Chemotherapy aided by opening of the blood-brain barrier with intra-arterial infusion of hyperosmolar mannitol improves the outcome in primary central nervous system lymphoma. Proper opening of the blood-brain barrier is crucial for the treatment, yet there are no means available for its real-time monitoring. The intact blood-brain barrier maintains a mV-level electrical potential difference between blood and brain tissue, giving rise to a measurable electrical signal at the scalp. Therefore, we used direct-current electroencephalography (DC-EEG) to characterize the spatiotemporal behavior of scalp-recorded slow electrical signals during blood-brain barrier opening. Nine anesthetized patients receiving chemotherapy were monitored continuously during 47 blood-brain barrier openings induced by carotid or vertebral artery mannitol infusion. Left or right carotid artery mannitol infusion generated a strongly lateralized DC-EEG response that began with a 2 min negative shift of up to 2000 μV followed by a positive shift lasting up to 20 min above the infused carotid artery territory, whereas contralateral responses were of opposite polarity. Vertebral artery mannitol infusion gave rise to a minimally lateralized and more uniformly distributed slow negative response with a posterior-frontal gradient. Simultaneously performed near-infrared spectroscopy detected a multiphasic response beginning with mannitol-bolus induced dilution of blood and ending in a prolonged increase in the oxy/deoxyhemoglobin ratio. The pronounced DC-EEG shifts are readily accounted for by opening and sealing of the blood-brain barrier. These data show that DC-EEG is a promising real-time monitoring tool for blood-brain barrier disruption augmented drug delivery. The blood-brain barrier (BBB) inhibits the penetrance of hydrophilic and polar drugs into brain tissue and hinders effective use of treatments like methotrexate chemotherapy in the otherwise drug sensitive primary central nervous system lymphoma (PCNSL). It was recently shown that PCNSL relapses within 5 years in all subjects and over half of the subjects within 2 years with BONN intra-thecal reservoir treatment combined with multi-drug intravenous treatment and has dismal prognosis in a few months . HoweverThe most widely accepted view of the mechanism underlying hyperosmolar mannitol-induced BBBD accounts for the barrier breach by osmotic shrinkage of endothelial cells and consequent opening of tight junctions between the cells . The degVery low frequency  oscillations up to 1-2 mV in the electrical potential of mammalian brain tissue were observed for the first time in electro-cortical experiments on rabbits . In the In this study, we hypothesized that therapeutic BBBD induced by intra-arterial mannitol infusion could be monitored using scalp DC-EEG. To test the hypothesis, we measured DC-EEG during routine clinical treatment of PCNSL patients while they received chemotherapy augmented with BBBD that we perform 2 to 4 times per week. We also monitored the subjects with near-infrared spectroscopy (NIRS) in order to collect information on cerebral hemodynamic that is known to play a role in DC-EEG signal generation . We repoIn this study 47 consecutive BBBD treatments were monitored in 9 PCNSL patients . Sixteen of the infusions were introduced into the right internal carotid artery, thirteen into the left internal carotid artery and eighteen into the dominant vertebral artery. Patients were recruited in the study during 2014 and a written informed consent was obtained from each patient prior to the procedure in addition to routine clinical BBBD information. The study was carried out in accordance with the Declaration of Helsinki and approved by the Ethical Committee of Northern Ostrobothnia Hospital District, Oulu University Hospital (number 5/2014).The BBBD treatment for PCNSL in Oulu University Hospital is based on the original procedure of Neuwelt and coworkers ,31, and Before anaesthesia induction intravenous phenobarbital and midazolam are given. Anaesthesia is induced and maintained using propofol. Two to three minutes prior to intra-arterial mannitol infusion anaesthesia is deepened up to EEG suppression level (entropy 0) with a 250 mg intravenous thiopental bolus together with benzodiazepine. Atropine is given to counteract strong vasovagal effects of BBBD. Muscle relaxants are not used since they could impede detection of clinical seizures caused by the infusions.The BBBD treatment, adopted in 2007 from the pioneering Portland group led by Edward Neuwelt, is given to one of the internal carotid arteries or to the dominant vertebral artery . After aIntravenous contrast enhanced cone beam computed X-ray tomography (cbCT) (120 – 150 ml of Visipaque 270 mg/ml) is routinely used in our BBBD protocol to rule out excessive BBB opening that may lead to vasogenic edema requiring reversing cortisol treatment to close the BBB. Visipaque was given during carboplatin infusion and cbCT was performed immediately after the BBBD procedure was completed. The DC-EEG cap was removed for better image quality and region of interest measurements were performed on all major arterial territories to quantify the BBB status from cbCT data after the procedure using NeaView clinical analysis tool . The timing of the Visipaque application (>10 min after mannitol infusion) and the sensitivity of cbCT limit the use of the present cbCT data to the detection of prolonged or excessive BBB opening. Cortisol treatment was not needed in any of the 47 treatments included in the present study.The term DC-EEG refers to recording EEG without any high-pass filtering ,32,33. DUnless otherwise stated, EEG data were referenced to the common average and linear drifts were removed from all channels. This was done by subtracting off-line a linear trend from each individual channel after visual verification of the drift rate and its linearity throughout the entire recording period. Thereafter signals were downsampled to 1 Hz  and low-pass filtered using a 21-point moving average filter for detection of infraslow EEG signals . The specimen trace  is shownEach subject was measured with one NIRS channel placed on the forehead beneath the EEG-cap lead adjacent to Fp1 or Fp2 leads on the side of the infused artery using a source-detector distance of 3 cm. NIRS data was recorded using a NIRS measurement device utilizing wavelengths of 660 nm and 830 nm . The samCardiovascular signals  were collected simultaneously using GE Datex-Ohmeda S/5 Avance system for routine patient surveillance and for verification of BBBD-induced vasovagal changes (data not shown).Mannitol infusion induced a robust multiphasic response in EEG channels monitoring the affected arterial territory, with amplitudes that were orders of magnitude larger (up to 2 mV) than those of commonly observed EEG rhythms. In the middle of the infused carotid arterial territory (electrode F4 for right and F3 for left carotid artery infusion) the response to the 30 s mannitol infusion typically commenced with a negative peak lasting 1.5-2 minutes and coinciding with the first (and second) pass of the intra-arterial mannitol bolus . This inThe BBBD procedure includes an intravenous thiopental bolus 2 min prior to mannitol. Thiopental caused a negative baseline shift of about 100 μV at F4 by the time of the mannitol application, and it completely abolished neuronal activity seen at frequencies > 0.5 Hz . After aThe spatiotemporal characteristics of the DC-EEG shifts were analysed using more heavily low-pass filtered signals see . TypicalGrand average DC-EEG traces at opposite quarters of the scalp were calculated to illustrate characteristic responses evoked by a total of 47 intra-arterial mannitol infusions . On averA more detailed spatial mapping of the DC-EEG shifts is shown using topographic heat maps . The intBoth oxyhaemoglobin (HbO) and deoxyhaemoglobin (Hb) NIRS signals plummeted during the 30 s mannitol infusion Figs  and 2. TThe Hb signal behaved differently compared to HbO. After the mannitol-bolus induced initial fall, Hb temporarily increased towards its original value. This was followed by a prolonged decrease below the original baseline and a very slow recovery towards the original pre-bolus level.The NIRS measurements were performed always on the forehead due to imaging- and procedure-related limitations during the BBBD. The results were concordant between both carotid artery BBB disruptions, and the very prolonged cerebrovascular response was strikingly different compared to the gradually fading DC-EEG shifts. Obviously, a frontal NIRS cannot monitor the vertebral artery territory, and therefore the much less prominent NIRS responses evoked by vertebral artery mannitol infusion are shown only to illustrate the effects of diluted mannitol in a non-infused cortical area.We estimated whether excessive BBB opening occurred by comparing the enhancement of grey matter in the treated vascular territory vs. the non-treated arterial territories from cbCT data . The resThe results of this study demonstrate for the first time real-time monitoring of human therapeutic BBBD using scalp-recorded DC-EEG. BBBD was induced with intra-arterial mannitol infusion in anesthetized patients receiving chemotherapy for PCNSL. DC-EEG detected robust mV-level shifts providing spatiotemporal information on the course of the induced BBBD. Simultaneously measured NIRS detected dilution of blood upon intra-arterial mannitol infusion followed by marked alteration in oxygen extraction fraction. Since the intact BBB makes the brain parenchyma inaccessible to hydrophilic drugs, new diagnostic and therapeutic innovations involving BBBD may benefit from the methods described here.The BBB forms the first line defence of the brain against various forms of pathogens present in blood, and it also prevents targeting the brain parenchyma with systemic administration of hydrophilic medications. The interest in the role of BBB in brain pathology and in treating brain diseases has increased over the past few years ,39. PCNSThe ability to monitor the degree and duration of BBBD is crucial for the treatment of PCNSL as sub-optimal or excessive BBB opening increases mortality and complications . Previou+-solution [in vitro by arterial perfusion [The tight BBB maintains a trans-endothelial voltage between blood and brain tissue ,23,44. Tsolution . It is oerfusion ,49.Intra-arterial infusion of detergents like DHC can induce vascular thrombosis and be lethal in animal experiments, and some researchers suspect that other hyperosmolar solutions carry a potential for ischemia or cerebral haemorrhage . In braiNeuronal sources generate EEG signals with orders of magnitude smaller amplitudes which rules out their contribution to mV-level DC-EEG shifts. Furthermore, subjects in our study were in deep anaesthesia (with suppressed EEG activity at frequencies > 0.5 Hz) that was induced by intravenous thiopental 2 minutes prior to mannitol infusion . It has The time window observed in the DC-EEG potential response to mannitol is perfectly in line with the known BBB penetrance time window for large particles  that lasts maximally for 15 minutes following BBB disruption in animal models ,52. We dAs expected, the intravenous thiopental bolus-related DC-potential shift showed no lateralization. In contrast to this, the responses upon mannitol infusion via either left or right carotid artery were strongly lateralized and they indicate differential local effects in the generation of the DC-EEG shift. That the contralateral side showed a response of opposite polarity is at least partly a consequence of using the common average reference montage, but qualitatively similar results were seen with other montages (data not shown). Accordingly, vertebral artery mannitol infusions resulted in responses with a frontal-posterior distribution and little lateralization. These responses were smaller in amplitude, which is consistent with the location of the infused arterial territory and the mechanism that couples BBB-generated signals to scalp . Thus thChanges in brain hemodynamics induced by jugular vein compression, Valsalva or Müller manoeuvres, show good correlation between NIRS vs. DC-EEG shifts generated by the intact BBB . TherefoRecently, several diseases have been linked to the disruption of the protective properties of the BBB. Ischemia, degenerative diseases, inflammatory diseases, neoplasms and homeostatic disturbances all compromise the integrity of the BBB . On the Our results demonstrate the feasibility of DC-EEG for real-time monitoring of induced transient BBBD in anesthetized human patients receiving chemotherapy for PCNSL. In addition to providing valuable real-time information on BBBD during PCNSL treatment, our present results and the DC-EEG method may be exploited when devising novel therapeutic strategies involving BBBD-aided pharmacotherapy of brain diseases.


