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Nous rapportons une observation originale de maladie de Destombes-Rosai-Dorfman multifocale et réfractaire, associée à un syndrome myélodysplasique. Le traitement de la myélodysplasie a permis une très bonne réponse prolongée des 2 pathologies. Un patient de 35 ans était exploré pour une exophtalmie bilatérale, rapportée histologiquement à une maladie de maladie de Destombes-Rosai-Dorfman. Le bilan d'extension retrouvait une atteinte sinusienne, rénale et ganglionnaire. Il était traité successivement, sans succès, par corticoïdes, colchicine, méthotrexate, infliximab, cladribine et tociluzimab. L'apparition secondaire d'une myélodysplasie (AREB score IPSS intermédiaire-2) faisait proposer un traitement d'induction par aracytine et idarubicine, avec entretien par azacytidine pendant 2 ans. Avec un recul de 5 ans, le patient est en rémission du syndrome myélodysplasique et de la maladie de Destombes-Rosai-Dorfman. Notre observation discute l'intérêt du traitement du syndrome myélodysplasique, dans la prise en charge des manifestations extra-hématologiques associées. We report an original observation of multifocal refractory Destombes-Rosai-Dorfman disease associated with a myelodysplastic syndrome. The treatment of myelodysplasia allowed a good and prolonged response of both pathologies. A 35-year-old patient was investigated for bilateral exophthalmia, histologically related to Destombes-Rosai-Dorfman disease. The extension workup showed sinus, kidney and lymph node involvement. It was treated unsuccessfully with corticosteroids, colchicine, methotrexate, infliximab, cladribine and tociluzimab. The secondary appearance of myelodysplasia (AREB IPSS score intermediate-2) led to induction treatment with aracytin and idarubicin, and maintenance with azacytidine for 2 years. With 5 years of follow-up, the patient is in remission both of the myelodysplastic syndrome and Destombes-Rosai-Dorfman disease. Our observation discusses the interest of the treatment of myelodysplastic syndrome for the management of associated extra-hematological manifestations. [ABSTRACT FROM AUTHOR]

Spinal cord and neuron injury result in loss of muscle function, sensation, or autonomic function in the body. Tet1 produces 5-hydroxymethylcytosin. The conversion was proposed as the initial step of deoxyribonucleic acid demethylation in mammals. However, effects of Tet1 expression and hydroxymethylation status on neuron injury remain unclear. Therefore the current study was designed to explore effects of Tet1 expression and hydroxymethylation status on cell survival and gene expression after neuron injury. Mouse models of spinal cord injury and cell model of neuron injury were created. Animals were sacrificed, and injured spinal cord tissue was harvested. Neuron-like cells were cultured after scratch injury. Hydroxymethylated deoxyribonucleic acid concentration was detected, and Tet1 expression was examined by qPCR. Neuron-like cells were divided into 3 groups: control, injury, and azacytidine + injury (before injury, cells were pretreated with azacytidine) groups. Culture supernatant was collected, and lactate dehydrogenase concentration was detected. Meanwhile, injured neuron-like cells were divided into 3 groups: negative control, Tet1 overexpression, and Tet1 interference. Relative expression of Tet1, BDNF, NTF3, A20, FLIP, HSP70, HSP90, HSP27, and Bcl2 in neuron-like cells was detected by qPCR. In addition, neuron-like cells were divided into 7 groups. Tet1 expression and deoxyribonucleic acid hydroxymethylation increased initially and decreased thereafter after neuron injury in both animal and cell models. Percentages of dead cells increased significantly in neuron-like cells after injury. The percentages of dead cells markedly decreased in injured neuron-like cells that were pretreated with azacytidine. The percentages of dead cells increased markedly in the Tet1 interference group and decreased significantly in the Tet1 overexpression group. Expression of Tet1, BDNF, A20, FLIP, HSP70, HSP90, and Bcl2 decreased significantly after injury. Azacytidine pretreatment in injured neuron-like cells markedly increased expression of Tet1, BDNF, NTF3, A20, FLIP, HSP70, HSP90, HSP27, and Bcl2. Moreover, Tet1 interference significantly decreased the expression of Tet1, BDNF, A20, FLIP, HSP70, and HSP90 in neuron-like cells, whereas Tet1 overexpression markedly increased the expression of Tet1, BDNF, NTF3, A20, FLIP, HSP70, HSP90, HSP27, and Bcl2. BDNF interference significantly increased percentages of dead cells after injury. BDNF interference also markedly decreased the protection of azacytidine and Tet1 overexpression against cell death. Tet1 overexpression and demethylation caused by azacytidine protect neurons against cell death after injury by increasing expression of genes involved in cell survival. [ABSTRACT FROM AUTHOR]

