bluuebunny/crossref_metadata_2025 huggingface dataset:
Magnesium ion disrupts LAP surface re‐association of Listeria monocytogenes by dissociation of InlB
Authors: Liu Dongqi , Bai Xingjian , Tenguria Shivendra , Bailey Talyor , Drolia Rishi , Singh Atul K. , Amalaradjou Mary Anne Roshni , Seleem Mohamed , Bhunia Arun K.
Published: April 2020

Listeria monocytogenes crosses intestinal epithelial barrier for the systemic spread, which is facilitated in part, by Listeria adhesion protein (LAP). LAP, a housekeeping enzyme called acetaldehyde alcohol dehydrogenase, is present in both pathogenic and nonpathogenic Listeria. LAP is secreted with the aid of SecA2 translocon, and its’ re‐association on the surface of pathogenic Listeria i.e., L. monocytogenes is critical for its function. However, the cell wall component(s) involved in LAP interaction is unknown, which was investigated.

Using L. monocytogenes F4244 (WT), an isogenic lap− strain and purified recombinant LAP in the Caco‐2 cell adhesion assay and Western blot, we examined LAP re‐association on lap− cells. Extraneous added LAP restored adhesion capacity of lap−, but not of L. innocua to Caco‐2 cells, suggesting the presence of LAP‐interacting receptor(s) only on pathogenic bacteria.

To locate LAP interaction partners, co‐immunoprecipitation of WT cell wall fraction using anti‐LAP mAb was analyzed by SDS‐PAGE. Selected protein bands were subjected to MALDI‐TOF protein identification showing involvement of internalin B (InlB).

Next, we investigated the effect of MgCl2 pretreatment on LAP re‐association on the cell surface, cell‐free supernatant, and consequent bacterial adhesion. Mg2+ is a wall teichoic acid (WTA) charge neutralizing agent that reduced levels of InlB binding on the WT cell wall, resulting in surface disassociation of LAP. However, only partial surface reduction of LAP was observed in ΔinlB strain suggesting more than one ligand is involved.

In conclusion, our results indicate that both InlB and WTA may participate in LAP re‐association on L. monocytogenes cell surface, which is essential for its pathogenic activities.

Support or Funding Information

The technical assistance of Uma Aryal, Valerie Ryan, and Aaron Gall and funding from USDA‐ARS project number 59‐8072‐6‐001 through the Center for Food Safety Engineering at Purdue University are acknowledged.

LAP Surface Ligand Fishing Expedition

As the experiment scheme shown (A), several Co‐immunoprecipated candidates were visualized by Sliver staining. Red arrow point to a potential receptor around 70 kDa (B). Abundance of each identified protein was graphed according to relative protein abundance (C). Internalin B is the first protein present only in pathogenic strain.

Figure 1

MgCl2 Treatment Affects LAP Dissociation and Bacterial Adhesion

WB protein analysis in Supernatant and Cell Wall Fraction (A), growth curve (B) and adhesion (C) of LmWT grow with additional MgCl2 in TSBYE.

Figure 2
High expression of Nucleobindin‐2 is associated with poor prognosis in gastric cancer
Authors: Okada Junichi , Okada Shuichi , Yamada Masanobu
Published: April 2020

Nucleobindin‐2 (NUCB2) is a 396‐amino acid protein, cleaved into the N‐terminal nesfatin‐11–82, nesfatin‐285–163 and the C‐terminal nesfatin‐3166–396. NUCB2 contains a signal peptide, a leucine zipper structure, two Ca2+ binding EF‐hand domains, and has a wide variety of basic cellular functions. NUCB2 is also a precursor protein of nesfatin‐1, which was originally identified in hypothalamic nuclei, and which is a regulatory factor involved in the central control of food intake and energy balance.

There are several reports indicating that NUCB2 is also expressed in various human peripheral tissues. Moreover, recent studies have reported that high levels of NUCB2 mRNA and protein are a potent prognostic factor for prostate cancer, endometrial carcinoma, and breast cancer. However, the clinicopathological significance of NUCB2 expression in gastric cancer has still not been elucidated. Therefore, we examined NUCB2 expression in a large number of gastric cancer patients, using immunohistochemistry, to explore its clinicopathological significance. To explore this, we aimed to investigate the NUCB2 expression in gastric cancer tissues and adjacent non‐tumor tissues and its potential relevance to clinicopathological factors and prognosis using immunohistochemistry analysis. In our study, NUCB2 level in gastric cancer tissues was higher than in non‐tumor tissues. A high expression of NUCB2 is significantly associated with tumor depth, lymph node metastasis, lymphatic invasion, venous invasion and clinical stage. Furthermore, the expression level of NUCB2 protein was independent predictor of progression‐free survival.

In summary, NUCB2 might play a crucial role in gastric cancer development and could serve as an independent predictor of prognosis of gastric cancer patients.

Progression‐free survival and overall survival curves of 328 gastric cancer patients divided into two groups according to NUCB2 expression levels. The significant differences were given as p = 0.0214 for progression‐free survival and p=0.0211 for overall survival.

Figure 1
Effects of Obesity and Sleep Apnea on Sympathetic Neural Activity during Early Pregnancy in Humans: A Pilot Study
Authors: Hsieh Yi-Jin , Yoo Jeung-Ki , Parker Rosemary S. , Clark Amanda M. , Takeda Ryosuke , Fu Qi
Published: April 2020

Maternal obesity is a major risk factor for adverse pregnancy outcomes and this increased risk is attributed, at least in part, to obstructive sleep apnea (OSA; defined as an Apnea and Hypopnea Index ≥5). However, whether obesity and OSA increase the risk of adverse outcomes through sympathetic neural mechanisms is unknown. We hypothesized that obese women with OSA would have augmented sympathetic nerve activity during pregnancy.