Furthermore, a Tsn homolog called TAP-binding protein-related (TAPBPR) has only recently been demonstrated to function as a second MHC I-specific chaperone and peptide proofreader. Although TAPBPR is PLC-independent and has a distinct allomorph specificity, it is likely to share a common catalytic mechanism with Tsn. This review focuses on the current knowledge of the multivalent protein–protein interactions and the concomitant dynamic molecular processes underlying peptide-proofreading catalysis. We do not only derive a model that highlights the common mechanistic principles shared by the MHC I editors Tsn and TAPBPR, and the MHC II editor HLA-DM, but also illustrate the distinct quality control strategies employed by these chaperones to sample epitopes. Unraveling the mechanistic underpinnings of catalyzed peptide proofreading will be crucial for a thorough understanding of many aspects of immune recognition, from infection control and tumor immunity to autoimmune diseases and transplant rejection.The adaptive immune system is able to detect and destroy cells that are malignantly transformed or infected by intracellular pathogens. Specific immune responses against these cells are elicited by antigenic peptides that are presented on major histocompatibility complex class I (MHC I) molecules and recognized by cytotoxic T lymphocytes at the cell surface. Since these MHC I-presented peptides are generated in the cytosol by proteasomal protein degradation, they can be metaphorically described as a window providing immune cells with insights into the state of the cellular proteome. A crucial element of MHC I antigen presentation is the peptide-loading complex (PLC), a multisubunit machinery, which contains as key constituents the transporter associated with antigen processing (TAP) and the MHC I-specific chaperone tapasin (Tsn). While TAP recognizes and shuttles the cytosolic antigenic peptides into the endoplasmic reticulum (ER), Tsn samples peptides in the ER for their ability to form stable complexes with MHC I, a process called peptide proofreading or peptide editing. Through its selection of peptides that improve MHC I stability, Tsn contributes to the hierarchy of immunodominant peptide epitopes. Despite the fact that it concerns a key event in adaptive immunity, insights into the catalytic mechanism of peptide proofreading carried out by Tsn have only lately been gained  However, the peptides are not indiscriminately loaded onto MHC I molecules, but rather selected for high affinity and stability in an optimization step called peptide proofreading or editing (Presentation of antigenic peptides on major histocompatibility complex class I (MHC I) molecules is fundamental to the recognition of infected and cancerous cells by the immune system . Peptide editing . While T editing , is cata editing –9 and TA editing . The sel editing .This review will describe the major players in catalyzed peptide proofreading, in particular Tsn, with a focus on the molecular mechanisms of the proofreading activity, the associated protein plasticity, and the dynamics of the interaction partners. Key information about these aspects of antigen presentation has recently been obtained mainly by structural, biochemical, and computational studies.via a disulfide bond  peptide epitopes up to 10-fold . The α2-1 helix is embraced and stabilized by Tsn in a conformation that maintains the peptide-binding groove in an open state. The MD simulations predict that the C-terminal interface consists of the CD8-binding site of the MHC and a cluster of basic Tsn residues  can communicate with the peptide-binding groove might indicate that the predicted C-terminal interface of the Tsn–MHC I complex plays an active role during catalysis. A correlation between conformational flexibility and Tsn dependence similar to the HLA-B*44:02/B*44:05 pair has been described for the two allomorphs HLA-B*27:05 and HLA-B*27:09  they preferentially bind and stabilize MHC molecules that are empty or loaded with low-affinity peptides, (ii) they accelerate peptide exchange, favoring high-affinity epitopes, (iii) and they achieve the chaperoning effect and selection of high-affinity peptides by directly interacting with structural elements flanking the peptide-binding groove. But while Tsn and TAPBPR presumably engage the MHC binding groove at the C-terminus of the peptide, DM acts at the N-terminus of the peptide Figure  and has 2-1 helix of MHC I, whereas in the case of DM, exchange catalyst and peptide mainly compete for residues around the P1 pocket of the MHC II molecule (Figure In essence, both types of exchange catalysts lower the energy barrier for peptide dissociation and stabilize the empty binding groove in a state which only high-affinity epitopes are able to go past. However, in the case of Tsn and TAPBPR, catalysis seems to involve a tug-of-war over the αe Figure .The recently obtained insights into peptide proofreading described in this review have significantly advanced our knowledge of this fundamental process in adaptive immunity and underline the importance of malleable structural elements and plasticity in the involved protein interaction partners. However, a comprehensive understanding of the molecular determinants in MHC I peptide editing will only be achieved once an experimentally determined structure of a Tsn–MHC I and/or TAPBPR–MHC I complex is available. Crystal structures of these two complexes will also help to fully comprehend the fact that different MHC I allomorphs exhibit varying degrees of Tsn/TAPBPR dependence and that Tsn and TAPBPR have different allomorph specificities. As has previously been noted , Tsn andCT and RT wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There was an error in the Competing Interests Statement. The second sentence of the Competing Interests Statement should read: "The lt-NES cell technology is proprietary , and lt-NES® is a registered trademark of LIFE & BRAIN GmbH."


