bluuebunny/crossref_metadata_2025_split huggingface dataset:
Redesigned Upstream Processing Enables a 24‐hour Workflow from E. coli Cells to Cell‐free Protein Synthesis
Authors: So Byungcheol 
Published: April 2020

Cell‐free protein synthesis (CFPS) platforms have undergone numerous workflow improvements to enable diverse applications in research, biomanufacturing, point‐of‐care detection, therapeutics, and education using affordable laboratory equipment and reagents. The Escherichia coli cell extract‐based platform, being one of the most affordable and versatile CFPS platforms, has been broadly adopted. In spite of the promise of simplicity, the cell‐free platform remains technically nuanced, posing challenges to reproducible implementation and broad adoption. Additionally, while the CFPS reaction itself can be implemented on‐demand, the upstream processing of cells to generate crude cell lysate remains time‐intensive, representing one of the largest sources of cost associated with the biotechnology. To circumvent the lengthy and tedious upstream workflow, we have redesigned the processes by developing a long‐lasting autoinduction media formulation for cell‐free that obviates human intervention between inoculation and harvest. Cell‐free autoinduction (CFAI) media supports these advantages through the production of highly robust cell extracts from high cell density cultures nearing stationary phase of growth. Growth of cells to high density and autoinduction of T7 RNAP expression can be achieved by incubation overnight, eliminating the need for user intervention for the entirety of the process. The total mass of cells obtained is substantially increased, which directly results in a 400% increase in total extract volume obtained compared to past workflows. Based on these advances, we outline a new upstream processing workflow allowing researchers to go from cells on a streak plate to completing CFPS reactions within 24 hours while maintaining robust reaction yields of sfGFP (>1 mg/ml). We hope this advance improves the time and cost‐efficiency for existing CFPS researchers, increase the simplicity and reproducibility, and reduce the barrier‐to‐entry for new researchers interested in implementing CFPS. Lastly, we have completed preliminary metabolomics analysis to elucidate the mechanisms by which CFAI‐grown cells are capable of producing robust extracts.Support or Funding InformationAuthors would like to acknowledge Dr. Jennifer VanderKelen and Andrea Laubscher for technical support, Wesley Kao, Nicole Gregorio, Phillip Smith, and Logan Burrington for helpful discussions. Authors also acknowledge funding support from the Bill and Linda Frost Fund, Center for Applications in Biotechnology’s Chevron Biotechnology Applied Research Endowment Grant, Cal Poly Research, Scholarly, and Creative Activities Grant Program (RSCA 2017), and the National Science Foundation (NSF‐1708919). MZL would like to acknowledge the California State University Graduate Grant.
Role of MCM8/9 in DNA Fork Protection
Authors: Klinzing Kathleen N. , Griffin Wezley C. , McKinzey David R. , Trakselis Michael A. 
Published: April 2020

Efficiency of DNA replication is crucial to cell survival. Recognition of DNA damage by replication complexes results in impedance of this process until the damage is repaired. Fork stalling and reversal are important for the repair of DNA lesions. Stalled forks must be resolved quickly in order to prevent fork collapse resulting in double‐strand breaks (DSBs). Several proteins are recruited to mitigate damage to DNA prior to DSB formation. MCM8/9 is believed to play a role in both protection of DNA at stalled forks and in repair of DSBs through a helicase activity. Mutation of MCM8 or MCM9 has been attributed to development of cancer and infertility. The MCM8/9 complex recruits Rad51 to double stranded break sites, but its role in fork stalling remains unknown.A CRISPR‐Cas9 generated MCM9 knockout cell line was used to assess MCM8/9‐mediated protection of stalled or reversed forks from nucleolytic degradation using DNA fiber assays. Two halogenated thymine analogs, CldU and IdU, are added to the nucleotide pool to monitor discrete DNA replication tracks within HEK293T cells. After CldU and IdU pulses, DNA replication is stalled through the addition of hydroxyurea (HU) to allow for fork reversal. The IdU/CldU length ratio measured from DNA fiber analysis can reveal fork protection status. MCM9 knockout cells had a significant reduction in IdU/CldU track ratios demonstrating its importance in maintaining replication fork stability following stalling by HU. These results suggest that MCM8/9 plays a crucial but unidentified role in maintenance of replication fork integrity and protection of stalled forks during replication. Current and future experiments will focus on the direct interactions and effects of knockdowns of other known fork protection proteins with MCM9 to more definitely determine MCM8/9 function in maintaining the human genome.Support or Funding InformationThis work is supported by the National Institutes of Health (NIH) GM135791.
Microfluidic 3D Model of Heterochronic Parabiosis to Study Systemic Regulation of Skeletal Muscle Aging
Authors: Choi Jeongmoon , Lee Yunki , Lee Nanhee , Han Woojin M. , Mohiuddin Mahir , Shin Eunjung , Anderson Shannon , Park Ki Dong , Jang Young C. 
Published: April 2020

