Over the past few decades, the systemic therapy of breast cancer (early and advanced) has changed considerably. For the past 40–50 years, and since the discovery and further therapeutic use of tamoxifen, a selective estrogen receptor modulator, breast cancer treatment has become the model for the development and success of tailored medical treatment. Much still needs to be done in improving outcomes for all patients with breast cancer, and especially for those who have advanced breast cancer, a challenging area for medical oncologists. Ongoing international clinical trials are currently evaluating new therapeutic approaches and identifying specific biological subsets that could determine a patient’s ability to respond to particular chemotherapeutic drugs. Therapeutic approaches for breast cancer have changed over the past few decades, and the use of systemic therapy for early and advanced disease tailored to the individual patient holds the promise of delivering treatment to those in need and who could benefit the most. In this report, emerging therapeutic options for patients with endocrine-dependent breast cancer, monoclonal antibodies for those with HER2-positive disease, and newer available systemic chemotherapeutic agents will be discussed.The worldwide incidence of breast cancer continues to increase, and approximately 1.7 million new cases are diagnosed yearly. It also remains a leading cause of death with approximately 520,000 deaths/year, as reported by the World Health Organization in an updated breast cancer fact sheet of 20152. The options for the treatment of hormone-sensitive breast cancer have expanded in recent years with a number of other effective endocrine agents that reduce estrogen biosynthesis: the aromatase inhibitors (AIs) such as letrozole, exemestane, and anastrozole, gonadotropin-releasing hormone agonists (GnRH) such as goserelin and leuprolide, and selective estrogen receptor downregulators (SERDs) such as fulvestrant. Since the use of these compounds, alone or in combination, a substantial improvement in prolongation of disease-free intervals and survival outcomes has been reported.Hormonal therapy for those patients with breast cancer expressing the endocrine receptors estrogen and progesterone has been utilized for longer than 30 years with the administration of tamoxifen, a selective estrogen receptor modulator (SERM) and probably the first targeted therapy widely used in cancer treatment4. In the ATLAS (adjuvant tamoxifen: longer against shorter) randomized trial3, nearly 13,000 women with early breast cancer who completed 5 years of treatment with tamoxifen were randomized to continue tamoxifen for a total of 10 years or to stop at 5 years. The results obtained showed that for those patients continuing tamoxifen to 10 years, a further reduction in recurrence and mortality happened, in particular after year 10. These results, according to the ATLAS Collaborative Group investigators, suggest that 10 years of tamoxifen treatment can approximately halve breast cancer mortality during the second decade after diagnosis.Recently, two international multicentric clinical trials reported the continuous use of tamoxifen or an AI for a prolonged period of time (10 years) for early breast cancer endocrine-positive patients4 extended treatment with a non-steroidal AI (letrozole) to 10 years for postmenopausal endocrine-positive early breast cancer patients. Patients who received 5 years with an AI monotherapy upfront or after tamoxifen therapy were randomized to an extra 5 years. More than 1,900 patients were enrolled, and the extension of the AI to 10 years resulted in significantly higher rates of disease-free survival and lower incidence of contralateral breast cancer. As expected, bone-related side effects occurred more frequently among patients receiving letrozole than placebo.Another recently published study (MA17R)These longer tamoxifen and AI prospective clinical studies, together with earlier published clinical trials, confirmed that women at a higher risk of developing recurrence or metastatic disease should strongly consider continuation of their adjuvant therapy to 10 years without any long-term worsening of their quality of life.5.Despite hormonal standard therapy, approximately 20–30% of patients with endocrine-responsive breast cancer will suffer recurrences and the development of metastatic disease as they experience a biochemical mechanism of endocrine resistance. Recent ASCO guidelines on endocrine therapy for advanced breast cancer reaffirm many of the principles that should be considered when making treatment decisions in this patient populationSignificant progress has been made recently in this area, and newer therapeutic targets have been developed against a number of hormonal resistance mechanisms. Inhibitors of these pathways are under development to improve the efficacy of hormonal therapy, and some have already become FDA approved and commercially available.9. Everolimus, together with a steroidal AI (exemestane), has shown promising benefits in international clinical trials. It is FDA approved and available around the world. The regulatory approval was based on data from the Bolero trials, of which Bolero 2 has been completed. Based on doubling of the progression-free survival and prolongation of survival , the FDA approved itIt is known that resistance to endocrine therapy, and to some chemotherapy drugs, is usually associated with a number of different molecular mechanisms. PI3K/AKT/mTOR is the most commonly altered pathway in hormonal-positive breast cancer, and there are a number of agents targeting this specific pathway10.Pan class PI3K inhibitors such as buparlisib, pictilisib, alpelisib, and taselisib continue to be investigated, as they hold the potential to overcome hormone resistance and improve responses. Promising activity has already been shown in the clinic, but there are some limitations, mainly due to dose-limiting toxicities De novo or acquired resistance to adjuvant therapy and metastatic breast cancer remains an important therapeutic challenge. Cyclin D1 interacts with cyclin-dependent kinases 4 and 6 (CDK 4 and 6) in an active protein complex that promotes cell proliferation. Cyclin D1 is probably the most frequently overexpressed gene in primary breast cancer (approximately 15% of breast cancer patients). Therefore, CDK 4 and 6 represent potential therapeutic targets for endocrine-responsive breast cancer. Inhibition of the CDK 4/6 pathways is possible by small molecule inhibitor drugs such as palbociclib, ribociclib, and abemaciclib14.14. The hope remains that, in the future, reliable biomarkers providing oncologists with objective tools to make the best therapeutic decisions will become commonly available.Palbociclib was the first anti-CDK 4/6 drug approved by the FDA based on the Paloma clinical trials. Ribociclib has recently been approved . Abemaciclib has recently been granted breakthrough therapy designation by the FDA. This was based on data of an early clinical trial (MONARCH 1) that confirmed the efficacy and safety of abemaciclib in women with advanced or metastatic, refractory hormonal-positive breast cancer. Although the most common grade 4 toxicity is neutropenia (in 5% of patients), it is usually manageable and no febrile neutropenia was reported16.Since the introduction of the first anti-HER2 targeted therapy (trastuzumab) approximately 16 years ago, many other emerging monoclonal antibodies have been investigated in prospective randomized trials. Trastuzumab has significantly increased the survival of breast cancer patients with HER2-positive disease in the neoadjuvant, adjuvant, and metastatic groups of patients17. Different clinical studies in phase II and III have confirmed the value of TDM-1 administration in second- or third-line therapy for advanced breast cancer18.Newer drugs such as pertuzumab in combination with trastuzumab and chemotherapy, and TDM-1, an antibody drug conjugate, have shown a proven, consistent benefit with prolongation of disease-free intervals but not always an overall survival benefit. Final results from the CLEOPATRA study showed that the combination of two targeted agents, trastuzumab and pertuzumab, significantly prolonged survival in patients with HER2-positive advanced breast cancer when compared to trastuzumab alone. All patients received chemotherapy with docetaxel together with the targeted drugs19.Neratinib is an oral small tyrosine kinase inhibitor molecule initially tested in patients with HER2-positive metastatic breast cancer that showed antitumor activity in previously treated patients. The ExteNET phase III trial of neratinib versus placebo after adjuvant treatment with trastuzumab accrued more than 2,400 patients. The results demonstrated that neratinib after trastuzumab resulted in a 49% reduction of risk (recurrence or death) with a two-year disease-free interval advantage when compared to placebo. The downside of this drug is the relatively high incidence of side effects . It remains one of the most promising new drugs for HER2-positive breast cancer, and efforts to reduce and control its known side effects will ultimately allow for a safer use of this novel drug23.Unfortunately, most of these drugs are very costly and because of this they are not readily available worldwide. Efforts to ensure that treatments remain cost effective in environments of limited resources should be implemented. An alternative solution is to develop a cheaper, equally effective alternative, a biosimilar agent. Biosimilars are not biochemically identical to the original and must be tested in clinical trials to ensure that they are not inferior 23.A recent study performed with MYL-14010, a trastuzumab biosimilar, was presented at ASCO 2016. The clinical trial (HERITAGE) was a double blind, prospective randomized trial that enrolled over 450 patients with HER2-positive advanced breast cancer and who never received prior chemotherapy or an anti-HER2 agent for their metastatic disease. The conclusion by the investigators was that an equivalent efficacy was demonstrated between the biosimilar MYL-14010 and trastuzumab, with similar safety, immunogenicity, and pharmacokineticsFor those patients with advanced HER2-positive breast cancer, the treatment scenario is evolving rapidly as many effective targeted therapies are being developed and combinations of drugs are shown to induce a higher benefit.For patients with HER2-negative metastatic disease, the treatment approach should be tailored according to a number of prognostic factors, which include endocrine tumor status, other possible actionable targets , hormonal status, prior therapies (adjuvant and metastatic), disease-free interval, comorbidities, performance status, burden of metastatic disease, patient preferences, prior toxicities, geographic accessibility to the closest cancer treatment center, and available as well as affordable cancer therapies.24 has established itself as a major international developer of guidelines in the treatment of advanced disease based on the most up-to-date evidence that can be used worldwide and in different healthcare settings. The new consensus guidelines will be published during the last quarter of this year.Over the past few years, the International Consensus Conference for Advanced Breast Cancer (ABC)26. Further investigation of these novel immunotherapy drugs is warranted, and new clinical trials are currently ongoing.During the last few years, immunotherapy has been shown to induce remarkable responses in the treatment of advanced malignant melanoma, non-small-cell lung cancer, and bladder cancer. Several new drugs (anti-CTLA4 and anti-PD-1 and PDL-1) have been approved by the FDA. Clinical trials with this new group of drugs for patients with advanced breast cancer have shown induction of moderate overall response rates. In some subsets of patients (those with triple-negative disease and those expressing PDL-1), an improved clinical activity was seen29. The results confirmed that genomic risk assessment can reduce the need for chemotherapy in up to 46% of patients who might otherwise receive it on the basis of their clinical and pathologic assessment alone.Are all patients with early breast cancer benefiting from adjuvant chemotherapy? With this question in mind, global investigators, motivated by the development of new genomic profiling tests (Oncotype Dx and Mammaprint), have been focusing on how to personalize therapies so that only the necessary treatment is delivered. Recent publications have demonstrated how genomic information provided by the use of a 21-gene assay (Oncotype Dx) or a 70-gene assay (Mammaprint), combined with the patient’s clinicopathologic status, can come up with a risk score predicting the need for adjuvant chemotherapyOncotype Dx and Mammaprint are first-generation genomic signature tests able to better predict early relapses. PAM50 and Endopredict are second-generation genomic signatures helpful in predicting late relapses, guiding clinicians during the 5–10-year follow-up of those patients.The advent of new drugs targeting specific actionable targets has led to considerable progress in the treatment of breast cancer over the past few years. Challenges remain, such as resistance to systemic therapy (endocrine and others), high cost of treatments, and limited availability in many countries of appropriate cancer services.We must continue to find ways to improve our available technology to provide proper guidance for those living with the disease, and for those at high risk of developing it, and to develop new, more effective therapies to substantially improve breast cancer patients’ outcomes worldwide. Tailoring treatments to the individual patient holds the promise of guiding them as they face difficult treatment decisions in an effort to improve their long-term outcomes.


