bluuebunny/crossref_metadata_2025 huggingface dataset:
Secular changes in growth of Polish schoolboys from 1955 to 1988 in historic context
Authors: Bielecki Emily Marcene , Haas Jere D , Hulanicka Barbara
Published: June 2021

Background

Anthropometric data, especially in children, has been widely used to evaluate the effects of socioeconomic conditions on growth and well‐being.

Objective

The aim of this analysis is to describe the changes in height, weight and body mass index (BMI) in Polish schoolboys born between 1937 and 1981 from a historic perspective.

Methods

Data from four nation‐wide cross‐sectional surveys (1955, 1966, 1978, and 1988) were analyzed. Height, weight and BMI were converted to standardized scores using the LMS method applied to the 2000 National Center for Health Statistics growth reference.

Results

In each consecutive survey year schoolboys (7–18 years) are generally taller and heavier than the same aged boys in the previous survey year. Within survey years, there is a decline with age into puberty in height Z‐scores. The prevalence of obesity is low and underweight appears to have greater relative importance.

Conclusions

Historical context of economic changes supports that adolescents measured within each survey year likely experienced an adverse environment during their first few years of life. This was partly compensated during later growth in selected birth cohorts that were measured over a 10 to 12 year interval. Some, but not all of the age dependant decline in the Z‐scores can be explained as an artifact of the reference.

Research support came from Fulbright and the NS Meinig Professorship at Cornell.

Grant Funding Source: Research support came from Fulbright and the NS Meinig Professorship at Cornell.
Visualization of virtual screening results on tiled display walls (TDW)
Authors: Lau Christopher D. , Levesque Marshall J. , Date Susumu , Chien Shu , Haga Jason H.
Published: June 2021

Virtual screening has facilitated the drug discovery process through the use of molecular simulations to computationally identify inhibitors and has yielded a tremendous amount of data. These results cannot be utilized in laboratory experiments until they are analyzed manually to identify anomalies, redundant chemical structures, and false positives. For this reason, a new, high‐throughput method to analyze the results of virtual screenings is needed, thereby accelerating the drug discovery process.

We have developed, deployed, and tested a modified version of ViewDock (named ViewDock TDW) on two separate TDW configurations. This tool allows a user to simultaneously visualize, group, compare, and analyze dozens of ligand‐receptor interactions on a TDW. To achieve this it was necessary to modify its communication with the molecular visualization program Chimera and create several other shell, python and perl scripts to enable the graphic user interface. ViewDock TDW preserves the functionality of the Chimera software, allowing for rotation, zoom, view angle, protein surface visualization, and calculation of hydrogen bonds. The program also enables global control over all ligand‐receptor models as well as specific control for commands to be sent to individuals or groups of ligand‐receptor pairs on the TDW. This program will help greatly in the visualization and analysis of virtual screening data.
In‐silico identification of high potential SSH‐2 specific inhibitors
Authors: Mui Matthew K. , Levesque Marshall J. , Chien Shu , Haga Jason H.
Published: June 2021

Cell growth and movement are controlled in part through the activation of a dual specificity phosphatase (DSP) called Slingshot‐2 (SSH‐2). SSH‐2 is known to contribute to the progression of cancer and Alzheimer's disease. For this reason, finding a specific inhibitor for SSH‐2 may have a profound impact in clinical treatments for different diseases. This study eliminates the need to manually test a very large chemical compound space by employing grid computer technologies in virtual molecular docking experiments to identify a list of high potential SSH‐2 inhibitors.

Binding affinities were determined in the in‐silico experiments using a previously established grid‐computing workflow that uses the molecular docking simulation software, DOCK 6.0. The results from five DSP screenings, specifically SSH‐2, VHR, VH3, PTEN and KAP, suggest that 3‐[(4,5‐dimethoxy‐3‐oxo‐1H‐isobenzofuran‐1‐yl)amino]‐4‐methyl‐benzoic acid shows the highest affinity for SSH‐2, but lowest affinity for the other DSPs, among the best 100 SSH‐2 binding compounds. These results indicate that this compound has high specificity towards SSH‐2. Although these results must be confirmed through wet bench testing, this study eliminates compounds that are not likely to bind or show high specificity for SSH‐2 and provides a foundation for the analysis of molecular interactions of chemicals with SSH‐2.
Mechanisms of hypoxia‐induced intrapulmonary arteriovenous shunting in healthy humans at rest: arterial oxygen saturation or pulmonary artery systolic pressure?
Authors: Lovering Andrew T , Yang Ximeng , Elliott Jonathan E , Beasley Kara M , Hawn Jerold A , Laurie Steven S
Published: June 2021

Acute, brief (5–10 min) exposure to hypoxia (fraction of inspired oxygen (FIO2) = 0.12) has been shown to open intrapulmonary arteriovenous (IPAV) shunt pathways in some subjects, but not all, at rest. We set out to determine the threshold FIO2 necessary to open large diameter IPAV pathways in all humans at rest and determine if the detection of IPAV shunt was correlated with arterial oxygen saturation (SpO2) or pulmonary artery systolic pressure (PASP). Ten healthy subjects (5 female) breathed four levels of hypoxia (FIO2=0.16, 0.14, 0.12, 0.10) at rest in ascending or descending order for 30 min, with each FIO2 separated by a 15 min normoxic break. Saline contrast echocardiography was used to detect the passage of bubbles through IPAV shunts into the left heart. The number of bubbles appearing in the left heart was scored on a 0–5 scale with a bubble score ≥2 defined as IPAV shunt. PASP was determined from the peak velocity of the tricuspid regurgitation. IPAV shunt occurred after 30 min of breathing an FIO2=0.16, 0.14, 0.12, and 0.10 in 1/10, 6/10, 7/10, and 10/10 subjects, respectively. Shunt score and SpO2 were well correlated (Spearman correlation, rs=−0.77, p<0.01) whereas shunt score and PASP were poorly correlated (rs=0.14, p=0.397). Our results demonstrate that hypoxia‐induced IPAV shunt can occur in all subjects at rest and that it is primarily mediated by arterial O2 saturation rather than PASP. Funding: OHSU MRF 0820
Nonselective cation channel function in sheep pulmonary arteries is affected by postnatal maturation and chronic hypoxia
Authors: Papamatheakis Demosthenes G , Vemulakonda Srilakshmi , Blood Quintin , Merritt Travis T , Lauw Sidney , Longo Lawrence D , Wilson Sean M
Published: June 2021

