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Alcoholic hepatitis accelerates early hepatobiliary cancer by increasing stemness and miR-122-mediated HIF-1alpha activation

Thu, 06/30/2016 - 6:48pm

Alcohol-related hepatocellular carcinoma (HCC) develops with advanced alcoholic liver disease and liver fibrosis. Using adult mice, we evaluate the effect of alcoholic steatohepatitis on early hepatobiliary carcinoma after initiation by diethyl-nitrosamine (DEN). Here we show that alcohol-fed DEN-injected mice have higher ALT and liver-to-body weight ratio compared to pair-fed DEN-injected mice. Alcohol feeding results in steatohepatitis indicated by increased pro-inflammatory cytokines and fibrotic genes. MRI and liver histology of alcohol+DEN mice shows hepatobiliary cysts, early hepatic neoplasia and increase in serum alpha-fetoprotein. Proliferation makers (BrdU, cyclin D1, p53) and cancer stem cell markers (CD133 and nanog) are significantly up-regulated in livers of alcohol-fed DEN-injected mice compared to controls. In livers with tumors, loss of miR-122 expression with a significant up-regulation of miR-122 target HIF-1alpha is seen. We conclude that alcoholic steatohepatitis accelerates hepatobiliary tumors with characteristic molecular features of HCC by up-regulating inflammation, cell proliferation, stemness, and miR-122 loss.

P-Rex1 Promotes Resistance to VEGF/VEGFR-Targeted Therapy in Prostate Cancer

Thu, 06/30/2016 - 6:47pm

Autocrine VEGF signaling is critical for sustaining prostate and other cancer stem cells (CSCs), and it is a potential therapeutic target, but we observed that CSCs isolated from prostate tumors are resistant to anti-VEGF (bevacizumab) and anti-VEGFR (sunitinib) therapy. Intriguingly, resistance is mediated by VEGF/neuropilin signaling, which is not inhibited by bevacizumab and sunitinib, and it involves the induction of P-Rex1, a Rac GEF, and consequent Rac1-mediated ERK activation. This induction of P-Rex1 is dependent on Myc. CSCs isolated from the PTEN(pc-/-) transgenic model of prostate cancer exhibit Rac1-dependent resistance to bevacizumab. Rac1 inhibition or P-Rex1 downregulation increases the sensitivity of prostate tumors to bevacizumab. These data reveal that prostate tumors harbor cells with stem cell properties that are resistant to inhibitors of VEGF/VEGFR signaling. Combining the use of available VEGF/VEGFR-targeted therapies with P-Rex1 or Rac1 inhibition should improve the efficacy of these therapies significantly.

Biochemical and functional characterization of Plasmodium falciparum DNA polymerase delta

Thu, 06/30/2016 - 6:47pm

BACKGROUND: Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase delta is an essential enzyme required for chromosomal DNA replication and repair, and therefore may be a potential target for anti-malarial drug development. However, little is known of the characteristics and function of this P. falciparum enzyme. METHODS: The coding sequences of DNA polymerase delta catalytic subunit (PfPoldelta-cat), DNA polymerase delta small subunit (PfPoldeltaS) and proliferating cell nuclear antigen (PfPCNA) from chloroquine- and pyrimethamine-resistant P. falciparum strain K1 were amplified, cloned into an expression vector and expressed in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE and identified by LC-MS/MS. PfPoldelta-cat was biochemically characterized. The roles of PfPoldeltaS and PfPCNA in PfPoldelta-cat function were investigated. In addition, inhibitory effects of 11 compounds were tested on PfPoldelta-cat activity and on in vitro parasite growth using SYBR Green I assay. RESULTS: The purified recombinant protein PfPoldelta-cat, PfPoldeltaS and PfPCNA showed on SDS-PAGE the expected size of 143, 57 and 34 kDa, respectively. Predicted amino acid sequence of the PfPoldelta-cat and PfPoldeltaS had 59.2 and 24.7 % similarity respectively to that of the human counterpart. The PfPoldelta-cat possessed both DNA polymerase and 3'-5' exonuclease activities. It used both Mg(2+) and Mn(2+) as cofactors and was inhibited by high KCl salt ( > 200 mM). PfPoldeltaS stimulated PfPoldelta-cat activity threefolds and up to fourfolds when PfPCNA was included in the assay. Only two compounds were potent inhibitors of PfPoldelta-cat, namely, butylphenyl-dGTP (BuPdGTP; IC50 of 38 microM) and 7-acetoxypentyl-(3, 4 dichlorobenzyl) guanine (7-acetoxypentyl-DCBG; IC50 of 55 microM). The latter compound showed higher inhibition on parasite growth (IC50 of 4.1 microM). CONCLUSIONS: Recombinant PfPoldelta-cat, PfPoldeltaS and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA increased DNA polymerase activity of PfPoldelta-cat. The high sensitivity of PfPoldelta to BuPdGTP can be used to differentiate parasite enzyme from mammalian and human counterparts. Interestingly, 7-acetoxypentyl-DCBG showed inhibitory effects on both enzyme activity and parasite growth. Thus, 7-acetoxypentyl-DCBG is a potential candidate for future development of a new class of anti-malarial agents targeting parasite replicative DNA polymerase.