Net survival is the most common measure of cancer prognosis and has been used to study differentials in cancer survival between ethnic or racial population subgroups. However, net survival ignores competing risks of deaths and so provides incomplete prognostic information for cancer patients, and when comparing survival between populations with different all-cause mortality. Another prognosis measure, “crude probability of death”, which takes competing risk of death into account, overcomes this limitation. Similar to net survival, it can be calculated using either life tables (using Cronin-Feuer method) or cause of death data (using Fine-Gray method). The aim of this study is two-fold: (1) to compare the multivariable results produced by different survival analysis methods; and (2) to compare the Cronin-Feuer with the Fine-Gray methods, in estimating the cancer and non-cancer death probability of both Indigenous and non-Indigenous cancer patients and the Indigenous cancer disparities.Cancer survival was investigated for 9,595 people (18.5% Indigenous) diagnosed with cancer in the Northern Territory of Australia between 1991 and 2009. The Cox proportional hazard model along with Poisson and Fine-Gray regression were used in the multivariable analysis. The crude probabilities of cancer and non-cancer methods were estimated in two ways: first, using cause of death data with the Fine-Gray method, and second, using life tables with the Cronin-Feuer method.Multivariable regression using the relative survival, cause-specific survival, and competing risk analysis produced similar results. In the presence of competing risks, the Cronin-Feuer method produced similar results to Fine-Gray in the estimation of cancer death probability  and non-cancer death probabilities . Cronin-Feuer estimated much lower non-cancer death probabilities than Fine-Gray for non-Indigenous patients with head and neck cancers and lung cancers (both smoking-related cancers).Despite the limitations of the Cronin-Feuer method, it is a reasonable alternative to the Fine-Gray method for assessing the Indigenous survival differential in the presence of competing risks when valid and reliable subgroup-specific life tables are available and cause of death data are unavailable or unreliable.The online version of this article (doi:10.1186/s12963-016-0118-9) contains supplementary material, which is available to authorized users. In Australia, Indigenous cancer patients have lower net survival than non-Indigenous patients , but theNet survival is the measure most commonly used to compare cancer prognosis in different populations  , 3–9 becFor this purpose, “crude probability of death” is a more appropriate measure of prognosis than net survival because it takes competing risk into account and enables us to identify whether survival disparities are caused by cancer deaths, non-cancer deaths, or both. Crude probability of cancer death (hereafter referred to as “cancer death probability”) is the probability of death from cancer in the presence of other causes; while crude probability of non-cancer death (hereafter referred to as “non-cancer death probability”) is the probability of death from other causes in the presence of cancer.Both net survival and crude probability of death can be estimated in two different ways: one based on cause of death to classify each death as a cancer or non-cancer death, the other based on life tables to compare the number of deaths in cancer patients with the number “expected” based in total population mortality rates Table . Cause oLife table-based methods are commonly used for comparing cancer survival between population groups because of deficiencies in cause of death data; such deficiencies may be greater for disadvantaged subgroups. However, life tables are often not available for such subgroups because of uncertainty about population estimates and absent or incomplete identification of subgroup members in death registrations. Australia has high-quality cause of death data but incomplete life tables for the Indigenous population, except for the Northern Territory (NT) where Indigenous Australians are very high prevalence (30% of the NT population compared to 2–4% in other states) and consequently are accurately identified in deaths data and have reliable population estimates . IndigenThe aim of this study is two-fold: (1) to compare the multivariable results  produced by relative survival, cause-specific survival, and competing risk (Fine-Gray) survival analysis methods; and (2) to compare the Cronin-Feuer with the Fine-Gray survival analysis methods, in estimating the cancer and non-cancer death probability of both Indigenous and non-Indigenous cancer patients and the Indigenous cancer disparities in the presence of competing causes of death.Cancer registrations data for all NT residents diagnosed with cancer between 1 January 1991 and 31 December 2009 was obtained from the NTCR for the following data items: sex; date of birth; Indigenous status; remoteness of residence (urban or remote); date of diagnosis; cancer site, coded according to the International Classification of Diseases 10 (ICD-10); date of death; and underlying cause of death. Vital status was verified by matching the NTCR dataset to the National Death Index for deaths occurring up to 31 December 2011 . For the survival analysis of site-specific cancers, female breast (C50), colorectal (C18-20), lung (C33-34), and head and neck (C1-C14 & C30-32) cancers were chosen because there were sufficient numbers of both Indigenous and non-Indigenous cases for the analysis, and they are Australia’s designated national “priority” cancers  , and Fine-Gray regression were used to investigate the Indigenous survival disparity; multivariable analysis was not available for the Cronin-Feuer method. The same independent variables were included in all final models: Indigenous status (Indigenous compared to non-Indigenous); age at diagnosis (per year); gender ; and cancer site. Since the effect of age at diagnosis was found to be different for Indigenous compared with non-Indigenous patients, an interaction term for Indigenous status by age at diagnosis (base age 55 years) was added to the models. For the Cox proportional hazard regression, scaled Schoenfeld residuals were used to check if the proportional hazards assumptions of each variable were satisfied. As the proportional hazards assumption was not met for Indigenous status, a step function of Indigenous status with follow-up time  was also included in the model.In the multivariable analysis for time trends, as all subjects had at least 2 years of potential follow-up, the follow-up was limited to the first 2 years after diagnosis so that the shorter follow-up time for subjects diagnosed late in the study period did not bias time trends. Regression models included the same terms as above, plus year of diagnosis.All analyses were conducted using Stata/SE 13 . Ethics approval was obtained from the Human Research Ethics Committee of the Menzies School of Health Research and NT Department of Health. Approval to use the cancer registrations data was obtained from the NT Cancer Registry.There were 9,595 invasive cancer cases between 1991 and 2009 that fulfilled the study criteria  was lower for Indigenous than non-Indigenous patients at both 1 and 5 years after diagnosis for breast, colorectal, and head and neck cancers Table .Table 3OThe multivariable results for all cancers combined (adjusted for cancer site) were similar for all terms included in the model for each of the three methods: relative survival analysis using Poisson regression; cause-specific analysis using Cox proportional hazards regression; and competing risk analysis using Fine-Gray regression Tables  and 5. TThe measures of crude probabilities of deaths are presented as proportion of cases that have died at 5 years after diagnosis Table  from canThis study is the first to examine the implications of various survival methods to assess the survival inequalities faced by Indigenous cancer patients in the NT, in particular the impact of competing causes of death. Multivariable regression using relative survival (Poisson model), cause-specific survival (Cox regression) and competing risk analysis (Fine-Gray model) produced similar results. For all cancers combined, death rates were higher for Indigenous, male and older cancer patients for both cancer and non-cancer death. In the absence of competing risk, Indigenous people have lower 5-year net survival than non-Indigenous people for all three cancers .Crude probability of death potentially offers a more understandable way than net survival to communicate cancer prognosis to patients and clinicians, showing the different mortality risk profiles (cancer and non-cancer death probabilities) among the Indigenous and non-Indigenous populations, while acknowledging that cancer patients can also die of non-cancer causes . It enabIn the presence of competing risks, the Cronin-Feuer method produced similar results to the Fine-Gray method in the estimation of cancer death probability  and non-cancer death probabilities  5 years after cancer diagnosis. The Cronin-Feuer method estimated much lower non-cancer death probabilities than the Fine-Gray method for non-Indigenous patients with head and neck cancers and lung cancer, which are both smoking-related cancers.Comparison of death probabilities estimated using the Cronin-Feuer method and Fine-Gray models provides insights into “external factors” affecting measures of survival for various cancer types such as smoking-related cancers. The death probabilities estimated using the Cronin-Feuer method can be seen as the death probabilities that the cancer patients were “expected to have”, in the hypothetical scenario where cancer patients have the same death risks as the general population, unaffected by “external factors” that cancer patients experienced such as unhealthy behaviors, access to health care, or screening effects. The death probabilities estimated using cause of death data and the Fine-Gray method can be seen as the “observed death probabilities” in which cancer patients might have different risks of death to the general population due to various “external factors”. Therefore, higher (or lower) non-cancer death probabilities estimated by the Fine-Gray model imply higher (or lower) non-cancer death risks in cancer patients than the general population.When analyzing smoking-related cancers, caution is required for prognosis measures calculated using general population life tables such as relative survival and the Cronin-Feuer method –20, whicPrevious studies suggested that relative survival tends to underestimate social inequalities in cancer survival –23. Our The lower estimates of cancer death probabilities of the Fine-Gray method might indicate that cancer survival for breast cancer patients is better than expected . The lower estimates of non-cancer death probabilities of the Fine-Gray method might indicate lower non-cancer 5-year death risk in breast cancer patients than the background population, supported by the findings of another study  (not speWhile net survival is useful for reporting trends in cancer survival, comparing different groups of cancer patients and investigating the impact of various factors on cancer treatment, crude probability of death is important when communicating risks to patients  during c