Non selective cation channels (NSCC) are important to pulmonary arterial (PA) pharmacomechanical coupling. Their activities and expression of the underlying Transient Receptor Potential (TRP) channel isoforms are implicated in the changes in PA contractility due to chronic hypoxia (CH) and postnatal maturity. Hence, we evaluated the combined influence of CH and postnatal maturity on NSCC function. We examined serotonin (5‐HT) elicited contraction in PA rings isolated from near term sheep fetuses (~140 days) and adult ewes maintained under normoxic (300m) or CH conditions (3801m) for 100+ days. To facilitate the delineation of potential molecular candidates, PA rings were contracted in the absence and presence of various NSCC antagonists including 50 μM SKF 96365 with 10 μM nifedipine (SKF+NIF), 10–30 μM KB‐R7943 (KBR), 100 μM flufenamate (FFA), 50–100 μM 2‐APB, 10 μM LOE 908 (LOE), or 100 μM spermine (SPE). SKF+NIF blocked contraction most prominently, followed by KBR, and then FFA. These inhibitory actions were attenuated to varying extents in normoxic and CH fetal PA. LOE and SPE modestly reduced PA tension in normoxic adult while 2‐APB preferentially reduced contractility in normoxic fetus and adult. This pharmacological profiling suggests that CH and postnatal maturation affect the functional expression of select TRPC and TRPM channels. NIH P01HD031226, R01HD003807 (LDL)
The role of CaL in sheep pulmonary arteries is altered by chronic hypoxia and postnatal maturation
Authors: Vemulakonda Srilakshmi , Papamatheakis Demosthenes G , Blood Quintin , Merritt Travis , Longo Lawrence D. , Wilson Sean M
Published: June 2021

L‐type Ca2+ channels (CaL) are important to electro‐ and pharmaco ‐mechanical coupling in pulmonary arteries (PA). Evidence indicates the role of CaL to PA contraction is potentially reduced by chronic hypoxia (CH) but increased after birth. We tested the hypotheses that these couplings are blunted by CH and accentuated following birth by examining contraction in PA rings isolated from near term sheep fetuses (~140 days) and adult ewes maintained under normoxic (300 m) or CH conditions (3801 m) for 100+ days. PA rings were depolarized with cumulative [K+] doses, from 5 to 125 mM or stimulated with 10 μM serotonin (5‐HT). The estimated V1/2 for K+‐induced contraction was ~ 10 mV more negative in PA from normoxic ewes relative to normoxic fetuses or CH fetuses or ewes. CaL inhibition with 10 μM verapamil (VER), diltiazem (DIL) or nifedipine (NIF) reduced, but did not ablate, K+‐dependent contraction. NIF reduced 5‐HT elicited tension more significantly in rings from normoxic ewes relative to fetuses or CH ewes and fetuses. VER inhibited 5‐HT elicited tension more than DIL or NIF. Overall, electromechanical coupling appears restrained before birth and by CH and non‐CaL‐related pathways contribute to this coupling process. VER possibly blocks non‐CaL pathways during pharmacomechanical but not electromechanical coupling, which may provide insights regarding its therapeutic actions. NIH P01HD031226, R01HD003807 (LDL)
Respiratory syncytial virus (RSV) induces airway insensitivity to β‐agonists in BALB/c mice
Authors: Davis Ian Christopher , Traylor Zachary P. , Yu Erin N. Z.
Published: June 2021

Beta‐adrenergic agonists (β‐agonists) are commonly used to treat RSV bronchiolitis, but are generally ineffective. We have previously shown that RSV strain A2 inhibits bronchoalveolar epithelial responses to β‐agonists in BALB/c mice. The aim of the current study was to determine whether RSV also induces airway insensitivity to β‐agonists. Total lung resistance was measured in anesthetized female BALB/c mice on a flexiVent ventilator. RSV A2 did not induce airway hyperresponsiveness to increasing doses of nebulized methacholine (MCH) at any timepoint. Prenebulization with the β‐agonist terbutaline (100 μM) significantly attenuated bronchoconstrictive responses to MCH in uninfected mice and in mice infected with RSV for 4–8 days. However, in mice infected with replication‐competent, but not UV‐inactivated, RSV for 2 days, significant terbutaline insensitivity was found. Terbutaline insensitivity could be reversed by I.P. treatment with neutralizing anti‐CXCR2 antibodies (which reduced BAL neutrophil counts, but not viral replication), or by post‐infection nebulization with neutralizing anti‐KC or anti‐CXCR2 antibodies, and could be replicated in normal, uninfected mice by nebulized recombinant KC. These data suggest that KC/CXCR2‐mediated heterologous desensitization of β‐adrenergic receptors may underlie the modest utility of these drugs as bronchodilators in RSV bronchiolitis.
High‐Frequency Electrical Stimulation (HES) of Vagus Nerves Reduces Airway Resistance (RL) in Response to Methacholine (MCh) in Anesthetized Guinea Pigs
Authors: Xu Fadi , Zhuang Jianguo , Bailet Daniel , Curtis Robert , Zhang Cancan
Published: June 2021

