bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kv1.3 channel is a potential marker for B acute lymphoblastic leukemia
Authors: Valle Juan Salvador , Castellanos Ramon , Olivas Miguel Ángel , Pottosin Igor , Dobrovinskaya Oxana , Schnoor Michael 
Published: April 2020

Acute lymphoblastic leukemia (ALL) is a public health problem worldwide and is the leading cause of death in children under 14 years. Current ALL treatment is efficient in 80% of cases, but 5% of patients may relapse, with a life expectancy less than 5 years. Thus, the search for new therapeutic targets and diagnostic biomarkers needs to be continued. In ALL, different mutations have been identified providing leukemic cells with high metabolic rate, survival due to evasion of apoptosis, proliferation, and drug resistance. Many of these events are directly or indirectly related to Ca2+ signaling that relies on plasma membrane Ca2+ channels. The magnitude and duration of Ca2+ signals are regulated by the function of K+ channels in the same membrane. In healthy lymphocytes, two K+ channels are expressed, the voltage‐sensitive Kv1.3 and the calcium‐activated KCa3.1 ones. Kv1.3 channel in healthy B and T cells regulate their membrane potential, cell volume and entry into the cell cycle, whereas KCa3.1 channel activity is associated with cytokine secretion, differentiation and proliferation. T‐ALL cell lines CCRF‐CEM and MOLT‐3 are reminiscent of activated T‐cells, with a high density of Kv1.3 and KCa3.1 currents. Much less is known about K+ channels expression in B‐ALL. Here, we analyzed the expression of Kv1.3 channel in B‐ALL cells lines, REH and RS4:11, and freshly isolated CD19+ cells from healthy donors, activated with 1 μg/mL of PMA and 500 nM of ionomicin. Kv1.3 mRNA levels were similar in all cell models as revealed by real time RT‐PCR. Western blot assays confirmed the presence of the Kv1.3 protein in all cell models. Immunofluorescence assays localized Kv1.3 channel in the plasma membrane of CD19+ as well as of REH and RS4:11 cells. Patch‐clamp recordings in whole cell configuration revealed typical voltage‐dependent Kv1.3 activity in CD19+ cells, which was totally abolished by 1 nM of margatoxin, a specific blocker of Kv1.3 channel. However, zero Kv current was detected in REH and RS4:11. Thus, albeit being present in the plasma membrane in B‐ALL cells, Kv1.3 channel protein does not mediate K+ fluxes. It may play additional non‐canonical roles, e.g. in signaling. Its loss of ionotropic function may be linked to the leukemic event and could serve as a reliable diagnostic tool, whereas conventional RT‐PCR method will be ineffective.Support or Funding InformationSupported by CINVESTAV (MS) and CONACyT grants CB‐238689 (OD), CB‐220793 (IP), Fronteras in Ciencia 140 (OD).
Mutants in mitochondrial DNA repair genes in Arabidopsis thaliana generated by using CRISPR‐Cas9
Authors: Campbell David , Wright Janet , Christensen Alan 
Published: April 2020

The genomes of plant mitochondria exhibit unique mutation rates and structural variability. Compared to their animal counterparts, plant mitochondria have low mutation rates, high recombination rates, and non‐circular genomes. Recent evidence suggests the prevalence of double‐strand break repair (DSBR) in plant mitochondria as one possible cause of these traits. Interestingly, all of the genes required for plant mitochondrial DSBR are located in the nucleus.In order to study DSBR, it is necessary to be able to reliably generate single and double‐mutants for DSBR genes. Using traditional methods, such as T‐DNA insertions, double mutant generation has proven cumbersome due to the occasional difficulty in PCR‐based identification of single mutants, as well as the low map distance between several genes of interest. CRISPR‐induced knockouts allow us to overcome some of these difficulties and generate a greater variety of mutants in a shorter period of time. In this study, we selected five proteins related to the processing of double strand breaks (DSBs) in plant mitochondria for mutagenesis: MSH1, RECA3, RNaseH1, SUV3, and OTP87. We prepared CRISPR Cas9 vectors to produce knockouts for each of these genes in Arabidopsis thaliana. The reliable production of mutants for these 5 genes will allow for in depth study of DSBR in the future. CRISPR Cas9 constructs were designed to produce two guide RNAs for each gene, targeting two different exons in the gene of interest, to give a better chance of successful knockouts, or deletions, between the two sites. These constructs were then transformed into Agrobacterium. Successful transformants were selected using a spectinomycin resistance gene in the construct. We then used a standard floral dip protocol to generate mutant Arabidopsis plants. PCR analysis confirmed Arabidopsis knockout mutations in all five lines.The mutants generated in this study will allow us to investigate several avenues of research on DSBR. We plan to expose plants with knockouts in either one or two DSBR genes to ciprofloxacin, an antibiotic which induces double strand breaks in plant mitochondrial DNA. From there we can observe the mitochondrial genome recombination rates using PCR and Illumina sequencing for each of the mutants.Support or Funding InformationThis work was supported in part by grants from the National Science Foundation to A.C.C. (MCB‐1413152 and MCB‐1933590) and a grant from the UNL UCARE program (26‐0101‐8001‐001)
Homing Endonuclease and Protein Splicing Activity of Inteins from Thermophilic Archaea
Authors: Tarrant Seanan P. , Jankowski Bella J. , Comeau Hannah Y. , Wibben Amanda C. , Mills Kenneth V. 
Published: April 2020