Picea glauca (Moench) Voss x P. engelmannii Parry ex Engelm.) trees, representing 25 OP families, growing on three sites in Interior British Columbia, Canada. The use of the genomic realized relationship permitted genetic variance decomposition to additive, dominance, and epistatic genetic variances, and their interactions with the environment, producing more accurate narrow-sense heritability and breeding value estimates as compared to the pedigree-based counterpart. The impact of retaining (random folding) vs. removing (family folding) genetic similarity between the training and validation populations on the predictive accuracy of genomic selection was illustrated and highlighted the former caveats and latter advantages. Moreover, GBLUP models allowed breeding value prediction for individuals from families that were not included in the developed models, which was not possible with the ABLUP. Response to selection differences between the ABLUP and GBLUP models indicated the presence of systematic genetic gain overestimation of 35 and 63% for HT and WD, respectively, mainly caused by the inflated estimates of additive genetic variance and individuals’ breeding values given by the ABLUP models. Extending the OP genomic-based models from single to multisite made the analysis applicable to existing OP testing programs.The advantages of open-pollinated (OP) family testing over controlled crossing  are the potential to screen and rank a large number of parents and offspring with minimal cost and efforts; however, the method produces inflated genetic parameters as the actual sibling relatedness within OP families rarely meets the half-sib relatedness assumption. Here, we demonstrate the unsurpassed utility of OP testing after shifting the analytical mode from pedigree- (ABLUP) to genomic-based (GBLUP) relationship using phenotypic tree height (HT) and wood density (WD) and genotypic (30k SNPs) data for 1126 38-year-old Interior spruce ( A-matrix; Wright G-matrix) which can be used as a substitute to the A-matrix in quantitative genetics analyses   and the realized genomic relationship G-matrix (the observed relationships). More specifically, we evaluated the genomic selection predictive ability after removing genetic relatedness between the training and validation populations during the cross-validation process and determined the response to selection differences between the expected and realized relationships for the studied attributes. This OP progeny test permitted extending the single-site analysis to a more generalized multiple-site model that partitioned the genetic variance components into additive and nonadditive effects as well as accounting for and determining the extent of genotype × environment interaction, thus allowing covering a more complex genetic structure.Here, using 25 Interior spruce OP families grown in a replicated block design over three sites in British Columbia, Canada, we compared the genetic variance estimates generated from the average numerator relationship Picea glauca (Moench) Voss), Engelmann spruce (Picea engelmannii Parry), and their natural hybrids, and because of their similar growing habitat and silvicultural requirements, they are often collectively treated as one complex species ; WD (g·cm−3) was determined from bark-to-bark wood cores using X-ray scanning ; the cores were extracted from each tree at breast height in the north-south direction by 5-mm increment borers.Interior spruce is a complex of white spruce  was theZ is the rescaled genotype matrix following M − P; M is the genotype matrix containing genotypes coded as 0, 1, and 2 according to the number of alternative alleles; and P is the vector of twice the allelic frequency p ; a is the vector of random additive genetic effects following a ~ N interaction effects following axe ~ N is the vector of random block (replication) nested within the site effect following r(s) ~ N were obtained from the model described above, but the average numerator relationship matrix A is substituted by the marker-based relationship matrix Gadd. The extended model for the dominance term (GBLUP-AD) is performed as follows:d is the vector of the random dominance effect following d ~ N were obtained by solving the mixed models following:The narrow-sense heritability estimate was estimated as mentclass2pt{minimR2) was used , while the standard error (SE) of the predictions (SEPs) of the BVs was used to assess the precision of the BVs.Finally, to compare the relative quality of the goodness-of-fit for the said models, the variance explained by each model ) and the prediction accuracy  and predicted BVs from cross-validation (PBV-CV)) for the four models were estimated using 10-fold CV and five replicates. To assess the role of relatedness between the training and validation populations on the genomic selection predictive accuracy, two folding scenarios were used, namely, random  and family (removed relatedness as entire families were absent during the CV process) folding. In each replicate, the data was divided into 10-folds according to the used folding scenario, 9-folds was assigned as the training population, while the last fold was used as the validation population to estimate PBV-CV. The five replicates were used to estimate the SE of the correlation. Model pairwise prediction accuracy was also estimated between the four models in order to evaluate the ability of predicting each other. In this case, accuracy was estimated as the Pearson product-moment correlation between EBV-all of one model and PBV-CV of the other model (see above).Replications within-site variance components were consistent across the four models and accounted in each case for a relatively small variance component for both height HT 1.63–2.07%) and wood density (WD 6.91–8.62%)   can only be expressed through the terms “additive variance × environment,” and these were the first and third highest variance components, accounting for 34.73 and 16.98% of the total variance for HT and WD, respectively (Table R2) and the fitted line plots . Moving from ABLUP to the GBLUP-A was characterized by the lack of improvement for the two model comparison methods   model for HT , reflecting the presence of significant additive × additive genetic variances and indicating that GBLUP-ADE is the best model for WD. The R2 differences between HT and WD reflect the different genetic architecture of the two traits   for estimating genetic parameters and variance components; thus, the degree of genetic advance is dependent on the accuracy and precision of these parameters, specifically heritability and individuals’ breeding values. The utilized mating design determines mainly which genetic component can be generated (additive and dominance), and in some cases, additional efforts such as combining full-sib families with replicated clonal trials are required to estimate epistatic genetic variance (Foster and Shaw G-matrix) among individuals, irrespective of their genealogy, and substitute the A-matrix in estimating genetic variance components particularly in the population of forest trees (Denis and Bouvet R2 term (variance explained by the model) and the fitted line plots  Table . The threl Table ; Fig. 1 el Table .R2 value supporting better model fit (Table The GBLUP-AD model produced surprising results with a significant dominance variance for HT relative to the additive variance with a higher it Table . This wait Table . The sigit Table . Additioit Table  reportedP. glauca x P. engelmannii) in British Columbia , which was extended to include first-order interactions, gave exactly the same results as GBLUP-AD for HT indicating the absence of all kinds of epistatic interactions; this model subsequently showed no improvement in all goodness-of-fit measures and precision estimates Table  and 2. Tes Table , the fites Table  and 2. Aes Table  and in les Table . Thus, tes Table  and the The advantage of GBLUP models is their use of the realized genomic relationship among individuals regardless of their genealogy, while the ABLUP is mainly dependent on the pedigree-structure created by mating designs. Additionally, to capture the additive relatedness among individuals, the realized genomic relationship matrix is also capturing the linkage disequilibrium (LD) between the SNPs and quantitative trait loci (QTLs) and their co-segregation  and dominance as well epistasis variances (GBLUP-ADE in the case of WD) resulted in improving the breeding values’ estimates precision Fig. ; howeverThe observed predictive accuracy differences between the random (retaining genetic similarity) and family (excluding genetic similarity) folding are of great importance Table . Our resR = h2S; Falconer et al. The reported multisite genetic variance decomposition along with the selection of the best model for each studied trait is expected to improve the genetic variance partition (see above) as well as the individuals’ breeding values. We compared the ranking of the top 50 individuals for HT and WD between the pedigree-based ABLUP and the genomic-based GBLUP models  Fig. . Only 78The utilization of the genomic-based relationship in tree improvement testing and evaluation programs is implemented, and its usefulness, in particular for the simplest OP testing mode, is demonstrated. The proposed approach of genetic variance decomposition was illustrated, and additional estimates such as dominance and epistatic genetic variances as well as their interactions with the environment were obtained. The incorporation of genomic information in the analysis provided more accurate genetic variance estimates, particularly for those that could not be derived using the traditional OP pedigree-based analysis. The negative role of genetic similarity between training and validation populations on the predictive accuracy of genomic selection as well as examples of the unrealistic genetic gain estimates derived from pedigree-based analyses was illustrated. Finally, extending the GBLUP model to include multisite makes the analysis applicable to existing OP testing programs.


