Apoptosis plays an important role in the myocardial loss after acute myocardial infarction and participates in the process of subsequent left ventricular remodeling and development of symptomatic heart failure. Finding a sensitive apoptotic marker that would help in prognostic stratification of patients after acute myocardial infarction and offer new therapeutic strategies is thus of a great importance. Several studies suggest that tumor necrosis factor-related apoptosis inducing ligand (TRAIL) represents a very promising marker of prognosis in patients with acute myocardial infarction. This review article provides an overview of current knowledge on the role of apoptosis in ischemic heart disease and highlights potentially beneficial apoptotic markers in clinical practice. Ischemic heart disease (IHD) is a leading cause of death worldwide. Acute myocardial infarction (AMI) is the most common first manifestation of IHD and represents a major cause of morbidity and mortality. In the past decades, treatment of patients with AMI has significantly improved. However, more patients subsequently suffer from left ventricular (LV) dysfunction and heart failure . AlthougApoptosis, a form of programmed cell death, represents a highly regulated and energy-requiring process by which activation of specific signaling cascades leads to a cell death . ApoptosSeveral recent studies have also demonstrated an important role of apoptosis in ischemic heart disease. Apoptosis significantly contributes to myocyte cell death in AMI and occurs predominantly in the peri-infarcted region , 15. HigApoptosis can be initiated through two main pathways: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. Both pathways converge on the same terminal pathway. Markers of apoptosis will be introduced thereinafter according to the pathway they are involved in.Death receptor pathway is significantly involved in inducing myocardial apoptosis in IHD. The mechanism of death receptor pathway has been extensively reviewed in the literature. Briefly, after an apoptotic ligand binds to its death receptor, death-inducing signaling complex is formed, resulting in caspase-8 activation . CaspaseIn experimental studies, apoptosis-stimulating fragment (Fas) is described as a critical mediator of cardiomyocyte apoptosis during ischemia/reperfusion (I/R) injury . HoweverIn the study by Fertin et al. levels of FasL were measured in patients 1 month after MI and several echocardiographic studies were performed up to 1-year post MI to evaluate LV volumes. LV remodeling was documented by a significant increase of LV volumes; however, changes in LV volumes were not associated with FasL levels .Similar findings were documented by Nilsson et al. who measured levels of both sFas and sFasL in patients with ST-elevation myocardial infarction (STEMI) prior to percutaneous coronary intervention (PCI) and 24 h after the procedure. Cardiac MRI was used to evaluate infarct size and LV dysfunction 5 days and 4 months after STEMI. No correlation was found between sFas or FasL and infarct size or LV dysfunction .In line with this, serum levels of sFas measured in patients with acute coronary syndromes were not associated with patient prognosis during 6-month follow-up .In conclusion, sFas levels are significantly higher in patients with acute myocardial infarction but failed to correlate with the infarct size, LV dysfunction, or prognosis. Thus the role of Fas-mediated apoptosis in IHD is yet unclear.However, sFas levels seem to be helpful as a prognostic marker in patients with various forms of heart failure. Prognostic value of sFas was demonstrated in patients with dilated cardiomyopathy, decompensated heart failure, as well as in patients with compensated heart failure –26. HighExperimental studies also demonstrate an important role of tumor necrosis factor alpha (TNF-α) in mediating apoptosis of cardiomyocytes during I/R. TNF-α is a proinflammatory cytokine with various biological functions. After binding to its receptor, TNF-α can induce apoptosis in cardiomyocytes, as shown in an in vitro rat study . TNF-α sStudies in heart failure patients demonstrate that high levels of TNF-α are expressed within failing human myocardium, suggesting its role in the progression of heart failure , 31. HowLevels of TNF-α and its receptors are elevated also in patients with acute myocardial infarction and can predict infarct size, LV dysfunction and prognosis , 34, 35.TNFR1 has proven to have a prognostic value in patients after acute myocardial infarction. Valgimigli et al. show that plasma levels of soluble TNFR1 are a predictor of mortality and new onset of heart failure in patients with AMI . Ueland Other studies suggest that also TNF-α has a prognostic value in patients with AMI –40. HoweThese findings suggest that involvement of TNF-α in ischemic heart disease is very complex and includes a wide range of biological processes—both harmful and beneficial. Further research is needed to clarify the exact molecular mechanism of TNF-α in IHD and find possible ways to inhibit only the negative TNF-α effects.Since TNF-α is involved in IHD pathological pathway, TNF-α gene polymorphisms and their association with the risk of myocardial infarction and coronary artery disease (CAD) have been extensively studied. A meta-analysis investigating relationship between TNF-α gene polymorphisms and CAD risk, however, showed no association .TNF-related apoptosis stimulating ligand (TRAIL) is a member of TNF superfamily that can induce apoptosis. After binding to its receptors TRAIL-R1 and TRAIL-R2, TRAIL initiates intracellular signaling cascade resulting in the apoptotic cell death , 44. In Several clinical studies report that levels of TRAIL are decreased in patients with acute myocardial infarction , 49, 50.Secchierro et al. measured TRAIL in patients with acute myocardial infarction in serial serum samples during hospitalization and in a 12 month-follow-up. Serum levels of TRAIL were significantly decreased in AMI patients at baseline  and low TRAIL levels at patient discharge were associated with increased incidence of cardiac death and heart failure at the 12-month follow-up, even after adjustment for demographic and clinical risk parameters. Thus low TRAIL levels represent a potential predictor of cardiovascular events following acute myocardial infarction .A study by Osmancik et al. provided similar findings. TRAIL levels were measured in acute coronary syndrome patients, who were then followed for 6 months. Low serum TRAIL concentrations were the strongest significant and independent predictor of the composite endpoint of death and hospitalization for heart failure .Low TRAIL levels can predict worse prognosis also in patients with chronic heart failure and in older patients with cardiovascular disease , 51.In conclusion, serum TRAIL levels seem to represent an important predictor of prognosis in patients with acute myocardial infarction. Low TRAIL levels are associated with worse prognosis of AMI patients while higher TRAIL levels seem to be protective. It is unclear whether decreased TRAIL levels reflect reduced production or increased consumption. Metalloproteinase 2, which level is elevated in patients with acute coronary syndromes, can cleave TRAIL, as shown in an in vitro study . This coThe intracellular signaling cascade of mitochondrial pathway is shown in Fig. Experimental studies show that cardiac specific overexpression of Bcl-2, an inhibitor of apoptosis, significantly reduces infarct size after I/R injury. This reduction of I/R injury correlates with the reduction of cardiomyocyte apoptosis , 55. ExpTerminal apoptotic pathway is common for both extrinsic and intrinsic pathway. Activation of execution caspases results in degradation of cellular constituents. This is followed by fragmentation into apoptotic bodies that are quickly removed by phagocytes .The most important caspase of the terminal apoptotic pathway is caspase-3. Overexpression of cardiac specific caspase-3 in transgenic mice showed increased infarct size and pronounced susceptibility to die after I/R injury . Vice veThe cleaved caspase-3 p17 peptide was shown to escape from apoptotic cancer cells and was detectable in extracellular medium after induction of apoptosis, suggesting the concept that p17 peptide can escape also into circulation from apoptotic cells in patients. This presumption was used in a clinical study with 27 STEMI patients undergoing PCI . p17 pepSince apoptosis is significantly involved in myocardial injury after AMI, inhibition of apoptosis represents an appealing target for a therapeutic intervention.TNF-α inhibition therapy was examined in large clinical trials with heart failure patients, using TNF-α antagonist infliximab or TNF-α antibody etanercept. Anti-TNF-α therapy, however, provided no evidence of clinical benefit to heart failure patients.Two trials RECOVER and RENAISSANCE evaluated a TNF-α antagonist etanercept in 2000 patients with chronic heart failure. Both trials were terminated prematurely because etanercept failed to show clinically relevant benefit on the patient clinical status, the rate of death or hospitalization due to chronic heart failure .ATTACH trial evaluated a TNF-α antibody infliximab in 150 patients with moderate-to-severe heart failure. Results showed that short-term inhibition of TNF-α with infliximab did not improve and high dose adversely affected the clinical condition of examined patients .In patients with acute myocardial infarction, TNF-α antagonist etanercept was used in one trial with 26 patients. Etanercept reduced systemic inflammation markers but increased platelet activation. TNF-α antagonism thus provided no evidence of immediate benefit for AMI patients . Effect These findings suggest that involvement of TNF-α in the pathogenesis of ischemic heart disease and development of heart failure includes various biological processes and simple inhibition of all TNF-α effects does not provide clinical benefit to cardiac patients.On the other hand, TNF-α inhibition therapy brought a revolution in the treatment of several autoimmune diseases, such as rheumatoid arthritis, psoriasis and non-specific inflammatory bowel diseases. Clinical trials indicate that anti-TNF-α therapy in patients with psoriasis is associated with significantly decreased risk of myocardial infarction, compared to treatment with topical agents , 63. TheNo clinical trials have been done using TRAIL as a therapeutic target yet.Apoptotic cell death represents a significant contributor to myocardial damage in patients with acute myocardial infarction and participates in the process of subsequent LV remodeling and development of heart failure. Finding a sensitive marker of apoptosis that would help predict prognosis of AMI patients is of a great importance. Among intracellular apoptotic mediators, the only one evaluated in serum of patients with AMI is fragment of caspase-3 p17 peptide. Serum levels of p17 peptide brought some hopeful results in one study with STEMI patients; however, further research is needed to prove p17 peptide as a substantial marker of apoptosis. Among extracellular markers of apoptosis, several soluble forms of receptors and their ligands were evaluated in clinical studies with AMI patients. The most promising apoptotic marker seems to be TRAIL. Serum TRAIL levels represent an important predictor of prognosis in AMI patients. Low TRAIL levels are associated with worse prognosis and high TRAIL levels seem to be beneficial. So far, it is unclear whether decreased TRAIL levels represent a reduced production or an increased consumption. Deeper understanding of the exact molecular mechanism of TRAIL may offer a new target for therapeutic intervention in patients with acute myocardial infarction.