Protein splicing is a post translational, self‐catalyzed reaction by which an intein removes itself from flanking polypeptides and ligates those polypeptides together. We are interested in the splicing and homing endonuclease activities of the intein that interrupt the DNA polymerase II in the extreme thermophiles Thermococcus barophilus and Thermococcus kodakarensis. Given that T. kodakarensis is a surface‐dwelling organism whereas the T. barophilus is a piezophile that lives in deep‐sea thermal vents, yet both inteins are highly similar in sequence, we are interested in how temperature and pressure may affect enzyme activity. Additionally, we are interested in the role of the two conserved C‐terminal residues of the intein. We have found that the homing endonuclease domain of the T. kodakarensis intein is more active at a lower concentration and lower temperatures than that of the T. barophilus intein. Protein splicing of both inteins can be induced by in vitro incubation of an isolated, unspliced precursor at elevated temperature, which may also be influenced by hydrostatic pressure.Support or Funding InformationThis work was supported by NSF grant MCB‐1517138, a Henry Dreyfus Teacher‐Scholar Award, and by NIH Grant 1R15GM132817‐0
Exercise Training Restores K+ Channel Contributions to Hydrogen Peroxide‐Mediated Dilation Independent of PKG Dimerization in Collateral‐Dependent Coronary Arterioles
Authors: Johnson Kalen A. , Ramirez Cristina R. , Bray Jeffrey F. , Heaps Cristine L. 
Published: April 2020

In coronary artery disease (CAD), the ability of the coronary circulation to deliver blood to meet oxygen demands of the heart is often impaired. Exercise training has been established to mitigate the negative effects of CAD by preserving reactivity of the coronary microcirculation. We have previously reported that exercise increases the contribution of hydrogen peroxide (H2O2) to enhanced endothelium‐dependent vasodilation in collateral‐dependent coronary arterioles. Others have shown that cGMP‐dependent protein kinase (PKG) can be activated via oxidation by H2O2, inducing dimer formation. The PKG dimer has been shown to act on substrates, such as K+ channels, to contribute to vascular smooth muscle relaxation and vasodilation. In the current study, we tested the hypothesis that exercise training‐enhanced H2O2‐induced vasodilation is mediated through enhanced PKG dimerization and subsequent activation of Kv and BKca channels in coronary arterioles isolated from ischemic myocardium. An ameroid occluder was surgically placed on the proximal left circumflex coronary artery of female Yucatan miniature swine to induce gradual occlusion while the left anterior descending artery was left unoccluded to serve as a control. Eight weeks postoperatively, pigs were assigned to sedentary (n=14) or exercise training (n=13; progressive treadmill training for 13 weeks) regimens. Coronary arterioles (75–150 μm) and small arteries (250–400 um) were isolated from myocardium of the left circumflex and left anterior descending regions. Immunoblot analysis indicated that H2O2 induced PKG dimer formation in both porcine coronary arterioles and small arteries. Chronic coronary artery occlusion appeared to diminish H2O2‐mediated PKG dimer formation in collateral‐dependent arterioles and the reduced dimerization persisted with exercise training. H2O2‐mediated vasodilation tended to be impaired in arterioles isolated from the collateral‐dependent regions of sedentary pigs and was partially restored with exercise training. It also appears that both Kv and BKCa channels contributed to H2O2‐mediated dilation after exercise training. Our data demonstrate that H2O2 stimulates PKG dimer formation in both porcine coronary arterioles and small arteries. Contrary to our hypothesis, H2O2‐induced PKG dimerization is not enhanced by exercise training in either nonoccluded or collateral‐dependent arterioles. However, both Kv and BKCa channels appear to contribute to exercise training‐enhanced H2O2‐mediated dilation in collateral‐dependent coronary arterioles.Support or Funding InformationNational Institutes of Health R01‐HL139903
Drawing Our Feelings: An Approach to the First Day of Cadaver Dissection
Authors: Zumwalt Ann 
Published: April 2020