Asthmatic patients are characterized by cholinergic‐mediated airway hyperreactivity and bronchoconstriction. Tachyphylaxis and safety‐related side effects of the drugs used to treat asthma are clinical challenges. Because vagotomy blunts the RL response to MCh and HES blocks nerve conduction, we hypothesized that HES of the cervical vagus nerve would reduce the RL responses to MCh, especially in ovalbumin (Ova)‐sensitized guinea pigs, an established animal model of asthma. Both saline (Sal)‐ and Ova‐treated animals were anesthetized, paralyzed, and ventilated. RL and its responses to 10 s of low frequency electrical stimulation (LES, 50 Hz) and HES (≥1 KHz, 0.5 V; 0.2‐ms pulse duration) were recorded before and after MCh (100 μg/ml/min for 2 min) inhalation. Only LES significantly increased RL in both groups of animals before MCh. After MCh, the RL was increased 2.5‐fold higher in the Ova‐ than Sal‐treated animals and LES still enhanced RL, but HES suppressed the RL response more greatly in the Ova‐ (49%) than the Sal‐treated animals (35%). These evoked RL responses completely followed the electrical stimulation in an on‐and‐off manner with little effect on cardiovascular activity. Our data suggest that HES is a potentially useful approach in alleviating asthmatic bronchoconstriction.
Dynamic [Ca2+]i regulation in human airway smooth muscle by STIM and Orai1 proteins
Authors: Thompson Michael A , Pabelick Christina M , Prakash Y S , Sieck Gary C
Published: June 2021

Regulation of intracellular Ca2+ in human airway smooth muscle (ASM) involves sarcoplasmic reticulum (SR) Ca2+ release/reuptake and plasma membrane (PM) Ca2+ influx, including store‐operated Ca2+ entry (SOCE) following SR Ca2+ depletion. Studies in human ASM have shown that siRNA suppression of stromal interaction molecule 1 (STIM1) and Orai1 decrease SOCE; however, their exact role in regulating Ca2+ are still under investigation. In human ASM, STIM1 and STIM2 were found exclusively in SR membrane, and Orai1 only in PM (especially caveolae). GFP‐tagged STIM1 transfection showed intracellular/SR localization at baseline, with increased peri‐PM aggregation following store depletion with 10ƒÝM cyclopiazonic acid (CPA). Such aggregation was prevented by microtubule inhibition (nocodazole), but not actin inhibition (phalloidin). FRET analysis revealed co‐localization of STIM1 with TRPC3 and Orai1 in CPA‐stimulated cells. In fura‐2 loaded ASM cells, SOCE was significantly reduced by STIM1 and Orai1 siRNA (less so by STIM2 siRNA). However, basal Ca2+ and agonist (ACh, histamine)‐induced Ca2+ responses were substantially modulated by STIM2 suppression. These results indicate that in human ASM, STIM1 mediates SR‐PM interactions in regulating SOCE, while STIM2 modulates sub‐maximal agonist‐induced changes in Ca2+.

Supported by NIH grant HL74309 (GCS), HL090595 (CMP) and HL088029 (YSP).
Protein kinase C activation induces low frequency Ca2+ oscillations, Ca2+ sensitization and contraction in airway smooth muscle
Authors: Mukherjee Seema , Willis Ray E , Pressley Thomas A , Perez‐Zoghbi Jose F
Published: June 2021

Protein kinase C (PKC) is an important regulator of smooth muscle cell (SMC) contraction; however the mechanisms and the PKC isoforms responsible for this regulation in small intrapulmonary airways are unknown. We have investigated the effects of pharmacological stimulation of PKC on Ca2+ signalling, Ca2+ sensitivity, and contraction in airway SMCs using mouse lung slices and phase‐contrast and confocal microscopy. Activation of PKC with phorbol 12‐myristate 13‐acetate (PMA, 1–10 μM) induced low frequency Ca2+ oscillations and transient airway contractions (twitching). However, a strong and sustained airway contraction was induced by PMA in Ca2+‐permeabilized lung slices with caffeine and ryanodine. This contraction was blocked by the PKC inhibitor GF‐109203X (1 μM), but not by the Rho kinase inhibitor Y‐27632 (10 μM). Furthermore, PMA treatment increased KCl‐induced airway contraction. Using RT‐PCR and specific oligonucleotide primers, the presence of mRNAs encoding PKCβ, PKCγ, and PKCζ were identified in the airways. In conclusion, our results suggest that activation of specific PKC isoforms induce airway contraction by stimulating Ca2+ oscillations and Ca2+ sensitivity in SMCs.
Impact of BDNF/TrkB signaling on recovery of phrenic activity after cervical spinal cord injury in rats
Authors: Zhan Wen‐Zhi , Mantilla Carlos B , Sieck Gary C
Published: June 2021

Upper cervical spinal cord injury disrupts descending inspiratory‐related drive to phrenic motoneurons (PMN) paralyzing the diaphragm muscle (DIAm). There is gradual recovery of rhythmic DIAm activity ipsilateral to injury suggesting strengthened spared synaptic inputs to PMN. Neurotrophins play an important role in regulating synaptic function. Thus, we hypothesized that intrathecal brain‐derived neurotrophic factor (BDNF) enhances recovery of rhythmic phrenic activity after C2 spinal cord hemisection (C2HS), whereas quenching neurotrophins with TrkB‐IgG impairs functional recovery. An intrathecal catheter was placed at C4 to chronically infuse artificial CSF, BDNF or TrkB‐Fc using an osmotic pump. DIAm EMG electrodes were implanted bilaterally to verify complete hemisection at the time of C2HS and 3D post‐C2HS. In the artificial CSF group, functional recovery was evident in 33% of C2HS rats at 7D and 56% of rats at 14D post‐injury. Intrathecal BDNF treatment enhanced functional recovery, with 88% of rats at 7D and 100% at 14D post‐C2HS displaying rhythmic phrenic activity. In contrast, intrathecal TrkB‐Fc treatment completely prevented functional recovery up to 14D post‐C2HS. Our results demonstrate that BDNF/TrkB signaling at the level of the PMN pool is both sufficient and necessary for functional recovery of rhythmic phrenic activity after C2HS.

Supported by NIH grant AR51173.
High Frequency Spinal Cord Stimulation (HF‐SCS) to Provide Ventilatory Support in an Animal Model of Spinal Cord Injury
Authors: DiMarco Anthony F. , Kowalski Krzysztof E.
Published: June 2021

Objective

HF‐SCS is a novel technique of inspiratory muscle activation via spinal cord pathways. The purpose of this study was to evaluate the utility of HF‐SCS to provide long‐term ventilatory support utilizing intercostal/accessory muscle stimulation alone.

