bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhancer regulation by H3K4me1 methyltransferases MLL3/MLL4
Authors: Ge Kai 
Published: May 2022

Enhancers control cell type‐specific gene expression and are marked by H3K4me1. Active enhancers are further marked by H3K27ac. We identified MLL3/MLL4 as major H3K4me1 methyltransferases and CBP/p300 as the H3K27 acetyltransferases in mammalian cells (EMBO J 2011; eLife 2013). During differentiation of adipocytes, myocytes and ES cells, MLL3/MLL4 co‐localize with lineage‐determining transcription factors (LDTFs), CBP/p300, Brd4 and MED1 on active enhancers. MLL3/MLL4 are required for enhancer activation, enhancer‐promoter interaction, cell type‐specific gene expression, and cell differentiation (eLife 2013; PNAS 2016; Nat Comm 2017). MLL3/MLL4 control cell differentiation by orchestrating CBP/p300‐mediated enhancer activation (NAR 2017). Ectopic expression of H3.3K4M, an inhibitor of H3K4 methylation, or deletion of the enzymatic SET domain, destabilizes MLL3/MLL4 proteins, prevents enhancer activation, and impairs cell differentiation and tissue development (NAR 2019).Using proteomic approaches, we found that endogenous MLL4 complex associates with SWI/SNF chromatin remodeling complex components in cells. Using adipogenesis as a model system, we establish an interdependent relationship between SWI/SNF complex BAF and MLL4 in promoting cell type‐specific enhancer activation by LDTFs, which rectifies seemingly conflicting results from previous studies (Nat Comm 2021).We showed that MLL4 protein, rather than MLL4‐mediated H3K4me1, controls p300 recruitment to enhancers during ES cell differentiation, suggesting that MLL4 regulates enhancer activation independent of H3K4me1 (PNAS 2016). We have generated enzyme‐dead MLL3/MLL4 double knockin ES cells and mice by CRISPR. Initial analyses indicate that MLL3/MLL4 regulate ES cell differentiation and mouse embryonic development through both enzymatic activity‐dependent and ‐independent mechanisms.
Applying Crystallography and 19F NMR to investigate dynamics and partner protein interactions in a long chain Flavodoxin
Authors: Khan Sharique , Ansari Ahmadullah , Miller Anne‐Frances 
Published: May 2022

Flavodoxin (Fld) is a small FMN containing protein that is involved in single electron transfer. The long‐chain flavodoxin in Rhodopseudomonas palustris bacteria replaces ferredoxin as a low‐potential electron carrier when iron is scarce. Thus it is proposed to interact with the bifurcating electron transfer flavoprotein (ETF) that yields low‐potential electrons. A surface loop on Fld interacts with another of Fld's partner proteins, so we hypothesize that it also mediates Fld's interaction with ETF. To monitor interactions with ETF directly and investigate dynamics in this loop, we are using 19F NMR in solution. 19F is hyperresponsive to changes in its chemical environment with a chemical shift range of >300 ppm. To provide a static reference point and assess structural heterogeneity, we are also exploiting X‐ray crystallography.In this study we selectively fluorinated the five tyrosine residues in Fld. We obtained resonance assignments from 19F spectra of Fld variants in which individual tyrosine residues have been replaced. We obtain well resolved signals for each residue, but the resonances' linewidths indicate dynamics that affects some resonances more than others. Y90 residue displays two resonances demonstrating two different conformations that interconvert slowly on an NMR time scale. Meanwhile the crystal structure solved at 2.1Å resolution reveals two molecules per asymmetric unit providing two perspectives on the details of the structure. The crystal symmetry is monoclinic in contrast to most of the other Flds, which are orthorhombic. Interestingly, the long loop bearing Y121 and Y123 is not well resolved in chain B of the crystal structure, and the NMR line of Y123 is exceptionally broad, both indicating that the loop is dynamic and capable of altering its conformation to accomodate binding to a partner protein.Future directions include monitoring the changes in the 19F NMR of the Fld when titrated with the partner protein, temperature dependence of the NMR spectrum and relaxation studies to evaluate time scales of motions.
Heterologous expression of methyl‐coenzyme M reductase reveals the importance of organism‐specific accessory proteins for proper assembly
Authors: Gendron Aleksei , Allen Kylie 
Published: May 2022