Mouse models of folate-responsive neural tube defects (NTDs) indicate that impaired de novo thymidylate (dTMP) synthesis through changes in SHMT expression is causative in folate-responsive NTDs. We have created a hybrid computational model comprised of ordinary differential equations and stochastic simulation. We investigated whether the de novo dTMP synthesis pathway was sensitive to perturbations in FOCM that are known to be associated with human NTDs. This computational model shows that de novo dTMP synthesis is highly sensitive to the common MTHFR C677T polymorphism and that the effect of the polymorphism on FOCM is greater in folate deficiency. Computational simulations indicate that the MTHFR C677T polymorphism and folate deficiency interact to increase the stochastic behavior of the FOCM network, with the greatest instability observed for reactions catalyzed by serine hydroxymethyltransferase (SHMT). Furthermore, we show that de novo dTMP synthesis does not occur in the cytosol at rates sufficient for DNA replication, supporting empirical data indicating that impaired nuclear de novo dTMP synthesis results in uracil misincorporation into DNA.Folate-mediated one-carbon metabolism (FOCM) is an interconnected network of metabolic pathways, including those required for the  Furthermore, enzymes in FOCM have been successful targets for the development of antineoplastic pharmaceutical agents including0020methotrexate and 5-flurouracil6. FOCM in the cytoplasm is composed of three interconnected biosynthetic pathways, which include de novo thymidylate (dTMP) synthesis, de novo purine synthesis and homocysteine remethylation to methionine  competition among the pathways for a limiting pool of folate cofactors10, (b) long-range and indirect regulatory processes, (c) formation of multi-enzyme complexes, (d) cellular compartmentation, (e) interactions with other metabolic pathways, (f) nutritional status (g) penetrant genetic variants11. Mathematical models have been developed to assess this complexity and gain an understanding of the cause-and-effect relationships that regulate FOCM functioning in health and disease. The overall goal is to provide an understanding of function of the entire system in silico that can be used to accelerate discovery and guide the design of biological experimentation.Perturbations in folate-mediated one-carbon metabolism (FOCM) are associated with numerous pathologies including neural tube defects (NTDs)de novo purine and thymidylate biosynthesis in L1210 cells15. These models considered FOCM reactions as occurring in a common cellular compartment, described the reactions in terms of Michaelis-Menten kinetics, and investigated biological consequences of inhibiting de novo purine and dTMP biosynthesis. Other models have been constructed to simulate the homocysteine remethylation in liver18. Using data from experimental animals, non-trivial mathematical functions were adopted to address long-range inhibition and activation processes involving folates and other metabolites that cannot be translated into simple Michaelis-Menten equations17. These models can reproduce changes in metabolite concentrations in response to nutritional deficiencies and the effects of gene variants, thus corroborating human clinical and epidemiological data. These models have also integrated other pathways such as glutathione metabolism and polyamine synthesis, studying the influence of indirect effects beyond FOCM20.Early models of FOCM quantified steady-state concentrations of folates, and predicted how methotrexate and 5-fluorouracil affect the rates of 20. Sensitivity analysis is used to summarize the effects on metabolite concentrations and reaction velocities in response to changes in inputs or enzyme activities21. Here we present a hybrid stochastic model for simulating the FOCM dynamics where state-of-the art deterministic simulation (based on ODEs) has been coupled with exact stochastic simulation to assess metabolite variabilities in the FOCM network at steady state. A detailed description of this combined approach can be found in Methods. The deterministic approach used in isolation can only provide a rough estimate of FOCM model dynamics, because the deterministic approach is limited when enzyme substrates, such as folate cofactors, are present in the cells at low micromolar concentrations, and because reactions within the network occur randomly at discrete time points. FOCM is expected to exhibit variability (i.e. stochasticity) in its behavior22. Capturing system stochasticity is essential when substrate concentrations are low and limiting, but requires consideration of molecules as discrete entities, rather than describing concentrations as continuous variables through ODEs23. Simulation strategies that combine both deterministic and stochastic approaches can give a more accurate and more detailed understanding of FOCM network functioning and stability. In contrast to approaches based solely on deterministic simulation, these studies can be used to assess the contributions of factors such as genetic variation and nutritional status on the stochastic behavior of individual pathways within the network, thereby aiding in establishing which system inputs (i.e. nutrition) and outputs (i.e. biomarkers) are most closely associated with human health outcomes.These existing FOCM models describe the continuous flux of metabolite concentrations in terms of ordinary differential equations (ODEs) with time as an independent variablede novo dTMP biosynthesis and homocysteine remethylation pathways is believed to underlie FOCM-associated pathologies including NTDs  using parameters for folate monoglutamates, which are not the physiological form of folate cofactors in cells. Under these conditions, the variant allele decreased concentrations of 5mTHF and SAM and increased the concentrations of homocysteine, SAH, and rates of dTMP and purine biosynthesis21, which is inconsistent with current understanding of biochemical changes associated with NTD risk. The effect on the redistribution of folate cofactors towards 10fTHF that is associated with the 677 T variant, or its impact on other pathways within the network, was not reported21.Dysregulation of the partitioning of one-carbon units in the form of 5,10-methylenetetrahydrofolate (CH2F) cofactors between the de novo dTMP biosynthesis38, and the effects of known genetic and nutritional variables that impact movement of CH2F through the network. Existing models are limited by adopting kinetic parameters determined from the use of folate monoglutamate substrates20. Folate polyglutamates are the functional form of folate cofactors in cells and have much higher affinity for their respective FOCM enzymes than the corresponding monoglutamate forms of folate40. Therefore, we updated the parameters in the deterministic model to include the physiologically-relevant polyglutamate forms of folate cofactors and demonstrate that it faithfully recapitulates existing data in the literature , with a SAM/SAH ratio around one being achieved in CC homozygotes compared to 0.24 in TT homozygotes  an increased flux through both the 10fTHF synthetase activity leading to increased synthesis (FTS), and (2) an increased flux through the cyclohydrolase/dehydrogenase activity of MTHFD1 which converts CH2F to CHF to 10fTHF . Decreased MTHFR activity, which lowers cellular 5mTHF levels  and intracellular folate concentrations; the 677 T variant lowers intracellular folate levels. Therefore, the impact of this variant on FOCM was modeled at two different levels of folate Tables  . Model steady states were obtained under four different conditions that differed by MTHFR 677 genotype (CC and TT case) and intracellular folate levels (replete and low). Interestingly, the most stable steady state , see Methods for details), was the CC case with folate replete concentrations, consistent with numerous epidemiological studies associating the MTHFR C677T genotype with folate-related pathologies  are present in both the cytosol and recently have been found to function in the nucleus. In the nucleus, they comprise a multi-enzyme complex at sites of DNA synthesis that may be critical to limit rates of uracil misincorporation into DNA, but regulatory mechanisms remain unknown45. These enzymes are modified by the Small Ubiquitin-like MOdifier (SUMO) protein at the G1/S boundary, which permits their nuclear translocation during S-phase of the cell cycle46. One study showed that when nuclear translocation of this complex is impaired in a mouse model over-expressing SHMT1, rates of uracil misincorporation into DNA increased several fold45. In this model, SHMT1 protein levels were elevated several fold in the liver, yet its localization was restricted to the cytoplasm and nuclear SHMT1 levels were depleted compared to wild-type mice45. Furthermore, nuclei isolated from SHMT1 overexpressing mice exhibited lower rates of de novo dTMP synthesis compared to nuclei isolated from wild-type mice45. This suggests that de novo dTMP synthesis occurs when the enzymes are present in the multi-enzyme complex within the nucleus in mammals. However, no definitive experiment has been performed that identifies the relative contribution of nuclear and cytosolic dTMP synthesis to overall dTMP synthesis. Interestingly, S. cerevesiae do not import the dTMP synthesis pathway into the nucleus47.Mouse models implicate SHMT1 and impaired de novo dTMP pathway was required to meet cellular demands for dTTP during DNA replication, rates of dTMP synthesis were modeled for mammalian cells using standard Michaelis-Menten kinetics (Table 49), the rate of dTMP synthesis required for faithful cell replication is 7.8 µM/min  is 0.5 µM/min, indicating that yeast synthesize dTMP at a rate that is more than 3-fold greater than necessary for adequate dTMP synthesis . Furthermore, in response to DNA damage, yeast increase dNTP concentrations 6–8 fold54 and E. coli increase dNTP concentrations 1.8–3.7 fold55, but dNTP concentrations do not increase after DNA damage in mammals57.To determine if nuclear import of the cs Table ; Table 452 Table . The ratUnderstanding the dynamics of FOCM and its responsiveness to both genetic and environmental perturbations is the key to understanding the etiology of folate-related pathologies. Computational models and related simulations permit an identification of the most sensitive reactions within the network that exhibit the greatest degree of stochastic behavior leading to variability in network outputs. Furthermore, computational models allow an understanding of how both genetics and environmental factors can enhance or repress stochastic behavior at defined locations within the network, accelerating the development of diagnostics to identify those at risk for folate-related pathologies as well as lead to the development of targeted nutritional interventions for