Twenty‐five women within 10 weeks of gestation were studied; 13 of them were normal‐weight (BMI 18.5–24.9 kg/m2) while 12 were obese (BMI ≥30 kg/m2). Home‐based sleep testing was performed using a portable device (WatchPat) and 24‐hour ambulatory blood pressure (BP) was monitored in all subjects. Muscle sympathetic nerve activity (MSNA), beta‐by‐beat BP, and heart rate were continuously recorded during supine rest in our laboratory.

We found that obese pregnant women had higher 24‐hour systolic (mean±SD; 129±13 vs 108±5 mmHg, P<0.001) and diastolic (72±9 vs 62±4 mmHg, P=0.0013) BP compared with normal‐weight pregnant women. Resting MSNA trended to be greater in obese than normal‐weight women (burst frequency: 31±20 vs 19±7 bursts/min, P=0.063; burst incidence: 40±26 vs 26±10 bursts/100 heartbeats, P=0.108; total activity: 358±283 vs 189±95 a.u./min, P=0.075). Preliminary data showed that among obese pregnant women, those with OSA (n=3) had much higher systolic (140±8 vs 125±12 mmHg)and diastolic (82±3 vs 69±8 mmHg) BP compared to those without OSA. In addition, MSNA appeared to be greater in obese women with OSA versus those without OSA (57±12 vs 20±10 bursts/min; 72±21 vs 26±11 bursts/100 heartbeats).

These preliminary results indicate that obesity and OSA may be associated with augmented sympathetic activity and elevated blood pressure early on during pregnancy, leading to the development of adverse maternal‐fetal outcomes.

Support or Funding Information

Supported by the NIH R01 grant (HL142605).

24 hour ambulatory blood pressure was greater in obese pregnant women with sleep apnea than pregnant women with obesity only.

Figure 1

Impact of sleep apnea and maternal obesity on resting MSNA during early pregnancy.

Figure 2
Sildenafil reverses the right ventricular hypertrophy induced by hypoxia but does not bring down the expression of MHC‐β to normal value
Authors: Al-Janabi Mukhallad M. , Khatib Said Y.
Published: April 2020

Introduction

Myosin is the major player in structure and function of cardiac muscle. There are two sub‐types of myosin heavy chain in cardiac muscle; myosin heavy chain‐α (MHC‐α) and myosin heavy chain‐β (MHC‐β). The contractile properties of the cardiac muscle are determined largely by types and the expression of the myosin heavy chain (MHC) which is altered by many physiological and pathological conditions.

Objectives

In this study we investigated the effect of hypoxia and sildenafil treatment on myosin heavy chain isoform in hy poxia ‐ induced hypertrophied right ventricle.

Methods

Right ventricular hypertrophy was induced in 45 mice by exposing them to hypoxic stimulus (11% ambient oxygen) in normobaric chamber for 20 days. The animals were distributed randomly into three groups: first group served as control (CO), second group exposed to hypoxia for 20 days without sildenafil treatment (HY) and third group was given sildenafil orally at dose of 30 mg.Kg−1.day−1 plus exposure to hypoxia for 20 days (HS). MHC isoforms were calculated using two ELISA kits containing antibodies against α and β MHC and by SDS‐PAGE (Mybiosource‐USA). The HY group in comparison with CO group, showed a significant increase in right ventricle weight/left ventricle plus septum ratio (Fulton’s ratio). HS group showed significant decrease in Fulton’s ratio compared with HY but not with CO. Expression of MHC‐β isoform was significantly increased in HY group compared with CO. There was no significant difference in MHC‐β between HY group and HS group. Expression of more beta myosin heavy in hypoxic group is beneficial to the heart since hearts with more MHC‐β have more economic contraction.

Conclusion

sildenafil reversed the right ventricular hypertrophy induced by hypoxia but did not bring down the expression of MHC‐β to normal value.

Support or Funding Information

Deanship of research at Jordan University of Science & Technology

Effects of hypoxia and hypoxia with sildenafil on myosin heavy chain isoforms.

Expression of cardiac myosin heavy chains in different groups: Hypoxia caused expression of more MHC‐β and sildenafil failed in revering the changes caused by hypoxia. Control (CO), hypoxia for 20 days without treatment (HY) and hypoxia for 20 days plus treatment with sildenafil (HS). *** P < 0.01 HY and HS vs CO

Figure 1
Improved Difluoroboron β‐Diketonate Poly(lactic acid) Nanoparticles for Monitoring Wound Oxygenation
Authors: Bruce Anthony C. , Zhuang Meng , DeRosa Christopher A. , Corliss Bruce A. , Fraser Cassandra L. , Peirce Shayn M.
Published: April 2020

We have previously demonstrated the ability of a difluoroboron β‐diketonate poly(lactic acid) material, BF2nbm(I)PLA, hereafter nbm, to detect tissue oxygen levels in murine cutaneous wound beds
1. This relies on oxygen‐insensitive fluorescence (F) as an internal standard and oxygen‐sensitive phosphorescence (P) as a sensor. In an effort to improve imaging agent clearance and ease of manufacture, as well as to optimize emission wavelengths to color channels of RGB cameras, a new dithienyl dye, BF2dtm(I)C12H25‐, hereafter dtm, with red phosphorescence was developed. The dtm dye was mixed with a blue fluorescent dye, BF2dbm(F)OCH12H25, hereafter dbm, and coprecipitated with PLA to form oxygen sensing nanoparticles.

