Loss of epigenetic suppression of retrotransposons with oncogenic potential in aging mammary luminal epithelial cells.

A primary function of DNA methylation in mammalian genomes is to repress transposable elements (TEs). The widespread methylation loss that is commonly observed in cancer cells results in the loss of epigenetic repression of TEs. The aging process is similarly characterized by changes to the methylome. However, the impact of these epigenomic alterations on TE silencing and the functional consequences of this have remained unclear. To assess the epigenetic regulation of TEs in aging, we profiled DNA methylation in human mammary luminal epithelial cells (LEps)-a key cell lineage implicated in age-related breast cancers-from younger and older women. We report here that several TE subfamilies function as regulatory elements in normal LEps, and a subset of these display consistent methylation changes with age. Methylation changes at these TEs occurred at lineage-specific transcription factor binding sites, consistent with loss of lineage specificity. Whereas TEs mainly showed methylation loss, CpG islands (CGIs) that are targets of the Polycomb repressive complex 2 (PRC2) show a gain of methylation in aging cells. Many TEs with methylation loss in aging LEps have evidence of regulatory activity in breast cancer samples. We furthermore show that methylation changes at TEs impact the regulation of genes associated with luminal breast cancers. These results indicate that aging leads to DNA methylation changes at TEs that undermine the maintenance of lineage specificity, potentially increasing susceptibility to breast cancer.

Neoantigen T-Cell Receptor Gene Therapy in Pancreatic Cancer.

A patient with progressive metastatic pancreatic cancer was treated with a single infusion of 16.2×109 autologous T cells that had been genetically engineered to clonally express two allogeneic HLA-C*08:02-restricted T-cell receptors (TCRs) targeting mutant KRAS G12D expressed by the tumors. The patient had regression of visceral metastases (overall partial response of 72% according to the Response Evaluation Criteria in Solid Tumors, version 1.1); the response was ongoing at 6 months. The engineered T cells constituted more than 2% of all the circulating peripheral-blood T cells 6 months after the cell transfer. In this patient, TCR gene therapy targeting the KRAS G12D driver mutation mediated the objective regression of metastatic pancreatic cancer. (Funded by the Providence Portland Medical Foundation.).

Long-Read Sequencing with Hierarchical Clustering for Antiretroviral Resistance Profiling of Mixed Human Immunodeficiency Virus Quasispecies.

BACKGROUND: HIV infections often develop drug resistance mutations (DRMs), which can increase the risk of virological failure. However, it has been difficult to determine if minor mutations occur in the same genome or in different virions using Sanger sequencing and short-read sequencing methods. Oxford Nanopore Technologies (ONT) sequencing may improve antiretroviral resistance profiling by allowing for long-read clustering. METHODS: A new ONT sequencing-based method for profiling DRMs in HIV quasispecies was developed and validated. The method used hierarchical clustering of long amplicons that cover regions associated with different types of antiretroviral drugs. A gradient series of an HIV plasmid and 2 plasma samples was prepared to validate the clustering performance. The ONT results were compared to those obtained with Sanger sequencing and Illumina sequencing in 77 HIV-positive plasma samples to evaluate the diagnostic performance. RESULTS: In the validation study, the abundance of detected quasispecies was concordant with the predicted result with the R2 of > 0.99. During the diagnostic evaluation, 59/77 samples were successfully sequenced for DRMs. Among 18 failed samples, 17 were below the limit of detection of 303.9 copies/µL. Based on the receiver operating characteristic analysis, the ONT workflow achieved an F1 score of 0.96 with a cutoff of 0.4 variant allele frequency. Four cases were found to have quasispecies with DRMs, in which 2 harbored quasispecies with more than one class of DRMs. Treatment modifications were recommended for these cases. CONCLUSIONS: Long-read sequencing coupled with hierarchical clustering could differentiate the quasispecies resistance profiles in HIV-infected samples, providing a clearer picture for medical care.