From 2012 to 2016, California experienced one of the worst droughts since the start of observational records. As in previous dry periods, precipitation-inducing winter storms were steered away from California by a persistent atmospheric ridging system in the North Pacific. Here we identify a new link between Arctic sea-ice loss and the North Pacific geopotential ridge development. In a two-step teleconnection, sea-ice changes lead to reorganization of tropical convection that in turn triggers an anticyclonic response over the North Pacific, resulting in significant drying over California. These findings suggest that the ability of climate models to accurately estimate future precipitation changes over California is also linked to the fidelity with which future sea-ice changes are simulated. We conclude that sea-ice loss of the magnitude expected in the next decades could substantially impact California’s precipitation, thus highlighting another mechanism by which human-caused climate change could exacerbate future California droughts. Persistent atmospheric ridging in the North Pacific steered storms away and led to the California drought of 2012-16. Here the authors use simulations to show that sea-ice changes trigger reorganization of tropical convection resulting in drying over California. These uncertainties are largely a consequence of the sensitivity of Californian rainfall to both tropical and mid-latitude atmospheric circulation changes, such as variability in the El Niño/Southern Oscillation (ENSO) and shifts in the Pacific jet stream4. California’s winter precipitation has decreased over the past two decades, and between 2012 and 2016 California has entered into one of the most severe droughts on record9. The impacts of reduced rainfall have been intensified by anomalously high temperatures that have enhanced potential evapotranspiration5. Several studies posit that California’s drought has an anthropogenic component arising from increased temperatures, with the likelihood of such warming-enhanced droughts expected to increase in the future11.There is considerable uncertainty in model projections of twenty-first century precipitation changes over California6. A La Niña event in 2011/12 and anomalously warm sea-surface temperatures (SST) over the west tropical Pacific in 2012/13 and 2013/14 may have helped sustain the North Pacific geopotential ridge8. Climate model simulations forced with observed SSTs alone were unable to capture all the important features of the recent precipitation decline, suggesting that California’s recent precipitation deficit may in part be attributable to other factors, such as internal atmospheric variability8.The exceptionally dry conditions during the winters of 2012–2015 were accompanied by a prominent dynamical feature: a persistent geopotential ridge located in the North Pacific. This ridge pushed storm tracks further north, resulting in wetter than normal conditions over the northwest and substantial drying over the southwest of the United States12. Most of the existing literature points to anomalous tropical SSTs and convection as dynamical drivers of the geopotential changes in the North Pacific14. However, at least one study has implicated Arctic sea-ice loss as a possible cause of the drier winters over the American southwest18. One proposed physical mechanism involves surface warming and geopotential height increase over the newly open ocean areas in the Arctic, which in turn drives anomalous ridging over the North Pacific by perturbing high-latitude planetary wave patterns15. Although this mechanism is not considered to be a primary driver of California’s precipitation variability, scientific interest in the “sea-ice hypothesis” has been heightened by the pronounced Arctic sea-ice loss over the satellite era and the dramatic sea-ice retreat expected by the end of the twenty-first century19. Motivated by this, we focus on elucidating the possible mechanisms by which large-scale sea-ice cover changes could affect precipitation over California.A positive geopotential anomaly in the North Pacific is not unique to the recent drought. Similar anomalies are known to be associated with precipitation decline across the American southwest20), thereby obtaining an ensemble of simulations with a seasonally ice-free Arctic (“low Arctic ice” simulations). These are compared to control simulations representative of sea-ice conditions at the end of the twentieth century. A comparison between “low Arctic ice” and control simulations allows us to isolate the atmospheric response that is solely associated with changes in sea-ice cover. Neither the SSTs nor the sea-ice cover are prescribed in our model simulations. We are therefore able to account for atmosphere–surface ocean interactions and SST changes necessary for propagation of high-to-low latitude atmospheric teleconnections23. Unlike previous studies that impose artificial energy fluxes to achieve sea-ice loss25, our sea-ice parameter perturbations allow for energy budget conservation.A novel feature of our approach is that we sample the uncertainties in selected sea-ice physics parameters  coupled to a mixed-layer ocean model. We do not investigate how this initial atmosphere–surface ocean response may become modulated by the slower deep-ocean response over longer  time periods as in refs. 25. We verify that at the short timescales of interest here (<50 years after the sea-ice loss), an AGCM coupled to a slab ocean yields a qualitatively similar short-term response to that produced by the same AGCM coupled to a full ocean model (AOGCM) (see Supplementary Discussion). Our results show that the consequences of Arctic sea-ice decline are not restricted to the Arctic region alone. Loss of Arctic sea-ice cover of the magnitude expected in the next few decades, could induce large-scale atmospheric circulation changes across the Northern hemisphere, resulting in significant drying over California.Our primary interest is in the fast atmospheric response to sea-ice changes in the first decades after the sea-ice loss has occurred. This fast response is mediated by the surface ocean, and typically dominates the overall response during the first years or decades after the initial high-latitude perturbation2 threshold used to define a nearly ice-free Arctic19. By comparison, CMIP-5 simulations of twenty-first century climate and extrapolations of observed changes in ice volume place the time horizon for a nearly ice-free Arctic roughly between 2020 and 204019. The smallest and largest differences between the “low Arctic ice” and control Arctic sea-ice area  in response to sea-ice changes. Experimental configurations that impose artificial surface energy fluxes in order to remove sea-ice cover necessarily yield a substantially larger surface energy imbalance than the one described here.In the first step of this teleconnection, sea-ice loss alters the high-latitude energy budget  can provide a compensation for energy budget perturbations elsewhere23. This is not dissimilar to the observed changes in tropical TOA fluxes during ENSO-related convection changes that are also capable of affecting the global energy budget32.Tropical compensation of high-latitude energy budget perturbations has been noted in other studies employing AGCMs coupled to a slab ocean model23. Specifically, over the northern tropics, in the region of the northeasterly trades, the progression of cold (warm) SST anomalies will lead to an increase (decrease) in the surface wind strengths. This results in increased (decreased) evaporative cooling, thus allowing for further propagation of cold (warm) anomalies toward the equator. Over the southern tropics, the response is the opposite, due to the background southeasterly flow. Negative (positive) wind strength and latent heat flux anomalies over the northern (southern) tropics in response to Arctic sea-ice loss are shown in Supplementary Fig. The mechanism by which high-latitude changes propagate into the tropics and induce tropical convection responses involves two processes: (i) a southward advection of the initial high-latitude temperature perturbation over the mid-latitudes through transport and mixing; and (ii) a wind/evaporation/sea-surface temperature feedback in the region of the northeasterly trades. The tropical response to high-latitude sea-ice loss in our model simulations is consistent with these findings  in the equatorial Pacific initiates a northward-propagating Rossby wavetrain. The characteristics of this wavetrain are positive stream function and geopotential anomalies immediately outside the region of increased convection (anticyclonic flow), and negative anomalies in the extratropical Pacific (cyclonic flow). We find that this mechanism also operates in our simulations.We suggest that the tropical Pacific convection changes described above play a key role in driving the North Pacific circulation changes. Linkages between the equatorial Pacific convection anomalies and extratropical (North Pacific) atmospheric circulation changes are found in both observational and modeling studiesDuring the DJF season, the area of strongest decrease in high cloud cover is located over the central tropical Pacific  California Fig. . Arctic 13. While such tropical and subtropical convection changes could be a consequence of natural variability, our analysis indicates that they can also be forced from the high latitudes by substantial sea-ice loss. Our simulations imply that two hitherto separate hypotheses—that Californian rainfall is primarily influenced by tropical convection changes 11 and that Arctic sea-ice loss can drive precipitation changes over California (“Arctic sea-ice loss hypothesis”)30—are not easily separable. Our study demonstrates that the impacts of Arctic sea-ice changes require understanding complex atmospheric teleconnections that can propagate back and forth between the extratropics and the tropics.The schematic in Fig. 40. This is the opposite to the precipitation response accompanying Arctic sea-ice loss. In accord with previous studies36 and the mechanism advanced here, increased convection should in turn drive a northward-propagating Rossby wavetrain consisting of an anticyclonic anomaly in the tropical Pacific and a cyclonic anomaly in the North Pacific. This cyclonic flow is associated with negative geopotential height anomalies over the North Pacific  leading to wetter conditions over California. Simulations in which the sea-ice physics parameter perturbations are prescribed in the Southern hemisphere only  and no high northern latitude changes, it provides additional support for our conjecture that the sea-ice changes can influence North Pacific geopotential height through tropical convection changes. More generally, any high-latitude perturbation (northern or southern hemispheric warming or cooling) that impacts the position of the tropical Pacific ITCZ, will have an impact on California’s rainfall.Many aspects of the observed mean state of the atmosphere during the recent 2012–2016 California drought are qualitatively consistent with our simulated response to Arctic sea-ice changes. ERA-Interim estimates of the precipitation, geopotential, and OLR anomalies between 2012 and 2015 (relative to the period 1979–2010) are shown in Supplementary Fig. 41. Another important factor may be the 2014 phase shift of the Pacific Decadal Oscillation from negative to positive. However, this shift would be expected to alleviate drought conditions over California—not intensify them42. Finally, we note that other hemispherically asymmetric forcings may also trigger—via the same physical pathways described here—tropical convection changes and teleconnections affecting California’s precipitation. Possible examples include asymmetric forcing by anthropogenic aerosols, volcanic eruptions, and solar irradiance variations. All of these forcings can alter the inter-hemispheric temperature gradient43, thereby causing shifts in the location of tropical convection and initiating “far-field” precipitation changes over California.This consistency does not, however, constitute compelling evidence that the 2012–2016 California drought is attributable to Arctic sea-ice changes. Rather, it illustrates that some of the atmospheric features of the droughts driven by Arctic sea-ice loss may resemble those of the most recent California drought. The intensification of dry conditions over California since late 2012 may have also been affected by several other factors not discussed in this study, such as the appearance of a large warm SST anomaly off the west coast of North America—though this appears to be a consequence rather than a cause of the altered atmospheric circulations causing the drought44. Abrupt high-latitude temperature increase during the last interglacial (the Dansgaard–Oeschger and Bølling–Allerød warmings) are manifest in the tropics through a northward shift in the position of the ITCZ47. Conversely, the signature of cold stadials and Younger Dryas cooling in the Arctic is matched by a southward shift of tropical precipitation across all ocean basins48. These ITCZ shifts are consistent with