Methyl‐coenzyme M reductase (MCR) is the key rate‐determining enzyme of methanogenesis as well as the anaerobic oxidation of methane, the essential energy metabolisms of methanogenic archaea and anaerobic methanotrophs (ANME), respectively. MCR is a dimer of heterotrimers with a 2α, 2β, 2γ configuration, and requires the nickel tetrapyrrole prosthetic group, coenzyme F430. The requirement of a unique cofactor, various unusual post translational modifications, and many remaining questions surrounding assembly and activation of MCR has so far largely limited in vitro experiments to native enzymes. To allow further investigation into the catalytic properties and mechanistic aspects of different MCRs, as well as facilitate the development of optimized biocatalytic systems to convert methane to more usable liquid fuels and other valuable compounds, we are developing methods for the heterologous expression of recombinant MCRs in Methanococcus maripaludis, a model methanogen with robust genetic tools.In methanogens, MCR is encoded in the highly conserved MCR gene cluster mcrBDCGA, which encodes two accessory proteins (McrD and McrC) in addition to the MCR‐encoding genes (McrA, McrB, and McrG). The accessory proteins are proposed to be involved in the assembly and activation of MCR. Interestingly, most ANME lack one or more accessory proteins in their MCR gene clusters. We have created a series of MCR expression constructs containing the MCR operons from several ANME organisms as well as several methanogens, with and without accessory protein(s). All constructs contain a his‐tag on the C‐terminus of McrA which allows the purification and determination of proper assembly. We have successfully purified a recombinant ANME‐2d MCR that is assembled and binds F430, whereas in the case of the ANME‐1 MCR, we can only recover the his‐tagged McrA. Since the ANME‐2d MCR operon contains McrD and the ANME‐1 operon does not, this result supports the importance of McrD for proper assembly in vivo. Combined with other results from the expression of recombinant methanogenic MCRs, our results indicate that MCR accessory proteins are organism specific. Current work is focused on demonstrating the role of McrD through in vitro binding studies as well as further elucidating the importance of mcrDand mcrC for recombinant expression of diverse MCRs.
Profiling Metabolic and Signaling Phenotype of Bladder Cancer Cell Lines
Authors: Ducci Giacomo , Pasquale Valentina , Rota Stefano , Arrigoni Edoardo , Campioni Gloria , Ventura Elisa , Giordano Antonio , Morrione Andrea , Sacco Elena , Vanoni Marco 
Published: May 2022

Bladder cancer (BC) is one of the most common malignancies worldwide[1]. Most patients are diagnosed with non‐muscle invasive BC, which is associated with frequent recurrence. The prognosis for early‐stage bladder tumors is generally good; however, patients often progress to invasive disease resulting in a much less favorable outcome[2,3]. 3D cultures constitute more clinically relevant models than monolayers for studying cancer as spheroids recapitulate in vivo structures, cell‐cell interactions, nutrients and oxygen gradients[4]. In this study we conducted a comparative analysis of metabolism and cellular features of a panel of BC cell lines covering different stages/grades, grown in monolayer cultures and spheroids. Samples were analysed at different timepoints for cellular features, like spheroid morphology and viability, by quantitative imaging using Operetta CLS high‐throughput microscope. Here we show that high‐grade cell lines had an increased capacity to form viable spheroids. In addition, we used Seahorse technology to assess bioenergetic parameters, e.g. mitochondrial respiration and glycolysis, and demonstrated that high‐grade cell lines grown in monolayer cultures present higher basal and maximal glycolysis compared to low‐grade cells, while mitochondrial respiration showed no obvious correlation with pathological grade. No obvious correlation was observed in the use of either mitochondrial respiration or glycolysis and pathological grade during spheroid formation, although some high‐grade cell lines, like HT1376, showed a preferential use of respiration for ATP production. Our laboratories have previously established the role of the progranulin/EphA2 axis in regulating motility, invasion and in vivo tumour formation in bladder cancer[6] but whether this pathway regulates the capacity of bladder cancer cells to form spheroids has not been established. Significantly, we provide now preliminary evidence that genetic targeting of either progranulin or EphA2 by CRISPR/Cas9 approaches modulated spheroid formation of invasive bladder cancer cells. Understanding the pathways and mechanisms regulating spheroid formation and metabolic changes of bladder cancer cells might contribute to the identification of novel targets and specific therapies for bladder cancer.1. C. Yeung, et al (2014), Pharmacoeconomics, 32, 1093–1104.2. M. Babjuk, et al. (2017), Eur. Urol., 71, 447–461.3. J. Alfred Witjes, T. Lebret, E. M. Compérat, N. C. Cowan, et al. (2017), Eur. Urol., 71, 462–475.4. I. Vasyutin, L. Zerihun, C. Ivan, A. Atala. (2019), Anticancer Res., 39, 1105–1118.5. V. Pasquale, G. Ducci, G. Campioni, A. Ventrici, et al. (2020), Cells, 9, 1–26.6. S. Buraschi, T. Neill, S.‐Q. Xu, C. Palladino, et al. (2020), Matrix Biol., 93, 10–24.
Pilot Study on the Influence of Race and Sex on beat‐to‐beat Blood Pressure Variability in Healthy Young Adults
Authors: Jeong Soolim , Linder Braxton A. , Tharpe McKenna A. , Hutchison Zach J. , Barnett Alex M. , Culver Meral N. , Nichols Olivia I. , Fuller‐Rowell Thomas E. , Robinson Austin T. 
Published: May 2022