Drosophila Vision Depends on Carcinine Uptake by an Organic Cation Transporter

Thu, 06/30/2016 - 6:47pm

Recycling of neurotransmitters is essential for sustained neuronal signaling, yet recycling pathways for various transmitters, including histamine, remain poorly understood. In the first visual ganglion (lamina) of Drosophila, photoreceptor-released histamine is taken up into perisynaptic glia, converted to carcinine, and delivered back to the photoreceptor for histamine regeneration. Here, we identify an organic cation transporter, CarT (carcinine transporter), that transports carcinine into photoreceptors during histamine recycling. CarT mediated in vitro uptake of carcinine. Deletion of the CarT gene caused an accumulation of carcinine in laminar glia accompanied by a reduction in histamine, resulting in abolished photoreceptor signal transmission and blindness in behavioral assays. These defects were rescued by expression of CarT cDNA in photoreceptors, and they were reproduced by photoreceptor-specific CarT knockdown. Our findings suggest a common role for the conserved family of CarT-like transporters in maintaining histamine homeostasis in both mammalian and fly brains.

Cross-Sectional Associations of Computed Tomography (CT)-Derived Adipose Tissue Density and Adipokines: The Framingham Heart Study

Thu, 06/30/2016 - 6:47pm

BACKGROUND: Excess accumulation of abdominal subcutaneous (SAT) and visceral adipose tissue (VAT) is associated with adverse levels of adipokines and cardiovascular disease risk. Whether fat quality is associated with adipokines has not been firmly established. This study examined the association between abdominal SAT and VAT density, an indirect measure of fat quality, with a panel of metabolic regulatory biomarkers secreted by adipose tissue or the liver independently of absolute fat volumes. METHODS AND RESULTS: We evaluated 1829 Framingham Heart Study participants (44.9% women). Abdominal SAT and VAT density was estimated indirectly by adipose tissue attenuation using computed tomography. Adipokines included adiponectin, leptin receptor, leptin, fatty acid-binding protein 4 (FABP-4), retinol-binding protein 4 (RBP-4), and fetuin-A. Fat density was associated with all the biomarkers evaluated, except fetuin-A. Lower fat density (ie, more-negative fat attenuation) was associated with lower adiponectin and leptin receptor, but higher leptin and FABP-4 levels (all P < 0.0001). SAT density was inversely associated with RPB-4 in both sexes, whereas the association between VAT density and RPB-4 was only observed in men (P < 0.0001). In women, after additional adjustment for respective fat volume, SAT density retained the significant associations with adiponectin, leptin, FABP-4, and RBP-4; and VAT density with adiponectin only (all P<0.0001). In men, significant associations were maintained upon additional adjustment for respective fat volume (P < 0.005). CONCLUSIONS: Lower abdominal fat density was associated with a profile of biomarkers suggestive of greater cardiometabolic risk. These observations support that fat density may be a valid biomarker of cardiometabolic risk.

Promoter-enhancer looping at the PPARgamma2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

Thu, 06/30/2016 - 6:47pm

PPARgamma2 is a critical lineage-determining transcription factor that is essential for adipogenic differentiation. Here we report characterization of the three-dimensional structure of the PPARgamma2 locus after the onset of adipogenic differentiation and the mechanisms by which it forms. We identified a differentiation-dependent loop between the PPARgamma2 promoter and an enhancer sequence 10 kb upstream that forms at the onset of PPARgamma2 expression. The arginine methyltransferase Prmt5 was required for loop formation, and overexpression of Prmt5 resulted in premature loop formation and earlier onset of PPARgamma2 expression. Kinetic studies of regulatory factor interactions at the PPARgamma2 promoter and enhancer revealed enhanced interaction of Prmt5 with the promoter that preceded stable association of Prmt5 with enhancer sequences. Prmt5 knockdown prevented binding of both MED1, a subunit of Mediator complex that facilitates enhancer-promoter interactions, and Brg1, the ATPase of the mammalian SWI/SNF chromatin remodeling enzyme required for PPARgamma2 activation and adipogenic differentiation. The data indicate a dynamic association of Prmt5 with the regulatory sequences of the PPARgamma2 gene that facilitates differentiation-dependent, three-dimensional organization of the locus. In addition, other differentiation-specific, long-range chromatin interactions showed Prmt5-dependence, indicating a more general role for Prmt5 in mediating higher-order chromatin connections in differentiating adipocytes.