Her2 is a transmembrane protein with tyrosine kinase activity, overexpressed in several cancers including BC. Various studies in different populations have shown association of Her2 variants with susceptibility to BC, however these results were inconsistent, inconclusive and controversial. To obtain a common conclusive finding, we performed meta-analysis of 35 case-control studies reported earlier including 19, 220 cases and 22, 306 controls. We observed significant association of Her2Ile655Val polymorphism with susceptibility to development of breast cancer . Subgroup analysis indicated a significant association with susceptibility to breast cancer in African and Asian populations. However, such association was not observed in other ethnic groups. Our findings suggested that Her2Ile655Val polymorphism is associated with breast cancer risk in overall, Asian and African populations, and can be used as diagnostic marker for BC.Breast cancer (BC) is one of the most common types of cancer in women worldwide. Several factors including genetic and environmental have been linked with susceptibility to development of BC.  It has been estimated that 252,710 new cases of invasive breast cancer will be diagnosed in 2017 among women in US alone. Although, breast cancer is most common in females it also rarely diagnosed in male individuals and 2,470 males are estimated to be diagnosed with breast cancer in 2017 in United States2. Among the overall cancer deaths worldwide, approximately 60% of deaths occur in developing countries including India. In Indian woman, majority of cancer related deaths are due to breast cancer1.Breast cancer (BC) is second leading cause of cancer deaths worldwide and approximately 1.7 million new cases are being diagnosed every year and 521,900 deaths occurred in 2012 alone globally3. Estrogen receptor (ER) plays an important role in breast cancer progression and treatment. Approximately 20–30% breast cancers are of Human epidermal growth factor receptor2 (Her2) positive and are highly aggressive in nature4. High levels of Her2 expression was also observed in tamoxifen resistant breast cancers. Human epidermal growth factor receptor family members are a group of molecules having tyrosine kinase activity with no natural ligand found till date. Heterodimerization among the family members leads to autophosphorylation of cytoplasmic domain which leads to cell proliferation7. Her2 is highly expressed in various cancers types viz. breast, endometrial, ovarian, colon, lung, prostrate and cervical cancers. Role of ERBB2/Her2 in physiological processes  as well as in carcinogenesis and metastasis has been well investigated10. Her2 plays major role in the regulation of several pathways such as Raf/Ras/MAPK and PI3K/AKT pathways11. Receptor mediated signaling pathways has pivotal role in the regulation of normal cell function, growth and division. However disruption of these pathways might lead to several cancers15. Her2 positive breast cancers show poor survival rate, treatment with tyrosine inhibitors showing promising results in harboring these aggressive tumors16. Trastuzumab a monoclonal antibody specifically binds to Her2 and disrupts the downstream pathways of Her2 and it is effectively used for the treatment of Her2 positive breast cancers19. However several patients developed resistance to trastuzumab over a period of time20. Recent studies suggested that Her2 Ile655Val polymorphism is associated with cardiac toxicity. Moreover, it has been identified that both the Her2 Ala1170Pro polymorphisms also responsible for increasing the risk of cardiac toxicity in women administrated with trastuzumab22.BC is highly heterogeneous and ~60–70% is of estrogen receptor positive which responds to anti-hormone therapy25. Cell cycle regulatory role of Her2 and its importance in prognosis of breast cancer clearly indicates that polymorphism in coding region of Her2 might be associated with either cancer susceptibility risk or resistance. One such single nucleotide transition mutation in transmembrane domain coding region of Her2 at codon 655  was well investigated in different populations in relation to risk of breast cancer61. Milikan et al.40 reported the association of Valine allele at Her2 655 codon with breast cancer risk. Whereas, Baxter et al.29 and Xie et al.60 found no association with breast cancer risk in women aged <40 years, post menopausal respectively. However, few researchers performed meta-analysis and tried to conclude the possible correlation of Her2 polymorphism with breast cancer risk. Tao et al.56 showed no association in overall analysis, however mild association of Her2 polymorphism with susceptibility to breast cancer in Asian ethnic group was suggested. Another meta-analysis by Chen et al.62 including 32 case control studies revealed comparable distribution of Her2Ile655Val variants among cases and controls in Caucasian, American and European population. Interestingly, Asian ethnic group showed significant association of breast cancer risk with Her2Ile655Val polymorphism. In the present meta-analysis, total of 35 case-control studies were analyzed and investigated for possible association of Her2Ile655Val polymorphism with development of breast cancer. Furthermore, we subgrouped included reports according to ethnicity and the association was analyzed .Genetic epidemiological studies indicated association between single nucleotide polymorphisms and different cancersHer2Ile655Val polymorphism with breast cancer risk, we have performed meta-analysis using 35 case-control eligible studies including 19, 220 cases and 22, 306 controls. Genotype and allele frequency for case and control of each eligible study was extracted and the characteristics of each study are shown in Table To understand association of 2 statistics were used. I2 more than 50 (I2 > 50) with significant p-value (p < 0.05) considered to be presence of heterogeneity among included studies. Among the models tested, heterogeneity was observed in allele comparison, heterozygous and dominant genetic models. However, other genetic comparison models such as recessive and homozygous were homogeneous. Observations of heterogeneity Q test and I2 statistics of each model are shown in Table To evaluate the heterogeneity among the studies Q test with IBegg’s funnel plot and egger’s regression test was performed to assess the publication bias within the studies included in meta-analysis. Results are imputed in Table Her2 polymorphism with increased risk of breast cancer. The overall allele model revealed association between Her2 polymorphism and breast cancer risk with Odds ratio (OR) = 1.130, 95% confidence interval (CI) = 1.051–1.216, p = 0.001. Furthermore, both dominant and heterozygous models showed significant association of Her2 Ile655Val polymorphism with increased risk of breast cancer . However, comparison of genotypes in other genetics models didn’t show significant association   are playing an important role in various cancer types and are capable of serving as diagnostic tools25. One such single nucleotide polymorphism with substitution of isoleucine with valine at codon 655 in transmembrane region of Her2 has been found to be playing an important role in development of cancer72. The transmembrane domain region of Her2 with valine at 655 domain region stabilizes the formation of protein dimer and thus predisposing to an auto-activity of the receptor73. The hydrophobicity and conformational stability of the hydrophobic domains such as transmembrane domains may alter due to Isoleucine to valine change74. Her2Ile655Val polymorphism was well studied for association with breast cancer risk, whereas other polymorphism at 1170 codon of Her2 (Pro1170Ala) was correlated with cardiotoxicity75. Her2Ile655Val polymorphism is not only associated with breast cancer risk but also associated with other cancers such as ovarian and endometrial cancers77. However, these results are inconsistent and a stringent and powerful analysis is required to conclude the association with breast cancer. In the present study we have analyzed the association of Her2Ile655Val polymorphism with increased breast cancer susceptibility using powerful tool comprehensive meta-analysis (CMA). Overall allele comparison genetic model results suggest that valine allele in Her2 655 codon favors the development of breast cancer in worldwide population. Heterozygous, dominant models also prove that Her2 polymorphism is associated with increased risk of breast cancer. Whereas, subgroup analysis showing different results for different ethnic population. Earlier reports by Wang et al.58 and Chen et al.62 demonstrated association of Her2 valine allele with breast cancer risk in Caucasian population. In contrast, our study failed to show such link. The present study has several advantages over earlier reports. We have included more number of studies in the current meta-analysis including larger number of cases and controls.Human epidermal growth factor family members are a group of receptors with tyrosine kinase activity which affects cell proliferation and survivalet al.56 reported the association of Her2 polymorphism with breast cancer risk in Asian population whereas later Wang et al.58 and Chen et al.62 showed no such association with breast cancer. In this present meta-analysis, we have performed subgroup analysis and demonstrated that valine allele is associated with breast cancer risk in Asian population. In addition to that Val-Val + Ile-Val vs Ile-Ile model also prove the susceptibility of Her2 polymorphism with breast cancer. We also observed that subjects with valine/valine genotype are susceptible for the development of breast cancer in African population. These results are in agreement with the study demonstrated by Wang et al.58; however other studies failed to show such association with breast cancer susceptibility. Ethnic groups such as American, European and Afro-American are not showing such association with breast cancer risk. Our present meta-analysis includes all the studies in which either Taqman or RFLP used as detection method. Frank et al. suggested the biasness in the methods used for the detection of polymorphism and suggested that Taqman method is capable of producing false results78. We excluded the studies in which Taqman method used as detection method and performed the analysis. Studies which used other than Taqman method for the detection of polymorphism showed significant association with breast cancer risk in all models.Tao Her2Ile655Val polymorphism is associated with breast cancer risk in Asian, African population but not in other ethnic groups such as Caucasian, European, American and Afro-American. These results suggest that Her2Ile655Val polymorphism could be considered as possible susceptible bio marker for the detection of breast cancer.In conclusion, our present meta-analysis demonstrated that valine allele is susceptible in overall worldwide population and Asian ethnic group. Her2Ile655Val polymorphism”, “Herceptin receptor polymorphism”, “rs1136201” and breast cancer. In addition to the preliminary online database search we have checked the cross references for the potential publications, those possibly missed in preliminary search. Our present study includes recently published (earliest by 2017) 35 case-control studies with 19, 220 cases and 22, 306 controls for Her2Ile655Val polymorphism  using either single or combination of given keywords i.e., “Her2Ile655Val polymorphism with breast cancer risk and (e) studies representing original data. The studies excluded based on the criteria given below: (a) studies published in other languages except English, (b) studies having only case samples, (c) representing risk of other cancers, (d) without genotypic distribution and allele frequency data and (e) reviews and abstracts.The studies which met all the criteria given below have been included in the present meta-analysis: (a) studies published in English, (b) must have case-control or cohort design, (c) have available genotype frequency of both the cases and controls or have odds ratio (OR) and 95% confidence interval (CI) values, (d) evaluating the association of The data extraction was performed by three independent authors  independently and the disagreement about the studies between the authors was resolved and came to a conclusion by conducting a group discussion within the authors. We followed previously established data form to extract the data from the studies and the following data was extracted from each article: first author’s name, year of publication, country, ethnicity, number of case and control samples, genotype distribution, allele frequency for each case and control.https://www.meta-analysis.com/pages/comparisons.php. CMA v3 is a powerful tool to analyze and has several advantages over other software available for computational meta-analysis. Combined odds ratio with 95% CI was calculated and was taken into consideration to apprise the association of Her2 polymorphism with breast cancer risk. Chi-Squared based Q test was performed to analyze the heterogeneity and p-value < 0.05 was considered as significant. In case of no significant heterogeneity fixed effect model was used to assess the combined OR. In contrast, Random effect model was considered to calculate the combined odds ratio with 95% CI among the studies. I2 statistics was used to quantify inter study variability, greater I2 value depicts greater degree of heterogeneity. Publication bias was examined using Begg’s funnel plot. Egger’s linear regression test was employed to analyze and measure the asymmetry of Begg’s funnel plot and the significance of intercept was assessed by t-test. Intercept considering p-value < 0.05 was considered as significant and the publication bias was reduced using “trim and fill” method.The current meta-analysis was performed using comprehensive meta-analysis version 3 software (CMA v3) All those named as authors confirmed the availability of data and materials.Supplementary Info


