bluuebunny/crossref_metadata_2025 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 Information

Authors 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 Information

This work is supported by the National Institutes of Health (NIH) GM135791.
Sialic Acid Alterations in Mouse Models of Atrophy and Hypertrophy
Authors: Busam Sara , Goines Briana N. , Weisleder Noah L. , Crowe Kelly E.
Published: April 2020

Sialic acids (SAs) are negatively charged nine‐carbon sugars that are typically found at the ends of glycan chains on glycolipids and glycoproteins. As such, they play important structural and signaling roles for the cell. Skeletal muscle seems to be particularly sensitive to SA perturbations; in GNE myopathy, hypomorphic mutations in a key enzyme of SA biosynthesis leads to muscle weakness and wasting, and SA abundance has been shown to correlate with muscle function in mice. Additionally, enzymatic activity of sialidases, which removes SAs, is altered in both atrophy and hypertrophy. However, linkage‐specific SA changes have not yet been described in atrophy and hypertrophy. Here, we have used a panel of lectins to probe alterations in SA abundance in mouse models of hind‐limb suspension atrophy and hind‐limb overload‐induced hypertrophy.
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 Information

NIH 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 Information

Research 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 Information

Supported 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 Information

HL130577
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 Information

This 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 Information

This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases, award R01 AR072328
Dietary Sugars, bacterial colonization and the behavior of fruit flies
Authors: Wu Janina L. , Cao Xixi , Yang Chan
Published: April 2020

Cavity remains prevalent in young children today. One major contributor is the modern diet, specifically the large amounts of dietary sugars consumed. Another key factor is bacteria in our mouth that eat these sugars. Streptococcus mutans among other acid producing bacteria is one of the leading microbes that initiate and promote cavity (tooth decay). It metabolizes sugars to produce plaque, which can build up over time and simultaneously increase acid production that causes cavity. So studying how quickly different sugars affect Streptococcus mutans colonization is important to understand the development of cavity and its impact on host behavior. My hypothesis was that S. mutans breaks down some sugars more efficiently and as a result, can colonize more quickly with some than others and produce more acid, thus impacting host behavior.

To study bacterial colonization, I used a fruit fly model because fruit flies like sugars and Streptococcus mutans can readily colonize fruit flies when is mixed with sugars. Each sugar solution was mixed with green fluorescent protein (GFP) labeled S. mutans and fed fruit flies. We then waited for 6 days to let flies eat and grow at room temperature. After this incubation period, we took flies out every 2 days to image the green color under the fluorescent microscope, which indicates S. mutans colonization. On the final day, we took all of the remaining flies, ground them up, and performed appropriate dilutions to plate and count bacterial colonies, which represents bacterial colonization of flies. Colonization of flies fed with five different sugars by S. mutans was monitored by direct imaging and counting bacterial colony forming units. Flies fed with sucrose exhibited the best colonization, while flies fed with xylitol did not show any colonization. The pH values of S. mutans cultures (pH adjusted to 7.5) fed with different sugars were monitored using a pH meter. With all sugars, the pH dropped significantly after five minutes. The pH dropped below 4.5 with sucrose that supports the best colonization. The pH dropped at an average of 2.91 during the 30 minutes testing period, suggesting sugars and bacteria fuel acid production.

Furthermore, flies colonized by S. mutans fed with sucrose were monitored for their behavior (locomotion) using beam breaks and analyzed by DAMsystems308 software. Sucrose fed flies exhibited more bacterial colonization and became hyperactive. Overall the data indicated that S. mutans utilizes some dietary sugars more efficiently and as a result, can colonize better, produce more acid and become more active. Our studies demonstrated that the interactions between bacteria and sugars are important for the development of cavity and impact host behavior.
TMAO Activates Carotid Endothelial Inflammasomes Leading to Enhanced Neointimal Formation in Nlrp3 Mice
Authors: Boini Krishna M. , Puchchakayala Goverdhan , Zhang Yang , Koka Saisudha
Published: April 2020