precipitation changes inferred from high-resolution, precisely dated speleothem records: speleothems across the southwestern United States indicate shifts to drier conditions during the Dansgaard–Oeschger warmings50, the Bølling–Allerød warming, and the termination of the Younger Dryas cooling51. Similarly, during the Heinrich 1 and Younger Dryas cold periods, wetter conditions are indicated by lake-level high stands in the Great Basin and glaciation in central and southern California53, while at least one modeling study has implicated North Atlantic cooling and a corresponding southward ITCZ shift as a possible cause of increased precipitation over the western United States54.It is of interest to consider whether paleoclimatic evidence provides support for our model-inferred two-step teleconnection Fig. . We focu55. In addition, volcanic activity may have also had an impact on tropical precipitation during this time56. However, proxy data for the last millennium does yield information regarding the second teleconnection step—the link between the ITCZ location and southwestern rainfall. The southward shift of the ITCZ in the transition from the medieval climate anomaly (MCA) to the little ice age (LIA) is well documented58. The effect of this shift is apparent in the contrast between the dry MCA and the wet LIA in the western US lake levels59, from trees growing on lake beds exposed by low lake levels61, and in pollen records62. Interestingly, a brief southward shift in the ITCZ position between ~AD 1050 and 125057 is clearly recorded in the American southwest as a wet interval in the midst of the dry MCA. Great Basin lakes expanded briefly during this period64, concurrent with drowning of trees growing below modern water levels61 and increased soil moisture in southern California (as inferred from pollen records)62. Such periods during the MCA and LIA provide some support for the second teleconnection step. In the absence of more reliable sea-ice data sets, however, we cannot confirm if such covariance relationships between the ITCZ location and southwestern rainfall are directly attributable to sea-ice changes, or could instead be due to other factors .The timing, magnitude, and drivers of sea-ice changes during the last millennium are less well understood30. Additional analysis of the short-term (first 50 years) response of an AOGCM response to sea-ice changes in a complementary, energy-conserving setup confirms our AGCM/slab ocean findings  the different timescales of the response; and (ii) studies that attempt to isolate the impacts of sea-ice changes by imposing large artificial energy perturbations in order to achieve the sea-ice loss25, and investigations that achieve sea-ice changes in an energy-conserving manner (ref. 30 and the current study).Finally, we address the suitability of our experimental configuration (an AGCM coupled to a slab ocean) for studying the fast atmosphere–surface ocean-mediated response to sea-ice changes. In our simulations, key response features are manifest within the first two decades  response to Arctic sea-ice loss described in our study is consistent with the response found in previous AOGCM studies that allow for ocean dynamics and deep-ocean changes. The long-term centennial climate response to sea-ice changes may, however, differ from the fast response described here. While investigations with other climate models are necessary to further confirm these findings, our results strongly suggest that what happens in the Arctic does not stay in the Arctic and provide a physically plausible pathway by which high-latitude sea-ice forcing may mediate tropical climate, and thereby influence California’s rainfall.68. Our study thus identifies yet another pathway by which human activities could affect the occurrence of future droughts over California—through human-induced Arctic sea-ice decline.As a final remark, we note that the pronounced Arctic sea-ice loss over the satellite era is likely human-induced, arising from anthropogenic warming caused by greenhouse gas increases69. The model configuration incorporates the Community Atmosphere Model version 4 (CAM4)70, Community Land Model version 4 (CLM4)71, and Los Alamos Sea Ice Model version 4 (Community Ice CodeE 4—CICE4)72 coupled to a mixed-layer (slab) ocean. The atmosphere and land models are run on a 1.9° × 2.5° (latitude × longitude) finite volume grid with 26 atmospheric levels in the vertical dimension. The ice and ocean models use a 1° displaced pole grid. The greenhouse gas concentrations, solar insolation, and orbital parameters are representative of the year 2000 values for all simulations. The prescribed ocean heat flux (“q-flux”), salinity, temperature, and velocity fields required for the mixed-layer ocean model simulations are derived from a fully coupled CCSM simulation using the full-depth ocean.We use the Community Climate System Model (CCSM) version 4In order to isolate the atmospheric impacts of sea-ice loss, we compare two sets of experiments that differ only in the amount of sea-ice cover. For this purpose, we have developed a novel experimental configuration in which we alter the sea-ice cover without imposing artificial energy fluxes in the high latitudes as done in the previous studies. When sea-ice loss is achieved by imposing non-physical heat flux perturbations, energy is not conserved. This makes it difficult to determine if the observed atmospheric response originates solely from the sea-ice changes or if it is also impacted by the imposed energy flux perturbations. Analyzed response represents cumulative effect of sea-ice extent and thickness changes.20. The selected sea-ice physics parameters are shown in Supplementary Table Sea-ice loss in our model simulations is achieved through sea-ice physics parameter perturbations. We use an ensemble of 70 Latin hypercube simulations to select the sea-ice physics parameters that have the strongest impact on the resulting sea-ice extentt-test in order to account for different sample size and variances between the control and “low Arctic ice” simulations. Prior to t-test calculations, the equivalent sample size is determined at each grid point .Statistical significance is estimated using Welch’s https://esgf-node.llnl.gov/projects/psipps/. Provided are monthly means of the following CAM4 variables: ice fraction (ICEFRAC), surface pressure (PS), surface temperature (TS), upper tropospheric temperature (TROP_T), total precipitation (PRECT), geopotential (Z3), horizontal and meridional wind speed components (U and V), vertical (pressure) velocity (OMEGA), net longwave flux at the atmosphere top (FLNT), and high cloud cover (CLDHGH); all for years 20–40 of model integration. Additional data can be made available on request.Simulation output processed for the purpose of this study has been made public and can be obtained at Perturbed sea-ice physics parameter simulations require changes to the following parameters: snow grain radius tuning parameter, thermal conductivity of snow, and snow melt maximum radius. Their respective values are given in Supplementary Table Peer Review FileSupplementary Information


Epidemiological studies have found that triple-negative breast cancer (TNBC) and TN inflammatory breast cancer (IBC) are associated with lower frequency and duration of breast-feeding compared to non-TNBC and non-TN IBC, respectively. Limited breast-feeding could reflect abrupt or premature involution and contribute to a “primed” stroma that is permissive to the migration of cancer cells typical of IBC. We hypothesized that gene expression related to abrupt mammary gland involution after forced weaning may be enriched in the tissues of IBC patients and, if so, provide a potential correlation between limited breast-feeding and the development of aggressive breast cancer.We utilized the Short Time-series Expression Miner (STEM) program to cluster significant signatures from two independent studies that analyzed gene expression at multiple time-points of mouse mammary gland involution. Using 10 significant signatures, we performed gene ontology analysis and gene set enrichment analysis (GSEA) on training and validation sets from human breast cancer gene expression data to identify specific genes that are enriched in IBC compared to non-IBC and in TN compared to non-TN in IBC and non-IBC groups.Examining the combined data, we identified 10 involution gene clusters (Inv1-10) that share time-dependent regulation after forced weaning. Inv5 was the only cluster significantly enriched in IBC in the training and validation set  and only by unadjusted p-values . Eight genes in Inv5 are upregulated in both the training and validation sets in IBC. Combining the training and validation sets, both Inv5 and Inv6 have nominal p-values <0.05 and q-values 0.39 and 0.20, respectively. The time course for both clusters includes genes that change within 12 hours after forced weaning.in silico study suggest correlation between molecular events during abrupt involution and aggressive breast cancer. Specifically, candidate genes from Inv5 merit functional investigation regarding the role of limited breast-feeding in IBC development.Results from this  Involution is a term that has been described as the reverse of development . Post-lagression –12.Breast cancer is the most common cancer and the second leading cause of cancer mortality among women in the United States . In the JNK1). Specifically, we identified 3 genes–Involucrin (IVL), Cluster of Differentiation 79B (CD79B), and leptin (LEP)–that were significantly enriched in IBC compared to non-IBC. Thus, it is possible that these genes play a role in IBC development and progression.Given the correlation between minimal breast-feeding and aggressive breast cancers, we hypothesized that gene expression signatures of the abrupt post-lactational involution stage of mammary gland development persist, are complicit in the development and progression of these cancers, and are therefore enriched in TNBC and IBC bulk tumor samples. To test our hypothesis, we identified gene expression signatures for the abrupt post-lactational involution stages of mammary gland development using gene expression data from post-natal mouse mammary gland development and evaluated whether or not these gene expression signatures were enriched in IBC versus non-IBC as well as TNBC versus non-TNBC. We found significant enrichment of one post-lactational involution gene signature in IBC compared to non-IBC. This enriched signature represents genes showing initial up-regulation and later down-regulation during the involution process and significant overlap with genes upregulated in vascular smooth muscle cells (VSMC) by c-Jun N-terminal protein kinase  analysis  with quaTests of differences in expression were performed with the limma package (version 3.22.1)  from theErnst et al. presented an algorithm specifically designed for clustering time-series expression data  and deveMus musculus gene products available from Mouse Genome Informatics. Enrichment analysis for GO annotations was performed using a hypergeometric distribution in the STEM program, and multiple hypothesis correction was done using a randomization test. For gene-ontology enrichment with this program, p-values were corrected with 500 randomizations and were considered significant with an FDR of <0.05.An ontology-based analysis was performed on genes of significant clusters identified through the STEM program. We used gene ontology (GO) annotations for Gene expression data for IBC and non-IBC