A promising alternative to address the problem of acquired drug resistance is to rely on combination therapies. Identification of the right combinations is often accomplished through trial and error, a labor and resource intensive process whose scale quickly escalates as more drugs can be combined. To address this problem, we present a broad computational approach for predicting synergistic combinations using easily obtainable single drug efficacy, no detailed mechanistic understanding of drug function, and limited drug combination testing. When applied to mutant BRAF melanoma, we found that our approach exhibited significant predictive power. Additionally, we validated previously untested synergy predictions involving anticancer molecules. As additional large combinatorial screens become available, this methodology could prove to be impactful for identification of drug synergy in context of other types of cancers. While targeted therapies have achieved remarkable antitumor responses, resistance to targeted agents frequently develops and renders the targeted drug ineffective. Combination therapies have been successful in delaying resistance and overcoming resistance. Additional goals of combination therapy are to achieve enhanced effectiveness through drug synergy and to reduce the frequency and severity of adverse events through lower individual drug dosage levels. However the identification of synergistic drug combinations is often a labor and resource intensive process. Therefore a systematic method for identifying optimal combinations would be highly impactful. Here we present a computational method for predicting synergistic and effective drug combinations using only single drug efficacy information. We have applied and validated our method on a high-throughput drug screen of 780 combinations involving 40 individual molecules in the context of mutant BRAF melanoma. Additionally we have made predictions and validated 11 previously untested drug combinations with a diverse set of outcomes. Targeted therapies designed to specifically target molecules involved in carcinogenesis have achieved remarkable antitumor efficacy. In melanoma, over half of patients are reported to harbor activating mutations in the BRAF oncogene , 2. BRAFCombination therapy has been proposed for preventing and overcoming resistance. This is thought to be a promising option because resistance to the combinatorial therapy would require either acquisition of multiple mutations rapidly  or an inDrug synergy can occur through a variety of mechanisms. These include enhancement of bioavailability, through inhibition of parallel pathways, and cheGiven the poor prognosis of BRAF melanoma and the rapid rate at which resistance develops and tumors progress, there is an urgent need to identify suitable combinations. The first drug combination for treatment of advanced melanoma was approved in January 2014 and involved the BRAF inhibitor, dabrafenib, and the MEK inhibitor trametinib. This combination was pursued due to the great response rates of the individual drugs with the goal of preventing drug resistance. Indeed, it has been observed that the combination delays the development of resistance and prolongs progression free and overall survival. HoweverExisting methods that have been developed to predict synergistic combinations have generally relied on mechanistic insights. However they have been applied only in limited specific contexts, such as in B cells . Furtheret al.  and maintained in a 37° incubator at 5% CO2.Cells were pipetted into 384-well plates at 750 cells per well using a multi-channel pipette (Eppendorf) in 20 ul basal medium, and placed in an 37° incubator at 5% CO2 overnight prior to exposure to the agent. Drug stock plates for single therapy agents were created by serial dilution, generally 1:10 from 10 mmol/L stock. Using a multi-channel pipette, 2.5 ul drug volume from drug stock plates were added to the 384-well cell plates. A total of 0.1% and 10% DMSO was used as negative and positive controls, respectively.For combinatorial, dual-agent screens, a 2.5 ul volume of Drug X was plated at GI-50 concentration (determined from single-agent screen), and 2.5 ul volume from drug stock plate of Drug Y was added in a range of concentrations. Initial dual agent screening was carried out in 384-well plates at 750 cells per well in 20 ul basal medium Further exploration screening were carried out in 96-well plates, in 100ul basal medium with 3500 cells per well.All experiments were carried out in triplicates at the Weill Cornell Medicine’s Meyer Cancer Center . Cells were exposed to drugs for 72 hours, followed by GI measurement by CellTiter-Glo ATP detection assay (Promega) following the manufacturer’s recommended. A 20 ul volume of basal medium served as a background for luminescence.A combination was considered to be effective if the third quantile value of observed growth inhibition values was at least 70%. Synergistic combinations were those that achieved a Chou-Talalay synergy score of -1 in at least one dosage-level pair.S1 FigFor each drug pair, we combined the drug pair’s efficacies by taking the mean (μ) and difference (Δ) in 27 mutant BRAF (red), mutant RAS (green) and WT (blue) melanoma cell lines.(TIF)Click here for additional data file.S2 FigMean decrease Gini coefficient observed upon feature removal for the top 30 features for the (left) BRAF effectiveness model and (right) BRAF synergy model.(TIF)Click here for additional data file.S3 Figet al., for 274 predicted effective or ineffective drug combinations. C We focused on a subset of 7 drugs to experimentally follow-up on our predictions for previously untested combinations.A We applied our trained model to make predictions for 10,395 additional untested combinations. B Comparison of growth inhibition levels, as reported by Friedman (TIF)Click here for additional data file.


An early Cambrian greenhouse climate constrained by combining oxygen isotope and climate modeling data approaches. 18O) record before the evolution of euconodonts. We show that previously overlooked sources of fossil biogenic phosphate have the potential to fill this gap. Pristine phosphatic microfossils from the Comley Limestones, UK, yield a robust δ18O signature, suggesting sea surface temperatures of 20° to 25°C at high southern paleolatitudes (~65°S to 70°S) between ~514 and 509 million years ago. These sea temperatures are consistent with the distribution of coeval evaporite and calcrete deposits, peak continental weathering rates, and also our climate model simulations for this interval. Our results support an early Cambrian greenhouse climate comparable to those of the late Mesozoic and early Cenozoic, offering a framework for exploring the interplay between biotic and environmental controls on Cambrian animal diversification.The oceans of the early Cambrian (~541 to 509 million years ago) were the setting for a marked diversification of animal life. However, sea temperatures—a key component of the early Cambrian marine environment—remain unconstrained, in part because of a substantial time gap in the stable oxygen isotope (δ Representative specimens from each bulk subset were subjected to a rigorous protocol to assess the preservation of biogenic phosphate. We could consistently distinguish subsets of pristine and altered SSFs using optical microscopy, confirmed by combining high-resolution scanning electron microscopy (SEM) and energy dispersive x-ray (EDX) spectroscopy to assess micro- and ultrastructural preservation and the distribution of diagenetically sensitive elements . In particular, brachiopod specimens taken from pristine subsets were found to have compact laminae comprising phosphate spherules tens of nanometres in diameter, whereas the compact laminae of specimens taken from altered subsets were recrystallized as micrometer-scale phosphate prisms .To ensure that isotope data recovered from the phosphatic SSFs reflect a Cambrian paleoenvironmental signal rather than later diagenetic conditions, we restricted our analyses to taxa known to have produced phosphatic skeletons based on phylogenetic and geological criteria (e prisms . To helpTorellella) yielded δ18Ophos values of +13.9 to +15.2 per mil (‰) Vienna standard mean ocean water (VSMOW). In contrast, three linguliformean brachiopod samples, identified a priori as being affected by diagenetic alteration, yielded lighter values of +13.3 to +14.3‰  of pristine phosphatic microfossils  to those of the phosphate hardground (+12.8 to +14.0‰) suggests that these altered SSFs may record early diagenetic conditions or at least have followed a similar diagenetic pathway as the hardground. In contrast, pristine Torellella samples yielded δ18Ophos values of 14.6 to 14.7‰, comparable to the pristine brachiopods (+13.9 to +15.2‰) and heavier values than those from the diagenetically altered brachiopods and the hardground samples. Most Torellella specimens were identified as pristine, and altered Torellella specimens were too scarce to assemble samples for bulk analysis .In paired analyses of linguliformean brachiopods from the same sample, identified during our preanalysis screening as preserving either pristine or demonstrably altered phosphate, the pristine brachiopods consistently yielded heavier δ18O values of compact and porous laminae, with compact laminae consistently heavier, by at least approximately 2‰, than porous laminae in the same specimen  measured by the trisilver bulk phosphate method. Because phosphate-oxygen temperature equations are based on trisilver δ18Ophos analyses  analysis shows that oxygen isotopic preservation varies in the same manner as bulk elemental composition within individual brachiopod specimens . SIMS analysis consistently distinguishes between the δspecimen . Bulk δ1analyses  is likel18Ophos values that reflect shallow marine conditions on early Cambrian Avalonia. The δ18Ophos data incorporate signals from the oxygen isotopic composition of contemporaneous sea water (δ18Osw), the temperature of the water in which the animal lived, and biological fractionation . The convergence of phosphate oxygen isotope temperature equations based on both marine invertebrate and nonmammalian marine vertebrate biominerals indicates that phosphate oxygen vital effects are small in comparison with analytical uncertainty T balance  of 20° to 25°C for the Comley Limestones  witmestones . This is18Osw typically used in Paleozoic paleoclimate studies  of northern Gondwana  or even mid-paleolatitudes  during the early Cambrian . This greenhouse gas forcing is in line with Cambrian pCO2  estimates from GEOCARB suite of models .Overall, our new data provide the first quantitative constraints on early Cambrian climate, corroborating qualitative geological data and geochemical arguments that also support interpretations of this as a greenhouse world. Our new data fill an extensive time gap in the paleotemperature record of the beginning of the Phanerozoic eon and provide environmental context to a time when the animal-rich marine ecosystems of the Phanerozoic were first evolving. Using quantitative data to inform environmental and climate models will enable more rigorous interrogation of first-order hypotheses surrounding the ecological revolutions of the Cambrian Period, and these data can be recovered from globally widespread phosphatic Cambrian microfossils.2 to Ac5 and Ad) recognized on the basis of their petrographic and paleontological characteristics and separated by erosional disconformities that represent depositional hiatuses . The residues were subsequently examined using a binocular microscope, and fossil specimens were picked out with a brush and deionized water. SSFs were assembled into 30-mg taxon-specific bulk samples, with each sample comprising several tens to a few hundred individual specimens.Blocks of the Comley Limestones were macerated in buffered (10 to 15%) acetic acid using a standard extraction protocol, modified after Jeppsson Torellella specimens were deemed pristine: light blue-gray in color, thick-walled, and with visible growth structures both within the tube walls and on the exterior surface. Although altered Torellella specimens exist , no lithological sample yielded sufficient of these for bulk isotope analysis. The actual preservation state of samples was confirmed following the protocol outlined below.Specimens were assembled into “pristine” and “altered” samples based on their appearance under optical microscopy. Pristine brachiopod specimens had a translucent light-brown appearance under reflected light, whereas altered specimens were opaque and appeared very dark brown to black (fig. S2). Specimens intermediate between these end-member states were excluded from further analysis. The vast majority of Because it was not possible to assess the preservation of every fossil in a bulk isotope sample, individual representative specimens were taken from bulk samples and examined to determine the typical preservation of each sample. Fractured surfaces of individual specimens were examined using high-magnification SEM to investigate the extent of recrystallization or overgrowth by diagenetic phosphate. Polished cross sections of individual