Recent studies have shown that gut microbial metabolite, trimethylamine‐N‐Oxide (TMAO), is a biomarker for atherosclerosis. However, the mechanisms of how TMAO induces atherosclerosis are still unclear. The present study tested whether TMAO induces Nlrp3 inflammasome activation and consequent endothelial injury leading to atherosclerotic lesion development in the carotid arterial wall. Confocal microscopy showed that colocalization of Nlrp3 with ASC significantly increased in the intima of the carotid arteries of TMAO‐treated Nlrp3+/+ mice, suggesting the formation of Nlrp3 inflammasomes in the endothelium of these arteries. In Nlrp3−/− mice, however, such colocalization was much less detected. In consistent with inflammasome formation, TMAO‐induced IL‐1β production in the intima was abolished in Nlrp3−/− mice. Furthermore, we found that TMAO treatment decreased ZO2, VE‐cadherin and occuludin expression in carotid arteries of Nlrp3+/+ mice but not in Nlrp3−/− mice, signifying tight junction disruption in the carotid arteries of Nlrp3+/+ mice. In addition, we found that TMAO treatment had significantly increased vascular permeability to intravenously injected Evans blue dye compared to vehicle treated Nlrp3+/+ mice. However, such TMAO‐induced vascular permeability was attenuated in Nlrp3−/− mice, suggesting that Nlrp3 inflammasome activation plays an important role in TMAO‐induced vascular leakage in mouse hearts. Furthermore, we also found that TMAO markedly increased neointimal formation in partially ligated left carotid arteries (PLCA) of Nlrp3+/+ mice. However, such TMAO‐enhanced neointimal formation and increased I/M ratio in PLCA was markedly attenuated in Nlrp3−/− mice. These results suggest that the formation and activation of NLRP3 inflammasomes by TMAO may be an important mechanism in initiating endothelial inflammatory response leading to arterial inflammation and endothelial dysfunction and consequent atherosclerosis in mice.

Support or Funding Information

R01DK104031 and R56HL143809
Translation Initiation Rates Govern Protein Self‐Assembly In Vivo
Authors: Halfmann Randal , Venkatesan Shriram , Wu Jianzheng
Published: April 2020

Living proteomes are necessarily far from equilibrium. Otherwise cells could not use the energy released by phase separation to organize protein activities in space and time. It is paradoxical, then, that reducing protein influx into the proteome ‐‐ which should promote equilibration ‐‐ instead prolongs the lives of organisms from yeast to human. We therefore reasoned that kinetic barriers must exist to delay equilibration, and that these decrease with protein influx. We further hypothesized that the probabilistic nucleation of protein self‐assemblies ‐‐ or deposits ‐‐ could underlie those barriers. To investigate the effects of influx on protein deposition, we probed phase boundaries and nucleation barriers for structurally diverse protein assemblies at different rates of translation initiation in living cells. We discovered that at protein concentrations that were globally supersaturated with respect to deposition as amyloids or other ordered phases, rapid translation promoted nucleation and thereby equilibration of that protein. We tentatively attribute this effect to unexpectedly slow diffusion of proteins away from sites of translation. The resulting local accumulation appears to facilitate protein clustering into nucleation competent multimers, even at global concentrations that are subsaturated with respect to liquid‐liquid phase separation. Finally, we show that longevity‐enhancing reductions in global protein initiation, as occur during stress responses or calorie restriction, similarly sharpen phase boundaries and nucleation barriers. Together, these observations suggest that living proteomes are supersaturated with respect to highly ordered, collectively lethal deposits.

Support or Funding Information

This work was funded by NIH Director’s Early Independence Award DP5‐OD009152, NIH grant P30 AG035982, and the Stowers Institute for Medical Research.
Neurochemical Substrates of Menthol Actions in Reward‐Addiction Brain Areas.
Authors: Davila-Garcia Martha Isabel , Wang Paul , Lin Stephen , Dekhordi Ozra
Published: April 2020

Menthol, a commonly used flavoring additive in cigarettes has been found to facilitate smoke initiation and nicotine addiction. Menthol masks the harshness of nicotine, increases its absorption, and provides a cooling sensation that appeals to many smokers. In addition to its peripheral sensory impact, recent studies have demonstrated the presence of menthol in the CNS after in vivo exposure. This implies that menthol may also impact nicotine addiction through central mechanisms. In the present study in mice, we hypothesized that dopaminergic and GABAergic neurons of the reward‐addiction circuitry are among the initial targets of acute menthol in the CNS. We tested this hypothesis by utilizing c‐Fos immunohistochemical technique together with double labeling to identify anatomical location of menthol activated cells with respect to tyrosine hydroxylase (TH) immunoreactive (TH‐IR) dopaminergic and GAD67‐GFP positive GABAergic cells. Menthol (80 mM, IP), produced c‐Fos activation at multiple sites of the mesocorticolimbic reward‐addiction pathways including several structures previously shown to be activated by nicotine such as the periaqueductal gray (PAG), dorsal raphe (DR), ventral tegmental area (VTA), amygdala, nucleus accumbens (NAcc), anterior olfactory nucleus (AON) and cingulate‐orbitofrontal cortex (A25/MO). However, none of the dopaminergic neurons in VTA, PAG/DR region or hypothalamus were activated by IP administration of menthol. Moreover, GABAergic cells of VTA, NAcc, or A25/MO were not the initial targets of menthol at these sites. In summary, the present study identifies several sites, including those of the mesocorticolimbic reward‐addiction pathways that are activated by acute IP administration of menthol, however, none contained dopaminergic and GABAergic neurons.