disease prevention.Shmt1 knockout mouse model  exhibits impaired de novo dTMP synthesis in the absence of perturbations of homocysteine remethylation. It also recapitulates risk for NTDs in humans. Specifically, the mouse model exhibits folate-responsive NTDs that occur with minimal perturbation in FOCM, and exhibit low and variable penetrance34. In fact, most if not all, folate-related pathologies whose etiology involves interactions among genetic risk variants and nutrient exposures also exhibit low and/or variable clinical presentation. Understanding the stochastic behavior of the various reactions within FOCM that results in increased variability in FOCM network outputs is essential to understand which enzymes in the network contribute to folate-related pathologies.The 58. The polyglutamate chain (N = 3 glutamate and higher) increases the affinity of folate cofactors for many folate-dependent enzymes by one to two orders of magnitude40. Models that include kinetic parameters derived from the use of folate monoglutamates can limit model reliability. Here we established a hierarchy of criteria to select a more homogeneous set of kinetic parameters (i.e. Km and Vmax) by referring, when possible, to L1210 cells because of the richness and quality of the data used to derive kinetic parameters. Furthermore, our preference was to select kinetic coefficients generated using folate polyglutamate cofactors and purified proteins from animal models closest to humans, as the variability in kinetic parameters among mammals is much less than the differences observed between folate monoglutamate and polyglutamate cofactor substrates.Existing FOCM models rely on the limited quantity of kinetic data present in the literature, and the performance of the model will be dependent upon the kinetic parameters chosen to include in the model. Much of the available kinetic data for FOCM enzymes present in the literature was collected using the commercially available monoglutamate folate substrates, with few studies using the physiologically relevant polyglutamate forms of the cofactor. In the cell, newly transported monoglutamate folates are converted to folate polyglutamates, containing 3 to 7 polyglutamate moieties, though the action of folylpolyglutamate synthetaseMTHFR 677 T variant are accompanied by elevated levels of 10fTHF, which has been observed in animal models and in humans29 .The deterministic simulation was based on ODEs, where reactions were described in terms of Michaelis-Menten equations consistent with the original model of Reed60 was employed to quantify the level of stochasticity in the considered FOCM steady states. We refer to the Supplementary Material for a detailed description of how the ODE model has been translated to a reaction based stochastic one. According to the seminal work of Gillespie59, exact stochastic simulation allows simulation of each reaction event asynchronously when such reaction event is most probable to occur. To allow this, the simulation algorithm computes a propensity function aj(x) at each simulation step for each modeled reaction Rj, where x is the current state of the system providing the abundances of all modeled species at the considered time. The propensity value of a reaction has a direct link with the probability of its execution, that is, reactions with higher propensity are more likely to be fired in the near future. To evaluate when the next reaction event will occur, also the total sum of propensities 15–1016 reaction events per unit of time during the stochastic simulation, due to stiffness of the system. To circumvent the problem, we relied on the concept of total propensity to evaluate the stability of the steady state, by assuming that a steady state is more stable when it has a lower value of a0(x), that is, a lower averaged number of reaction events that can perturb the equilibrium of the steady state.The set of ODEs was further translated into a stochastic reaction-based model and a hybrid simulation approachin silico experiments were performed to determine if the model could recapitulate empirical data generated in MCF-7 cells by Herbig et al.10 focusing on the effect of glycine on FOCM  and S-adenosylhomocysteine (SAH) levels. In summary, the empirical data revealed that as glycine concentrations increase intracellular 10fTHF levels increase at the expense of 5mTHF levels that decrease. Furthermore, as glycine concentrations increase SAM levels are depleted and SAH levels rise. These changes were interpreted by the effects of glycine concentration driving the reversible SHMT reaction in the direction of serine synthesis10.To validate the FOCM model, OCM Fig. . Glycineet al. study at this glycine concentration.We simulated the effect of glycine on folate distribution, SAM, and SAH concentrations using the computational model for values of glycine ranging from 0 to 10 mM Fig. . The treSupplementary Data


BCL2, the founding member of a family of apoptotic proteins and archetype of cell death inhibitors, have been tremendously insightful, culminating in the recent successful clinical translation of new therapeutic approaches exploiting cell death pathways  translocation carried by patients with the follicular variant of B-cell lymphoma, from which it takes its name.1 Such chromosomal rearrangement places the immunoglobulin heavy-chain enhancers near the BCL2 promoter resulting in BCL2 overexpression. The discovery of BCL2, and its function as inhibitor of apoptosis by Vaux,2 laid the foundation for the identification of cell death mechanisms across different organisms. It also heralded the key discovery of a new cancer mechanism: some genes contribute to cancer development by inducing cell death escape, rather than cell proliferation, which afterward was recognized as one of the cancer hallmarks.3 Here Croce and colleagues recall those early steps and show how the same powerful approach of positional cloning led, years later, to the identification of the first microRNAs known to underlie cancer development.4 They were investigating the 13q chromosomal deletion frequently found in patients with chronic lymphocytic leukemia (CLL), the most common human leukemia, in the search for an elusive candidate cancer gene. The analysis of a few CLL specimens allowed them to narrow down the deletion to a 30 kb region which, disappointingly, did not contain any protein-coding gene. This region however contained a cluster of two microRNAs, miR-15a and miR-16, on which they zeroed in. When they found BCL2 listed as a top predicted target of miR-15/-16, it turned as an immediate pick among proteins involved in B-cell transformation. Indeed they went on demonstrating that the BCL2 overexpression observed in over 70% of CLLs was mainly caused by the loss of miR-15/-16, occurring through deletion or downregulation. Here the authors hail the encouraging results on the efficacy of the BCL2 targeting drug ABT-199 (venetoclax) approved by the FDA in 2016 for the treatment of refractory CLL, and raise important questions as to whether other crucial targets of miR-15/-16 have a role in CLL and could be tackled in combination with BCL2-targeting drugs and whether regulation of miR-15/-16 itself might be translated in the future into a potential pharmaceutical approach against CLL.4BCL2 protein family has grown larger over the last years counting 30 members each containing from one to four BCL2 homology (BH) domains and categorized into three functional classes: the pro-survival members  recognized as ‘guardians’ the pro-apoptotic ‘effectors’ ; and the pro-apoptotic ‘BH3-only’ proteins acting as ‘initiators’ . BCL2 family proteins control the mitochondrial apoptotic pathway, which is engaged both by physiological stimuli during development or by a myriad of cytotoxic cues. The interaction between BCL2 pro- and anti-apoptotic family members sets the apoptotic threshold acting as a tripartite switch that determines the life/death decision:6 once the balance is tipped toward the formation of BAX and BAK oligomers, the cell is committed to suicide. BAX and BAK assembly in fact leads to the formation of pores within the mitochondrial outer membrane causing its permeabilization (MOMP) and providing a getaway for apoptogenic molecules, whose release from the mitochondrial intermembrane space into the cytosol, triggers, in turn, a caspase cascade ultimately leading to cell death.The 8 On the basis of this functional dichotomy, BH3-only proteins have been further classified into ‘sensitizers’, which lead to MOMP indirectly (such as BAD and NOXA), and ‘activators’, which directly trigger BAX and BAK oligomerization (such as BID and BIM). The boundaries between these two classes however are blurred, with some activator BH3-only proteins reportedly capable of binding both pore formers and anti-apoptotic proteins, giving rise to different models of apoptotic switch regulation by the BCL2 family, as discussed in.9 Moreover, the BH3 domain is not a simple passe-partout for all the BCL2 family member interactions: other residues or motifs might allow specific interactions, stressing the importance of the use of full-length proteins rather than peptides limited to the BH3 region, and also of physiological protein concentrations, to determine relevant interactions, as argued by Andrews and colleagues in this issue.7 Here the authors highlight how BCL2 protein interactions depend on protein concentration and on the affinity between members that are both largely affected by protein interaction with intracellular membranes, which are often neglected experimentally.7 Adding further complexity, the cellular context, the nature of apoptotic cues (in terms of stress type and intensity) and posttranslational modifications all contribute to dictating the interactions among the three subgroups of the BCL2 family.7Mechanistic insights from structural studies and sequence homology have allowed to draw a clear picture of the interactions between BCL2 family members, although there are still many open questions. The multi-BH domain family members  adopt a globular structure with a characteristic hydrophobic surface groove that mediates the interaction with the BH3 domain of the pro-apoptotic family members. Owing to these intrinsic properties, in healthy cells, pro-survival members keep their pro-apoptotic relatives in check. Following apoptotic cues however, the upregulation of BH3-only proteins favors their binding to pro-survival members, which sets free the pore formers BAX and BAK. Some BH3-only proteins can also directly activate the pore formers, inducing MOMP.8 The precise sequence of events and interactions leading to the formation of BAX/BAK homo-oligomers at the MOM, their transient interaction with BH3-only proteins and the nature, whether lipidic or proteinaceous, of the pores themselves, still need to be elucidated as discussed herein.8 Also, Kalkavan and