Retinal Organoids from an AIPL1 CRISPR/Cas9 Knockout Cell Line Successfully Recapitulate the Molecular Features of LCA4 Disease.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is expressed in photoreceptors where it facilitates the assembly of phosphodiesterase 6 (PDE6) which hydrolyses cGMP within the phototransduction cascade. Genetic variations in AIPL1 cause type 4 Leber congenital amaurosis (LCA4), which presents as rapid loss of vision in early childhood. Limited in vitro LCA4 models are available, and these rely on patient-derived cells harbouring patient-specific AIPL1 mutations. While valuable, the use and scalability of individual patient-derived LCA4 models may be limited by ethical considerations, access to patient samples and prohibitive costs. To model the functional consequences of patient-independent AIPL1 mutations, CRISPR/Cas9 was implemented to produce an isogenic induced pluripotent stem cell line harbouring a frameshift mutation in the first exon of AIPL1. Retinal organoids were generated using these cells, which retained AIPL1 gene transcription, but AIPL1 protein was undetectable. AIPL1 knockout resulted in a decrease in rod photoreceptor-specific PDE6α and ß, and increased cGMP levels, suggesting downstream dysregulation of the phototransduction cascade. The retinal model described here provides a novel platform to assess functional consequences of AIPL1 silencing and measure the rescue of molecular features by potential therapeutic approaches targeting mutation-independent pathogenesis.

Duet: SNP-assisted structural variant calling and phasing using Oxford nanopore sequencing.

BACKGROUND: Whole genome sequencing using the long-read Oxford Nanopore Technologies (ONT) MinION sequencer provides a cost-effective option for structural variant (SV) detection in clinical applications. Despite the advantage of using long reads, however, accurate SV calling and phasing are still challenging. RESULTS: We introduce Duet, an SV detection tool optimized for SV calling and phasing using ONT data. The tool uses novel features integrated from both SV signatures and single-nucleotide polymorphism signatures, which can accurately distinguish SV haplotype from a false signal. Duet was benchmarked against state-of-the-art tools on multiple ONT sequencing datasets of sequencing coverage ranging from 8× to 40×. At low sequencing coverage of 8×, Duet performs better than all other tools in SV calling, SV genotyping and SV phasing. When the sequencing coverage is higher (20× to 40×), the F1-score for SV phasing is further improved in comparison to the performance of other tools, while its performance of SV genotyping and SV calling remains higher than other tools. CONCLUSION: Duet can perform accurate SV calling, SV genotyping and SV phasing using low-coverage ONT data, making it very useful for low-coverage genomes. It has great performance when scaled to high-coverage genomes, which is adaptable to various clinical applications. Duet is open source and is available at https://github.com/yekaizhou/duet .

Induced Pluripotent Stem Cells and Genome-Editing Tools in Determining Gene Function and Therapy for Inherited Retinal Disorders.

Inherited retinal disorders (IRDs) affect millions of people worldwide and are a major cause of irreversible blindness. Therapies based on drugs, gene augmentation or transplantation approaches have been widely investigated and proposed. Among gene therapies for retinal degenerative diseases, the fast-evolving genome-editing CRISPR/Cas technology has emerged as a new potential treatment. The CRISPR/Cas system has been developed as a powerful genome-editing tool in ophthalmic studies and has been applied not only to gain proof of principle for gene therapies in vivo, but has also been extensively used in basic research to model diseases-in-a-dish. Indeed, the CRISPR/Cas technology has been exploited to genetically modify human induced pluripotent stem cells (iPSCs) to model retinal disorders in vitro, to test in vitro drugs and therapies and to provide a cell source for autologous transplantation. In this review, we will focus on the technological advances in iPSC-based cellular reprogramming and gene editing technologies to create human in vitro models that accurately recapitulate IRD mechanisms towards the development of treatments for retinal degenerative diseases.

LTRs activated by Epstein-Barr virus-induced transformation of B cells alter the transcriptome.