Muscle satellite cells (MuSCs) play an indispensable role in skeletal muscle regeneration after injury. However, their regenerative capacity declines with aging. Intriguingly, studies using parabiosis, a surgical technique that attaches two animals to share blood circulation, demonstrated that circulating factors from young animals can rejuvenate the regenerative capacity of aged animals. However, the exact identity of these putative youthful factors has yet to be elucidated. Due to the complexity of in vivo parabiosis, reliable identification of systemic rejuvenation factors remains a major hurdle. To overcome this challenge, we employed microengineering technologies and developed a 3D microfluidic circuit with biophysical and biochemical properties similar to in vivo muscle microenvironment. Here, we present a 3D micro‐vascularized muscle‐on‐a‐chip (VMoC) that exhibits key characteristics of the native MuSC niche, where FACS purified quiescent MuSCs are cultured in between myofibers and 3D hydrogel (extracellular matrix mimetic) underneath vascular endothelial cells. With the VMoC, we further integrated it to model parabiosis by co‐culturing cells/sera from young and aged animals. By adding the serum from young, aged, and Sod1−/− (oxidative stress model) mice to VMoCs, we demonstrate distinct myogenic activities of young MuSCs; VMoCs with aged and Sod1−/− sera showed significantly reduced myogenesis as measured by fusion index. Next, we replicated heterochronic parabiosis using our integrated chip‐to‐chip platform to assess the rejuvenating effects of young serum/cells. Interestingly, similar to in vivo parabiosis, when aged VMoCs were continuously exposed to the young systemic environment, the myogenic activity of aged MuSCs were significantly boosted. In addition, genes that regulate different steps of adult myogenesis (Desmin, MyoD, and Myogenin) were up‐regulated in the aged VMoC exposed to the young systemic environment. Furthermore, this rejuvenation effect in myogenesis was correlated with increased VEGF, which has been shown to play a crucial role in MuSC function in parabiosed VMoC. Collectively, our parabiosis‐on‐a‐chip platform will provide a state‐of‐the‐art pre‐clinical testing tool that will facilitate our understanding of the dynamic regulation of circulating humoral factors and potential drug discovery. More importantly, our approach can be translated into human clinical studies by replacing the murine cells with the corresponding human cells, emulating human parabiosis on‐a‐chip that blood transfusion cannot recapitulate.Support or Funding InformationNIH R21AR072287 (YJ), NIH R03AG062976 (YJ), and NIH DP2HL142050 (YK)
Targeting Lysine‐specific Demethylase 1 (LSD1) in an Experimental Model of Pulmonary Fibrosis
Authors: Shenoy Vinayak , Shenoy Anitha , Lu Jianrong 
Published: April 2020