Methods

In 4 anesthetized dogs after bilateral phrenicotomy, electrical stimulation (300Hz, 1.2mA, 12 breath/min, 1.2s inspiratory time) was applied via a single electrode placed ventrally at the T2 spinal level over an arbitrary period of 5.5 hours.

Results

Mean airway pressures and inspired volumes were 29±1cmH2O and 445±7ml during the first and 27±1cmH2O and 450±12ml during the final hour of chronic pacing, respectively (NS). Throughout the period of stimulation, airway pressure generation and inspired volume production remained unchanged. Mean end‐tidal PCO2 and SaO2 were 40±1mmHg and 95±1% and 40±1mmHg and 96±1% during the first and final hour of pacing, respectively (NS). To further assess the capacity of the system to maintain artificial ventilation, stimulus parameters were then increased to induce hyperventilation for an additional 30 min. This resulted in a sustained fall in end‐tidal PCO2 to 30±1 mmHg (p<0.05).

Conclusion

HF‐SCS has the potential to provide complete ventilatory support in subjects with ventilator‐dependent tetraplegia who lack adequate phrenic nerve function for diaphragm pacing.

Support: NIH‐NINDS (R01NS064157)
Glycinergic (Gly) Neurons are Activated by Acute Tracheal Occlusion (TO) in the Nucleus Tractus Solitarius (NTS)
Authors: Tsai Hsiu‐Wen , Davenport Paul W
Published: June 2021

The brainstem respiratory network includes the NTS, ventral respiratory group (VRG) and pontine respiratory group. It was demonstrated that acute TOs activated neurons in the NTS and VRG using c‐Fos. It is unknown if the activated neurons are excitatory or inhibitory. We hypothesized that inhibitory Gly neurons are activated in the NTS and VRG after acute TO. Experiments were performed in anesthetized rats, divided into experimental and sham groups with and without acute TO, respectively. The acute TOs were applied 2–3 breaths with 10–15 unoccluded breaths separating each TO presentation. The TO presentations were applied for total 10 mins. The animal was allowed 90 mins unobstructed breathing after completion of the occlusion trial. The rat was sacrificed, the brains perfused and harvested for fluorescent immunostaining. The GLYT2 (marker for Gly neurons) and c‐Fos were used for double immunofluorescence staining. The results showed the percentage of co‐labeled GLYT2 and c‐Fos neurons in the NTS of experimental group (76.5%) was greater than the sham group (43%). The co‐labeling of GLYT2 and c‐Fos were not different in the VRG. These results suggest that acute TO activated inhibitory Gly neurons in the NTS.
Adaptations of a Novel Microbial Fuel Cell Design for Electrical Production and Bacterial Load Reduction
Authors: Yoho Rachel Ann
Published: June 2021

Nine microbial fuel cells were constructed using different cathode materials and carbon cloth anodes for voltage production from a pig manure substrate. The cathode materials included: copper, brass, carbon cloth, titanium, sheet lead, galvanized low carbon steel, low carbon steel, aluminum foil‐faced fiberglass, and stainless steel. The open circuit voltage (OCV) of each of these cells was collected over time. The internal resistance (Rint) was measured using a decade resistance box at periodic intervals. From this information, the current (I) could be calculated. Data collection on six of the cells was discontinued due to low voltage production. The remaining three cells (copper, brass, and carbon cloth) continuously generated an OCV for over eighteen weeks. As compared to the discontinued cells, the copper and brass cathode cells produced consistently higher voltages (approximately 0.65 V and 0.55 V) and had currents in the range of 15 to 25 mA and 8 to 10 mA, respectively, for the majority of their life spans. The carbon cloth cathode cell produced high voltages (approximately 0.68 V), but displayed a current of less than 10 mA. These three cells displayed an average bacterial load reduction of approximately 93.6%. This information can be applied towards the development of more efficient microbial fuel cells with higher OCV and current outputs, and cells with increasing efficacy at reducing the bacterial load.
JSim: software for multiple hypothesis testing and model refinement
Authors: Butterworth Erik , Bassingthwaighte James B
Published: June 2021

JSim software expedites the hypothesis formulation/testing cycle for quantitative modeling. JSim's Mathematical Modeling Language allows concise, intuitive formulation of quantitative models with wide scope (ODEs, PDEs, DAEs, implicit eqns, events). The model compiler checks for mathematical and physical unit consistency thus eliminating common model formulation errors. Compiler generated multiprocessor computations are fast and support real‐time graphical monitoring. Menu‐driven looping, sensitivity, optimization and curve fitting facilities allow quick visualisation of model behaviour and statistical validation against experimental data. Menu selectable numeric methods (9 ODE solvers, 3 PDE, 8 optimizers) allow tuning to balance accuracy & performance. JSim “projects”, consisting of (often multiple) models, parameters sets, signal generators, numeric data sets, notes and plot pages facilitate comparison of multiple hypothesis, and enable easy sharing with collaborators. Documentation (100pp) and models (250+ locally curated, 50,000+ contributed) in MML, SBML and CellML posted at physiome.org Available for Windows, Mac, Linux, WWW applet and as open source. (NIH support T15HL88516, EB08407).
Vagally‐mediated gastrointestinal reflexes are modulated by central glucose levels
Authors: Browning Kirsteen N
Published: June 2021

The vagally‐mediated receptive relaxation reflex is activated upon distension of the esophagus and induces gastric relaxation and delays gastric emptying. Many gastrointestinal (GI) hormones exert dramatic control over vagally‐mediated GI functions via brainstem sites of action. The aim of this study was to determine whether glucose‐induced alterations in GI reflexes are achieved, in part, by brainstem sites of action.