Results of a Culturally Adapted Internet-Enhanced Physical Activity Pilot Intervention for Overweight and Obese Young Adult African American Women

Thu, 06/30/2016 - 6:47pm

PURPOSE: This study evaluated a culturally relevant, social cognitive theory-based, Internet-enhanced physical activity (PA) pilot intervention developed for overweight/obese African American (AA) female college students.

DESIGN: Using a 3-month, single group, pretest-posttest design, participants accessed a culturally relevant PA promotion website and engaged in four moderate-intensity PA sessions each week.

RESULTS: Study completers (n = 25, mean age = 21.9 years) reported a decrease in sedentary screen time (p < .0001); however, no changes in moderate-to-vigorous PA were reported (p = .150). A significant increase in self-regulation for PA (p < .0001) and marginally significant increases in social support (p = .052) and outcome expectations (p = .057) for PA were observed. No changes in body mass index (p = .162), PA enjoyment (p = .151), or exercise self-efficacy (p = .086) were reported.

CONCLUSIONS: Findings of this exploratory study show some preliminary support for Internet-enhanced approaches to promote PA among overweight/obese AA women.

IMPLICATIONS FOR PRACTICE: Future studies with larger samples are needed to further explore culturally relevant Internet-enhanced PA programs in this underserved population.

The carboxyl terminal mutational hotspot of the ciliary disease protein RPGRORF15 (retinitis pigmentosa GTPase regulator) is glutamylated in vivo

Thu, 06/30/2016 - 6:47pm

Mutations inRPGR(ORF15)(retinitis pigmentosa GTPase regulator) are a major cause of inherited retinal degenerative diseases. RPGR(ORF15)(1152 residues) is a ciliary protein involved in regulating the composition and function of photoreceptor cilia. The mutational hotspot in RPGR(ORF15)is an unusual C-terminal domain encoded by exon ORF15, which is rich in polyglutamates and glycine residues (Glu-Gly domain) followed by a short stretch of basic amino acid residues (RPGR(C2)domain; residues 1072-1152). However, the properties of the ORF15-encoded domain and its involvement in the pathogenesis of the disease are unclear. Here we show that RPGR(ORF15)is glutamylated at the C-terminus, as determined by binding to GT335, which recognizes glutamylated substrates. This reactivity is lost in two mouse mutants ofRpgr, which do not express RPGR(ORF15)due to disease-causing mutations in exon ORF15. Our results indicate that RPGR(ORF15)is posttranslationally glutamylated in the Glu-Gly domain and that the GT335 antibody predominantly recognizes RPGR(ORF15)in photoreceptor cilia.

Guided Tour of MedlinePlus Health Information

Thu, 06/30/2016 - 2:07pm

Black and Latino men in Central Massachusetts are an underserved population suffering significant health disparities. This presentation was part of training provided by the National Network of Libraries of Medicine/New England Region grant, Accessing Health Information for African American, African, and Latino Men: MedlinePlus training for the Barbershop Health Network and the Hector Reyes House, Worcester, MA. Train-the-trainer sessions were presented June 22, June 25, and July 8, 2015 to the staff from both organizations to learn how and where to access reliable consumer health information and, in turn, share these resources with their clients.

The IFITMs Inhibit Zika Virus Replication

Mon, 06/27/2016 - 3:51pm

Zika virus has emerged as a severe health threat with a rapidly expanding range. The IFITM family of restriction factors inhibits the replication of a broad range of viruses, including the closely related flaviruses West Nile virus and dengue virus. Here, we show that IFITM1 and IFITM3 inhibit Zika virus infection early in the viral life cycle. Moreover, IFITM3 can prevent Zika-virus-induced cell death. These results suggest that strategies to boost the actions and/or levels of the IFITMs might be useful for inhibiting a broad range of emerging viruses.