The objective of this study was to verify the utility of the newly developed dtm/dbm nanoparticles for sensing tissue oxygen levels in murine wound beds. To achieve this, we monitored the relative tissue oxygen levels of healing full‐thickness cutaneous wounds in C57BL/6J mice over the course of one week with both dtm/dbm and nbm using an RGB camera mounted to a microscope and custom MATLAB software.

We found the new dtm/dbm nanoparticles yielded nearly identical trends over time in wound bed F/P versus nbm. The dtm/dbm nanoparticles did not impair wound closure and demonstrated less adhesion to healing tissues relative to nbm. Dtm/dbm retains the tissue oxygen‐sensing capability of nbm while having fewer synthetic steps, better phosphorescence alignment with red RGB channel, improved solubility, and less retention in wound tissue than nbm.

Support or Funding Information

UVA‐Coulter Translational Research Project #155234

Verification of dtm/dbm in murine cutaneous wounds

A. Dtm/dbm generated the same trends in wound bed tissue oxygenation over time as nbm. B. Dtm/dbm did not impair wound healing versus nbm or saline control. C. Dtm/dbm displayed reduced particle retention in wounds after multiple applications. Scale bar = 1 mm

Figure 1
Electronic Depth Adjustment in an Automatic Chest Compression Device for Rodents – Design and Implementation of a LabVIEW‐based Control Unit
Authors: Riess Jonathan D. , Balzer Claudius , Riess Matthias L. , Cleveland William J.
Published: April 2020

Introduction

Despite best efforts of cardiopulmonary resuscitation (CPR), many patients still die or suffer severe cardio‐cerebral damage following cardiac arrest (CA). The quality of manual chest compressions (CC) rarely adheres to current guidelines with regards to CC rate, depth and fraction of time spent during CPR. Automated mechanical CC devices offer a reliable improvement in both the clinical arena and in research models. We describe the mechanical and programming aspects of the development of a depth controller for a pneumatic CC device for CPR in rodent CA model.

Methods & Results

In order to electronically control the amplitude/depth of the vertical CC piston (Weil Institute of Critical Care Medicine, Palm Springs, CA; Fig
1) that is pneumatically displaced between 0 and maximally 2 cm 200 times min−1 (duty cycle 1:1), a horizontally moving wedge was connected to a rack gearbox driven by a low‐voltage motor (Fischertechnik, Waldachtal, Germany) along a fixed rack. The unit was connected to a horizontally sliding potentiometer to monitor the exact position of the wedge and, therefore, the piston displacement which itself was monitored through a second, vertically sliding potentiometer. Both potentiometers were connected to an USB 6343 data acquisition system (DAQ, National Instruments, Austin, TX). The motor was connected to two analogue outputs of the DAQ (for movement and direction) via a commercially available low‐voltage amplifier. Stepwise movement of the wedge in either direction could be controlled by a momentary rocker switch connected to two analogue input channels of the DAQ (for deeper and shallower compressions) and its LabVIEW software which was programmed to allow precise control of the wedge’s position.

Conclusions

We report the design and construction of an inexpensive, LabVIEW‐based control unit to electronically adjust CC depth in a rodent model of CA and CPR. Neither the hardware, nor the software required to perform this highly specialized application were commercially available. This custom‐designed and ‐built device achieved its purpose of allowing electronic control and recording of the CC depth in this rodent model with high reliability and precision. Moreover, it constitutes the necessary basis of pre‐programmed or feedback‐assisted adjustments of variable CC depth in the future to improve neurologically favorable outcome after CA.

Support or Funding Information

This work was supported, in part, by institutional funds, and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service.

Pneumatic chest compression device for rodents with added control unit for electronic depth adjustment.

Figure 1
Development of picomolar liver receptor homolog‐1 agonist
Authors: Cato Michael Lee , Cornelison Jeffery , Flynn Autumn , D’Agostino Emma , Jui Nathan , Ortlund Eric
Published: April 2020

Nuclear receptors are ligand‐regulated transcription factors that alter target gene expression in response to the binding of lipophilic signaling molecules. Liver receptor homolog‐1 (LRH‐1) is a nuclear receptor that has shown promise as an anti‐diabetic therapeutic in murine studies and regulates bile acid biosynthesis, steroidogenesis, and glucose homeostasis, making it an attractive target for treating a variety of metabolic diseases. Although phospholipids are the putative endogenous ligands for LRH‐1, they are insoluble, readily remodeled and hydrolyzed, and are only efficacious at very high concentrations, making development of synthetic agonists critical for targeting LRH‐1 in clinical and laboratory contexts. Using structural data that delineated the binding pose of the only class of synthetic agonists known for LRH‐1, we have conducted structure‐activity relationship studies to enhance potency, efficacy, and solubility of small molecules. Specifically, we targeted residues deep within the binding pocket that radically enhance potency, which has led to the first low‐nanomolar LRH‐1 agonist (“6N”). We have also targeted residues contacted by the headgroups of activating phospholipids as a complementary strategy for activation. These modifications resulted in our most efficacious small molecule (“10CA”). To capture the characteristics of both compounds (6N and 10CA), we have synthesized a chimeric small molecule that includes the key moieties of 6N and 10CA responsible for high affinity and efficacy, respectively. Excitingly, this new hybrid compound binds with picomolar affinity, is more efficacious than 10CA, and elicits LRH‐1 target gene expression to a greater degree than either 6N or 10CA alone. The hybrid molecule is our most effective agonist to date, providing a critical tool for both studying LRH‐1 biology and treating a range of metabolic disorders.

Support or Funding Information

NIH‐5T32GM008367‐29 (BCDB Training Grant) | NIH‐5R01DK115213‐02 | Emory Catalyst Program

Binding mode and residue contacts of leading agonists.

Figure 1

Binding and in‐cell reporter data demonstrating affinity, potency, and efficacy of the hybrid compound relative to our two leading agonists.