Myeloid malignancies such as myelodysplastic syndrome (MDS) & acute myeloid leukemia (AML) are clonal diseases that emerge and progress due to the expansion of disease-initiating aberrant hematopoietic stem cells, that are not eliminated by conventional cytotoxic therapies. Hypomethylating agents(HMA), azacytidine and decitabine are the first line agents for treatment of MDS and a combination with BCL-2 inhibitor, venetoclax, is approved for AML induction in patients above 75 years and is also actively being investigated for use in high risk MDS. Resistance to these drugs has become a significant clinical challenge in treatment of myeloid malignancies. In this review, we discuss molecular mechanisms underlying the development of resistance to HMA and venetoclax. Insights into these mechanisms can help identify potential biomarkers for resistance prediction, aid in the development of combination therapies and strategies to prevent resistance and advance the field of cancer therapeutics. [ABSTRACT FROM AUTHOR]

We established an azacytidine (AzaC)-resistant human acute myeloid leukemia (AML) cell line (SKM-1/AzaC) by culturing SKM-1 cells in the presence of increasing amounts of AzaC for six months. Because AzaC is not a substrate of P-glycoprotein (a product of the ABCB1 gene; ABCB1), ABCB1 was not responsible for AzaC resistance; nevertheless, it was notably upregulated in SKM-1/AzaC cells. In addition, the transcription of the Nfkb1 gene, which encodes a member of the canonical NF-kappaB regulatory pathway, was downregulated, and the transcription of the Nfkb2 gene, which encodes a member of the non-canonical NF-kappaB regulatory pathway, was upregulated in SKM-1/AzaC cells. Here, we investigate whether miRNA-27a and miRNA-138 (both of which are known to be regulators of ABCB1 expression) are involved in the regulation of ABCB1 expression in SKM-1/AzaC cells. We observed decreased levels of miRNA-27a but of not miRNA-138 in SKM-1/AzaC cells compared with SKM-1 cells. The transfection of SKM-1/AzaC cells with a miRNA-27a mimic induced the downregulation of the ABCB1 mRNA. This was associated with an increase in Nfkb1 and a decrease in Nfkb2 transcript levels in SKM-1/AzaC cells. Taken together, these data indicate that the downregulation of miRNA-27a is involved in the upregulation of ABCB1 expression in SKM-1/AzaC cells, and this effect is associated with a switch between the canonical and non-canonical NF-kappaB pathways. [ABSTRACT FROM AUTHOR]

To highlight the role of azacytidine (AZA) in patients with myeloproliferative neoplasms developing blast phase (MPN-BP), we evaluated retrospectively 19 patients [M/F 15/4, median age 71.3 years, interquartile range (IQR) 64.5–77.7] reported in the database of our cooperative group. Median time from diagnosis to BP evolution was 52.7 months (IQR 11.2–181.8). All patients were treated with AZA at the standard dosage of 75 mg/m 2 . Two patients died early after 5-AZA initiation from pulmonary fungal infection and respiratory failure respectively, 4 patients had a disease progression, 4 patients a stable disease, 3 patients had an hematological improvement, 1 patient a partial response and 5 pts (26.3%) a complete response (CR) after 4, 4, 4, 5, and 12 months. The median cumulative survival from BP evolution was 9.9 months (95%CI 6.6–13.1): the comparison with an historical cohort of 72 patients with MPN-BP treated with approaches other than AZA (median cumulative survival 3.1 months, 95%CI 1.1–5.0) showed a significant advantage for patients treated with AZA ( p = 0.02). Our data confirm the relative efficacy and safety of AZA in this group of patients with otherwise dismal prognosis, underlining the possible achievement of long-lasting responses in a sizeable portion of them. [ABSTRACT FROM AUTHOR]