An invertebrate smooth muscle with striated muscle myosin filaments

Mon, 06/27/2016 - 3:33pm

Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and actin filaments are in register, whereas in smooth muscles, the actin filaments are attached to dense bodies and the myosin and actin filaments are out of register. The structure of the filaments in smooth muscles is also different from that in striated muscles. Here we have studied the structure of myosin filaments from the smooth muscles of the human parasite Schistosoma mansoni. We find, surprisingly, that they are indistinguishable from those in an arthropod striated muscle. This structural similarity is supported by sequence comparison between the schistosome myosin II heavy chain and known striated muscle myosins. In contrast, the actin filaments of schistosomes are similar to those of smooth muscles, lacking troponin-dependent regulation. We conclude that schistosome muscles are hybrids, containing striated muscle-like myosin filaments and smooth muscle-like actin filaments in a smooth muscle architecture. This surprising finding has broad significance for understanding how muscles are built and how they evolved, and challenges the paradigm that smooth and striated muscles always have distinctly different components.

Intraflagellar transport is essential for mammalian spermiogenesis but is absent in mature sperm

Mon, 06/27/2016 - 3:33pm

Drosophila sperm are unusual in that they do not require the intraflagellar transport (IFT) system for assembly of their flagella. In the mouse, the IFT proteins are very abundant in testis, but we here show that mature sperm are completely devoid of them, making the importance of IFT to mammalian sperm development unclear. To address this question, we characterized spermiogenesis and fertility in the Ift88(Tg737Rpw) mouse. This mouse has a hypomorphic mutation in the gene encoding the IFT88 subunit of the IFT particle. This mutation is highly disruptive to ciliary assembly in other organs. Ift88(-/-) mice are completely sterile. They produce approximately 350-fold fewer sperm than wild-type mice, and the remaining sperm completely lack or have very short flagella. The short flagella rarely have axonemes but assemble ectopic microtubules and outer dense fibers and accumulate improperly assembled fibrous sheath proteins. Thus IFT is essential for the formation but not the maintenance of mammalian sperm flagella.

Pacemaker-induced transient asynchrony suppresses heart failure progression

Mon, 06/27/2016 - 3:33pm

Uncoordinated contraction from electromechanical delay worsens heart failure pathophysiology and prognosis, but restoring coordination with biventricular pacing, known as cardiac resynchronization therapy (CRT), improves both. However, not every patient qualifies for CRT. We show that heart failure with synchronous contraction is improved by inducing dyssynchrony for 6 hours daily by right ventricular pacing using an intracardiac pacing device, in a process we call pacemaker-induced transient asynchrony (PITA). In dogs with heart failure induced by 6 weeks of atrial tachypacing, PITA (starting on week 3) suppressed progressive cardiac dilation as well as chamber and myocyte dysfunction. PITA enhanced beta-adrenergic responsiveness in vivo and normalized it in myocytes. Myofilament calcium response declined in dogs with synchronous heart failure, which was accompanied by sarcomere disarray and generation of myofibers with severely reduced function, and these changes were absent in PITA-treated hearts. The benefits of PITA were not replicated when the same number of right ventricular paced beats was randomly distributed throughout the day, indicating that continuity of dyssynchrony exposure is necessary to trigger the beneficial biological response upon resynchronization. These results suggest that PITA could bring the benefits of CRT to the many heart failure patients with synchronous contraction who are not CRT candidates.

An approach to improve the resolution of helical filaments with a large axial rise and flexible subunits

Mon, 06/27/2016 - 3:33pm

Single particle analysis is widely used for three-dimensional reconstruction of helical filaments. Near-atomic resolution has been obtained for several well-ordered filaments. However, it is still a challenge to achieve high resolution for filaments with flexible subunits and a large axial rise per subunit relative to pixel size. Here, we describe an approach that improves the resolution in such cases. In filaments with a large axial rise, many segments must be shifted a long distance along the filament axis to match with a reference projection, potentially causing loss of alignment accuracy and hence resolution. In our study of myosin filaments, we overcame this problem by pre-determining the axial positions of myosin head crowns within segments to decrease the alignment error. In addition, homogeneous, well-ordered segments were selected from the raw data set by checking the assigned azimuthal rotation angle of segments in each filament against those expected for perfect helical symmetry. These procedures improved the resolution of the filament reconstruction from 30 A to 13 A. This approach could be useful in other helical filaments with a large axial rise and/or flexible subunits.