Green examine the role of BOK, which seems to function as an additional, non-canonical MOMP effector, acting independently of BAX and BAK in certain contexts.8The mechanisms acting upstream, during and after MOMP are also being intensively investigated as each step could offer therapeutic opportunities to turn the apoptotic cell machinery into a precision medicine strategy. In particular, targeted release of BAX and BAK ability to perforate the MOM might be harnessed as a powerful killing approach. However, the exact mechanisms underlying pore formation are still a matter of debate.8But what happens next, after the MOM is pierced? Is the release of molecules (such as citochrome c and SMAC/DIABLO), which reside within the inner mitochondrial space, coordinated? Or are they released independently? Which factors regulate their release is equally a matter of current investigation and unravelling these mechanisms could help to identify patients who could benefit most from the use of SMAC mimetics.8Another challenge brought up here by Green is to understand which features enable the cell to complete its deadly task. Once thought to be an ‘all or nothing’ phenomenon, the output of MOMP seems to be more nuanced: most but not all mitochondria within a cell can undergo MOMP (incomplete MOMP) or just a few mitochondria can be permeated (minority MOMP) when exposed to sublethal cellular stress. In these cases, the outcome of the cell seems likely dependent on the amount of caspase activation but cell survival could come at the high cost of bearing damaged DNA. In the long run, while from an evolutionary perspective such stress-induced genetic diversity could be advantageous, it could also promote tumorigenesis or cancer resistance.10 While the successful induction of apoptosis is crucial to eliminate undesired cells, it is also critical to protect long-lived cell types and progenitor cells during development. Indeed, we now know that several pro-survival BCL2 family members have developmental roles in a variety of tissue types. Indeed, Opferman and Khotari, in this issue, describe how gene-targeting approaches in animal models have proved to be instrumental to our understanding of how BCL2-mediated mechanisms preserve specific cell lineages at different stages during development.11 Our basic understanding of such developmental functions will be important to establish whether the BCL2 family of proteins can be targeted therapeutically 11 and to anticipate potential toxicities .6Interestingly, as traced back by Strasser and Vaux in this issue, the origin of the BCL2 family might have occurred with the appearance of the apoptotic effectors in unicellular organisms. Apoptosis in unicellular organisms could have likely provided an advantage serving as an altruistic defense against pathogens. According to this account, subsequent gene duplication and mutations might have given rise to the other family members in multicellular organisms endowing them with a deadly tool to shape development, regulate cell number homeostasis and respond to stress.6 Although various hurdles might hamper their application, especially for solid tumors, the road ahead has begun to be paved: Montero and Letai9 show how BH3 profiling, by dissecting the cell apoptotic assets, can reliably identify tumor dependency on specific pro-survival proteins guiding precisely the therapeutic use of BH3 mimetics, many of which are in the clinical development pipeline.12 Overall, it seems that the best has yet to come for turning death into better lives.As mentioned above, the wealth of information on the BCL2 family structure and function provided a fertile ground for the development of small molecules BH3 mimetics, which mimic the binding of BH3 peptides to the hydrophobic groove of anti-apoptotic proteins thereby displacing both BH3-only proteins and active BAX/BAK from the constraint of pro-survival members. Somewhat surprisingly, considering that cancer cells are usually resistant to apoptosis, many tumors are more prone to die than their normal counterparts owing to the high expression of pro-apoptotic proteins induced by multiple stresses upon transformation. To survive, cancer cells become addicted to the expression of anti-apoptotic BCL2 proteins but such addiction turns into their Achille’s heel. Indeed, inhibition of BCL2 through BH3 mimetics opened a new era in cancer therapy providing the anticancer arsenal with a new class of agents with enormous potential and considerable advantages as discussed by Adams and Cory.


Alcohol and nicotine addiction are frequently co-morbid and share common genetic factors and molecular mechanisms, such as the involvement of the nicotinic acetylcholine receptors . EpidemiTo capture the molecular and behavioral interactions that occur with voluntary alcohol and nicotine consumption, we developed a method for assessing chronic, voluntary oral alcohol and nicotine co-consumption in mice . In our Many studies investigating oral nicotine consumption in mice utilize sweeteners to make nicotine more palatable and to encourage consumption. Taste preferences differ across strains of mice and correspond to differences in drug consumption; however, our work and others suggest that sweeteners are not necessary for C57BL/6 mice to consume consistent and biologically relevant amounts of nicotine [An understudied factor that we are able to examine with our model is the effect of sex in the interactions between voluntary alcohol and nicotine consumption. In our study, female C57BL/6 mice consume greater amounts of nicotine than males at the higher concentrations tested (40 and 50 μg/mL free base nicotine), and consistently drink more 20% alcohol per gram of body weight in all experiments. In continuous 2-bottle choice experiments, where C57BL/6 mice have access to one bottle of drug and one bottle of water, the preference for 20% alcohol or 30 μg/mL nicotine versus water was similar between the sexes. In our 3-bottle choice experiments, the presence of nicotine decreases alcohol consumption in male, but not female mice, suggesting that female mice may process the combined rewarding effects of alcohol and nicotine differently than males. We also find that in our intermittent co-consumption experiment, the withdrawal-associated behaviors observed 24 hours after the drug bottles are removed differ by sex. The reason for the sex discrepancy in the amount of drug consumption and the associated withdrawal signs is unclear. Estradiol has been shown to increase dopamine release and turnover in the striatum and to down-regulate D2 dopamine receptors , which mIn conclusion, our key findings are that C57BL/6 male and female mice readily co-consume both unsweetened alcohol and nicotine in 2- and 3- bottle choice procedures. There are significant sex differences in drug consumption that are highlighted by our experiments, with females consuming more drug than males. Co-consumption of alcohol and nicotine together decreases alcohol intake in males but not females, and intermittent co-consumption results in physical dependence in mice, with males and females displaying different withdrawal signs after chronic consumption. Our models capture the interactions between alcohol and nicotine that occur during voluntary co-consumption, and have greater clinical relevance to human co-morbid alcohol and nicotine addiction compared with models that use passive, investigator-administered drugs.


Current diagnostic models of psychosis have been questioned since Kraepelin’s original dichotomy of dementia praecox and manic depression. Indeed, increasing evidence has suggested that a dimensional approach might be a valid alternative platform for research. However, while an increasing number of studies have investigated how environmental risk factors for affective and non-affective psychosis map onto symptom dimensions, only a few have examined these dimensions in relation to genetic variants as summarised by Polygenic Risk Score (PRS). Furthermore, no studies have examined the putative effect of PRS for Schizophrenia (SZ), Bipolar Disorder (BP), and Major Depressive Disorder (MDD) on previously identified general and specific symptom dimensions. At the same time, only one study has investigated how symptoms vary according to epidemiological factors such as living in urban neighbourhoods. The objectives of this study were to: 1) test whether a bi-factor model statistically fits the conceptualization of psychosis as composed of general and specific dimensions; 2) examine the extent to which SZ, BP, and MDD PRSs explain the phenotypic variance due to general and specific dimensions; 3) test the hypothesis that the general psychosis dimension would be more severe in highly urban environments.We used clinical and epidemiological data from the EUropean network of national schizophrenia networks studying Gene-Environment Interactions (EUGEI) study, including 2322 First Episode Psychosis (FEP) patients recruited in 17 sites across 6 countries. Genetic variants were collectively analyzed for 800 individuals.The following analysis steps were performed:1) Psychopathology items were analysed using multidimensional item response modelling in MPlus to estimate unidimensional, multidimensional, and bi-factor models of psychosis. Model fit statistics included Log-Likelihood, and Akaike and Bayesian Information Criteria to compare these models.2) SZ, BP, and MDD PRSs for general and specific dimensions were built using PRSice. Summary statistics from large case-control mega-analyses from the Psychiatric Genomics Consortium were used as base data sets and general and specific dimension scores were used as discovery data sets. Individuals’ number of risk alleles in the discovery sample was weighted by the log odds ratio from the base samples, accounting for population stratification, and summed into the three PRSs.3) Multilevel regression analysis was used in STATA 14 to examine the variance in general dimension due to the population density levels across the sites.A bi-factor solution, composed of one general and five specific symptom dimensions, showed the best model fit statistics.Higher SZ PRS score was associated with higher scores on positive dimensions ; higher BP PRS was associated with higher scores on mania dimension ; higher MDD PRS was associated with lower scores on negative dimension . No trends of association were found for SZ, BP, or MDD PRSs and the general psychosis dimension.The transdiagnostic symptom dimension score was elevated in people living in more densely populated sites .Our results suggest that a) symptom dimension structure at FEP is best represented by the bi-factor model; b) in FEP patients, there is a trend of associations between SZ PRS and positive dimension, and between BP PRS and mania dimension; and c) elevated level of transdiagnostic symptomatology was observed in more densely populated sites.