specimens embedded in epoxy resin were examined using EDX spectroscopy to investigate the extent of chemical alteration and particularly the distribution of any such alteration within individual specimens.Torellella  outlines only.The shells of modern linguliformean brachiopods comprised alternating dense layers of biophosphate (compact laminae) and organic-rich layers with much less biophosphate (porous laminae of various morphologies). The compact laminae, which constituted most of the biomineralized material, were composed of densely packed calcium phosphate spherules measuring from tens to a few hundred nanometers in diameter  before being ground and polished with silicon carbide paper, diamond paste, and γ-alumina, before being thoroughly rinsed with ethanol and deionized water. EDX analyses of these specimens enabled us to map element distributions across different biological domains (compact and porous laminae). In brachiopod specimens taken from pristine samples, we found that elements indicative of alteration  were restricted to porous laminae and were excluded from compact laminae . In EDX 18O diagenetic gradient. These were acquired by microdrilling approximately 30 mg of powder from an irregular phosphatic hardground (HG-A) horizon in the lower part of the Comley Limestones and from a phosphatic pebble (HG-B) a few centimeters above the hardground. The in situ hardground is up to 3 cm deep, with an angular upper surface, and separates two distinct lithologies: an underlying trilobite-rich sandy limestone and an overlying glauconite-rich sandy limestone with few fossils. The overlying glauconite-rich unit also contains subrounded to angular phosphatic pebble-sized clasts. In places, the uppermost part of the hardground includes laminae of glauconite and quartz clasts.Sedimentary phosphate samples were also taken for bulk isotope analysis to provide a δPetrographic observations, including the lithological differences between the underlying and overlying units, the irregular upper surface, the preservation of calcareous fossils between the phosphate groundmass, and the fine glauconite and quartz laminae near the top of the horizon, suggested an early diagenetic origin of the phosphate, with occasional interruption of phosphate precipitation at the sediment-water interface and, ultimately, exposure of the hardground on the sea bed. Phosphate clasts were incorporated into the horizon above the hardground and were interpreted as deriving from the hardground during its exposure on the sea bed after its formation.18O values were reported with respect to VSMOW. Bulk isotope data were obtained from 30-mg fossil samples, with each sample comprising several tens of individual specimens, treated to solubilize PO4 anions and precipitated as silver phosphate  and 18O (~4 × 106 cps) monitored simultaneously on dual Faraday cups (L’2 and H’2). Each analysis began with 50-s presputtering time, followed by automatic secondary beam and entrance slit centring, before data collection in two 10-cycle blocks. Each SIMS run began with 10 Durango analyses, followed by alternating analysis sets of five unknowns (samples) and five standards (Durango apatites), dropping to three standards when beam stability was good. Linear regressions were applied to each analysis run to correct for instrument drift. Mean external precision was derived from the SD of Durango analyses following linear regression corrections for long-term (session duration) drift. This value was reported for each unknown (sample) analysis and ranged from ±0.11 to 0.41‰. The Durango apatite standards were fragments of a larger crystal, supplied by EIMF, whose isotopic composition  was independently verified by trisilver phosphate analysis at NIGF before this project began.SIMS analyses were made using the CAMECA IMS-1270 ion microprobe. A primary beam of Csz-coordinate ocean GCM giving 1.4° × 2.8° resolution on a regular longitude-latitude grid. The coupled model had no flux corrections, and its short turnaround time allowed millennial-scale integration.We used the three-dimensionally coupled ocean-atmosphere FOAM version 1.5 (−2)  temperature. The last 50 years of the model run were used to build the climatology files used for analysis.We used the Cambrian continental configuration from BugPlates for 510 Ma ago (


Patients treated with dialysis have high rates of brain infarcts, brain atrophy, and white matter disease. There are limited data regarding the presence of more subtle damage to brain white matter.p value of <0.05.In the Cognition and Dialysis Study, we compared brain structure using diffusion tensor imaging in hemodialysis (HD) patients to individuals without known kidney disease, using tract based spatial statistics (TBSS) to compare Fractional Anisotropy (FA) and Mean Diffusivity (MD). Statistical comparison of each overlaid voxel was age controlled using a permutation based corrected Thirty-four HD patients and twenty six controls (52 vs 51 years for HD vs control) had adequate magnetic resonance imaging for analysis. The HD group had fewer women (38% vs 23%) and a higher prevalence of diabetes (29% vs 8%), heart failure (29% vs 0%) and clinical stroke (15% vs 0%). Hemodialysis patients had significantly lower FA across multiple white matter fiber tracts, with fronto-temporal connections, the genu of the corpus callosum and the fornix more significantly affected than posterior regions of the brain. Similarly, HD patients had significantly higher mean diffusivity in multiple anterior brain regions. Results remained similar when those with a prior history of stroke were excluded.In HD patients, there is more white matter disease in the anterior than posterior parts of the brain compared to controls without kidney disease. This pattern of injury is most similar to that seen in aging, suggesting that developing chronic kidney disease and ultimately kidney failure may result in a phenotype consistent with accelerated aging. Anatomic brain abnormalities, including stroke and brain atrophy, are common in patients with kidney failure –5. PatieThrough measurement of water diffusivity in the brain, diffusion tensor imaging (DTI) allows for the characterization of alterations to white matter tracts in addition to localization of white matter damage to specific brain regions . In thosDialysis patients are at particularly high risk for stroke  and cognAll patients receiving hemodialysis at five Dialysis Clinic Inc. (DCI) units and one hospital-based dialysis unit in the greater Boston, MA area who enrolled at baseline in the Cognition and Dialysis Study  (01/21/02 within 1 year of MRI (80%) or through review of the medical record if an eGFR was not available (20%). The Tufts MC Institutional Review Board approved the study, and all participants signed informed consent allowing for review of individual medical records as well as participation in the study.Controls were recruited from Tufts Medical Center (Tufts MC) and were approached if they were already scheduled for a brain MRI for another indication and were between 18 and 75 years of age. After obtaining the originally scheduled MRI scan, an additional scan to obtain DT images was performed. Among controls, the most common indications for undergoing a MRI were headache (56%), vertigo (8%), and facial pain (8%). Exclusion criteria were known kidney disease, a presentation with symptoms or signs of stroke or history of stroke, psychiatric disease, dementia, other serious neurological disorders and history of malignancy. Self-reported demographic data on age, sex, race, and history of diabetes, hypertension, coronary artery disease, and congestive heart failure were collected at time of enrollment. Confirmation of the absence of kidney disease was determined through documentation of estimated glomerular filtration rate  of grea-T Philips scanner using a single-shot, spin-echo, echo-planar sequence with 16 independent directions. Eleven subjects in the hemodialysis group and eleven subjects in the control group were excluded from analysis due to an inadequate MRI field of view, which is required for the normalization process to utilize tract based spatial statistics [Magnetic resonance imaging was performed on 45 participants in the hemodialysis group and 37 participants in the control group. Diffusion tensor imaging was performed on a 3atistics . Thirty Diffusion tensor imaging allows for the measurement of water displacement within the brain, yielding two primary outcomes which provide information on white matter integrity: fractional anisotropy and mean diffusivity . Fractiohttps://fsl.fmrib.ox.ac.uk/fsl/fslwiki). Raw DTI data was preprocessed for correcting eddy-currents and head motion using the diffusion toolbox (part of FSL). Then, fractional anisotropy (FA) and mean diffusivity (MD) were calculated and used for the TBSS processing. TBSS processing includes, 1) a creation of a standard brain template consisting of a three dimensional skeleton highlighting white matter fiber tracts, 2) spatial normalization of subject data into the standard space and 3) performing voxel-wise statistical comparisons along the major white matter pathways [Diffusion tensor imaging processing was performed using the FSL software package  and all hypothesis tests were two-sided, with a p < 0.05 considered as significant. Voxel-wise comparison between the hemodialysis and control groups was performed using TBSS. All TBSS analyses were controlled for age and used a permutation-based inference method of nonparametric statistical thresholding; [p value of <0.05 considered significant.Demographic characteristics for the hemodialysis group and control group were reported as means with standard deviations or percentages and were compared using Fisher exact tests or holding;  this metTo examine how cardiovascular disease and its risk factors within hemodialysis patients impact white matter integrity, we divided the hemodialysis group into two subgroups based on vascular disease history and risk factors . Group A includes those HD patients with two or more risk factors while Group B includes those with one or zero risk factors. Each subgroup was then compared to the control group without history of kidney disease.To examine how a prior history of clinical stroke impacted differences in white matter disease, we repeated analyses removing those HD patients with a prior reported history of stroke and compared them to the control group.Thirty-four HD patients and twenty six controls had adequate imaging for analysis. The mean age was similar (52 years vs 51 years for HD vs control), while the HD group had fewer women (38% vs 23%), more participants who were black (38% vs 19%) and a higher rates of diabetes (29% vs 8%), heart failure (29% vs 0%), stroke (15% vs 0%) and hypertension (85% vs 35%) . This pattern of injury seen within HD patients is most similar to that seen with aging , 26, desMultiple prior studies have demonstrated an increased prevalence and severity of white matter changes in both patients with chronic kidney disease as well as those with end-stage renal disease requiring dialysis , 5, 6. Hincrease in FA over one year. This result appears contrary to both our findings and those previously published, which generally have agreed that decreased FA is consistent white matter injury. Our study confirms that HD patients have decreased FA and increased MD compared to controls, but differs from these prior studies by demonstrating a predominantly frontal pattern of white matter injury. The strengths of our study include the use of high resolution 3 T MRI and use of TBSS analysis, a more diverse cohort than prior DTI studies, and ascertainment of cardiovascular risk factors. The inclusion of participants of differing races, as well as those with multiple cardiovascular disease risk factors may account for the spatial differences in white matter damage. In fact, a DTI study of ESRD patients in the United States who ultimately underwent renal transplantation also demonstrated anterior pattern of white matter damage, which showed improvement after transplantation [There are currently limited and inconsistent data on diffusion tensor imaging in patients receiving hemodialysis. Three prior studies of DTI in a population of HD patients have been published. Chou et al. performed DTI in 28 HD patients and 25 age matched controls using a 1.5 T MR scanner, demonstrating increased MD and decreased FA in the HD group versus controls . A studyantation . LimitatThere are several possible explanations for our findings. First, white matter damage/disease is reported to be a possible precursor for clinical vascular disease, with strong associations seen between the presence of white matter disease and future risk of transient ischemic attack or stroke , 9. PatiThe pattern of anterior white matter damage is most similar to that observed in studies of the effect of aging on white matter integrity , 26. PriDiffusion tensor imaging performed within a group of patients receiving hemodialysis versus controls without kidney disease demonstrated significant alterations in white matter integrity. Damage was primarily located within anterior brain regions, in contrast to posterior regions which were on average more similar to the control group. These findings may occur either due to a propensity for cerebrovascular disease, through hemodynamic effects mediated by hemodialysis, or be mediated by kidney failure itself. Future studies should focus on the pathophysiology and risk factors that may lead to white matter damage in kidney disease patients as well as methods to prevent or limit brain injury.