Figure 2
Exercise‐mediated Wall Shear Stress Attenuates NLRP3 Inflammasome‐induced Endothelial Dysfunction in Obesity
Authors: Lee Jonghae , Hong Junyoung , Umetani Michihisa , Juttu Aishwarya , Park Yoonjung
Published: April 2020

PURPOSE

NLRP3 inflammasome plays a pivotal role in obesity‐induced vascular pathology through caspase‐1‐mediated IL‐1β processing, while precise mechanism by which exercise ameliorates the inflammasome remains unknown. Thus, we investigated the effect of exercise on endothelial dysfunction in obesity via NLRP3 inflammasome‐dependent mechanisms.

METHODS

C57BL/6J male mice were provided a control low‐fat diet (LFD; 4.5% calories from fat) or a high‐fat diet (HFD; 45% calories from fat) with a running wheel access (LF‐RUN or HF‐RUN) or a sedentary no‐wheel condition (LF‐SED or HF‐SED) for 12 to 14 weeks. Endothelium‐dependent vasodilation in response to increasing acetylcholine (ACh, 1 nmol/L to 10 μmol/L) was assessed using intact mesentery arteries isolated from mice. Mouse microvascular endothelial cell (MVEC) line EOMA was treated with palmitate (PA, 20 μmol/L) and laminar shear stress (LSS, 20 dynes/cm2): Static control (STT), PA, LSS, and LSS+PA. The NLRP3 inflammasome axis, including NLRP3, ASC, caspase‐1, p20, and IL‐1β in mice aorta and MVECs was examined by RT‐qPCR, FLICA and DAB staining and western blot.

RESULTS

HFD significantly increased obesity‐associated parameters while these were attenuated by running exercise. Obesity greatly decreased ACh‐induced endothelium‐dependent vasodilation compared to LF‐SED. Exercise improved reduced vasodilator response to ACh in obese mice. Incubation of caspase‐1 inhibitor (10 μmol/L) to the isolated vessel restored obesity‐induced impairment of vasodilator response similar to one of LF‐SED, LF‐RUN, and HF‐RUN. Exercise‐induced decrease of caspase‐1 and IL‐1β in endothelial cells of obese mice aorta was determined by immunohistochemistry using FLICA and DAB, respectively. Likewise, in in vitro experiment, LSS diminished inflammasome components in PA‐treated MVECs.

CONCLUSION

These findings suggest that exercise attenuates against obesity‐induced endothelial dysfunction through a shear stress‐dependent mechanism. Our finding suggests that exercise may be a therapeutic strategy for inflammation‐related vascular dysfunction in obesity.

Exercise attenuates IL‐1β in endothelium of obese mice aorta.

Immunohistochemical analysis of IL‐1β expression (dark brown precipitate) in the endothelial layer of the aorta. Summarized data showing area percentage of the endothelium positive for IL‐1β in aorta (n=4–6). Values are means ± SEM. * P<.05 vs. LFSED; # P<.05 vs. HF‐SED.

Figure 1

Inhibition of caspase‐1 abolishes endothelial dysfunction in mesentery arteries of obese mice.

Incubation of the caspase‐1 inhibitor, Ac‐YVAD‐cmk, in isolated mesentery arteries from obese mice showing improved ACh‐induced endothelium‐dependent vasodilation in HF‐SED + Ac‐YVAD‐cmk (n = 3) similar to the levels of LF‐SED (n=8), HF‐RUN (n = 9) and HF‐RUN + Ac‐YVAD‐cmk (n = 7). Values are means ± SEM. ## P<.01 vs. HF‐SED.

Figure 2
Discovery of novel compound promotes neurogenesis by activation of mTOR signaling
Authors: Ashraf-Uz-Zaman Md , Vaidya Bhuvaneshwar , Abbruscato Thomas , German Nadezhda
Published: April 2020

Neurodegenerative disorders are continually increasing in incidence due to higher life expectancy. Currently, limited therapeutic strategies are available for affected patients, most targeting symptomatic treatment. mTOR pathway is identified as important player in neurogenesis that modulates healthy neuron growth and differentiation. Moreover, many neurodegenerative disorders are associated with downregulated mTOR signaling. However, mTOR activation can also lead to uncontrolled cell proliferation, promoting cancer. This careful balance limits the development of mTOR as a target for modulation of neuronal growth. Recently, selected classes of antidepressants were reported to activate mTOR pathways, causing dendritic outgrowth and increased synaptic protein levels. Although results were promising, they require treatment with up to 100 μM of tested compounds.

To identify potential utilization of penfluridol, an anti‐psychotic agent, as a neuron growth modulator, we have screened this compound and its analogs using reported methodology. Although no activity was observed for PFL, one of its analogs, PF331, promoted neurogenesis in primary neurons. In particular, treatment with PF331 (100 nM,1 μM) for 5 days during reoxygenation (post‐stroke conditions) resulted in improved neurite outgrowth. At the same time, no effect on the viability of the primary neurons was observed under normoxic conditions and during the reperfusion stage. Further, we have established that observed activity was not related to the overstimulation of mTOR pathway, but rather to the compound’s ability to restore mTOR activation to the pre‐stress levels. We hypothesized that PF331 will have minimal impact on cancer proliferation and confirmed it in vivo, where our compound had no effect on cancer growth. Stroke is one of the pathological conditions characterized by hypoxia‐induced damage to neurons. Hence, we have proposed to evaluate our compound for the ability to lessen hypoxia‐induced damage using in‐vivo stroke model with prior confirmation of the ability of PF331 to cross the BBB in vivo and to achieve desired levels in the brain.

