ mutant than in a rad9 mutant  or collapsed (replisome disassembled), and to reflect the need for some replisome component to be replaced or modified in order to continue elongation. These crippled forks are then recognized and restored by a There are alternative models to explain the role of the DNA damage response pathway in maintaining the 5ORIΔ-ΔR fragment, which has a large inter-origin gap. The simplest is that the DNA damage response monitors the completion of replication. However, the evidence for such a checkpoint is not compelling see . Debate Another model to explain the role of the DNA damage response pathway in maintaining fragments with large inter-origin gaps suggests that forks stall at sites of endogenous DNA damage and are stabilized by this pathway. The 5ORIΔ-ΔR and ΔL-6ORIΔ fragments would be especially sensitive to such events in the absence of the DNA damage response because the stalled forks would collapse. In the case of the 0ORIΔ-ΔR fragment, which has a full complement of replication origins, a collapsed fork could be rescued by a converging fork from an adjacent origin. In contrast, the 5ORIΔ-ΔR fragment has fewer initiation events, so a collapsed fork would be rescued less often by a converging fork, resulting in an elevated loss rate in a DNA damage checkpoint mutant. Consistent with this suggestion, our analysis of individual 5ORIΔ-ΔR molecules in wild type cells suggests that replication initiates at only one or two places per molecule, but at different places on different molecules .rad9 and rad24Δ mutants is also consistent with this endogenous damage model, as a collapsed leftward-moving fork in the 5ORIΔ chromosome can be rescued by a fork initiating at one of the dormant origins near HML. Our finding that mec1Δ confers an Ofm phenotype while tel1Δ does not is also consistent with this model because MEC1 plays a more important role in the tolerance of DNA damage than does TEL1The enhanced stability of the full-length 5ORIΔ chromosome compared to the ΔL-6ORIΔ fragment in the RAD53 than on CHK1CHK1 makes a more important contribution than RAD53 to 5ORIΔ-ΔR fragment maintenance, suggesting that the DNA damage response is not simply stabilizing forks in response to damage. While Segurado and Diffley CHK1 in stabilizing replication forks, that function was detected only in the absence of both RAD53 and EXO1, which encodes a nuclease responsible for fork collapse in the absence of RAD53. Thus, it seems unlikely that this explains the contribution of CHK1 to 5ORIΔ-ΔR fragment maintenance. Another problem is that deletions of genes, whose products are required for mismatch repair, repair of UV damage, and homologous recombination, did not increase the loss rate of the 5ORIΔ-ΔR fragment in the primary screen, as would have been expected if DNA damage-provoked fork collapse was responsible for loss of this fragment.However, this endogenous damage model is challenged by findings that fork stabilization at sites of DNA damage and survival are more strongly dependent on S. cerevisiae. A median inter-origin distance of 36 kb was estimated from visualization of replicating molecules by electron microscopy . pol32Δ mutants, which lack a nonessential subunit of DNA polymerase Δ, also show fork progression defects, which may explain their Ofm phenotype In addition to the checkpoint genes, our Ofm mutants included deletions of two other genes from the DNA Integrity Network, In summary, we have identified a set of genes whose products facilitate replication of large inter-origin gaps. This set is enriched in components of the DNA damage and replication stress signaling pathways. Replication of large inter-origin gaps shows several surprising features: Dependence on the DNA-damage-specific mediator, Rad9p, rather than the replication-stress-specific mediator, Mrc1p; a stronger dependence on the effector kinase, Chk1p than Rad53p, and no dependence on homologous recombinationYeast strains are listed in natMX gene, amplified from pAG25 ADE2 locus; sequences of primers are available upon request. Individual G418-resistant knockouts were moved into the YKN10 background by transformation with a PCR product amplified from the appropriate strain