cases were obtained from the NCBI Gene Expression Omnibus (GSE22597) and the EBI ArrayExpress (E-MTAB-1006 and E-MTAB-1547) and collected through the World IBC Consortium . These dWe also examined benign-appearing breast tissues from both IBC and non-IBC patients, 44 in total (19 with IBC and 25 with non-IBC) for further analysis. All were treated with neoadjuvant chemotherapy and mastectomy from March 2004 –May 2012. Clinical details for all patients was recorded as part of an institutional database or prospective tumor registry. All patients gave written informed consent to banking surplus tissue for future research prior to study enrollment. This specific study was separately approved by the appropriate institutional review board of The University of Texas MD Anderson Cancer Center to examine these banked tissues and correlate findings to clinical demographics.Raw gene expression profiles were preprocessed using GCRMA analysis  with quaWe employed the previously described methods for gene expression studies from normal adjacent breast tissue , 30. BriInvolution-specific gene signatures were identified from the results of STEM cluster analysis conducted on post-natal mouse mammary gland development as previously described here. We identified orthologous genes for genes that were found to form significant clusters in STEM cluster analysis by using ENSEMBL gene id on the orthologous data downloaded from the ENSEMBL website. Involution-specific gene signatures have also been reported in the study by Stein et al. . We downWe used GSEA algorithm as mentioned in  to evaluA training set is a set of data used to discover potentially predictive relationships, and a validation set is used to assess the strength and utility of said predictive relationships. We divided the merged dataset into the training set to run the GSEA and into the validation set to validate the GSEA results for reproducibility. We used the stratified random sampling method with inclusion of information on IBC status, TN status, and age at diagnosis (<50 years or > = 50 years) to divide the merged dataset into the training and validation sets .We used the limma package  from thep-value of significant clusters out of 50 possible cluster profiles. Patterns indicate the log2 fold change in expression of genes in clusters compared to the lactation day 10 levels. Given the limited overlap between the two datasets, we sought to identify relevant gene clusters over time from the two published studies together. We used the STEM algorithm developed by Ernst et al. , 27 to cTo use the involution-specific gene signatures to conduct gene set analysis on human IBC and non-IBC gene expression profiles, we identified human orthologous genes for genes of significant clusters identified through STEM . OrtholoOut of 10 gene signatures developed through the STEM clustering using data from Clarkson et al. , 7 signaOut of 10 gene signatures developed through STEM clustering using data from Clarkson et al. , we founUnfortunately there are not additional samples from which to validate the findings beyond the report above. Nevertheless, we speculated that the relevant signatures might be expressed in gene expression data from histopathologically normal breast isolated from IBC (N = 19) and non-IBC (N = 25) mastectomy patients given our hypothesis that the unique normal tissue changes influence the symptoms of IBC.Out of 10 gene signatures developed through STEM clustering using data from Clarkson et al. , we founThis again demonstrates enrichment of involution signatures in human breast tissues from women with breast cancer, but the significant enrichment pattern is different in normal versus tumor. Again, this is limited by a small sample size . Of the three genes mentioned from Inv5 tumor data as upregulated, only Leptin is upregulated in IBC in normal breast tissue. Of note, Inv1 genes with 2-fold enrichment are the following: CYP4B1, ACADL, PCK1, RASA3, CDO1, ABCA1 . We focused on genes that were differentially expressed during time periods spanning from the last day of lactation (day 10) to the fourth day of involution by greater than 2-fold (p <0.05) and performed STEM cluster analysis to discern time-varied expression patterns. We identified 10 time-based gene clusters that represent this time period, Inv1-10. Broadly, many are enriched versus depleted in IBC samples, but only Inv5 is significantly enriched in both the training and validation set based on nominal p-values, and note that FDR adjusted p-values are not significant for this cluster. Nevertheless, given the limitations of the data and the lack of larger datasets or similar extensive gene array studies of the involuting human breast, these hypothesis-generating findings may merit further study.Up to now, the two most comprehensive studies examining global gene expression on the post-lactational mammary gland have been conducted by Clarkson et al. , 5 and SWe examined the enrichment of post-lactational mammary gland involution gene expression patterns in TNBC and IBC using the GSEA method. First, we used 10 significant time varied gene expression patterns that we found in the dataset from Clarkson et al. (3). We found that only one gene expression pattern was enriched in IBC compared to non-IBC in both training and validation sets at the nominal p-value. None of these gene expression patterns was enriched in TNBC compared to non-TN BC for both IBC and non-IBC groups in both training and validation sets. Second, we used 13 gene expression patterns on post-lactational involution as reported by Stein et al (9) and found that none of these gene expression patterns was significantly enriched in IBC compared to non-IBC and TN BC compared to non-TN BC in both training and validation sets. To investigate further, we examined the overlap between the involution-specific signatures and the IBC-like signature (79 genes) . We founIVL), Cluster of Differentiation 79B (CD79B), and leptin (LEP)–were significantly enriched in IBC compared to non-IBC in both training and validation data sets in our analysis. Involucrin is a transglutaminase substrate protein present in keratinocytes of epidermis and other stratified squamous epithelia [OB) gene, an important regulator of energy balance and necessary for normal mammary gland development [One gene signature that showed nominal enrichment in IBC compared to non-IBC, Inv5, contained genes that showed initial up-regulation and later down-regulation during the involution process. This might suggest that genes that upregulate during an initial phase of involution after abrupt weaning might not be turning off and, therefore, could be responsible for facilitating an IBC-like phenotype after a tumor-initiating event. We examined the overlap of this gene expression pattern with the existing gene signatures using the Molecular Signatures Database (MSigDB) v4.0 . We founpithelia . Tsuda epithelia  investigelopment . In ER-pelopment . Thus, oA major strength of our study is the use of the largest series of IBC samples ever reported by the World IBC Consortium. Furthermore, this work is novel in part due to the use of updated Chip Definition Files from the BrainArray  during pIVL, CD79B, and LEP in particular may play in role in IBC development after abrupt or involution. Given the limitations of this study, however, additional research is warranted before a concrete conclusion can be made.Major limitations of our study include the cross-sectional analysis of enrichment of involution specific signatures in breast cancer and array-based measurement of gene expression profiles, which limit the detection of differentially expressed genes with lower levels of expression. Also, although having the parity status of the patients within the World IBC consortium would be valuable to our study, that information is not recorded or available to us and, as such, we cannot determine or comment on whether parity-related effects persist or were present prior to the time of analysis. Further, there are surprisingly few TNBC in the cohort given the established over-representation of these subtypes in IBC which may influence the findings overall and regarding TNBC versus non-TNBC. Additionally, the studies by Clarkson et al (3) and Stein et al (4) did not include a corresponding group that underwent non-abrupt involution or that was not force weaned, as the authors did not distinguish between abrupt involution versus the normal involution process. Thus, the correlation between limited nursing and abrupt involution signatures remains unstudied here. Although inclusion of such a control group in these studies would be useful to our analysis, we can speculate that Inv5 and In conclusion, our results provide some evidence that molecular events after abrupt involution are identifiable in IBC patient tissues from the uninvolved breast and tumor; however, they are hypothesis-generating given the potential for false discovery after multiple comparisons as well as the other noted limitations of our study. Whether or not Inv5 or Inv6 related genes or signaling are upregulated in the normal tissues around IBC tumors, and if breast-feeding or abrupt cessation of breast-feeding contributes to the persistence of related genes in the normal breast will be investigated in future studies.S1 Table(DOCX)Click here for additional data file.S2 TableBP, biological process; CC; cellular components.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


A long non-coding RNA (lncRNA), named myocardial infarction associated transcript (MIAT), has been documented to confer risk of myocardial infarction (MI). The aim of this study is to elucidate the pathophysiological role of MIAT in regulation of cardiac fibrosis. In a mouse model of MI, we found that MIAT was remarkably up-regulated, which was accompanied by cardiac interstitial fibrosis. MIAT up-regulation in MI was accompanied by deregulation of some fibrosis-related regulators: down-regulation of miR-24 and up-regulation of Furin and TGF-β1. Most notably, knockdown of endogenous MIAT by its siRNA reduced cardiac fibrosis and improved cardiac function and restored the deregulated expression of the fibrosis-related regulators. In cardiac fibroblasts treated with serum or angiotensin II, similar up-regulation of MIAT and down-regulation of miR-24 were consistently observed. These changes promoted fibroblasts proliferation and collagen accumulation, whereas knockdown of MIAT by siRNA or overexpression of miR-24 with its mimic abrogated the fibrogenesis. Our study therefore has identified MIAT as the first pro-fibrotic lncRNA in heart and unraveled the role of MIAT in the pathogenesis of MI. These findings also promise that normalization of MIAT level may prove to be a therapeutic option for the treatment of MI-induced cardiac fibrosis and the associated cardiac dysfunction. On one hand, generation and maintenance of the extracellular matrix (ECM) in the scar is essential for preventing dilatation of the infarct zone leading to HF. On the other hand, ECM deposition at regions remote from the infarct area can result in excessive collagen production thereby cardiac stiffness that constitutes the basis of the development of HF. Because of this delicate balance between good and bad, delicate regulation of cardiac fibrosis is absolutely required by highly evolved creatures like our human beings. Indeed, cardiac fibrosis is an intricate process orchestrated by a wide spectrum of causal factors and regulatory factors and their mediated signaling pathways. Thorough understanding