Idiopathic pulmonary fibrosis (IPF) is a chronic disease that is characterized by abundant deposition of extracellular matrix proteins in the lungs, which ultimately leads to respiratory failure and death. The existing treatment options neither arrest disease progression nor provide significant improvement in lung function. Hence, it is essential that we identify novel targets or develop add‐on pharmacological agents to treat this fatal disease. Epithelial to mesenchymal transition (EMT) plays a key role in the pathogenesis of IPF, wherein alveolar epithelial cells lose part of their characteristics, while gaining mesenchymal markers. Inhibition of the EMT process could be a potential approach to treat lung fibrosis. Accumulating evidence suggests that inhibition of lysine‐specific demethylase 1 (LSD1), a chromatin‐modifying enzyme that regulates gene transcription prevents EMT and fibrogenesis. Here, we hypothesized that inhibition of LSD1 by tranylcypromine protects against bleomycin‐induced lung fibrosis.A single instillation of Bleomycin (Bleo; 2.5mg/Kg) in 8‐weeks old rats was used to induce lung fibrosis. Drug treatment with tranylcypromine was commenced on the same day as Bleo administration using two different doses (1mg/Kg and 2 mg/Kg ip). After two weeks, rats were instrumented to measure right ventricular systolic pressure (RVSP) and cardiac function. Bleo injected animals developed secondary pulmonary hypertension as evidenced by increase in RVSP in comparison with control rats (RVSP; 25.4±0.71 vs 52.7±2.5, mmHg) with a concomitant development of right ventricular hypertrophy (RVH; 0.27±0.01 vs 0.45±0.06). Significant attenuation of RVSP (1mg/kg: 52.7± 2.5 vs 42.3± 3.1; 2mg/kg: 36.2±1.8) and RVH (1 mg/kg: 0.47±0.06 vs 0.4+0.08; 2mg/Kg: 0.34±0.01) was observed with both the doses of tranylcypromine; however, 2mg/Kg dose showed better effects. Bleo‐induced lung fibrosis was also reduced by drug treatment as evaluated by picro‐sirius red staining. Collectively, our findings reveal that inhibition of LSD1 by tranylcypromine protects against bleo‐induced cardiopulmonary damage.
Histone mRNA Uridylation: Oligo(U) Tail, or Stem‐Loop?
Authors: Lackey Patrick E. , Shine Morgan , Harris Sarah , Mihailescu Mihaela-Rita 
Published: April 2020

Histone mRNAs are tightly cell‐cycle regulated mRNAs, with their expression and degradation both tied tightly to S‐Phase and DNA replication. This regulation is controlled by histone mRNA’s unique 3′ end; instead of a poly(A) tail, the histone message ends with a 3′ stem‐loop. This stem‐loop and its protein binding partner, SLBP, are both important factors in controlling the expression and degradation of histone mRNA. At the beginning of degradation, the human 3′ exonuclease (3′hEXO) forms a complex with SLBP and the stem‐loop, trimming 2 to 7 nucleotides from the 3′ end of the histone message. While the exact function of these uridylated intermediates and their role in histone mRNA metabolism is unknown, we used nuclear magnetic resonance (NMR) to show that a nine‐membered stem‐loop may still form even after seven nucleotides are removed and replaced with five uridines. We have also demonstrated that this uridylated stem‐loop is capable of binding SLBP and 3′hEXO, and have further characterized these interactions and the conformation of the uridylated stem‐loop during the interactions with various biophysical methods, including UV‐Vis thermal denaturation and circular dichroism.Support or Funding InformationResearch by M. Shine and S. Harris was partially supported by NSF‐REU (CHE‐1659821)
SPARC: The Emergence Of Cardioprotection From The Vagus Nerve Mediated Brain‐Heart Network
Authors: Gorky Jonathan , Vadigepalli Rajanikanth , Schwaber James S. 
Published: April 2020

Heart disease remains the number one cause of mortality in the world in spite of significant efforts aimed at treatment. The use of vagal stimulation in the treatment of heart failure has shown mixed successes, suggesting that the treatment has potential, but that the mechanism incompletely understood. Vagal activity confers a robust cardioprotective effect in both humans and animal models, preferentially originating specifically from the dorsal motor nucleus of the vagus (DMV) and deriving a significant benefit from intact gut projections, not just cardiac. In order to examine the DMV response to heart failure, myocardial infarction was induced in male Sprague Dawley rats. DMV neurons were isolated in small pools of single cells using laser capture microdissection 1 week and 3 weeks after infarction and their gene expression assayed. The results show a transcriptional shift towards a neurosecretory phenotype starting at 1 week and increasing in recruitment of neurons to 3 weeks. This was evidenced by the upregulation of ion channels associated with a pacemaker phenotype, an upregulation several neuropeptides. This response to chronic ischemic damage in the heart demonstrates that neurons of the DMV are capable of adaptive responses in the context of peripheral organ damage. The LAD ligation shift appears mediated in part by upregulation of Pax4a, a transcription factor most active during stem cell development of neurosecretory cells during embryonic development. This phenotype is characterized by upregulation of Cacna1d (Cav1.3) and Hcn2 along with increased expression of Cck and Sst. This work suggests that the neurons of the DMV adaptively respond to the dynamics present in the periphery, elucidating the means by which the nature of vagal activity responds to heart failure. The autonomic nervous system plays a significant role in the pathogenesis of cardiovascular disease. Through demonstration of shifting neuronal phenotypes in central neurons in response to peripheral stimuli, we suggest that neuron peptide or neurotransmitter phenotypes are not static in adult rodents. This suggests that even “reflexes” are modifiable dynamic systems. With such plasticity in the transcriptional programming existing in autonomic brain regions there can be new potential therapeutic interventions for cardiovascular disease aimed at leveraging the autonomic nervous system.Support or Funding InformationSupported by the NIH Common Fund SPARC Program award OT2 OD023848 and NIH NHLBI Multiscale Modeling award U01 HL133360.
Impact of chronic hyperleptinemia on placental ischemia‐induced hypertension in pregnant rats
Authors: Spradley Frank T. , Wilson Barbara A. , Martin Hunter L. , Palei Ana C. 
Published: April 2020