from the ORF deletion collection (Open Biosystems) using the locus specific A and D primers (www-sequence.stanford.edu/group/yeast_deletion_project/Deletion_primers_PCR_sizes.txt). The mrc1Δ::NAT allele was introduced into the YKN10 background using primers and a template generously provided by K. Sugimoto (UMDNJ). This allele was converted to mrc1Δ::KAN by transforming YJT294 with NotI-cut pFA-KanMX4 his3-Δ367 alleles were generated by fusion PCR and introduced by two-step gene replacement bar1-Δ1327 allele carries a BglII-BsrGI deletion that removes 1327 bp within the open reading frame.YJT242 was created by transforming Y7029 with a PCR product carrying the ade2Δ::natMX mutation, which causes the accumulation of a red pigment in colonies grown on limiting adenine and confers nourseothricin resistance, was mated to the array of viable deletion mutants, each marked with kanMX, which confers G418 resistance. The resulting diploids were then sporulated, and double mutant ade2Δ::natR xxxΔ::kanR haploid MATa progeny were selected. The array of double-mutant strains was mated to F510αA1–4, the donor strain, carrying the 5ORIΔ-ΔR derivative of chromosome III marked with ADE2  strains generated by SGA analysis are Leu−, Ade−, canavanine-resistant, and thialysine-resistant. Any diploids that form between the donor strain and the ade2Δ::natR xxxΔ::kanR double mutant strains are Leu+ and Ade+, but canavanine-sensitive and thialysine-sensitive because the can1Δ and lyp1Δ mutations are recessive. The desired chromoduction event results in cells that are Leu+ and Ade+, because they carry the 5ORIΔ-ΔR fragment, and canavanine- and thialysine-resistant, because they carry the can1Δ and lyp1Δ mutations. Medium lacking leucine, adenine, arginine, and lysine and containing both canavanine and thialysine selects for these cells. A preliminary screen using approximately 100 strains selected from the viable deletion collection was carried out to determine conditions for the chromoduction. We found that pinning the array of double mutants at the density found in a standard 384 well plate was necessary to ensure efficient mating of the donor strain to the array.In our version of the screen, a strain carrying an ith ADE2 . Becausedeletion . The corThe screen was done in duplicate, and chromoductants from the duplicate arrays were streaked side-by-side on a single plate with limiting adenine for scoring sectoring patterns see . This prChromosome loss rates were determined by fluctuation analysis using the colony isolation method NdeI, ClaI+EcoRV, or FspI+SphI+ClaI, subjected to BND-cellulose (Sigma) chromatography, electrophoresed on neutral-neutral 2D gels, blotted, and hybridized as described ARS301 was the1.3-kb EcoRI-XhoI fragment from p78_4.6; the probe for ARS302/ARS303/ARS320 was the 1.9-kb EcoRI-HindIII fragment from p78_5.2; the probe for ARS304 was the 3.5-kb PshAI-BamHI fragment from D10B; the probe for ARS314 was the 1.8-kb HindIII fragment from pH 1.8 32P] dATP (Perkin Elmer) using the Megaprime DNA-labeling system . Images were acquired on a Molecular Dynamics Typhoon 9410, and the exposure was adjusted using ImageQuant 5.2 software. Quantitations of bubble-shaped and Y-shaped replication intermediates were determined using the polygon tool and the line tool of ImageQuant 5.2.Genomic DNA was prepared from log-phase cultures as described Colonies were photographed after ∼5 days of growth at 30°C on limiting adenine plates. Images were acquired as TIFF files with a Nikon D-100 camera fitted with an AF Micro-Nikkor 60 mm f/2.8 D lens. Images were cropped and adjusted for color balance and brightness/contrast in Photoshop.cs v8.0 (Adobe Systems).Figure S1aro7Δ, ddc1Δ, and rad17Δ strains. The 5ORIΔ-ΔR fragment was introduced into aro7Δ, ddc1Δ and rad17Δ strains by chromoduction, and the chromoductants were streaked on plates with limiting adenine and photographed after growth for 5 days.Sectoring patterns of (1.70 MB TIF)Click here for additional data file.Figure S2rad52 epistasis group. Top panels: 5ORIΔ-ΔR chromoductants of rad50Δ, rad51Δ, rad52Δ, rad54Δ, rad55Δ, rad57Δ, rad59Δ, rdh54Δ, mre11Δ and xrs2Δ strains isolated in the whole genome screen were streaked on