Nr3c1) and brain-derived neurotrophic factor (Bdnf). These epigenetic changes have been linked to changes in brain gene expression, stress reactivity, and behavior, and often times, these effects are shown to be dependent on the gestational window of exposure, sex, and exposure level. Although evidence from human studies is more limited, gestational exposure to environmental risks in humans is associated with epigenetic changes in peripheral tissues, and future studies are required to understand whether we can use peripheral biomarkers to predict neurobehavioral outcomes. An extensive research effort combining well-designed human and animal studies, with comprehensive epigenomic analyses of peripheral and brain tissues over time, will be necessary to improve our understanding of the epigenetic basis of neurodevelopmental disorders.Prenatal adverse environments, such as maternal stress, toxicological exposures, and viral infections, can disrupt normal brain development and contribute to neurodevelopmental disorders, including schizophrenia, depression, and autism. Increasing evidence shows that these short- and long-term effects of prenatal exposures on brain structure and function are mediated by epigenetic mechanisms. Animal studies demonstrate that prenatal exposure to stress, toxins, viral mimetics, and drugs induces lasting epigenetic changes in the brain, including genes encoding glucocorticoid receptor ( Th. Th40]. Gfap promoter has to be demethylated to allow Gfap gene expression, and hence, astrocyte differentiation; all non-expressing cells, including neurons, show high levels of DNA methylation within this region , an analogue of double-stranded RNA, to pregnant dams. Basil et al.  used thi(Gad)1 and Gad2, the two genes that encode GABA-synthesizing enzymes GAD67 and GAD65, respectively. The gene expression changes were accompanied by increased DNA methylation and MeCP2 binding to the Gad1 regulatory region, and with prenatal immune activation-induced impairments in working memory and social interaction. This study provided important evidence that lasting epigenetic changes within the GABA-relevant genes, such as Gad1 and Gad2, may be an important molecular mechanism linking prenatal infection to GABAergic dysfunction and associated behavioral and cognitive abnormalities in the offspring.Labouesse et al.  focused Nrgn1), neurexin 2 (Nrxn2), and neuronal differentiation 6 (Neurod6). On the other hand, differentially methylated regions following the GD9 immune-activation were primarily associated with genes involved in Wnt signalling, whereas the GD17 immune activation was primarily linked to epigenetic changes in genes involved in the differentiation of the GABAergic neuronal system. Importantly, for a subset of genes, the authors confirmed that the DNA methylation differences correlated with changes in gene expression, further suggesting that the altered methylation levels were functionally relevant. It is important to note that the genes differentially methylated in the adult PFC were not differentially methylated in the PFC at postnatal day (PD) 1 of the prenatally immune-challenged animals. This finding provides additional evidence that, rather than inducing permanent effects on specific CpG sites, an early-life insult may affect the developmental programming of the machinery that regulates DNA methylation throughout life, allowing the DNA methylation status of genes to be regulated in an age- and state-dependent manner.Finally, the most recent study by Richetto et al.  considerMaternal exposure to drugs, both therapeutic and recreational, has been linked to neurobehavioral consequences for the offspring, and the possible mediating role of epigenetic mechanisms has been proposed . The effDRD2) has been shown to be reduced in adult drug addicts [DRD2 gene expression in the fetal NAc at approximately 20 weeks of gestation [An interesting study explored the role of epigenetic mechanisms, specifically histone modifications H3K4me3 and H3K9me2, in the neurodevelopmental effects of Δ-9-tetrahydrocannabinol (THC), the main active component of marijuana or cannabis . Materna addicts . In addiestation .Drd2 gene regulation in the NAc that persists into adulthood, and whether epigenetic mechanisms are involved in this effect. Importantly, gestational exposure to THC led to reduced Drd2 gene expression in the offspring’s NAc, both at PD2  and in adulthood (PD62). Decreased Drd2 expression in adult NAc was associated with the following epigenetic changes: a decreased histone mark H3K4me3 typically associated with active promoters and an increased H3K9me2 typically associated with a repressed chromatin state; changes in these two marks were further associated with a decreased binding of the RNA polymerase II at the Drd2 gene promoter. Moreover, the adult offspring prenatally exposed to THC also exhibited a reduced number of D2 receptors in the NAc and an increased sensitivity to opiate reward. In summary, this study provides evidence that prenatal cannabis exposure can alter the developmental epigenetic programming of D2DR expression within the brain’s reward pathway, resulting in a lasting reduction of D2 receptors that may contribute to addiction vulnerability. Interestingly, human genetic studies have also linked genetic polymorphisms in the D2DR gene to addiction [D2DR gene expression that may promote vulnerability to addiction.DiNieri et al.  used a rddiction , implyinAnother study explored maternal cocaine exposure during the second and third gestational week and the associated epigenetic consequences for the offspring . MaternavyA allele as an epigenetic biosensor: Avy mice (the vyA/a genotype) contain a meta-stable, DNA methylation-sensitive vyA allele in the Agouti gene locus, which determines the coat color. DNA methylation levels at CpG sites within vyA are established during early embryogenesis and are probabilistic events, resulting in a wide distribution of the coat color of vyA mice, ranging from pure yellow (hypomethylation of the vyA) to pseudoagouti brown (hypermethylation of the vyA). Kaminen-Ahola et al. [vy mice exhibited postnatal growth restriction and changes in craniofacial morphology, similar to fetal alcohol syndrome in humans, implying that the expression of genes other than vyA was also affected by prenatal ethanol exposure. This study provides evidence that the epigenome is vulnerable to ethanol during early development and suggests that epigenetic mechanisms may contribute to ethanol-induced neurodevelopmental effects.The effects of maternal exposure to alcohol on the offspring’s epigenome and brain structure have also been explored . High lea et al.  demonstrIn summary, several studies show that prenatal exposure to recreational drugs, such as marijuana, cocaine, and alcohol, may induce lasting epigenetic changes in the offspring which, in turn, may contribute to behavioral alterations and increased psychiatric risk in the prenatally-exposed offspring.In humans, it is very challenging to establish the epigenetic link between prenatal environmental exposures and behavioral/psychiatric disorders occurring later in life. First, humans are always exposed to a mixture of environmental agents and it is difficult to clearly dissociate the contribution of a single factor to any neurodevelopmental outcome. Second, the brain as a target tissue is inaccessible in living humans and hence, for now, we have to rely on epigenomic studies of peripheral tissues. It is fortunate that increasing evidence shows that the epigenetic profiling of peripheral tissues such as blood or buccal cells may be informative for behavioral outcomes ,88. In tThe first experimental evidence that in utero environmental conditions can cause long-lasting epigenetic changes in humans was provided by the famed Dutch Famine Study. This is a seminal epidemiological study that established important associations between the prenatal environment and long-term mental health outcomes ,91. The In the case of the Dutch Famine study, at least two risk factors may have interacted to affect neurodevelopment in the offspring: (i) malnutrition due to the famine; and (ii) maternal stress due to the famine, war, and many other stressful events associated with this tragic life situation. Regarding the increased risk of SCZ, the role of nutritional deprivation is likely to be prevailing , and amoImportantly, periconceptional exposure to famine was associated with widespread DNA methylation changes in the blood, when the individuals prenatally exposed to famine were compared to their unexposed, same-sex siblings at approximately 60 years of age ,96. The NR3C1), which is associated with the HPA axis. For instance, an analysis of cord blood samples from children born to mothers suffering from depression during the third semester of pregnancy demonstrated increased DNA methylation of the NR3C1 gene, when compared to controls [N3RC1 methylation in fetal cord blood were further found to predict infant HPA reactivity (cortisol response to stress) at three months of age, suggesting a possible functional consequence of this epigenetic variation. In another study, maternal depressive symptoms during pregnancy predicted not only increased NR3C1 DNA methylation in buccal cells of male infants but also decreased BDNF IV DNA methylation in both male and female infants [BDNF in response to maternal adversity, as previously shown by animal studies [N3RC1 DNA methylation beyond infancy [N3RC1 DNA methylation levels in the whole blood samples of 10–19 year-old offspring. In these studies [N3RC1 gene occurs during developmental epigenetic programming. Together with the animal studies, these findings further support the hypothesis that maternal stress during pregnancy can alter in utero epigenetic programming, contributing to neurodevelopmental and behavioral deficits in the offspring.Several human studies provided a link between prenatal maternal stress or maternal depression and epigenetic changes within the gene encoding glucocorticoid receptor , ChIP-seq , and ATAC-seq , provide a better resolution and coverage, and allow for more accurate profiling of epigenetic modifications at the level of the whole genome. In addition to this, a cell-type specific epigenomic profiling  in the bIn summary, a global research effort combining well-designed human and animal studies, with comprehensive epigenomic analyses of peripheral and brain tissues over time, will be necessary to improve our understanding of the epigenetic basis of neurodevelopmental disorders; an effort which we believe will be very rewarding.