of the regulation of cardiac fibrosis is therefore imminently desirable for more rational management of MI and MI-induced development of HF associated with fibrosis.Among various fibrosis-regulating pathways, the transforming growth factor β1 (TGF-β1) signaling pathway is of paramount importance7Myocardial infarction associated transcript (MIAT) has been reported to be associated with the pathogenesis of myocardial infarction (MI)To investigate if MIAT up-regulation in MI can produce any consequences on the pathological process or is merely a consequence of myocardial ischemia, we assessed the effects of MIAT knockdown on cardiac function in the context of MI. We first injected the lentiviral vectors carrying siMIAT (Len-siMIAT) into left ventricular chamber of mice and then occluded the left descending coronary artery (LAD). We then performed echocardiographic measurements 4 weeks after siMIAT administration. The aberrant upregulation of MIAT in MI hearts was significantly inhibited by the administration of Len-siMIAT  for 24 h, and found that MIAT expression was up-regulated by 3.1 ± 0.7 and 5.8 ± 1.5 times respectively compared with control group . In addiThe efficacy of siMIAT to knockdown endogenous MIAT was examined in the NMCFs. Of the 5 siRNA for MIAT, 2 siRNAs (siMIAT-3 and siMIAT-5) reduced the expression of MIAT by approximately 60% and 52%, while siMIAT-1, siMIAT-2, and siMIAT-4 did not produce such effects in NMCFs. siNC (negative control siRNA) showed no appreciable effects on MIAT levels . As expeRecent studies have demonstrated that lncRNAs can act as competing endogenous RNAs (ceRNAs) or endogenous sponge RNAs to bind miRNAs by sequence complementarity so as to absorb them and decrease the availability of the functional miRNAs in the cell16We used luciferase reporter gene assay to establish the direct functional interactions between MIAT and miR-24. As illustrated in The above results strongly suggested that up-regulation of MIAT in the setting of MI could dramatically reduce the availability of functional miR-24 in the heart, or more specifically in CFs. This thought prompted us to continue our investigation on the changes of expression of Furin, a target gene for miR-24. Our data demonstrated that Furin was significantly up-regulated in its expression at the protein level in peri-infarct region of the MI hearts . In sharFurin is a proprotein convertase enzyme that process latent precursor proteins into their biologically active products and it plays an important role in TGF-β1 activation  leading to cardiac fibrogenesisin vivo model and by AngII in an in vitro cellular model with cardiac fibroblasts.LncRNAs have been increasingly recognized for their roles in shaping cardiac structure and function and defining the pathogenesis of cardiovascular disease. Like miRNAs, these regulatory molecules are often able to cause well-defined phenotypical changes of cells, tissues, organs or even the whole organisms. A number of lncRNAs have recently been identified for their roles in cardiovascular pathology1719et al.in vivo and in vitro models, which by working together with miR-489 and Myd88 constitutes a novel cardiac hypertrophy regulating system. Subsequently, the same group reported identification of another lncRNA named cardiac apoptosis-related lncRNA (CARL) that was shown to inhibit anoxia-induced mitochondrial fission and apoptosis in cardiomyocytes by relieving miR-539-dependent PHB2 repressionet al.Though a number of lncRNAs have been linked to cardiovascular disease through genetic linkage studies182223The mechanisms by which lncRNAs regulate gene expression are diverse and largely unpredictable based on their sequence information, except for their interactions with miRNAs. LncRNAs often contains sequence domains complementary to miRNAs, which confers their ability to bind miRNAs and serve as sponge of these small molecules so as to limit the availability of the targeted miRNAs16171911MI can result in death of ischemic cardiac tissue followed by ECM deposition/cardiac fibrosis and dysfunction of myocardial contraction. The adverse structural and functional remodeling processes during MI can eventually lead to heart failure. Preventing pathological fibrogenesis therefore has been regarded as a promising strategy for alleviating the remodeling processes thereby HF. Our data showed that preventing MIAT up-regulation by knocking down its expression significantly lessened the MI-induced cardiac fibrosis and restored the impaired cardiac function. At the cellular level, MIAT knockdown by a means of gene silencing techniques significantly inhibited cardiac fibroblast proliferation and collagen accumulation induced by high serum or AngII. These findings provided a new strategy for the management of MI hearts through targeting MIAT to limit cardiac fibrogenesis so as to retard the development of HF. From a broader perspective, MIAT may be considered a therapeutic target for other cardiac conditions associated with fibrosis. This conception should spur us into more future studies with rigorous and vigorous experimentations on MIAT and other lncRNAs as well.One of the limitations of the present study is the lack of artificial gain-of-function approaches to experimentally address the adverse effects of MIAT. This is primarily due to the technical difficulty of manipulating a large size RNA like MIAT (8760 bp) for overexpression. Nevertheless, the fact that MIAT was “overexpressed” in MI hearts could be regarded as pathological gain-of-function. It should be noted that while our study focused on cardiac fibrosis in the context of MI, it does not exclude any other unknown cellular functions and pathological roles that MIAT can possibly elicit in the heart. Additionally, the mechanisms of MIAT function are likely multiplex and require more studies to elucidate.et al. demonstrated that miR-133 and miR-30 participate in the process of cardiac fibrosis in hypertension-induced heart failureWith regard to the mechanisms by which MIAT takes its effects in cardiac fibrosis, our study focused on miR-24 only leaving other miRNAs  were provided by the Experimental Animal Center of the Harbin Medical University. Food and water were freely accessible by mice. All experimental procedures were performed in accordance with and approved by the Institutional Animal Care and Use Committee of the Harbin Medical University.Mice were anesthetized with Avertin  and their chests were opened to expose the hearts. The left descending coronary artery (LAD) was ligated with a 7/0 nylon suture at 2 mm below the border between left atrium and ventricle to induce myocardial infarction. Myocardial ischemia was confirmed by significantly S-T segment elevation with electrocardiographic measurements. The sham-operated mice for control underwent the same experimental procedures as the MI group but without ligation of LAD.After surgery mice were monitored daily for signs of infection and state of health and activity. The mice were anesthetized for echocardiography and hemodynamic measurements and sacrificed by cervical dislocation at 1, 2 and 4 weeks after MI.8 TU/ml) carrying siMIAT or siNC was injected into the left ventricular chamber and was forced into the coronary arteries when aortic artery was temporarily occluded for 15 s. Sham group mice received DMEM 70 μl for control. After 15 min of injection, myocardial infarction was induced in the transfected mice.MIAT siRNAs (siMIAT) and the negative control siRNA (siNC) were designed and purchased from Invitrogen . Their sequences are provided in Four weeks after injection of lentivirus, transthoracic echocardiography was performed to monitor changes of the left ventricular function using ultrasound machine Vevo2100 high-resolution imaging system  equipped with a 10-MH2 phased-array transducer with the M-mode recordings. Ventricular parameters were measured and analyzed including left ventricular internal diastolic diameter (LVIDd), left ventricular internal systolic diameter (LVIDs), interventricular septum diastolic thickness (IVSDT) and interventricular septum systolic thickness (IVSST), left ventricular ejection fraction (EF), and fractional shortening (FS). The mice were killed with overdose sodium pentobarbital after measurements and the hearts were quickly removed and placed in liquid nitrogen or 4% paraformaldehyde for later uses.Mice were anesthetized using Avertin  and the hearts were collected and fixed in 4% paraformalin for 24 h, embedded with paraffin, then cut into 5 μm thick cross-sectional slices along the center of the fibrotic scar. The slices were stained by Masson trichrome. Fibrotic tissue was stained blue and myocardium stained red. The fibrotic areas are calculated with image analysis software . We used the percentage of fibrotic tissue area (blue area) to the myocardial surface area (red area) and fibrotic area (blue area) to obtain the collagen volume fraction for evaluation of the cardiac fibrosis.NMCFs were isolated as described previously in detail4) were seeded on a 96-well plates and treated as designated. At 80% confluence, 15 μl MTT solution  was added into each well and the plates were incubated for 4 h. Then 200 μl of DMSO was added into each well to dissolve the formazan. The optical density was measured at 490 nm using a microplate reader .Cell viability was assessed by measuring the mitochondrial dependent reduction of 3--2,5-diphenyltetrazolium bromide (MTT), as previously describedTotal protein samples were extracted from NMCFs or left ventricular peri-infarct region  and corresponding regions of sham-operated hearts were collected 1, 2, 4 weeks after MI. Immunoblotting analysis was performed as already described elsewhere−^^Ct and normalized to β-actin as an internal control. The sequences of primers were synthesized by Guangzhou Ribo Biotechnology Co. Ltd  and are listed in Total RNA samples were extracted from cultured neonatal mouse CFs or left ventricular infarct border zone of heart using Trizol reagent . For each sample, 0.5 μg of the total RNA was converted to cDNA using high-capacity cDNA reverse transcription kit . RNA levels of MIAT, miR-24, collagen I (Col I) and collagen III (Col III) were detected by SYBR Green method on the ABI 7500 fast real-time PCR system . After circle reaction, the threshold cycle (Ct) was determined and the relative quantitative expression of MIAT, miR-24, and mRNAs was calculated using method 2A fragment of the mouse MIAT gene containing the miR-24 binding site (designated Luc-wt-MIAT) was PCR amplified, and so was a fragment with nucleotide replacement mutation (designated Luc-mut-MIAT). For luciferase reporter gene assay, HEK293 cells were cultured in 24-well culture plates and were transfected with varying constructs as to be specified below. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System .NMCFs after a variety of treatments were lysed in 0.05 M Tris buffer (pH 7.5), and the total collagen was extracted with picrosirius red in saturated picric acid. Collagen contents were measured using essentially the same procedures as previously reportedP < 0.05 was taken to indicate a statistically significant difference.Data are expressed as mean ± SEM in the present study. Statistical analyses were performed using one-way ANOVA and Student’s test. A two-tailed How to cite this article: Qu, X. et al. MIAT Is a Pro-fibrotic Long Non-coding RNA Governing Cardiac Fibrosis in Post-infarct Myocardium. Sci. Rep.7, 42657; doi: 10.1038/srep42657 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.