Obesity is a risk factor for preeclampsia, which is new‐onset hypertension during pregnancy. The mechanisms linking obesity and preeclampsia are poorly understood. The adipokine, leptin, is elevated in the circulation in proportion to fat mass and elicits hypertension when infused into non‐pregnant animals. However, less is studied about how hyperleptinemia impacts blood pressure regulation during pregnancy. A contributing factor to hypertension during preeclampsia is placental ischemia. Therefore, we tested the hypothesis that chronic hyperleptinemia exaggerates placental ischemia‐induced hypertension. On gestational day 14, timed‐pregnant Sprague‐Dawley rats were implanted with osmotic pumps to intravenously deliver recombinant rat leptin at 1.0 ug/kg per min or vehicle and placental ischemia was produced by the reduced uterine perfusion pressure (RUPP) procedure or Sham surgery. By day 19, leptin levels were elevated (P<0.05) in Sham (leptin‐infused, N=9: 41.0±2.8 ng/mL vs vehicle, N=23: 0.8±0.1 ng/mL) and RUPP (leptin‐infused, N=9: 29.7±7.2 ng/mL vs vehicle, N=17: 1.0±0.1 ng/mL). RUPP elicited hypertension (Sham+vehicle: 100±1 mmHg vs RUPP+vehicle: 122±3 mmHg mean arterial blood pressure, P<0.05). In contrast to our hypothesis, hyperleptinemia attenuated RUPP hypertension (RUPP+leptin: 109±4 mmHg, P<0.05 vs RUPP+vehicle) and did not alter blood pressure in Sham rats (Sham+leptin: 100±6 mmHg vs Sham+vehicle). Therefore, we examined whether hyperleptinemia was vasoprotective by promoting vasorelaxation during pregnancy in isolated third‐order mesenteric arteries. Although endothelial‐dependent vasorelaxation to acetylcholine (measured as logEC50 sensitivity) was similar within Sham (vehicle: −6.8±0.1 M vs leptin: −6.9±0.2 M) and RUPP (vehicle: −7.0±0.2 M vs leptin: −7.0±0.3 M) groups, endothelial‐independent vasorelaxation to the nitric oxide (NO)‐donor, sodium nitroprusside, was increased (P<0.05) in both Sham (vehicle: −6.0±0.2 M vs leptin: −6.8±0.3 M) and RUPP (vehicle: −6.9±0.2 M vs leptin: −7.6±0.01 M) groups. This suggested that NO‐cyclic guanosine monophosphate (cGMP) signaling was increased. Plasma levels of cGMP were elevated (P<0.05) within Sham (vehicle: 1.6±0.3 pmol/mL vs leptin: 3.9±0.6 pmol/mL) and RUPP (vehicle: 3.6±0.8 pmol/mL vs leptin: 5.8±1.1 pmol/mL) groups. In contrast to our original hypothesis, chronic hyperleptinemia does not produce hypertension during pregnancy likely via promoting vascular NO‐cGMP signaling.Support or Funding InformationHL130577
Translation of Clinical Dysphagia Scales for Use in Translational Studies
Authors: Schneider Kellyanna , Strecker Courtney , Iceman Kimberly , Pitts Teresa 
Published: April 2020