The cMyBP-C HCM variant L348P enhances thin filament activation through an increased shift in tropomyosin position

Mon, 06/27/2016 - 3:33pm

Mutations in cardiac myosin binding protein C (cMyBP-C), a thick filament protein that modulates contraction of the heart, are a leading cause of hypertrophic cardiomyopathy (HCM). Electron microscopy and 3D reconstruction of thin filaments decorated with cMyBP-C N-terminal fragments suggest that one mechanism of this modulation involves the interaction of cMyBP-C's N-terminal domains with thin filaments to enhance their Ca(2+)-sensitivity by displacement of tropomyosin from its blocked (low Ca(2+)) to its closed (high Ca(2+)) position. The extent of this tropomyosin shift is reduced when cMyBP-C N-terminal domains are phosphorylated. In the current study, we have examined L348P, a sequence variant of cMyBP-C first identified in a screen of patients with HCM. In L348P, leucine 348 is replaced by proline in cMyBP-C's regulatory M-domain, resulting in an increase in cMyBP-C's ability to enhance thin filament Ca(2+)-sensitization. Our goal here was to determine the structural basis for this enhancement by carrying out 3D reconstruction of thin filaments decorated with L348P-mutant cMyBP-C. When thin filaments were decorated with wild type N-terminal domains at low Ca(2+), tropomyosin moved from the blocked to the closed position, as found previously. In contrast, the L348P mutant caused a significantly larger tropomyosin shift, to approximately the open position, consistent with its enhancement of Ca(2+)-sensitization. Phosphorylated wild type fragments showed a smaller shift than unphosphorylated fragments, whereas the shift induced by the L348P mutant was not affected by phosphorylation. We conclude that the L348P mutation causes a gain of function by enhancing tropomyosin displacement on the thin filament in a phosphorylation-independent way.

Use of HCA in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns

Mon, 06/27/2016 - 3:33pm

Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production.

Finding Shangri-La: Limiting the Impact of Senescence on Aging

Mon, 06/27/2016 - 3:32pm

Senescence plays an important role in the age-associated decline of tissue functions. Recent studies now show that targeting senescent cells can enhance the functions of stem/progenitor cells in aged mice and extend lifespan.

Phosphorylation and calcium antagonistically tune myosin-binding protein C's structure and function

Mon, 06/27/2016 - 3:32pm

During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C's N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain's extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C's inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C's calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C's phosphorylation state.

Targeting the chromatin remodeling enzyme BRG1 increases the efficacy of chemotherapy drugs in breast cancer cells

Mon, 06/27/2016 - 3:32pm

Brahma related gene product 1 (BRG1) is an ATPase that drives the catalytic activity of a subset of the mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is overexpressed in most human breast cancer tumors without evidence of mutation and is required for breast cancer cell proliferation. We demonstrate that knockdown of BRG1 sensitized triple negative breast cancer cells to chemotherapeutic drugs used to treat breast cancer. An inhibitor of the BRG1 bromodomain had no effect on breast cancer cell viability, but an inhibitory molecule that targets the BRG1 ATPase activity recapitulated the increased drug efficacy observed in the presence of BRG1 knockdown. We further demonstrate that inhibition of BRG1 ATPase activity blocks the induction of ABC transporter genes by these chemotherapeutic drugs and that BRG1 binds to ABC transporter gene promoters. This inhibition increased intracellular concentrations of the drugs, providing a likely mechanism for the increased chemosensitivity. Since ABC transporters and their induction by chemotherapy drugs are a major cause of chemoresistance and treatment failure, these results support the idea that targeting the enzymatic activity of BRG1 would be an effective adjuvant therapy for breast cancer.

Global gene expression profiling of JMJD6- and JMJD4-depleted mouse NIH3T3 fibroblasts

Mon, 06/27/2016 - 3:32pm

Emerging evidence suggests Jumonji domain-containing proteins are epigenetic regulators in diverse biological processes including cellular differentiation and proliferation. RNA interference-based analyses combined with gene expression profiling can effectively characterize the cellular functions of these enzymes. We found that the depletion of Jumonji domain-containing protein 6 (JMJD6) and its paralog protein Jumonji domain-containing protein 4 (JMJD4) individually by small hairpin RNAs (shRNAs) slowed cell proliferation of mouse NIH3T3 fibroblasts. We subsequently performed gene expression profiling on both JMJD6- and JMJD4-depleted mouse NIH3T3 fibroblasts using the Affymetrix GeneChip Mouse Exon 1.0 ST Array. Here we report the gene profiling datasets along with the experimental procedures. The information can be used to further investigate how JMJD6 and JMJD4 affect gene expression and cellular physiology.