A balloon was used to distend the esophagus of adult rats and the resulting gastric relaxation measured via miniature strain gauges affixed to the stomach. Increasing blood glucose levels decreased the magnitude of the receptive relaxation from 0.6±0.1g to 0.4±0.1g and 0.3±0.1g at 3±0.2mM glucose, 6±0.5 and 10±1.8mM blood glucose, respectively (n=3). Similarly, altering brainstem glucose directly (via 4th ventricular application 3, 4.5 or 10mM glucose) decreased the receptive relaxation by 5±3, 15±0 and 20±3%, respectively (n=3). The glucose‐induced decreased receptive relaxation was blocked by brainstem application of the 5‐HT3 antagonist, ondansetron (30nmoles; 15±0.3% reduction to 10mM glucose vs 0±0.1% reduction in the presence of ondansetron, n=3).

These results suggest that alterations in brainstem glucose levels, either directly or indirectly, exerts profound effects upon vagally‐mediated GI reflexes possibly via mechanism involving alterations in brainstem 5‐HT3 levels

Supported by DK 55530
Influence of Perfluorocarbons on Systemic Parameters of Transgenic Sickle Cell Mice
Authors: Andrade Gabriela Chagas , Pedro Flavia P. , Torres Ivo P.
Published: June 2021

Transgenic (Tg) sickle cell disease (SCD) mice have been used to study SCD pathophysiology. Perfluorocarbon (PFC) is a synthetic oxygen (O2) carrier that can improve the blood O2 content. Thus, physiological responses to hypoxia (hyp)/reoxygenation (reox) were studied in Tg SCD mice treated with PFC. Anesthetized Tg mice were subjected to 60 min of hyp (FiO2=13%) followed by 90 min of reox (FiO2=50 %). PFC (Oxycyte, Oxygen Biotherapeutics, Inc, 6ml/kg BW) was injected at the end of the hyp. O2 saturation (SO2), heart rate (HR), and breath rate (BR) were measured each 5 min. Hemoglobin concentration (Hb), hematocrit (HCT) and venous PO2 were measured at the end of the experiment. Homozygous SCD mice (n=11) presented Hb= 6.7 ± 1.2 g/dL (mean ± SD) and HCT=31.1 ± 3.9 %, heterozygous (n=16) showed Hb=12.7 ± 1.5 g/dL and HCT=39.5 ± 3.5 %), while control mice (CB57BL6) (n=4) showed Hb=14.5 ± 1.3 g/dL and HCT=48.7 ± 3.3 %. PFC‐treated homozygous mice (n=6) showed lower SO2 (p<0.02) than the saline group (99.1 ± 0.8 % and 99.6± 0.3 %, respectively) but BR and PO2 were significantly higher in PFC‐treated mice (141 ± 8 bpm vs 134 ± 11 bpm and 51 ± 6 mm Hg vs 41 ± 9 mm Hg, respectively). HR in the PFC group was also significantly higher than in saline‐treated mice (593 ± 16 bpm vs 564 ± 16bpm). The results suggest that PFC therapy may improve cardiac and respiratory responses to hyp/reox in these severely anemic SCD mice.

Support: Oxygen Biotherapeutics, Inc.
Enhancement of Membrane Cholesterol Attenuates the Leukocyte Shear Stress Response
Authors: ZHANG XIAOYAN , Hurng Jonathan , Chan Darby , Rateri Debra , Daugherty Alan , Schmid‐Schonbein Geert , Shin Hainsworth
Published: June 2021

We tested the hypothesis that elevated membrane cholesterol of leukocytes (associated with elevated plasma cholesterol levels due to hypercholesterolemia) influences their sensitivity to shear. For this purpose, we assessed the shear stress regulation of pseudopod activity of human neutrophils after membrane cholesterol‐modifying treatments and of leukocytes from hypercholesterolemic mice. Our results show that, compared to naive cells, migrating human neutrophils with enhanced membrane cholesterol exhibited impaired pseudopod retraction during micropipette shear (τmax~2 dyn/cm2, 2 min). Moreover, significantly elevated (>4‐fold) blood cholesterol levels in LDL receptor‐deficient (LDLR−/−) mice fed a high fat diet (WD) for 2 wks compared to those in LDLR−/− (ND) or wild‐type (WT) mice on regular chow correlated with an impaired shear response; suspended neutrophils from WD did not exhibit significant reductions in pseudopod activity after exposure to cone‐plate shear (5 dyn/cm2, 5 min). Although plasma cholesterol levels in ND were ~2‐fold higher than those in WT, neutrophils from ND still responded to shear with reduced pseudopod activity. Combined, these results suggest that elevated membrane cholesterol impairs the leukocyte shear response, a perturbation that may contribute to chronic leukocyte activity in hypercholesterolemia. Funded by the NIH, the UKY Research Support Fund and the AHA.
LINE‐1 DNA methylation in Colombian school children is associated with birth weight and maternal BMI, and predicts physical growth
Authors: Villamor Eduardo , Rozek Laura S , VanZomeren‐Dohm Adrienne A , Dolinoy Dana , Marin Constanza , Mora‐Plazas Mercedes , Baylin Ana
Published: June 2021

Early developmental exposures involve epigenetic modifications, such as DNA methylation, that influence subsequent health outcomes. We quantified CpG methylation (%5‐methylated cytosines, 5mC) of long interspersed nucleotide elements (LINE)‐1, an indicator of global DNA methylation, in blood DNA samples from 97 participants of an ongoing schoolchildren cohort in Bogota, Colombia, and examined its associations with sociodemographic factors and child growth. Mean age was 9.6 y; 49% were boys. Mean %5mC was 80.3% (range=71.0, 88.3). %5mC was 1.7% higher in boys than girls (P=0.01); it was inversely associated with birth weight (−0.1% per 100 g, P=0.01) and with current maternal BMI <18.5 kg/m2 (−2.2% vs. normal BMI, P=0.003). In longitudinal analyses over a median 2.5 y follow‐up, LINE‐1 methylation inversely predicted height and BMI growth velocities in boys (−0.1 cm/y, P=0.05; and −0.03 kg/m2/y, P=0.02, per 1 %5mC, respectively), but not in girls. These results highlight the importance of sex‐specific analyses when evaluating epigenetic effects. Future studies should consider analyses of gene‐specific methylation profiles to better understand the functional consequences of global hypomethylation on child health.
Visceral adipose tissue DNA methylation at dipeptidyl peptidase‐4 gene locus is associated with gene expression and plasma lipid levels in severe obesity
Authors: Turcot Valerie , Bouchard Luigi , Tchernof Andre , Deshaies Yves , Marceau Simon , Biron Simon , Lescelleur Odette , Berthio Laurent , Vohl Marie‐Claude
Published: June 2021

Dipeptidyl peptidase‐4 (DPP4) is overexpressed in visceral adipose tissue of severely obese subjects with the metabolic syndrome (MS) as compared to those without MS.