Drosophila melanogaster serves as a powerful organism to study the developmental control of DNA replication in various cell cycle contexts in a variety of differentiated cell and tissue types. Additionally, Drosophila has provided several developmentally regulated replication models to dissect the molecular mechanisms that underlie replication-based copy number changes in the genome, which include differential underreplication and gene amplification. Here, we review key findings and our current understanding of the developmental control of DNA replication in the contexts of the archetypal replication program as well as of underreplication and differential gene amplification. We focus on the use of these latter two replication systems to delineate many of the molecular mechanisms that underlie the developmental control of replication initiation and fork elongation.Proper control of DNA replication is critical to ensure genomic integrity during cell proliferation. In addition, differential regulation of the DNA replication program during development can change gene copy number to influence cell size and gene expression.  Drosophila has provided powerful developmental systems to study both replication initiation and elongation at the cellular and molecular levels 2–7 replicative helicase complex. In budding yeast, Cdt1 and MCM2–7 form a stable complex in cell lysates and are recruited to origins of replication together  and Dbf4-Dependent Kinase (DDK), the MCM2–7 complex is joined by CDC45 and the Go-Ichi-Ni-San (GINS) complex, a four-membered complex composed of Sld5, Psf1, Psf2, and Psf3. Together, the CDC45/MCM2–7/GINS (CMG) complex forms the functional replicative helicase .Despite the conservation of the proteins governing initiation of DNA replication in eukaryotes, there are complexities in the control of metazoan DNA replication. At the most fundamental level, it remains to be determined what dictates a replication origin and where ORC will bind in metazoans . This hain vitro has been described  coupled to microarray analysis revealed that distinct regions of the genome are replicated at different times during S phase. Most origins could be classified as early or late replicating origins with minimal overlap , are associated with the localization of members of the SAGA histone acetyltransferase complex as well as with OSA, a member of the Brahma (SWI/SNF) chromatin remodeling complex (su(Hw) mutants, enrichment of these factors is decreased at these insulator binding sites, concomitant with a higher enrichment of histone H3. Interestingly, ORC3 enrichment at these sites also is decreased in the su(Hw) mutant (Su(Hw) may recruit these chromatin remodeling factors to create a platform for ORC binding. Similar associations are observed with the CTCF, GAF, and BEAF32 chromatin insulator proteins, thus general chromatin remodeling may be associated with ORC binding (Su(Hw) coimmunoprecipitates with ORC3, and artificial tethering of Su(Hw) to an ectopic site is sufficient for the recruitment of chromatin remodeling factors as well as ORC  that continues through larval and adult development . The neuDrosophila. Nearly all larval tissues and many adult tissues in Drosophila have increased ploidy that is achieved via the endocycle. The replicated DNA duplex copies are held in register to produce polytene chromosomes with stereotypic banding patterns in most Drosophila endocycling tissues. The most well studied of these polyploid tissues is the larval salivary gland, which undergoes ∼10 endocycles during larval development to obtain a final ploidy of roughly 1024C  relative to overall ploidy of the cell , as all underreplicated regions become fully replicated in the SuUR mutant , further supporting the fact that SUUR is recruited to active replication forks , γH2Av .SuUR mutants are viable and exhibit normal morphology and fertility  are chromosomal locations characterized by recurrent breaks, gaps, and constrictions on metaphase chromosomes upon replication stress , are located at distinct sites within the follicle cell genome and are referred to by their cytological locations. The level of gene amplification varies, ranging from 60- to 80-fold amplification at DAFC-66D to 4-fold amplification at several amplicons  for activity -mediated degradation during S phase   do not exhibit hampered fork progression . Surprisingly, mutants for the key HR factors BRCA2 and SpnA  tools. A crucial question to be solved is how genomic regions are established that lack ORC binding. Another is whether genomic rearrangements resulting from underreplication serve biological functions.In S phase in dividing or endocycling cells, replication timing is regulated such that some genomic regions replicate early in S phase while others regulate late, a property shared between Both the differential replication systems in which gene copy number is decreased through underreplication and in which copy number is increased through gene amplification have permitted metazoan replication fork progression and destabilization to be visualized and analyzed. This led to the identification of the chromatin protein SUUR as a repressor of replication and inhibitor of fork progression and has uncovered links between this protein and other chromatin proteins as well as replication components. Further insights into the tissue specificity of underreplicated domains and the mechanisms of their designation will be critical to our understanding of how chromatin configuration can affect the elongation phase of DNA replication. These principles will be applicable to mammalian cells and thus to our understanding of common chromosomal fragile sites.Both underreplication and gene amplification lead to genome instability, in the former due to replication fork instability and in the latter due to replication fork collisions. The double-strand breaks that result from these events can lead to genomic rearrangements. These models are powerful in defining repair mechanisms that can restore fork progression to prevent rearrangements, with important implications for genome stability in mammalian cells.


The development of next generation sequencing (NGS) techniques has enabled researchers to study and understand the world of microorganisms from broader and deeper perspectives. The contemporary advances in DNA sequencing technologies have not only enabled finer characterization of bacterial genomes but also provided deeper taxonomic identification of complex microbiomes which in its genomic essence is the combined genetic material of the microorganisms inhabiting an environment, whether the environment be a particular body econiche  or a food manufacturing facility econiche . To date, 16S rDNA sequencing, metagenomics and metatranscriptomics are the three basic sequencing strategies used in the taxonomic identification and characterization of food-related microbiomes. These sequencing strategies have used different NGS platforms for DNA and RNA sequence identification. Traditionally, 16S rDNA sequencing has played a key role in understanding the taxonomic composition of a food-related microbiome. Recently, metagenomic approaches have resulted in improved understanding of a microbiome by providing a species-level/strain-level characterization. Further, metatranscriptomic approaches have contributed to the functional characterization of the complex interactions between different microbial communities within a single microbiome. Many studies have highlighted the use of NGS techniques in investigating the microbiome of fermented foods. However, the utilization of NGS techniques in studying the microbiome of non-fermented foods are limited. This review provides a brief overview of the advances in DNA sequencing chemistries as the technology progressed from first, next and third generations and highlights how NGS provided a deeper understanding of food-related microbiomes with special focus on non-fermented foods. It is well known that foodborne diseases cause considerable morbidity and mortality in humans particularly in immunocompromised individuals and in young children , play important roles in any food matrix ranging from fermentation, contamination and spoilage. Deep taxonomic understanding of the microorganisms and their communities is required either to enhance desired food processes like fermentation or to mitigate detrimental occurrences like contamination and spoilage. Historically, conventional techniques including the classical Gram stain along with individual biochemical characteristics are used for the isolation, identification and characterization of bacteria from clinical, food or environmental origins. Even though considered as the “gold standard,” culture dependent techniques can only detect 0.1% of a complex community, such as that found in the human intestinal microbiota. Therefore, to extend the understanding of an ecological niche, such as food, techniques are needed to identify or characterize microorganisms and predict the functional interactions of different microbiological communities present in the sample. To this end, contemporary advances in multi-omic technologies have enabled microbial community profiling, monitoring population fluctuations in different microbial ecosystems and characterization of different microbial species in food matrices.The rapid development of nucleic acid sequencing technologies over the past four decades has improved the capacity to characterize the microbiomes of complex matrices associated with food or environmental samples. The ubiquitous nature and specificity of nucleic acids make the molecule an ideal target for bacterial or microbiome characterization. Utilizing significant advancements in sequencing chemistries, DNA sequencing gradually evolved from low throughput DNA fragment sequencing to high throughput next generation (NGS) and third generation sequencing techniques , next generation sequencing (NGS high throughput sequencing) and the third generation of sequencing (single molecule long read sequencing) , corresponding to the nucleotide identity at the terminal position. DNA fragments are then separated in a high resolution electrophoretic capillary containing polymer gel, and upon exit from the gel, the fluorescent label is excited by an argon laser and the emission spectrum is captured. Read lengths of approximately 1,000 base pairs were obtained with an accuracy of 99.99%. However, low throughput results together with high operational costs, restricted the application of this method.The first DNA sequencing strategy was the in vitro construction of the sequencing library; (2) in vitro clonal amplification of DNA fragments; (3) array based sequencing enabling DNA fragments to be multiplexed (4) solid phase immobilization of DNA. Based on the different methods used to immobilize DNA on a solid substrate, three technologies were commercialized; (a) high throughput pyrosequencing on beads, (b) sequencing by ligation on beads and (c) sequencing by synthesis on a glass substrate.The advantages of NGS over Sanger sequencing can be summarized as follows (1) (a) High throughput pyrosequencing on beads. The first next-generation high throughput sequencer to be made commercially available was the 454 GS20 pyrosequencing platform (Roche)  Sequencing by ligation on beads. SOLiD technology was based on the Multiplex Polony Sequencing technology  Sequencing by synthesis on a glass solid phase surface. The Illumina Genome Analyser (SOLEXA) was described in 2006 and 2008  technology (Pacific Biosciences)  was released in 2014 through the MinION Access Programme (MAP). Here, electrophoresis is used to move the DNA/RNA molecule through a nanopore. This system involves the