Broad domain promoters and super enhancers are regulatory elements that govern cell-specific functions and harbor disease-associated sequence variants. These elements are characterized by distinct epigenomic profiles, such as expanded deposition of histone marks H3K27ac for super enhancers and H3K4me3 for broad domains, however little is known about how they interact with each other and the rest of the genome in three-dimensional chromatin space. Using network theory methods, we studied chromatin interactions between broad domains and super enhancers in three ENCODE cell lines  obtained via ChIA-PET, Hi-C, and Hi-CHIP assays. In these networks, broad domains and super enhancers interact more frequently with each other compared to their typical counterparts. Network measures and graphlets revealed distinct connectivity patterns associated with these regulatory elements that are robust across cell types and alternative assays. Machine learning models showed that these connectivity patterns could effectively discriminate broad domains from typical promoters and super enhancers from typical enhancers. Finally, targets of broad domains in these networks were enriched in disease-causing SNPs of cognate cell types. Taken together these results suggest a robust and unique organization of the chromatin around broad domains and super enhancers: loci critical for pathologies and cell-specific functions. Super enhancers are demarcated by high levels of enhancer-associated histone modification mark H3 lysine 27 acetylation (H3K27ac) and are catalogued in 86 human cell and tissue types using this mark2. Moreover, super enhancers have been shown to harbor Single Nucleotide Polymorphisms (SNPs) associated with the diseases of the cognate cell type, including cancer4. Pharmacological molecules have been used to effectively and specifically target super enhancer domains at oncogenes5, further reinforcing their significance for disease biology. Similarly, cell type-specific promoters  are associated with expanded deposition of histone H3 lysine 4 tri-methylation (H3K4me3) mark - a signature conserved across diverse cell types (>99 in human cells) and organisms3. Shortening of broad domains has been observed in cancer cells at tumor suppressor genes, enabling the discovery of novel tumor suppressors4. Recently, super enhancers and broad domains overlapping super enhancers were shown to be more associated with chromatin interactions than their typical counterparts6 suggesting a unique organization of chromatin around cell-specific loci.Cell-type-specific functions of super enhancers and broad domains have been extensively studied and well established across diverse cell types and organisms7. Advances in genomewide chromatin interaction profiling have shown that many regulatory elements  that are distal on the linear genome map are actually in close physical proximity with each other as a result of the 3D chromatin structure10. Among these technologies, the Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) combines chromatin immunoprecipitation with chromatin conformation capture to identify chromatin interactions that are mediated by a protein8, such as RNA Polymerase II (Pol2) which mediates interactions between promoters and enhancers11. More recently, an alternative method has been developed, HiChIP12, to detect protein-centric chromatin interactions12 using 100-fold less input material, providing an opportunity to generate such maps in primary human cells and tissues. These datasets, particularly the ones capturing protein-mediated promoter and enhancer interactions enable genomewide study of chromatin interactions between broad domains and super enhancers.Chromatin structure plays a major role in governing cellular functions in a cell type- and condition-specific manner14, super enhancer2, and broad domain3 definitions in the corresponding cell types 17, we showed that these chromatin connectivity patterns can effectively discriminate broad domains from regular promoters and super enhancers from regular enhancers. Our results suggest a unique and conserved chromatin organization around critical regulatory elements. Finally, we studied the clinical relevance of these annotated chromatin interaction networks by demonstrating that enhancers targeting broad domains harbor more SNPs associated to diseases of the cognate cell type.This study utilizes advanced computational methods to uncover how broad domains and super enhancers interact in the 3D chromatin space, in particular, whether they are associated with distinct connectivity patterns, whether these patterns are conserved across cell types and assays, and whether they are predictive of the cell-specific nature of promoters and enhancers. For this, we built chromatin interaction networks using diverse assays  in three ENCODE cell lines: MCF-7 (breast adenocarcinoma), K562 (chronic myeloid leukemia), and GM12878 (lymphoblastoid cell line). These networks were annotated using ChromHMM states18 and HiChIP12 (targeting cohesion subunit Smc1a) data in GM12878. These networks consisted of 20–50 thousand network nodes/edges and thousands of connected components . In Pol2 ChIA-PET networks, a majority of nodes (68–80%) overlapped promoters and enhancers, showing the utility of Pol2-mediated ChIA-PET interactions to capture interactions between regulatory elements7 , K562 (derived from chronic myelogenous leukemia cells), and GM12878 (lymphoblastoid cell line) and using Hi-Cnts Fig. . Next, nts7 Fig. , in agrei.e., broad domains, typical promoters, super enhancers, typical enhancers, and other annotations) and compared against theoretical expectations (Methods). These analyses showed that in Pol2 ChIA-PET networks, broad domains were more connected to all other nodes than theoretically expected (2.9 times more than expected) in all three cell lines  beyond immediate interactions. However, enumerating all possible configurations in a large network is computationally intractable (subgraph isomorphism problem). To effectively and systematically uncover chromatin interaction patterns associated with cell-specific regulatory elements, we utilized graphlets15. Graphlets are small, connected, and non-isomorphic  subnetworks within a large network that enables systematically studying and quantifying the local network structure around a node of interest. Topologically distinct nodes within a graphlet are referred to as orbits. We studied the local structure of chromatin interaction networks using all possible graphlets composed of two to five nodes, which encompasses 73 orbits  than regular enhancers  broad domains from regular promoters and ii) super enhancers from regular enhancers. The discriminative power of these models was quantified using receiver operating characteristic (ROC) curves and area under these curves (AUC), where a perfect predictor  has an AUC score of 1.To determine whether chromatin connectivity patterns of regulatory elements can be predictive of their cell-specific activity, we used machine-learning models based on support vector machines (SVM) Fig. . Each nee.g., orbit cluster scores) with other genomic features improved the predictive ability of these models, suggesting that chromatin interaction networks and connectivity patterns harbor functional and non-redundant information. Prediction of super enhancers in HiChIP and Hi-C was as effective (AUC = 0.81 and 0.76 respectively)   and high degree, reinforcing the importance of tight connectivity around super enhancers  targeting broad domains were significantly enriched in breast-cancer associated SNPs  with index SNP rs11780156 (r2 > 0.7), a known breast-cancer associated variant  is robust to the disruption of any single enhancer element within the enhancer cluster. Such a tightened connectivity pattern might also be critical in establishing and maintaining robust  expression patterns associated with broad domain genes3. Our machine learning models showed that connectivity patterns of super enhancers and broad domains are predictive of their cell-specific nature.In this study, we showed that broad domains and super enhancers have distinct connectivity patterns in chromatin interaction networks that are conserved across cell types and can be captured using diverse assays. Broad domains tend to be central nodes in these networks and are frequently targeted by multiple constituent enhancers that belong to a super enhancer domain. A recent study employed CRISPR/Cas9 to delete enhancers and super enhancers in mouse embryonic stem cells (mESCs) and suggested that super enhancer clusters act in a redundant manner to fine tune the expression of their target genes25. Our study is in alignment with these findings where we observed increased connectivity for super enhancer nodes in comparison to regular enhancers using three different assays  and in three different cell types . Our study furthered our knowledge on how super enhancers are connected in these networks and revealed that super enhancers form clique-like structures and typically connect to broad domains and other super enhancer elements within the same domain. Broad domains were associated with more chromatin interactions compared to typical H3K4me3 domains6. Our study established that in comparison to regular promoters, broad domains had more interactions overall. Moreover, these interactions were especially connecting them to super enhancers. Increased targeting of broad domains by super enhancers likely ensures robust and increased expression of these important genes in cognate cells. Furthermore, we showed that enhancers targeting broad domains in chromatin interaction networks harbor disease-associated SNPs, further reinforcing the importance of studying these genes and their regulators in the context of human diseases.Recent studies revealed that regulatory elements with frequent chromatin interactions  are enriched in super enhancers and harbor more GWAS SNPsA challenge we faced in our analyses was the difficulty of representing super enhancers that span long genomic regions (10–20 kb) in our networks, which would harbor more interactions merely due to their genomic coverage. We overcame this challenge by defining the network nodes using open chromatin regions , which represent putative active regulatory elements. This methodology also enabled the study of individual enhancer elements within a super enhancer domain. We observed that a super enhancer element typically is not a single and expanded regulatory element but a combination of constituent active enhancers that are in close proximity to each other in both linear and 3D space, and they frequently interact with one another and with their target gene to regulate that gene’s expression levels  and to systematically quantify differences in the connectivity of different regulatory elements. Our findings revealed that chromatin connectivity patterns around super enhancers and broad domains are non-stochastic and conserved across cell types and can be captured via different assays. However, we acknowledge the caveat that genome-wide chromatin interaction maps analyzed here are generated from millions of cells. Therefore it is not possible to dissect whether the connectivity patterns observed for super enhancers and broad domains take place in individual cells. Advances in single-cell chromatin interaction