Endogenous retroviruses (ERVs) are ancient viral elements that have accumulated in the genome through retrotransposition events. Although they have lost their ability to transpose, many of the long terminal repeats (LTRs) that originally flanked full-length ERVs maintain the ability to regulate transcription. While these elements are typically repressed in somatic cells, they can function as transcriptional enhancers and promoters when this repression is lost. Epstein-Barr virus (EBV), which transforms primary B cells into continuously proliferating cells, is a tumor virus associated with lymphomas. We report here that transformation of primary B cells by EBV leads to genome-wide activation of LTR enhancers and promoters. The activation of LTRs coincides with local DNA hypomethylation and binding by transcription factors such as RUNX3, EBF1, and EBNA2. The set of activated LTRs is unique to transformed B cells compared with other cell lines known to have activated LTRs. Furthermore, we found that LTR activation impacts the B cell transcriptome by up-regulating transcripts driven by cryptic LTR promoters. These transcripts include genes important to oncogenesis of Hodgkin lymphoma and other cancers, such as HUWE1/HECTH9 These data suggest that the activation of LTRs by EBV-induced transformation is important to the pathology of EBV-associated cancers. Altogether, our results indicate that EBV-induced transformation of B cells alters endogenous retroviral element activity, thereby impacting host gene regulatory networks and oncogenic potential.

Novel Long Noncoding RNA, Macrophage Inflammation-Suppressing Transcript (MIST), Regulates Macrophage Activation During Obesity.

OBJECTIVE: Systemic low-grade inflammation associated with obesity and metabolic syndrome is a strong risk factor for the development of diabetes mellitus and associated cardiovascular complications. This inflammatory state is caused by release of proinflammatory cytokines by macrophages, especially in adipose tissue. Long noncoding RNAs regulate macrophage activation and inflammatory gene networks, but their role in macrophage dysfunction during diet-induced obesity has been largely unexplored. Approach and Results: We sequenced total RNA from peritoneal macrophages isolated from mice fed either high-fat diet or standard diet and performed de novo transcriptome assembly to identify novel differentially expressed mRNAs and long noncoding RNAs. A top candidate long noncoding RNA, macrophage inflammation-suppressing transcript (Mist), was downregulated in both peritoneal macrophages and adipose tissue macrophages from high-fat diet-fed mice. GapmeR-mediated Mist knockdown in vitro and in vivo upregulated expression of genes associated with immune response and inflammation and increased modified LDL (low-density lipoprotein) uptake in macrophages. Conversely, Mist overexpression decreased basal and LPS (lipopolysaccharide)-induced expression of inflammatory response genes and decreased modified LDL uptake. RNA-pull down coupled with mass spectrometry showed that Mist interacts with PARP1 (poly [ADP]-ribose polymerase-1). Disruption of this RNA-protein interaction increased PARP1 recruitment and chromatin PARylation at promoters of inflammatory genes, resulting in increased gene expression. Furthermore, human orthologous MIST was also downregulated by proinflammatory stimuli, and its expression in human adipose tissue macrophages inversely correlated with obesity and insulin resistance. CONCLUSIONS: Mist is a novel protective long noncoding RNA, and its loss during obesity contributes to metabolic dysfunction and proinflammatory phenotype of macrophages via epigenetic mechanisms.

A Novel Angiotensin II-Induced Long Noncoding RNA Giver Regulates Oxidative Stress, Inflammation, and Proliferation in Vascular Smooth Muscle Cells.