RATIONALEThe cadaver dissection experience can elicit intense emotions in students who are taking anatomy for the first time. Students are often disinclined to share these feelings with others because they believe they are the only ones experiencing such feelings and/or do not want to appear less able than their student colleagues to cope. Discussing anticipatory feelings about the dissection process can reduce students’ feelings of isolation, imposter syndrome, and simple nervousness by revealing that most students share similar experiences and that there is a range of normal anticipatory feelings.METHODSFor the last 10 years at Boston University School of Medicine we have employed the following exercise on the first day of anatomy: Students are asked to draw their answers to the question, “How do you feel about the experience of dissecting a human body?” After the drawing exercise the course director presents a slideshow of drawings from the same exercise by previous classes. The slideshow demonstrates that there are common themes exhibited by students in every class: mixed feelings, all alone/nervous, physiologic reactions, journey, knowledge, enthusiasm, and the humanity of their donor. The students are then invited to share comments or ask questions in a large group discussion format. Participation in this session is typically lively, likely because the drawing exercise and slideshow demonstrate to individual students that their responses are normal and expected.RESULTS & CONCLUSIONSThe combination of exercises illustrates for students that there is a variety of normal reactions to the dissection experience. The exercise opens opportunities for dialogue that are otherwise less likely to occur. The overall result is a culture within the class that is open to acknowledging and discussing the complex feelings elicited by dissection.
DMSO inhibits human cancer cells and downregulates the expression of cdk2 and cyclin A
Authors: Villarroel Ariana , Duff Angela , Hu Tang 
Published: April 2020

Dimethyl sulfoxide (DMSO) has many applications in basic research. For example, it has been broadly used as an efficient solvent for many chemicals with poor solubility in water and as a common cryoprotectant (5–10%) that prevents cellular damage during freeze‐thaw process. DMSO is also a prescription medicine that provides rapidly, temporary relief of pain in patients with headache, arthritis, and skeletal tissue injuries. However, little known is its effect on cell viability and proliferation. In this study, we found that DMSO significantly inhibited proliferation of human myeloid leukemia (TF‐1a and MV4‐11) and epithelia cancer (Hep‐G2) cells in culture, measured by XTT assays and microscope examinations. The initial inhibition was observed when DMSO was added to the cell culture at a concentration of 2%. The inhibition was substantial and appeared dose‐dependent, with a maximal inhibition of the cell growth being observed in the cells treated with 5–6% of DMSO. It is a little surprising that more than 6% DMSO (up to 12%) did not further inhibit the cell proliferation and cell viability, generating a hyperbolic dose‐response curve. DMSO at 1% or less had no significant inhibitory effect on the cells. Cytology examination showed that both MV4‐11 and TF‐1a cells have a relatively homogeneous population with smooth cytoplasmic border. These cells have a large nucleus to cytoplasm ratio. Addition of DMSO induced clear cellular damages: cytoplasm shrank or broken; the nuclei appeared as fragments or dots; and the cell body decreased in size. Western blot analysis showed that DMSO downregulated the expression of CDK2 and cyclin A in TF‐1a and MV4‐11 cells after 24 h incubation. The inhibition of CDK2 and cyclin A is also dosedependent. Thus, our data demonstrate that DMSO has a significant inhibitory effect on the growth and viability of the cancer cells tested. This inhibition is linked to cell cycle control and apoptotic molecules. The outcome from this study also suggests that as a solvent the final concentration of DMSO in cell culture should be no more than 1%. In summary, our study provides a supplementary information on both function and mechanism by which DMSO affects human cancer cells.Support or Funding InformationBarry University
FoxO1 inhibition attenuate diabetes‐related vascular remodeling by reducing chronic inflammatory states
Authors: Xie Xiang , Xia Zhengyuan , Irwin Michael. G 
Published: April 2020