profiling techniques will be essential in studying these patterns at the single cell level. As a step towards this direction, Hi-ChIP27 were obtained from29. Accession numbers for these datasets are as follows: MCF-7 (GSE39495), K562 (GSE39495), and GM12878 (GSE72816). ChIA-PET Tool27 interactions were preferred over the alternative Mango30, since calls obtained via Mango were sparse and did not include as many broad domain or super enhancer interactions  defined from DNAse-seq open chromatin peaks, which reduces false positive calls and likely captures active regulatory loci. These peaks where then used to define the nodes of the interaction networks, which were constructed using QuIN31. MCF-7, K562, and GM12878 open chromatin peaks were called using MACS2 software32 (version 2.1) after pooling replicates (GSE32970 and GSE2969228). For network building, we used 250 bp extension and removed inter-chromosome interactions and edges spanning distances greater than 1 Mb. Network nodes were first annotated using ChromHMM states14. If a node overlapped with multiple ChromHMM annotations, we applied the following priority schema to dissolve ambiguities in annotations due to the genomic segmentation framework in ChromHMM: 1) enhancers/promoters 2) insulators 3) poised promoters 4) repressed elements 5) transcribed elements 6) low signal. If a node was annotated both as a promoter and an enhancer, known transcription start site (TSS) definitions were used to define promoter nodes found within 2 kb of a known TSS. Broad domains and super enhancers annotations were then assigned respectively to promoter and enhancer nodes using the previously defined broad domain3 and super enhancer2 regions in corresponding cells. If a broad domain was found to overlap multiple promoter nodes, the promoter node with the largest overlap with the broad domain in terms of base pairs (bps) was assigned as the broad domain node. In the case of ties, both promoters were labeled as broad domain nodes.Pol2 and CTCF ChIA-PET chromatin interactions called using ChIA-PET Toolns Table . To mini12 (GSE80820) (targeting cohesion subunit Smc1a) and Hi-C18 (GSE63525) data in GM12878 cell line. Valid interaction pairs from HiChIP biological and technical replicates were pooled and filtered by extending 250 bp in both directions, keeping only the pairs with both ends overlapping a DNase-seq peak. Significant HiChIP interactions between peaks were called based on the hyper-geometric distribution as described in27 and filtered using the Benjamini-Hochberg procedure (FDR < 0.05). Significant Hi-C intra-chromosomal interactions at 1 kb resolution were identified using in-house software implemented based on the HiCCUPS method18. For each contact, expected values based on donut, vertical, horizontal and lower left filters were calculated using parameters P = 20 and W = 40 to calculate a P-value based on the Poisson distribution and filtered based on the Benjamini-Hochberg procedure (FDR < 0.025). A more stringent FDR cutoff is used for Hi-C data to make it more comparable with other assays, since Hi-C data were more deeply sequenced than the others. The intersection of these four filters was used to identify the final set of contacts. Network construction was performed similar to ChIA-PET data using 0 bp and 1,250 bp extension parameters for HiChIP and Hi-C networks, respectively. These values where chosen to account for differences in read extension introduced in interaction calling steps. HiChIP edges were further filtered to remove edges with less than 4 supporting valid pairs.We called interaction pairs from HiChIP33. These data were filtered to exclude samples that were generated under an experimental treatment or audited for quality concerns. This resulted in 23 cell and tissues types  that were used to quantify the degree of cell-specificity of gene expression levels. For this, we calculated the cell-type specificity measure (SPM)34 for each gene in the three cell types. SPM was used to quantify cell-specificity of genes associated to regular and broad domain promoters, as well as target genes of regular and super enhancers. For super enhancers and enhancers, we considered both nearest TSS definitions (up to 10 kb) as well as promoter targets identified from ChIA-PET networks. Network targets were chosen by selecting promoter/broad domain targets of up to 4 edges using breadth first search between the enhancer and promoter node, stopping at the first promoter node found. If a network node was associated with multiple genes, the average SPM measure of all associated genes was used.RNA-seq data was obtained from the ENCODE project repository for all available cell lines and adult tissuesA” and all nodes annotated as “B” is calculated as:Ci represents the set of all nodes in chromosome i, |ECi| represent the number of observed edges within the node subset Ci and gd is the genomic distance from node x to node y. The expected frequency obtained corrects for both analyzing only intra-chromosomal interactions as well as the limitation that connected nodes within the network are within 1 Mb. For nodes with the same annotation, the expected value was adjusted accordingly to account for two nodes being selected from the same set.We calculated the enrichment of interactions between every pair of annotations  by calculating a theoretical expectation of interaction frequency and by comparing against the observed interaction frequency for this annotation pair. More formally, expected interaction frequency between all nodes annotated as “To study whether super enhancer interactions are restricted within a domain, we generated interactions networks again by merging super enhancer nodes that belong to the same super enhancer domain and representing the whole domain as a single node in the network. Then we counted the number of edges that are connecting these merged nodes with the rest of the nodes in the network and repeated the interaction enrichment analyses.31. Connectivity degree refers to the number of edges connected to a node. Closeness centrality was computed as:nd is the network distance  between nodes u and v, and Nc represents the set of nodes within the connected component containing v. A connected component is defined as a collection of nodes where every node pair is connected to each other through some path  such that nodes are not connected to other nodes outside this collection. Harmonic centrality was computed as follows:sp denotes all shortest paths between nodes u and x that include node v. Normalized centrality measures of a node were calculated with respect to the size of the network component |Nc|.Network metrics were calculated using QuINComparisons of connectivity degree measures of promoters to broad domains and super enhancer to enhancers were performed using the Wilcoxon rank sum test. The Wilcoxon rank sum test is a non-parametric test that does not assume normally distributed measurements. Corresponding n-values for each annotation subset and network are reported in Table i.e., topologically distinct) subnetworks components that can be used to decompose and describe a large network15. Topologically distinct nodes of a graphlet are referred to as orbits. There exist 73 possible orbits for all graphlets of size 2–5, which were counted using the Cytoscape plugin GraphletCounter35. For each ChIA-PET network node, a vector of 73 orbit counts was obtained. The proportion of each orbit with respect to all 73 orbits counted was calculated to normalize each vector. To identify orbits that are topologically similar, we performed hierarchical clustering based on Spearman rank correlation coefficient on orbit scores of ChIA-PET network nodes. Seven orbit clusters were obtained at a Spearman cutoff of 0.3  network related and (2) genomic-data related. We extracted sixteen network related features, which included network measures  (n = 8), connected component size, and graphlet cluster scores (n = 7). For genomic-data related features, we extracted and used seven features: open chromatin peak length in bps , the ratio of a node’s size to the average size of all nodes connected to it, the average PET of a node’s edges, and gene definition-related features (n = 4), which include the distance to the closest upstream and downstream TSS and the direction of transcription for these two genes. SVM17 models were trained with a radial basis function (RBF) kernel using scikit-learn Python libraries38. Grid search was used to tune hyper-parameters of the SVM models (C and gamma). For feature ranking, we implemented forward feature selection using a greedy algorithm that implemented a stepwise addition of features to the model, keeping the best-performing feature in the model for each round until all features were included. Matthews correlation coefficient (MCC) was used as a performance measure both during hyper-parameter tuning and feature selection analyses, since this measure is effective for unbalanced class sizes .We used support vector machines (SVMs) due to their efficacy in handling high dimensional and noisy data39. Index SNPs from the GWAS catalogue20 were used for enrichment analyses (n = 657 phenotypes) along with the SNPs that are in high linkage disequilibrium (LD) with the index SNPs (R2 > = 0.7). Benjamini-Hochberg FDR method was used for multiple hypothesis correction of enrichment p-values.We tested for enrichment of GWAS SNPs in enhancer loci targeting regular and broad domain promoters using GREGORhttps://github.com/UcarLab/CellSpecificNetworks.Datasets and scripts are available on the Ucar Lab github website: Supplementary FiguresTable S1Table S2Table S3Table S4


Intact oxaliplatin and Pt(DACH)Cl2 were measured in incubation solutions by HPLC-UV. A clinical study determined changes in plasma concentrations of calcium and magnesium in cancer patients and their impact on oxaliplatin clearance. Kinetic analyses modelled oxaliplatin degradation reactions in vitro and contributions to oxaliplatin clearance in vivo. Calcium and magnesium accelerated oxaliplatin degradation to Pt(DACH)Cl2 in chloride-containing solutions in vitro. Kinetic models based on calcium and magnesium binding to a monochloro-monooxalato ring-opened anionic oxaliplatin intermediate fitted the in vitro degradation time-course data. In cancer patients, calcium and magnesium plasma concentrations varied and were increased by giving calcium gluconate and magnesium sulfate infusions, but did not alter or correlate with oxaliplatin clearance. The intrinsic in vitro clearance of oxaliplatin attributed to chloride-, calcium- and magnesium-mediated degradation predicted contributions of <2.5% to the total in vivo clearance of oxaliplatin. In conclusion, calcium and magnesium accelerate the in vitro degradation of oxaliplatin by binding to a monochloro-monooxalato ring-opened anionic intermediate. Kinetic analysis of in vitro oxaliplatin stability data can be used for in vitro prediction of potential effects on oxaliplatin clearance in vivo.This study evaluated the impact of calcium and magnesium on the  It, in combination with a fluoropyrimidine, has become the standard chemotherapy for colorectal cancer in both adjuvant and palliative settings7. However, its neurotoxicity is a major treatment-limiting adverse effect resulting in a dose reduction or early termination of the drug treatment