RATIONALE: AngII (angiotensin II)-mediated vascular smooth muscle cell (VSMC) dysfunction plays a major role in hypertension. Long noncoding RNAs have elicited much interest, but their molecular roles in AngII actions and hypertension are unclear. OBJECTIVE: To investigate the regulation and functions of a novel long noncoding RNA growth factor- and proinflammatory cytokine-induced vascular cell-expressed RNA ( Giver), in AngII-mediated VSMC dysfunction. METHODS AND RESULTS: RNA-sequencing and real-time quantitative polymerase chain reactions revealed that treatment of rat VSMC with AngII increased the expression of Giver and Nr4a3, an adjacent gene encoding a nuclear receptor. Similar changes were observed in rat and mouse aortas treated ex vivo with AngII. RNA-FISH (fluorescence in situ hybridization) and subcellular fractionation showed predominantly nuclear localization of Giver. AngII increased Giver expression via recruitment of Nr4a3 to Giver promoter. Microarray profiling and real-time quantitative polymerase chain reaction validation in VSMC showed that Giver knockdown attenuated the expression of genes involved in oxidative stress ( Nox1) and inflammation ( Il6, Ccl2, Tnf) but increased Nr4a3. Conversely, endogenous Giver overexpression showed opposite effects supporting its role in oxidative stress and inflammation. Chromatin immunoprecipitation assays showed Giver overexpression also increased Pol II (RNA polymerase II) enrichment and decreased repressive histone modification histone H3 trimethylation on lysine 27 at Nox1 and inflammatory gene promoters. Accordingly, Giver knockdown inhibited AngII-induced oxidative stress and proliferation in rat VSMC. RNA-pulldown combined with mass spectrometry showed Giver interacts with nuclear and chromatin remodeling proteins and corepressors, including NONO (non-pou domain-containing octamer-binding protein). Moreover, NONO knockdown elicited similar effects as Giver knockdown on the expression of key Giver-regulated genes. Notably, GIVER and NR4A3 were increased in AngII-treated human VSMC and in arteries from hypertensive patients but attenuated in hypertensive patients treated with ACE (angiotensin-converting enzyme) inhibitors or angiotensin receptor blockers. Furthermore, human GIVER also exhibits partial functional conservation with rat Giver. CONCLUSIONS: Giver and its regulator Nr4a3 are important players in AngII-mediated VSMC dysfunction and could be novel targets for antihypertensive therapy.

Notch and Aryl Hydrocarbon Receptor Signaling Impact Definitive Hematopoiesis from Human Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) stand to revolutionize the way we study human development, model disease, and eventually, treat patients. However, these cell sources produce progeny that retain embryonic and/or fetal characteristics. The failure to mature to definitive, adult-type cells is a major barrier for iPSC-based disease modeling and drug discovery. To directly address these concerns, we have developed a chemically defined, serum and feeder-free-directed differentiation platform to generate hematopoietic stem-progenitor cells (HSPCs) and resultant adult-type progeny from iPSCs. This system allows for strict control of signaling pathways over time through growth factor and/or small molecule modulation. Through direct comparison with our previously described protocol for the production of primitive wave hematopoietic cells, we demonstrate that induced HSPCs are enhanced for erythroid and myeloid colony forming potential, and strikingly, resultant erythroid-lineage cells display enhanced expression of adult ß globin indicating definitive pathway patterning. Using this system, we demonstrate the stage-specific roles of two key signaling pathways, Notch and the aryl hydrocarbon receptor (AHR), in the derivation of definitive hematopoietic cells. We illustrate the stage-specific necessity of Notch signaling in the emergence of hematopoietic progenitors and downstream definitive, adult-type erythroblasts. We also show that genetic or small molecule inhibition of the AHR results in the increased production of CD34+ CD45+ HSPCs while conversely, activation of the same receptor results in a block of hematopoietic cell emergence. Results presented here should have broad implications for hematopoietic stem cell transplantation and future clinical translation of iPSC-derived blood cells. Stem Cells 2018;36:1004-1019.

Understanding the effect of repeated administration of meloxicam on feline renal cortex and medulla: A lipidomics and metabolomics approach.