plates with limiting adenine and photographed after growth for 5 days. aro7Δ and rad9Δ chromoductants were included as controls. Lower panels: Chromoductants were tested for sensitivity to phleomycin, which induces double-stranded breaks, to confirm that the strains carried the expected deletions. Cultures of the strains shown in the top panels were grown overnight in YEPD, serially diluted and spotted on YEPD plates (control) and plates with 0.1 and 1.0 µg/ml phleomycin. YEPD plates and 0.1 µg/ml phleomycin plates photographed after 3 days, 1.0 g/ml phleomycin plates after 5 days. Each of the strains, with the exception of rad59Δ and rdh54Δ, showed sensitivity, indicating that the sensitive strains carried the expected deletions. rad59 mutants have been reported to be 10,000-fold less sensitive to gamma irradiation than rad52 mutants [Bai et al], so the lack of sensitivity of the strain we tested was expected. The sensitivity of rdh54Δ to gamma irradiation had not been previously tested but it had been shown not to be sensitive to HO-induced double strand breaks [Klein et al]; we found that an authentic rdh54Δ mutant was also not sensitive to phleomycin.  Sectoring patterns of mutants in the (6.45 MB TIF)Click here for additional data file.Table S1Results of secondary screen for Ofm mutants.(0.73 MB PDF)Click here for additional data file.Table S2Strains used in this study.(0.06 MB DOC)Click here for additional data file.Table S3Quantitation of 2D gels.(0.03 MB DOC)Click here for additional data file.Table S4Appearance of rarely-sectored colonies in strains carrying the ΔL-6ORIΔ fragment.(0.03 MB DOC)Click here for additional data file.


Pancreas cancer is a dreaded disease with high mortality, despite progress in surgical and oncological treatments in recent years. The field is hampered by a lack of good prognostic and predictive tumour biomarkers to be used during follow-up of patients.The circulating level of type IV collagen was measured by ELISA in pancreas cancer patients and controls. The expression pattern of type IV collagen in normal pancreas, pancreas cancer tissue and in pancreas cancer cell lines was studied by immunofluorescence and Western blot techniques.in vitro, which in part can explain the high type IV collagen expression observed in pancreas cancer tissue, and the increased circulating levels in pancreas cancer patients. Of clinical importance, our results show that the circulating level of type IV collagen after surgery is strongly related to prognosis in patients treated for pancreas cancer by pancreatico-duodenectomy with curative intent. Persisting high levels of circulating type IV collagen after surgery indicates a quick relapse in disease and poor survival.Patients with pancreas cancer have significantly increased circulating levels of type IV collagen. In pancreas cancer tissue high levels of type IV collagen expression was found in close proximity to cancer cells in the tumour stroma. Furthermore, pancreas cancer cells were found to produce and secrete type IV collagen Our results most importantly show that stroma related substances can be evaluated as potential cancer biomarkers, and thereby underline the importance of the tumour microenvironment also in this context. Pancreas cancer is still one of the most feared malignancies with a poor prognosis. The overall median survival is 3–5 months, and the 5-year survival rate is only 3% –α6(IV), which appear in different combinations to form triple helical molecules called protomers (α(IV)-chains, which renders different functional and structural properties to the BMs in that tissue.Type IV collagen is an ECM protein found in all basement membranes (BM) (α1(IV)-, α2(IV)- and α3(IV)-chains, respectively) have been shown to inhibit angiogenesis and tumour growth in vivo (α4(IV)-, α5(IV)- and α6(IV)-chains were found to have antiangiogenic features in vitro -chains in normal pancreas and pancreatic cancer tissue, as well as analyse the expression of type IV collagen by pancreas cancer cells in vitro. We also measure the circulating levels of type IV collagen in patients with pancreas cancer before and after treatment, and introduce type IV collagen as a promising future biomarker