Support or Funding Information

This work was supported by grants from Charles and Mary Latham Funds and Howard University Bridge Funds and Pilot Study Awards Program (BFPSAP). The authors acknowledge use of the resources provided by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number G12MD007597.
Geldanamycin Alters Abundance and Composition of Low Molecular Weight Proteins and Multiprotein Complexes as Observed by Size Exclusion Column Chromatography
Authors: Sherwood Susannah , Murphy Patrick
Published: April 2020

Size exclusion chromatography (SEC) is a commonly used and minimally disruptive liquid chromatography‐based technique that separates unbound cytosolic proteins and multiprotein complexes according to their perceived molecular weights. The purpose of this project was to develop a method using SEC to partially purify and isolate biomolecular complexes, including those containing the essential molecular chaperone protein hsp90, in the presence and absence of the hsp90 inhibitor geldanamycin. Using an automated SEC program and S‐200 column, unbound proteins and protein complexes of differing molecular weights were isolated from reticulocyte lysate incubated in the presence and absence of geldanamycin. In addition to hsp90 eluting in a complex of ~250–300 kDa, consistent with it being part of a multiprotein chaperone machinery, other predominant protein elution peaks corresponded to ~26 kDa and ~16 kDa. Interestingly, incubating cell lysate with geldanamycin depleted nearly all of the ~26 kDa peak, indicating an unexpected hsp90 effect. Continued examination of eluate fractions from + and − geldanamycin‐incubated lysates has allowed us to further characterize these geldanamycin‐sensitive low molecular weight proteins as we examine the multitude of intracellular hsp90 functional activities.

Support or Funding Information

Funding was provided by the Seattle University College of Science & Engineering undergraduate research program.
Glutamatergic Neurotransmission at Rat Phrenic Motor Neurons
Authors: Mantilla Carlos B. , Rana Sabhya , Zhan Wen-Zhi , Sieck Gary C.
Published: April 2020

Glutamatergic (Glu) synaptic inputs at phrenic motor neurons (PhMNs) predominantly comprise descending axons from respiratory centers in the ventrolateral medulla. Descending synaptic inputs provide the drive for inspiratory as well as various other behaviors that result in higher force generation. We recently examined pre‐synaptic Glu terminals (VGLUT1 and VGLUT2‐positive) around the somal membrane of PhMNs grouped according to size using a robust confocal imaging‐based technique and semi‐automated image processing, and found an ~10% higher density of Glu terminals at PhMNs in the lower tertile of somal surface area. In parallel studies, we determined the density of AMPA and NMDA mRNA expression in PhMNs grouped according to size, and found that PhMNs in the lower tertile of somal surface area display ~25% higher density of AMPA (Gria2) and NMDA (Grin1) mRNA expression compared to PhMNs in the upper tertile of somal surface area. Assuming an orderly recruitment of motor units based on their intrinsic electrophysiological properties, PhMNs in the lower tertile of somal surface area are likely recruited first in order to accomplish lower force behaviors necessary for inspiration in normoxic as well as in hypoxic (10% O2) and hypercapnic (5% CO2) conditions. Thus, differences in glutamatergic neurotransmission across PhMNs of increasing size support the orderly recruitment of motor units, promoting recruitment of fatigue resistant units for inspiratory behaviors. In adult male rats, unilateral C2 hemisection (C2SH) abolishes inspiratory‐related diaphragm muscle (DIAm) activity ipsilateral to injury, but DIAm activity during higher force behaviors persists, reflecting the contribution of spared inputs, likely contralateral. Accordingly, we hypothesized that C2SH primarily disrupts Glu synaptic inputs to smaller PhMNs, whereas Glu synaptic inputs to larger PhMNs are preserved. By 7 days following unilateral C2SH, there was a ~45% reduction in ipsilateral Glu synaptic input to PhMNs compared to a ~20% reduction contralaterally. Ipsilateral to C2SH, there was an ~60% reduction in presynaptic Glu terminals at PhMNs in lower tertile of somal surface area compared to ~30% reduction at PhMNs in the upper tertile of size. Contralateral to C2SH, the reduction in presynaptic Glu terminals was similar across PhMNs grouped according to somal surface area. These results are consistent with a more pronounced effect of C2SH on inspiratory behaviors of the DIAm, and the preservation of higher force behaviors after C2SH. These results also indicate that Glu synaptic input to PhMNs varies depending on motor neuron size and likely reflects different motor control, including possibly distinct central pattern generator and premotor circuits, onto PhMNs recruited for motor behaviors with varying levels of force generation.