Repeated administration of meloxicam can cause kidney damage in cats by mechanisms that remain unclear. Metabolomics and lipidomics are powerful, noninvasive approaches used to investigate tissue response to drug exposure. Thus, the objective of this study was to assess the effects of meloxicam on the feline kidney using untargeted metabolomics and lipidomics approaches. Female young-adult purpose-breed cats were allocated into the control (n = 4) and meloxicam (n = 4) groups. Cats in the control and meloxicam groups were treated daily with saline and meloxicam at 0.3 mg/kg subcutaneously for 17 days, respectively. Renal cortices and medullas were collected at the end of the treatment period. Random forest and metabolic pathway analyses were used to identify metabolites that discriminate meloxicam-treated from saline-treated cats and to identify disturbed metabolic pathways in renal tissue. Our results revealed that the repeated administration of meloxicam to cats altered the kidney metabolome and lipidome and suggest that at least 40 metabolic pathways were altered in the renal cortex and medulla. These metabolic pathways included lipid, amino acid, carbohydrate, nucleotide and energy metabolisms, and metabolism of cofactors and vitamins. This is the first study using a pharmacometabonomics approach for studying the molecular effects of meloxicam on feline kidneys.

Sustainability of Clinical Benefits Gained During a Multidisciplinary Diabetes Shared Medical Appointment After Patients Return to Usual Care.

IN BRIEF This study examined whether clinical benefits gained while participating in interdisciplinary diabetes shared medical appointments (SMAs) of finite duration (three to four monthly visits) are sustained after patients return to usual care. There are currently no publications confirming sustained clinical benefits beyond 9 months after SMA discharge without continued booster sessions to maintain benefits. At the end of the study, it was confirmed that both diabetes and cardiovascular benefits gained during multidisciplinary diabetes SMAs were sustained after patients were discharged to usual care without booster sessions for up to 3 years. The only exceptions were a statistically significant decrease in diastolic blood pressure at each yearly time point and a decrease in the percentage of patients meeting an A1C goal of <9% at year 2.

Impact of Parent Practices of Infant Positioning on Head Orientation Profile and Development of Positional Plagiocephaly in Healthy Term Infants.

AIMS: The influence of infant positioning on the development of head orientation and plagiocephaly is not clear. This study explored the relationship between infant body and head positioning, with the development of asymmetrical head orientation and/or positional plagiocephaly. Methods: Clinician measurement of head orientation profile and parent-reported infant positioning data were collected for 94 healthy term infants at 3, 6, and 9 weeks of age. Plagiocephaly was measured at 9 weeks with the modified Cranial Vault Asymmetry Index. RESULTS: More severe plagiocephaly was associated with longer supine-sleep-maximum (p = 0.001) and longer supine-lying-total (p = 0.014) at 6 weeks. Prone positioning was not associated with plagiocephaly. Parent-reported head asymmetry during awake and sleep time at 3 weeks identified infants with clinician-measured head asymmetry at 9 weeks. Better symmetry in head turning was associated with more side-lying-total time by 9 weeks (p = 0.013). CONCLUSIONS: Our results showed that infant positioning is associated with early head orientation and plagiocephaly development. Early parent-reported asymmetry during awake and sleep time is an important indicator for the need for professional assessment and advice. A Plagiocephaly Prevention Strategy and Plagiocephaly Screening Pathway are provided for clinicians and parents.

C. elegans HAM-1 functions in the nucleus to regulate asymmetric neuroblast division.

All 302 neurons in the C. elegans hermaphrodite arise through asymmetric division of neuroblasts. During embryogenesis, the C. elegans ham-1 gene is required for several asymmetric neuroblast divisions in lineages that generate both neural and apoptotic cells. By antibody staining, endogenous HAM-1 is found exclusively at the cell cortex in many cells during embryogenesis and is asymmetrically localized in dividing cells. Here we show that in transgenic embryos expressing a functional GFP::HAM-1 fusion protein, GFP expression is also detected in the nucleus, in addition to the cell cortex. Consistent with the nuclear localization is the presence of a putative DNA binding winged-helix domain within the N-terminus of HAM-1. Through a deletion analysis we determined that the C-terminus of the protein is required for nuclear localization and we identified two nuclear localization sequences (NLSs). A subcellular fractionation experiment from wild type embryos, followed by Western blotting, revealed that endogenous HAM-1 is primarily found in the nucleus. Our analysis also showed that the N-terminus is necessary for cortical localization. While ham-1 function is essential for asymmetric division in the lineage that generates the PLM mechanosensory neuron, we showed that cortical localization may not required. Thus, our results suggest that there is a nuclear function for HAM-1 in regulating asymmetric neuroblast division and that the requirement for cortical localization may be lineage dependent.