use of electrolytic solutions and the application of a constant electric field. As the nucleic acid passes through the nanopore, the change in the current pattern and magnitude is measured. During the library preparation step, double stranded DNA is sheared using a Covaris g-TUBE and fragmented DNA is repaired using a PreCR step. Blunt ended DNA molecules are then created using an end repair step before a poly A tail is added to the 3′-OH end. Two adaptors are then added to the DNA, a Y adapter  and a hair pin adaptor. A motor protein unzips the double stranded DNA at the Y adapter and feeds the DNA as a single strand through the nanopore. Base calling is then performed and a read length of a few hundred thousand base pairs is achieved with an accuracy ranging from 65 to 88%. If information from only one strand is used, base calling is 1-dimensional (1D), otherwise it is 2-dimensional (2D) system  content found in a sample, independent of its origin. Template DNA contained in a sample of interest is subject to sequencing directly, without any prior marker gene amplification step. Metagenomic data not only provides an in-depth taxonomic identification of the microbiome but can also simultaneously compare the relative abundance of all organisms present in the microbiome. Substantial amounts of sequencing data generated using a metagenomic approach is then queried against databases, such as k-mer  using a group of seven phrases as follows: (1) 16S rDNA sequencing food microbiome, (2) 16S rRNA sequencing food microbiome (3) 16S metagenetics, (4) metagenomic food sequencing, (5) metagenomic food, (6) metatranscriptomic food and (7) metatranscriptomic food sequencing.For the purpose of this review, a literature search was performed on the current NCBI PubMed database  along with pathogenic bacteria  in the raw material can potentially contaminate the resident microbiome of bulk milk tank in which the raw material is stored representing a threat to the entire downstream production line. The influence of the ecology of processing environments on food quality was shown in a more elaborate study involving bulk milk tankers , nisin treatment and others are often used to extend the shelf life of many perishable food products. The microbiome undergoes considerable compositional change when food products are stored under defined conditions. It is possible that these fluctuations in the microbiome may finally affect the quality of the food product. NGS techniques have been increasingly applied to study how these variations contribute to improved shelf life. The most common response noted in refrigerated food products was a reduction in bacterial diversity associated with the microbiome. A 16S rDNA sequencing approach noted this reduction in bacterial diversity in refrigerated spinach  and/or NCTC  collections  strategies are not routinely used for microbiome studies. Recently, a third generation sequencing MinION platform was used to sequence full length 16S rDNA amplicons generated from a synthetic community of ten microbial species with varying relative abundance (Li et al., YC, SF, and SS organized the draft and wrote the manuscript. YC, SF, SP, KJ, and SS participated in the critical revision of the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Histone deacetylase (HDAC) inhibitors  are FDA Previous understanding of the action of HDAC inhibitors has been based on both the demonstration that they alter gene expression through their impact on histone acetylation  and the Recent studies by Ding et al.  provide . [BCL2 gene-rearranged lymphomas  have amino acid altering BCL2 mutations that, at least in some instances, are able to enhance the anti-apoptotic effects of the corresponding protein. The study by Ding et al. [BCL2 mutations are more resistant to DNA damaging treatments, including ionizing radiation, etoposide and cytarabine, than lymphoid lines without BCL2 mutations. In contrast, BCL2-mutant and BCL2-wild type lines were equally sensitive to HDAC inhibitors, possibly reflecting the ability of the upregulated BIM to induce apoptosis whether BCL2 is mutated or not.These observations have potential implications not only for the mechanism of action of HDAC inhibitors, but also for overcoming drug resistance in lymphoid malignancies. Previous observations of Correia et al.  demonstrg et al.  reported. have several implications. First, they suggest that high levels of RASGRP1 might be a potential biomarker for susceptibility of individual lymphoid neoplasms to HDAC inhibitors. Second, these findings suggest that further preclinical and possible clinical study of HDAC inhibitors might be beneficial in BCL2-mutant lymphoid malignancies that fail to respond to conventional therapy and/or BH3 mimetics. In short, further study of RASGRP1 as a determinant of sensitivity to inhibitors of both HSP90 and histone deacetylases might be a fruitful area of future investigation.Because the study of Ding et al. focused on sensitivity of malignant B cell lymphoma and did not examine cell lines derived from either peripheral T cell lymphoma or multiple myeloma, the two diseases for which HDAC inhibitors are currently approved, there is still more work to be done to determine whether the mechanism shown in Fig.


HSP90AB1, HSP27, GPX1, XRCC1, BAG-1, HHR23A, FAP48, and C-FOS. No gender-specific differences or relationship to number of missions flown was observed. This study provides a first assessment of transcriptomic changes occurring in the whole blood of astronauts in response to spaceflight.Astronauts are exposed to a unique combination of stressors during spaceflight, which leads to alterations in their physiology and potentially increases their susceptibility to disease, including infectious diseases. To evaluate the potential impact of the spaceflight environment on the regulation of molecular pathways mediating cellular stress responses, we performed a first-of-its-kind pilot study to assess spaceflight-related gene-expression changes in the whole blood of astronauts. Using an array comprised of 234 well-characterized stress-response genes, we profiled transcriptomic changes in six astronauts (four men and two women) from blood preserved before and immediately following the spaceflight. Differentially regulated transcripts included those important for DNA repair, oxidative stress, and protein folding/degradation, including  Targeted spaceflight-associated changes in cytokine and antibody expression have been previously reported for astronauts,1–3 and more recently Terada et al reported the effects of spaceflight on hair follicle gene expression in astronauts.6 To evaluate the potential impact of the spaceflight environment on molecular pathways mediating cellular stress responses, we performed a first-of-its-kind pilot study to assess spaceflight-related changes in the expression of stress-response genes in the whole blood of astronauts in response to spaceflight.During spaceflight, astronauts experience a variety of physical and psychological stressors, including acceleration at launch, isolation, confinement, high levels of ionizing radiation, sleep deprivation, and microgravity, which have been linked to a variety of physiological changes, including dampened immune function, bone and muscle loss, and increased viral reactivation.5Transcriptional profiling of 234 well-characterized stress-response genes was performed using total RNA isolated from whole blood obtained from six consenting astronauts 10 days before launch aboard the space shuttle and 2–3 h after return to Earth . Four men between the ages of 38 and 47 (mean=43 years) and two women between the ages of 38 and 44 (mean=41 years) participated in the study. Each of these astronauts flew aboard one of four space shuttle missions ranging between 10 and 13 days duration that took place during a 2-year period. Previously published studies using blood and saliva samples from these same crew members before, during and just after spaceflight indicated that all six astronauts displayed increases in Epstein Barr virus reactivation both during and immediately post flight, which is consistent with these individuals being in a stressed state.7 High variability was also observed in gene-expression studies using astronaut hair follicles.6 We found six transcripts that displayed significant (P<0.05) changes in expression in the crew in response to spaceflight when compared with pre-flight levels (n of 1 in each condition (spaceflight and ground). However, one advantage in the Twins Study over the present one is the longitudinal sample collection that could be performed during the year that Kelly spent in space. It will be intriguing to compare the results from gene-expression analyses between the two studies.Microarray analysis revealed some variation in the gene-expression patterns displayed across individual crew members. This is not surprising given the smaller study size due to the exceptionally rare opportunity for this type of sample collection, as well as other studies that have shown that there can be a wide range of individual and temporal variability in gene-expression patterns in human blood.t levels . Two addXRCC1 and HHR23A), oxidative stress (GPX1), and chaperones which have key roles in protein folding and/or proteasomal degradation (HSP27 and HSP90AB1). The finding that genes encoding DNA-repair proteins are downregulated supports previous studies which showed post-flight increases in chromosomal aberrations in lymphocytes of astronauts8 and indications of increased DNA damage in astronauts after long-duration spaceflight.9 Kumari et al previously reported that exposure of human lymphocytes to simulated microgravity over the course of 7 days led to increased DNA damage.10 This damage was accompanied by progressive decreases in the expression of a number of representative DNA-repair genes, leading the authors to postulate that impaired DNA-repair capacity could lead to increased damage and mutations. In a separate study, it was reported that 1 week of hindlimb unloading in male BALB/c mice led to the downregulation of multiple DNA-repair genes including ERCC 1, ERCC 3, ERCC5, ERCC6, and XPC in the testes of BALB/c mice.11Genes altered in expression encode proteins of known importance for DNA repair , an enzyme that protects cells from oxidative damage, modulates the immune response (including delayed type hypersensitivity/DTH), and has been associated with increased viral titers in herpes viral infections.12 Given that spaceflight depresses the DTH response,2 alters cellular oxidative functions,13–17 and increases herpes viral reactivation in astronauts on orbit,5 additional detailed studies related to glutathione and oxidative stress may fundamentally advance our understanding of mechanisms underlying risks to crew health during a mission. Several previous studies have indicated the potential for alterations in oxidative stress and antioxidant defense status in response to spaceflight and spaceflight-analog models in humans and animals.13–16 Although the reported magnitude and direction of the expression/activity levels of specific redox enzymes (including GPX1) were variable across these studies  the overall findings support the potential for increased oxidative stress and/or decreased antioxidant defense capacity in response to spaceflight. It is also interesting to note that GPX1 is a selenoprotein and decreased serum selenium levels have been reported in astronauts post landing as compared with pre-launch.9 Therefore, additional insight into GPX1 and other oxidative defense mechanisms may serve as a guiding principle for establishing nutritional requirements to ensure health safety and performance of