Pathways for tolerating and repairing DNA–protein crosslinks (DPCs) are poorly defined. We used transposon mutagenesis and candidate gene approaches to identify DPC-hypersensitive Escherichia coli mutants. DPCs were induced by azacytidine (aza-C) treatment in cells overexpressing cytosine methyltransferase; hypersensitivity was verified to depend on methyltransferase expression. We isolated hypersensitive mutants that were uncovered in previous studies ( recA , recBC , recG , and uvrD ), hypersensitive mutants that apparently activate phage Mu Gam expression, and novel hypersensitive mutants in genes involved in DNA metabolism, cell division, and tRNA modification ( dinG , ftsK , xerD , dnaJ , hflC , miaA , mnmE , mnmG , and ssrA ). Inactivation of SbcCD, which can cleave DNA at protein–DNA complexes, did not cause hypersensitivity. We previously showed that tmRNA pathway defects cause aza-C hypersensitivity, implying that DPCs block coupled transcription/translation complexes. Here, we show that mutants in tRNA modification functions miaA , mnmE and mnmG cause defects in aza-C-induced tmRNA tagging, explaining their hypersensitivity. In order for tmRNA to access a stalled ribosome, the mRNA must be cleaved or released from RNA polymerase. Mutational inactivation of functions involved in mRNA processing and RNA polymerase elongation/release (RNase II, RNaseD, RNase PH, RNase LS, Rep, HepA, GreA, GreB) did not cause aza-C hypersensitivity; the mechanism of tmRNA access remains unclear. [ABSTRACT FROM AUTHOR]

• The first study about azacytidine-dsDNA interaction using different techniques. • Binding constant and thermodynamic profile of the binding event is evaluated. • The binding constant values are calculated as 2.420 × 104 and 3.266 × 104 M−1 at 25 °C. • Molecular docking suggested that azacytidine-dsDNA interaction is groove binding. This study examines the interaction between pyrimidine nucleoside analogue azacytidine, an anti-leukemic drug, and DNA by employing electrochemical, UV–vis spectroscopy, fluorescence spectroscopy and molecular docking techniques. In the electrochemical technique, azacytidine and dsDNA interaction was investigated in two different ways: (1) in solution and (2) with a biosensor using differential pulse voltammetry (DPV) at a glassy carbon electrode. The interaction between azacytidine and dsDNA at increasing interaction times was investigated in line with the changes in adenine and guanine oxidation signals. In addition, interaction studies of polyguanine-azacytidine and polyadenine-azacytidine were performed with DPV. The binding constant values were calculated as 2.420 × 104 M−1 and 3.266 × 104 M−1 at 25 °C using UV and fluorescence spectroscopy, respectively. In conclusion, based on electrochemical and spectroscopic methods as well as molecular docking studies, it was predicted that azacytidine can bind to dsDNA via groove binding. [ABSTRACT FROM AUTHOR]

Abstract: Data derived from clinical trials consistently show a prolongation of overall survival of late-stage MDS patients with the introduction of azacytidine. Nevertheless, the applicability of the above results to real-world clinical settings may be questionable due to the strict design, the controlled medical environment, and the limited patient sample of explanatory studies. We retrospectively compared the outcome of two well-balanced groups of late-stage MDS patients. The first consisted of 46 patients treated with azacytidine (AZA cohort) and the second of 41 patients treated with other agents (non-AZA cohort). Patients in the AZA cohort displayed superior survival compared to the non-AZA ones. However, subgroup analysis revealed that azacytidine conferred a significant survival advantage only in patients with AML–MDS and those who attained a CR at any time after treatment initiation, while all other patients displayed comparable outcome with the non-AZA cohort. Larger series are needed to determine which patients benefit most from azacytidine therapy. [Copyright &y& Elsevier]