Persistent Chromatin Modifications Induced by High Fat Diet.

Obesity is a highly heritable complex disease that results from the interaction of multiple genetic and environmental factors. Formerly obese individuals are susceptible to metabolic disorders later in life, even after lifestyle changes are made to mitigate the obese state. This is reminiscent of the metabolic memory phenomenon originally observed for persistent complications in diabetic patients, despite subsequent glycemic control. Epigenetic modifications represent a potential mediator of this observed memory. We previously demonstrated that a high fat diet leads to changes in chromatin accessibility in the mouse liver. The regions of greatest chromatin changes in accessibility are largely strain-dependent, indicating a genetic component in diet-induced chromatin alterations. We have now examined the persistence of diet-induced chromatin accessibility changes upon diet reversal in two strains of mice. We find that a substantial fraction of loci that undergo chromatin accessibility changes with a high fat diet remains in the remodeled state after diet reversal in C57BL/6J mice. In contrast, the vast majority of diet-induced chromatin accessibility changes in A/J mice are transient. Our data also indicate that the persistent chromatin accessibility changes observed in C57BL/6J mice are associated with specific transcription factors and histone post-translational modifications. The persistent loci identified here are likely to be contributing to the overall phenotype and are attractive targets for therapeutic intervention.

Functional Long Non-coding RNAs in Vascular Smooth Muscle Cells.

Increasing evidence shows that long non-coding RNAs (lncRNAs) are not "transcriptional noise" but function in a myriad of biological processes. As such, this rapidly growing class of RNAs is important in both development and disease. Vascular smooth muscle cells are integral cells of the blood vessel wall. They are responsible for relaxation and contraction of the blood vessel and respond to hemodynamic as well as environmental signals to regulate blood pressure. Pathophysiological changes to these cells such as hyperproliferation, hypertrophy, migration, and inflammation contribute to cardiovascular diseases (CVDs) such as restenosis, hypertension, and atherosclerosis. Understanding the molecular mechanisms involved in these pathophysiological changes to VSMCs is paramount to developing therapeutic treatments for various cardiovascular disorders. Recent studies have shown that lncRNAs are key players in the regulation of VSMC functions and phenotype and, perhaps also, in the development of VSMC-related diseases. This chapter describes our current understanding of the functions of lncRNAs in VSMCs. It highlights the emerging role of lncRNAs in VSMC proliferation and apoptosis, their role in contractile and migratory phenotype of VSMCs, and their potential role in VSMC disease states.

Role of telehealth in perioperative medicine for regional and rural patients in Queensland.

BACKGROUND: Australians living in regional, rural and remote areas face a myriad of complexities resulting in a trend to poorer health outcomes. Telehealth is being utilised as an alternative mode of service delivery to overcome such barriers. However, there is limited published information concerning the use of telehealth in perioperative medicine. AIMS: To review the performance of the telehealth preoperative assessment service at Mater Hospital Brisbane to determine last-minute cancellation rates secondary to medical reasons. METHODS: A retrospective chart audit of all patients referred to perioperative medicine telehealth consultation services was provided at Mater Hospital Brisbane. RESULTS: During a 1-year period, 229 patients across eight surgical subspecialties were referred for telehealth assessment (85 females, 144 males) with a median age of 67 years. Patients lived a median distance of 1597 km from Brisbane. Of the 229 patients included in the study, 7 (3.1%) experienced last-minute cancellations. From these data, the last-minute cancellation rate due to medical reasons was 1.3%, which is consistent with the international average. CONCLUSION: The Mater Hospital Perioperative Medicine Telehealth Programme is a means of providing comprehensive perioperative assessment to regional, rural and remote patients that result in reduced last-minute surgical cancellations and surgery within or below the national recommended guidelines.