BACKGROUNDAugmented beat‐to‐beat blood pressure (BP) variability is prognostic of target organ damage and cardiovascular events. A recent study demonstrated greater BP variability in healthy young Black males compared to White males, but whether these differences appear in females remains unclear. Therefore, we sought to examine race and sex differences in beat‐to‐beat BP variability in healthy young Black and White adults.METHODS52 adults including 13 Black females (age 21.5 ± 0.8 yrs, BMI 27.0 ± 4.2 kg/m2, resting BP 126 ± 10/78 ± 12 mmHg; Mean ± SD), 13 White females (21.2 ± 0.7 yrs, 24.0 ± 3.8 kg/m2, 116 ± 8/66 ± 11 mmHg), 9 Black males (21.6 ± 0.7 yrs, 25.5 ± 4.3 kg/m2, 131 ± 11/75 ± 11 mmHg), and 17 White males (21.5 ± 0.7 yrs, 25.0 ± 3.7 kg/m2, 123 ± 11/67 ± 9 mmHg) participated in this study. Non‐invasive beat‐to‐beat BP was continuously measured at the participant’s middle finger via photoplethysmography (Finapres NOVA) for 10 minutes while sitting semi‐recumbent in a dimly lit room. The finger pressure was calibrated using resting oscillometric brachial BP. Heart rate was measured via 3‐lead electrocardiogram (ECG) and BP was gated to R‐waves from the ECG signal. BP variability was assessed with the average real variability (ARV) index and standard deviation (SD) of all BP readings. Statistical procedures included two‐way analysis of variance (sex x race) with Bonferroni post‐hoc test when appropriate (a priori α < 0.05).RESULTSNo significant differences existed for systolic BP ARV (sex p= 0.85, race p= 0.41, interaction p= 0.63; Black females 2.02 ± 0.64, White females 1.84 ± 0.44, Black males 1.98 ± 0.24, White males 1.93 ± 0.44) or diastolic BP ARV (sex p= 0.31, race p= 0.76, interaction p= 0.51). For systolic BP SD, there was a significant main effect of sex (p= 0.008) suggesting greater systolic BP variability in males than females, and a trend for race by sex interaction (p= 0.08) but no difference for race (p= 0.79); (Black females 4.80 ± 1.07, White females 5.80 ± 2.10, Black males 7.77 ± 3.89, White males 6.42 ± 1.74). However, there were no significant differences for diastolic BP SD (sex p= 0.19, race p= 0.55, interaction p= 0.46).CONCLUSIONThese preliminary findings suggest males have significantly greater systolic BP variability than females from systolic BP ARV. No significant differences were identified for diastolic BP SD and BP ARV (systolic and diastolic) by race and sex, but additional data are needed.
In vitroevaluation of anti‐aging and regenerative properties of dermatan sulfate for skin care
Authors: Galvez‐Martin Patricia , Martinez‐Puig Daniel , Soto‐Fernández Cristina , Romero‐Rueda Jessica 
Published: May 2022