In conclusion, PF331 has shown the ability to promote neurite outgrowth in primary neurons and restore mTOR activation in the hypoxic in vitro model. Because of its biological activity and favorable in vivo PK profile, we envision that this compound is a potential candidate for the treatment of neurodegenerative diseases, such as stroke.

Evaluation of the effect of PF331 on primary neurons

Evaluation of the effect of PF331 on of primary neurons. Neurons were subjected to 3 hrs of OGD followed by 4 (A and B)/ 5 (C) days of oxygenation to mimic ischemia reperfusion injury. (A–B) OGD reperfusion (OGDR) down‐regulates mTOR and ERK compared to normoxic (Nor) condition whereas treatment with Compound PF331 activates mTOR and ERK. (C) PF331 facilitates dendritic outgrowth and increased number of synapses.

Figure 1
Ca(II) and Zn(II) synergistically modulate the structure and self‐assembly of Calgranulin C (S100A12)
Authors: Wang Qian , Aleshintsev Aleksey , Jose Aneesha , Aramini James , Brenowitz Michael , Gupta Rupal
Published: April 2020

S100A12 is a member of the Ca(II) binding S100 family of proteins that functions within the human innate immune system. As a metal‐chelator, S100A12 binds both Ca(II) and Zn(II) at structurally distinct sites. Ca(II) binding enhances the Zn(II) binding affinity and the mechanism of the mode of action of Ca(II) is not fully understood. Here, using solution and solid state NMR spectroscopy, we have conducted structural characterization of S100A12 upon Ca(II) and Zn(II) binding. We demonstrate that distinct conformational changes are introduced in helix II and the hinge domain of the protein upon Zn(II) binding to Ca(II)‐S100A12, suggesting that both ions modulate the structure of S100A12. Moreover, Ca(II)‐S100A12 exhibits a broader pH range for Zn(II) binding compared to the apo protein by restricting conformational changes to Zn(II) binding residue, D25. In conclusion, our results indicate that Ca(II) stabilizes Zn(II) binding affinity by modulating structural changes. These findings will be of value for understanding of the nutrient‐sequestration effect of neutrophils and the interaction between the S100A12 and other receptors such as receptor for advanced glycation endproducts (RAGE) and toll‐like receptor 4 (TLR).

Support or Funding Information

This work was supported by the City University of New York startup funds, the National Institutes of Health (R15GM131338) and PSC CUNY award (62234‐00 50) to Rupal Gupta. NMR data presented herein were collected (in part) at the City University of New York’s Advanced Science Research Center (CUNY ASRC) Biomolecular NMR Facility.

MAS NMR spectra of apo and Zn2+‐S100A12. a) Left: DARR (14.1 T) and NCACX (16.4 T) 2D spectra apo S100A12 with resonance assignments; Right: strip plots from CBCACONH (blue) and HNCACB (orange) solution NMR spectra for residues T26‐S28. Signals belonging to an assigned residue in MAS and solution spectra are connected with the same color; b) overlay of Zn2+‐(orange) and apo‐(blue) S100A12 DARR spectra acquired at 14.1 T and; c)–e) expansions of b) with assignments of the perturbed residues.

Figure 1
Companions of Arterial Compliance in Male and Female Heart Failure Patients
Authors: Kerkhof Peter L. , Heyndrickx Guy R. , Li John K-J.
Published: April 2020

Arterial compliance (C) is defined as the ratio of stroke volume (SV) to pulse pressure (PP). Reflecting a major risk factor in terms of overall vascular stiffness, the estimation of C is clearly of clinical importance. As SV and PP each refer to a difference, their numerical values leave some uncertainty which can only be compensated by considering the Pythagorean mean. Therefore we explore the impact of these companion metrics, denoted as SVC and PPC, respectively.

We retrospectively analyze the sex‐specific impact of SVC and PPC in heart failure (HF) patients, using central (aortic) pressure, combined with ventricular volume determinations by 2D angiocardiography. After obtaining informed consent, data were collected in 197 HF patients (130 men) who underwent cardiac catheterization at the Cardiovascular Center in Aalst (Belgium). SV is calculated as end‐diastolic volume (EDV) minus end‐systolic volume (ESV).

Average indexed (i) SVi = 50.46 (±19.65) mL/m2 and average PP = 71.53 (± 27.09), resulting in average C = 0.80 (± 0.45) mL/mmHg.m2. However, PP and PPC are higher (P<0.0001) in women compared to men, yielding 84.8 ± 29.9 vs 64.7 ± 22.7 mmHg, and 167.8 ± 34.1 vs 150.0 ± 26.6 mmHg, respectively. SVi (P=0.45) and SViC (P=0.15) are not different for the sexes. Apart from sex‐related differences it is also important to consider the vast ranges occupied by the companions: 31.4 to 220.4 mL/m2 for SViC, and 79.9 to 282.3 mmHg for PPC. This divergence means that the isolated use of C only provides an incomplete presentation.

In terms of hemodynamics the PPC can be interpreted as mean arterial pressure (MAP), as the two yield high correlations: R=0.948 for men and R=0.950 for women. Thus, PP and MAP emerge as complementary metrics. The SViC can be identified as a surrogate measure of preload, as this metric correlates with EDVi, yielding R=0.852 for men and R=0.952 for women. Therefore, plotting SVi versus its companion is similar to creating an ensemble‐based Starling curve.

For women ESVi is smaller (P=0.003) than in men, implying that all derived metrics such as effective arterial elastance, ejection fraction, SViC and ventriculo‐arterial coupling are affected in a sex‐specific manner.

We conclude that compliance data should be interpreted in a sex‐specific manner, and that the isolated use of C precludes full insight if the contributions of associated companions are neglected.