and long term toxicity that may adversely affect patient’s quality of life for many months to years9. Clinically, most patients undergoing oxaliplatin therapy typically experience cold-induced peripheral paraesthesia, and some experience muscle cramps, throat or jaw tightness, and pharyngolaryngeal dysesthesia immediately after the oxaliplatin infusion9. Abnormal spontaneous high-frequency motor unit action potentials are detectable on electromyography, reflecting hyper-excitability of these peripheral nerves12. The mechanism of this acute neurotoxicity of oxaliplatin has been proposed to be related to the prolonged opening of calcium-dependent voltage-gated sodium channels resulting from chelation of calcium by oxalate16. The prolonged opening of these sodium channels may induce cellular stress, which in addition to the accumulation of platinum compounds in the dorsal root ganglial cells, may cause damage to cell body and axon19, and contribute to the development of chronic peripheral sensory neurotoxicity of oxaliplatin. It has also been suggested that oxaliplatin and/or its metabolites may increase calcium influx into the cytosol of peripheral neurons, which in turn may enhance the activation of sodium and potassium channels, calcium regulated transcription factors and intracellular signalling involving calcium-dependent protein kinases21. On the basis of a retrospective clinical study suggesting neuroprotective effects of calcium gluconate and magnesium sulfate (CaGluc/MgSO4) infusions given concurrently with oxaliplatin therapy22 and supporting preclinical data23, these infusions became routinely used without further confirmatory prospective randomised trials or evaluation of any potential pharmacokinetic interactions with oxaliplatin. Since 2013, after two prospective randomised trials definitively showing a lack of neuroprotective effects of these infusions against both acute and chronic forms of oxaliplatin neurotoxicity25, they are now commonly omitted from the routine clinical practice26.Calcium and magnesium have been implicated in the mechanism and clinical prevention of oxaliplatin-induced neurotoxicity. Oxaliplatin -cyclohexane-1,2-diamine)platinum(II)) is an important anticancer drug for the treatment of many cancers of the gastrointestinal tractin vitro degradation and in vivo clearance of oxaliplatin was previously unknown. Once administered oxaliplatin has been proposed to undergo rapid non-enzymatic biotransformation reactions with water and nucleophiles such as chloride, methionine and glutathione29. It is not known to be subject to CYP-P450 mediated metabolism. The initial reaction in oxaliplatin biotransformation can involve displacement of the oxalate group by water and chloride, resulting in formation of reactive species such as monochloro-, dichloro-, and diaquo-DACH platinum, where DACH is the common abbreviation for cyclohexane-1,2-diamine29. Initial degradation of oxaliplatin in the presence of chloride in vitro was previously suggested to lead to formation of an intermediate species, monochloro-monooxalato ring-opened complex, [Pt(DACH)oxCl]−30 Cl2 concentrations at pre-defined time points over the incubation period. Data were presented as plots of concentration versus time and the mass balance of the reaction oxCl]−. In contrast, oxaliplatin remained stable in water oxCl]− followed by an irreversible second step producing Pt(DACH)Cl2 oxCl]− intermediate were decreased in 75 mM CaCl2 and 75 mM MgCl2 as compared to 150 mM NaCl. Finally, the values for the k2 rate constant for the loss of the monodentate oxalate ligand from [Pt(DACH)oxCl]− leading to formation of Pt(DACH)Cl2 were increased in 75 mM CaCl2 and 75 mM MgCl2 as compared to 150 mM NaCl. Thus, the observed increase in degradation is not due to an increased rate of the initial oxalate ring opening, but rather is due to a combination of a decrease in the reverse rate of oxalate ring closure combined with an increase in the rate of loss of oxalate.The parameters λ−, and in doing so, decreased the rate of oxalate ring closure and increased the rate of loss of oxalate from the ring-opened intermediate. The kinetics of the reaction mechanism were therefore modelled further by including an additional metal bonded intermediate species as shown in the shaded area of Fig. −1,M) was zero, then the calculated values of the equilibrium constant (K) for the reversible conversion of [Pt(DACH)oxCl]− to the metal bonded intermediate were 20 M−1 for Ca2+ and 30 M−1 for Mg2+. If k−1,M was not zero then the equilibrium constants would be higher. These equilibrium constant values indicate that in 75 mM CaCl2 or MgCl2 60% or 70% respectively of the [Pt(DACH)oxCl]− is ion-paired with the metal. Using these equilibrium constant values, the values of the rate constant for the conversion of the metal bonded intermediate to Pt(DACH)Cl2  were calculated to be 3.3 hr−1 and 1.5 hr−1 for calcium and magnesium, respectively.These findings suggested that calcium and magnesium did not interact significantly with oxaliplatin at the concentrations studied, but bound to the monodentate oxalate-containing complex [Pt(DACH)oxCl]in vitro findings described above suggested that calcium and magnesium may alter the in vivo clearance of oxaliplatin by accelerating its degradation via binding to [Pt(DACH)oxCl]−. Plasma calcium and magnesium concentrations vary between patients, on different occasions, and are altered in the presence of cancer, associated disease processes and treatments37. Until recently, CaGluc/MgSO4 infusions had been routinely given concurrently with oxaliplatin for the purpose of limiting neurotoxicity26 but their effects on oxaliplatin clearance or plasma calcium and magnesium levels had not been well studied. In a randomised placebo-controlled crossover clinical study, we sought to understand how variation in plasma calcium and magnesium concentrations, and the administration of CaGluc/MgSO4 infusions, influenced oxaliplatin clearance in cancer patients receiving chemotherapy, by measuring plasma concentrations of calcium and magnesium before and after CaGluc/MgSO4 and placebo infusions given with oxaliplatin. We previously reported that oxaliplatin clearance was not altered by giving CaGluc/MgSO4 infusions24. The mean oxaliplatin clearance was 35.3 L/hr (SD 9.8) with CaGluc/MgSO4 infusions and 33.6 L/hr (7.7) with placebo infusions (p = 0.17). Here we report that plasma concentrations of calcium and magnesium were significantly higher after the first CaGluc/MgSO4 infusions than at baseline  . Plasma magnesium concentration, however, remained significantly higher  than baseline when the second infusion was due and were increased further following the second infusion . There were no significant changes in plasma calcium and magnesium levels after the placebo infusions.The 4 or placebo infusions were plotted against their corresponding maximal plasma calcium and magnesium levels for each study patient  and 19 (100%) patients for calcium and magnesium, respectively. According to Common Toxicity Criteria Adverse Effect version 4.0 grading, elevated plasma calcium were severity grade 1 in 3 patients (16%) and grade 2 in 1 patient (5%), while elevated plasma magnesium was severity grade 1 in 12 patients (63%) and grade 3 in 7 patients (37%). Pearson co-efficient correlation analysis showed that oxaliplatin clearance did not correlate with plasma calcium (p = 0.33) or magnesium (p = 0.60) concentrations.Oxaliplatin clearance values measured on treatment cycles given with CaGluc/MgSOin vivo clearance of oxaliplatin in patients, an in vitro – in vivo extrapolation method was developed. First, the in vitro intrinsic clearances of oxaliplatin attributable to calcium-, magnesium- and chloride-mediated degradation were calculated. Non-compartmental analysis was used to calculate the AUC0−infinity for oxaliplatin concentration versus time for each experimental condition. Then the total in vitro clearance of oxaliplatin for each condition was obtained by dividing the amount of oxaliplatin added to the solution per unit volume by the AUC0−infinity. Oxaliplatin clearance attributable to calcium- or magnesium- mediated degradation was then calculated by subtracting the clearance attributable to chloride from the total in vitro clearance value for the relevant experimental condition. Calculated values for oxaliplatin clearance attributable to chloride, calcium and magnesium were then plotted against the concentrations of the relevant ion in the incubation solution and analysed by linear regression. The in vitro intrinsic clearance attributable to chloride, calcium and magnesium was taken from the slope of the linear regression fit to its data as shown in Table in vivo setting by calculating the total extracellular fluid (ECF) content of calcium, magnesium and chloride from estimates of ECF volume and measured plasma concentrations of these ions in patients from our clinical study. In vivo clearance predictions were then made by multiplying the in vitro intrinsic clearance value by the calculated ECF content value of each ion.To estimate potential contributions of calcium-, magnesium- and chloride-mediated degradation to the in vitro intrinsic clearance of oxaliplatin attributable to calcium-, magnesium- and chloride-mediated degradation was calculated to be 0.0039, 0.0023 and 0.00037 L/hr/mmol, respectively , calcium-, magnesium- and chloride-mediated degradation was predicted to account for less than 2.5% of the total clearance of oxaliplatin in vivo.The −, that forms in chloride-containing physiological solutions in vitro. To our knowledge, calcium and magnesium have not been previously reported to alter oxaliplatin degradation or bind to its degradation intermediates. We showed that the in vitro degradation of oxaliplatin to Pt(DACH)Cl2 was faster in the presence of calcium and magnesium by directly quantifying both intact oxaliplatin and Pt(DACH)Cl2 using HPLC-UV. Mass balance analysis revealed a deficit after accounting for these two compounds consistent with the transient formation of an intermediate species. Further evidence came from kinetic analyses and rate constants calculated for the reaction scheme in Fig. −, resulting in a decreased rate of oxalate ring closure, and an increased loss of monodentate oxalate leading to formation of Pt(DACH)Cl2. Oxaliplatin was unstable in the presence of chloride as previous reported by Jerremalm et al.30 but our findings also provide a mechanism of how calcium and magnesium influence this process of oxaliplatin degradation. This new information may have important implications for understanding how oxaliplatin behaves under in vivo conditions when other reactants or cations are present.This study showed that calcium and magnesium accelerate oxaliplatin degradation by binding to the monochloro-monooxalato ring-opened anionic oxaliplatin intermediate, [Pt(DACH)oxCl]in vivo clearance of oxaliplatin from in vitro studies of oxaliplatin degradation. It is, to our best knowledge, the first time that in vitro – in vivo extrapolation methods have been applied to predicting effects on oxaliplatin clearance. The concept was based on methods which are already established for in vitro - in vivo prediction of drug clearance mediated by hepatic microsomal metabolism by CYP-P450 and other enzymes, and which are commonly used in pre-clinical