IntroductionDermatan sulfate (DS) is a linear polysaccharide classified as a sulfated glycosaminoglycan (GAGs). GAGs form an integral part of the skin and make up 0.1–0.3% of total skin weight. DS is covalently attached to the core proteins to form proteoglycans (PGs). PGs predominates in dermis presenting higher level of DS than other sulfated GAGs.The intrinsic aging processes of the skin entails the reduction of total sulfated GAGs levels causing a thinning of the dermis and modifying the quality of the skin. DS possess strong biological activity that can be modulated to improve skin quality.This study was performed to determine the effects of DS on Human Dermal Fibroblasts (HDF) to evaluate its anti‐aging and regenerative properties.MethodsHuman Dermal Fibroblasts (HDF) were isolated from a sample of the foreskin of a neonatal caucasian donor (Lonza). The effect of the DS on cell proliferation was assessed at 24 h and 48 h in HDF, using the method bromodeoxyuridine (BrdU) incorporation, detected by ELISA. The cell migration was evaluated in HDF at 48 h using the Oris™ Cell Migration Assay (Platypus Technologies). To monitor the migration progress, the cells were labelled with calcein. The wound healing capacity of DS was evaluated in HDF using the scratch test. The inductive capacity on Extracellular matrix proteins (ECMp) synthesis was evaluated by immunolocalization‐ELISA quantifying type I and type III collagen and elastin.ResultsThe results showed that DS induced HDF proliferation after 24 h and 48 h of treatment at all tested concentrations. A dose‐dependent increase in proliferation was observed after 48 h of treatment of these cells, reaching a maximum increase of 5.10 (± 0.82) fold at the highest concentration tested (5 mg/mL) compared to uHDF. Regarding HDF cell migration, no significant differences were observed at 24 h after treatment with DS.The results showed a significant improvement in the in vitro wound healing capacity of HDF treated with 0.25 and 1 mg/mL of DS, with an increase in the healed area of 30.28% (± 4.27%) and 23.76% (± 8.87%) respectively, compared to uHDF.Regarding the ECMp synthesis‐stimulating capacity of DS, the results showed a significant increase of both type III Collagen (50.98 ± 34.48% increase) and Elastin (32.25 ± 2.61%) levels in HDF treated for 72 h with 1 mg/mL of DS compared to uHDF. HDF treated with 0.25 mg/mL also showed higher type I Collagen production than uHDF, although the increase (30.96 ±7.04%) was not statistically significant.ConclusionsThe results showed that DS improves the proliferation and regeneration of HDF present in dermis. In addition, DS increases the synthesis of ECMp as collagen type III and elastin, promoting the maintenance and functionality of the dermis. All these properties support the regenerative and anti‐aging activity of DS acting against intrinsic aging in dermis.
Anatomical Substract of the Temporal Muscle Myofascial Trigger Points in Fresh Cadavers
Authors: Garrido Luis C. F. , Jacomo Alfredo L. , Saleh Samir O. , Hojaij Flávio , Andrade Mauro , Akamatsu Flávia E. 
Published: May 2022

BackgroundMyofascial syndrome is a common cause of chronic musculoskeletal pain and is characterized by myofascial trigger points (MTPs). MTPs are clinically identified by palpation of a muscle or fascial taut band. Although pathophysiology of MTPs remains unclear, coincidence between them and muscle motor plate at the innervation zone (IZ) have been reported. The temporal muscle is related to headache and temporomandibular disorders and pain radiates from hyperalgesic points to distant areas symptomatic patients. Clinical reports usually describe four trigger points located in the temporal muscle and we aimed to identify anatomical correlation with muscle innervation.MethodsWe dissected the temporal muscles from five fresh cadavers to observe the exact point where nerve fibers penetrated the muscle belly. As muscle size varies among individuals, we calculated the relative entry point of the nerve into the muscle by defining six different areas in the muscle belly: three superior (I, II and III) and three inferior (IV, V and VI), beginning at the posterior margin of the muscle. Statistical analysis of anatomical data was obtained by Poisson distribution and logarithm link function followed by Bonferroni multiple comparisons. The comparison of points between the sides was made by Student’s t‐test. IBM‐SPSS for Windows version 20.0 software was used to perform the analysis, and Microsoft Excel 2003 software was used to tabulate the data. Data are presented as the mean ± standard deviation, and the level of significance was adjusted to 5% (p <0.05) for all tests.ResultsThe number of temporal nerve branches did not differ between sides. Areas II and V had the largest number of temporal nerve entry sites, with a mean of 3 (22%) and 5 (36%) points, respectively. Areas III and VI had a mean of 2 sites both (14%) and areas I and IV, one (7%).ConclusionIn accordance with the clinical literature, the branches of the temporal nerve in the temporal muscle corresponded to the described areas of the myofascial trigger points (MTPs). Anatomical correlations between MTPs may be a useful tool for a better understanding of the physiopathology of these disorders and may provide a rational basis for their treatment.
Resonance Raman of the nitrite reductase action of heme‐copper oxidoreductases
Authors: Varotsis Constantinos , Tselios Charalambos , Pinakoulaki Eftychia 
Published: May 2022