for identification of patients at high risk of quick relapse and poor prognosis after surgery.We show here the expression pattern of different n=9) and after surgery . In the survival analysis additional patients were added (n=5), where only samples after surgery were available. Postoperative plasma samples were collected 4 weeks or more after surgery. Control samples were collected from patients (n=8) admitted to the hospital for a non-malignant disease. Patient characteristics are shown in n=6) and controls (n=5) were snap frozen in liquid nitrogen and stored at −80°C until analysis. Informed consent was obtained from all patients. The ethical committee at the Medical faculty of Umeå University approved the study.Patients with pancreas cancer were admitted to the Department of Surgery, Umeå University Hospital and evaluated by CT scan, MRI and laparoscopy. Patients who were considered operable underwent a curatively aimed pancreatico–duodenectomy (Whipple's procedure). One patient was operated with an expanded Whipple's procedure , and one obtained intraoperative radiotherapy (IORT). Plasma samples were collected from these patients before  and streptomycin (100 mg l−1) and grown in an incubator at 37°C in an atmosphere of 5% CO2.Two well characterised human pancreatic adenocarcinoma cell lines were used, HPAC and CFPAC-1. Both cells lines were kindly provided by Professor Helena Edlund, Umeå University. HPAC  is a human pancreatic adenocarcinoma cell line of ductal origin from a woman with moderate to well differentiated pancreatic adenocarcinoma . CFPAC-1α1(IV)NC1  (α2(IV)7S , mouse-anti-α3(IV)NC1  (α5(IV)NC1  (α4(IV)- and α6(IV)-chains are commercially available. For detection of α5(IV)NC1, tissue was denatured with 6 M urea–0.1 M glycine before staining. Secondary antibodies used were; donkey-anti-sheep FITC , and donkey-anti-mouse TRITC  and donkey-anti-rabbit FITC . Sections were mounted with medium containing DAPI . Positive control staining for each antibody was performed on tissue known to express the antigen in question. As negative controls, primary antibodies were omitted and sections were incubated with secondary antibodies only.Hematoxylin and eosin (H&E) and Masson's trichrome staining (MTS) were performed according to standard protocols. Immunohistochemical staining was performed on frozen sections (six micrometer) and immunocytochemical staining on HPAC and CFPAC-1 cells cultured on Falcon Culture Slides  using methods described earlier (μg of protein from the cell layer and 25 μg of protein from the cell media were loaded onto a 12% SDS–polyacrylamide gel. As a positive control 500 ng of recombinant human arresten (for α1(IV)NC1) (α2(IV)NC1) (α1(IV)- and α2(IV)-chains, was used as a primary antibody, and peroxidase-conjugated goat-anti-rabbit-IgG  as secondary antibody. Proteins were detected by chemiluminescense (ECL) .HPAC and CFPAC cells were cultured to subconfluency, then serum-starved for 1–2 days. Cells were lysed as described earlier . Samples were run in duplicates and according to the manufacturer's protocol. In these ELISA-assays two monoclonal antibodies (clones 4H12 and 1D3) directed against the collagenous domain of type IV collagen are used - and α2(IV)-chains are highly expressed in vascular basement membranes (VBM) and epithelial basement membranes (α3(IV)- and α5(IV)-chains could not be detected (data not shown). In pancreas cancer the tissue morphology is changed, with no organised ductal structures, and strong α1(IV)- and α2(IV)-chain expression is now observed close to the cancer cells in the tumour stroma and the VBMs (α3(IV)- and α5(IV)-chains of type IV collagen were not expressed in pancreas cancer (data not shown).We analysed the expression pattern of the different embranes , whereasthe VBMs . Interesin vitro studies on two well characterised pancreas cancer cell lines HPAC and CFPAC-1. Western blot analysis of cell lysate and media, and immunocytochemical staining, show that both α1(IV) and α2(IV), the two α-chains that were strongly expressed in the tumour stroma in pancreas cancer tissue, are produced by the cancer cells and secreted to the media in both cell lines  when compared with the controls, and no