Videofluoroscopy is the gold standard for clinical measurement of swallow disorders (dysphagia). Patients are presented with foods and liquids of varying consistencies in order to evaluate the risk of aspiration. The Modified Barium Swallow Impairment Profile (MBSImP) was developed in 2008 and has since become the primary tool to define the degree of dysphagia impairment. In order to determine the utility of the MBSImP in animals with cervical spinal cord injury, animals were presented with tuna water and pate cat food laced with 40% barium sulfate by volume. Data was collected using a miniature c‐arm collecting at 30 frames per second, and exams were performed pre and post cervical spinal cord injury. Out of the 17 components scored on the MBSImP, only 10 components are usable for animals. Additional components/variables are needed due to their anatomical and physiological differences. More specifically, the pharyngeal phase of the swallow in cats shows a threshold in the pharyngeal distention before a full swallow is completed as well as numerous “rocking” motions to transport the bolus posteriorly from the oral cavity. Another difference shows that component 9 (Anterior Hyoid Excursion) is harder to detect due to the slight displacement of the hyoid. However, we did determine that penetration and aspiration could be reliably detected post‐injury using the 30‐frames per second recording. These results support the use of clinical techniques; however, the addition of continuous variables would assist in the measurement of small changes across time.Support or Funding InformationThis work was supported by NIH grant NS110169 and the Craig H. Neilsen Foundation Pilot Research Grant 546714.
Intraoperative Hemidiaphragm Stimulation Offsets Slow‐Twitch Muscle Fiber Atrophy and Contractile Dysfunction
Authors: Bresciani Guilherme , Mankowski Robert , Ferreira Leonardo , Leeuwenburgh Christiaan , Arnaoutakis George , Martin Tomas , Xue Wei , Martin Daniel , Beaver Thomas , Smith Barbara K. 
Published: April 2020

Prolonged mechanical ventilation induces considerable atrophy of slow‐ and fast‐twitch fibers in the human diaphragm. In pilot work, we detected early atrophy and specific force decrement in fast‐twitch fibers of patients following an average 4.6‐hour thoracic operations. We now present interim data from a larger controlled trial of diaphragm stimulation as a means to reduce slow‐ and fast‐twitch‐ fiber atrophy and metabolic derangements that occur with mechanical ventilation. We hypothesized that intraoperative hemidiaphragm would mitigate both slow‐ and fast‐twitch fiber contractile dysfunction in the human diaphragm. Twelve adults (6F, age 62 ±12 years) consented to participate in this IRB‐approved study. During cardiothoracic surgical procedures, one hemidiaphragm underwent 1 minute of electrical phrenic nerve stimulation every 30 minutes (30 twitches/minute, 1.5 milliseconds pulse duration, amplitude variable to produce a strong twitch contraction, up to 25 mA), while the contralateral hemidiaphragm remained inactive. Thirty minutes following the last stimulation bout, biopsies were obtained from both hemidiaphragms. Muscle fibers were buffered, permeabilized, and separated for evaluation of cross‐sectional area and force‐generating properties. A mixed effects model with random intercept for each subject was used to evaluate the fixed effects of stimulation and fiber type on single‐fiber force mechanics, and significance was p<0.05. Subjects underwent an average 7 ±2 hemidiaphragm stimulations at 17 ±2 mA, and biopsies were collected after 4.75 ±1.03 hours of mechanical ventilation. Cross‐sectional area of unstimulated slow‐twitch fibers was significantly smaller than stimulated slow‐twitch fibers (5632 ±349 vs 4619 ±247 μM2), with no effect on fast‐twitch fibers (5631 ±350 vs 5768 ±351 μM2), [p<0.005]. Likewise, stimulation resulted in significantly higher specific force of slow‐twitch (106 ±6 vs 97 ±6 kN/m2), but not fast‐twitch diaphragm single fibers (126 ±6 vs 130 ±6 kN/m2), [p<0.001]. Stimulation did not appreciably alter fiber calcium‐sensitivity properties of either fiber type. Our data suggest that stimulation attenuates diaphragm slow‐twitch fiber atrophy and contractile dysfunction induced by mechanical ventilation. Fiber type‐specific changes with short periods of mechanical ventilation may be attributed to predominant slow fiber recruitment during tidal breathing.Support or Funding InformationThis work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases, award R01 AR072328