Aim

To test whether methylation of DPP4 is associated with DPP4 mRNA levels and MS‐phenotypes.

Methods

Greater omentum fat tissue DNA was extracted in 92 severely obese women undergoing biliopancreatic derivation to treat morbid obesity. Women were premenopausal, non‐diabetic and not taking medications to treat MS features. Cytosine methylation levels (%) in DPP4 CpG island (Human build 36.3; chr2: 162638045‐162639319 reverse strand) was assessed by pyrosequencing of sodium bisulfite treated DNA.

Results

Mean methylation levels of combined CpG sites no.94–102 (%Meth) were calculated. The %Meth correlated negatively with DPP4 mRNA levels (r=0.25, p<0.05). Subjects with %Meth corresponding to the second tertile had lower total‐ and LDL‐cholesterol levels (PANOVA<0.05; adjusted for age, smoking, body mass index).

Conclusions

In visceral adipose tissue of severely obese women, lower methylation levels of specific DPP4 CpG sites were associated with higher mRNA levels. Methylation levels were also associated with blood lipids.

Grant Funding Source: Canadian Institutes of Health Research
The costs of getting too hot: Proteins that take the heat from global warming
Authors: Tomanek Lars
Published: June 2021

The heat shock response (HSR) varies in animals that occupy different thermal environments. Some organisms occupying stable thermal environments do not have an inducible HSR. Organisms that occupy highly variable thermal environments (variations up to > 20°C) induce the HSR at times frequently and within the range of body temperatures they normally experience, suggesting that the response is part of their biochemical strategy to occupy this thermal niche. In comparison, organisms occupying moderately variable thermal environments (<10°C) activate the HSR at temperatures above those they normally experience in their habitats. They have a wider temperature range above their body temperature range over which they can synthesize Hsps. Due to this variation in the HSR, species from stable and highly variable environments are likely to be more affected by climate change than species from moderately variable environments. Furthermore, animals that occupy different thermal environments also vary in their proteomic response to heat stress; results that provide insights into the metabolic costs of sublethal levels of thermal stress.
A fitness cost for thermal tolerance in a marine copepod: Implication on biological effects of global warming
Authors: Leung Kenneth Mei Yee
Published: June 2021

On rocky shores, the marine copepod Tigriopus japonicus is an inhabitant of shallow supralittoral tidepools and able to cope with a certain degree of thermal stress. However, different geographical populations of T. japonicus may have different extents of thermal tolerance. This study used a two‐generation experiment to compare the thermal tolerance of a temperate South Korean population (SK) and a tropical Hong Kong population (HK) of T. japonicus. 81 mate‐guarding pairs (F0; 27 pairs each) were raised in three temperatures: 15, 20 and 25°C for 30 days. The first brood of each female was cultured in the same medium and their offspring were crossed with others exposed to the same temperature. The results showed that F0 of SK copepods demonstrated a lower lethal temperature (LT) at 42.9°C than F0 of HK copepods (LT: 45.3°C). At 25°C, although F1 of SK copepods showed an improved thermal tolerance, their reproductive output was significantly less than that of the HK's F1 group. Also, expression of heat shock proteins in the SK group was significantly higher than that in HK group after heat stress at 30°C for 90 minutes. Our results suggest that multigenerational development of thermal tolerance in SK copepod is possible, but at the expense of population fitness. Such observations have profound implications on the long‐term biological consequence of global warming on T. japonicus populations along the Western Pacific coasts.
Restoration of fatty acid but not glucose utilization in the infarcted heart following stem cell therapy
Authors: Hughey Curtis , Ma Lianli , James Freyja , Young Pampee Paul , Wasserman David , Rottman Jeff , Shearer Jane
Published: June 2021

Aim

To determine whether stem cell therapy reduces abnormal cardiac substrate utilization following a myocardial infarction (MI).

Methods

Three treatment groups, SHAM, MI and MI‐STEM receiving intramyocardial injection of mesenchymal stem cells. Echocardiography was performed at 0, 1 and 3wk. C57BL/6J mice underwent a hyperinsulinemic‐euglycemic clamp 4wk following treatment (n=8–11). To assess indices of whole body and cardiac long chain fatty acid (LFCA) and glucose utilization (R(f), R(g); umol/g wet weight/min), 2‐deoxy[(3)H]glucose and [125I]‐15‐(rho‐iodophenyl)‐3‐R,S‐methylpentadecanoic acid were administered. Both the peri‐infarct (PI) region and a non‐involved (NI) area of the left ventricle were examined.

Results

No differences in whole body substrate utilization or insulin sensitivity, as assessed by glucose infusion rate were apparent between treatments. In the PI region, MI resulted in declines in R(f) compared to SHAM (0.25 vs 0.44). This decline was mitigated in MI‐STEM (0.33 vs 0.44). In contrast, R(g) was diminished in both the MI and MI‐STEM groups in the PI (1.77 vs 2.0 vs 3.87) compared to the SHAM. Additionally, R(f) and R(g) in the NI was not different between groups.