Abstract: Stem cells in cell based therapy for cardiac injury is being potentially considered. However, genetic regulatory networks involved in cardiac differentiation are not clearly understood. Among stem cell differentiation models, mouse P19 embryonic carcinoma (EC) cells, are employed for studying (epi)genetic regulation of cardiomyocyte differentiation. Here, we comprehensively assessed cardiogenic differentiation potential of 5-azacytidine (Aza) on P19 EC-cells, associated gene expression profiles and the changes in DNA methylation, histone acetylation and activated-ERK signaling status during differentiation. Initial exposure of Aza to cultured EC-cells leads to an efficient (55%) differentiation to cardiomyocyte-rich embryoid bodies with a threefold (16.8%) increase in the cTnI+ cardiomyocytes. Expression levels of cardiac-specific gene markers i.e., Isl-1, BMP-2, GATA-4, and α-MHC were up-regulated following Aza induction, accompanied by differential changes in their methylation status particularly that of BMP-2 and α-MHC. Additionally, increases in the levels of acetylated-H3 and pERK were observed during Aza-induced cardiac differentiation. These studies demonstrate that Aza is a potent cardiac inducer when treated during the initial phase of differentiation of mouse P19 EC-cells and its effect is brought about epigenetically and co-ordinatedly by hypo-methylation and histone acetylation-mediated hyper-expression of cardiogenesis-associated genes and involving activation of ERK signaling. [Copyright &y& Elsevier]

Abstract: A controlled azacytidine release system based on drug encapsulation with a polymer material has been prepared and characterized. The drug systems were prepared by precipitation from solutions, using supercritical CO2 antisolvent technique operating in a semi-continuous mode. Azacytidine was dissolved in dimethylsulfoxide and poly-lactic acid in methylene chloride. The two solutions were mixed before being sprayed into the supercritical reactor. Experimental conditions were 40°C for temperature, 11MPa for pressure, and a CO2 flow rate of 30mlmin−1. The precipitated drug–polymer particles were further characterized to determine the percentage of encapsulated drug and establish the delivery kinetics under various release conditions. A sustained delivery of the drug over a period of various hours was obtained. Besides, an improved stability of the coated drug with respect to the pure azacytidine was found, thus proving the suitability of this approach for dealing with unstable compounds. [Copyright &y& Elsevier]

Abstract: Increased expression of genes, silenced by methylation of their promoters, could have relevance for increasing effects of not only interferons (IFNs) but also APO2L/TRAIL, cytotoxics and immunotherapeutics for melanoma and other malignancies. A resistant melanoma cell line, A375, lacked APO2L/TRAIL or apoptosis induction by either IFN-α2 or IFN-β. However, apoptosis was induced by IFNs in A375 cells by 5-aza,2′-deoxycytidine (5-Aza-dC), evaluated based upon the postulate that promoter methylation might be silencing pro-apopoptotic IFN-stimulated genes (ISGs). RASSF1A, commonly methylated at high frequency in many tumors including melanoma, which we discovered to be also an IFN-regulated gene, was increased by 5-Aza-dC. RASSF1A was important in enhancing apoptotic effects of not only IFNs and APO2L/TRAIL but also cisplatin. Unraveling epigenetic regulatory mechanisms, as yet only partially identified, will result in new biological insights and improved strategies for therapeutic use of IFNs or ISGs such as APO2L/TRAIL. [Copyright &y& Elsevier]

Summary: Myelodysplastic syndromes (MDS) are a set of oligoclonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis that manifest clinically as anemia, neutropenia, and/or thrombocytopenia of variable severity. The result often is transfusion-dependent anemia, an increased risk of infection or hemorrhage, and a potential to progress to acute myelogenous leukemia (AML). Although progression to acute leukemia can lead to death in patients with MDS, many deaths are consequences of cytopenias and marrow failure in the absence of transformation. Approximately 2/3 of patients succumb to the disease within 3–4 years after presentation, and individuals with high-risk MDS generally survive about 1 year. Given that the disease is more prevalent in the elderly who often have comorbid conditions, the current treatment of MDS consists mainly of supportive care. Curative treatments are restricted to younger, healthy individuals with histocompatible (HLA)-matched donors for allogenic transplant or those able to undergo intensive chemotherapeutic regimens. However, understanding of the pathophysiology of MDS and identification of potential cellular and molecular targets in recent years has led to novel therapeutic approaches. Encouraging results using these heterogeneous therapeutic approaches alone or in combination in Phase I and II trials, have, in turn, called into question previous classification systems and have confirmed the need for an all-encompassing molecular, diagnostic and prognostic staging system. [Copyright &y& Elsevier]