Arterial compliance can be defined as the ratio of stroke volume index (SVi) to pulse pressure (PP). As SVi and PP are differences between paired measurements, it is required to consider their companion, derived from the Pythagorean mean. This figure illustrates the distribution of the companions in 197 heart failure patients.

Figure 1
Understanding the Pathogenesis of Human Kidney Stone Disease by Spatially Mapping its Proteome
Authors: Canela Victor-Hugo , Bledsoe Sharon B. , Worcester Elaine , El-Achkar Tarek M. , Williams James C.
Published: April 2020

Background

Urolithiasis affects approximately one‐in‐eleven people in the United States, and more than half of affected individuals will have recurrence within ten years of the first episode. A detailed hypothesis of the mechanisms of stone disease etiology remains elusive, and thus difficult to treat and prevent. The present study aims to advance the understanding of the pathogenesis of stone disease by determining the pattern of protein organization within the matrix of human kidney stones.

Methods

The methodological approach of this work relies on an innovative technique to perform histological sectioning of calcium oxalate (CaOx) stones following demineralization. Multi‐photon imaging and label‐free proteomics were used on fluorescently‐identified, laser micro‐dissected specific regions to assess proteome identity and signaling across spatial coordinates within the stone‐matrix.

Results

The average area of laser micro‐dissected samples for proteomic analysis was 1.64×106μm2, and these samples yielded an average of 629 distinct proteins. Dissection of broad regions of CaOx stone by LMD yielded similar proteins as found in larger specimens of pulverized CaOx stone powder. Proteins identified in LMD and pulverized samples included those involved in cell injury and repair as well as important mediators of the immune system. Fatty acid synthase, uromodulin, myosin‐9, osteopontin‐D, and complement C3 are examples of proteins abundant in the stone‐matrix that have been linked to inflammation and regulation of the immune system. More recent results show brilliant autofluorescence of decalcified CaOx stones, which will allow LMD of distinctive regions of the stone without staining.

Conclusion

This is the first demonstration of laser microdissection applied to kidney stones. Proteomic analysis in the different regions along with the interface region will provide invaluable information in understanding the growth of CaOx. These studies will further insight into the underlying signaling events that could become therapeutic targets to prevent stone growth.

Support or Funding Information

Source of Funding: P01 DK056788

LMD of human kidney stones.

Demineralized stone‐matrix on LMD slides were stained with methylene blue (before). Laser microdissection of layers in the stone matrix were successfully collected in phosphate buffer for protein extraction and proteomic analysis (after). Dissection of broad regions of CaOx stone by LMD yielded similar proteins as found in larger specimens of pulverized CaOx stone powder.

Figure 1
Upconversion Nanoparticles Induce Lung Inflammatory, Immunologic and Pulmonary Injury in Vivo
Authors: Chan Ming-Hsien , Hsiao Michael
Published: April 2020

Introduction

Engineered nanoparticles which are developed for multiple uses are defined as those in which the one‐dimensional length is in the range of 1 – 100 nm. Nanoparticles enter the exposure pathway of the organism and are now known to enter the path via the respiratory tract, skin penetration, oral ingestion, and via injection. As an emerging optical nanomaterial, the study of the effects of upconversion nanoparticles (UCNP) on organisms has not yet been discussed. Recently, many studies use this UCNP for lung cancer treatment, so it can be detecting the deposition of the lungs, which will lead to the poisoning of lung tissue.

Challenge

The main effects on the lung tissue caused by the entry of the respiratory tract into the organism are cytotoxicity, inflammatory response, early pulmonary fibrosis, oxidative stress, inhibition of the immune system, increased chance of infection by microorganisms, and even cancer. But the toxicity test of nanoparticles. Technically, there is still no standard test method.

Action

To elucidate the UCNP‐induced cytotoxicity at the molecular level, we try to measure the gene expression changes in human THP‐1 derived macrophages exposed to UCNP for 24 h evaluating be affymetrix microarrays. To analyze the complicated genome‐wide gene expression data, we introduced a computational method to decipher the molecular cytotoxic mechanisms induced by UCNP exposure on the macrophages.

Resolution

Based on the comprehensive and detailed molecular characterization of UCNP‐induced cytotoxicity, to our knowledge, it has not been achieved previously, and this study provides an important insight into the molecular mechanisms involved in UCNP‐induced lung inflammatory, immunologic and pulmonary Injury.

Support or Funding Information

This research was supported by Academia Sinica [AS‐SUMMIT‐108] to MH and Academia Sinica Joint Program Office [AS‐NTU‐108‐01] to MH.

The actual operation of IT.

Figure 1

(a) The schematic imaging shows the IT drug administration. (b) The PLNs persistent luminescence can achieve an in vivo test that arrived at 6 hr. (c) The truly tumor size evaluation of lung tissue. (d) The luminescence of CL1‐5 cell line and PLN fluorescence signal

Figure 2
Electrochemical Reduction of FAD for NSMOA‐Catalyzed Epoxidation Reactions
Authors: Amongre Robert Adongo , Gassner George
Published: April 2020

Styrene monooxygenase (SMO) is a two‐component flavoprotein composed of an NADH‐dependent flavin‐reductase (SMOB) and FAD‐dependent epoxidase (NSMOA). The SMO‐catalyzed enantioselective epoxidation of styrene has potential applications in green‐chemical aqueous phase synthesis and environmental remediation. In the present work, we investigate the possibility of eliminating the NADH‐dependence of this two‐component biocatalyst by developing an electrochemical system that can be substituted for SMOB as the source of reduced FAD as the rate‐limiting step in the styrene epoxidation reaction.