Conclusions

Stem cell therapy assists in reducing the metabolic abnormalities following a MI. Specifically, it minimizes MI‐induced declines in R(f) but not R(g).
A Pathological Role for Protease‐Activated Receptor 2 in the Heart
Authors: Pawlinski Rafal , Antoniak Silvio , Spring Denise , Verrier Edward D. , Blaxall Burns C. , Mackman Nigel
Published: June 2021

We investigate the role of protease‐activated receptor 2 (PAR2) in heart pathology. PAR2 mRNA expression was analyzed by real‐time PCR in heart biopsies from patients with heart failure (HF). The role of PAR2 was investigated in mouse model of cardiac ischemia‐reperfusion (I/R) injury. Finally, we examined the effect of a cardiomyocyte‐specific overexpression of PAR2 in mice (TgPAR2). We found increased PAR2 mRNA levels in patients with ischemic HF (3.0±0.7fold) and idiopathic HF (4.3±0.6fold; P<.05 vs. controls). These levels returned to baseline after implantation of a LVAD. Interestingly, the infarct size was reduced in the hearts of PAR2−/− mice (24.3±1.7% vs. 35.8±3.4%; P<.05 vs. WT) after 2 h of reperfusion. This injury reduction was associated with less expression of pro‐inflammatory genes. PAR2−/− were protected from I/R injury‐induced heart remodeling determined by ECHO (PAR2−/− vs. WT: LV diameter: 1.43±0.06mm vs. 1.78±0.09mm; fractional shortening (FS): 49.3±1.2% vs. 40.9±1.6%; P<.05). Finally, we observed that 1 year old TgPAR2 mice developed heart hypertrophy with increased HW/BW ratio. ECHO analysis revealed that TgPAR2 mice had reduced heart function (FS: 38.3±5.1% vs. 24.5±6.7%; WT vs. TgPAR2; P<.05). These results indicate that PAR2 in the injured heart enhances inflammation and infarct size that leads to hypertrophy and HF.

This study was supported by the AHA and NIH.
Integrative approach to study the molecular basis of post‐myocardial infarction remodeling in porcine heart
Authors: Kuster Diederik , Verhoeven Adrie , Merkus Daphne , Duncker Dirk
Published: June 2021

After myocardial infarction (MI), the left ventricle (LV) undergoes remodelling in an attempt to maintain cardiac performance. Despite the apparent appropriateness, LV remodeling is an independent risk factor for heart failure. Although much is known about the role of individual proteins in cardiac remodeling, an integrative unbiased approach has been lacking, especially in large animal models, such as the pig. Therefore, we studied gene expression with microarray and transcription factor (TF) activity with Protein/DNA array and linked these to in vivo measurements of LV remodeling and function in pigs, by using network analysis. Three weeks after MI, LV remodeling and dysfunction as well as blunting of the β‐adrenergic response were noted in vivo. At gene expression level, matrix metalloproteinases were upregulated and the β‐adrenergic/cAMP signaling was disturbed. Network analysis suggested a role for inflammation and calcineurin signaling. Also several transcription factors were activated or deactivated. Linking these TFs to microarray data suggested an important role for glucocorticoid receptor (GR).

The present study shows that an integrative approach, linking in vivo observations with changes in gene expression and TF activity leads to new insights in the molecular basis of post‐MI remodeling in a large animal.

This study was supported by a grant from the Netherlands Heart Foundation (2005B234)
Decrease IL33 expression in cardiac fibroblasts with high concentration of glucose leads to collagen IV production: role of PKCβ
Authors: Su Zhaoliang , Xu Hu , Zhang Jingchao , Martin Claudio M. , Rui Tao
Published: June 2021

Diabetic cardiomyopathy (DiCM) is one of major complications of diabetes mellitus. Increase in collagen production by cardiac fibroblasts has been implicated in the development of the DiCM. In the present study, we assessed the role of PKCβ/IL‐33 pathway in the high glucose (HG)‐induced collagen production in cardiac fibroblasts.

Methods

Cultures cardiac fibroblasts were exposed to medium containing high concentration of glucose. The PKCβ phosphorylation status, IL33 and collagen production were assessed with Western or real‐time RT‐PCR.

Results

Treatment of the cardiac fibroblasts with HG led to a decrease in IL33 mRNA and protein expression which was associated with an increase in collagen IV production. Administration of IL‐33 prevented HG‐induced collagen IV production. Expose of cardiac fibroblasts to HG resulted in PKCβ;activation as indicated by increase in phosphorylation of PKCβ;Furthermore, inhibition of PKCβ;with an inhibitor prevented the increase in collagen IV production in HG‐treated cardiac fibroblasts.

Conclusion

Our results suggest that PKCβ/IL33 pathway plays an important role in regulation of HG‐induced collagen IV production in cardiac fibroblasts. (CIHR MOP‐81303)
Statins improve innate resistance to pneumococcal pneumonia via lung macrophage NOS3
Authors: Yang Zhiping , Ghosh Sanjukta , Kobzik Lester
Published: June 2021

Statins have been linked epidemiologically to better outcomes in pneumonia, but the mechanisms are unclear. We investigated the role of statins in boosting innate immune resistance to bacterial pneumonia using a mouse model. In bacterial pneumonia induced by Strep. pneumoniae (serotype 3, 100K CFU i.n.), pravastatin (50 mg/kg i.p.) caused improved bacterial clearance (% bacterial left alive in lungs at 24 hours, mean ± SD: control, 65 ± 22 vs statin‐treated, 17 ± 11; n = 7 female mice/grp), reduced BAL neutrophilia and improved survival. The mechanisms were linked to activation of lung macrophage NOS3, based on statin‐induced activation of macrophage NOS3 (increased phosphorylation of serine 1177 on Western blot analysis), statin‐mediated enhancement of macrophage killing of pneumococci in vitro and absence of a beneficial effect of statins in NOS3‐deficient mice. In addition, statin treatment of mice with secondary bacterial pneumonia after influenza resulted in prolonged survival. The data support the potential benefit of short‐term therapy with statins to boost innate resistance to pneumococcal pneumonia. Supported by NIH HL083436, ES11008, ES00002.
Stereologic sampling and quantitation of Mycobacterium tuberculosis (M.tb.) lesions in rhesus monkey lungs
Authors: Hyde Dallas M , Adamson Lou , Canfield Don , Tarara Ross , Hirst Linda , Christianson Miles , Wisner Eric , Ventimiglia Frank , Luciw Paul
Published: June 2021