Abstract: It has been observed that decrease of DNA methyltransferase 1 (DNMT1) activity is associated with low content of the CD3-zeta (ζ) chain in T cell receptor (TCR)/CD3 complex of T cells in systemic lupus erythematosus (SLE) patients. The CD3-ζ chain plays a pivotal role in intracellular signal transmission between TCR/CD3 complex and nuclei. The compounds 5′-azacytidine (AZC) and procainamide (PCA) belong to inhibitors of DNMT1, whose low activity correlates with increase in transcription of various genes. Using the reverse-transcription and real-time quantitative PCR (RQ-PCR) analysis, we indicated that AZC and PCA did not profoundly affect on CD3-ζ chain transcription in Jurkat T leukemia cells clone E6-1. However, the flowcytometric analysis revealed that AZC and PCA decreased intracellular contents of CD3-ζ chain in these cells in dose dependent manner. Our results suggest that decrease of DNMT1 activity may alter intracellular signal transmission without effect on transcription level of CD3-ζ chain. [Copyright &y& Elsevier]

Patients with myelodysplastic syndromes (MDS) who were anemic and/or thrombocytopenic were treated with 5-azacytidine (5-AZA) at a dose of 75 mg/m2 per day SQ×7 days. This cycle was repeated every 28 days. Forty-eight patients who received at least one cycle of 5-AZA were evaluable for response. Hematological toxicity was mild and consisted of thrombocytopenia and leukopenia. Extramedullary toxicity was uncommon and consisted of pneumonia, arthralgia, diarrhea, and injection site irritation. Eighteen of the 46 transfusion dependent patients became transfusion independent (39%). Median duration of response was 7 months with three patients continuing beyond 2 years. French Anglo British (FAB) classification and the International Scoring System (ISS) did not predict response to 5-AZA. However, a decrease in the white blood cells (WBC) during the initial cycle of 5-AZA correlated with a higher response rate. [Copyright &y& Elsevier]

Older patients with acute myeloid leukemia (AML) face a dismal prognosis. Although venetoclax-based therapy has led to improved outcomes among unfit older patients with AML, it is not curative and its efficacy and long-term outcomes among fit older patients is unclear. This review provides insights into factors that influence treatment choices among older patients with AML and what we would need to know for venetoclax-based therapy to replace standard intensive 7 + 3 induction therapy. [ABSTRACT FROM AUTHOR]

The BH-3 mimetic venetoclax overcomes apoptosis and therapy resistance caused by high expression of BCL2 or loss of BH3-only protein function. Although a promising therapy for hematologic malignancies, increased expression of anti-apoptotic MCL-1 or BCL-XL, as well as other resistance mechanisms prevent a durable response to venetoclax. Recent studies demonstrate that agents targeting epigenetic mechanisms such as DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, histone methyltransferase EZH2 inhibitors, or bromodomain reader protein inhibitors may disable oncogenic gene expression signatures responsible for venetoclax resistance. Combination therapies including venetoclax and epigenetic therapies are effective in preclinical models and the subject of many current clinical trials. Here we review epigenetic strategies to overcome venetoclax resistance mechanisms in hematologic malignancies. • Mutations affecting epigenetic regulation of gene expression are frequent in cancer and impact apoptosis mechanisms. • Venetoclax is a promising therapy for malignancies, but altered expression of BCL2 family members can cause resistance. • Combinations of venetoclax with epigenetic therapies have been approved and demonstrated effective in clinical trials. [ABSTRACT FROM AUTHOR]

Abstract: DNA demethylation agent 5-azacytidine has been widely described in literature as an effective chemical stimulus used to promote cardiomyogenic differentiation in various cell types, ranging from embryonic stem cells, P19 cells, bone marrow-derived mesenchymal stem cells, and recently to adipose-derived stem cells. The purpose of this study was to examine the effects of 5-azacytidine on human adipose precursor cell differentiation along the cardiomyogenic lineage. [Copyright &y& Elsevier]

Abstract: For patients with acute myeloid leukemia refractory to intensive chemotherapy prognosis is very poor and treatment options are limited. 5-Azacytidine, a demethylating drug, is effective in the treatment of myelodysplastic syndromes when administered at a low-dose, subcutaneously. We report a case of a patient with AML refractory to induction chemotherapy as well as to two high-dose salvage regimens. The patient achieved CR through monotherapy with low-dose azacytidine. [Copyright &y& Elsevier]