In this work, a miniature spectroelectrochemical cell was constructed and interfaced with a potentiostat that enabled the study of electrode‐mediated flavin‐reduction reactions by cyclic voltammetry and diode array‐based UV‐vis spectroscopy under anaerobic and aerobic conditions. Different results were obtained depending on the composition of the working electrode in terms of the kinetics of FAD desorption from the electrode surface and the applied electrical potential required to achieve FAD reduction. Glassy carbon and graphene‐based working electrodes are observed to readily establish and remain in electrical communication with FAD, but the kinetics of FAD reduction in bulk solution is limited by the slow kinetics of FAD desorption from the surface of these working electrodes. Under aerobic conditions, oxygen diffuses within and reacts with reduced FAD adsorbed to the electrode surface in a reaction which is rapid compared with the kinetics of the desorption of reduced FAD. The implementation of more hydrophilic working electrodes prepared as composites of graphene and polyethylene glycol and the inclusion of electron carriers with oxidation reduction‐potentials in the range of FAD significantly improves the rate FAD desorption and reduction in bulk solution where the rapid kinetics of association of reduced FAD to the epoxidase (NSMOA) can efficiently out compete undesirable side reactions of reduced FAD with dissolved oxygen. In this work, we address the central challenges associated with the electrochemical reduction preparation and transmission of reduced FAD to styrene monooxygenase and report how this can be achieved efficiently by using a combination of cyclic voltammetry, electron mediators and an electrode material with rapid FAD desorption characteristics.

Support or Funding Information

This research is founded by NIH MBRS‐RISE (R25‐GM059298).

Cyclic voltammetry of FAD at the surface of a glassy carbon electrode and saturation plot of the flavin interaction at the electrode surface.

Figure 1

Absorbance spectra recording the reduction of FAD (Left) and consumption of styrene (right) by UV‐Vis spectroscopy.

Figure 2
Single Nuclear Transcriptomes of Raphe Cell Types May be Altered with Repeated Seizures
Authors: Osmani Wasif , Duffy Erin , Manis Anna , Kasmani Moujtaba , Khatun Achia , Mouradian Gary , Forster Hubert , Hodges Matthew
Published: April 2020

Epilepsy affects millions of individuals globally and is one of the most common neurological disorders in the US. A proportion of these individuals who suffer from intractable epilepsy (unresponsive to current treatments) are at an elevated risk for SUDEP (Sudden Unexpected Death in Epileptic Patients), which is more common in males than females. There is general agreement that post‐ictal cardiorespiratory suppression progressively leads to SUDEP. However, the pathophysiology remains unclear. Research has shown neuroinflammation playing a role in seizures and epilepsy. Moreover, two key modulators of brainstem cardiorespiratory control centers, serotonin (5HT) and adenosine, have been shown to play roles in recurrent seizures and animal models of epilepsy. The link between neuroinflammation and these mediators has not been studied. We hypothesized that repeated seizures may lead to a progressive brainstem pathology initiated by neuroinflammation leading to a SUDEP event. We exposed SSkcnj16 −/− rats to daily sound‐induced seizures for 3, 7, or 10 days for comparison to naïve (no seizures) SSkcnj16 −/− rats. Brainstems were rapidly extracted, flash‐frozen, and tissue punches obtained from the Raphe Magnus and processed for extraction of cell nuclei. Single‐nuclear RNAseq (sn‐RNAseq) was performed using a 10x Genomics protocol and sequenced (NovaSeq). Results from the modest numbers of nuclei recovered (~3000 per sample) showed high map rates (80%) to the rat genome. Sequence data was processed through CellRangr and Seurat to generate tSNE and UMAP plots to show clustering of cell types, from which microglia (Arhgap15 expression), astrocytes (Slc4a4 expression), and serotonergic neurons (Tph2 expression) were identified. Additional differential expression analyses within these cell types across conditions (increasing seizure number) are ongoing. Finally, we show for the first time, application of sn‐RNAseq to identify brainstem cell types affected by seizures in a rat model, with the ultimate goal to link neuroinflammation and pathophysiology to gain additional insights into pathophysiology behind SUDEP.

Support or Funding Information

NIH HL 122358

UMAP of clusters generated through Seurat package on R. 12 clusters were generated and identified by genes highly expressed in that specific cluster. Clusters noted here of note are the microglia population, astrocyte population and serotonergic neuron population. Different seizure conditions shown as well.

Figure 1
SPARC: Mechanophysiological analysis of anorectal function using simulated feces
Authors: Sun Daming , Donghua Liao , Gregersen Hans
Published: April 2020

Introduction

Defecation is a complex process that may easily get disturbed. Defecatory disorders are commonly diagnosed with anorectal manometry and the balloon expulsion test. Recently we developed a simulated electronic stool named Fecobionics that integrates several tests and simultaneously assesses pressures, orientation, bending and geometry during expulsion of the device. The aim was to use new analytical tools to calculate frictional force and tension during expulsion of Fecobionics in normal subjects.

Methods

The 12‐cm‐long 12mm OD Fecobionics contained pressure sensors at the front, rear and inside a bag, two motion processor units for measurement of orientation and bending, and impedance rings for measurement of eight cross‐sectional areas/diameters. The geometry of the bag was computed from the cross‐sectional area measurements. Eight presumed normal subjects (4F/4M) defecated Fecobionics. High‐resolution anorectal manometry and the balloon expulsion test confirm that the subjects were normal. The bending angle of the device, the frictional force between the probe and the surrounding tissue, the membrane tension, and the probe expulsion velocity were calculated.