TB is a global infectious killer. Detection systems for TB are not efficient, TB drugs are over 40 years old, and the BCG vaccine dates back over 80 years; hence, there is an urgent need to develop methods to find faster, easier and more effective diagnostics, drugs and vaccines to control TB. Research in nonhuman primate models addresses these needs. Six healthy macaques were intratracheally inoculated with 500 CFU of the clinical M.tb. Erdman strain and followed for disease progression. M.tb. infection was confirmed in all 6 monkeys. At 3 months after inoculation, 2 animals developed significant TB symptoms and were euthanized and necropsied. The remaining 4 animals were necropsied at 6 months. Lungs slices (5 mm thick), overlaid with a Plexiglas template with 5 mm holes, were used to cut cores for the volume of TB lesions and bacterial burden. Lung cores were fixed, embedded, sectioned and stained with H and E. We estimated the volume of TB lesion by stereologic point counting, and homogenates of lung cores were serially diluted and plated to measure CFU. The volume TB lesions and the number of M.tb. CFU were estimated from lung core samples. Our results show that a Plexiglas overlay and circular knives enables a stratified random sampling of TB infected lung tissue in a BSL‐3 environment that produces accurate and efficient estimation of the amount of TB lesions and the number of TB bacteria.

NCRR RR000169 and Aeras Foundation
Morphometric analysis of bleomycin‐induced alveolar epithelium type 2 cell injury and cell shape changes during repair in murine lungs
Authors: Wang Ping Ming , Martin William J
Published: June 2021

Bleomycin is known to induce diffuse alveolar damage including injury to alveolar type 2 epithelial (AT2) cells, the progenitor cell for the alveolar type 1 cell. There is a need to quantitatively assess injury to alveolar epithelial cells. To address this, we performed morphometric analysis of lung tissue from female mice given bleomycin (3.33 U/kg) or saline intratracheally. Lung were isolated and cryosectioned at day 2, 4, 7, 9, 11, or 14 post bleomycin. We identified AT2 cells immunostained with anti‐pro‐SPC and quantified the pro‐SPC (+) cells using image software (MetaMorph) from 8 randomly selected regions of lung sections. Average AT2 cell counts were markedly reduced 27–43% from the control level at day 2, 4, 9, and 11 post bleomycin (P<0.05, n=4 for each time point). These were consistent with morphologic evidence of bleomycin induced alveolar epithelial injury. However, the total pixel area per imaging region, average pixel area, and length of individual SPC (+) AT2 cells increased from day 2 to 14 post bleomycin (P<0.05, ANOVA) and the shape factor decreased (P<0.0001, ANOVA), consistent with morphologic transition of many AT2 cells from a cuboidal shape to the more flattened shape of AT1 cells. Our results suggest that bleomycin‐induced acute lung injury and repair can be quantified at the cellular level that may permit an improved approach to dissecting the underlying mechanisms of injury and repair.
Functional Expression of the Purinergic Receptor P2Y14 in the Human Lung Microvascular Endothelial Cells (HLMVEC)
Authors: Umapathy Nagavedi Siddaramappa , Hick Richard Todd , Yu Yanfang , Verin Alexander D
Published: June 2021

The P2Y14 is a Gi‐coupled receptor that inhibits the synthesis of cAMP, and is activated by UDP‐glucose, UDP‐galactose, and UDP‐N‐acetylglucosamine. Recent studies indicate the role of P2Y14 in modulating the immune function in T‐lymphocytes and secretion of the proinflammatory cytokine IL‐8 in airway epithelial cells. Furthermore, extracellular release of UDP‐glucose was observed in thrombin‐treated astrocytes suggesting that P2Y14 may be activated in inflammation. Our data indicate for the first time that P2Y14 is expressed in endothelial cells (HLMVEC) as evidenced by RT‐PCR, Western blot, and Immunofluorescence studies. The cAMP assay indicates that P2Y14 is functional in HLMVEC as an addition of the P2Y14‐specific substrate, UDP‐glucose, inhibited the forskolin‐induced cAMP production. Signaling studies show treatment of HLMVEC with UDP‐glucose activates the Raf‐1/MEK‐1/ERK1/2 pathway. The ECIS (Electrical cell‐substrate impedance sensing) data indicate that pretreatment of HLMVEC with UDP‐glucose stimulated wound healing suggesting the participation of P2Y14 in endothelial repair after acute lung injury. P2Y14 has been linked to the neuroimmune function and we hypothesize that it could be regulated by immunologic challenge in modulating the immune system and detailed studies are necessary in order to establish its role in physiological/pathophysiological state of the lung endothelium. This work was supported by NIHLBI grant HL083327 (ADV) and MCG PSRP grant (NSU).
Chemical‐genetic activation of the Unfolded Protein Response reduces protein misfolding
Authors: Chiang Wei‐Chieh , Joseph Victory , Messah Carissa , Lin Jonathan H
Published: June 2021

Unfolded Protein Response (UPR) comprises a set of intracellular signaling pathways ‐ controlled by Ire1, Perk, and Atf6 genes ‐ that detect protein misfolding in the endoplasmic reticulum (ER) and activate a broad transcriptional program that restores cellular proteostasis. UPR transcriptional targets include chaperones, ER‐associated protein degradation (ERAD) and the ubiquitin‐proteasome system components, and ER structural and transport proteins. Many human diseases arise from protein misfolding. We hypothesized that UPR signaling could be harnessed to prevent protein misfolding. To test this, we developed chemical‐genetic tools that allowed us to selectively activate individual UPR pathways. We then assessed how individual UPR pathways affected rhodopsin protein bearing a proline to histidine missense mutation at residue 23 (P23H). P23H rhodopsin was previously demonstrated to form insoluble aggregates that are retained in the ER and ultimately cause cell death and hereditary Retinitis Pigmentosa. We found that artificial UPR signaling selectively removed misfolded rhodopsin from the ER and enhanced its degradation. This suggests that UPR signaling pathways may be used to treat protein misfolding diseases.

(Supported by NIH EY018313, Fight for Sight Foundation, Cystic Fibrosis Foundation, and American Federation for Aging Research)