Support or Funding Information

NIH Grants R01 HL96750 and HL146114
Neural‐Like Plasticity in Airway Epithelia
Authors: Reznikov Leah R. , Liao Yan-Shin , Guevara Maria
Published: April 2020

Many cells exhibit plasticity. In neurons, plasticity is associated with learning and memory. Here we describe plasticity in epithelia similar to neurons. In neurons, a strong or repeated stimulus often results in enhanced release of neurotransmitters via a process known as sensitization. We discovered that intra‐airway acid challenge in pigs resulted in a sensitization‐like augmentation of mucus secretion in airway epithelia that persisted in vitro. Treating airway epithelia from normal pigs with neurotransmitter secretagogues in vitro also induced a long‐lasting augmentation of mucus secretion. Inhibition of protein synthesis or cAMP‐responsive element‐binding (CREB) protein, which are required for neuronal memory, prevented secretagogue‐mediated increases in mucus secretion in airway epithelia, whereas inhibition of CREB in vivo mitigated some features of acid‐induced sensitization. These observations suggest new properties of airway epithelia that require further characterization and might be of critical importance in airway health and disease.

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R01HL153730 and OT2OD023854
Sheathing the Hybrid Cleaver: Exploring Substrate‐Envelope‐Guided Drug Design with HIV‐1 RNase H
Authors: Wilcox Abigail E. , Cota Ahnika , Woodson S. Alice , Shor Audrey C.
Published: April 2020

Since the start if the epidemic in 1981, nearly 75 million people have been infected with the Human Immunodeficiency Virus (HIV). Despite significant medical advancements scientist have yet to find a cure for the disease and its subtypes. Many patients are treated with the FDA approved HAART (highly active antiretroviral therapy), in which several antiretroviral are given in attempt to control HIV reproduction and its quickly mutating enzymes. The advanced adaptability of the virus emphasizes the need for novel approaches to be taken to develop antivirals that are more robust in preventing drug resistance. We sought to investigate the strategies of substrate‐envelope‐guided design by studying variants of HIV‐1 RNase H. RNase H is an endonuclease that cleaves RNA from the RNA/DNA hybrids formed during reverse transcription of the viral genome. The approach taken to complete this study included considering RNase H structure, function, and mechanism. Variants were categorized and mapped to the structure of the enzyme. Resistance variants that extended beyond the substrate envelope were excluded in developing the proposed substrate envelope to target for inhibitor design. Applying the substrate‐envelope hypothesis developed by Dr. Cynthia Schiffer’s lab group afforded consideration of the method, while acquiring an advanced understanding of the structure and function of this significant domain within the larger, complex reverse transcription enzyme of HIV. Furthermore, RNase H active site serves as an emerging target of next generation therapeutic targets for treatments of HIV. Studying the evolutionary dynamics of this adaptive virus should be considered for the rational design of potent inhibitors of this protein, significant to HIV reproduction.

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The CREST Project is funded by NSF‐DUE 1022793 and NSF‐DUE 1323414.
SPARC: A Road Map for Vagus Nerve Stimulation: Evidence of Vagotopy in a Swine Model
Authors: Settell Megan L. , McConico Andrea L. , Nicolai Evan N. , Knudsen Bruce E. , Shoffstall Andrew , Ross Erika K. , Pelot Nicole A. , Grill Warren M. , Johnson Aaron J. , Ludwig Kip A.
Published: April 2020