Results

Data examples are shown in figure
1. The bending angle and pressures changed during expulsion of the device with the maximum pressure recorded at the rear. The change in bending angle during defecation was approximately 40 degrees. The averaged circumferential tension, longitudinal tension and friction force change were associated with the pressure difference between the rear and the front ends (R2=0.878±0.011, p<0.005; R2=0.885±0.01, p<0.005; R2=0.411±0.007, p<0.005; respectively). The peak tensions and friction force immediately before expulsion of the rear were significantly higher than that when the front entered into the anal canal (circumferential and longitudinal tension p<0.005, and friction force p<0.05).

Conclusions

Fecobionics obtained reliable data under physiological conditions. Mechanical features such as frictional force and membrane tension were assessable during Fecobionics expulsion using the developed analysis.

Support or Funding Information

NIH SPARC Funding

Top panels: The geometry and the membrane tension distributions of the Fecobionics during expulsion. Bottom panel: The recorded pressures and bending angles in the Fecobionics during expulsion.

Figure 1

Acknowledgements

.

Abbey Chen, Cherry Wong, Wingwah Leung, Kaori Futaba, Tony Mak and Simon Ng are thanked for providing data.
The Concept of Tonicity: Problems and Resolutions
Authors: Kuang Serena Y. , Walter Stefan , Yang Xiaoqi , Li Xiaonan
Published: April 2020

Introduction

In this presentation, we address 6 major problems in the current understanding of the concept of tonicity that uses inconsistent descriptions/definitions of tonicity rooted in the problems with the current definitions of osmolarity (conventional osmolarity, see our abstract #2, “The Concept of Osmolarity: Problems and Resolutions” in EB2020). We then resolve these problems using two new concepts we defined in the abstract #2: 1) OC0 (the initial osmotic concentration (the concentration of the impermeant solute particles (SP)) in S‐m‐H2O (a simple osmosis system consisting of a solution (S) compartment and a water compartment separated by a selectively permeable membrane (either water‐permeable (only permeable to water) or SP‐permeable (permeable to water and some solute particle species). 2) S1‐m‐S2 (a composite osmosis system consisting of two solutions separated by a selectively permeable membrane).

Method

logical reasoning.

Results

1) To understand tonicity, a composite S1‐m‐S2 needs to be deconstructed into two simple osmosis systems that are mirrored (Fig
1). 2) When OC0 is used to eliminate the use of both conventional osmolarity and effective osmolarity (see the abstract #2), the meanings of the two sets of osmolarity and tonicity‐related terms (hyper/iso/hypo‐osmotic and hyper/iso/hypotonic) become consistent: If a solution is hyper/iso/hypo‐osmotic to another solution, it must also be hyper/iso/hypotonic to another solution. 3) Tonicity is a score‐like, relational concept that reflects a comparison between two OC0 in a composite osmosis system (S1‐m‐S2). It does not “belong” to a solution, but between two solutions. Its owner is a given composite osmosis system (S1‐m‐S2). 4) Tonicity can be expressed in multiple ways. 5) Tonicity has three important properties (graphical illustration will be provided in the poster): membrane‐dependency inherited from the membrane‐dependency of OC0 (see the abstract #2), reciprocity, and reference‐solution dependency or relativity. 6) Tonicity has a physical nature (Fig
2): either the ΔOC0 (the concentration gradient between two OC0 across the membrane) or the Δπ(S1‐S2) (the osmotic pressure gradient between S1 and S2). 7) The relationship among CTSP, OC0, and tonicity is illustrated in Fig
2. CTSP is the molar concentration of the total solute particles (TSP) measured in mM, not mOsm/L, defined in the abstract #2.

Deconstruction of a composite S1‐m‐S2 into two simple osmosis systems: S1‐m‐H2O + H2O‐m‐S2.

Figure 1

A panoramic view of the concepts of CTSP, OC0, tonicity as the cause of osmosis (the ΔOC0(SX‐SY)), and tonicity as the effect of osmosis (reflected as the Δπ). Water is symbolized by the rope in the tug‐of‐war analogy. σ: reflection coefficient.

Figure 2
The helical arrangement of nitrilase enzymes determines their substrate size
Authors: Woodward Jeremy David , Kirykowicz Angela Mary , Mulelu Andani Errol , Markus  , Piotrowski  , Sewell Trevor David
Published: April 2020

Nitrilases are commercially important biocatalysts found in diverse taxa that catalyse the hydrolysis of nitriles to amides and acids. In vitro they form rings, terminating spirals or long filaments that seem to be necessary for activity. In vivo, NIT1 assemblies have been observed in Arabidopsis. Substrate size is correlated with the helical twist of nitrilase filaments and can be altered by changing a single residue by site‐directed mutagenesis or by mixing with an excess of enzyme in a different helical conformation. We have determined the first close‐to‐atomic resolution structure of an active nitrilase by cryo‐EM and identified substrate‐specifying residues for site saturation directed evolution. In silico docking of the substrate, β‐L‐cyanoalanine, into the binding pocket led to the identification of a loop that limits the maximum length of a bound substrate and shifts position as a function of helical twist. We propose that the overall size of the binding pocket is specified by this loop, while binding pocket residues alter selectivity for different functional groups. We propose that this mechanism allows nitrilases to rapidly neofunctionalise according to cellular requirements.

Support or Funding Information

We acknowledge Diamond for access and support of the Cryo‐EM facilities at the UK national electron bio‐imaging centre (eBIC), (proposal EM20975‐1), funded by the Wellcome Trust, MRC, and BBSRC. Y. Qayiya, N. Arendse, M. Jaffer, B. Weber, and S. Neumann for technical assistance. This work was funded by the South Africa/Germany Cooperation in Science and Technology Joint Research Grant, the Deutsche Forschungsgemeinschaft (PI 424/1, PI 424/3), Research Career Advancement Fellowship from the National Research Foundation of South Africa (92556) and GCRF‐START (UK Science and Technology Facilities Council grant ref. ST/R002754/1).