Several themes have emerged in the studies of biological control of nitrite and nitric‐oxide chemistry.[1‐2] Apparently, evolution has clearly selected means by which to take advantage of the unique NO2 and NO chemistry, and a number of enzymes have evolved that are able to activate NO2/NO kinetically, and control their redox chemistry. In bacterial respiration the reduction of NO2/NO to N2O by terminal oxidoreductases supports the hypothesis of a common evolutionary origin of bacterial denitrification and aerobic respiration. Comparison of the enzymes responsible for the activation of NO in denitrification and bacterial respiration may provide the means to identify conserved structural features, which can be assumed to be involved in basic functions common to both classes of enzymes. The most commonly encountered metals in the biological reduction of NO2 to NO and subsequently to N2O are iron and copper in mononuclear, binuclear, and dinuclear centers. In P450Nor the electrons needed for the reduction of NO are directly transferred from NADH. This way, a single molecule of NO binds at the heme Fe, and addition of two electrons to heme Fe3+‐NO yields the two electron reduced species Fe2+‐N=O‐. A second NO moleculeattacks the N atom of the ferrous‐NO species to transiently yield hyponitrite (HONNO–), and thus the N‐N bond formation. Cleavage of the N‐O bond produces the ferric enzyme, N2O and H2O. Resonance Raman studies of the NO2‐ligand binding properties of heme‐copper oxidases will be presented.References.

Discrete Ligand Binding and Electron Transfer Properties of ba3‐Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and …C Koutsoupakis, T Soulimane, C. Varotsis Accounts of chemical research 52 (5), 1380‐139, 2019.
The structure of a ferrous heme‐nitro species in the binuclear heme a3/CuBcenter of ba3‐cytochrome c oxidase as determined by resonance Raman spectroscopy A. Loullis, MR Noor, T Soulimane, E Pinakoulaki Chemical Communications 51 (2), 286‐289, 2015.
AJAP1 Gene Silencing Alters Protein Expression in Epithelial Cells
Authors: Gerick Alexander , Osuji Nnedi , Jeffers Cameron , Hatcher Cathy J. 
Published: May 2022

IntroductionThe epicardial layer of the heart is formed during cardiovascular development from a transitory structure known as the proepicardium (PE). Proepicardial cells migrate out of the PE to surround and adhere to the myocardium to form a layer of epithelial cells that compose the epicardium. A subset of the epicardial cells delaminate and migrate into the subepicardial space where they undergo epithelial‐mesenchymal transition (EMT) to become epicardial‐derived cells (EPDCs). These EPDCs invade the myocardium and differentiate into a subset of cells that make up the coronary vessels. During this entire process, the epicardium receives input from several transcription factors including Tbx5. Mice with a conditional deletion of Tbx5 exhibit impaired epicardium formation and coronary vessel development. Tbx5‐deficient mouse hearts have a significant decrease in mRNA expression of adherens junction associated protein (Ajap1). Our studies show that knockdown of endogenous AJAP1 (AJAP1 KD) mRNA expression in epithelial cells leads to increased cell adhesion, reduced cell migration, enhanced expression of epithelial markers of EMT, and altered expression of transcripts associated with adherens junctions, tight junctions and EMT. Specifically, our qPCR array data shows a significant alteration in ZEB2 mRNA expression in AJAP1 KD cells versus negative control cells.Study ObjectiveThe goal of this study was to examine the role of AJAP1 in regulating protein expression of ZEB2 as it may be necessary for epicardium formation. Based on previous cell function and mRNA expression data, we hypothesize that the ZEB2 protein is an important functional mediator of AJAP1 in epithelial cells for undergoing EMT.MethodsWe utilized the primary human mammary epithelial cell line (HMEpiC) as an in vitro system to examine contributions of AJAP1 to epithelial cell biology. We silenced AJAP1 mRNA expression in HMEpiCs using small interfering RNAs and examined protein expression of ZEB2. We compared ZEB2 expression in negative control versus AJAP1 KD HMEpiCs.ResultsReduced expression of AJAP1transcripts produced noticeable changes in protein expression of ZEB2 in epithelial cells, and it had significant effects on expression of adherens junction‐related transcripts. Western blot analysis revealed an increase in ZEB2 protein expression in AJAP1 KD versus negative control HMEpiCs.ConclusionOur data indicate that AJAP1 contributes to epithelial cell function, and it does this potentially by mediating ZEB2 protein expression. ZEB2 is known to regulate EMT, which is a biological process that is crucial for the formation of the epicardium and its subsequent formation of the coronary vasculature.