• Low concentration 5-Aza-C improved maturation of pig oocytes. • 5-Aza-C treated oocytes had improved ability to develop in vitro after IVF or PA. • Enucleated 5-Aza-C treated oocytes had better reprogramming ability upon SCNT. • Enucleated 5-Aza-C treated oocytes may be a better cytoplast for SCNT. Treatment of donor cells and/or cloned embryos with cytidine analogues, having an Aza group at its 5th carbon (5-Aza), such as 5-Azacytidine (5-Aza-C) or 5-Aza-2′-deoxycytidine (5-Aza-dC) improves the in vitro development of cloned embryos produced by somatic cell nuclear transfer (SCNT). In vitro maturation (IVM) of immature pig oocytes treated with 5-Aza-C not only results in greater (P < 0.05) meiotic maturation to the MII stage but also enhances the capacity of 5-Aza-C treated oocytes for early embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF) or SCNT in a dose-dependent manner (0–10 μM). Cloned embryos generated from 5-Aza-C (0.01 μM) treated oocytes had an increased capacity to develop to the blastocyst stage (14.1 ± 1.5% compared with 9.6 ± 1.8%), greater probability of hatching (61.8 ± 1.5% compared with 45.0 ± 3.9%) and contained a greater number of cells per blastocyst (38.5 ± 4.4 compared with 30.5 ± 3.4) than those produced from non-treated control oocytes (P < 0.05). Data from the present study indicate that treatment of oocytes with 5-Aza-C may be an important approach to enhance the meiotic maturation and subsequent in vitro development of pig embryos. Future studies should be conducted to determine the underlying mechanism of improved early embryonic development of 5-Aza-C treated oocytes. [ABSTRACT FROM AUTHOR]

Summary In the developing pancreas, transient Neurog3 -expressing progenitors give rise to four major islet cell types: α, β, δ, and γ; when and how the Neurog3+ cells choose cell fate is unknown. Using single-cell RNA-seq, trajectory analysis, and combinatorial lineage tracing, we showed here that the Neurog3+ cells co-expressing Myt1 (i.e., Myt1+Neurog3+) were biased toward β cell fate, while those not simultaneously expressing Myt1 (Myt1-Neurog3+) favored α fate. Myt1 manipulation only marginally affected α versus β cell specification, suggesting Myt1 as a marker but not determinant for islet-cell-type specification. The Myt1+Neurog3+ cells displayed higher Dnmt1 expression and enhancer methylation at Arx , an α-fate-promoting gene. Inhibiting Dnmts in pancreatic progenitors promoted α cell specification, while Dnmt1 overexpression or Arx enhancer hypermethylation favored β cell production. Moreover, the pancreatic progenitors contained distinct Arx enhancer methylation states without transcriptionally definable sub-populations, a phenotype independent of Neurog3 activity. These data suggest that Neurog3-independent methylation on fate-determining gene enhancers specifies distinct endocrine-cell programs. Highlights • Neurog3-null cells have distinct DNA methylation states but uniform transcriptome • Neurog3+ cells have distinct transcriptome, DNA methylation state, and cell fate • β-biasing Neurog3+ progenitors co-express Myt1 and have higher Arx enhancer methylation • Increased methylation in Arx promoter drives progenitors to the β cell lineage During embryogenesis, seemingly equivalent progenitors produce distinct cell types. The mechanisms driving this divergence are unknown. Liu et al. show that the pancreatic progenitors before endocrine determination are transcriptionally uniform but epigenetically distinct pools with different DNA methylation in gene enhancers. This epigenetic variation biases progenitors toward specific islet-cell fate. [ABSTRACT FROM AUTHOR]

Despite improvements to hematopoietic stem cell transplantation over the past several decades, further advances are necessary to achieve: improved control of toxicities like graft-versus-host disease; enhanced immunologic reconstitution posttransplantation; and reduction in relapse risk via enhancement of graft-versus-tumor responses. Achieving these disparate hematopoietic stem cell transplantation goals will likely require the introduction of novel therapeutic agents to the current armamentarium. In this article, we outline preclinical and early-phase clinical data indicating the potential of proteasome-inhibitor therapy (bortezomib), hypomethylating agent therapy (azacytidine), and histone deacetylase-inhibitor therapy (vorinostat) to help improve hematopoietic stem cell transplantation outcomes. [ABSTRACT FROM AUTHOR]