studies of the metabolic stability of new chemical entities32. However, oxaliplatin is not subject to hepatic metabolism, but degrades via leaving group displacement reactions, although little is known about their contributions to the in vivo clearance of oxaliplatin. To estimate effects on the in vivo clearance of oxaliplatin, we first obtained experimentally determined values for the in vitro intrinsic clearance of oxaliplatin attributable to calcium-, magnesium- and chloride-mediated oxaliplatin degradation, calculated from kinetic analyses of in vitro oxaliplatin stability data. Then, scaling factors were used to estimate the contributions of these processes to the in vivo clearance of oxaliplatin. This experimental approach has potential for providing new insights into mechanisms of oxaliplatin clearance. Previously, for example, it was suggested that oxaliplatin may initially react with water or chloride during its in vivo biotransformation29. However, we found oxaliplatin to be stable in water in vitro, and that chloride-mediated oxaliplatin degradation was predicted to contribute only 1.9% to the total in vivo clearance of oxaliplatin. This stability of oxaliplatin in pure water or in chloride may be due to slow oxalate ring opening and/or fast oxalate ring closing. These findings suggest that oxaliplatin degradation mediated only by water or chloride may contribute less than previously thought to the total clearance of oxaliplatin in vivo, as suggested by a low formation of Pt(DACH)Cl2 from oxaliplatin under in vivo conditions38.This current study provides a new experimental approach for predicting effects on the 4 infusions. Post-infusion elevations in plasma calcium were CTCAE severity grade 1 in three patients and grade 2 in one patient, while elevations in plasma magnesium were CTCAE grade 1 in twelve patients and grade 3 in seven patients. There has not been a previous study, which we are aware of, showing these effects on plasma calcium and magnesium levels in patients immediately or soon after these infusions prior to this current study. In our study, plasma calcium and magnesium levels were measured only at baseline, 20 minutes after the first CaGluc/MgSO4 infusions and immediately after the second CaGluc/MgSO4 infusion. We did not fully evaluate the time-course, duration, extent or clinical safety concerns related to these treatment-associated elevations in plasma calcium and magnesium levels. Previously, Gamelin et al.39 reported no difference in plasma calcium and magnesium levels after the end of oxaliplatin infusion given without CaGluc/MgSO4 infusions. Their reported values were very similar to those found in the current study at baseline or after placebo infusions. This clinical study also provided an opportunity to explore potential relationships between plasma calcium and magnesium levels and the clearance of oxaliplatin. Plasma calcium and magnesium varied widely between different patients and treatment cycles but did not correlate with oxaliplatin clearance. This finding was in keeping with the in vitro prediction that calcium- and magnesium-mediated degradation contributed a relative small amount (<2.5%) to the total in vivo clearance of oxaliplatin.In cancer patients receiving oxaliplatin chemotherapy, we found that plasma calcium and magnesium concentrations were increased significantly after the infusions of calcium gluconate and magnesium sulfate given immediately before and after oxaliplatin chemotherapy. These infusions were, until recently, routinely used in the clinic for the purpose of reducing neurotoxicity. Statistically significant increases in plasma calcium and magnesium occurred following each of the two CaGluc/MgSOin vitro prediction of effects on the in vivo clearance of oxaliplatin could assist with translating new treatments to clinical evaluation for preventing oxaliplatin neurotoxicity. Many new candidate treatments for preventing neurotoxicity of oxaliplatin are being identified by preclinical studies, for example, L-type calcium channel blockers20 and calcium/calmodulin-dependent kinase inhibitors21. Our method could identify the potential for deleterious pharmacokinetic interactions with oxaliplatin prior to clinical studies. In this way, this in vitro assessment will complement trial design and endpoints we recently proposed for the early clinical evaluation of investigational treatments for preventing oxaliplatin neurotoxicity40.The method we describe for the in vitro degradation of oxaliplatin by binding to a monochloro-monooxalato ring-opened anionic intermediate. Kinetic analyses of in vitro oxaliplatin stability data predicted the contributions of calcium-, magnesium- and chloride-mediated degradation to the total in vivo clearance of oxaliplatin in patients. In vitro-in vivo extrapolation methods can be used in future studies for the in vitro prediction of potential effects on oxaliplatin clearance in vivo.In conclusion, calcium and magnesium accelerate the 2 were obtained from Sigma-Aldrich . Hydroxyethylpiperazine-N’-2-ethane sulfonic acid (‘HEPES’) was obtained from Gibco-BRL Life Technologies . Powder sodium chloride (NaCl) and magnesium chloride (MgCl2) anhydrous were obtained from Sigma Aldrich . Calcium chloride (CaCl2) was obtained from Riedel-de Haen AG (Germany). Methanol of chromatographic grade, triflic acid (98% reagent grade) was obtained from Sigma Aldrich . All solutions and mobile phase were prepared using Milli-Q grade water .Oxaliplatin and Pt(DACH)Cl2 , and MgCl2  in HEPES buffer at pH 7.3 and 37 °C. A temperature regulated water-bath  was used to maintain temperature during incubation. Samples for analysis were taken at 0, 5, 10, 20, 30, and 60 minutes then hourly thereafter until 8 hours. To detect oxaliplatin and its degradation product, Pt(DACH)Cl2, these incubation samples were analysed using a Hewlett Packard HP1200 HPLC online system. This included a binary pump, a degasser and an autosampler , a Waters µBondapak C18 3.9 × 300 mm column  with a guard column . The UV detector used was Millipore Waters Lambda-max model 480 LC Spectrophometer . The HPLC separations were performed at room temperature using a mobile phase containing 3% methanol in Milli-Q water (adjusted to pH 2.5 with triflic acid). The flow rate was 0.5 mL/min. The UV wavelength monitored was 210 nm with a reference of 550 nm. The injection volume of all samples was 50 µL. Data acquisition and processing were performed using HP4500 ChemStation and HP1200 Agilent ChemStation offline software B.04.01 . The samples were analysed immediately after they were taken from the incubation solutions whenever possible, otherwise they were snap frozen using liquid nitrogen and thawed within a minute before being injected onto the HPLC for analysis to avoid continuation of degradation of oxaliplatin at higher temperatures. All samples from each incubation study were analysed on the same day using the same mobile phase. Concentrations of oxaliplatin and Pt(DACH)Cl2 were determined by using their respective calibration curves to convert the areas under the chromatographic peaks to concentration values. The concentrations of the intermediate species were estimated from a mass balance after accounting for oxaliplatin and Pt(DACH)Cl2. The kinetics of degradation reactions were modelled as described below.To study the stability of oxaliplatin in the presence of chloride, calcium and magnesium under physiological conditions, oxaliplatin (100 μM prepared in Milli-Q water) was incubated in water alone, NaCl , CaCl24, a randomised double-blind placebo-controlled design was used to evaluate the effects of calcium gluconate (1 g) and magnesium sulfate (1 g) (CaGluc/MgSO4) infusions on the pharmacokinetics and acute neurotoxicity of oxaliplatin. Each patient undergoing oxaliplatin chemotherapy was given either CaGluc/MgSO4 or placebo infusions immediately before and after oxaliplatin infusion on cycle 1 then the opposite study infusion on cycle 2. This study was approved by the Northern Y Regional Ethics Committee  and was conducted in accordance with its guidelines and regulations. Informed consent was obtained from all study participants. The plasma pharmacokinetic samples were collected at 13 predefined times including at baseline, during and 3 hours post oxaliplatin infusion. The samples used for this current study were taken at baseline, 20 minutes post first CaGluc/MgSO4 or placebo infusion, immediately prior and post second CaGluc/MgSO4 or placebo infusions. The plasma was immediately prepared by centrifugation at 4 °C and 5000 G for 5 minutes, then snap-frozen using liquid nitrogen. The samples were stored at −80 °C until analysis. Plasma calcium  and magnesium concentrations were measured using Cobas 8000 modular analyser  at LabPlus, Auckland City Hospital . Data were presented as measured values and the levels of calcium and magnesium were graded using Common Toxicity Criteria Adverse Effect (CTCAE) version 4. Plasma calcium and magnesium concentrations were compared between baseline and post CaGluc/MgSO4 or placebo infusions by repeated measures one-way ANOVA analysis. Correlation between calcium and magnesium concentrations and observed oxaliplatin clearance for each patient obtained from our previous study24 was assessed by Pearson correlation analysis. All statistical analyses were performed using PRISM 6, GraphPad, San Diego, USA.In our previously reported clinical study2 at each sampling time. Rate constants were obtained by least-square fitting the analytical expressions for the reaction scheme to the three species simultaneously Clin vitro intrinsic clearance of oxaliplatin attributable to chloride-, calcium- and magnesium-mediated degradation, the following steps were undertaken: (1) AUC0−infinity for oxaliplatin concentration versus time for each in vitro incubation experimental condition was calculated using non-compartmental analysis; (2) total in vitro clearance of oxaliplatin for each condition was calculated by dividing the amount of oxaliplatin in the incubation solution per unit volume by the corresponding AUC0−infinity; (3) oxaliplatin clearance attributable to calcium- and magnesium-mediated degradation was calculated by subtracting the clearance attributable to chloride from the total in vitro clearance value for each condition; (4) calculated values for oxaliplatin clearance attributable to these ions were then plotted against the concentrations of the corresponding ions in the incubation solution and analysed by linear regression; and (5) finally, the in vitro intrinsic clearance attributable to chloride, calcium and magnesium was taken from the slope of the linear regression fit to its data  contents of chloride, calcium and magnesium in each patient. First, total body water was estimated as 60% of body weight in men and 50% in women, and the ECF volume was estimated as 40% of total body water. The ECF content of chloride, calcium and magnesium was then calculated by multiplying the measured plasma concentrations of these ions by the estimated ECF volume in each patient. Finally, in vivo clearance of oxaliplatin mediated by each of chloride, calcium and magnesium was calculated using the following formula: in vivo CL = in vitro CLint x ECF content, where units for CL = L/h; CLint = L/h/mmol; and ECF content = mmol).To extrapolate Supplementary Figure