the crew during human exploration of space.18 When we compared these transcriptomic findings from blood samples to a previously published study using hair samples,6 we did not find similarities in the genes that were differentially regulated pre- and post flight, which could be due to a variety of experimental differences, including sample source (whole blood versus hair follicles) and mission duration .Of particular interest was the downregulation of 1–3 future studies designed to establish direct links between specific physiological/cellular adaptations underlying these transcriptomic changes in response to the microgravity environment will be important. It is important to note that no direct correlation can be made between gene-expression changes observed in this study and the disease status or potential for the development of disease in these individuals. This study provides an initial foundation into the molecular genetic response profiles of astronauts during spaceflight from which additional research into alterations in crew health and performance can be investigated. This kind of research could lead to human health countermeasures to mitigate risk during the transition from short-to-long-duration flight and could potentially translate to health benefits for the general public.To our knowledge, this study represents the first report of transcriptomic changes occurring in the blood of astronauts in response to spaceflight. Several transcripts encoding stress-response genes were suppressed in the crew after exposure to the microgravity environment, including those important for DNA repair, oxidative stress, detoxification, and protein folding/degradation. These processes are vital for maintaining human health by mediating cellular pathways that serve to protect against both environmental and physiological stressors and have been implicated in a broad spectrum of diseases. Since changes in gene expression in peripheral blood may be attributed to a number of factors, including changes in the distribution of blood cellular subsets (which has been detected post flight in the crew),


In addition, when analyzing the prognostic factors, death due to other cancers before disease progression is considered a competing risk6.Therapeutic improvements for cancers in general have resulted in a growing population of cancer survivors at risk of developing secondary primary malignancies (SPMs) due to a variety of biological factors such as cancer predisposition syndromes, environmental factors, immune impairments, and late effects of genotoxic therapies. Patients with a history of other primary malignancies have the possibility of dose-reduced treatment because of the chronic health problems induced by previous cancer treatment or previous cancer damage7. While the recent treatment progress has markedly improved overall long-term outcomes for DLBCL by the advent of the immunochemotherapy containing rituximab1, the clinical and prognostic impacts of a history of past cancer or co-existence of another synchronous cancer in “secondary” or “concomitant” DLBCL has been rarely investigated in the recent era. To answer this question, we performed a retrospective analysis of 809 DLBCL patients who were diagnosed histologically and treated between January 2006 and February 2016 at institutes in the Kyoto Clinical Hematology Study Group (KOTOSG). The criteria for multiple primary malignancy (MPM) used were proposed by Warren and Gates: i) tumors have definite features of malignancy, ii) tumors are separate and distinct from the index tumor, and iii) the possibility of a tumor being a metastasis of the index tumor is ruled out8. Based on the Surveillance Epidemiology and End Results Program definition for the chronicity of MPM9, the term “synchronous” refers to a condition where more than two malignancies, including DLBCL, are detected within two months, while “past” indicates a metachronous condition where the tumors are detected more than two months apart. This study was approved by the Institutional Review Boards.This could also be the case with diffuse large B-cell lymphoma (DLBCL), the most prevalent subtype of non-Hodgkin lymphomaP < 0.001). There was no significant difference in disease stage or international prognostic index (IPI)-defined disease risk between patients with and without MPM. Although small adjustments of the therapeutic regimen were allowed at the doctor’s discretion, more than 85% of patients received a rituximab plus CHOP  (R-CHOP)-like regimen regardless of the presence of MPM. In the 123 DLBCL patients with MPM, 103, 16, and 4 had one, two, and three other primary malignancies, respectively, before treatment for DLBCL. Stomach cancer was the most common, followed by colorectal cancer, lung cancer, prostate cancer, and breast cancer. Hematologic malignancies were less common than solid cancers as both past cancer or synchronous cancer  that occurred more than two months before the diagnosis of DLBCL); those with synchronous cancer ; and patients without another cancer. Among them, 123 (15.2%) were defined as having MPM, including 94 with past cancer and 29 with synchronous cancer (Supplementary Table P = 0.015)   and PFS , along with age, clinical stage, and IPI disease risk  for all 809 patients, and was significantly shorter in DLBCL patients with MPM compared to those without MPM  Fig. . There w01) Fig. . Given t10. Due to the retrospective study design, we could not analyze the precise pathologic features of previous cancers, treatment efficacy for past cancer, or the detailed adverse events with chemotherapy for past cancer before DLBCL treatment, however, the types and frequencies of other cancers in our cohort of DLBCL patients with MPM, mostly reflected those in cancer surveillance research in Japan11. The type of past or synchronous malignancy did not differ significantly among patients according to the IPI risk  of 809 patients were diagnosed with DLBCL with synchronous cancer, indicating that careful systemic screening, including that of the upper aerodigestive tract is needed at the time of initial diagnosis of DLBCL.The frequency of SPM should depend on the prognosis of the first malignancy. For instance, SPM should be less frequent in patients with cancers with a poor survival rate, such as pancreatic carcinoma, but more frequent in cancers with better prognosis, such as low grade thyroid cancer15, we speculate that a paraneoplastic effect occasionally promotes disease progression and causes early death due to DLBCL, especially in IPI-high risk patients. In addition, it is also conceivable that a proportion of patients with MPM in our cohort harbored unverified mechanisms, such as inherited gene abnormalities associated with tumor development or immune surveillance, or environmental factors that underlie development of MPM and cause treatment-refractory disease.While the choices of treatment regimen did not differ significantly between DLBCL patients with and without MPM, our multivariate analysis revealed that the negative prognostic impact of MPM was significant only in IPI-high risk DLBCL. Given that synchronous MPM including DLBCL is sometimes associated with an aggressive clinical course and a dismal prognosisIn conclusion, approximately 15% of DLBCL patients had the history of MPM in our cohort. While our study did not provide the evidence for the need for chemotherapeutic modification because of the presence of MPM in DLBCL, careful attention is needed especially for IPI-high risk patients with MPM to improve their survival, as the cumulated death rate was higher in those patients, perhaps due to various patient-oriented and MPM-associated factors, including treatment failure for DLBCL.Supplemental table 1Supplemental table 2Supplemental table 3Supplemental table 4Supplemental table 5Supplemental figure 1Supplemental figure 2Related Manuscript File


Finally, the total nitric oxide assay for NO production in the cultured medium was evaluated. An enhanced concentration of intracellular Ca2+ and ROS were observed, while the concentration of extracellular NO also increased after nsPEFs treatment. Such experimental results demonstrated that nsPEFs with appropriate pulse parameters could effectively enhance cell proliferation on PIEC cells, and the cell proliferation associated strongly with the changes of intracellular Ca2+ concertation, ROS and NO production induced by nsPEFs treatment. This in vitro preliminary study indicates that as a novel physical doping, the nsPEFs have potential in stimulating endothelial cells to accelerate stent endothelialization.Currently, nanosecond pulsed electric fields (nsPEFs) with short pulse duration and non-thermal effects have various potential applications in medicine and biology, especially in tumor ablation. Additionally, there are a few investigations on its proliferative effects in the normal cell. Clinically, proliferation of endothelial cells can perhaps accelerate the stent endothelialization and reduce the risk of acute thrombosis. To explore the feasibility using nsPEFs to induce proliferation of endothelial cells, in this study, porcine iliac endothelial (PIEC) cell line was cultured and tested by CCK-8 assay after nsPEFs treatment. The results reflected that nsPEFs with low field strength  had a significant proliferative effect with an increase in the PIEC cell growth of 16% after a 48 hour’ post-treatment. To further understand the mechanism of cell proliferation, intracellular Ca Coronary artery stenosis is one of the major cardiovascular diseases, which cause myocardial infarction and peripheral artery disease worldwide . ImplantDifferent methods have been attempted to accelerate the endothelialization on the stent surface. Wu  et al. dHaloxylon ammodendron seeds i response was due to the release of Ca2+ from the intracellular store at a low electric field strength and that the increase in [Ca2+]i was caused by both an internal release and an influx across the plasma membrane at a high electric field strength. Our results aligned with previous theoretical and experimental studies. At a low field strength of 5 kV / cm, the pulse only acts on the intracellular endoplasmic reticulum, so that the endoplasmic reticulum calcium library was released. At a high field strength of 20 kV / cm, the pulse not only leads to the release of calcium in the endoplasmic reticulum, but also causes cell membrane damage, leading to calcium influx in the culture medium. The calcium signal created by the entry of external calcium and its release from internal stores related to the cell proliferation by activating the immediate early genes responsible for inducing resting cells (G0) to re-enter the cell cycle [We examined the intracellular free calcium levels after exposure to nsPEFs and found that intracellular free calcium concentrations increased after nsPEFs treatment compared to the control group. Previous studies , 27 have2-), hydrogen peroxide (H2O2), and hydroxyl radical (HO•), consist of radical and non-radical oxygen species formed by the partial reduction of oxygen. As “two-faced” molecules, ROS are involved in various complex signaling pathways and are critical to the fate of both healthy and diseased cells. Additionally, a low level of ROS can directly interact with critical signing molecular as a means to initiate signaling in a broad variety of cellular processes, such as proliferation and survival. Meanwhile, higher levels of ROS cause disruptions of cellular processes through the non-specific attack of proteins, lipids, and DNA, which can cause cell dysfunction [Reactive oxygen species such as superoxide anion Click here for additional data file.S2 Fig(TIF)Click here for additional data file.