Positional plagiocephaly is associated with sternocleidomastoid muscle activation in healthy term infants.

PURPOSE: To explore the relationship between sternocleidomastoid activation and positional plagiocephaly in healthy full term infants. METHODS: Participants were 82 infants from a regionally based-longitudinal study of infant development. Sternocleidomastoid (SCM) activation was assessed using active head-righting responses of body-on-head with and against gravity and head-on-body against gravity at 3, 6 and 9 weeks. Plagiocephaly was assessed using the Modified Cranial Vault Asymmetry Index (mCVAI) at 9 weeks. RESULTS: More severe plagiocephaly was associated with more severe asymmetry in active head-righting responses at all ages (p < 0.001). Greater right-sided occipital flatness was related to stronger contralateral/left SCM activation at 3 and at 9 weeks (p = 0.008). Greater left-sided occipital flatness was related to stronger contralateral/right SCM activation at 3 weeks (p = 0.004). In infants with any right-sided occipital flatness, the mCVAI was greater in infants with asymmetrical gravity assisted body-on-head responses at 3 weeks (mCVAI = 4.31 (2.01)%, 95% CI 2.87-5.75) compared to those with symmetrical responses (mCVAI = 2.64 (1.66)%, 95% CI 2.06-3.22) (p = 0.011). CONCLUSIONS: Sternocleidomastoid activation asymmetry is a significant contributor to plagiocephaly development by 9 weeks of age due to stronger contralateral SCM activation. Active head-righting responses are appropriate to assess sternocleidomastoid activation in infants under 2 months of age.

The core autophagy protein ATG4B is a potential biomarker and therapeutic target in CML stem/progenitor cells.

Previous studies demonstrated that imatinib mesylate (IM) induces autophagy in chronic myeloid leukemia (CML) and that this process is critical to cell survival upon therapy. However, it is not known if the autophagic process differs at basal levels between CML patients and healthy individuals and if pretreatment CML cells harbor unique autophagy characteristics that could predict patients' clinical outcomes. We now demonstrate that several key autophagy genes are differentially expressed in CD34(+) hematopoietic stem/progenitor cells, with the highest transcript levels detected for ATG4B, and that the transcript and protein expression levels of ATG4 family members, ATG5 and BECLIN-1 are significantly increased in CD34(+) cells from chronic-phase CML patients (P < .05). Importantly, ATG4B is differentially expressed in pretreatment CML stem/progenitor cells from subsequent IM responders vs IM nonresponders (P < .05). Knockdown of ATG4B suppresses autophagy, impairs the survival of CML stem/progenitor cells and sensitizes them to IM treatment. Moreover, deregulated expression of ATG4B in CD34(+) CML cells inversely correlates with transcript levels of miR-34a, and ATG4B is shown to be a direct target of miR-34a. This study identifies ATG4B as a potential biomarker for predicting therapeutic response in treatment-naïve CML stem/progenitor cells and uncovers ATG4B as a possible drug target in these cells.

Chromatin Modifications Associated With Diabetes and Obesity.

The incidence of obesity across the globe has doubled over the past several decades, leading to escalating rates of diabetes mellitus, cardiovascular disease, and other complications. Given this dramatic rise in disease incidence, understanding the cause of these diseases is therefore of paramount importance. Metabolic diseases, such as obesity and diabetes mellitus, result from a multitude of genetic and environmental factors. Although the genetic basis of these diseases has been extensively studied, the molecular pathways whereby environmental factors influence disease progression are only beginning to be understood. One manner by which environmental factors can contribute to disease progression is through modifications to chromatin. The highly structured packaging of the genome into the nucleus through chromatin has been shown to be fundamental to tissue-specific gene regulation. Modifications to chromatin can regulate gene expression and are involved in a myriad of biological functions, and hence, disruption of these modifications is central to many human diseases. These modifications can furthermore be epigenetic in nature, thereby contributing to prolonged disease risk. Recent work has demonstrated that modifications to chromatin are associated with the progression of both diabetes mellitus and obesity, which is the subject of this review.