Cervical vagal nerve stimulation (VNS) is an FDA approved treatment for epilepsy and depression and is currently being evaluated for treatment of numerous other disorders including pain, anxiety, and inflammation. VNS also received European market approval for heart failure, bronchoconstriction, and diabetes; however, approval for these indications was based on studies in animals, such as dogs and rats, whose vagal nerve size may not have been representative of humans. When subjected to double‐blinded, randomized, sham controlled studies for subsequent U.S. approval, many of these CE‐marked VNS indications failed to meet their primary efficacy endpoints. More than 100,000 patients have undergone VNS, but despite the efficacy of vagal nerve stimulation in a subset of patients, many fail to reach stimulation levels that result in alleviation of symptoms without triggering unwanted side effects. Post hoc studies of the patient population suggested that VNS may not engage the intended fiber pathways in humans, despite engagement in preclinical studies in mouse, rat, or dog. Stimulation parameters were limited in these studies as higher parameters caused side effects such as cough, voice alteration, and dyspnea. Discrepancies between stimulation amplitude needed for intended effect versus unwanted side effect could be directly related to the relative size of the vagus nerve across animal models, variations in fascicle organization, or variations in branching patterns.

To address translating improvements in VNS from the lab to the clinic, we evaluated the swine as a potential model for approximating the clinical environment. The size and fascicular organization of the swine vagus nerve is more representative of the human nerve than previously used models. We used a combination of histology, microdissection, and ultrasound to establish a distinct bimodal organization of groups of fascicles stemming from one large fascicle at the inferior (nodose) ganglion, an organization we have termed ‘vagotopy’. We demonstrated that with the use of ultrasound we can visualize the fascicular organization in vivo, an approach that could be used clinically to target stimulation of either sensory fibers or parasympathetic efferents, depending on therapeutic intent. These advances in a clinically relevant model not only inform electrode placement for target engagement, but provide an approach for reduction of side effects.

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Acknowledgements

Funded by TL1TR002380 andThe Defense Advanced Research Projects Agency (DARPA) Biological Technologies Office (BTO) Targeted Neuroplasticity Training Program under the auspices of Doug Weber and Tristan McClure‐Begley through the Space and Naval Warfare Systems Command (SPAWAR) Systems Center with (SSC) Pacific grants no. N66001‐17‐2‐4010, and the NIH SPARC Program Award OT2 OD025340.
Investigating the effects of Progressive Right Ventricular Remodeling on Systolic and Diastolic RV Function in a Longitudinal Animal Model Study of Pulmonary Arterial Hypertension
Authors: Kwan Ethan Dillon , Zhang Xiaoyan , Velez-Rendon Daniela , Stowe Jennifer , Valdez-Jasso Daniela
Published: April 2020

Pulmonary Arterial Hypertension (PAH) is a progressive vasculopathy which increases pulmonary vascular resistance and induces a pressure overload on the right ventricle, however the mechanisms by which the RV adapts to pressure overload is not well understood
. The purpose of this study was to characterize the mechanistic progression of earlier‐stage right ventricular compensated remodeling in an animal model of PAH. In a longitudinal study of the Sugen‐Hypoxia animal model of PAH, invasive RV catherization measurements were used to assess right heart function, including RV pressure and volume relations, hemodynamic parameters, and systolic and diastolic elastance measures. Because RV ejection fraction has been found to be transiently maintained in early stage remodeling, this study also sought to distinguish the contributions of systolic and diastolic volume preservation or increases in stroke volume to this maintenance
. To elucidate the mechanistic contributions to changes in organ‐level cardiac function over the course of PAH progression, a computational model was developed to differentiate between contributions due to RV geometric remodeling such as chamber dilatation and wall thickening from contributions due to intrinsic sarcomere changes such as myocardial contractility. Over the course of PAH progression, pulmonary vascular resistance and end systolic and end diastolic pressures continuously increased, while end systolic and diastolic volumes seemed to maintain. Ejection fraction and stroke volume also seemed to maintain over this period of compensated RV remodeling. Computational model analysis of RV myocardial wall mechanics and RV geometry seemed to indicate that changes in sarcomere mechanical properties were required to predict both end‐systolic and end‐diastolic PV relations throughout the study time points. Passive material parameters increased between early weeks 3–4 to mid weeks 5–6 to late weeks 8–10. Active material parameters indicated that large increases in myocardial inotropy, in part due to increases in intracellular calcium concentration and sarcomere length, occurred in order to maintain RV compensation after four weeks, and remained high through ten weeks. Ongoing biochemical analysis and tissue mechanical testing of the extracellular matrix indicate that significant contribution of fibrotic collagen deposition and stiffening of the ECM in the later weeks may explain increase in diastolic stiffness. Distinguishing between the specific mechanistic contributors to RV compensated remodeling can help identify the mediators of the transition between early stage compensated RV remodeling and later stage decompensated RV remodeling.

Support or Funding Information

National Institutes of Health NHLBI 1T32HL 105373