statistically significant change was observed when comparing with the levels before surgery. However, the variation within the postoperative group was high, which prompted us to plot the levels before and after surgery for individual patients (Circulating levels of type IV collagen fragments were measured with ELISA in control patients and pancreas cancer patients before and after surgery . A signipatients . It turn−1), and those with lower levels (<200 ng ml−1) can also be illustrated in a survival curve -, α2(IV)-, α5(IV)- and α6(IV)-chains are expressed in normal pancreas, whereas the α5(IV)- and α6(IV)-chains are lost from the BM in the pancreatic duct in cancer (α1(IV)- and α2(IV)-chain staining in the BM surrounding the acini structures and distal ducts in the exocrine pancreas. This suggests the presence of the α1α2α2 protomer of type IV collagen in this part of the normal pancreatic tissue, as we could not see any expression of the α3(IV)- and α5(IV)-chains.It has previously been shown that in the BM of the pancreatic ducts α1(IV)- and α2(IV)-chains were expressed in the direct proximity to the cancer cells in the tumour stroma. No expression of the α3(IV)- and α5(IV)-chains were observed. This is well in line with earlier studies of the expression pattern of type IV collagen in pancreatic adenocarcinomas (α1(IV)- and α2(IV)-chain expression was found in the areas of tumour desmoplasia, indicating that type IV collagen has a distinct expression pattern when compared with the fibrillar collagens found in this area.In pancreas cancer, high levels of in vitro experiments on two pancreas cancer cell lines, we could verify that pancreas adenocarcinoma cells produce and secrete type IV collagen. Our results therefore indicate that the expression pattern of type IV collagen observed in pancreas cancer tissue is a result of remodelling of pre-existing type IV collagen, together with de novo production by the cancer cells. It has been shown in vitro in other human cancer cells, such as glioblastoma and melanoma cell lines, that type IV collagen is produced by the cancer cell  are involved in remodelling the basement membrane . We haveThis is the first description of increased circulating type IV collagen levels in patients with pancreas cancer. We believe that this increase is due to the breakdown of existing BMs by the tumour, combined with type IV collagen production and secretion by the cancer cells. Furthermore, we show that circulating type IV collagen levels have prognostic value when predicting prognosis after surgery with curative intent. On the other hand, preoperative circulating type IV collagen levels do not predict prognosis. This is most likely due to the fact that preoperative level of circulating type IV collagen corresponds to the total tumour load. One can speculate that a large primary tumour can produce high levels of type IV collagen, because of both matrix remodeling and expression by the cancer cells, but it can nevertheless be a locally growing tumour with good prognosis. After successful removal of the primary tumour, the type IV collagen levels instead reflect the metastatic tumour load, and high levels in the postoperative situation therefore could correspond to spread disease and poor prognosis. Our findings indicate that by measuring circulating levels of type IV collagen soon after surgery, patients at high risk of quick relapse and poor prognosis can be identified in an early stage and be offered more aggressive adjuvant therapy.α1β1 integrin functioning as a type IV collagen receptor in pancreas cancer cells (Attachment to the BM is crucial for the survival and growth of epithelial cells. Integrin receptors play a role in anchoring the cells to the ECM and in providing survival signalling. If the epithelial cell looses contact with the BM, a cascade of events is initiated eventually leading to apoptosis (er cells . In thisMost importantly our study shows that a tumour stroma-derived substance could be a promising future tumour biomarker in evaluating prognosis and predict the effect of cancer treatment. This underlines the importance of expanding the horizon beyond the cancer cell, when searching for novel tumour biomarkers.