Diabetic vascular complications result in poor outcomes, even after optimized interventions. Forkhead transcriptional factor(FoxO1) contributes to diabetic cardiomyopathy, yet the role of FoxO1 in diabetic vascular complications is poorly understood. We found that arterial wall hypertrophy occurred in the carotid artery of diabetic rats, which was associated with significant increases in wall thickness and cross‐sectional area as compared to non‐diabetic control group. The immunofluorescence results showed that α‐smooth muscle specific protein expression was downregulated in diabetic carotid media which was restored by treatment with the selective FoxO1 inhibitor AS1842856. The mRNA expressions of pro‐inflammatory biomarkers IL‐1 and TNF‐α in diabetic carotid arteries were increased as compared with control and were decreased following AS1842856 treatment. Also, the NLRP3 protein expression in diabetic carotid arteries was increased as compared with control group and reverted by AS1842856 treatment. It is concluded that our findings identify FoxO1 as a critical trigger for diabetes‐related adverse vascular remodeling and define the FoxO1 as a novel checkpoint for NLRP3 inflammasome‐dependent low‐grade chronic inflammatory states during diabetic adverse vascular remodeling progression, which may provide a promising therapeutic target to treat the diabetic cardiovascular disease.Support or Funding InformationThe work was supported by the NSFC grant (81770831) and in part by the General Research Fund (17118619M, 17123718M, Research Grants Council of Hong Kong).
The Effects of Quercetin on Apoptosis and Cell Proliferation in A375 Melanoma Cells
Authors: Martinez P.J. , Byrd Shere Kuss 
Published: April 2020

Quercetin, a bioflavonoid, is known to have various health benefits and has been used as a treatment for several forms of cancer. The aim of our research was to understand quercetin effects on cell growth and apoptosis pathways in a melanoma cell line. A dose/toxicity curve was done to determine cell viability in relation to a 24 hr treatment with quercetin concentrations ranging from 25–150μM. To test the effects of quercetin on cell proliferation, melanoma cells were grown to confluence then wounded while being treated for 24 hr with 50μM quercetin. DMSO was used as a vehicle control. Average initial wound distance was measured at three sites along the wound. Wound closure (cell proliferation) was re‐measured after 24 hr and the rate of closure was used as an index of cell proliferative ability. Results of the wound assay showed 40% less regrowth for the cells treated with quercetin, indicating inhibition of proliferative ability. To determine whether the inhibition of proliferation was due to an increase in quercetin‐induced apoptosis, we used Annexin V/PI and flow cytometry to quantify early and late apoptosis following treatment and found that the increase in apoptotic cells mirrored the loss in proliferative ability. These results show that quercetin should be considered as an adjunct treatment to prevent proliferation of melanoma.Support or Funding InformationColorado Alliance for Minority Participation
Terbutaline, Forskolin and cAMP Reduce Secretion of Aqueous Humor and Inhibit Intracellular Calcium Release in Bovine Cultured Ciliary Epithelium
Authors: Shahidullah Mohammad , Rafiq Kazi , Sikder Mahmudul Hasan , Ferdous Jannatul , Wilson William Stuart , Delamere Nicholas A. 
Published: April 2020

In order to elucidate the involvement of cyclic AMP and intracellular Ca2+ concentration [Ca2+]i, in the modulation of aqueous humor (AH) secretion, we studied the effects of terbutaline, forskolin and 8‐Br‐cAMP on AH secretion in the isolated intact bovine eye and on ATP‐induced increase in [Ca2+]i in cultured ciliary epithelial (CE) cells. Drug effects on AH secretion were measured by fluorescein dilution. Drug effects on [Ca2+]i were studied by the Fura‐2 fluorescence ratiometric measurement. Terbutaline (100 nmol–100 μM), forskolin (30 nM–100 μM) or 8‐Br‐cAMP (100 nM – 10 μM), administered at fixed concentrations in the arterial perfusate of the intact eye, produced significant reductions in AH secretion. The AH reducing effect of terbutaline was blocked by a selective inhibitor of protein kinase A (KT‐5720). ATP (100 μM) caused a rapid, transient (peak) increase in [Ca2+]i followed by a small but sustained plateau phase lasting more than 5 minutes. Preincubation of the cells (6 min) with terbutaline, forskolin or 8‐Br‐cAMP significantly reduced the peak calcium response to ATP. Each of the drugs, on the other hand, augmented the sustained plateau phase of the calcium response. KT‐5720 partially reversed the inhibitory effect of terbutaline on the peak and totally inhibited its effect on the plateau phase. Taken together, our data support the notion that AH secretion in the bovine eye can be manipulated through cyclic AMP, operating through protein kinase A and that interaction of [Ca2+]i and cAMP may play a role in modulating AH secretion.Support or Funding InformationFight for Sight, London
Radiation‐induced Changes in Stimulant Pharmacokinetics Relevant to Long‐distance Space Travel
Authors: Hambuchen Michael D. , Mazur Anna , Findley David L. , McGill Mitchell R. 
Published: April 2020

A round trip to Mars or a 10 year stay on the planet’s surface is estimated to be associated with 1 or 3 Gray (Gy) whole body radiation exposure, respectively. Radiation exposure is known to alter the pharmacokinetics of some medications. The objective of these experiments was to determine how radiation exposure may alter drug pharmacokinetics in male Wistar rats using methamphetamine (METH) as a probe. METH was chosen due to its well established pharmacokinetics in rats and the relevance of stimulant administration for alertness in space travel. Rats were exposed to 0, 1, or 3 Gy X‐ray radiation followed by measurement of clearance‐related serum biomarkers (e.g., alanine aminotransferase, serum creatinine, etc.), 1 mg/kg subcutaneous (SC) METH pharmacokinetics, and 1 mg/kg SC METH‐induced locomotor activity (which is correlated with brain METH concentrations). While radiation dose‐dependently reduced rat total body weight and spleen weight, there were no significant changes in serum biomarkers, METH pharmacokinetic parameters, or METH‐induced locomotor activity. In conclusion, acute Mars travel relevant radiation exposures did not alter METH disposition, but longer‐term studies of gradual radiation exposure on stimulant pharmacokinetics are needed.Support or Funding InformationNASA West Virginia EPSCoR Research Seed Grant
Astrocytes play an active role in persistence of respiratory augmentation in the recovery phase after hypoxic exposure
Authors: Fukushi Isato , Kono Yosuke , Takeda Kotaro , Yokota Shigefumi , Onimaru Hiroshi , Pokorski Mieczyslaw , Okada Yasumasa 
Published: April 2020

Hypoxia induces shortness of breath, and augments respiration, and the respiratory augmentation persists for a while even in the post‐hypoxic recovery phase. However, the mechanism of posthypoxic persistent respiratory augmentation has not been fully elucidated. We propose a hypothesis that astrocytes are involved in the central mechanism of post‐hypoxic persistent respiratory augmentation. To investigate this hypothesis, we conducted two kinds of experimental analyses. First, we examined astrocytic involvement in hypoxic ventilatory responses in the in vitro experiments. In this experiment, we used the medulla‐spinal cord preparation isolated from newborn rats. Briefly, the medulla and spinal cord were together isolated from neonatal rats under anesthesia. The preparations were fixed in a recording chamber and superfused firstly with oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (aCSF), secondly with hypoxic (95% N2, 5% CO2) aCSF for 5 min, and thirdly again with oxygenated aCSF for 20 min. We compared respiratory frequencies in the post‐hypoxic recovery phase in preparations without and with administration of arundic acid (500 μM), a blocker of astrocytic activation. Second, we examined astrocytic involvement in hypoxic ventilatory responses in the in vivo experiments. In these experiments, unanesthetized adult mice were exposed to various levels of hypoxia, and ventilatory responses were analyzed by whole body plethysmography. Effects of arundic acid on ventilation were investigated. In results, in in vitro experiments, hypoxia increased respiratory frequency in both groups. During superfusion with oxygenated aCSF after hypoxic exposure, respiratory frequency in the group without arundic acid was still persistently increased. On the other hand, respiratory frequency in the group with arundic acid was gradually decreased in the post‐hypoxic phase, and it was eventually depressed and less than that of the pre‐hypoxic phase in the later post‐hypoxic phase. In in vivo experiments, moderately strong hypoxia increased ventilation, and ventilatory augmentation persisted even after cessation of hypoxia loading. Arundic acid blocked persistence of post‐hypoxic ventilatory augmentation. Stronger hypoxia induced biphasic responses, i.e., initial ventilatory augmentation followed by ventilatory depression. Arundic acid strengthened hypoxic ventilatory depression. We conclude that astrocytes play an important role in persistence of post‐hypoxic respiratory augmentation.Support or Funding InformationJSPS KAKENHI numbers 17H05540, 17K08559 and 18K17783